Saturday, 7th November 2015
|17.00 – 21.00||Secretariat Meeting||E' Carina|
Sunday, 8th November 2015
|09.00 – 16.00||Registration Open at RIKEN|| Koryuto Hall, RIKEN
|09.30 – 11.45||Bioinformatics workshop||Koryuto Hall, RIKEN|
|11.30 – 13.55||Optional RIKEN lab tours
(11.55 - 12.45 / 12.30 - 13.20 / 13.05 - 13.55)
|meet at Koryuto Hall, RIKEN|
|11.45 – 13.30||Lunch|| Cafeteria 2nd floor Koryuto Hall
|13.30 – 18.00||Trainee Symposium
||Koryuto Hall, RIKEN|
|19.00 – 21.00||Registration Open at Welcome Reception|| Fisherman's Market
|19.30 – 21.00||Welcome Reception||Fisherman's Market|
Monday, 9th November 2015 (all events at Yokohama Port Opening Memorial Hall)
|09.00 – 19.30
||Poster Space Open (Odd Posters on Display)
|| Rooms No. 1 & 7
|09.15 – 9.30||Opening Greetings|
|9.30 – 11.00||Plenary Session:
Human Disease Models & Immunology I
|11.00 – 11.30||Break||Rooms No. 4, 6 & 9|
|11.30 – 13.00||Plenary Session:
Neuroscience, Development & Stem Cells
|13.00 – 14.00||Lunch|| Rooms No. 4, 6 & 9 and Sei-ren
|13.00 – 14.00||Mentor Lunch|| E' Carina
|14.00 – 15.00||Plenary Session:
Genomics and Computational Analysis I
|15.00 – 16.00||IMGS Business Meeting (All are encouraged to participate)|
|16.00 – 18.00||Exhibition & Posters (Odd)|| Rooms No. 1 & 7
|18.00 – 19.00||Plenary Session:
Human Disease Models & Immunology II
|19.30 – 21.30||Systems Genetics Workshop (Optional)|
|19.30 – 21.30||Scientific Literature Curation (Optional)|
|Evening free for delegates' own dinner plans|
Tuesday, 10th November 2015
|09.00 – 19.30
||Poster Space Open (Even Posters on Display)||Rooms No. 1 & 7|
|09.15 – 10.45||Plenary Session:
Human Disease Models & Immunology III
|10.45 – 11.35||Mini Session:
Is the mouse still needed as a human disease model?
|11.35 – 12.00||Break||Rooms No. 4, 6 & 9|
|12.00 – 13.00||Plenary Session:
Epigenomics and noncoding RNAs I
|13.00 – 14.00||Lunch||Rooms No. 4, 6 & 9 and Sei-ren|
|13.00 – 14.00||Nomenclature Lunch Meeting||Anteroom|
|13.00 – 14.00||Luncheon seminar|| La bar a vin 52
|14.00 – 14.45||Keynote Lecture: Masayo Takahashi|
|14.45 – 15.30||Plenary Session:
Advances in Genome Editing
|15.30 - 17.30||Exhibition and Posters (Even)/Break||Rooms No. 1 & 7|
|17.30 – 19.00||Plenary Session:
Genomics and Computational Analysis II
|19.30 – 21.30||Gene Enrichment Analysis Workshop (Optional)|
|19.30 – 21.30||FANTOM Workshop (Optional)|
|Evening free for delegates' own dinner plans|
|Wednesday, 11th November 2015|
|09.15 – 10.30||Plenary Session:|
|Large-scale resources I|
|DMDS Lecture: Janan Eppig|
|10.30 – 11.00||Break||Rooms No. 4, 6 & 9|
|11.00 – 12.00||Verne Chapman Lecture: John Mattick|
|12.00 – 13.00||Plenary Session:
Epigenomics and noncoding RNAs II
|13.00 – 14.00||Lunch||Rooms No. 6 & 9 and Sei-ren|
|13.00 – 14.00||Lunchtime Secretariat Meeting||Anteroom|
|13.00 – 14.00||Luncheon seminar||La bar a vin 52|
|13.00 – 14.00||Lunchtime Mammalian Genome Editorial Board Meeting|| Room. No. 4
|14.00 – 14.45||Keynote Lecture: Hideyuki Okano|
|14.45 – 15.30||Plenary Session:
Large-scale resources II
|15.30 – 16.00||Break||Rooms No. 4, 6 & 9|
|16.00 – 17.00||Plenary Session:
Genomics and Computational Analysis III
|18.00||Bus from YPOMH to Conference Dinner|
|19.30||Conference Dinner and Awards Ceremony|| The Manyo Club
Saturday, 7th November 2015
|17.00 – 21.00||Secretariat Meeting||E' Carina|
Sunday, 8th November 2015
|09.00 – 16.00||Registration Open at RIKEN||Koryuto Hall at RIKEN
|09.30 – 11.45||Bioinformatics workshop||Koryuto Hall at RIKEN|
|11.30 – 14.00||Optional RIKEN lab tours
(11.55-12.45 / 12.30-13.20 / 13.05-13.55)
|meet at Koryuto Hall at RIKEN|
|11.45 – 13.30||Lunch||Cafeteria 2nd floor Koryuto Hall
|13.30 – 18.00||Trainee Symposium
||Koryuto Hall at RIKEN|
|13.30||TS-01: "Development of innovative therapeutic strategy using DNA minor groove binder-drug conjugate in MYCN-driven neuroblastoma", Hiroyuki Yoda|
|13.45||TS-02: "Mapping SARS-Coronavirus susceptibility alleles using the Collaborative Cross", Lisa Gralinski|
|14.00||TS-03: "Transcriptional control of hibernation: first insights on comparative genomics of dormice", Guzel Gazizova|
|14.15||TS-04: "Combination of selective breeding and genome-wide SNP analysis revealed the genetic loci associated with tame behavior in mice ", Yuki Matsumoto|
|14.30||TS-05: "Conservation and evolution of splicing patterns during postnatal development of prefrontal cortex in primates", Pavel Mazin|
|14.45||TS-06: "Conservative miRNA target analysis: are we limiting our discoveries of neuronal miRNA function?", Belinda J Goldie|
|15.00||TS-07: "Dissecting the regulation of olfactory receptor expression in the mouse.", Ximena Ibarra-Soria|
|15.15||TS-08: "Horses: an underutilized animal model", Brandon Velie|
|15.30 – 16:00||Break|
|16.00||TS-09: "Higher expression of Adcyap1 gene is associated with altered behavioral and prolonged physiological responses to stress in wild-derived MSM mice", Akira Tanave|
|16.15||TS-10: "Analysis of three human genomic loci associated with Tetralogy of Fallot.", Gennadiy Tenin|
|16.30||TS-11: "Cognitive Endophenotypes of Modern and Extinct Hominins Associated With NTNG Gene Paralogs", Pavel Prosselkov|
|16.45||TS-12: "Skin Megagenetics - Novel skin phenotypes revealed by a genome-wide mouse reverse genetic screen", Kifayathullah Liakath-Ali|
|17.00||TS-13: "KRAS mutation specific alkylating pyrrole-imidazole polyamide, KR12 showed significant anti-tumor efficacy and preferential localization in KRAS mutant xenografts without adverse effects", Takahiro Inoue|
|17.15||TS-14: "Development of HTS system to optimize SINEUPs, antisense long-noncoding RNAs that increase translation of target mRNAs", Hazuki Takahashi|
|17.30||TS-15: "Cell-cell-communication in cancer", Riti Roy|
|17.45||TS-16: "ENU mutagenesis identifies a novel molecular pathomechanism of severe immunodeficiency", Irina Treise|
|18.15 –||Bus from RIKEN to Welcome Reception|
|19.00 – 21.00||Registration Open at Welcome Reception|| Fisherman's Market
|19.30 – 21.00||Welcome Reception||Fisherman's Market|
Monday, 9th November 2015 (all events at Yokohama Port Opening Memorial Hall)
|09.00 – 19.30
||Poster Space Open (odd numbered posters)|| Rooms 1 & 7
|09.15 – 9.30||Opening Greetings|
|9.30 – 11.00||Plenary Session:
Human Disease Models & Immunology I
|9.30||O-01: "Patient Derived Xenografts (PDX) Models of Human Breast Cancer: A Platform for Precision Oncology", Carol J Bult|
|9.45||O-02: "Novel ENU-induced ankyrin 1 mutations reveal complex role of erythrocyte cytoskeleton plays during malaria infection", Gaetan Burgio|
|10.00||O-03: "Genetic mechanism of aerobic capacity and metabolic disease in Rat model", Yu Wang|
|10.15||O-04: "Analysis of murine resistant and susceptible transcriptomes in plague", Jean Jaubert|
|10.30||O-05: "Emergence of extreme phenotypes and new disease models in the Collaborative Cross: from bronchiectasis to parkinsonism", Fernando Pardo-Manuel de Villena|
|10.45||Invited talk from Trainee Session|
|11.00 – 11.30||Break|| Rooms 4, 6 & 9
|11.30 – 13.00||Plenary Session:
Neuroscience, Development & Stem Cells
|11.30||O-06: "Mouse embryonic stem cell lines derived in 2i culture conditions demonstrate X Chromosome silencing and extreme male sex bias.", Anne Czechanski|
|11.45||O-07: "Endogenous L1 Retrotransposition in the Mammalian Early Embryo and Primordial Germline", Sandra R. Richardson|
|12.00||O-08: "The complex transcriptional landscape of the C9orf72 gene locus the most common cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)", Patrizia Rizzu|
|12.15||O-09: "Dissection of Genetic Redundancy: A Novel Role for FGF Signaling During Mouse Eye Development", Yasuhide Furuta|
|12.30||O-10: "Molecular biomarkers of neurodegenerative disease in the olfactory epithelium", Gabriela Sanchez-Andrade|
|12.45||Invited talk from Trainee Session|
|13.00 – 14.00||Lunch|| Rooms 4, 6 & 9 and Sei-ren
|13.00 – 14.00||Mentor Lunch||E' Carina|
|14.00 – 15.00||Plenary Session:
Genomics and Computational Analysis I
|14.00||O-11: "A tale of two gene sets: low and high variability in single cell RNA-seq data", Anna Mantsoki|
|14.15||O-12: "Single-molecule RNA sequencing in single cells.", Charles Plessy|
|14.30||O-13: "Digital expression profiling of Purkinje neurons and dendrites in subcellular resolution", Anton Kratz|
|14.45||Invited talk from Trainee Session|
|15.00 – 16.00||IMGS Business Meeting (All are encouraged to participate)|
|16.00 – 18.00||Exhibition & Posters (Odd)||Rooms 1 & 7|
|18.00 – 19.00||Plenary Session:
Human Disease Models & Immunology II
|18.00||O-14: "Time-dependent host response to influenza A virus infection in Collaborative Cross founder strains and lines", Heike Kollmus|
|18.15||O-15: "A forward-reverse systems genetics approach to understand host-pathogen genetic interactions", Clare Smith|
|18.30||O-16: "Mapping thrombosis modifier genes by bulk exome sequencing mice from a sensitized ENU mutagenesis screen ", Kart Tomberg|
|18.45||Invited talk from Trainee Session|
|19.00 – 19.30||Remove Posters||Rooms 1 & 7|
|19.30 – 21.30||Systems Genetics Workshop (Optional)|
|19.30 – 21.30||Scientific Literature Curation (Optional)|
|Evening free for delegates' own dinner plans|
Tuesday, 10th November 2015
|09.00 – 19.30
||Poster Space Open (even numbered posters)||Rooms 1 & 7|
|09.15 – 10.45||Plenary Session:
Human Disease Models & Immunology III
|9.15||O-17: "A systematic functional screening approach to identify candidate genes obtained through large scale whole genome and exome sequencing of human disease cohorts.", Peter Heutink|
|9.30||O-18: "Natural allelic variation of interleukin 21 receptor modulates ischemic stroke outcomes", Han Kyu Lee|
|9.45||O-19: "Identification of novel susceptibility genes for aggressive prostate cancer using Diversity Outbred mice", Nigel Crawford|
|10.00||O-20: "Strong epistatic control of development of leishmaniasis.", Tatyana Kobets|
|10.15||O-21: "Metabolic regulation by the MECP2 transcriptional repressor complex points to new therapeutic targets in Rett syndrome", Stephanie M Kyle|
|10.30||O-22: "Mouse Models of Human Diaphragmatic Birth Defects: The mesothelium performs a fundamental role in proper formation of the diaphragm", Kate Ackerman|
|10.45 – 11.35||Mini Session "Is the mouse still needed as a human disease model?"|
|O-23: "Genomic responses in mouse models greatly mimic human inflammatory diseases", Tsuyoshi Miyakawa|
|11.35 – 12.00||Break||Rooms 4, 6 & 9|
|12.00 – 13.00||Plenary Session:
Epigenomics and noncoding RNAs I
|12.00||O-24: "Bipartite structure of the inactive mouse X Chromosome", Christine M Disteche|
|12.15||O-25: "Genetic and dietary effects on gametic selection at fertilization", Joseph Nadeau|
|12.30||O-26: "Epigenetic inheritance of diet induced obesity and diabetes via oocyte and sperm", Johannes Beckers|
|12.45||O-27: "Exploring regulatory networks through omics data", S Sethi|
|13.00 – 14.00||Lunch|| Rooms 4, 6 & 9 and Sei-ren
|13.00 – 14.00||Nomenclature Lunch Meeting||Anteroom|
|13.00 – 14.00||Luncheon seminar||La bar a vin 52|
|14.00 – 14.45||Keynote Lecture:|
|O-28: "Retinal cell therapy using iPS cells", Masayo Takahashi|
|14.45 – 15.30||Plenary Session:
Advances in Genome Editing
|14.45||O-29: "CRISPR/Cas9 genome editing in rodents: In vivo and in vitro applications", Marie-Christine Birling|
|15.00||O-30: "CRISPR/Cas9-mediated plasmid knock-in and replacement of genomic region with single stranded oligonucleotides in rodents", Kazuto Yoshimi|
|15.15||O-31: "CRISPR-Driven Replacement of a Mouse Tumor Suppressor with 25-kbp of the Orthologous Human Gene", David Bergstrom|
|15.30 - 17.30||Exhibition and Posters (Even)/Break||Rooms 1 & 7|
|17.30 – 19.00||Plenary Session:
Genomics and Computational Analysis II
|17.30||O-32: "The Estimation of Selective Effects Using Large Scale Population Data Identifies Genes Required For Normal Mammalian Development.", David Beier|
|17.45||O-33: "The landscape of replication associated mutations in the human and mouse germlines", Martin Taylor|
|18.00||O-34: "Paradoxical evolution of a large segmental duplication in mouse", Andrew P Morgan|
|18.15||O-35: "The frequent evolutionary birth and death of functional promoters in mouse and human", Robert Young|
|18.30||O-36: "Elucidation of the Underlying Mechanism of the Induction of Pluripotency Genes by SAHA-Conjugated Pyrrole-Imidazole Polyamides by Computational Genomic Analysis", Jason Lin|
|18.45||O-37: "Capybara genome sequencing offers deeper insights into rodent evolution", Isaac Adeyemi Babarinde|
|19.00 – 19.30||Remove Posters||Rooms 1 & 7|
|19.30 – 21.30||Gene Enrichment Analysis Workshop (Optional)|
|19.30 – 21.30||FANTOM Workshop (Optional)|
|Evening free for delegates' own dinner plans|
|Wednesday, 11th November 2015|
|09.15 – 10.30||Plenary Session:
Large-scale resources I
|9.15||O-38: DMDS Lecture "The Short Story of a Long Tale", Janan Eppig|
|9.45||O-39: "Informatics for the International Mouse Phenotyping Consortium- a Platform for Phenotypic and Translational Discovery", Terrence F Meehan|
|10.00||O-40: "Systemic metabolic phenotyping in the German Mouse Clinic in search of new mouse models for metabolic disorders", Jan Rozman|
|10.15||O-41: "Genetic architecture of behavior in an advanced intercross line of mice", Natalia M. Gonzales|
|10.30 – 11.00||Break||Rooms 4, 6 & 9|
|11.00 – 12.00||Verne Chapman Lecture:|
|O-42: "The hidden layer of regulatory RNA in mammalian genome biology", John Mattick|
|12.00 – 13.00||Plenary Session:
Epigenomics and noncoding RNAs II
|12.00||O-43: "In vivo profiling of the promoter- and enhancer landscape of inflammatory bowel disease ", Albin Sandelin|
|12.15||O-44: "Enhancers lead waves of coordinated transcription in transitioning mammalian cells", Erik Arner|
|12.30||O-45: "Network architecture of microRNA regulation in mammalian cells", Michiel De Hoon|
|12.45||O-46: "Regulated mobilization of Retrotransposable elements in cell identity, reprogramming and disease", Valerio Orlando|
|13.00 – 14.00||Lunch|| Rooms 6 & 9 and Sei-ren
|13.00 – 14.00||Lunchtime Secretariat Meeting||Anteroom|
|13.00 – 13.45||Luncheon seminar|| La bar a vin 52
|13.00 – 14.00||Lunchtime Mammalian Genome Editorial Board Meeting|| Room 4
|14.00 – 14.45||Keynote Lecture:|
|O-47: "Modeling Psychiatric/Neurological disorders using iPS cell technologies and transgenic non-human primates.", Hideyuki Okano|
|14.45 – 15.30||Plenary Session:
Large-scale resources II
|14.45||O-48: "GENCODE: revealing transcriptional complexity in Human and Mouse", Mark Thomas|
|15.00||O-49: "FANTOM6: Functional elucidation of lncRNA", Jay W Shin|
|15.15||O-50: "Multiple mouse reference genomes and strain specific gene annotations", Thomas Keane|
|15.30 – 16.00||Break||Rooms 4, 6 & 9|
|16.00 – 17.00||Plenary Session:
Genomics and Computational Analysis III
|16.00||O-51: "A survey of genome rearrangements in human evolution", Martin Frith|
|16.15||O-52: "An experimental approach to elucidate enigmatic isochore evolution by using ENU mutagenesis", Satoshi Oota|
|16.30||O-53: "Post-translational mechanisms buffer protein abundance against transcriptional variation.", Steven Munger|
|16.45||O-54: "Analysis of energy demands during lactation in mouse models", Peter Williamson|
|18.00||Bus from YPOMH to Conference Dinner|
|19.30 – 22.30||Conference Dinner and Awards Ceremony|| The Manyo Club
|P-001||"Abnormal Innate Immune Responses of ENU-induced Ali18 and Ali14 Mutant Mice Lead to Autoinflammatory Syndrome-like Phenotypes", Koichiro Abe|
|P-002||"Xist/Tsix expression dynamics during mouse peri-implantation development revealed by whole-mount 3D RNA-FISH", Kuniya Abe|
|P-003||"Towards the Creation of Reference Transcription Start Site Set (refTSS)", Imad Abugessaisa|
|P-004||"Somatic variations in healthy skin fibroblasts and their relation to cancer and aging", Alexej Abyzov|
|P-005||"Elevated canonical Wnt signalling disrupts development of the embryonic midline and can cause Heterotaxy", Ruth Arkell|
|P-006||"Targeted reduction of highly abundant transcript with pseudo-random primers", Ophelie Arnaud|
|P-007||"F5-explorer: an interactive webserver for quick gene-oriented browsing of FANTOM5 data ", Frederik Otzen Bagger|
|P-008||"Multimer formation explains allelic suppression of PRDM9 recombination hotspots ", Christopher Baker|
|P-009||"Influence of diet on metabolic syndrome in four genetically diverse mouse strains", William Barrington|
|P-010||"Characterization and mapping of colimba, a new spontaneous mouse mutation with hair coat abnormalities.", Fernando Benavides|
|P-011||"Cilia and Ciliopathogies: Impact of new knowledge on our understanding of biology and human disease", Judith Blake|
|P-012||"Single-cell transcriptomes of fluorescent, ubiquitination-based cell cycle indicator cells", Michael Boettcher|
|P-013||"Analysis of ENU mutant mice indicates the existence of non-exomic thrombosis modifier mutations", Marisa A Brake|
|P-014||"Shape-based morphometric analysis of homozygous lethal embryos imaged by micro-CT", James M Brown|
|P-015||"The JAX Synteny Browser: A new visualization tool for mouse-human comparative genomics", Carol J Bult|
|P-016||"Modeling Binding Affinity of the Multiple Zinc-Finger Protein PRDM9", Gregory W Carter|
|P-017||"Mapping Genomic Distributions of Combinatorial Histone Modifications at the Single Molecule Level", Jen-Chien Chang|
|P-018||"Zebrafish serves as a disease model system to investigate the embryonic development effects of human parvovirus B19- NS1 and VP1u protein", Ju Chang-Chien|
|P-019||"Investigation of defective spermiogenesis in a human XLID mouse model", Chun-Yu Chen|
|P-020||"MECOM (EVI1 or PRDM3) maintains neuronal stem cell self-renewal through chromatin control over RBPJ recruitment.", Elaine KY Chung|
|P-021||"The ORFeome Collaboration: A community resource for expression of most human protein-coding genes", The ORFeome Collaboration|
|P-022||"KCNQ1 and CFTR act as tumor suppressors in colorectal cancer", RT Cormier|
|P-023||"Whole-exome sequencing of ENU-induced mutant mice with BALB/c background", TA de Souza|
|P-024||"A selfish genetic element drives recurrent selective sweeps in the house mouse", John P Didion|
|P-025||"Implication of truncated CABLES1 in agenesis of the corpus callosum", TH Tra Dinh|
|P-026||"Association Analysis of -1997 Polymorphism in Upstream Regulatory Site of COL1A1 with Low Bone Mineral Density: A Study on Iranian Post-menopausal Women", Mina Ebrahimi-Rad|
|P-027||"Genetic control of extreme Influenza disease", Martin T Ferris|
|P-028||"The environmental toxicant Tetrabromobisphenol-A promotes adipogenesis by downregulating THY1 (CD90) in human mescenchymal stem cells.", E"Lissa Flores|
|P-029||"Genetics of Meiotic Recombination Rate", Jiri Forejt|
|P-030||"Meiotic Arrest, Crossover Paterns, and Hybrid Sterility of [BALB/cJxJF1/Ms] F1 Male Mice", Yasuhiro Fujiwara|
|P-031||"ENU mutation cataloging in the RINEN ENU mutant mouse library using whole exome analysis with Ion Proton sequencer.", Ryutaro Fukumura|
|P-032||"Correlation of Trp53cor1 and Trp53 expression in the Trp53cor1 gene trap mouse line", Riki Furuhata|
|P-033||"Maternal malnutrition alters gene expression, genomic methylation and behavioral phenotypes of progeny", Tamio Furuse|
|P-034||"Transcriptional control of hibernation: first insights on comparative genomics of dormice", Guzel Gazizova|
|P-035||"Conservative miRNA target analysis: are we limiting our discoveries of neuronal miRNA function?", Belinda J Goldie|
|P-036||"Needs of fundamental revision of mouse genome reference sequences.", Yoichi Gondo|
|P-037||"Mapping SARS-Coronavirus susceptibility alleles using the Collaborative Cross", Lisa Gralinski|
|P-038||"SINEUPs: a new class of natural and synthetic antisense long non-coding RNAs that activate translation.", Stefano Gustincich|
|P-039||"Genome-wide mapping of hyper-acetylated chromatin with a novel antibody in lung cancer cells", Lusy Handoko|
|P-040||"Up-regulation of non-coding RNAs in adult and pediatric liver cancers", Kosuke Hashimoto|
|P-041||"Dissecting the contribution of genes from the 17q21.31 region in the Koolen-deVries deletion syndrome using the mouse", Yann Herault|
|P-042||"Effects of early-life exposure to Western diet on adult activity levels and associated behavioral and physiological traits in mice", Layla Hiramatsu|
|P-043||"An atlas of 5’ complete transcripts reveals the genomic origins and expression landscape of human long non-coding RNAs", Chung-Chau Hon|
|P-044||"CAGE revealed novel biomarker of periodontitis-associated fibroblasts", Masafumi Horie|
|P-045||"Evidence of a direct role for endothelial cells in the development of acute leukemias", Viive Howell|
|P-046||"Identification of key regulators contributing to the different responses of transforming growth factor beta in A549 cells by single-cell transcriptome", Yi Huang|
|P-047||"Dissecting the regulation of olfactory receptor expression in the mouse.", Ximena Ibarra-Soria|
|P-048||"KRAS mutation specific alkylating pyrrole-imidazole polyamide, KR12 showed significant anti-tumor efficacy and preferential localization in KRAS mutant xenografts without adverse effects", Takahiro Inoue|
|P-049||"Optimization of a Mathematical Model for Explanation of Biological Functions Using Monte-Carlo Methods and Parallel Computing", Kazuo Ishii|
|P-050||"Molecular and functional characterization of Angelman syndrome patient-derived iPSCs and neurons", Mitsuru Ishikawa|
|P-051||"Recurrent Transcriptome Alterations Across Multiple Cancer Types", Bogumil Kaczkowski|
|P-052||"Analysis of mutational landscape in protein domains across a broad spectrum of pathological conditions", Alexander Kanapin|
|P-053||"Variation in transcriptional response to 1,25-dihydroxyvitamin D3 and bacterial lipopolysaccharide in primary human monocytes", Silvia Kariuki|
|P-054||"Versatile instrument for efficient single cell collection and deposition", Stanislav Karsten|
|P-055||"The function of non-coding RNA in stem cell maintenance and differentiation", Kaori Kashi|
|P-056||"SEC23A functionally compensates for SEC23B deficiency in mice", Rami Khoriaty|
|P-057||"Developing mouse models of open angle glaucoma using large scale ENU mutagenesis", Stephen C Kneeland|
|P-058||"Establishing new mouse resource, wild-derived heterogeneous stock, which is useful for genetic analysis of tameness and other complex traits", Tsuyoshi Koide|
|P-059||"Does inter-subspecific and -specific swapping of Prdm9 ZFA affect recombination and reproduction in mice?", Hiromitsu Kono|
|P-060||"C1 CAGE: Quantifying coding and non-coding RNAs at single-cell and single molecule level", Tsukasa Kouno|
|P-061||"Targeting PIK3CA gene by Pyrrole Imidazole polyamide seco-CBI conjugates in cervical cancer ", Sakthisri Krishnamurthy|
|P-062||"Role of a novel asRNA in human white adipose differentiation and metabolism", Hiroko Kushige|
|P-063||"Transcriptome analysis of controlled and therapy-resistant childhood asthma reveals distinct gene expression profiles", Andrew T Kwon|
|P-064||"A mouse model of development syndrome associated with kaptin (Kptn) mutations", MO Levitin|
|P-065||"Collateral damage: Identification and characterisation of spontaneous mutations causing deafness from a targeted knockout programme", Morag Lewis|
|P-066||"The long non-coding RNA NEAT1 in cell biology, cancer and paraspeckle function", Ruohan Li|
|P-067||"Alkaline ceramidase 1 (Acer1) is indispensable for mammalian skin homeostasis and thermoregulation", Kifayathullah Liakath-Ali|
|P-068||"Skin Megagenetics - Novel skin phenotypes revealed by a genome-wide mouse reverse genetic screen", Kifayathullah Liakath-Ali|
|P-069||"Mapping histon modification marks for activated enhancers genome wide by ChIP of articular cartilage and underlying subchondral bone in human osteoartritic knees", Ye Liu|
|P-070||"Gateways to the FANTOM5 promoter level mammalian expression atlas", Marina Lizio|
|P-071||"Diversity Outbred Mice Indicate Idiosyncratic Drug-Induced Liver Injury Potential", LE Lyn-Cook|
|P-072||"Age-related retinal abnormalities and Bloom Syndrome", Erica Macke|
|P-073||"Homeostasis of motifs for transcription factors binding withstanding cancer somatic mutations", Vsevolod Makeev|
|P-074||"Production of a truncated protein from the Gli3 gene with a frameshift mutation, which is introduced by the CRISPR/Cas9 system", Shigeru Makino|
|P-075||"Development of Semantic Web/RDF based integrated database of experimental animals", Hiroshi Masuya|
|P-076||"Combination of selective breeding and genome-wide SNP analysis revealed the genetic loci associated with tame behavior in mice ", Yuki Matsumoto|
|P-077||"Conservation and evolution of splicing patterns during postnatal development of prefrontal cortex in primates", Pavel Mazin|
|P-078||"Site-Directed Endonuclease Mutagenesis: Naming Mutations", Monica McAndrews|
|P-079||"EpiFACTORS: a comprehensive database of human epigenetic factors and complexes", Yulia Medvedeva|
|P-080||"Cell-cycle classification at the single-cell level with Random Forest", Mickael Mendez|
|P-081||"Updates about the Collaborative Cross and Features of the Systems Genetics Core Facility at UNC", Darla R Miller|
|P-082||"CRISPR/Cas9-based generation of knockdown mice using long single-stranded DNA", Hiromi Miura|
|P-083||"The Integrated Transcriptome Analysis of Adipocyte and Osteoblast Differentiation", Yosuke Mizuno|
|P-084||"Reliable and efficient analysis of non-coding DNA elements in vivo at the mouse Tyr locus using CRISPR-Cas9 mutagenesis", L Montoliu|
|P-085||"Identification of novel chimeric transcripts associated with human-specific retroposed gene copies", Saori Mori|
|P-086||"Gene-trap mutagenesis is useful for analysis of long intergenic non-coding RNA genes.", Mai Nakahara|
|P-087||"Exploring new gene integration sites for gene knock-in by gene-trapping strategy.", Isamu Nanchi|
|P-088||"Identification of Genetic Susceptibility Loci to Alveoler Bone Loss Affected by Type 2 Diabetes Induced by High-Fat-Food Using Collaborative Cross Mice", Aysar Nashef|
|P-089||"Efficient State of the Art generating of mutant mouse models under full cost accounting conditions in relation to the 3 R's and personnel management", Ronald Naumann|
|P-090||"Rat models for complex human disease: utilizing phenotypes and genotypes to identify the desired strains", Rajni Nigam|
|P-091||"Single-cell data integration platform", Shuhei Noguchi|
|P-092||"rRNA depletion for low quantity RNA-seq involving coding and non-coding RNA", Shohei Noma|
|P-093||"Partition Heritability of Variants in Gene Regulatory Regions for Complex Traits in Mice", Hiroko Ohmiya|
|P-094||"A Spontaneous and Novel Pax3 Mutant Mouse That Models Waardenburg Syndrome and Neural Tube Defects", Tetsuo Ohnishi|
|P-095||"GONAD: a novel CRISPR/Cas9 genome editing method that does not require ex vivo handling of fertilized eggs", Masato Ohtsuka|
|P-096||"A cancer modifier role for Parathyroid Hormone in mouse skin carcinogenesis", Kazuhiro Okumura|
|P-097||"A new framework to analyze a mouse aberrant gait pattern by using a neuro-musculoskeletal model", Satoshi Oota|
|P-098||"Genetic dissection of Rift Valley fever pathogenesis: Rfvs2 on mouse Chromosome 11 impacts tolerance to early onset hepatitis", Jean-Jacques Panthier|
|P-099||"Myeloid cell specific interferon hyporesponse contributes to Rift Valley fever susceptibility and virus induced sepsis", Jean-Jacques Panthier|
|P-100||"Epigenetic Studies Reveal the Role of Genetic Background in Corticosteroid Response in Mouse Hepatocytes", Petko M Petkov|
|P-101||"Transcriptome analysis of FACS-sorted single cells with nanoCAGE", Stephane Poulain|
|P-102||"Cognitive Endophenotypes of Modern and Extinct Hominins Associated With NTNG Gene Paralogs", Pavel Prosselkov|
|P-103||"A draft network of ligand-receptor mediated multicellular signaling in human", Jordan A Ramilowski|
|P-104||"Insights into aberrant methylation enhancer regions in hepatocellular carcinoma", Claire Renard-Guillet|
|P-105||"Serotonin receptor HTR3A in the development of sacral autonomic and sensory ganglia", K Elaine Ritter|
|P-106||"Cell-cell-communication in cancer", Riti Roy|
|P-107||"Computer Simulation of Scoliosis-like Phenotypes: Skeletal Analysis of Unbalanced Vertebral Bone Growth in ENU-induced KTA41 Mutant Mice", Nobuho Sagawa|
|P-108||"Genetic analysis of Stmm3 locus controlling tumor progression in a Japanese wild-derived mouse strain, MSM/Ms", Megumi Saito|
|P-109||"Nuclease-Mediated Conditional Allele in a Gene Refractory to Gene Targeting in ES Cells", Thomas Saunders|
|P-110||"A natural antisense RNA to the protein phosphatase 1 regulatory subunit 12A (PPP1R12A) functions as a SINEUP in human cells", Aleks Schein|
|P-111||"Water-soluble fullerene [C60] derivative causes myogenic differentiation of human tissue-derived mesenchymal stem cells.", Vasilina A. Sergeeva|
|P-112||"Visualization and Analysis of Cell and Tissue Omics data with ZENBU genome browser system", Jessica Severin|
|P-113||"Live Imaging Analysis of Mouse Development during DVE migration", Go Shioi|
|P-114||"The Genetic Regulation of Serpine1, Plasminogen Activator Inhibitor-1 in the LEWES/EiJ Mouse Strain ", Amy E Siebert|
|P-115||"Using RGD Genome and Phenotype Resources to Find and Assess a Model for Human Disease", Jennifer R Smith|
|P-116||"A Mutation in Greb1l Results in Multiple Organogenesis Defects", Olivia Sommers|
|P-117||"CARDIOGENE : deciphering the genetic mechanisms of cardiovascular diseases", Tania Sorg|
|P-118||"A combinatorial approach for targeted therapy of triple negative breast cancers: interference peptides against transcription factors, chemotherapy and nanoparticles", Anabel Sorolla|
|P-119||"Identification of novel hypomorphic and null mutations in Nrf1 and Klf1 derived from a genetic screen for modifiers of alpha-globin transgene variegation", Anabel Sorolla|
|P-120||"GUDMAP – GenitoUrinary Development Molecular Anatomy Project, an open resource. ", E Michelle Southard-Smith|
|P-121||"Identification of Active Signaling pathways in Neural Crest Derived Progenitors that Innervate the Lower Urinary Tract", E Michelle Southard-Smith|
|P-122||"WNT inhibition facilitates the establishment of stable and homogeneous EpiSCs", Michihiko Sugimoto|
|P-123||"Analysis of long non-coding RNAs functions in the human genome", Supat Thongjuea|
|P-124||"Redefining the transcriptional regulatory dynamics of classically and alternatively activated macrophages by deepCAGE transcriptomics", Harukazu Suzuki|
|P-125||"Genetic mapping of metabolic traits using the Diversity Outbred mouse population: Are we there yet?", Karen L Svenson|
|P-126||"Genome resequencing of wild mice-derived inbred strains originated from four subspecies of Mus musculus", Toyoyuki Takada|
|P-127||"The recessive congenital cataract in nat mice caused by mislocalization of the MIP", Gou Takahashi|
|P-128||"Development of HTS system to optimize SINEUPs, antisense long-noncoding RNAs that increase translation of target mRNAs", Hazuki Takahashi|
|P-129||"Site-directed DNA demethylation by transcription factor and Manipulation of DNA demethylation", Takahiro Suzuki|
|P-130||"Targeting the mutation site of oncogenic KRAS by KR12 identifies synthetic lethal interactions in colon cancer cells", Atsushi Takatori|
|P-131||"Exploring of novel mouse models for human disease with comprehensive mouse phenotyping data", Nobuhiko Tanaka|
|P-132||"Higher expression of Adcyap1 gene is associated with altered behavioral and prolonged physiological responses to stress in wild-derived MSM mice", Akira Tanave|
|P-133||"Valproic acid-induced vertebral malformations correlate with global expression changes in developmental regulators", Sho Tanimoto|
|P-134||"SATB1 orchestrates expression of lineage specifying genes during positive selection of thymocytes", Ichiro Taniuchi|
|P-135||"Engineering the mouse genome using CRISPR/Cas9", Lydia Teboul|
|P-136||"Analysis of three human genomic loci associated with Tetralogy of Fallot.", Gennadiy Tenin|
|P-137||"PRDM14 promotes epigenetic changes that lead to driver mutations at Notch1 in inducible mouse models of T-ALL", Lauren J Tracey|
|P-138||"ENU mutagenesis identifies a novel molecular pathomechanism of severe immunodeficiency", Irina Treise|
|P-139||"Aromatase inhibitors counteract tamoxifen-induced activation of breast cancer stem cells", Jie-Heng Tsai|
|P-140||"Germline mutation rates and mutation accumulation lines in mice", Arikuni Uchimura|
|P-141||"Granulosa Cell-Specific or Global PTEN Mutations in Combination with Transgenic FSH Expression Fails to Induce Ovarian Tumors", Dannielle H Upton|
|P-142||"Genomic analyses of repetitive elements in the context of early mouse development", J.M Vaquerizas|
|P-143||"Loss of MECP2 results in lung defects in a mouse model for Rett syndrome", Neeti Vashi|
|P-144||"Horses: an underutilized animal model", Brandon Velie|
|P-145||"IsoformSwitchAnalyzeR: Enabling Identification and Analysis of Isoform Switches, with Functional Consequences, from RNA-sequencing data", Kristoffer Vitting-Seerup|
|P-146||"Coexpression networks identify brain region-specific enhancer RNAs in the human brain", Irina Voineagu|
|P-147||"Challenging to RDoC matrix in behavior phenotype of mouse models for human psychiatric disorders", Shigeharu Wakana|
|P-148||"Targeting genes via architecture rather than recognition motifs: The FOS/NFY case", Edgar Wingender|
|P-149||"microRNA expression during erythroid differentiation", Louise Winteringham|
|P-150||"Phenoview: A tool for comparative visualisation of genotype-phenotype relationships", Gagarine Yaikhom|
|P-151||"Development of innovative therapeutic strategy using DNA minor groove binder-drug conjugate in MYCN-driven neuroblastoma", Hiroyuki Yoda|
|P-152||"Peroxiredoxin 2 Promotes HRAS*G12V-Induced Hepatic Tumorigenesis Through Reciprocal Regulation With Forkhead Box M1", Dae-Yeul Yu|
|P-153||"A mutation in TBC/LysM associated domain containing TLDC1 causes craniofacial abnormalities in mice", Rong Zeng|
|P-154||"Diversification of Behavior and Postsynaptic Properties by Netrin G Presynaptic Adhesion Family Proteins", Qi Zhang|
|P-155||"Genome-wide DNA methylation profile implicates potential cartilage regeneration at the late stage of knee osteoarthritis", Yanfei Zhang|
|P-156||"Genetic resistance to congenital hypothyroidism rescues neurosensory and conductive hearing impairment", SA Camper|
|P-157||Polycomb PRC1 and PRC2 complexes contribution to MYC-mediated Hepatocarcinoma, J Gimenez|
Student and Post-Doc Presentations
TS-01/P-151: Development of innovative therapeutic strategy using DNA minor groove binder-drug conjugate in MYCN-driven neuroblastoma
Hiroyuki Yoda*1,2, Atsushi Takatori1, Takahiro Inoue1,2, Takayoshi Watanabe1, Nobuko Koshikawa1, and Hiroki Nagase1
1Division of Cancer Genetics, Chiba Cancer Center Research Institute
2Department of Molecular Biology & Oncology, Graduate School of Medical & Pharmaceutical Sciences, Chiba University
The MYC family gene, MYCN, is a basic helix-loop-helix leucine zipper transcription factor, which is associated with diverse cellular processes including growth, proliferation, death, differentiation, metabolism, self-renewal and pluripotency. Amplification of MYCN initiates a dynamic process of genomic instability that is linked to tumor initiation. In neuroblastoma, a childhood tumor of the peripheral nervous system, MYCN amplification is found in ~25% of neuroblastoma patients and correlates with poor prognosis. Although development of MYCN inhibitor has been considered to be attractive for MYCN-driven neuroblastoma, no drugs have yet progressed to clinical application. In this study, we have developed a MYCN targeting Pyrrole-Imidazole polyamide-alkylating drug conjugate (MYCN-Y), designed to bind directly to minor groove of genomic DNA within the coding region of MYCN. Treatment of MYCN-amplified neuroblastoma cells with MYCN-Y significantly suppressed MYCN expression at the mRNA and protein levels. Accordingly, MYCN-Y caused much higher cell death in MYCN-amplified cells than those in MYCN-non-amplified cells, leading to cell cycle arrest and apoptosis in response to DNA damage. Intriguingly, FISH analyses demonstrated that MYCN-Y impairs&nbnbsp;the fluorescence intensity of a probe specific for MYCN gene loci (2p24.3), suggesting that MYCN-Y directly binds to target genomic sites and abrogates DNA binding of MYCN probe by alkylating its predetermined target site. Moreover, MYCN-Y strikingly inhibited tumor progression in human neuroblastoma xenograft mouse models. Taken together, our findings suggest that MYCN-Y is promising and an innovative therapeutic drug candidate for aggressive neuroblastomas with MYCN amplification.
TS-02/P-037: Mapping SARS-Coronavirus susceptibility alleles using the Collaborative Cross
Lisa Gralinski*1, Vineet Menachery1, Martin T Ferris2,3, Anne Beall3, Jessica Plante1, Jacob Kocher1, Alexandra Schaefer1, Fernando Pardo‑Manuel de Villena2, and Ralph Baric1,3
1Department of Epidemiology, University of North Carolina at Chapel Hill, USA
2Department of Genetics, University of North Carolina at Chapel Hill, USA
3Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, USA
New systems genetics approaches are needed to rapidly identify host genes and genetic networks that regulate complex disease outcomes. Using genetically diverse animals from incipient lines of the Collaborative Cross mouse panel, we demonstrate a greatly expanded range of phenotypes relative to classical mouse models of SARS-CoV infection including lung pathology, weight loss and viral titer. We selected two surviving strains, CC003/Unc and CC053/Unc, with highly divergent susceptibilities to SARS-CoV for further study. CC003/Unc is highly resistant to SARS-CoV-induced disease as measured by weight loss, despite having a high viral load (2.3x10^6 PFU per lung) at four days post infection (DPI). In contract, CC053/Unc is highly susceptible to SARS-CoV infection and exhibits extreme weight loss or death despite having a low viral load (4.3x10^3 PFU per lung) at 4 DPI. F1 progeny display an intermediate weight loss phenotype. 300 F2 mice were bred and infected with SARS-CoV; initial phenotyping included measuring daily weight loss, lung titer and lung hemorrhage at D4. Samples have also been collected for future analysis of lung pathology and serum cytokine response. Approximately 7% of mice succumbed to infection and at 4 DPI mice ranged from losing 25% of their body weight to gaining 5% of their starting body weight. Lung titers range from 1x10^3 PFU per lung to 6.7x10^6 PFU per lung. Hemorrhage was significantly correlated with weight loss (p<0.001) but not with viral load. We are currently genotyping these F2 mice in preparation for QTL mapping to identify host genome regions containing polymorphisms significantly associated with SARS-CoV-induced disease response.
TS-03/P-034: Transcriptional control of hibernation: first insights on comparative genomics of dormice
Guzel Gazizova*1, Kristina Kitaeva1, Oksana Tyapkina2, Maria Logacheva1,3, Olga Bondareva4, Leniz Nurullin2, Ivan Vikhlyantsev5, Akihiko Ishihara6, Noriaki Ishioka7, and Oleg Gusev1,8
1Kazan Federal University, Kazan, Russia
2KIBB KSC RAS, Kazan, Russia
3Lomonosov Moscow State University, Moscow, Russia
4Zoological Institute RAS, Saint-Petersburg, Russia
5ITEB RAS, Puschino, Russia
6Kyoto University, Kyoto, Japan
7ISAS, Sagamihara, Japan
8RIKEN, Yokohama, Japan
Hibernation is the hypometabolic state, allowing survival of some species of mammals under severe environmental conditions such as low temperature and food restriction. Remarkably, hibernating animals can stay immobilized for prolonged periods without loss of muscle strength and mass. Genetic control of hypometabolic-associated processes is of key interest, but remains poorly studied, due to the lack of systematic analysis of the activity of genomes of hibernators. In order to establish new genetic model for studying hibernation in mammals, we initiated dormice genome consortium, to sequence and compare genome structure and activity in several species of dormice different in ability to hibernation. At the current step, we analyzed transcriptome profile in two types of muscles (m. soleus, m. EDL) and spinal cord of edible and forest dormice, by using HiSeq Illumina platform and assembled de novo transcriptomes for both species. We determined 49 887 and 52 779 transcripts in edible and forest dormice respectively. We estimated genes, which were upregulated and downregulated during hibernation: most of genes altered by hibernation in edible dormice were found in both muscle types, while in forest dormice the major changes were observed only m. soleus muscle. The key genes, altered in response to hibernation encode ankyrin repeat domain-containing protein, myosin-binding protein and several transcription factors. Finally, using multi-sequence comparison we have analyzed phylogenetic status of dormice in mammals.
The work is performed according to the Russian Government Program of Competitive Growth of Kazan Federal University and supported by RFBR JSPS_a №14-04-92116.
TS-04/P-076: Combination of selective breeding and genome-wide SNP analysis revealed the genetic loci associated with tame behavior in mice
Yuki Matsumoto*1,2,3, Hirofumi Nakaoka4, Jo Nishino5, Tatsuhiko Goto6, and Tsuyoshi Koide1,2
1Department of Genetics, SOKENDAI
2Mouse Genomics Resource Laboratory, National Institute of Genetics
3JSPS Research Fellow
4Division of Human Genetics, National Institute of Genetics
5Graduate School of Medicine, Nagoya University
6College of Agriculture, Ibaraki University
Tame behavior is one of the major elements in domestication and defined as increased interaction of animals with human. Tame behavior can be divided into two component, one is active tame and the other is passive tame. The two components of tame behavior in mice (Mus musculus) can be quantified by behavioral assay which our group previously established. To identify genes associated with active tame behavior which is defined as contacting human hand (contacting), we performed selective breeding for contacting using wild-derived heterogeneous stock (WHS), which is a mixed population derived from 8 wild mouse strains originated in various geographic regions. At the 8th generation of the selective breeding, we obtained 16,328 single nucleotide polymorphism (SNP) data of 32 WHS mice in selection population and the 8 founder strains of WHS by using SNP genotyping array. Because the allele frequency associated with contacting should be increased by selective breeding for contacting, and the loci can be identified by using the deviation from hypothetical allele frequency determined by computer simulation based on non-selection model. Therefore we used computer simulation that combined the information of the pedigree of WHS from generation 0 to 8th, polymorphism and position of each SNP, and determined genome-wide thresholds for significant increase of allele frequency. Then we applied the threshold to observed data of selected population. As a result, a candidate SNP on Chromosome 11 exceeded the threshold. Additionally, the result of association analysis using the SNP and contacting in control population was statistically significant. Three candidate genes in Mouse Genomics Informatics database were so far found in the locus which strongly linked with the candidate SNP, suggested that these genes might be associated with increased levels of contacting.
TS-05/P-077: Conservation and evolution of splicing patterns during postnatal development of prefrontal cortex in primates
Pavel Mazin*1,2, Philipp Khaitovich1, and Mikhail Gelfand2
1Skolkovo Institute of Science and Technology, Moscow, Russia
2IITP RAS, Bolshoy Karetny per. 19, Moscow, Russia
Alternative splicing (AS) allows single gene to produce several mRNA through differential exclusion of introns. AS is abundant in higher eucariotes and affects 95% of human genes. Here, we study splicing changes taking place during postnatal prefrontal cortex development based on RNA-Seq data from 74 humans, 44 chimpanzees and 50 macaques. Using these data we created unbiased, not-human-based gene annotation for all three genomes. It allows us to assess for the first time species- and age-related AS variability in primate brains on genome-wide level.
In agreement with previous studies, our results show that inter-species differences are the main source of the splicing variability. We show that 15% (5788) of alternative exons detected in our data are differentially spliced among the three species. In all three evolutionary lineages the main trend was towards increase in proportion of alternative versus constitutive exons. Up to 38% of species-related AS changes can be explained by single nucleotide substitutions in the corresponding splicing sites
6.4% (2477) of alternative exons exhibit significant splicing changes with age. We have found prominent enrichment in functions linked to the neuronal development among corresponding genes. Our analysis shown significantly higher sequence conservation of the age-related exons and their flanking regions than expected by chance. Using motif-enrichment analysis we have shown that several splicing factors such as MBNL2, RBM4, YBX1, RBFOX2, RBM8A are involved in age-related regulation of cassette exon splicing.
The frequency of the intron retention changes with age in 1029 (14% of tested) genes. There are about two-fold more age-related retained introns in human, than in the other species. Intron retention is more abundant in newborns and drops with age. We shown that the frequency of intron retention does anti-correlate with the gene expression level that points to the possible participation of the intron retention in the age-related gene expression regulation.
TS-06/P-035: Conservative miRNA target analysis: are we limiting our discoveries of neuronal miRNA function?
Belinda J Goldie*1,2, Dan O Wang1, and Murray J Cairns2,3
1Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University
2University of Newcastle, Australia
3Schizophrenia Research Institute, Sydney Australia
In studies involving miRNA-mediated gene silencing, much emphasis is placed on the reduction of potential “false positive” candidates by limiting target searches to conserved target sites and conserved miRNA. In contrast, the human brain is unique from other species due to higher order functions that are not conserved. Moreover, many studies of learning and memory as well as models of neurological dysfunction are conducted in animals such as rats and mice.
What are we missing out on?
We used genome-wide techniques to investigate the suitability of the human SH-SY5Y neuroblastoma cell line for modelling the expression profile of neurons and showed that use of an appropriate neuronal differentiation paradigm induced expression of mRNAs and miRNAs characteristic of mature neurons. Using this system we studied the subcellular localisation of both species of RNA as well as their responses to neuronal cues for differentiation and depolarisation. Surprisingly, many miRNAs demonstrating strong localisation preference for the neurites and nucleus, as well as those modulated by activity and released in exosomes, were human- or primate- specific. Bioinformatic target functional analyses of these miRNA were severely limited by tools only considering conserved target pairings. In particular, for human-specific miR-638 only 30 conserved targets were predicted. Manual curation of non-conserved targets increased this list to 1290, and revealed putative regulation of synaptic function and gene expression, and indeed over-expression of this miRNA altered the abundance of around 500 loci and severely impacted the neuronal phenotype. Together these findings highlight the importance of an inclusive approach to miRNA target analysis in the neuronal context.
TS-07/P-047: Dissecting the regulation of olfactory receptor expression in the mouse.
Ximena Ibarra‑Soria*1, Darren W. Logan1, and John C. Marioni1,2
1Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton-Cambridge, CB10 1SA, United Kingdom
2European Bioinformatics Institute (EMBL-EBI), European Molecular Biology Laboratory, Wellcome Trust Genome Campus, Hinxton-Cambridge, CB10 1SD, United Kingdom
Detection of odorants occurs in the main olfactory epithelium (MOE), which contains olfactory sensory neurons (OSNs) that express olfactory receptors (ORs). These bind the odorants and then transmit an electrical signal to the brain. Each OSN expresses only one OR, from a repertoire of over 1,200 genes, and silences all the others. Therefore, the mouse nose has over 1,200 different OSN types, each patterned by a different OR gene. High levels of genomic variation have been reported both in the mouse and human OR repertoire. This is thought to contribute to the unique sense of smell each individual has, but the mechanisms responsible are not known.
We have devised an RNAseq-based approach to quantify the OSN repertoire of three inbred strains of mice (C57BL/6, 129S5 and CAST/EiJ) via their OR gene expression levels. We found that each strain has a unique and reproducible distribution of OSNs in their noses, and that this is directly instructed by genomic variation.
Additionally, OR expression in the MOE is susceptible to olfactory experience. Exposure to an enriched olfactory environment results in the differential expression of dozens of OR genes in a reproducible and specific manner. These changes increase with time and are reversible. These data allows, for the first time, to comprehensively explore and dissect the effects of genetic and environmental variation in the regulation of OR expression and OSN repertoire. Together they generate an olfactory sensory system that is individually unique.
TS-08/P-144: Horses: an underutilized animal model
Brandon Velie*1, Kim Fegraeus1, Merina Shrestha1, Anouk Schurink5, Liesbeth Francois4, Anneleen Stinckens4, Sarah Blott3, Bart Ducro5, Nadine Buys4, Sofia Mikko1, June Swinburne2, Susanne Eriksson1, Carl‑Johan Rubin6, Jennifer Meadows6, Leif Andersson1,6,8, Lisa Andersson7, and Gabriella Lindgren1
1Department of Animal Breeding & Genetics, Swedish University of Agricultural Sciences, Uppsala, Sweden
2Animal DNA Diagnostics Ltd, Cambridgeshire, United Kingdom
3School of Veterinary Medicine & Science, University of Nottingham, Leicestershire, United Kingdom
4Department of Biosystems, Division of Gene Technology, University of Leuven, Leuven, Belgium
5Animal Breeding and Genomics Centre, Wageningen University & Research Centre, Wageningen, the Netherlands
6Department of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden
7Capilet Genetics AB, Oster Skogsta, Vasteras, Sweden
8Department of Veterinary Integrative Biosciences, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, Texas, United States of America
Horses provide an opportunity to study unique phenotypes that can lead to fundamental biological insights as well as help to decipher mechanisms underlying biological and disease processes. At present, we have three horse projects with preliminary results that may serve as models for investigating gene functions in mammals. A GWAS of equine insect bite hypersensitivity (IBH), an allergic recurrent seasonal dermatitis classed as a type I and type IV hypersensitive reaction, suggests the importance of two genomic regions on Chromosome 8 (ECA8). An increased knowledge of the genes involved in the manifestation of IBH is expected to not only improve prevention, diagnosis, and treatment of equine IBH, but may also broaden our understanding of the biology underlying type I and type IV hypersensitive reactions across species. Observed in a wide range of species including humans, a second project concerns polydactyly, a genetic defect that presents as an increased number of digits. Preliminary analyses of a family of ponies suggest a recessive mode of inheritance in horses. Through whole-genome re-sequencing of this family (n=5) we aimed to confirm this mode of inheritance and identify the causative locus. Additionally, Delta FST analyses of harness racing breeds have identified specific candidate regions that harbor genes selected for athletic performance. These regions contain genes known to be involved in energy metabolism and cell growth. Genes that regulate energy metabolism and other biological processes that impact racing performance have the potential to improve our understanding of metabolic defects and diseases in horses as well as in other species. At the meeting we will present results from the three aforementioned studies and comment on the fact that in some circumstances the horse may provide unique knowledge of biological pathways that may not otherwise be fully understood.
TS-09/P-132: Higher expression of Adcyap1 gene is associated with altered behavioral and prolonged physiological responses to stress in wild-derived MSM mice
Akira Tanave*1,2, Aki Takahashi3, Kenta Sumiyama4, and Tsuyoshi Koide1,5
1Mouse Genomics Resource Laboratory, National Institute of Genetics in Japan
2Transdisciplinary Research Integration Center in Japan
3University of Tsukuba in Japan
4RIKEN Quantitative Biology Center in Japan
5Department of Genetics, SOKENDAI
Stress response is behavioral and physiological responses to stressful situation, in which the phenotypic variation is closely linked with the genetic variation among individuals. Wild-derived mouse strain MSM/Ms (MSM) shows higher behavioral responses to stress than laboratory mice in open-field test. Our previous study revealed that the behavioral responses to stress in MSM mice are mapped on Chromosome 17 under the genetic background of laboratory C57BL/6 (B6) mice. Using a series of congenic strains, we successfully mapped a genetic locus into about 2.3 Mb region at the distal end of Chr17, in which only one protein-coding gene Adcyap1 (PACAP) is located. In this study, we examined the association of the Adcyap1 gene with the behavioral responses to stress. Although non-synonymous mutation was not found in the Adcyap1 gene between MSM and B6 strains, the Adcyap1 mRNA and protein levels were significantly increased in hypothalamus of the Adcyap1 congenic mice. This higher Adcyap1 expression was considered as one of the reasons the altered behavioral responses to stress because Adcyap1 is a neuropeptide that regulates stress responses. We next examined physiological response to acute restraint stress. The Adcyap1 congenic mice showed increased serum corticosterone levels similar with B6 mice immediately after the stress, but the increased serum corticosterone levels were significantly prolonged after 1-2 hour of the stress in the Adcyap1 congenic mice compared to B6 mice. These results suggests that the altered stress responses may be linked to the altered behavioral responses to stress. In addition, we developed B6 transgenic mice with Adcyap1 gene derived from MSM strain by using Tol2 transposon system. Now we are conducting behavioral analyses using the transgenic mice. From these results, we will provide some insights into the functional mechanisms of Adcyap1 that alters behavioral responses to stress through stress response pathway.
TS-10/P-136: Analysis of three human genomic loci associated with Tetralogy of Fallot
Gennadiy Tenin*, and Bernard Keavney
Institute of Cardiovascular Sciences, University of Manchester, Manchester, UK
Congenital heart diseases (CHD) are defects in the structure of the heart that are present at birth. They are amongst the most common birth defects and found in 9 out of 1000 births. Tetralogy of Fallot (TOF) is one of the most common cyanotic CHD and is considered to be a multigenic condition. TOF involves four anatomical abnormalities of the heart: pulmonary stenosis, overriding aorta, ventricular septal defect and right ventricular hypertrophy. Studies on animal models have linked the development of TOF to defects in cardiac septation and heart valve formation during early cardiogenesis. Our recent GWAS study found significant association of Tetralogy with three genomic loci: a 1.5 Mb haplotype on Chromosome 12q24.12 (contains 24 coding genes), two variants on 10p11.22 (in NRP1 gene) and two variants on 13q31.3 (in GPC5 gene). Analysis of eQTL data available for 5 human cell types did not reveal the expression of which genes might be affected by these variants. Analysis of the published data allowed us to eliminate some genes from the candidate list as non cardiac-related. We further narrowed down the candidate genes list upon the analysis of the mRNA expression patterns in the developing mouse heart. Several poorly characterized genes (eg Gpc5 and Gpc6) showed remarkable expression in the endocardial cushions of both outflow tract and atrioventricular canal, which play a critical role in cardiac septation. We then developed the in vitro mouse heart culture assay to test for the functional consequences of the gene knock down by siRNA silencing. All this allowed us to identify new cardiac genes which may be involved in the development of the septation defects found in the Tetralogy of Fallot.
TS-11/P-102: Cognitive Endophenotypes of Modern and Extinct Hominins Associated With NTNG Gene Paralogs
Pavel Prosselkov*1,2, Ryota Hashimoto3,4, Denis Polygalov5, Ohi Kazutaka3, Qi Zhang1, Thomas J. McHugh5, Masatoshi Takeda3,4, and Shigeyoshi Itohara1
1Laboratory of Behavioral Genetics, RIKEN BSI, Wakoshi, 351-0198 Saitama, Japan
2Grad School, Dep Vet Medicine, Faculty of Agriculture, Tokyo University, 113-8657 Tokyo
3Mol Res Cnt for Child Mental Dev, Uni Grad Sch Child Dev, Osaka Uni, 565-0871 Osaka
4Department of Psychiatry, Osaka Uni Grad School of Medicine, 565-0871 Osaka, Japan
5Laboratory of Circuit and Behavioral Physiology, RIKEN BSI, Wakoshi, 351-0198 Saitama
A pair of gene paralogs, NTNG1 and NTNG2, sharing identical gene and protein structures and encoding similar proteins, forms a functional complement subfunctionalising (SF) within cognitive domains forming cognitive endophenotypes, as detected by Intellectual Quotient (IQ) test. NTNG1 SNPs affect either verbal comprehension (VC, rs2218404) or processing speed (PS, rs96501) while NTNG2 SNPs (rs2149171 and rs2274855) affect working memory (WM) and perceptual organization (PO), and rs1105684 affects verbal and performance IQs (VIQ and PIQ). Regions of interest (ROIs) defined as 21 nu long loci symmetrically embedding the SNPs’ areas underwent dramatic evolutionary changes from mice through primates to human gene orthologs. Two G and one T alleles associated with higher IQ scores show an early evolutionary appearance: rs2218404 “G” of NTNG1 in extinct Mesolithic human Loschbour (8,000 yrs BC); rs2149171 “T” and rs2274855 “G” of NTNG2 in Neolithic human Ötzi (5,300 yrs BC) and chimpanzee (6.3 mln yrs ago), respectively. Two other mutations associated with lower IQ scores are also relatively young: processing speed (PS)-affecting C allele of rs96501 appears ~50,000 yrs BC; WM/PO - affecting A allele of rs2274855 - 8,000 yrs BC. Intensive evolutionary changes resulting in the accelerated evolution of the VC (rs2218404) and WM/PO (rs2274855) ROIs point towards their potential contribution to the human-specific traits. Protein sequence of NTNG1 is 100% conserved among the archaic and modern extinct hominins while NTNG2 underwent a recent selection sweep. It encodes a primate-specific S371A/V substitution (emerged ~50,000 yrs ago) and a modern human (5,300 yrs) T346A substitution (located 20 nu downstream of the WM/PO-affecting rs2275855). NTNG paralogs SF perturbate “structure drives function” concept and do not obey it neither at the protein nor gene level proposedly forming a so-called “Cognitive Complement (CC)” as a product of gene duplication and subsequent function bifurcation by the gene duplicates.
TS-12/P-068: Skin Megagenetics - Novel skin phenotypes revealed by a genome-wide mouse reverse genetic screen
Kifayathullah Liakath‑Ali*1,2,3, Valerie Vancollie4, Emma Heath1, Damian Smedley4, Jeanne Estabel4, David Sunter4, Tia DiTommaso5, Jacqueline White4, Ramiro Ramirez‑Solis4, Ian Smyth5, Karen Steel6, and Fiona Watt1
1Centre for Stem Cells and Regenerative Medicine, Kings College London, UK
2Department of Biochemistry, University of Cambridge, UK
3Wellcome Trust-Medical Research Council Stem Cell Institute, University of Cambridge, UK
4Wellcome Trust Sanger Institute, Cambridge, UK
5Department of Anatomy and Developmental Biology, Monash University, Australia
6Wolfson Centre for Age-Related Diseases, Kings College London, UK
Permanent stop-and-shop large scale mouse mutant resources provide an excellent platform to decipher tissue phenogenomics. A skin specific high throughput genetic screen on these resources would reveal novel genes involved in skin homeostasis. In this presentation, we show analysis of skin from 538 unselected knockout mouse mutants. We have optimized labeling methods to allow systematic annotation of hair follicle, sebaceous gland and interfollicular epidermal abnormalities using ontology terms from the Mammalian Phenotype Ontology. Of the 50 mutants with an epidermal phenotype, 9 map to human genetic conditions with skin abnormalities. Some mutant genes are expressed in the skin, whereas others are not, indicating systemic effects. In-depth analysis of three mutants, Krt76, Myo5a (a model of human Griscelli syndrome) and Mysm1, provides validation of the screen. High incident of sebaceous gland abnormalities was observed and one mutant showed dietary influence on sebaceous gland architecture. Computational analysis showed strong association of mutants with core signaling pathways such as EGFR, Notch and Wnt. This is the first large scale genome-wide tissue phenotype screen from the International Knockout Mouse Consortium and provides an open access resource for the scientific community.
TS-13/P-048: KRAS mutation specific alkylating pyrrole-imidazole polyamide, KR12 showed significant anti-tumor efficacy and preferential localization in KRAS mutant xenografts without adverse effects
Takahiro Inoue*1, Kiriko Hiraoka1, Hiroyuki Yoda1, Takayoshi Watanabe1, Atsushi Takatori1, Nobuko Koshikawa1, Shigeharu Wakana4, Toshikazu Bando3, Hiroshi Sugiyama3, Toshinori Ozaki2, and Hiroki Nagase1
1Lab. Cancer Genetics, Chiba Cancer Center Res. Inst.
2Lab. DNA Damage Signaling, Chiba Cancer Center Res. Inst.
3Dep. Chem., Grad. Sch. Sci., Univ. Kyoto.
4Japan Mouse Clinic, RIKEN BRC.
Constitutive active mutations of KRAS have been shown to be associated with malignant properties of tumors as well as a poor clinical outcome of the patients in colorectal cancers. Unfortunately, yet no effective anti-cancer drug(s) specifically targeting KRAS mutations have been developed. Hence, we synthesized an alkylating agent conjugated with the pyrrole-imidazole polyamide (KR12: PIP-CBI), which recognized KRAS G12D or G12V mutation at codon12. We have previously found anti-tumor effects of low dose KR12 (0.3mg/kg) by suppressing mutant KRAS expression in vitro and in vivo. However, it still remains elusive the KR12 pharmacokinetics, maximum effective/tolerated doses and safety in animals. To address those, we initially performed Modified SHIRPA (behavioral and functional analysis of mouse phenotype) to test phenotype of ICR mice 3 days and one month after 3 mg/kg KR12 single intravenous administration. Modified SHIRPA screening, and simultaneous hematology, blood chemistry and urine analyses exhibited no KR12 exposure related toxicological changes. Additionally, we examine the distribution of KR12 using FITC labeled PI polyamide. In vivo imaging of tumor-bearing mice 72 hours after the administration demonstrated the highest fluorescence intensity in the tumor. Since KR12 showed long lasting accumulation in xenografts we compared the effect of single and multiple administration of KR12 in the homozygous mutant SW480 (KRAS*G12V homozygous mutation) xenograft model. Surprisingly, single treatment of 0.3 mg/kg KR12 induced significant tumor volume reduction as those seen in weekly injections for five to eight weeks. These data suggest that substantial KR12 exposure is well tolerated in mice, and KR12 may accumulate in xenograft tumor tissues and thus induced significant tumor growth inhibition even at low doses.
TS-14/P-128: Development of HTS system to optimize SINEUPs, antisense long-noncoding RNAs that increase translation of target mRNAs
Hazuki Takahashi*1, Ana Kozhuharova1, Harshita Sharma1, Diego Cotella2, Claudio Santoro2, Silvia Zucchelli3, Stefano Gustincich3, and Piero Carninci1
1RIKEN Center for Life Science Technologies Japan
2Universita del Piemonte Orientale Italy
SINEUPs are antisense long non-coding RNAs that are able to specifically stimulate translation of potentially any gene of interest, ultimately leading to increased levels of the protein product. They derive from a new functional class of natural long non-coding RNA transcripts antisense to protein coding genes that we recently discovered. These molecules have been named SINEUPs since their function requires the activity of an embedded inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. SINEUP activity depends on the combination of two elements present in an RNA molecule: the overlapping region is indicated as the Binding Doman (BD) and is responsible of target specificity; the embedded Inverted SINEB2 element is the Effector Domain (ED) and is required for translation enhancement. In order to screen for SINEUPs with enhanced activity, we established a high throughput screening (HTS) system based on high-resolution automated fluorescent imaging using CeligoS platform. A series of variants were generated in the BD and ED of SINEUP-GFP and activity measured as mean of GFP fluorescence intensity. Our HTS analysis identified a number of short BD variants overlapping around the AUG Kozac region with improved activity. Moreover, with the same platform, we validated ED from additional natural SINEUPs that were found effective in enhancing protein translation. In summary, our automated HTS platform can be used for fast and efficient screening for SINEUP design and optimization and can be applied to virtually any target gene of interest.
TS-15/P-106: Cell-cell-communication in cancer
Riti Roy*1, Jordan A Ramilowski2, and Alistair Forrest1,2
1Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, the University of Western Australia, PO Box 7214, 6 Verdun Street, Nedlands, Perth, Western Australia 6009, Australia
2RIKEN Center for Life Science Technologies (Division of Genomic Technologies), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045 Japan.
To coordinate multicellular processes, cells need to communicate. One way they communicate is through receptor and ligand interactions. By studying the peptide ligands and plasma-membrane receptors expressed on different primary cell types we have generated the world’s first draft map of the cell-cell communication network CCCN (Ramilowski et al. 2015). Here we explore what happens to cell-cell communication in tumors (in particular cancer-cancer and cancer-normal signalling). Examining the expression profiles of receptors and ligands in cell lines profiled by the FANTOM5 project and tumors profiled by the TCGA we report on changes to the ligand and receptor repertoires expressed in cancer and relate this to mutation profiles. We also examine changes in cell-cell communication in cancer, with a systematic revaluation of cancer-autocrine, cancer-stroma, cancer-vascular and cancer-immune signalling. This area of research is important as it gives new insights into the complex network of cell-cell communication in tumors and has the potential to identify new therapeutic targets.
TS-16/P-138: ENU mutagenesis identifies a novel molecular pathomechanism of severe immunodeficiency
Irina Treise*1,2, Eva M. Huber3, Tanja Klein‑Rodewald1,7, Wolfgang Heinemeyer3, Thure Adler1,2, Birgit Rathkolb1,4, Christian Andres8, Thomas Wieland6, Tim M. Strom6, Kathy D. McCoy5, Andrew J. Macpherson5, Eckhard Wolf4, Markus Ollert8, Frauke Neff7, Valerie Gailus‑Durner1, Helmut Fuchs1, Martin Hrabe de Angelis1, Michael Groll3, and Dirk H. Busch2
1German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum Muenchen, Ingolstaedter Landstrasse 1, 85764 Neuherberg, Germany
2Institute for Medical Microbiology, Immunology and Hygiene, Technische Universitaet Muenchen, 81675 Munich, Germany
3Center for Integrated Protein Science at the Department Chemistry, Chair of Biochemistry, Technische Universitaet Muenchen, Lichtenbergstr. 4, 85747 Garching, Germany
4Institute for Molecular Animal Breeding and Biotechnology, Gene Center of the Ludwig-Maximilians-Universitaet Muenchen, Feodor-Lynen Str. 25, 81377 Munich, Germany
5University Clinic of Visceral Surgery and Medicine, Departement Klinische Forschung, University of Bern, Murtenstrasse 35, CH-3010 Bern, Switzerland
6Institute of Human Genetics, Helmholtz Zentrum Muenchen, Ingolsaedter Landstr. 1, 85764 Neuherberg, Germany
7Institute of Pathology, Helmholtz Zentrum Muenchen, Ingolsaedter Landstrasse 1, 85764 Neuherberg, Germany
8Department of Dermatology and Allergy Biederstein, Technische Universitaet Muenchen, Biedersteiner Str. 29, 80802 Munich, Germany
As part of the large-scale Munich ENU (N-ethyl-N-nitrosourea) mouse mutagenesis approach, the Immunology Screen of the German Mouse Clinic performed standardized immuno-phenotyping of mouse mutants with a particular focus on identifying mutants with clinical phenotypes. Thereby, we established and characterized a novel mouse mutant - TUB006 - with defects in both innate and adaptive immunity. Heterozygous TUB006 animals show significantly reduced T cell frequencies, and fail to induce a sufficient T cell response to Listeria monocytogenes, resulting in lethality upon low-dose infection. Homozygous TUB006 mutants display an early lethal phenotype presenting with severe combined immunodeficiency (SCID) lacking all three major types of lymphocytes: T cells, B cells and NK cells. Furthermore, homozygous mutants develop sterile autoinflammation characterized by granulocytosis and infiltration of neutrophil granulocytes into various organs. Whole exome sequencing unraveled the underlying point mutation in Psmb10, encoding a catalytic subunit of the immunoproteasome and thymoproteasome. Yeast mutagenesis and crystallographic data suggest that the severe TUB006-phenotype is caused by structural changes that prevent the biogenesis of functional immuno- and thymoproteasomes. The severe immunodeficiency in TUB006 mice is surprising, since Psmb10-knockout mice are healthy, displaying only a subtly altered T cell repertoire. Our data once more show the power of ENU to identify unknown gene functions by creating not only loss-of-function mutants but also gain-of-function, hypo-/hypermorphs or dominant-negative mutations. The identification of causative mutations in mice with clinical phenotypes can directly lead to the discovery of human disease genes. Given the high structural and functional conservation of proteasome subunits between mammals, our data point out the high potential of Psmb10 mutations to be of clinical relevance in humans with immunological defects of unknown etiology.
Panel: Is the mouse still needed as a human disease model?
The use of mice as human disease models is an interesting topic. Some scientists believe that the mouse is not appropriate as a model for innovative drug development and basic medical research. We think that the advantage of using mouse models for research on human diseases needs to be emphasized. Recently the article entitled "Inflammation debate reignites" is enclosed in the journal Science. The author claimed that the mouse was a poor model for inflammation research in the field of human medicine (Junhee Seok et al., PNAS, 2015). On the other hand, some Japanese scientists have reanalyzed data from the same paper using different assumptions and statistical techniques and concluded that the conclusions drawn were incorrect. Finally, they reported that the mouse models of inflammation do mimic the human conditions (Takeo et al., PNAS, 2015). These debates were included in the newspapers EurekAlert, SCIENCE CODEX, Medical Xpress, GEN News, and Science.
In this meeting, we will invite the Japanese scientist Dr. Miyawaka and ask him to describe his analysis of the inflammation-related data of the mouse and his opinion on the advantage of using the mouse as a human disease model. We also invite Drs. Martin Hrabé de Angelis and Gao Xiang to give commentary on his talk. Finally, we hope to promote a debate in the audience on this topic.
O-01: Patient Derived Xenografts (PDX) Models of Human Breast Cancer: A Platform for Precision Oncology
Carol J Bult*, James Keck, Susan Airhart, and Joel H Graber
The Jackson Laboratory, Bar Harbor, ME, USA
Precision oncology promises to deliver better outcomes for cancer patients by treating individuals with drugs that are selected based on the genetic and/or genomic profile of their tumor. This genome-guided therapy approach is proving effective in many cancers including, non small cell lung cancer, melanoma, lymphoma, leukemia and breast cancer, each of which has molecular subtypes that respond to targeted treatment. Triple-negative breast cancer (TNBC) patients have not benefited from tailored therapies because the tumors of these patients do not express the markers that make a targeted approach possible. Although TNBC patients often respond well to first line standard chemotherapy in a neoadjuvant setting, the rate of relapse is >50%. No standard treatment options exist for these patients for recurrent disease. Clinically relevant mouse models have the potential to accelerate understanding of the genetic and genomic basis of TNBC and to advance new treatment paradigms.
We have developed a resource of PDX models by implanting human tumors into immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (aka, NSG) mice. PDX models provide a powerful in vivo platform for testing standard of care and novel therapeutic options in TNBC patients. Human tumors that successfully engrafted were characterized for somatic mutations, copy number variants, and gene expression. Tumor bearing mice for the TNBC PDX models were treated with docetaxel, cisplatin, cyclophosphamide and doxorubicin. Preliminary computational analyses of tumor responses to these treatment regimes using a machine learning approach revealed systematic differences that can be correlated with genome properties of the tumors.
O-02: Novel ENU-induced ankyrin 1 mutations reveal complex role of erythrocyte cytoskeleton plays during malaria infection
Hong Ming Huang1, Andreas Greth1, Patrick Lelliott1, Shelley Lampkin1, Denis Bauer2,3, Brendan McMorran1, Simon Foote1, and Gaetan Burgio*1
1The John Curtin School of Medical Research, The Australian National University, ACT 2601, Australia
2Preventive Health Flagship, CSIRO, NSW 2113, Australia
3Computational Informatics, CSIRO, NSW 2113, Australia.
Malarial parasite resistance to all known antimalarial drugs is now the norm. Parasites develop resistance through modification of both target and intra-parasitic drug concentrations. We have developed a strategy to develop new therapies that will bypass both these mechanisms of resistance. ENU mutagenesis is used to introduce mutations into the germline of mice that are otherwise susceptible to murine malaria. Mice carrying protective mutations will survive a malarial challenge whereas all other mice will succumb. The genes harbouring the mutations are identified and assessed as potential antimalarial drug targets. At present we have identified 40 mutations conferring resistance to malarial infection and have 100 resistant lines. Here, we propose novel mechanisms of host resistance to malaria infection. Through our dominant large scale ENU screen for abnormal red blood cell count, 3 novel mutant alleles were identified in ankyrin 1, erythroid (Ank1). We hypothesized that depending on the location of mutation, varying degree of malaria susceptibility and perhaps different mechanisms of resistance could be observed. All of the mutants exhibit an abnormal red blood cell (RBC) count. However, some strains exhibited a shorter RBC half-life and deformed morphology responsible for hereditary spherocytosis disease. Most Ank1 mutant mice were resistant to the malaria parasite P. chabaudi. The RBCs from Ank1 mutant mice were resistant to parasite invasion, some might impair parasite growth and some were prone to clearance. Different Ank1 mutations cause changes on erythrocyte properties and malaria resistance with different severity. Truncation-causing mutations seem to affect parasite maturation while substitution causes increased clearance. This study provides the first evidence that multiple mutations in Ank1 can lead to different mechanisms of susceptibility to malaria in mice, and shows that Ank1 is essential for supporting the growth and the replication of Plasmodium spp within RBCs. Finally Ank1 is a promising targeted for anti-malarial therapies.
O-03: Genetic mechanism of aerobic capacity and metabolic disease in Rat model
Yu‑yu Ren1, Yu Wang*1, Lauren G Koch2, Steven L Britton2, Nathan R Qi3, Mary K. Treutelaar3, Charles F Burant3, and Jun Z Li1
1Department of Human Genetics
2Department of Anesthesiology
3Department of Internal Medicine, University of Michigan. Ann Arbor, MI, United States
Aerobic capacity refers to the max amount ability of oxygen consuming during exercise. It is a function of overall performance of cardiorespiratory system, and has strong association of risks of obesity, hypertension, and type-2 diabetes. Better understanding of the mechanism of aerobic capacity could promote the therapy of those disease in human. Existing studies on human populations have been largely limited to the complex background and finite sample size, yet further functional validation is insufficient to success in animal model. Indeed, animal model have a quite different genetic background. Here we adopted a rat model system, comprising with two lines with well established phenotype on running ability, to have a better understanding of the molecular mechanism of aerobic capacity and genetic dynamics during selection. The two lines, HCR/Mco (high-capability runners) and LCR/Mco (low-capability runners), shared a same founding group and inbred with rotational matings. Then, they were selected toward divergent running ability for more than 30 generations, and were both genetically and phenotypically heterogenous. Finally, the HCR/Mco lines displayed a significant improvement of aerobic capacity (the maximum distance of HCR/Mco is 9 fold of that of LCR/Mco). Meanwhile, dramatic phenotypic differences were observed on body weight, blood pressure, and life span between HCR/Mco and LCR/Mco lines. To decipher the genetic mechanism of aerobic capacity, we applied comprehensive tools, including custom SNP array and large scale sequencing to genotype F1 and F2. We genotyped 616 F2 and 56 F1 with ~800K custom Affymetrix arrays and archived an average read depth of ~37X on 40 samples with NGS sequencing. Combined with sequencing and microarray genotype data, we identified four regions highly associated ( LOD >= 5 ) with aerobic capacity. Furthermore, the dynamics of haplotype and recombination was described based on pedigree data. Our result provided a better understanding of metabolic mechanism and genetic background of HCR/Mco-LCR/Mco rat lines, which can serve as an excellent model for therapy of metabolic disease.
O-04: Analysis of murine resistant and susceptible transcriptomes in plague
Manal Khalife1,2,4, Odile Sismeiro3, Jean‑Yves Coppee3, Marie‑Agnes Dillies3, Bernd Jagla3, Elisabeth Carniel4, Christian Demeure4, Steven Munger5, Jean‑Jacques Panthier1,2, and Jean Jaubert*1,2
1Institut Pasteur, Mouse Functional Genetics Unit, F-75015 Paris, France
2CNRS URA 2578, F-75015 Paris, France
3Institut Pasteur, Transcriptome and Epigenome, F-75015 Paris, France
4Institut Pasteur, Yersinia Unit, F-75015 Paris, France
5The Jackson Laboratory, Bar Harbor, Maine 04609, US
Plague is caused by the Gram-negative bacterium Yersinia pestis. Laboratory mice such as C57BL/6J (B6) are susceptible to plague. We have described that wild-derived Mus spretus SEG/Pas (SEG) mice are exceptionally resistant (90%) to the virulent CO92 wild-type strain of Y. pestis in an experimental model of bubonic plague. We identified three genomic regions controlling this resistance, and characterized immunological, histological and cellular differences between SEG strain and B6 reference susceptible strain (Blanchet et al. 2011). To gain further insight in the understanding of this exceptional resistant phenotype, we have decided to explore by RNA-seq the modifications of the mouse transcriptome upon infection in both resistant (SEG) and susceptible (B6) context.
Spleen of control non-infected and infected mice were collected at day 3 post-infection. Day 3 was chosen as time-point for tissue collection as at day 4 there are already clear-cut histological differences in the spleen between the two strains and a marked expansion of a specific macrophage sub population (F4/80+ CD11b-) in SEG mice (Demeure et al. 2012). Important variations in spleen CFU counts were observed between same strain animals collected at day 3, in correlation with a previous study that suggested a high variability in the kinetics of infection (Nham et al. 2012). Animals were therefore divided in 2 groups per strain, based on their spleen CFU counts (Low with CFU count ~103 CFU/g and High with CFU count >105 CFU/g) and compared with non-infected animals. In order to avoid any species-specific transcripts misalignments, B6 and SEG RNAseq reads were aligned respectively on mm9 reference genome or a Mus spretus pseudogenome constructed with Seqnature software (Munger et al. 2014). Resistant SEG contrary to B6 susceptible mice display an important activation of genes implicated in innate immune response in Low group whereas both strains display such activation in High group.
O-05: Emergence of extreme phenotypes and new disease models in the Collaborative Cross: from bronchiectasis to parkinsonism
Fernando Pardo‑Manuel de Villena*
Department of Genetics, University of North Carolina, Chapel Hill, NC, USA
When the Collaborative Cross was conceived over a decade ago it was proposed that one of the key features of this genetic reference population would be the presence of novel pairwise combinations of alleles at many loci and that these unique combinations would lead to the emergence of both novel phenotypes and new models of human disease. That prediction was initially confirmed in 2014 with the report of spontaneous colitis in one CC inbred strain (CC011/Unc) and the mapping of multiple interacting QTL. Since then, there has been an explosion of such findings for a wide spectrum of phenotypes ranging from susceptibility to infectious agents to basic physiology. In addition, it has also become apparent that the vast majority of the CC lines that were initially started were incompatible with life or fertility and that completed CC inbred strains are generally “unfit”. Based on findings in both extinct and extant CC lines we propose that epistasis between alleles from different subspecies underlies the prevalence and severity of disease phenotypes in the CC. We will discuss this hypothesis in the light of our characterization of two new models of human disease: drug induced parkinsonism and severe bronchiectasis.
O-06: Mouse embryonic stem cell lines derived in 2i culture conditions demonstrate X Chromosome silencing and extreme male sex bias.
Anne Czechanski*, Candice Byers, Whitney Martin, and Laura G. Reinholdt
The Jackson Laboratory, Bar Harbor, ME 04609
The establishment of pluripotent mouse embryonic stem cells (mESC) from recalcitrant strains is now possible through derivation in the presence of the inhibitor cocktail commonly known as 2i. Together, CHIR99021, a glycogen synthase kinase-3B inhibitor, and PD0325901, a MEK inhibitor, overcome recalcitrance by shifting metastable mESCs from the primed to the pluripotent ground state. We have derived over 150 novel mESC lines using 2i culture conditions and observed a striking male sex bias (96%), regardless of strain background. These results are unexpected given the reported female bias in human ES cell derivation and more recent data suggesting that two X Chromosomes promote pluripotency through the same mechanism as 2i in mESCs. To understand the timing and etiology of male sex bias in 2i derivation conditions, we collected 500 blastocysts from Tg(CAG-EGFP)D4Nagy/J sires and sorted by sex on the basis of EGFP expression. 250 embryos per sex were plated in either 2i+LIF or serum+LIF and cultured under our standard protocols. Prior to disaggregation, no differences in the progression of male and female outgrowths were observed. However, by day 12 in culture, we observed attrition of 80% of female 2i lines, compared to 34% of male 2i lines. In contrast, no sex bias was observed in serum+LIF conditions. Coincident with attrition, emergent female 2i mESC lines exhibited slow growth and poor colony morphology. Quantitative analysis of EGFP expression confirmed loss of EFGP expression consistent with XCI in female lines, a prerequisite for exit from the pluripotent ground state. We hypothesize that the combination of X Chromosome dosage and 2i culture conditions prevent the establishment of a stable ground state in emergent mESC lines eventually leading to XCI in the majority of female mESC lines. We are currently exploring these events through transcriptional profiling of transitory female cell lines.
O-07: Endogenous L1 Retrotransposition in the Mammalian Early Embryo and Primordial Germline
Sandra R. Richardson*1, Daniel J. Gerhardt1, Francisco J. Sanchez‑Luque1,2, Patricia Gerdes1, J. Samuel Jesuadian1, Marie‑Jeanne H.C. Kempen1, Gabriela‑Oana Bodea1, and Geoffrey J. Faulkner1,3
1Mater Research Institute-University of Queensland, TRI Building, Woolloongabba QLD 4102, Australia
2Pfizer-Universidad de Granada-Junta de Andalucia Centre for Genomics and Oncological Research (Genyo). P.T. Ciencias de la Salud. Avda. de la Ilustracion 114, 18016 Granada, Spain
3Queensland Brain Institute, University of Queensland, Brisbane QLD 4072, Australia
Long Interspersed Element 1 (LINE-1 or L1) is a mobile genetic element or “jumping gene” presently active in mammals. The average human genome harbours ~80-100 active L1 copies, while mice contain ~3,000 per individual. By virtue of their replicative mobilisation strategy--a process termed retrotransposition--L1 sequences have accumulated to occupy approximately 17% and 18% of human and mouse genomic DNA, respectively. L1 insertions within and proximal to genes can impact gene expression in a variety of ways, and retrotransposition events are frequently associated with duplication, deletion, and rearrangement of genomic sequences. Thus, L1 is a potent endogenous mutagen and an arbiter of genome diversification. In order to exert an ongoing impact on the genomic landscape of a species, new L1 insertions must occur in cells that will contribute their genetic material to subsequent generations—ie, within the germ lineage or in the pluripotent cells of the early embryo, prior to germline specification. Previous studies have suggested that the early embryo is a prominent milieu for L1 retrotransposition; however, systematic study of the frequency and developmental timing of heritable L1 retrotransposition events has been technically challenging. Here, we have adapted retrotransposon capture sequencing (RC-seq) to detect retrotransposon insertions in mouse genomes, and applied this technique to identify de novo heritable L1 insertions in multi-generation pedigrees of C57BL/6 mice. We find that such insertions arise at a rate of approximately 1 in 8 mice. Using a PCR genotyping strategy to deduce the developmental timing of these events, we find evidence consistent with L1 retrotransposition in the early embryo resulting in somatic and germline genetic mosaicism, as well as in early primordial germ cells (PGCs), giving rise to germline-restricted genetic mosaicism. Our findings shed new light on the frequency and developmental origins of the ongoing retrotransposition events continuously shaping mammalian genomes.
O-08: The complex transcriptional landscape of the C9orf72 gene locus the most common cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)
Patrizia Rizzu*1, Cornelis Blauwendraat1, Sasja Heetveld1, Emily M Lynes1, Melissa Castillo‑Lizardo1, Ashutosh Dhingra1, Elwira Pyz1, Matthis Synofzik1,2, Margherita Francescatto1, and Peter Heutink1
1German Center for Neurodegenerative Diseases (DZNE), Tuebingen, Germany
2Department of Neurodegenerative Diseases, Hertie Institute for Clinical Brain Research, University of Tuebingen, Germany
The non-coding hexanucleotide repeat expansion (HRE) in the C9orf72 gene is a major cause for ALS/FTD. Several studies suggest the disease might occur through different, not necessarily exclusive mechanisms: a loss of function of C9orf72 due to haploinsufficiency or a gain of function mechanism mediated by aggregates of bidirectionally transcribed HRE-RNA and unconventionally non-ATG-translated di-peptide proteins.
The loss of function is supported by decreased C9orf72 mRNA expression in patients but emerging evidence suggests that the gain of function is sufficient for neurodegeneration. Haploinsufficiency, though not the major culprit, could be still detrimental to cells leading to defects in endosomal and autophagic processes. In this context it becomes important to fully understand how C9orf72 expression is regulated. We therefore first surveyed the C9orf72 locus in the context of the FANTOM5 project.
C9orf72 was very highly expressed in myeloblasts, as compared to brain and other tissues. The expression profile of C9orf72 transcription start sites (TSSs) showed a complex architecture with the two TSSs for the annotated C9orf72 transcripts composed by tag clusters differentially expressed, between myeloblasts and brain tissues hinting to a C9orf72 transcript specific cell and/or tissue function which is corroborated by our weighted gene co-expression analysis on myeloblasts and brain data.
We detected new non-annotated TSSs in the sense and antisense strand at the C9orf72 locus, 5’end of the annotated C9orf72 transcripts and we confirmed their expression in brain tissues and myeloblasts cells from patients and control donors. The TSSs antisense to C9orf72 are particularly interesting as they suggest the presence of natural head-to-head antisense transcripts to C9orf72 mRNA that can provide an additional level of regulation of gene expression. Our qPCR experiments indeed indicate that C9orf72 and the antisense transcripts negatively correlate in C9 and interestingly in other neurodegenerative diseases patients moreover suggesting C9orf72 plays a common role in neurodegeneration.
O-09: Dissection of Genetic Redundancy: A Novel Role for FGF Signaling During Mouse Eye Development
Takaya Abe, Yui Yamashita, Yoshiko Mukumoto, Atsumi Denda, Mari Kaneko, Megumi Watase, Hiroshi Kiyonari, and Yasuhide Furuta*
Laboratories of Animal Resource Development and Genetic Engineering (LARGE), RIKEN Center for Life Science Technologies (RIKEN CLST), Kobe, Japan
The fibroblast growth factor (FGF) signaling system comprises a battery of ligands and receptors that are promiscuous in their binding partners. Due to apparent functional redundancy, the collective function of endogenous FGF family ligands in embryonic development has not been fully understood genetically. To examine the role of FGF signaling in the developing eye, we are generating mice lacking the function of multiple Fgf gene family members, Fgf3, Fgf9, and Fgf15, expressed in the developing retina. Homozygous mutants lacking either of these genes individually exhibit subtle or no morphological defects during early eye development. While combining multiple mutations is required to better understand the function of these genes, generation of Fgf3;Fgf9;Fgf15 compound mutants by conventional genetic crosses have been hampered due to suboptimal fertility of Fgf9 mutants and a close genetic linkage between Fgf3 and Fgf15 loci on the same chromosome. To circumvent these problems, we have employed CRISPR/Cas9-mediated multi-gene targeting to generate compound mutant embryonic stem (ES) cells. Mutant ES cells were used to generate completely ES-derived F0 chimeras for direct phenotypic characterization. Preliminary phenotypic analyses have revealed that embryos lacking the functions of both Fgf9 and Fgf15 show misrouting of retinal ganglion cell (RGC) axons soon after the initiation of RGC differentiation. Subsequently, RGC axons fail to target the central retina, resulting in the absence of the optic nerve. These results suggest a previously unrecognized role of FGF signaling during retinal development, apparently controlling RGC axon behaviors to properly target the central retina for the formation of the optic nerve. Further phenotypic analyses of mutants with various Fgf3;Fgf9;Fgf15 compound genotype combinations will also be reported. These approaches have provided us with a stable source of compound mutants, and thus will allow for efficient genetic analyses of lethal multi-gene mutations.
O-10: Molecular biomarkers of neurodegenerative disease in the olfactory epithelium
Gabriela Sanchez‑Andrade*1, Rishika Kundra2, Sujeong Yang3, Michel Goedert4, Maria Grazia Spillantini3, Michele Vendruscolo2, and Darren W. Logan1
1Wellcome Trust Sanger Institute
2Department of Chemistry, University of Cambridge
3John van Geest Centre for Brain Repair, University of Cambridge
4MRC-LMB, University of Cambridge.
Olfactory dysfunction is one of the earliest and most prevalent symptoms of neurodegenerative disorders like Frontotemporal dementia, Parkinson’s and Alzheimer’s diseases. Odours are detected, in the first instance, by olfactory sensory neurons (OSNs) in the olfactory epithelium (OE) of the nose. OSNs are exposed to the external environment, making them accessible for biomedical studies. For these reasons, molecular analysis of the OE may reveal prodromal biomarkers of neurodegenerative brain diseases.
Here we present an olfactory analysis of a mouse model of Frontotemporal dementia and Parkinsonism linked to Chromosome 17 (FTDP-17). We find that the transgenic mice, which express a human mutant P301S MAPT (Tau) gene in all neurons, display severe olfactory deficits from an early age, even before the onset of motor or cognitive symptoms. We sequenced RNA obtained from the OE of these animals and compared it to wild-type controls. From ~17,000 genes expressed in the OE, over 2,500 genes were differentially expressed between mutants and control.
We established a reduced repertoire of genes, the “Cambridge Set”, to study based on the propensity of their protein product to aggregate in neurodegenerative diseases. We show the Cambridge Set can distinguish FTDP-17 mice from a range of healthy controls. Changes in the expression of these genes in OE are very similar to changes in brain areas most affected by the pathology, like the brain stem. These changes in the OE start occurring early, at the same stage when the olfactory bulb starts showing the pathology.
This study suggests that molecular analysis of just a few genes expressed in the OE may indicate the early stages of neurodegenerative disease. They may also show the progression of the pathology. This represents a promising approach to finding prodromal biomarkers of neurodegenerative diseases. We are currently working on applying this to humans.
O-11: A tale of two gene sets: low and high variability in single cell RNA-seq data
Anna Mantsoki*, Guillaume Devailly, and Anagha Joshi
The Roslin institute, University of Edinburgh, Easter bush campus, Midlothian,EH25 9RG.
Gene expression heterogeneity contributes to development as well as disease progression. Due to technological limitations, most studies to date have focused on differences in mean expression across experimental conditions, rather than differences in gene expression variance. The advent of single cell RNA sequencing has now made it feasible to study gene expression heterogeneity and to characterise genes with low and high coefficient of variation.
We collected single cell gene expression profiles for 32 human and 39 mouse embryonic stem cells and classified genes in three groups based on their coefficient of variation (CV) across single cells (Low, Mid and High CV). We systematically characterised diverse properties distinguishing Low and High CV gene sets.
Low CV genes were enriched for cell cycle genes with their gene expression tightly controlled at transcriptional level, and showed greater conservation of sequence and expression variance across species. In contrast, High CV genes were co-expressed with other High CV genes and were enriched for bivalent (H3K4me3 and H3K27me3) marked promoters and showed a weak enrichment for embryonic development and transcription control functional categories.
Taken together, this analysis demonstrates the highly divergent characteristics of Low and High CV genes with Low CV genes representing tightly controlled genes with specific function, and High CV genes explaining bivalency at least in part.
O-12: Single-molecule RNA sequencing in single cells.
RIKEN CLST, Division of Genomics Technologies, Yokohama, Japan
Single-cell transcriptome analysis aims to characterise exhaustively every functional RNA molecule in a cell, to accurately describe and model complex cell populations. Over the years, RIKEN has developed CAGE (Cap Analysis Gene Expression) to sequence the 5’ ends of RNA molecules, thereby identifying transcription start sites at single molecule resolution and quantifying their activity in a single experiment.
In our recent developments for single-cell CAGE analysis, we modified the nanoCAGE protocol, that captures 5’ ends using template-switching oligonucleotides. We added unique molecular identifiers to these oligonucleotides, to measure expression levels in transcript counts. To suppress the formation random primers dimers and rRNA signal, which waste sequencing reads, we developed “pseudo-random” primers, where similar sequences to rRNA and adaptors were removed. As few as 40 different primers are enough to prepare whole-transcriptome libraries.
To retrieve sequence information that associates novel promoters to downstream annotations, nanoCAGE libraries are designed for paired-end sequencing. In our original CAGEscan method, the information recovered was a single pair per transcript, and all the transcripts produced by the same promoter were aggregated into single "CAGEscan clusters". We gained single-molecule resolution by adding a fragmentation step using the Nextera tagmentation system. This way, PCR duplicates bearing the same unique molecular identifier produce a collection of pairs with identical 5’ ends but random 3’ ends, which are assembled into single-molecule “CAGEscan fragments” that we are validating on Nanopore sequencers.
We use this new protocol for the analysis of FACS-sorted single cells, and ported it to the Fluidigm C1 platform under the name “C1 CAGE”. Our new protocol is particularly useful for studies in mammalian systems that do not benefit from a high-quality genome annotation, and is also a unique tool for the study of non-coding RNAs in human and mouse systems at the single-cell level.
O-13: Digital expression profiling of Purkinje neurons and dendrites in subcellular resolution
Anton Kratz*1, Pascal Beguin2, Stephane Poulain1, Megumi Kaneko2, Takahiko Chimura2, Ana Maria Suzuki1, Atsuko Matsunaga2, Sachi Kato1, Nicolas Bertin1, Timo Lassmann1, Rejan Vigot2, Piero Carninci1, Charles Plessy1, and Thomas Launey2
1RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa, 230-0045 Japan
2RIKEN Brain Science Institute, Launey Research Unit, Wako, Saitama, 351-0198 Japan
Neuronal cells are not homogenously distributed and subtypes are intermingled with each other as well as with non-neuronal cells such as glia and blood vessel cells. When attempting to digitally profile the expression of a specific type of neuron, retrieving the RNA only of that cell type therefore poses a considerable challenge.
In previous work, we used a technology called translating ribosome affinity purification (TRAP) to isolate the ribosome-associated transcriptome—the translatome. We modified it to target any cell type that can be specifically infected by a modified adeno-associated virus, and applied it to Purkinje cells (PCs) in the rat cerebellum.
We obtained quantitative expression data in single-base-pair resolution by profiling the ribosome-associated, isolated RNA using the nanoCAGE protocol. Subsequent data analysis revealed the landscape of ribosome-associated RNA of PCs in different subcellular compartments: cytoplasm and rough endoplasmatic reticulum.
In neurons, protein translation occurs not only in the soma but also distally in dendrites near or within the dendritic spines. This distal translation is thought to be regulated in response to external stimuli including long-term depression and memory formation. We have applied TRAP to Purkinje dendrites and sequenced the isolated RNA with an improved nanoCAGE protocol including a tagmentation step, to address the increased difficulty of sequencing from dendrites, which contain even less RNA than Purkinje cell soma.
O-14: Time-dependent host response to influenza A virus infection in Collaborative Cross founder strains and lines
Heike Kollmus*1, Sarah Leist1, Carolin Pilzner1, Leonie Dengler1, Robert Geffers2, Rudi Balling3, and Klaus Schughart1,4,5
1Department of Infection Genetics, Helmholtz Centre for Infection Research, Braunschweig, Germany
2Genome Analytics, Helmholtz Centre for Infection Research, Braunschweig, Germany
3Luxembourg Centre for Systems Biomedicine, University of Luxembourg, Luxembourg
4University of Veterinary Medicine Hannover, Hannover, Germany
5University of Tennessee Health Science Center, Memphis, Tennessee, US
Influenza A virus is a zoonotic pathogen that poses a major threat to human and animal health. The course and outcome of influenza infection is influenced by viral virulence factors but also by differences in the host response. We studied the host response in the eight Collaborative Cross founder strains and several Collaborative Cross lines after influenza A H3N2 infections.
The founder strains exhibited a large diversity in their response to influenza infections with respect to survival, body weight loss, hematological parameters in the blood, relative lung weight and viral load. Strain was the main factor influencing body weight loss, thus indicating that genetic variation is the major cause for the different outcomes. Analysis of survival rates and mean time to death revealed three different groups of susceptibility phenotypes: highly susceptible (A/J, CAST/EiJ, WSB/EiJ), intermediate susceptible (C57BL/6J, 129S1/SvImJ, NOD/ShiLtJ) and highly resistant strains (NZO/HlLtJ, PWK/PhJ). Viral load was largely different between susceptible and resistant strains but not between different susceptible strains. CAST/EiJ mice showed a unique phenotype. Despite very high viral loads in their lungs, they exhibited low counts of infiltrating granulocytes and showed increased numbers of macrophages in the lung. Transcriptome analyses of peripheral blood cells and lungs confirmed an aberrant immune response in CAST/EiJ. The unique phenotype of the CAST/EiJ strain may provide a novel highly valuable model to understand abnormal immune responses to infections in humans.
Our studies of the host response to influenza infections in Collaborative Cross lines revealed further variation in phenotypes which are currently being further characterized by RNAseq transcriptional profiling.
O-15: A forward-reverse systems genetics approach to understand host-pathogen genetic interactions
Clare Smith*1, Bibhuti Mishra1, Jarukit Long1, Megan Proulx1, Jia Yao Phuah1, Gillian Beamer2, Richard Baker1, Martin T Ferris3, Robert Williams4, and Christopher Sassetti1,5
1Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, MA, USA
2Department of Infectious Diseases and Global Health, Cummings School of Veterinary Medicine, Tufts University, MA, USA
3Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, NC, USA
4Department of Anatomy and Neurobiology, University of Tennessee Health Sciences Center, Memphis, TN, USA
5Howard Hughes Medical Institute
Infection with Mycobacterium tuberculosis (Mtb) results in a spectrum of disease, ranging from asymptomatic to lethal disease. The underlying genetic causes driving the outcome to infection are unknown and likely involve a complex interplay of both host and pathogen factors. Here, we use a dual systems genetics approach to unravel the genetic interactions between host and pathogen, exploiting diverse panels of recombinant inbred mice and genome-wide bacterial genetics.
We infected 55 Collaborative Cross (CC) lines, 19 BXD lines and parental lines of both crosses with saturated libraries of Mtb transposon mutants to examine interactions between mouse genotype, extent of disease, and requirements for specific bacterial virulence functions. We quantified a variety of disease phenotypes, including bacterial burden, replication dynamics, wasting, and histopathology. The disease spectrum of the CC lines exceeded that seen in parental strains and standard inbred lines. This diversity was also apparent in the specific bacterial genes important for bacterial fitness through transposon sequencing (Tnseq) analysis of libraries from each mouse line. We identified bacterial genes differentially required in each unique host genetic background during infection. Quantitative trait loci (QTL) analysis is underway to identify host polymorphisms associated with specific disease alterations and bacterial mutant fitness traits, with several QTLs so far identified underlying differential control of bacterial burden.
The combination of a classic forward genetics approach to identify host susceptibility loci in conjunction with a reverse genetic approach to assess specific bacterial fitness requirements under different host genetic backgrounds will allow dissection of the host-pathogen crosstalk driving Mtb disease.
O-16: Mapping thrombosis modifier genes by bulk exome sequencing mice from a sensitized ENU mutagenesis screen
Kart Tomberg*1, Randal J Westrick2, David R Siemieniak3,4, Guojing Zhu3, Thomas Saunders5, and David Ginsburg1,3,4,6
1Department of Human Genetics, University of Michigan
2Department of Biological Sciences, Oakland University
3Life Sciences Institute, University of Michigan
4Howard Hughes Medical Institute, University of Michigan
5Transgenic Animal Model Core Laboratory, University of Michigan
6Department of Internal Medicine, University of Michigan
Only ~10% of individuals carrying the common venous thrombosis risk factor, Factor V Leiden (FVL) will develop venous thrombosis in their lifetime. In order to identify potential FVL modifier genes, we performed a sensitized dominant ENU mutagenesis screen, based on the perinatal synthetic lethal thrombosis previously observed in mice homozygous for FVL (F5tm2Dgi/F5tm2Dgi) and hemizygous for tissue factor pathway inhibitor (Tfpi+/Tfpitm1Gjb).
Out of ~9150 G1 (generation 1) offspring of mutagenized C57BL/6-F5tm2Dgi/F5tm2Dgi males and unmutagenized C57BL/6-F5tm2Dgi/+ Tfpi+/Tfpitm1Gjb females, we retrieved a total of 165 viable B6-F5tm2Dgi/F5tm2Dgi Tfpi+/Tfpitm1Gjb progeny (rescues). As an alternative to generating a pedigree and traditionally mapping the causal mutation for each G1 rescue progeny, whole exome sequencing was applied to DNA from 103 G1 and an additional 11 G2-G8 rescue progeny to identify candidate genes that are enriched for ENU mutations. A total of 3511 likely ENU coding variants (excluding synonymous SNVs) were identified in 3009 genes. After adjusting for coding region size, the ENU-induced mutation burden for 13 genes was significantly greater than expected by chance (p<0.0005, based on 10,000,000 permutations). Sanger sequencing validated 40 out of 41 variants within the top 13 genes. Validation of the top 6 genes (Arl6ip5, C6, Itgb6, Cpn1, Sntg1, Ces3b) is in progress, using CRISPR/CAS9 to introduce independent null alleles into mice.
Variation in these genes in humans could explain a significant portion of the incomplete penetrance and variable expressivity among patients with FVL, offer new insights into the overall regulation of hemostasis, and facilitate the development of future novel therapeutic interventions.
O-17: A systematic functional screening approach to identify candidate genes obtained through large scale whole genome and exome sequencing of human disease cohorts.
Peter Heutink*, and for the International Parkinsons Disease Genomics Consortium (IPDGC)
German Center for Neurodegenerative Diseases Tuebingen.
Genome sequencing is an efficient way to identify genes related to human diseases but it also generated a major problem; How do we interpret the large volumes of sequencing data and valuable biological information from trivial data? Large scale sequencing of patients identifies hundreds to thousants of potentially damaging variants in each individual and bioinformatic analysis alone is not sufficient to identify the causal variants that can be used as the starting point for building cellular or animal models to understand the pathogenic processes of disease. We have therefore developed a systematic screening approach to prioritise candidate genes from large scale datasets for functional follow up. As proof of principle we have generated whole exome sequencing data on ±1200 early onset Parkinson's Disease patients and > 2000 matched control samples. After QC we identified a total of 918,252 variants. Focussing on loss of function variants that are often associated with Autosomal Recessive Early Onset Parkinsons disease we identified 62 gene with homozygous or putative compound heterozygous loss of function variants absent in controls. In additional genetic datasets we identified further evidence for a subset of these genes and we performed systematic RNAi screens for all 62 genes using relevant assays for Parkinson's disease using three model systems; C. elegans; D. melanogaster and human neuroblastoma cell lines. As several molecular pathways have been shown to be involved in the disease process we used a variety of assays for cell viability/ alpha synuclein toxicity; mitochondrial function and morphology; intercellular translocation of PINK/Parkin. Based on our screens we have ranked our candidate genes for further follow up and have identified the molecular pathway in which each candidate gene is involved. Our approach is widely applicable and efficiently reduced the number of variants and genes to be used for in depth follow up studies.
O-18: Natural allelic variation of interleukin 21 receptor modulates ischemic stroke outcomes
Han Kyu Lee*1, Sehoon Keum1, Donald Lo2, and Douglas Marchuk1
1Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA
2Center for Drug Discovery and Department of Neurobiology, Duke University Medical Center, Durham, North Carolina, USA
Using quantitative trait locus (QTL) linkage analysis of cerebral infarct volume after middle cerebral artery occlusion (MCAO) we previously identified a locus on distal chromosome 7 that contributes to over 50% of the variation in infarct volume. This locus shares the same location with a locus that modulates variation in collateral vessel number. Here, using interval-specific ancestral SNP haplotype analysis we fine-mapped the locus to only 12 candidate genes. One gene, interleukin 21 receptor (Il21r), showed a significant difference in strain-specific transcription levels. To determine whether Il21r modulates infarction, we examined C57BL/6-Il21rtm1Wjl(Il21r KO) mice for their collateral vessel anatomy and cerebral infarct volume. While Il21r KO mice showed a moderate reduction in collateral vessel connections compared to wild-type littermates, cerebral infarct volume in Il21r KO mice was increased 2.3-fold. This suggests that Il21r has effects on both collateral vessel anatomy and innate neuroprotection. To examine the latter, we performed an ex vivo study of brain slices under in vitro oxygen deprivation (OD) and found that Il21r KO brain slices show an increase in OD induced neuronal cell death, showing that Il21r is also involved in collateral-independent neuroprotection. We determined that neuronal cell death is mediated by IL21-IL21R signaling transduction via STAT3. Interestingly, we also found a coding SNP difference in IL21R that segregates with infarct volume and collateral vessel anatomy across the strains, and determined that this sequence variation can also modulate receptor function to regulate neuronal cell death. Taken together, natural genetic variation in Il21r determines neuronal cell viability through modulation of receptor function, resulting in differential downstream signal transduction. Ultimately, these differences work in concert to modulate infarct volume in ischemic stroke. The identification of neuroprotective genes based on naturally occurring allelic variation will provide a path for the development of novel drug targets for ischemic stroke treatment.
O-19: Identification of novel susceptibility genes for aggressive prostate cancer using Diversity Outbred mice
Kendra Williams1, Jonathan Andreas1, Ying Hu2, Sujata Bupp1, Daniel M Gatti3, Gary A Churchill3, and Nigel Crawford*1
1Genetics and Molecular Biology Branch, NHGRI, National Institute of Health, Bethesda, MD 20892, USA
2Center for Biomedical Informatics and Information Technology, NCI, National Institute of Health , Rockville, MD 20850, USA
3The Jackson Laboratory, Bar Harbor, ME 04609, USA
Prostate cancer (PC) is the most commonly diagnosed non-cutaneous malignancy in men. However, <13% of cases are fatal, and more accurate means of quantifying aggressive PC risk are required to avoid over-treatment. We aimed to identify aggressive PC susceptibility genes using the C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of neuroendocrine PC, which represents a particularly aggressive form of this disease. We performed quantitative trait locus mapping (QTL) in a cohort of 392 (TRAMP x J:DO) F1 males to identify genetic regions associated with susceptibility to aggressive disease susceptibility. These analyses revealed two QTLs associated with metastasis achieved genome-wide significance (Chromosome 8 [LOD=8.94], and Chromosome 15 [LOD=7.87]). For the Chromosome 8 locus, analysis of strain-specific allelic effects indicated that linkage was driven by the 129S1/SvlmJ and PWK/PhJ strains; and for the Chromosome 15 locus, linkage was driven by the PWK/PhJ, NZO/HILtJ and WSB/EiJ strains. Trait-correlation and eQTL data derived from microarray analysis of 90 (TRAMP x J:DO) F1 tumors were integrated with strain-specific SNP data to identify 24 candidate metastasis susceptibility genes. The role of these genes in aggressive human PC was investigated via an in silico validation that utilized human tumor gene expression datasets and a human PC genome-wide association study. This approach led to the identification of 8 novel aggressive PC susceptibility genes. Among these genes were three type II Keratin gene family members (KRT5, KRT78, and KRT81). Interestingly this keratin cluster, which is located on human Chromosome 12q13, has been previously implicated in germline susceptibility to PC. Our analysis demonstrates the advantage of using systems genetics approaches and mouse models to gain insights into how hereditary variation influences susceptibility to aggressive PC. Ongoing work is focusing on the functional characterization of these genes to more fully comprehend their role in susceptibility to metastasis in PC.
O-20: Strong epistatic control of development of leishmaniasis.
Tatyana Kobets*1, Yahya Sohrabi1, Matyas Sima1,2, Valeriya Volkova1, Eliska Javorkova3, Jarmila Vojtiskova1, Tereza Pokorna1, Jan Bartunek1, Alena Zajicova3, Igor Grekov1, Tatana Jarosikova4, Martina Slapnickova1, Helena Havelkova1, Milena Svobodova2, Vladimir Holan3, Peter Demant5, and Marie Lipoldova1
1Laboratory of Molecular and Cellular Immunology, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague, Czech Republic
2Faculty of Science, Charles University, 128 44 Prague, Czech Republic
3Institute of Experimental Medicine Academy of Sciences of the Czech Republic, Videnska 1083, 14220 Prague, Czech Republic
4Faculty of Biomedical Engineering, Czech Technical University in Prague, Sitna 3105, 272 01 Kladno, Czech Republic
5Roswell Park Cancer Institute, Buffalo, New York 14263, USA
Leishmaniasis, the main health problem in a number of countries, is caused by a protozoan parasite Leishmania that infects mononuclear phagocytes. The immune response is determined by multiple factors, including parasite characteristics and host genetics.
We developed a unique model for analysis of epistasis based on two-way interaction between genomes of Leishmania-resistant mouse strains, O20 and C57BL/10 (B10). Transfer of genes of O20 into background of B10 generated an inbred strain B10.O20, highly susceptible to Leishmania. Contrastly, transfer of B10 genes into O20 background resulted in OcB recombinant congenic strains, carrying 6.2% or 12.5% of B10 genes. OcB11 and OcB31 strains exhibited significant susceptibility. Thus, susceptible phenotypes could develop from specific combinations of genes originating from resistant strains.
After Leishmania major infection, B10.O20 mice evolved large lesions, high parasite numbers in skin and lymph nodes, and massive infiltration of CD11b+Gr1+ cells in spleen. After stimulation with soluble Leishmania antigen (SLA), splenocytes of infected B10.O20 produce more Th1, Th2 and Th17 than B10 and O20, suggesting chronic inflammation with imbalance of several axes of immune response. In contrast, splenocytes of highly resistant O20 lacked response to SLA. Its intraperitoneal macrophages produced IL12, but not NO, suggesting a novel mechanism of resistance. As B10.O20 carries only 4.2% of O20-derived genes, it offers an opportunity to study this strong epistasis.
We also studied susceptibility to L. major in 15 OcB RC strains. Using F2 hybrids, derived from OcB43 (an OcB31 substrain), we detected that loci on Chromosomes 3 and 15 controlled parasite numbers in liver. Lesion was controlled by the interaction between loci on Chromosomes 2 and 3.
Combining detailed genetic analysis of these models with analysis of immunological and pathological parameters of infected mice and gene expression studies will provide a powerful tool to describe different mechanisms of resistance and susceptibility. Grant GACR 13-41002P.
O-21: Metabolic regulation by the MECP2 transcriptional repressor complex points to new therapeutic targets in Rett syndrome
Stephanie M Kyle*1,2, and Monica J Justice1
1The Hospital for Sick Children, Toronto, Ontario, Canada
2Baylor College of Medicine, Houston, Texas, USA
Metabolic dysregulation can lead to downstream pathogenesis in nearly all tissues and organ systems. In recent decades, a large body of data has implicated metabolic perturbations in neurological development and degeneration. In particular, dysregulation of cholesterol trafficking and biosynthesis are responsible for the onset of Neimann-Pick type C and Smith-Lemli Opitz syndrome, respectively. Furthermore, Fragile X syndrome, Alzheimer, Parkinson, and Huntington diseases have all been linked to aberrant cholesterol homeostasis. Rett syndrome (RTT) is a progressive neurodevelopmental disorder of females primarily caused by mutations in the X-linked gene encoding methyl-CpG-binding protein 2 (MECP2). To identify pathways in disease pathology for therapeutic intervention, we carried out a dominant random mutagenesis suppressor screen in Mecp2 null mice. One suppressor identifies a stop codon mutation in a rate-limiting enzyme in cholesterol biosynthesis, which ameliorates RTT-like symptoms and increases longevity in Mecp2 null mice by altering cholesterol homeostasis. Although RTT has been classically labeled a neurological disorder, these studies suggest that a metabolic component contributes to pathology. Here we show that Mecp2 deletion induces hyperlipidemia, fatty liver, and metabolic syndrome in mice. These metabolic phenotypes are strikingly similar to that in mice with a liver-specific knockout of histone deacetylase 3 (Hdac3), a potent regulator of lipogenesis and cholesterol biosynthesis. Consistently, we show that MECP2 and HDAC3 work in complex to suppress expression of the cholesterol enzyme identified in our screen, as well as other genes of the cholesterol and de novo lipogenesis pathways. Our data suggest a novel metabolic component in RTT, arising from loss of interaction between MECP2 and HDAC3. Concurrently, liver-specific deletion of Mecp2 is deleterious enough to cause fatty liver through aberrant lipogenic gene transcription. Our ongoing studies point to additional metabolic pathways that are prime targets in the pursuit of preventing morbidities associated with Rett syndrome.
O-22: Mouse Models of Human Diaphragmatic Birth Defects: The mesothelium performs a fundamental role in proper formation of the diaphragm
Nicole Paris, and Kate Ackerman*
Department of Pediatrics and Biomedical Genetics, University of Rochester Medical Center, Rochester, NY, 14642
Understanding genes and signaling pathways critical for normal diaphragm development is important for understanding human congenital defects of the diaphragm. Since many of the mouse genetic knock out models are embryonic lethal, we have worked to identify Cre Recombinase mouse lines useful for creating conditional deletion of genes required for diaphragm development. We have characterized an inducible Wt1 CreERT2 mouse line (STOCK-Wt1tm2(cre/ERT2)Wtp) and used it to understand whether β-catenin (CTNNB1) plays a critical role in diaphragm development. We have also used these models to investigate the role for the WT1+ mesothelium in normal diaphragm signaling and development.
We performed fate mapping of Wt1 expressing cells to the diaphragm after timed injections of tamoxifen (STOCK-Wt1tm2(cre/ERT2)Wtp x STOCK-Gt(ROSA)26Sortm1Sho), and we performed tissue specific, temporally regulated CTNNB1 loss-of-function and gain-of-function experiments with analysis of embryonic diaphragm development. These models primarily altered signaling in the diaphragmatic mesothelium. Loss of WT1 resulted in decreased WNT signaling and Ctnnb1 expression during a critical time period of development (E9.5-E12.5). Conditional loss of Ctnnb1 with Wt1tm2(cre/ERT2)Wtp mice caused large bilateral posterior diaphragm defects similar to the phenotype of Wt1 mutants while CTNNB1 activation rescued this phenotype. Although apoptosis and proliferation were affected, these genetic manipulations resulted in large changes in mesenchymal differentiation.
The diaphragmatic mesothelium functions to regulate normal diaphragm development. CTNNB1 is critical for diaphragm development during a defined window of time, and the mechanism of the diaphragm defects of the Wt1 and Ctnnb1 mutants involve disruption of basic cellular processes.
O-23: Genomic responses in mouse models greatly mimic human inflammatory diseases
Fujita Health University
The use of mice as animal models has long been considered essential in modern biomedical research, but the role of mouse models in research was challenged by a recent report that genomic responses in mouse models poorly mimic human inflammatory diseases (Seok et al., PNAS, 2013). We have been investigating the molecular basis of psychiatric disorders using gene expression analyses in mice models of the disorders. By applying the same analysis methods we have been using, we reevaluated the same gene expression datasets used in the study by Seok et al. Contrary to the previous findings, the gene expression levels in the mouse models showed extraordinarily significant correlations with those of the human conditions (Spearman's rank correlation coefficient: 0.43-0.68; genes changed in the same direction: 77-93%; P = 6.5 × 10(-11) to 1.2 × 10(-35)). Moreover, meta-analysis of those datasets revealed a number of pathways/biogroups commonly regulated by multiple conditions in humans and mice. These findings demonstrate that gene expression patterns in mouse models closely recapitulate those in human inflammatory conditions and strongly argue for the utility of mice as animal models of human disorders.
In this talk, I will introduce the debates ignited by the studies and also discuss the general significance of mice models of human diseases.
O-24: Bipartite structure of the inactive mouse X Chromosome
Xinxian Deng1,7, Wenxiu Ma2,7, Vijay Ramani2,7, Andrew Hill7, Fan Yang1,7, Ferhat Ay2,7, Joel B Berletch1,7, Carl Anthony Blau3,4,7, Jay Shendure2,7, Zhijun Duan3,4,7, William S Noble2,5,7, and Christine M Disteche*1,6,7
1Department of Pathology
2Department of Genome Sciences
3Institute for Stem Cell and Regenerative Medicine
4Division of Hematology
5Department of Computer Science and Engineering
6Department of Medicine
7University of Washington, Seattle, Washington, USA
We analyzed allele-specific chromatin contacts by a new Hi-C assay that uses DNase I for chromatin fragmentation to evaluate structural changes associated with X inactivation and imprinting in mouse F1 hybrid systems in which alleles can be distinguished based on single nucleotide polymorphisms. Both in vivo (brain) and in vitro (Patski cells) the two X Chromosomes have strikingly different 3D configurations. Two superdomains of frequent long-range intrachromosomal contacts separated by a hinge region are specifically observed on the inactive X Chromosome. Such a bipartite 3D organization has been also reported in human lymphoblastoid cells. We found that the genomic content of the superdomains is rearranged between human and mouse, but that part of the hinge region is conserved and located near the lncRNA Dxz4/DXZ4 locus that binds CTCF on the inactive X. In mouse, the hinge region also contains a minisatellite Ds-TR adjacent to a promoter with strong CTCF binding. Both Dxz4 and Ds-TR bind nucleophosmin and are enriched in nucleolus-associated chromatin, suggesting anchoring to the nucleolus. Genes that escape X inactivation and regions enriched in CTCF or RNA polymerase are preferentially located on the periphery of the inactive X while LINE1 elements are preferentially on the interior. Genes subject to silencing exhibit fewer detectable short-range intrachromosomal contacts than escape genes. This transcription-coupled pattern is also evident for imprinted genes, in which more chromatin contacts are detected on the expressed allele, suggesting greater constraint on the organization of expressed genomic regions.
O-25: Genetic and dietary effects on gametic selection at fertilization
Ghunwa Nakouzi1,2, Delphine Carouge3, Sabine Schaefer3, and Joseph Nadeau*3
1Center for Human Genetics, University Hospitals of Cleveland
2Department of Genetics, Case Western Reserve University School of Medicine, Cleveland
3Pacific Northwest Diabetes Research Institute, Seattle
Detecting Mendel’s Law of Segregation is usually based on gametes combining randomly at fertilization, independent of their genetic constitution. With rare exception such as t-haplotypes in mice, the X-chromosome in crosses with Mus spretus, and SD in Drosophila, Mendelian expectations hold. However, recent studies reveal new exceptions based on seemingly common dietary and genetic effects. Dietary folate supplementation reduces the incidence and severity of neural tube defects (NTDs) in humans and mouse models. In some models, dietary supplementation leads to highly non-random genotypic ratios that are usually interpreted as folate-induced embryonic lethality of mutant homozygotes and heterozygotes. In addition, litter sizes are not reduced and resorption rates are not increased, suggesting that lethality does not account for the unusual genotypic ratios. Instead, deviations from Mendelian expectations in intercrosses but not backcrosses suggest preferential fertilization between particular eggs and sperm. The second example involves the Deadend1 gene (Dnd1, miRNA control), Apobec1 complementation factor (A1cf), the Apobec1 cytidine deaminase gene (Apobec1, RNA editing) and other genes involved in RNA biology, which have heritable epigenetic effects on susceptibility to testicular germ cell tumors (TGCTs). Mutations in these genes, either alone or in combination, also lead to strong deviations from Mendelian expectations in intercrosses but not backcrosses. Again, neither litter sizes nor resorption rates are affected. Thus both folate supplementation as well as mutations in Dnd1, A1cf, Apobec1 and related genes lead to a strong bias for fertilization with wild-type rather than mutant alleles. We propose that anomalies in polyamine metabolism mediates the fertilization bias with folate supplementation of NTD mutants. Both the dietary and genetic discoveries must be more fully characterized and mechanisms identified because of their implications for our understanding of inheritance and fertilization as well as for public health policies concerning dietary supplementations to prevent common birth defects.
O-26: Epigenetic inheritance of diet induced obesity and diabetes via oocyte and sperm
Peter Huypens1, Daniela Dyckhoff1, Moya Wu1, Steffen Sass3, Susan Marschall1, Fabian Theis3,4, Martin Hrabe de Angelis1,2,5, and Johannes Beckers*1,2,5
1Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Munchen GmbH, 85764 Neuherberg, Germany
2German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany
3Institute of Computational Biology, Helmholtz Zentrum Munchen GmbH, 85764 Neuherberg, Germany
4Technische Universitat Munchen, Department of Mathematics, 85747 Garching, Germany
5Technische Universitat Munchen, Chair of Experimental Genetics, 85354 Freising, Germany
The inheritance of epigenetic information across generations has been controversial in mammals. Some reports provided initial evidence that a paternal high fat diet may propagate obesity and glucose intolerance in offspring, but potential confounders such as molecular factors present in seminal fluid or paternal-induced alterations in maternal care were not ruled out in these studies. We show in mice that a parental high fat diet renders offspring derived via in vitro fertilization (F1) more susceptible to develop excessive overweight and type 2 diabetes (T2D) in a gender and parent-of-origin specific mode. Female, but not male, offspring from obese parents became significantly more obese during a HFD challenge than female offspring from lean parents. Body weight trajectories and distribution patterns of individual body weights in female offspring from one obese and one lean parent demonstrate that paternal and maternal germline propagate obesity in a roughly equitable and additive fashion, but likely different mode of action. In contrast, a more deteriorated state of HFD-induced insulin resistance was observed in both F1 genders, albeit predominantly inherited via the maternal germline. Analyses of transcriptome and methylome signatures in gametes as well as the analysis of parental systemic factors that may contribute to the soma-to-germline information transfer are currently ongoing. We report for the first time epigenetic inheritance of an acquired metabolic disorder via mammalian oocytes and sperms excluding confounding factors. Such an epigenetic mode of inheritance may contribute to the observed pandemic increase in obesity and T2D prevalence rates, especially in an environment where nutrition is abundant.
O-27: Exploring regulatory networks through omics data
S Sethi*1, S Greenaway1, S Kumar1, J Fernandez2, M Simon1, and Ann‑Marie Mallon1
1MRC Harwell, Harwell Science and Innovation Campus, Harwell OX11 0RD, UK
2The Wellcome Trust Centre for Human Genetics, University of Oxford, OX3 7BN, UK
Transcriptional regulation involves a complex network of transcription factors (TFs) binding to DNA regulatory elements to control gene expression. In order to understand transcriptional regulation, we require the genomic locations of regulatory elements, the identity of factors that bind them, and the genes they target. ChIP-seq has been widely used to identify in vivo transcription factor binding sites (TFBSs), promoters and enhancers across the genome. DNase-Seq is used to identify DNASE1 hypersensitive sites (DHSs) across the genome indicating regions which have open chromatin and are accessible to DNA-binding proteins. RNA-Seq identifies co-expressed genes. My aim is to explore the publicly available data in ENCODE to study transcriptional regulation and to develop novel methods to analyse the phenodeviants at Mammalian Genetics Unit (MGU).
Here I present a novel method to identify known and novel TFBSs using DHSs. Using ChIP-seq data of various histone modifications from mouse ENCODE, we performed Chromatin segmentation in 22 tissues. This systematically characterized the mouse genome into regions of promoters and enhancers. Using this method, we observe that on average promoters, enhancers and insulators cover 1.25%, 4.5%, 0.8% of the mouse genome respectively. Overlying this data with DHSs, we discovered that TFBSs are highly enriched in DHSs as compared to promoter and enhancer regions suggesting DNase1 hypersensitivity data could be used as an alternative method for discovering enriched regulatory motifs.
We further implemented this method on RNA-Seq data from different mouse models of human diseases and found novel direct and indirect regulatory interactions. Future applications involve extending this model to include a phylogenetic module complexity analysis to detect conserved co-occurring TFBSs patterns within cis-regulatory modules.
O-28: Retinal cell therapy using iPS cells
RIKEN Center for Developmental Biology
The first in man application of iPS-derived cells started in September 2014 targeted the retinal disease called age-related macular degeneration (AMD). The grafted iPS-derived retinal pigment epithelial (RPE) cell sheet is survived well and good in color, that means no immune rejection occurred without immune suppression. Her visual acuity is stable, compare to the past history of deterioration even with multiple anti-VEGF injections. Primary endpoint, the safety was achieved at one year point.
We evaluated plasmid remnant & gene alteration using WGS, epigenetic characteristics and purity using single cell RT-PCR other than our original quality control (QC). From these experiences, we think we should do both but should distinguish between basic research and regulatory science in order to promote regenerative medicine promptly.
Since autologous transplantation is time consuming and the cost is high, it is necessary for making standard treatment to prepare allogeneic transplantation using HLA three loci homozygous iPS cell lines (iPS cell stocks) as well as autologous transplantation. It is known that RPE cells suppress the activation of T-cells, so that RPE cells appeared most suitable for such kind of allogeneic transplantation. We confirmed in vitro and in vivo that human iPS-derived RPE cells also have such function. It is possible that the rejection is considerably small by using the iPS-RPE cell with matched three loci of HLA.
O-29: CRISPR/Cas9 genome editing in rodents: In vivo and in vitro applications
Marie‑Christine Birling*1, Philippe Andre1, Sylvie Jacquot1, Laurence Schaeffer1, Marie Wattenhofer‑Donze1, Guillaume Pavlovic1, and Yann Herault1,2
1Institut Clinique de la Souris, PHENOMIN, CNRS UMR7104, INSERM U964, Universite de Strasbourg, 1 rue Laurent Fries BP 10142 Parc d"Innovation 67404 Illkirch, France
2Institut de Genetique Biologie Moleculaire et Cellulaire (IGBMC), CNRS, INSERM, Universite de Strasbourg, UMR7104, UMR964, Illkirch, France
The CRISPR/Cas9 system allows to generate insertions, deletions, duplications or substitutions at specific sites in rodents by simple pronuclear injection of the Cas9 mRNA or protein, one or more specific guide mRNA and a DNA template for specific modifications (when a specific modification is required). In many cases, this technology abolishes the need of embryonic stem cells.
We have obtained deletions through non-homologous end joining (NHEJ) with efficiencies up to 70% in both mouse and rat. We are currently working on improving the use of CRISPR/Cas9 for integrating mutations by homology- directed repair in order to be able to generate quicker and cost effective customized rodent models. We are also applying the CRISPR/Cas9 technology to the generation of mouse model that will be phenotyped in the IMPC (http://www.mousephenotype.org/). KO alleles are generated by deletion of one or more critical exon(s) in genes which are not currently available as targeted ES cells. Even more exciting, the CRISPR/Cas technology allows the generation, in an impressively fast way, of deletion/duplication of big genomic region (3-4 months versus to 3-4 years with the standard TAMERE route). We have easily achieved the deletion/ duplication of a 24 megabases genomic DNA fragment in rat.
We are also using successfully the CRISPR/Cas9 system in vitro in ES cells to improve the targeting efficiency for projects which failed previously. We have currently recovered 5 projects for which we were not able to obtain any targeted ES cells (in some case we screened previously more than 1500 ES cells) by standard electroporation. The simple addition of a plasmid expressing the Cas9 and a guide RNA recognizing the site of insertion of the selection cassette has dramatically improved the homologous recombination rate.
A few cases of both in vivo and in vitro experiments will be presented and discussed.
O-30: CRISPR/Cas9-mediated plasmid knock-in and replacement of genomic region with single stranded oligonucleotides in rodents
Kazuto Yoshimi*1, Takehito Kaneko2, Tsuyoshi Koide1, and Tomoji Mashimo3
1Mouse Genomics Resource Laboratory, National Institute of Genetics
2Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University
3Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University
Genome editing technologies such as ZFN, TALEN and CRISPR/Cas9 have enabled generating genetically modified animals within the past few years. Development of an efficient knock-in (KI) technology will facilitate easy and flexible genome engineering to introduce precise mutations or genetic modifications at any target sites of any cells from any strains and species.
We have reported the efficient generation of several types of KI rats using single-stranded oligodeoxyribonucleotides (ssODNs), such as SNP substitution, small DNA fragment insertion, and DNA fragment elimination of a 7kb retrotransposon element (Yoshimi K et al., Nat Commun 2014). It is easy to design and synthesize 80-160 base ssODNs donors, while the limitation of ssODN-mediated KI is the maximum length of the insert DNA, which is less than 100bp.
To generate the targeted KI with longer DNA fragments such as GFP reporter genes, we used CRISPR/Cas9 as “scissors” to cut at targeted sites in genome DNA and the plasmid DNA, and ssODNs as “paste” to ligate the ends of the cut sites. Co-microinjection of Cas9 mRNA, two gRNAs, two ssODNs and CAG-GFP donor plasmids into fertilized eggs in rats and mice resulted in pups carrying a CAG-GFP KI at the targeted Gt(Rosa26)Sor loci. The methods enabled not only efficient KI of plasmids without homology arms, including a 200kb bacterial artificial chromosome, but also the replacement of rat genes to human-specific genes.
These gene KI technologies are essentially applicable to any targeted site with any donor vector as it is in any species, which can provide the generation of genetically humanized models in a variety of species for understanding the mechanisms of human disease and physiological function.
O-31: CRISPR-Driven Replacement of a Mouse Tumor Suppressor with 25-kbp of the Orthologous Human Gene
Tiffany Leidy‑Davis, Kai Cheng, Meghan Schultz, Leslie Goodwin, Jiayuan Shi, Judy Morgan, and David Bergstrom*
Genetic Resource Science, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609 USA
Our interests lie in humanizing specific regions of the mouse genome using large segments of human DNA with extents of 10s to 100s of kilobase pairs. In the recent past, we achieved the targeted replacement of an 18-kbp segment of the mouse genome using a classic embryonic stem cell approach coupled with traditional recombineering and dual (G418/puromycin) selection.
To speed and simplify the humanization of mouse genes, here, we report the CRISPR-driven replacement (humanization) of an 18-kilobase pair (kbp) segment of a mouse tumor suppressor gene with an orthologous, disease-associated, 25-kbp segment of the corresponding human gene. Four Cas9/sgRNAs were designed to introduce redundant double-stranded breaks at each end of the 18-kbp mouse region. The four guides, along with a specially designed donor vector containing the 25-kbp segment of human sequence, were then introduced into C57BL/6 zygotes by microinjection.
Among 82 G0 founder animals, three were positive for the human sequence. Despite the possibility of mosaicism, loss-of-allele assays demonstrate a “hemizygote-like” copy number loss of the 18-kbp mouse segment in two of the G0 animals, and a “homozygote-like” copy number loss of the 18-kbp segment in the third. To date, the third G0 animal has given rise to progeny carrying either the 25-kbp humanized segment or an 18-kbp deletion but not both, consistent with this G0 animal carrying the 25-kbp humanized segment in trans to an 18-kbp deletion.
The new technique has numerous advantages including —
1) Obviating the need for antibiotic selection of embryonic stem cells,
2) Avoiding the recombinase-mediated excision of selection cassettes,
3) Expanding the physical size of CRISPR-driven knock-ins and gene replacements to ≥ 25-kbp,
4) Opening multiple strains and species to long range DNA modification.
The presentation details the specifics of our project, provides a brief overview of the technique, and describes progress to date.
O-32: The Estimation of Selective Effects Using Large Scale Population Data Identifies Genes Required For Normal Mammalian Development.
CA Cassa1, DM Jordan1, DJ Balick1, DP Nusinow1, SR Sunyaev1, and David Beier*2
1Division of Genetics, Harvard Medical School/Brigham and Women"s Hospital, Boston, MA, USA
2Center for Developmental Biology and Regenerative Medicine, Seattle Children"s Research Institute/Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA
The generation of large amounts of sequence data from various populations has enabled unbiased queries into the significance and potential consequences of DNA sequence variation in disease. We used the genome-wide distribution of expected and observed nonsense mutations in 60,706 patient exomes without severe Mendelian disorders from Exome Aggregation Consortium (ExAC) to estimate the strength of heterozygote selection for every human gene. We were specifically interested in those with high heterozygote selection; i.e., the cohort of genes for which one does not find nonsense mutations. Given that the sequenced population was viable, one may hypothesize that haploinsufficiency for these genes is not compatible with survival. Genes of this type are already well known; in humans they are often associated with defects in embryonic development. Notably, heterozygosity for many of these genes is tolerated in mice; while homozygous mutants have defects that recapitulate features of the human syndrome. This is fortuitous in that it enables the experimental characterization of molecular consequences of the mutation.
Indeed, the cohort of genes we identified with high heterozygote selection included many well-known human developmental disease genes. Importantly, our approach was predictive for genes with developmental effects, as the strength of heterozygote selection was highly correlated with the likelihood of recessive lethality for a set of 767 KOMP/EUCOMM alleles characterized by the Wellcome Trust/Sanger phenotyping program. We furthered characterized the gene set using a text-mining analysis; remarkably, a very large fraction of the high heterozygote selection cohort have little functional annotation. This provides a potentially robust opportunity for identifying novel genes with critical developmental roles. Further, given the empirical evidence that the high heterozygote selection gene set includes many that are causal for congenital defect syndromes, it is likely that analysis of uncharacterized genes in this cohort will be informative with respect to human disease.
O-33: The landscape of replication associated mutations in the human and mouse germlines
Lana Talmane1, Martin Reijns1, Harriet Kemp1, Robert Young1, James Ding1, Sophie Marion de Proce1, Ian Adams1, Rod Mitchell2, Wendy Bickmore1, Andrew Jackson1, and Martin Taylor*1
1MRC Human Genetics Unit, MRC IGMM, University of Edinburgh, UK
2MRC Centre for Reproductive Health, University of Edinburgh, UK
Genetic mutations provide the raw material for evolution, they are responsible for heritable disease and driving the development of cancer. We have shown that the binding of chromatin and regulatory proteins to DNA can interfere with replication and lead to regions with locally elevated mutation rates. Mechanistically this process appears to involve the trapping of DNA polymerase alpha synthesised DNA in the fully replicated genome; a process we have explored with a novel method, EmRiboSeq, that tracks replicative polymerase activity in vivo. Extending this work we have measured the patterns of chromatin accessibility and protein binding specifically in the mammalian germline and related it to the distribution of polymorphism and mutation, to reveal the terrain of replication associated mutations in mice and humans. This provides a means of adjusting neutral substitution rate estimates for fine-scale mutation rate fluctuation when identifying regions of selective constraint. We also identify likely hotspots of paternal lineage mutations within functional regulatory sites.
O-34: Paradoxical evolution of a large segmental duplication in mouse
Andrew P Morgan*1, Rachel C McMullan1, Amelia M‑F Clayshulte1, Timothy A Bell1, Grace Clark1, James M Holt2, Leonard McMillan2, and Fernando Pardo‑Manuel de Villena1
1Department of Genetics, University of North Carolina, Chapel Hill, NC, USA
2Department of Computer Science, University of North Carolina, Chapel Hill, NC, USA
Gene duplication and loss are major sources of genetic polymorphism in populations and important forces shaping the evolution of genome content and organization. We have reconstructed the evolutionary history of a 125 kbp duplicated segment, R2d, in the laboratory mouse (Mus musculus). The sequence is absent from the mouse reference genome but controls its own meiotic segregation in cis in a copy-number dependent manner. After an initial duplication event ~2 Mya in the common ancestor of M. musculus and M. spretus, the resulting paralogs (R2d1, R2d2) were subject to genetic drift, inter-locus gene conversion, further duplication and loss. We show that the R2d2 locus remains unstable: its copy number ranges from 0 to more than 80 in laboratory and wild mice sampled from around the globe, and mutation rate for new CNVs in laboratory populations exceeds 1% per generation. R2d encompasses a single protein-coding gene, Cwc22, which is expressed from all its paralogs and rapidly-evolving in rodents. Yet at the nucleotide level, sequence diversity in noncoding regions of R2d2 (but not R2d1) is significantly reduced relative to the genome-wide average. I will discuss implications for interpretation fo sequence divergence at other duplciated loci, and present evidence for an effect of parental age on de novo mutation rate for large CNVs at R2d2.
O-35: The frequent evolutionary birth and death of functional promoters in mouse and human
Robert Young*1, Yoshihide Hayashizaki2, Robin Andersson3, Albin Sandelin3, Hideya Kawaji2, Masayoshi Itoh2, Timo Lassmann4, Piero Carninci4, The FANTOM consortium4, Wendy Bickmore1, Alistair Forrest4,5, and Martin Taylor1
1MRC Human Genetics Unit, MRC Institute for Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh, EH4 2XU, UK
2RIKEN Preventive Medicine and Diagnosis Innovation Program, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan
3Department of Biology & Biotech Research and Innovation Centre, Copenhagen University, Ole Maaloes Vej 5, Copenhagen N, Denmark
4RIKEN Center for Life Science Technologies (Division of Genomic Technologies), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan
5Systems Biology and Genomics, Harry Perkins Institute of Medical Research, QEII Medical Centre, Nedlands, WA 6009, Australia
Promoters are central to the regulation of gene expression. Changes in gene regulation are thought to underlie much of the adaptive diversification between species and phenotypic variation within populations. In contrast to earlier work emphasizing the importance of enhancer evolution and subtle sequence changes at promoters, we show that dramatic changes such as the complete gain and loss (collectively turnover) of functional promoters are common. Using quantitative measures of transcription initiation in both humans and mice across 52 matched tissues we discriminate promoter sequence gains from losses and resolve the lineage of changes. We also identify expression divergence and functional turnover between orthologous promoters, finding only the latter is associated with local sequence changes. Promoter turnover has occurred at the majority (>56%) of protein-coding genes since humans and mice diverged. Tissue-restricted promoters are the most evolutionarily volatile where retrotransposition is an important, but not the sole source of innovation. There is considerable heterogeneity of turnover rates between promoters in different tissues, but the consistency of these in both lineages suggests the same biological systems are similarly inclined to transcriptional rewiring. The genes affected by promoter turnover show evidence of adaptive evolution. In mice, promoters are primarily lost through deletion of the promoter containing sequence; whereas in humans, many promoters appear to be gradually decaying with weak transcriptional output and relaxed selective constraint. Our results suggest that promoter gain and loss is an important process in the evolutionary rewiring of gene regulation and may be a significant source of phenotypic diversification.
O-36: Elucidation of the Underlying Mechanism of the Induction of Pluripotency Genes by SAHA-Conjugated Pyrrole-Imidazole Polyamides by Computational Genomic Analysis
Jason Lin*1, Hiroyuki Yoda1, Kiriko&nbnbsp;Hiraoka1, Takahiro Inoue1, Sakthisri Krishnamurthy1, Takayoshi Watanabe1, Atsushi Takatori1, Nobuko Koshikawa1, Seiya Imoto3, Satoru Miyano3, Hiroshi Sugiyama2, and Hiroki Nagase1
1Laboratory of Cancer Genetics, Chiba Cancer Center Research Institute
2Department of Chemistry, Graduate School of Science, Kyoto University
3Laboratory of DNA Information Analysis, Institute of Medical Science, University of Tokyo.
We have been actively developing highly sequence-specific pyrrole-imidazole DNA minor-groove binding polyamides as molecular switches of various biological triggers. Those polyamides demonstrate remarkable specificity in situ to DNA sequences, and further functionalization of this class of molecules provides a novel and effective approach in the development of molecular cancer therapy in vivo. Some of our recent advances include the development of a class of small molecules called SAHA-PIP comprising of histone deacteylase inhibitor SAHA and DNA binding pyrrole-imidazole polyamide capable of genome-wide epigenetic reprogramming. In mouse embryonic fibroblasts, we found that SAHA-PIP could induce multiple pluripotency-associated genes such as Rex1 and Cdh1; similar results were also observed in human dermal fibroblast cells. The ability for SAHA-PIP to turn the highly conserved genetic machinery of pluripotency greatly demonstrate the scientific and therapeutic potential of pyrrole-imidazole polyamides as an inhibitors delivery system for histone modifications.
Additionally, comparative genomic studies of SAHA-PIP binding site predictions, combined with microarray experiments, may further reveal the mechanistic insights on the critical genes involved in the epigenetic reprogramming process. We are currently utilizing various statistical and computational methods to understand the effect of SAHA-PIP on the induction of pluripotency, cellular reprogramming and oncogenesis. Our previous reports on the exceptional effect of SAHA-PIP have led us to explore the possibility of chromatin opening, gene activation and disrupting transcription factor binding events in the genome with a library of SAHA-conjugated pyrrole-imidazole polyamides. From comparing findings from the human and mouse genome, we may then understand the underlying biology behind cellular reprogramming and epigenetic regulation via SAHA-PIP.
O-37: Capybara genome sequencing offers deeper insights into rodent evolution
Isaac Adeyemi Babarinde*1,2, and Naruya Saitou1,2
1National Institute of Genetics, Mishima, Japan
2Graduate University for Advanced Studies, Mishima, Japan
Rodents are the mammalian order with the highest number of species and ecological success. Mouse and rat are two representative rodent species that have been extensively studied as models species. These two species are taxonomically close, belonging to Murinae subfamily. However, rodents have about thirty families classified into three morphologically different clades. Are the genomic and evolutionary features of mouse and rats true representatives of rodent order? In this study, we focused on evolutionary rate as an evolutionary feature. Using mouse and rat as representatives, higher evolutionary rate has been reported in rodents. The higher evolutionary rate has been attributed to shorter generation interval. Since generation interval is known to positively correlate with body size, we decided to sequence the whole genome of capybara, the largest living rodent species. Here, we report the first whole genome draft sequence of capybara. The assembled genome was about 2.4Gbp with the average depth of 15×. Capybara’s genomic CG content of ~40% is comparable to those of other rodent species. Using a combination of bioinformatics and statistical approaches, we extracted genomic regions with high quality. The extracted high-quality regions were more than 1.8Gbp and the average high-quality read depth was 8×. Using the high-quality regions, heterozygosity was estimated to be 0.0022. We performed comparative genomics with previously reported mammalian genomes. Using various estimates of evolutionary rates, we report some aspects of rodent evolution that are hitherto hidden. This study lays a foundation in understanding the genomics of capybara and offers deeper insights into rodent evolution.
O-38: The Short Story of a Long Tale
The Jackson Laboratory, Bar Harbor, ME 04609, USA
The mission of the Mouse Genome Informatics (MGI, www.informatics.jax.org) resource is to provide integrated genetic, genomic, and biological data about the laboratory mouse to facilitate the study of human health and disease. This mission is unchanged since its inception…But the dramatic changes in the scientific landscape ushered in by molecular biology, the sequencing of the human and mouse genomes, and the rapid advances in computer technology mean that the MGI of today is radically different than the MGI of 25+ years ago.
In this talk, I will
• begin at the beginning, in how I came to bioinformatics in the first place; and the initial collaborations
that formed MGI.
• show major change-points in MGI’s evolution and growth.
• fast forward to describe new changes to the user experience at the MGI web site (release date late
MGI is supported by NIH grants HG000330, HD064299, CA089713, OD011190, NS082666
O-39: Informatics for the International Mouse Phenotyping Consortium- a Platform for Phenotypic and Translational Discovery
Terrence F Meehan*1, and On Behalf of the Mouse Informatics Phenotyping Infrastructure ‑MPI21,2,3
1European Molecular Biology Laboratory - European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SD, UK MRC
2Mammalian Genetics Unit, MRC Harwell, Harwell Science and Innovation Campus, Harwell OX11 0RD, UK
3Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
The International Mouse Phenotyping Consortium (IMPC) is delivering new insights into biological mechanisms and human disease by generating and characterizing thousands of knockout mouse strains for genes with little or no known biological function. For each mouse, thousands of data points are collected according to a standardized, broad-based phenotyping pipeline and archived centrally within the IMPC-Data Coordinating Centre. Dedicated ‘data wranglers’ ensure quality of the data and an automated statistical analysis pipeline identifies mouse strains with abnormal phenotypes. As of August 2015, phenotype data from 2000 knockout strains is freely available and is providing global views into fertility, sexual dimorphism and the genetic mechanisms underlying a wide range of phenotype traits. Potential disease models are identified by orthologous gene and orthologous phenotype features and are used in prioritizing gene variant candidates for human genetic diseases. In addition, a dedicated pipeline is assessing dysmorphology in embryonic lethal strains by using state-of-the-art imaging analysis technologies. Users can freely access all data via an intuitive web portal that allows biologists and clinicians to easily find mouse strains with phenotypic traits relevant to their research. The community is invited to explore and provide feedback as we build this rich resource at: www.mousephenotype.org
O-40: Systemic metabolic phenotyping in the German Mouse Clinic in search of new mouse models for metabolic disorders
Jan Rozman*1,6, Robert Brommage1, Birgit Rathkolb1,2, Manuela Oestereicher1, Helmut Fuchs1, Valerie Gailus‑Durner1, Martin Klingenspor3,4, Eckhard Wolf2,6, and Martin Hrabe de Angelis1,5,6
1German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Germany
2Institute of Molecular Animal Breeding and Biotechnology, Gene Center, Ludwig-Maximilians-Universitaet Muenchen, Germany
3Molecular Nutritional Medicine, Else Kroener-Fresenius Center, Technische Universitaet Muenchen, Germany
4ZIEL Center for Nutrition and Food Sciences, Technische Universitaet Muenchen, Germany
5Chair of Experimental Genetics, Center of Life and Food Sciences Weihenstephan, Technische Universitaet Muenchen, Germany
6German Center for Diabetes Research (DZD)
The annotation of gene functions related to metabolic disorders such as obesity and type 2 diabetes is a key area in international large scale mouse phenotyping efforts. The German Mouse Clinic (GMC) is member of the International Mouse Phenotyping Consortium (IMPC) contributing to the global effort to generate a knockout mouse model for every protein-coding gene and provide phenotyping data to relate gene functions to human disease.
Several IMPC phenotyping assays address metabolic functions: clinical blood chemistry, insulin blood levels, indirect calorimetry, intraperitoneal glucose tolerance test, body composition analysis, combined SHIRPA and dysmorphology, as well as gross and histopathology.
We have particular interest in the areas regulation of energy metabolism and glucose homeostasis. Mechanisms involved in primary suppression or upregulation of metabolic rate are of special interest for the understanding of why energy balance regulation is impaired in obese or cachectic patients. We developed and applied data analysis tools that identify primary effects of single gene knock outs on metabolic regulation. In a preliminary survey of 99 mutant lines (>2,000 mice) from the GMC, 6% of the mutant lines were hypometabolic whereas 10% were hypermetabolic when adjusted for body mass differences.
Impaired glucose tolerance and hyperglycemia as diagnostic criteria for a pre-diabetic or diabetic state can be evaluated with a glucose tolerance test after overnight food deprivation. We evaluated the IMPC standard operating procedure and developed an easy and intuitive way of identifying genes related to dysfunctional glucose homeostasis. In a first step, evaluating fasting blood glucose values as the ratio of mutant to wildtype levels for >1200 lines identified outliers with either low or high fasting glycemia.
These initial steps in data analysis and functional annotation of genes indicate that the IMPC data resource offers a unique source of disease oriented phenotype information open to the scientific community.
O-41: Genetic architecture of behavior in an advanced intercross line of mice
Natalia M. Gonzales*1, Jungkyun Seo1, Shyam Gopalakrishnan3, and Abraham A. Palmer1,2
1Department of Human Genetics, University of Chicago, Chicago, IL 60637
2Department of Psychiatry and Behavioral Neuroscience, University of Chicago, Chicago, IL 60637
3Natural History Museum of Denmark, Copenhagen University, Copenhagen, Denmark
Mice are one of the primary model organisms used to study behavior. We are conducting a genome-wide association study of behavior in a LG/J x SM/J advanced intercross line (AIL) of mice (Aap:LG,SM-G50-56, derived from Jmc:LG,SM-G33). An AIL is generated by crossing two inbred strains for multiple generations and offers greater precision for mapping quantitative trait loci (QTL) than traditional genetic crosses.
We measured prepulse inhibition, locomotor activity, and a number of other traits in ~1,100 male and female AILs (Aap:LG,SM-G50-56). We genotyped ~2,000 AILs spanning over 12 generations (Aap:LG,SM-G34;G39-43;G50-56) using genotyping by sequencing (GBS) and used RNA-sequencing to map gene expression QTL (eQTL) in brain tissue from ~250 mice (Aap:LG,SM-G50-56).
Here we present preliminary eQTL identified in the hippocampus, striatum and prefrontal cortex and describe how we are integrating these data with QTL to identify genes underlying variation in locomotor activity and prepulse inhibition . We are also building an online database that includes all of the genotypes, phenotypes and RNA-sequencing data we have collected from the LG/J x SM/J AIL since G34. We will provide an example of how this resource is being used to replicate and refine QTL that we identified in previous studies.
Our results demonstrate that AIL mice can be used to identify genes that are involved in behavioral traits at a fraction of the cost and effort of a human mapping study. Integrating genotype, phenotype and gene expression data is a powerful approach that will accelerate the process of gene identification and provide insight into the biology of complex traits.
O-42: The hidden layer of regulatory RNA in mammalian genome biology
Garvan Institute of Medical Research, Sydney
It appears that the genomic programming of mammalian biology has been misunderstood for the past 50 years, because of the assumption that most genetic information is transacted by proteins. The mammalian genome contains only ~20,000 protein-coding genes, similar in number and with largely orthologous functions as those in other animals, including simple nematodes. On the other hand, the extent of non-protein-coding DNA increases with increasing developmental and cognitive complexity, reaching 98.5% in humans. Moreover, high throughput analyses have shown that the majority of the mammalian genome is dynamically transcribed during differentiation and development to produce tens if not hundreds of thousands of short and long non-protein-coding RNAs that show highly specific expression patterns and subcellular locations. Increasing numbers of these RNAs are being shown to have functions at many different levels of gene expression, including translational control and the guidance of epigenetic processes that underpin development, physiological adaptation, brain function and transgenerational communication, augmented by the superimposition of plasticity via RNA editing, RNA modification and retrotransposon mobilization. This suggests that there is there is a massive hidden layer of RNA-based information flux in mammalian genome biology and that the simple protein-centric operator-repressor model of ‘gene regulation’ derived from studies of bacteria is incorrect in highly organized and spatially specialized multicellular organisms. This in turn requires reassessment of the nature, hierarchies and scaling of the regulatory systems that control the evolution, 4-dimensional assembly and cognitive capacities of animals.
O-43: In vivo profiling of the promoter- and enhancer landscape of inflammatory bowel disease
Mette Boyd1, Morana Vitezic1, Jette Bornholdt1, Malte Thodberg1, Kristoffer Vitting‑Seerup1, Thilde Bagger Terkelsen1, Mehmet Coskun1,2, Robin Andersson1, Jesper Troelsen3, Ole Haagen Nielsen2, Jacob Bjerrum2, and Albin Sandelin*1
1Department of Biology and BRIC, University of Copenhagen, Copenhagen, Denmark
2Department of Gastroentorology, Medical Section, University of Copenhagen, Herlev Hospital, Herlev, Denmark
3Department of Science, Systems and Models, Roskilde University, Roskilde, Denmark
Coordinated gene regulation is essential for all aspects of cell biology, including development, differentiation and disease. Characterization of enhancers and promoters in disease has been difficult due to the lack of genome-wide methods suitable for the analysis of small tissue samples. Therefore, we know little about the regulation of genes in disease, and its variation between patients. Related to this, 85% of protein-coding genes show heritable variation in expression due to variance in gene regulation. Thus, localization of promoters and enhancers genome-wide within patient material is important for disease biology and genetics.
Because both promoter and enhancers are transcribed, they can be detected by RNA sequencing. Utilizing this, we have profiled promoter and enhancer usage in patients suffering from inflammatory bowel disease (IBD). IBD is a complex group of chronic inflammatory conditions in the gut. Crohn’s disease(CD) and Ulcerative Colitis (UC) are the two principal subtypes. Correct treatment depends on accurate sub-type diagnosis, which is challenging and expensive.
To this end, we have profiled the descending colon of 110 human subjects, stratified into disease subtypes and controls. We identified a promoter set that with high accuracy can distinguish the shared inflammatory response, and UC-or CD-specific profiles. The set included annotated promoters, alternative promoters and promoters of novel long non-coding RNAs.
Moreover, we identified over 20.000 enhancer regions that are active in these samples, where subsets are induced in general inflammation or in UC/CD specifically. These enhancers are often linked to known and novel IBD-induced genes, suggesting that they are important in the pathogenesis. Connected to this, IBD-associated SNPs were highly enriched in these regulatory regions, enabling subsequent identification of casual regulatory mutations.
To our knowledge, this is the first comprehensive profiling of enhancers and promoters in large patient cohorts for any disease.
O-44: Enhancers lead waves of coordinated transcription in transitioning mammalian cells
Erik Arner*1, Carsten O Daub1, Kristoffer Vitting‑Seerup2, Robin Andersson2, Kim M Summers3, Christine Wells4, David A Hume3, Alistair Forrest1, Albin Sandelin2, Piero Carninci1, and Yoshihide Hayashizaki5
1Div. of Genomic Technologies, Center for Life Science Technologies, RIKEN, Japan
2University of Copenhagen, Denmark
3Roslin Institute, University of Edinburgh, United Kingdom
4Institute for Infection Immunity and Inflammation, University of Glasgow, United Kingdom
5RIKEN Preventive Medicine and Diagnosis Innovation Program, Japan
Cellular differentiation requires coordinated induction of genes, facilitated by dynamic regulation of promoters and enhancers by transcription factors. Exploiting the fact that active promoters and enhancers are transcribed, and that genome-scale 5’RACE (CAGE) detects transcription start sites (TSS) including the bidirectional TSS characteristic of active enhancers, we simultaneously measured the activity of promoters and enhancers in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli, to dissect the relationship between dynamic changes in mRNA and eRNA. The time courses included stem cells (embryonic, induced pluripotent, trophoblastic and mesenchymal stem cells) and committed progenitors undergoing terminal differentiation towards mesodermal, endodermal and ectodermal fates, as well as fully differentiated primary cells and cell lines responding to stimuli (growth factors and pathogens). Enhancer RNAs dominated the earliest expression responses, followed by mRNAs encoding transcription factors and then by other transcripts. Binding sites for key lineage transcription factors were simultaneously over-represented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation, and the multitude of biological systems studied suggests that this phenomenon is a general feature of mammalian transcriptional regulation. This challenges previous models which suggested that linked enhancers and promoters are co-expressed over time.
O-45: Network architecture of microRNA regulation in mammalian cells
Michiel De Hoon*1,3, Derek De Rie1,4, Tanvir Alam9, Erik Arner1,3, Peter Arner24, Haitham Ashoor9, Gaby Astrom24, Magda Babina19, Nicolas Bertin1,3,28, Maxwell Burroughs8, Carsten O Daub1,3, Michael Detmar7, Alexandre Fort1,3, Dan Goldowitz13, Sven Guhl19, Jayson Harshbarger1,3, Akira Hasegawa1,3, Kosuke Hashimoto1,3, Hideya Kawaji1,2,3, Peter Klinken20, Timo Lassmann1,3,6, Charles Lecellier29, Weonju Lee11, Marina Lizio1,3, Vsevolod Makeev31, Anthony Mathelier30, Yulia Medvedeva25,26,27, Chris Mungall16, Shohei Noma1,3, Mitsuhiro Ohshima17, Helena Persson15, Filip Roudnicky7, Pal Saetrom12, Jessica Severin1,3, Kim M Summers10, Hiroshi Tarui1,3, Kristoffer Vitting‑Seerup5, Christine Wells23, Louise Winteringham20, Yoko Yamaguchi19, The FANTOM consortium, Albin Sandelin5, Michael Rehli21,22, Yoshihide Hayashizaki2,3, Piero Carninci1,3, and Alistair Forrest14
1RIKEN Center for Life Science Technologies (Division of Genomic Technologies), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045 Japan
2RIKEN Preventive Medicine and Diagnosis Innovation Program, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045 Japan
3RIKEN Omics Science Center (OSC), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045 Japan [RIKEN Omics Science Center ceased to exist as of April 1st 2013 due to RIKEN reorganization]
4Centre for Integrative Bioinformatics (IBIVU), VU University Amsterdam, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands
5The Bioinformatics Centre, Department of Biology and Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Ole Maaloes Vej 5, DK 2200 Copenhagen, Denmark
6Telethon Kids Institute, 100 Roberts Road, Subiaco, WA 6008, Australia
7Swiss Federal Institute of Technology (ETH) Zurich, Institute of Pharmaceutical Sciences, HCI H390, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland
8National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, 8600 Rockville Pike, Bethesda, MD 20894, USA
9Computational Bioscience Research Center, Computer, Electrical and Mathematical Sciences and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia
10The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Easter Bush, Edinburgh, Midlothian EH25 9RG, UK
11Department of Dermatology, Kyungpook National University School of Medicine, 130 Dongdeok-ro Jung-gu, Daegu 700-721, South Korea
12Department of Computer and Information Science, Norwegian University of Science and Technology, N-7489 Trondheim, Norway
13Department of Medical Genetics, University of British Columbia, 950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada
14Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, the University of Western Australia, Nedlands, Western Australia, Australia
15Division of Oncology and Pathology, Department of Clinical Sciences, Lund University, Medicon Village, building 404:B3, Scheelevaegen 2, SE-223 81 Lund, Sweden
16Genomics Division, Lawrence Berkeley National Laboratory, 84R01, 1 Cyclotron Road, Berkeley, California 94720, USA
17Department of Biochemistry, Ohu University School of Pharmaceutical Sciences Misumido 31-1, Tomitamachi, Koriyama, Fukushima 963-8611, Japan
18Department of Biochemistry, Nihon University School of Dentistry,1-8-13, Kanda-Surugadai, Chiyoda-ku, Tokyo 101-8310, Japan
19Department of Dermatology and Allergy, Charite Campus Mitte, Universitaetsmedizin Berlin, Chariteplatz 1, 10117 Berlin, Germany
20Harry Perkins Institute of Medical Research, and the Centre for Medical Research, University of Western Australia, QQ Block, QEII Medical Centre, Nedlands, Perth, Western Australia 6009, Australia
21Department of Internal Medicine III, University Hospital Regensburg, Regensburg, Germany
22Regensburg Centre for Interventional Immunology (RCI), Regensburg, Germany
23Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland, Brisbane St Lucia, Queensland 4072, Australia
24Department of Medicine, Karolinska Institutet at Karolinska University Hospital, Huddinge, SE-141 86 Huddinge, Sweden
25Josep Carreras Leukaemia Research Institute (IJC), Muntaner 383, 3r 2a, 08021 Barcelona,Spain
26Vavilov Institute of General Genetics, Russian Academy of Science, Gubkina str., 119333 Moscow, Russia
27Bioengineering Centre, Russian Academy of Science, pr. 60-letiya Oktyabrya, 7-1, 117312, Moscow, Russia
28Cancer Science Institute of Singapore, National University of Singapore, 14 Medical Dr, 117599 Singapore
29Institute of Molecular Genetics of Montpellier, CNRS-UMR 5535, 1919 Route de Mende, 34293 Montpellier, Cedex 5, France
30Centre for Molecular Medicine and Therapeutics (CMMT), University of British Columbia | Child & Family Research Institute, 980 West 28th Avenue, Room 3109 | V5Z 4H4, Vancouver, BC, Canada
31Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow 119991, Russia.
The regulatory network governing cellular behavior includes both transcription factors and miRNAs as key regulators. To understand the role of miRNAs in the integrated network architecture of cellular regulation, we analyze deep sequencing data of paired small RNA and Cap Analysis of Gene Expression (CAGE) libraries across a wide range of cell types, in particular human primary cells. We found that microRNAs can be divided into two classes based on their expression pattern: Cell type specific miRNAs that are highly expressed only in a few cell types, and ubiquitous miRNAs that are expressed in most cell types but depleted in particular cell types. Ubiquitous miRNAs preferentially target genes expressed in the cell types in which the miRNA is depleted, and may play a role in preventing inappropriate activation of specific transcriptional programs. In contrast, cell type specific miRNAs also target genes co-expressed with the miRNA, and may play a role in buffering gene expression. We find that promoter sequences of miRNAs are highly conserved and that miRNA expression is primarily regulated at the transcriptional level, with ubiquitous miRNAs preferentially regulated by repressors, and cell type specific miRNAs both by activators and repressors.
O-46: Regulated mobilization of Retrotransposable elements in cell identity, reprogramming and disease
Beatrice B Bodega1,2, Francesco Della Valle3, Loqmane Seridi3, Yanal Gosheh2, Federica Marasca3, Gregorio Alanis‑Lobato3, Manjula Thimma4, Sara Pinosio5, Valentina Saccone5, Sara Consalvi6, Jenny Martone6, Valentina Cazzella7, Marina Mora6, Irene Bozzoni5, Per Lorenzo Puri4, Michele Morgante3, Timothy Ravasi2, and Valerio Orlando*2,3
1Istituto Nazionale di Genetica Molecolare (INGM) Romeo and Enrica Invernizzi, Genome Biology Unit, Milan, Italy
2IRCSS Fondazione Santa Lucia, Epigenetics and Genome Reprogramming, Rome, Italy
3KAUST Environmental Epigenetics Research Program, Biological Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology, Kingdom of Saudi Arabia
4Institute of Applied Genomics, Udine, Italy
5IRCCS Fondazione Santa Lucia, Phamacology and Epigenetics, Rome, Italy
6Department of Biology and Biotechnology Charles Darwin, Sapienza University of Rome, and Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, Italy
7Division of Neuromuscular Diseases and Neuroimmunology, Istituto Nazionale Neurologico C. Besta, Milano, Italy
The rapid progress of genomics technologies, in particular high-resolution Cap Analysis Gene Expression (CAGE) for transcriptome analysis and whole genome sequencing (WGS) have been unveiling global regulatory features in the eukaryotic genome by which transposable elements (TEs) would provide substantial contribution to the genetic and epigenetic cell program. Whether the global TEs distribution is random or follows regulated developmental programs remains to be elucidated. To this aim we investigated L1 dynamics during differentiation of human primary muscle cells, finding that skeletal myogenesis supports a MyoD- and HDAC2/Dystrophin-NOS1 dependent activity of this class of repetitive elements resulting in the acquisition of L1 elements specifically at expressed skeletal muscle gene loci. We found that this phenomenon is impaired during differentiation of muscle cells derived from patients affected by Duchenne muscular Dystrophy (DMD), being HDAC2 aberrantly recruited at L1 promoter and their transcription repressed. Pharmacological rescue of DMD phenotype by HDAC inhibitors or gene therapy approach by exon-skipping is accompanied by normal L1 expression and CNV both in C57BL/6J-Dmdmdx model mice and in human DMD primary muscle cells. We found that during differentiation L1 and other TEs are transcribed following a characteristic profile made of successive waves of activation that closely matches enhancer elements and myogenic program. Interestingly this profile is flat in DMD cells as consequence of global defect in chromatin acetylation. In another work we investigated L1 dynamics in somatic cell reprogramming finding that L1 mobilization is required also for efficient reprogramming. We propose that L1 repetitive elements mobilization is an integral regulatory part of developmental and cell specialization programs and their epigenetic deregulation a key trait in loss of cell identity and disease.
O-47: Modeling Psychiatric/Neurological disorders using iPS cell technologies and transgenic non-human primates.
Dept of Physiology, Keio University Graduate School of Medicine, Japan
What makes the investigation of human psychiatric/psychiatric disorders so difficult? This could be attributed to the following reasons 1) Diseases model mice do not always recapitulate the pathophysiology of human diseases, 2) It is extremely difficult to investigate what is taking place in vivo at the onset of the disease due to the low accessibility to the pathological foci in the brain, and 3) The responsible neuronal circuits for the phenotype are not identified. In order to overcome these difficulties, we took advantage of iPS cell technologies and transgenic non-human primates for modeling human psychiatric/psychiatric disorders. So far, we have established iPS cells from the patients of about 40 human psychiatric/psychiatric disorders and characterized their pathophysiology. For example, in collaboration with the group of RIKEN BSI and University of Tokyo, we established iPS cells from the schizophrenia patients containing 22q11 deletions (Bundo et al., Neuron, 2014). Interestingly, we found that the copy number of a retrotransposon, long interspersed nuclear element-1 (L1), was increased in neurons induced from iPS cells from schizophrenia patients containing 22q11 deletions, indicating that hyperactive retrotransposition of L1 in neurons triggered by genetic risk factors may contribute to the susceptibility and pathophysiology of schizophrenia.
Furthermore, for faithfully modeling the human psychiatric/psychiatric disorders in vivo, we developed transgenic non-human primates (common marmosets) with germline transmission (Sasaki et al., Nature, 2009). In the present talk, we also wish to mention our recent data of generation of common marmoset transgenic models of neurodegenrerative diseases, including Parkinson disease, Alzheimer disease and ALS. Furthermore, we could generate knock-out technologies of common marmoset using genome editing technologies for the generation of transgenic marmoset model of autism and psychiatric disorders.
At the end, I will mention about Brain Mapping Projects in Japan, in which investigation of common marmoset brains plays key roles.
O-48: GENCODE: revealing transcriptional complexity in Human and Mouse
Mark Thomas*, Jennifer Harrow, and GENCODE Consortium
Wellcome Trust Sanger Institute, United Kingdom
GENCODE is now the default human gene set in both the UCSC and Ensembl genome browsers, combining computational and manual genome annotation approaches. With an emphasis on alternative splicing, our current release (v23) contains 198,619 transcripts based on EST and mRNA evidence from 19,797 protein coding and 15,931 long non-coding genes. The increasing availability of next-generation sequencing data from RNAseq, CAGEseq and PolyAseq, allows us to define transcribed regions with ever increasing accuracy; adding to the transcriptional complexity of the genome. Understanding this transcriptional complexity is important for the study of disease, especially now that CRISPR-Cas9 technologies are driving a genome-editing revolution. Identifying functional transcripts is particularly important, so as to differentiate them from transcripts arising via stochastic events or spliceosomal errors. The function of most protein coding transcripts is evident from the encoded protein, whereas the function of long non-coding transcripts is more difficult to determine. In an effort to improve our functional understanding of transcripts, we are using advances in ribosome profiling and mass spectrometry to assess the coding potential of transcripts. Combining these approaches not only highlights how differences in the precise TSS can influence the translational start of proteins, but has also allowed us to identify entirely novel proteins. The same approaches are currently being used to improve annotation of the mouse genome, which remains an important model for studying disease. As a result, we now have regular releases of the mouse GENCODE gene set for the C57BL/6 reference genome. This annotation will be extended to other laboratory mouse strains with de-novo genome assemblies through collaboration with the Sanger Mouse Genomes project.
O-49: FANTOM6: Functional elucidation of lncRNA
Jay W Shin*, Michiel De Hoon, and Piero Carninci
RIKEN Yokohama - Center for Life Science Technologies, Division of Genomics Technologies
FANTOM is an international research consortium initially to assign functional annotations to the full-length cDNAs that were collected during the Mouse Encyclopedia Project at RIKEN (fantom.gsc.riken.jp). FANTOM has since developed and expanded over time to encompass the fields of human transcriptomics. The recent FANTOM has unraveled a myriad of transcriptome diversity across 400 human cell types where many were considered non-coding RNAs (FANTOM5 consortium; Nature 2014). Cellular diversity of these lncRNA and their dynamic interplays between enhancers and promoters suggest much more investigation is needed to elucidate their function. In the 6th edition of FANTOM, we aim to broadly generate a reference set of profiles for multiple human cell types using the latest next generation sequencing (NGS) technologies followed by large-scale perturbation of lncRNAs and infer a molecular phenotype via CAGE analyses; in parallel, we will functionally characterize and classify lncRNAs using technologies complementary to CAGE. Here, I will introduce the next FANTOM and reveal our current pipeline to build our unique collection of functional lncRNA atlas.
O-50: Multiple mouse reference genomes and strain specific gene annotations
Benedict Paten1, Mario Stanke2, Son Pham3, David Thybert4, Laura G. Reinholdt5, Jennifer Harrow6, Kerstin Howe6, David Adams6, and Thomas Keane*6
1University of California, Santa Cruz
2University of Greifswald
3University of California, San Diego
4European Bioinformatics Institute, Hinxton, UK
5Jackson Laboratory, Bar Harbor, ME
6Wellcome Trust Sanger Institute, Hinxton, UK
The generation of the mouse reference genome sequence from the C57BL/6J strain was a major milestone in recent mouse genetics history. It has enabled a whole set of new applications and technologies. In phase 2 of the Mouse Genomes Project, we are producing accurate de novo genome sequences and strain specific gene annotation for 16 strains (129S1/SvImJ, A/J, ARK/J, BALB/cJ, C3H/HeJ, C57BL/6NJ, CAST/EiJ, CBA/J, DBA/2J, FVB/NJ, LP/J, NOD/ShiLtJ, NZO/HlLtJ, PWK/PhJ, SPRET/EiJ, and WSB/EiJ) from a mixture of short and long range illumina libraries, optical maps, and third generation sequencing. In 2015 we produced the first set of pseudo chromosome sequences for each strain. In a collaboration with Dovetail Genomics, we produced near chromosome length de novo scaffolds for CAST/EiJ, PWK/PhJ, and SPRET/EiJ. Extensive QC and validation is being carried out in preparation for full public release. Work is underway to produce strain specific gene sets by a combined comparative gene prediction approach using the C57BL/6J genes with strain specific evidence such as RNA-Seq and PacBio cDNA sequencing. Our initial analysis on Chromosome 11 has revealed new gene structures at small and large scale, not present in the existing Gencode C57BL/6J gene set. In complex and highly polymorphic complex loci such as the major urinary proteins (Mups), H2, and Irg, we have identified new allelic forms of these genes, gene shuffling, large translocations, and incorporation of open reading frames (ORFs) from other parts of the genome. We are working with the UCSC Genome Browser group to develop a comparative mouse strains browser to visualise the genomic structural differences in conjunction with the strain specific gene predictions.
O-51: A survey of genome rearrangements in human evolution
National Institute of Advanced Industrial Science and Technology (AIST), Tokyo, Japan
Genomes evolve in part by rearrangements, such as inversions and translocations, which reorder the DNA code in a drastic and perhaps irreversible way. Despite great interest, there has been no comprehensive survey of rearrangements in human or mammal evolution, beyond simple ones like inversions.
Here we survey all types of human rearrangements that have occurred since the last common ancestor with chimpanzee. We use a recent algorithm to align the human and chimp genomes, which finds orthologies more accurately and lacks bias against micro-rearrangements [Frith and Kawaguchi 2015]. We systematically infer rearrangement events from the alignments, and classify them based on the minimum number of DNA breaks needed to produce them. We sub-classify them by topology (inversion, interchromosomal translocation, etc.) We exclude chimp-specific rearrangements (including misassemblies) by checking their presence in human-orangutan alignments.
We find 291 rearrangements, including 193 inversions, but also complex ones requiring at least five DNA breaks. Many of the rearrangements seem to have arisen by DNA shattering into multiple fragments, which then re-joined in random order and orientation, with some fragments being lost. This resembles damage from radiation (e.g. natural radon gas or cosmic rays) repaired by non-homologous end-joining. It also resembles "chromothripsis", observed recently in cancer and other diseases. Some inversions were caused by shattering, others by recombination between inverted repeats, and it is usually easy to tell these apart. We reconstruct some shattering-and-joining events at the single-base level.
Most rearrangements do not overlap known exons. However, one interesting inversion swapped the upstream halves of two divergently-transcribed chymotrypsinogens, CTRB1 and CTRB2. This was accompanied by gene conversion, so that the upstream halves of both genes resemble the ancestral CTRB2.
This study reveals what kinds of rearrangements have occurred, and the likely mechanisms. We plan to check these rearrangements in ancient hominin (e.g. Neanderthal) DNA.
O-52: An experimental approach to elucidate enigmatic isochore evolution by using ENU mutagenesis
Satoshi Oota*1, Ryutaro Fukumura2, Naruya Saitou3, and Yoichi Gondo2
1Bioresource Information Division, RIKEN BioResource Center
2Mutagenesis and Genomics Team, RIKEN BioResource Center
3Division of Population Genetics, National Institute of Genetics
Historical events in the mouse genome are obviously key factors to determine various characteristics of mouse strains. For making use of the mouse resource, therefore, it is crucially important to elucidate the genome evolution. One of the big issues of the mammalian genome evolution is the “isochore” problem: a queer spatial structure of the genome in GC content. So far, no coherent explanations were achieved due to contradictory observations against the proposed models: i.e., selection, biased gene conversion, and mutation bias models. Today, the typical approach to study evolution is the “backward” analysis by using extant molecular data to infer the past by extrapolation. In the isochore evolutionary study, however, this conventional approach is insufficient because we have at least two kinds of issues to be considered: (1) the evolutionary signal of interest might be eroded by unknown evolutionary process; (2) we need to handle non-coding regions, to which it is difficult to apply conventional evolutionary models. Especially, (1) is a critical problem because subtle information in non-coding sequences may be easily worn out even during short-term evolution. To overcome those problems, we took advantage of ENU mutagenesis as a tool for the experimental evolution, accelerating evolutionary rate to make it possible to observe ongoing evolution through a large number of de novo mutations. We found bidirectional (diverged) mutation pressures that support the legendary Sueoka’s mutation bias theory. Our finding also has potential to explain the enigmatic isochore evolution.
O-53: Post-translational mechanisms buffer protein abundance against transcriptional variation.
Steven Munger*1, Joel Chick2, Petr Simecek1, Edward Huttlin2, Kwangbom Choi1, Daniel M Gatti1, Narayanan Raghupathy1, Karen L Svenson1, Steven Gygi2, and Gary A Churchill1
1The Jackson Laboratory, Bar Harbor, Maine USA
2Harvard Medical School, Cambridge, Massachusetts USA
Recent studies have reported low correlation between protein and mRNA abundance, which has led to the prediction of widespread buffering of protein expression against variation in mRNA levels. However, the relative importance of transcriptional and post-transcriptional modes of protein regulation remains poorly understood. Our study builds on earlier observations, integrating new techniques in mass spectrometry to expand the breadth of protein quantification, new methods to accurately quantitate transcript abundance in RNA-seq data, and new mouse models with extensive genetic variation to perturb mRNA and protein expression. We identified 1728 proteins that are regulated by local genetic variation; for 80% of these proteins, the causal variant acts proximally on transcript abundance, and consequently there is high correlation between protein and transcript abundance. Further, we identified 1362 proteins that are regulated by distant genetic variation. In stark contrast to local associations, nearly all distant loci appear to act on target protein abundance independent of their cognate mRNA via unknown post-transcriptional mechanism(s) – aka protein buffering. We applied a novel mediation approach to identify causal regulatory proteins and transcripts underlying these distant loci. Our analysis revealed an extensive network of protein-protein interactions that act to achieve stoichiometric balance of functionally related enzymes and subunits of multimeric complexes, and moreover provides new insights into the specific mechanisms of protein buffering.
O-54: Analysis of energy demands during lactation in mouse models
Jerry Wei1,2, Walter Olea2, Louise Hadsell2, Peter Thomson1, Darryl Hadsell2,3, and Peter Williamson*1
1Faculty of Veterinary Science, The University of Sydney, Sydney, NSW 2006. Australia
2USDA/ARS Children"s Nutrition Research Center, Department of Pediatrics
3Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston TX 77030
Redirection of available energy accompanies dynamic changes in physiological processes. Lactation places the greatest energy demand during the female life-cycle. This requires a sustained increase in maternal metabolism, often resulting in a change in body weight and body composition. Impaired regulatory responses to such a metabolic challenge may lead to lactation failure and have serious consequences for neonatal survival, or at least impact growth and development. We used a complex phenotyping strategy to measure variation across 32 inbred strains of mice from the diversity panel, during lactation. An in silico genome-wide association analysis was performed to identify regions that contribute to energy expenditure and body composition during lactation. Dams and their litters were studied for 8 days postpartum during second lactation. On day 1 postpartum (L1), 10 one-day old CD1 pups were cross-fostered to each lactating dam to standardise variation in pup suckling drivers during the study. Food intake and litter weight gain were recorded daily. Data for food intake and milk energy output (litter weight gain) between day 7 (L7) and day 8 postpartum (L8), were used to calculate energy balance. Body composition was measured using quantitative magnetic resonance (QMR) on L1 and again at L8 to assess changes during the study period. Genotyping was based on 132k SNP genotypes from across the inbred strains, and derived haplotag SNPs were then utilized for genome-wide association analysis. Significant strain differences (P < 0.001) were observed in food intake, milk energy output, maternal body weight variation, and body composition during lactation. GWAS revealed seven regions associated with change in body weight, five associated with fat mass change, three associated with milk energy output, 32 associated with food energy intake and 1 associated with energy balance. The study demonstrates utility of the mouse diversity panel in analysis of complex traits.
P-001: Abnormal Innate Immune Responses of ENU-induced Ali18 and Ali14 Mutant Mice Lead to Autoinflammatory Syndrome-like Phenotypes
Koichiro Abe*1, Satochi Nunomura2, Chisei Ra2, Atsushi Tajima3, Helmut Fuchs4, and Martin Hrabe de Angelis4
4Helmholtz Zenrum Muenchen
Autoinflammatory syndromes are associated with acute spontaneous inflammation such as painful abdomen and extremities. Unlike autoimmune diseases, autoinflammatory syndromes are caused by defects in the innate immunity. However, the molecular mechanism underlying pathogenesis of autoinflammtory syndromes is poorly understood. In a mouse ENU (N-ethyl-N-nitrosourea) mutagenesis screen, Ali18 and Ali14 dominant mutant strains were established because of spontaneous inflammation on peripheral paws. By genetic mapping and candidate sequencing, Ali18 and Ali14 are identified as gain-of-function mutations in a protein kinase and phospholipase C gamma2, respectively. Bone marrow transfer and other immunological experiments indicate that innate immune cells contribute to trigger autoinflammation in both Ali18 and Ali14 mutant mice. We assumed that mast cells are possible candidates to initiate autoinflammation in mutants, because they are bone marrow-derived, settle in various tissues, such like mucosal membrane and epidermis, and release various inflammatory mediators. To know contribution of mast cells to the inflammatory phenotypes, Ali18 and Ali14 mutant mice were crossed with W mutant mice as W/Wv alleles have almost no mast cells in tissues. Interestingly, double mutants with Ali18 and W/Wv (Ali18; W/Wv) showed no autoinflammation in peripheral paws, but Ali14; W/Wv alleles showed inflammation on paws. In addition, we analyzed activity of cultured mast cells derived from bone marrow of Ali18 and Ali14 mice. Although abnormal degranulation was not detected in mast cells from mutant mice, non-canonical mobilization of cytoplasmic calcium concentration was detected when stimulated by IgE cross-linking. The gene expression patterns of cytokines and chemokines in mast cell from mutant mice are under analysis. These results strongly suggest that mast cells are contribute to initiate autoinflammation in Ali18 mutant mice. In Ali14 mutant mice, however, partly mast cells but other myeloid cells and monocytes could be more important to trigger spontaneous inflammation.
P-002: Xist/Tsix expression dynamics during mouse peri-implantation development revealed by whole-mount 3D RNA-FISH
Hirosuke Shiura, and Kuniya Abe*
Technology & Development Team for Mammalian Genome Dynamics, RIKEN BioResource Center, Ibaraki 305-0074, Japan
During peri-implantation development in mice, the X Chromosome inactivation (XCI) status changes dynamically. However, the occurrence of these processes in vivo has not been explored in detail. To delineate the changes in XCI status in vivo, the expression of Xist and its antisense partner, Tsix, was examined via whole-mount RNA-FISH using strand-specific probes. In the embryonic cell lineage, Xist RNA began to disappear at embryonic day 3.75 (E3.75) and was lost completely by E4.5, whereas the derepression of Tsix from the silenced allele of the inactive X lagged behind the erasure of Xist. Tsix biallelic expression became dominant at E5.0, while the Xist cloud was visible again in some nuclei of E5.25 epiblasts. Considering that Tsix biallelic expression is a sign of epigenetic equivalency of two X Chromosomes, imprinted XCI reversal is likely completed by E5.0, and random XCI starts immediately after the completion of imprinted XCI erasure. Moreover, these results suggest that Tsix expression is dispensable for Xist downregulation during the erasure process. Intriguingly, epiblast cells exhibiting biallelic expression of Xist including two Xist clouds per nucleus were observed frequently (~15%) at E5.25 and E5.5. Although imprinted XCI appeared to be stable in the primitive endoderm–visceral endoderm lineage, transient loss of Xist clouds was noted in a subset of extraembryonic ectodermal cells, suggesting distinct features of XCI among the three different embryonic tissue layers. These results will serve as a basis for future functional studies of XCI regulation in vivo.
P-003: Towards the Creation of Reference Transcription Start Site Set (refTSS)
Imad Abugessaisa*1, Shuhei Noguchi1, Akira Hasegawa2, Hideya Kawaji3, and Takeya Kasukawa1
1RIKEN Center for Life Science Technologies Division of Genomic Technologies Life Science Accelerator Technology Group Large Scale Data Managing Unit
2RIKEN Center for Life Science Technologies Division of Genomic Technologies Life Science Accelerator Technology Group Genomics Algorithms Development Unit RIKEN Center for Life Science Technologies Center Director"s Strategic Program Molecular Network Control Research Project Molecular Network Control Genomics Unit
3RIKEN Research Cluster for Innovation RIKEN Preventive Medicine and Diagnosis Innovation Program RIKEN Center for Life Science Technologies Division of Genomic Technologies Life Science Accelerator Technology Group Genome Information Analysis Team
Reference data sets in genomics, such as reference genomes and reference genes, are of great importance to enable researchers to access integrated and annotated data with specific focus and scope, and to compare their own data against other data on the reference data set.
Several international efforts have been dedicated to generate transcription start site (TSS) and promoter data using sequence technologies such as CAGE. Despite the vast number of TSS data sets available, we still lack integrated reference TSSs.
Therefore, the refTSS project aims to construct a comprehensive TSS data set. Through manual and automated procedures, the refTSS will provide an integrated and well-annotated data set based on the published FANTOM5 promoter atlas and the publically available TSS and promoter data set. In particular, the refTSS platform will integrate various data sets based on TSS, such as TSS activity, gene annotation, regulation, epigenetics, and segmentation information.
Our approach to construct refTSS is based on three components. The first is the raw data sets for genomic coordination of TSSs, which consist of the 5’ end sequence information. The second is the annotation information, such as functional annotation and transcriptional regulation. Annotation information will be integrated by association of annotation to the individual TSS. The third is the human curation procedures required to confirm the identified TSS and the annotation.
This is an ongoing project. We have established the work flow and started verifying our approach. We applied remapping procedures on individual data sets (FANTOM5, EPD, and DBTSS) to the latest genome assembly. The remapping is followed by quality evaluation and TSS identification and integration.
The refTSS will be publically available via web portals. We envision refTSS as an important tool for enabling integrative analysis with different “players” within the TSS field.
P-004: Somatic variations in healthy skin fibroblasts and their relation to cancer and aging
Alexej Abyzov*1,2, Livia Tomasini2, Bo Zhou3, Nikolaos Vasmatzis1, Anahita Amiri2, Jessica Mariani2, Yanhong Wu1, Denise Walker1, Mariangela Amenduni2, Michael Wilson2, Mark Gerstein2, Julie Cunningham1, Jin Jen1, Sherman Weissman2, Alexander Urban3, and Flora Vaccarino2
1Mayo Clinic, Rochester, MN, 55905
2Yale University, New Haven, CT, 06520
3Stanford University, Palo Alto, CA, 94305.
Only a few studies have been conducted to understand natural somatic mosaicism, that is post-zygotic accumulation of mutations in cells of multicellular organisms. Fundamental knowledge about somatic mosaicism is not only crucial for finding determinants of cancer development and progression, but also for an understanding of various diseases and aging. For ten people, we have compared genomes of over twenty clonally derived human induced pluripotent stem cell (hiPSC) lines to the genomes of four primary skin fibroblast samples, parental to the hiPSC lines. The clonal nature of hiPSC lines allows the discovery of somatic genomic variants present in the founder cell, but not in all fibroblast cells, thereby providing a mean for a high-resolution analysis of single cell genomes. With this approach we found that, on average, an iPSC line derived from children manifests 400 single nucleotide variants (SNVs) not apparent in the fibroblasts. We next performed an in-depth re-evaluation of these candidate somatic variants in the fibroblasts with three orthogonal experimental techniques. These experiments confirmed that at least 20% (roughly 80 SNVs), and up to 70% (roughly 270 SNVs), of the candidate SNVs are mosaic somatic variants in fibroblast cells. The allele frequency of these SNVs ranged from 0.1% to a dozen percent in the fibroblast cell population, and the mutation spectrum was surprisingly similar to that observed in some cancers (Alexandrov et al., Nature, 2013). Analysis of hiPSC derived from fibroblasts of children’s parents indicates that parents have 1.5 to 2 times more mosaic SNVs per cell. These new discoveries emphasize a large degree of somatic mosaicism existing in healthy human tissues, provide the first evidence that mutational signatures observed in cancers could be attributed to a background somatic mosaicism in normal cells, and demonstrate that the amount of somatic mosaicism increases with age.
P-005: Elevated canonical Wnt signalling disrupts development of the embryonic midline and can cause Heterotaxy
Alaa Alzahrani, Koula Diamand, Jehangir Ahmed, Kristen Barratt, and Ruth Arkell*
Early Mammalian Development Laboratory, Research School of Biology, The Australian National University, Canberra, ACT 2601, Australia
Heterotaxy is a congenital abnormality where the internal thoraco-abdominal organs demonstrate abnormal arrangement across the left-right (L-R) axis of the body. It can affect the development of the heart, liver, lungs, intestines, and spleen. The L-R embryonic axis is established early in embryogenesis when unidirectional signals emanate from a specialised structure at the embryonic midline, called the node, to initiate distinct molecular pathways on the left and right sides of the developing embryo. The gene most commonly mutated in human cases of Heterotaxy is the X-linked ZIC3, but the mechanism by which the ZIC3 transcription factor prevents Heterotaxy remains unknown. A genetic screen for mutations that affect murine embryogenesis identified katun (Ka), a novel null allele of Zic3. The mutant embryos exhibit Heterotaxy and also incompletely penetrant, partial (posterior) axis duplications and anterior truncation. These latter two phenotypes are redolent of elevated canonical Wnt signalling and analysis of Ka embryos reveals ectopic expression of direct targets of Wnt/β-catenin mediated transcription in mutant embryos. ZIC3 is a member of the Zic family of transcriptional regulators and previous work has shown that ZIC proteins can inhibit Wnt/β-catenin mediated transcription when overexpressed in cell lines. This raises the possibility that dysregulated Wnt signalling may contribute to Heterotaxy. We have investigated this notion by analysis of the murine batface (Bfc) gain-of-function allele of β-catenin that results in elevated Wnt/β-catenin signalling. We find this strain exhibits incompletely penetrant defects of L-R axis formation, caused by a defective midline development, and synergises with the Zic3 Ka allele to produce an increased incidence of L-R axis defects. Moreover we find that human ZIC3-Heterotaxy associated mutations encode proteins that are defective in their ability to inhibit Wnt/β-catenin mediated transcription. Overall this provides strong evidence that Wnt dysregulation can contribute to human cases of Heterotaxy.
P-006: Targeted reduction of highly abundant transcript with pseudo-random primers
Ophelie Arnaud*, Stephane Poulain, Sachi Kato, and Charles Plessy
RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa, 230-0045 Japan.
Since a few years, several methods to study the single cell transcriptome have been developed. Most of them use poly-T oligonucleotides in the reverse transcription step, which limits the detection of non-poly(A) transcripts. However, given the importance of the non-coding RNA, random-priming the RNA should give a more complete transcriptome. Unfortunately, the use of random primers produces a high proportion of reads coming from ribosomal RNA, decreasing the depth of the sequencing. Moreover, because of the sequencing cost, the single cells are multiplexed, which limits the total number of sequenced read per single cell. As a consequence, only the most abundant transcripts in a cell are detected. Therefore, the depletion of the abundant and undesirable transcripts, like the ribosomal RNA one, before the sequencing will improve the detection of the lower expressed transcripts. However, as the RNA starting quantity is very low, an extra step (like Ribo-Zero) isn’t possible. Thus, we propose to use a selective set of random primers, the pseudo-random (PS) primers, allowing the detection of the non-poly(A) RNA while also limiting the detection of the ribosomal RNA. Here, we are presenting, the proof of concept of the PS primers in the nanoCAGE protocol, starting with 50ng of total RNA.
We have demonstrated that the use of only 40 PS primers instead of the 4096 random primers considerably decreased the reads coming from ribosomal RNA sequences without affected the transcriptome diversity or the number of genes detected. We have also demonstrated that this strategy can be applied to deplete a sample of other undesirable sequences like the sequences coming from the hemoglobin genes.
In conclusion, the pseudo-random primers are effective for eliminating specific and unrelated sequences without affecting the gene coverage. They are simple to design, and therefore are flexible tool for depleting any transcriptome libraries in abundant and undesirable sequences.
P-007: F5-explorer: an interactive webserver for quick gene-oriented browsing of FANTOM5 data
Frederik Otzen Bagger*1,2,3,4,7, Sina Hadi Sohi5, Mette Louise Trempenau1,2,4, Maria Dalby3, Damir Sasivarevic5, Bo Torben Porse1,2,4, Ole Winther5, The FANTOM consortium, Robin Andersson3, and Nicolas Rapin1,2,3,4
1The Finsen Laboratory, Rigshospitalet, Faculty of Health Sciences, University of Copenhagen, Denmark
2Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark
3The Bioinformatics Centre, Department of Biology, Faculty of Natural Sciences, University of Copenhagen
4Danish Stem Cell Centre (DanStem) Faculty of Health Sciences, University of Copenhagen, Denmark
5DTU Compute, Technical University of Denmark, Lyngby, Denmark
6The FANTOM Consortium
7current address: European Molecular Biology Laboratory - European Bioinformatics Institute (EMBL-EBI), Wellcome Trust Genome Campus, Hinxton, Cambridge, UK, and Department of Haematology, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0PT, UK
A large and comprehensive body of data has been laid out by the FANTOM5 project covering transcription start site usages across a wide range of cell and tissue types. Here, we present a gene-oriented interface for accessing and visualizing the data via a web-browser: the F5-explorer.
The F5-explorer offers a simple interface to plot expression levels of promoters or enhancers associated with a single queried gene. The platform utilizes annotations of samples to structured ontologies (Cell Ontology, Uberon, Disease Ontology) provided by the FANTOM consortium and a simplified tree-like hierarchical representation provided directly on the interface makes it possible quickly to browse different cell types or tissues of the human body. The interface does not encompass the full body of data, nor displays all details of the metadata and ontology annotations, but rather attempts to simplify and speed up the process of answering simple, yet important, biological questions for biologists with or without bioinformatics skills. For predetermined users, or users in need of a specific plot or comparison not available from the general default terms a shopping basket functionality has been built into the F5-explorer. This allows for creating any user-defined plot or comparison from the data and furthermore expands functionality of the interface to include the entirety of the FANTOM5 data.
In conclusion, we present a new gene-oriented interface for quick browsing of the FANTOM5 data. By metadata pruning and an interactive graphical user-interface, we present a portal to one of the most comprehensive and encompassing transcriptomics datasets available to date. We believe that this platform will further increase the availability and use of the FANTOM5 consortium effort and broaden its potential users.
The web interface is freely available and requires no login at: http://servers.binf.ku.dk/fantom5
P-008: Multimer formation explains allelic suppression of PRDM9 recombination hotspots
Christopher Baker*1, Pavlina Petkova1, Michael Walker1, Petr Flachs2, Ondrej Mihola2, Zdenek Trachtulec2, Petko M Petkov1, and Kenneth Paigen1
1Center for Genome Dynamics, The Jackson Laboratory, Bar Harbor, ME 04609, USA
2Laboratory of Germ Cell Development, Division BIOCEV, Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic, v. v. i., Videnska 1083, CZ14220 Prague, Czech Republic
Genetic recombination during meiosis functions to increase genetic diversity, promotes elimination of deleterious alleles, and helps assure proper segregation of chromatids. Mammalian recombination events are concentrated at specialized sites, termed hotspots, whose locations are determined by PRDM9, a zinc finger DNA-binding histone methyltransferase. Prdm9 is highly polymorphic with most alleles activating their own set of hotspots. Most mammalian populations exhibit high frequencies of heterozygosity; however, questions remain about the influences different alleles have in heterozygous individuals where the two variant forms of PRDM9 typically do not activate equivalent populations of hotspots. We now find that, in addition to activating its own hotspots, the presence of one Prdm9 allele can modify the activity of hotspots activated by the other allele. PRDM9 function is dosage sensitive; B6-Prdm9+/Prdm9tm1Ymat heterozygous null mice have reduced numbers and less active hotspots and increased numbers of aberrant germ cells. In mice and humans carrying two Prdm9 alleles, there is allelic competition; the stronger Prdm9 allele can partially or entirely suppress chromatin modification and recombination at hotspots of the weaker allele. In cell cultures, PRDM9 protein variants form functional heteromeric complexes which can bind hotspots sequences. When a heteromeric complex binds at a hotspot of one PRDM9 variant, the other PRDM9 variant, which would otherwise not bind, can still methylate hotspot nucleosomes. We propose that in heterozygous individuals the underlying molecular mechanism of allelic suppression results from formation of PRDM9 heteromers, where the DNA binding activity of one protein variant dominantly directs recombination initiation towards its own hotspots, effectively titrating down recombination by the other protein variant. In natural populations with many heterozygous individuals, allelic competition will influence the recombination landscape.
P-009: Influence of diet on metabolic syndrome in four genetically diverse mouse strains
William Barrington*1,3, Daniel Pomp2, Brian Bennett2, and David Threadgill3
1North Carolina State University and Texas A&M University
2University of North Carolina Chapel Hill
3Texas A&M University
Recent research has demonstrated the profound impact of altering dietary macronutrient composition on mouse health in C57BL/6J mice. However, there is little research exploring how relevant human diets affect health in genetically diverse mice. Our study addressed this gap in knowledge by assessing metabolic syndrome-related phenotypes in four genetically diverse mouse strains (A/J, C57BL/6J, FVB/NJ, NOD/ShiltJ) fed one of five human diets (Western, Mediterranean, Japanese, Hunter-Gatherer, and Ketogenic) or a standard mouse chow. Adiposity, blood pressure, liver triglycerides, blood parameters, and mitochondrial function were assessed. Each strain had substantially different reactions to the diets. A/J mice were generally resistant to negative impacts from all diets. C57BL/6J performed poorly on the high-fat Western diet, but had positive effects when fed a high-fat ketogenic diet. Oppositely, the ketogenic diet produced strong, negative effects on metabolic syndrome phenotypes in FVB/NJ mice that did better on a Western diet. Detailed effects on background and diet-dependent development of metabolic syndrome will be presented. The data indicates that there is profound individual variation in diet response that is not detectable in human epidemiological studies. Follow-up studies are underway to determine the genetic causes underlying these diverse diet responses.
P-010: Characterization and mapping of colimba, a new spontaneous mouse mutation with hair coat abnormalities.
David Bazaga1, Carlos Perez1,2, Joe Angel, and Fernando Benavides*1
1Department of Epigenetics and Molecular Carcinogenesis, The University of Texas MD Anderson Cancer Center, Smithville, Texas, USA
2Division of Pharmacology & Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas, USA
In this study we present colimba (col), a new autosomal recessive mutation with early growth retardation and sparse hair coat. The mutation appeared in a C57BL/6.Psl1Edba congenic strain and was maintained as a homozygous line. The initial mapping using (C57BL/6 x FVB/N)F2-col/col mice allowed us to localize the col locus to proximal Chromosome 1, between the centromere and marker D1Mit373 (26.4 Mb), a region of homology with human Chromosomes 8q11-13 and 6q12. The phenotype of col/col mice is obvious a few days after birth and includes retardation in post-natal development and partial alopecia. Histologically, the skin shows altered hair follicle cycle at several time points. No other histological lesions were observed. Furthermore, tumor multiplicity after chemical skin carcinogenesis is higher in mutant mice than wild-type littermates. There are no obvious functional candidate genes for the col mutation in the candidate region; however, we will present the analysis of complete exome sequencing.
P-011: Cilia and Ciliopathogies: Impact of new knowledge on our understanding of biology and human disease
Karen Christie, Janan Eppig, Cynthia Smith, Hiroaki Onada, Joel Richardson, and Judith Blake*
The Jackson Laboratory, Bar Harbor, ME USA
Interest in primary cilia has increased dramatically as it has become clear that ciliopathies are an underlying cause of numerous human diseases including for some types of retinitis pigmentosa , for polycystic kidney disease, and for cardiovascular pathologies. Once thought to be restricted to a few cell types, it is now apparent that primary cilia are found on almost all vertebrate cells and are critical to sonic hedgehog (Shh) signaling. Mouse models play a key role in developing our understanding of the role of cilia in control of Shh signaling in development throughout the embryo, and in ongoing maintenance of structures such as photoreceptors.
To maximize the utility of the wealth of experimental data generated by mouse ciliopathy models, we have engaged in a project to comprehensively annotate experimentally characterized ciliary genes of mouse using Gene Ontology (GO) terms to describe their molecular functions, biological roles, and cellular locations, using the SYSCILIA gold standard of known human ciliary components as a starting point. We are updating the GO to add new terms that represent recent advances in our understanding of ciliary biology. Comprehensive GO annotation of ciliary genes in the mouse will be a great resource for researchers engaged in high throughput studies or comparative genomic analysis across species. In addition, Mouse Genome Informatics (MGI) has tools, including the Human-Mouse: Disease Connection interface and MouseMine, to help researchers identify connections between mouse genes and human genes, and to identify relevant mouse models which may be useful in the study of a given disease. We are actively collaborating in the Cardiovascular Development Consortium (CvDC), Bench-to-Bassinet (B2B) program of the National Heart Lung and Blood Institute (NHLBI), and some of the results of that work relative to cilia processes will be presented.
Funded by HG002273 to GOC, HG000330 to MGD, and HL098188 for CvDC component.
P-012: Single-cell transcriptomes of fluorescent, ubiquitination-based cell cycle indicator cells
Michael Boettcher*1, Tsukasa Kouno1, Elo Madissoon2, Efthymios Motakis1, Imad Abugessaisa1, Sachi Kato1, Harukazu Suzuki1, Yoshihide Hayashizaki3, Takeya Kasukawa1, Piero Carninci1, Timo Lassmann4, Jay W Shin1, and Charles Plessy1
1RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa, 230-0045 Japan
2Karolinska Institutet, Department of Biosciences and Nutrition, SE-141 83 Huddinge, Sweden
3RIKEN Preventive Medicine & Diagnosis Innovation Program, Yokohama, Kanagawa, 230-0045 Japan
4Telethon Kids Institute, West Perth Western Australia 6872, Australia
Using HeLa cells that report their cell cycle phase through fluorescent, ubiquitination-based cell cycle indicators (Fucci), we produced a reference dataset of more than 270 curated single cells for which each single-cell’s transcriptome can be matched with cell cycle information via the fluorescence intensity of the transgenes in that cell. We developed a comprehensive open data management and quality control pipeline that enables users of our dataset to process all available sequence and image files in a highly reproducible way without prior knowledge of the underlying bioinformatical toolset. The final output of that pipeline is a customizable table with relevant metadata and quality information for each single cell. This metadata table can be easily used as input for sophisticated data analysis. Our workflow is also adjustable for usage with other single-cell datasets that consist of RNA-sequencing and fluorescence data. Currently, we use the Fucci dataset to create a model for cell cycle phase inferences, which can be applied to other single-cell transcriptomes without cell cycle phase reporting metadata.
P-013: Analysis of ENU mutant mice indicates the existence of non-exomic thrombosis modifier mutations
Marisa A Brake*1, Amy E Siebert1, Kart Tomberg2, Guojing Zhu2, David R Siemieniak2, and Randal J Westrick1
1Department of Biological Sciences, Oakland University, Rochester, Michigan, USA
2Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, USA
A variant of coagulation Factor V called Factor V Leiden (FVL) is a potent, yet incompletely penetrant, risk factor for venous thromboembolism (VTE). We sought to understand the genetic basis for this incomplete penetrance by using a FVL (F5tm2Dgi) mouse model. We identified a perinatal lethal phenotype in homozygous FVL mice also heterozygous deficient for tissue factor pathway inhibitor (F5tm2Dgi/F5tm2Dgi Tfpitm1Gjb/+). This life/death phenotype formed the basis for a binary sensitized whole genome ENU mutagenesis screen for dominant thrombosis suppressors. ENU mutagenized F5tm2Dgi/F5tm2Dgi Tfpitm1Gjb/+ males were bred to F5tm2Dgi/F5tm2Dgi females and surviving F5tm2Dgi/F5tm2Dgi Tfpitm1Gjb/+ offspring inherited an ENU induced mutation that suppresses the lethal phenotype. Screening of 6,754 progeny identified 98 mice with putative suppressors. Of these, 16 were successfully mated back to F5tm2Dgi/F5tm2Dgi to create thrombosis suppressor lines. Three of these lines, MF5L5, MF5L14, and MF5L22 exhibited penetrances of 58.9%, 33.9%, and 80.8%, respectively. Five mice from each of these lines were whole exome sequenced to identify candidate suppressor mutations. MF5L5 had 6 candidates, MF5L14 had 5 candidates, and MF5L22 had 22 candidates. To determine the actual suppressor mutation among the candidates, a minimum of 12 mice from multigenerational pedigrees were genotyped for the presence of the mutations. Kaplan-Meier survival curves for each putative modifier were insignificant. In addition, F5tm2Dgi/F5tm2Dgi Tfpitm1Gjb/+ mice with none of the original exomic candidates were observed in succeeding generations. Thus, whole genome sequencing is the best option for identifying the suppressor mutants in these three lines and sequencing of mice from each line is in progress. The identification of thrombosuppressive modifiers will provide novel insights into the pathways leading to VTE and facilitate novel therapeutic interventions.
P-014: Shape-based morphometric analysis of homozygous lethal embryos imaged by micro-CT
James M Brown*, Neil Horner, James Cleak, Sara Johnson, Zsombor Szoke‑Kovacs, Lydia Teboul, Carl Henrik Westerberg, and Ann‑Marie Mallon
Mammalian Genetics Unit, MRC Harwell, Harwell Science and Innovation Campus, Harwell OX11 0RD, UK
The International Mouse Phenotyping Consortium (IMPC) (https://www.mousephenotype.org) is a multi-national effort to functionally characterize each of the 20,000 genes in the mouse genome. Of the 2,400 knockout lines assessed so far, approximately 30% are embryonic or perinatal lethal. In order to establish the cause of lethality, 3D imaging techniques such as micro-CT, OPT and HREM are employed at several key developmental stages to capture aberrant embryo morphology.
The high-throughput nature of the screening process makes manual annotation of 3D imaging data infeasible, and subtle phenotypes may be overlooked by human experts. Automated methods for phenotype detection in 3D images often rely on image registration, where wild-type and mutant embryos are brought into spatial alignment with one another. Voxel or deformation-based morphometry is then used to elucidate statistically significant differences between the two groups. With the addition of an appropriately labelled atlas, differences in total organ volume may also be measured. Such an approach is highly amenable to further analysis, based on shape-based (surface) representations of organs.
Statistical shape models (SSMs) are used to extract the geometric variation from a set of input objects. Using such a model, individual shapes (such as organs) may be represented as deviations from the average shape, allowing for morphological differences to be readily identified. In this work, we propose a novel method for constructing multi-organ SSMs from micro-CT images of E14.5 embryos, having been registered towards an average atlas. The proposed method was applied to a number of knockout mouse lines with known phenotypes, and the results compared to those produced using existing methods. We demonstrate that an SSM-based approach provides a means of assessing embryo organ morphology that is complementary to existing methods, as well as providing additional information about abnormal organ morphology.
P-015: The JAX Synteny Browser: A new visualization tool for mouse-human comparative genomics
Paul Hale, Keith Sheppard, Mei Xiao, Joel Richardson, and Carol J Bult*
The Jackson Laboratory, Bar Harbor, Maine, 04609
We have developed a new web-based synteny browser to support visualization of conserved synteny between the mouse and human reference genomes. In contrast to existing synteny visualization tools, the JAX Synteny Browser is tightly integrated with the rich biological annotations of mouse genes available from the Mouse Genome Informatics (MGI) database. The browser allows users to navigate to specific regions of conserved synteny and then to highlight or filter the features displayed according to specific biological properties including gene function, phenotype, or disease. The browser supports four distinct levels for visualization: genome wide, chromosome, block, and feature level. All of the underlying data needed for the browser are expected to conform to common bioinformatics data format standards. For example, genome features are represented in GFF3; functional annotations are represented in a GAF format; variants are represented in VCF files, etc. The software has been developed in alignment with the philosophy behind other “generic model organism database (GMOD)” software products so that the visualization tool can be extended easily to other pairs of organisms.
Supported in part by NIH HG000330-P1 and P30 CA034196.
P-016: Modeling Binding Affinity of the Multiple Zinc-Finger Protein PRDM9
Alexander Fine1,2, Michael Walker1, Timothy Billings1, Kenneth Paigen1, Petko M Petkov1, and Gregory W Carter*1,2
1The Jackson Laboratory, Bar Harbor, ME 04609
2Tufts University School of Medicine, Boston, MA 02111
Mammalian genomes encode hundreds of zinc finger proteins (ZFPs) that bind DNA, but most have unknown functions. One well-characterized example, PRDM9, recognizes and binds with multiple tandem zinc fingers to regulate genomic locations of meiotic recombination. Over 20 alleles of PRDM9 are known in mice, each containing a unique array of zinc fingers. Each allele is expected to bind a different set of loci, providing a natural experimental system for investigating how ZFPs recognize DNA sequence. To study site selection, we used a novel, in vitro sequencing strategy called Affinity-Seq to assess binding of PRDM9 to genomic DNA. We found over 30,000 significant binding sites for the PRDM9Dom2 isoform and quantified the frequency of binding at each sequence. This enabled estimation of binding affinity at each site in addition to standard nucleotide frequencies. The vast majority (95%) of sites contained an allele-specific binding motif, suggesting a single binding sequence for each PRDM9 isoform. We identified a few core nucleotides required for binding. However, analysis of F1 hybrid mice suggested variability at all bases affects binding frequency. To assess the importance of each nucleotide, we performed linear regression to model effects on binding affinity. Quantitative data for thousands of sites allowed us to infer additive and interactive effects for all bases, revealing multiple nucleotide-nucleotide interactions that drive binding. We tested this model by performing Affinity-Seq on a second genome, providing thousands of natural polymorphisms for quantitative validation. Our work provides a detailed view of DNA binding by PRDM9 and unprecedented power to assess the complex rules of zinc finger binding specificity. Models based on these rules can potentially predict novel recombination hotspots and the functions of uncharacterized ZFPs.
P-017: Mapping Genomic Distributions of Combinatorial Histone Modifications at the Single Molecule Level
Jen‑Chien Chang*1, Takashi Umehara2, Keisuke Fujita3, Yuichi Taniguchi4, Toshio Yanagida3, and Akiko Minoda1
1Division of Genomic Technologies, RIKEN Center for Life Science Technologies
2Division of Structural and Synthetic Biology, RIKEN Center for Life Science Technologies
3Laboratory for Cell Dynamics Observation, RIKEN Quantitative Biology Center
4Laboratory for Single Cell Gene Dynamics, RIKEN Quantitative Biology Center
Post-translational modifications of histones, which mediate many nuclear processes, is one of the most well characterized epigenetic mechanisms. In recent years, it has become evident that specific combinations of histone modifications represent various chromatin states, and are associated with different regulatory functions. However, standard methods probe one histone modification at a time; thus, the combinatorial patterns at each histone or nucleosome are largely unknown. Here, we develop a novel method for mapping the genomic distribution of combinatorial histone modifications by integration of two platforms: single molecule fluorescent microscope and the next-generation sequencer. Once this method is established, we will first investigate the genomic localization of coexisting active and repressive histone marks in different cell types. Other applications include the colocalization of histone variants, DNA methylation, transcription factors, and chromatin-binding proteins. By combining other data sets such as RNA-seq, transcription factors ChIP-seq, and DNase Hypersensitivity assay, we aim to identify the functions of chromatin states, with the ultimate goal in providing a complete picture of how epigenome is modulated.
P-018: Zebrafish serves as a disease model system to investigate the embryonic development effects of human parvovirus B19- NS1 and VP1u protein
Ju Chang‑Chien*1,2, Jiann‑Jou Yang2, Tsai‑Ching Hsuc1, and Wei‑Jen Chen2
1Institute of Biochemistry, Microbiology and Immunology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan
2Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan
Parvovirus B19 (B19) is known as a human pathogen and has been associated with a variety of disorders including fetal anemia, cardiomegaly, pericardial effusion, hydropic or nonhydropic intrauterine fetal death (IUFD) and autoimmune diseases. To investigate the influences of B19 on embryo development, zebrafish animal model was adopted to elucidate the effects of B19-NS1 and B19-VP1u on embryonic development of zebrafish. Abnormal embryos development from 6 hpf to 96 hpf were observed after injected with B19-NS1 or VP1u expression plasmid. The abnormal phenotype revealed a progressive and defective rate in B19-NS1 or VP1u injected embryos as compared to the controls. In addition, embryos injected with B19-NS1 or B19-VP1u showed the heart edema or abnormal somite development at 96 hpf. However, higher mortality rate was detected in B19-VP1u injected embryos than those embryos injected with B19-NS1. In this study, our data suggests that both B19-VP1u and B19-NS1 proteins interfere the embryonic development and could provide a clue in understanding the possible pathogenic mechanism of B19 in embryonic development.
P-019: Investigation of defective spermiogenesis in a human XLID mouse model
Chun‑Yu Chen*, Chih‑Hsiang Yu, I‑Shing Yu, Yu‑Chen Hsu, Ming‑Shian Tsai, Meng‑Ni Fan, and Shu‑Wha Lin
Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, Taiwan
Generation of mature spermatozoa requires timely activation and removal of regulators to accommodate the goals in each distinct stage during spermatogenesis. Although it is known that ubiquitination plays important roles in the progression of spermatogenesis, it is unclear whether a key protein degradation regulator. Ubiquitination of target proteins occurs in the progression of germ cell development; however, key participators that maintain functional spermatogenesis remain largely undefined. We report a human X-linked intellectual disability (XLID)-associated cullin-ring-ligase plays crucial roles in post-meiotic sperm development. We found that this XLID-associated gene is consistently expressed in spermatogonia and dynamically expressed in post-meiotic spermatids but not expressed in meiotic spermatocytes. Mutant male mice are sterile and display a progressive loss of germ cells during spermiogenesis leading to oligoasthenospermia. Epididymides of mutant mice contained very low number of mature spermatozoa with pronounced morphological abnormalities. Although mitosis of spermatogonia and meiosis of spermatocytes appeared unaffected in mutant mice, a significant elevation of apoptosis is presented in spermatids during spermiogenesis from acrosome phase to cap phase. Ultrastructural pathological study confirmed that spermatids develop aberrant acrosome and nuclear morphology in the mutant mice. In conclusion, our findings reveal a critical role for this XLID-associated gene as an acrosomal formation and nuclear condensation regulator in haploid male germ cell differentiation and loss of this it in mice leads to male fertility.
P-020: MECOM (EVI1 or PRDM3) maintains neuronal stem cell self-renewal through chromatin control over RBPJ recruitment.
Elaine KY Chung*, Tobias Hohenauer, Fatma Urun, Chee Wee Tee, and Adrian W Moore
Lab for Genetic Control of Neuronal Architecture, RIKEN Brain Science Institute, Japan.
Stem cells divide to either self-renew or create precursor cells destined to differentiate. Failure to balance between these outcomes in the nervous system causes neurological disorders and cancer. NOTCH2 activation drives neuronal stem cell self-renewal by repressing proneural gene expression and altering the cell cycle. However, how pleiotropic NOTCH2 initiates this self-renewal program is unclear. Here we show that MECOM oncogenic locus (encoding EVI1, also called PRDM3), commonly overexpressed in leukemia, acts with NOTCH2 in olfactory neuronal stem cells to repress proneural gene expression and promote the G1/S transition. Upon activation, NOTCH2 utilizes the transcription factor RBPJ to activate transcription, and the target Hes1 is the core of the neuronal stem cell self-replication regulatory network. EVI1 binds but does not induce the Hes1 locus. Instead, we find that EVI1 controls histone methylation status and nucleosome positioning at the Hes1 locus. These changes in chromatin structure create a locus with high potential for RBPJ access, and upon NOTCH2 activation they amplify RBPJ binding and Hes1 induction. Our data define a chromatin control system that primes stem cells to respond to Notch through enabling activation of the self-renewal program.
P-021: The ORFeome Collaboration: A community resource for expression of most human protein-coding genes
The ORFeome Collaboration*
ORFeome Collaboration is an international consortium
We present the ORFeome Collaboration (OC) and the community resources this effort has generated: a genome-wide collection of high-quality, fully sequenced human open-reading-frame (ORF) cDNA clones for studies on human protein structure and function; a searchable, annotated database to locate available clones; and an international distribution network to provide the resource to the research community. The OC clone collection comprises one or more ORF clones for 73% of 20,906 RefSeq human genes and 79% of the 19,022 highly curated Consensus Coding DNA Sequence Project (CCDS) human genes. All clones are provided in a GatewayTM entry vector format for easy transfer of the ORF onto a large number of expression vectors covering most expression systems. The ORFeome Collaboration is out of today the most comprehensive collection of human ORF clones made available to the entire international research community to promote the functional annotation of protein coding genes and their gen products.
P-022: KCNQ1 and CFTR act as tumor suppressors in colorectal cancer
BLN Than1,11, JF Linnekamp2, JACM Goos3, SH den Uil3, RJA Fijneman3,4, MG O"Sullivan12, DA Largaespada5,9,10, A Schumann1, GA Meijer3,4, Y Zhang7, C Hodges8, TK Starr5,6,10, E Van Den Broek3, JP Medema2, PM Scott1, L Vermeulen2, and RT Cormier*1,10
1Department of Biomedical Sciences, University of Minnesota Medical School, Duluth, MN, USA
2Laboratory of Experimental Oncology and Radiobiology, Academic Medical Center, Amsterdam, The Netherlands
3Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands
4Netherlands Cancer Institute, Amsterdam, The Netherlands
5Department of Genetics, Cell Biology and Development, University of Minnesota Medical School, Minneapolis, MN, USA
6Department of Obstetrics, Gynecology and Women"s Health, University of Minnesota Medical School, Minneapolis, MN, USA
7University of Minnesota Supercomputing Institute, Minneapolis, MN, USA
8Department of Pediatrics, Case Western Reserve University, Cleveland, OH, USA
9Department of Pediatrics, University of Minnesota Medical School, Minneapolis, MN, USA
10University of Minnesota Masonic Cancer Center, Minneapolis, MN, USA
11Department of Gastroenterology, Weill Cornell Medical College, New York, NY, USA
12College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA
A role for the ion channel genes Kcnq1 and Cftr in colorectal cancer (CRC) was first indicated by their identification as candidate driver genes in Sleeping Beauty transposon-mediated forward genetic screens in mice. Follow-up mouse genetic studies conducted in the C57BL/6J-ApcMin background showed that Kcnq1 deficiency significantly enhanced ApcMin tumor multiplicity, with some adenomas progressing to adenocarcinoma. ApcMin Cftr(Cftrtm1Cwr/Cftrtm1Cwr)-deficient mice also showed significant increase in tumor multiplicity and progression and loss of Cftr alone was sufficient for development of tumors in >60% of Apc+/+ Cftrtm1Cwr/Cftrtm1Cwr(Cftr KO) mice.
Further, we found that maintenance of KCNQ1 protein expression was associated with improved survival in patients undergoing resection for Stage IV colorectal cancer liver metastases with a significant increase in median overall survival of 23 months. This finding was confirmed by enhanced disease-free survival (DFS) in Stage II and Stage III CRC patients. A similar enhancement of DFS was observed in early stage CRC patients who maintained CFTR expression.
To investigate the basis of protective effects of KCNQ1 and CFTR in CRC we examined global gene expression in Kcnq1(Kcnq1tm1Kpfe/Kcnq1tm1Kpfe) and Cftr-deficient mouse colon and small intestine. Among top gene clusters were genes involved in innate and adaptive immune responses, ion channels, mucins, cancer cell migration and invasion, stress responses and intestinal stem cell-related genes. In tumors isolated from Apc+/+ Cftr KO mice we found upregulation of Wnt/beta-catenin target genes and intestinal stem cell genes, a phenotype similar to that observed in ApcMin mice. To investigate the roles of Kcnq1 and Cftr in the stem cell compartment we examined intestinal organoid cultures from knockout mice and found that organoid outgrowth and delayed differentiation was enhanced in Kcnq1-/- and Cftr-/- colons compared with wild type colons.
Overall, our data identify KCNQ1 and CFTR as CRC tumor suppressor genes that can act as prognostic biomarkers and potential therapeutic targets.
P-023: Whole-exome sequencing of ENU-induced mutant mice with BALB/c background
TA de Souza*, S Ienne, MAC Rocha de Carvalho2, CF Menck3, and SG Massironi2
1Core Facility for Scientific Research, University of Sao Paulo, Brazil
2Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil
3Department of Microbiology, Institute of Biomedical Sciences, University of Sao Paulo, Brazil
Mutant mice are valuable tools for understanding the mechanisms of basic and complex biological processes. Mutagens, as ENU (N-ethyl-N-nitrosourea), were used to induce random mutations in germinative mouse cells, generating mutant lineages with potentially interesting phenotypes. A key challenge is to identify and associate causal variants in DNA to phenotypes in models. Considering that most of all known ENU-induced causal mutations are found in exons, we sequenced exomes from eight ENU-induced mutant mice with BALB/c background presenting interesting phenotypes. A hybridization-based mouse whole-exome capture system was used to construct libraries for massive parallel sequencing. Reads were mapped to mouse reference genome then an SNP calling pipeline was used to create a raw SNP list for each ENU-induced mutant mice and for inbred strains C57BL/6 and BALB/c used as controls. Here we report the optimization of an SNV filtering strategy for discovery of candidates for ENU-induced mutations in mice. Filtering steps were based upon previously mapped regions, type of inheritance, and uniqueness compared to dbSNP and controls. Using this method, we found strong candidate mutations for all mice sequenced, including potential new models for diseases in humans. Putative causative mutations were evaluated by impact prediction tools and validated by Sanger sequencing. Functional investigations of mutant gene products are still in course. In addition, we are also investigating SNP and small INDELs profiles of our C57BL/6 and BALB/c inbred strains, maintained for decades in our breeding facilities. Taken together, our results will be an important source of information for biomedical researchers and to the field of laboratory mouse genetics.
Financial Support: FAPESP (São Paulo, SP, Brazil).
P-024: A selfish genetic element drives recurrent selective sweeps in the house mouse
John P Didion*1, Andrew P Morgan1, Liran Yadgary1, Daniel Pomp1, Gary A Churchill2, and Fernando Pardo‑Manuel de Villena1
1Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, US
2The Jackson Laboratory, Bar Harbor, ME, US
A selective sweep is the result of strong positive selection rapidly driving newly occurring or standing genetic variants to fixation, and can dramatically alter the pattern and distribution of allelic diversity in a population or species. Population-level sequencing data have enabled discoveries of selective sweeps associated with genes involved in recent adaptations in many species. In contrast, much debate but little empirical evidence addresses whether “selfish” genes are capable of fixation, thereby leaving signatures identical to classical selective sweeps – despite being neutral or deleterious to organismal fitness. Previously, we described the novel R2d2 locus, a large copy-number variant that causes non-random segregation of mouse Chromosome 2 during female meiosis due to meiotic drive. Here, we show that R2d2 has driven recurrent selective sweeps while having no discernable effect on fitness. We tested multiple closed breeding populations from 6 outbred backgrounds and found that alleles of R2d2 with high copy number (R2d2HC) rapidly increase in frequency, and in most cases become fixed in significantly fewer generations than can be explained by genetic drift. A survey of 15 natural mouse populations in Europe and the United States revealed that R2d2HC alleles are circulating at intermediate frequencies in the wild; moreover, patterns of local haplotype diversity are consistent with recent positive selection. Our results provide direct evidence of populations actively undergoing selective sweeps driven by a selfish genetic element, and demonstrate that meiotic drive can rapidly alter the genomic landscape in favor of mutations with neutral or even negative effect on overall Darwinian fitness. This has broad implications for evolutionary studies: loci that are implicated as drivers of selective sweeps require independent evidence to determine the type of sweep - hard, soft, or selfish.
P-025: Implication of truncated CABLES1 in agenesis of the corpus callosum
TH Tra Dinh*1, Seiya Mizuno1, Hiroyoshi Iseki1, Saori Mizuno‑Iijima1, Jun‑Dal Kim2, Junji Ishida2, Satoshi Kunita3, Akiyoshi Fukamizu2, Fumihiro Sugiyama1, and Ken‑ichi Yagami1
1Laboratory Animal Resource Center, University of Tsukuba, Tsukuba, Japan
2Life Science Center, Tsukuba Advanced Research Alliance, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan
3Center for Experimental Medicine, Jichi Medical University, Shimotsuke, Japan
Agenesis of the corpus callosum (ACC) is a congenital abnormality of the brain structure. More than 60 genes are involved in the corpus callosum development. However, the molecular mechanisms underlying ACC are not fully understood. Previously, we produced a novel transgenic mouse strain, named as BALB/cAJci-Tg(Actb-rtTA,Actb-tTS)138Utr or TAS, carrying genes of the tetracycline-inducible expression system that are not involved in brain development, and inherited ACC was observed in the brains of all homozygous TAS mice. Although ACC was probably induced by transgene insertion mutation, the causative gene and the molecular mechanism of its pathogenesis remain unclear.
In this study, we first performed interphase three-color fluorescence in situ hybridization (FISH) analysis and determined that transgenes were inserted into ~12.0Mb distal to the centromere of Chromosome 18. Gene expression analysis and genomic PCR walking showed that the genomic region containing exon 4 of Cables1 was deleted by transgene insertion and the other exons of Cables1 were intact. The mutant allele was designated as Cables1Tg(Actb-rtTA,Actb-tTS)138Utr aka Cables1TAS. Interestingly, Cables1TAS mRNA consisted of exons 1–3 of Cables1 and part of the transgene that encoded a novel truncated CABLES1 protein. Homozygous TAS mice exhibited mRNA expression of Cables1TAS in the fetal cerebrum, but not that of wild-type Cables1.
To investigate whether a dominant negative effect of Cables1TAS or complete loss of function of CABLES1 gives rise to ACC, we produced Cables1-null mutant mice (BALB/c-Cables1tm1Utr). ACC was not observed in Cables1-null mutant mice, suggesting that a dominant negative effect of CABLES1TAS impairs callosal formation. Moreover, ACC frequency in Cables1+/CablesTAS mice was significantly lower than that in Cables1-/CablesTAS mice, indicating that wild-type CABLES1 interfered with the dominant negative effect of Cables1TAS. This study indicated that truncated CABLES1 causes ACC and wild-type CABLES1 contributes to callosal formation.
P-026: Association Analysis of -1997 Polymorphism in Upstream Regulatory Site of COL1A1 with Low Bone Mineral Density: A Study on Iranian Post-menopausal Women
Mina Ebrahimi‑Rad*1, Fatemeh Mirkhanim2, Mohammadmehdi Emam3, and Reza Saghiri1
1Biochemistry Department, Pasteur Institute of Iran, Tehran.Iran
2Department of Science,Parand Branch,Islamic Azad University,Parand,Iran
3Rheumatology Department, Loghman Hakim Hospital, Shaheed Beheshti University of Medical Sciences, Tehran,Iran
4Biochemistry Department, Pasteur Institute of Iran, Tehran,Iran
Osteoporosis is a growing public health problem in the most developed countries and a common age-related disease with low bone mass density (BMD) and increased fracture risk. Some of the fractures lead to morbidity and mortality especially in women. Many different factors are involved in osteoporosis pathogenesis, among which, genetic markers have been found to have a great impact (75-80%) on bone turn over and loss, predisposing to osteoporosis. Several genes have been studied as candidate genes in osteoporosis pathogenesis, including COL1A1, coding for the major bone matrix protein. This study was designed to analyze the correlation between the polymorphism of COL1A1 gene regulatory upstream site at promoter region (-1997 G/T) and BMD variation in post-menopausal women. 424 Iranian post-menopausal women in the age range of 45-67 years old were enrolled into the study, considering the inclusion and exclusion criteria. Control healthy group (N:211, T-score ≥ -1) and patient group (N:213), including osteoporotic (T-score ≤ -2.5) and severely osteopenic (T-score> -2.5) were analyzed. The decisions were made based on clinical examinations, BMD measurements by Dual-energy X-ray Absorptiometry (DXA), and the data obtained through a questionnaire, consisting of the necessary data.The subjects were genotyped by using PCR-Fragment Length Polymorphism (PCR-RFLP). Software Stata version 11 was applied to statistically analyze the collected data. The association between the specific genotypes, the low BMD and the disease was analyzed by using One-way ANOVA for quantitative variables. Although, for qualitative variables, test was applied in which P≤ 0.05 was considered to be significant. Our data showed that there was no significant linkage between the -1997 G/T polymorphism in the COL1A1 upstream regulatory region with the variation of bone mineral density (BMD) in Iranian postmenopausal women (for ee genotype versus EE+Ee the OR was 0.8, with 95% confidence CI was found to be 0.18-1.67) .
P-027: Genetic control of extreme Influenza disease
Martin T Ferris*1, Kelsey E Noll1, Clayton R Morrison1, Ande West1, Alan C Whitmore1, Richard R Green2, and Mark T Heise1
1Department of Genetics, University of North Carolina at Chapel Hill
2Department of Microbiology, University of Washington, Seattle WA
Genetic variation within immune pathways has increasingly been seen as a critical player in a variety of human disease conditions. To understand how this variation in immune response pathways drives viral disease response to multiple respiratory diseases, we have used F1 crosses of the Collaborative Cross recombinant inbred panel. We have identified a wide range of immune response phenotypes to a number of pathogens, including Influenza A Virus. Importantly, we have identified a CC line, CC017/UNC that shows extreme susceptibility to the human pandemic A/California/04/09 virus. To better understand the underlying host pathways driving this extreme response, we have conducted in depth phenotyping of CC017/UNC and the classic inbred strain DBA/2J, which is also highly susceptible to influenza; furthermore we have generated and phenotyped an F2 cross between CC017/UNC and DBA/2J. We found that while C57BL/6J animals show a transient weight loss and >85% survival through 12 days post infection, CC017/UNC and DBA/2J strains show 100% mortality between 5-7 days post infection, with divergent disease etiologies. Furthermore, at 4 days post infection, all 3 strains show an influx of immune cells into the lungs, but there are strain-specific differences in neutrophil and t-cell populations following infection. Transcriptional analysis further confirmed an upregulation of phagocytic and granulocytic associated responses in both DBA/2J and CC017/UNC following infection. The F2 mapping population displays a wide range of Influenza response phenotypes, including animals that survive with only mild disease signs; a phenotype not seen within the parent strains.
P-028: The environmental toxicant Tetrabromobisphenol-A promotes adipogenesis by downregulating THY1 (CD90) in human mescenchymal stem cells.
E"Lissa Flores*1,2,4, Collynn Woeller2,4, and Richard Phipps2,3,4
1Translational Biomedical Science, Clinical and Translational Institute
2Department of Environmental Medicine
3Department of Microbiology and Immunology
4University of Rochester. Rochester, NY
Obesity is characterized by excessive formation of adipocytes (adipogenesis) and increases in adipocyte size. Adipocytes produce pro-inflammatory adipokines and cytokines such as IL6 that promote the inflammatory state seen in obesity. Environmental factors are emerging as a cause of the drastic rise in obesity over the past decades and these require further study. Tetrabromobisphenol-A (TBBPA) is an environmental toxicant now called an “obesogen” as it can disrupt the endocrine system and increase adipogenesis. The mechanisms of action whereby TBBPA and other environmental toxicants promote fat cell formation and obesity remain unclear. THY1 (also called CD90) is a cell surface protein. THY1 has been widely used as a cell surface marker and is expressed on various subsets of cells, such as fibroblasts and stem cells. We have previously shown that only THY1-/low fibroblasts can differentiate into adipocytes while THY1+/high fibroblasts cannot, indicating THY1 can determine cell fate and is thus more than a cell marker. We hypothesized that TBBPA promotes adipogenesis by decreasing THY1 expression on human mesenchymal stem cells (hMSCs). Our data show that TBBPA promotes adipogenesis and decreases THY1 mRNA and protein levels in hMSCs. Furthermore, TBBPA also reduces cell surface expression of THY1 in hMSCs. Adipogenesis was measured by visualization of lipid droplets and expression of the key adipocyte marker, fatty acid binding protein 4 (FABP4). Interestingly, even transient TBBPA exposure in hMSCs resulted in continued reduction of THY1 expression, even after TBBPA exposure was stopped. Therefore, developmental or even transient exposure to TBBPA could affect THY1 levels on stem cells and alter physiology long after exposure, which may contribute to the obesity epidemic.
P-029: Genetics of Meiotic Recombination Rate
Barbora Faltusova1, Vaclav Gergelits1, Maria Balcova1, Tanmoy Bhattacharyya1,2, Ondrej Mihola1, Corrina Knopf1,3, Vladana Fotopulosova1, Zdenek Trachtulec1, Sona Gregorova1, and Jiri Forejt*1
1Institute of Molecular Genetics AS CR, Division Biocev, Prague, Czech Republic
2Current address: The Jackson Laboratory, Bar Harbor, Maine, USA
3Current address: Medical University Vienna Center of Medical Physics and Biomedical Engineering, Vienna, Austria
During differentiation of germ cells into gametes, a maternal and a paternal copy of each chromosome have to find each other, recombine, pair and synapse in order to ensure proper chromosome segregation into the gametes. Because of the unique ability to identify homologous DNA sequences between homologous chromosomes, meiotic recombination is an essential step in proper chromosome pairing and synapsis in the majority of species. However, when the paternal and maternal sets of chromosomes come from different (sub)species, the recognition of homologs can be disturbed and result in sterility of male hybrids. Here we investigated the meiotic recombination rate at the global genome-wide level and its possible relation to hybrid sterility in mouse strains derived from subspecies Mus m. musculus (Mmm) and Mus m. domesticus (Mmd). Using immunofluorescence microscopy we quantified the foci of MLH1 DNA mismatch repair protein, the cytological counterparts of reciprocal crossovers, in a panel of inter-subspecific chromosome substitution strains. Two autosomes, Chr 7 and Chr 11, significantly modified the meiotic recombination rate, but the strongest modifier, designated meiotic recombination 1, Meir1, emerged in the 4.7 Mb Hstx2 genomic locus on Chr X. Mapping Meir1 to a narrow candidate interval on Chr X is an important first step towards positional cloning of the respective gene responsible for variation in the global recombination rate between closely related mouse subspecies.
P-030: Meiotic Arrest, Crossover Paterns, and Hybrid Sterility of [BALB/cJxJF1/Ms] F1 Male Mice
Yasuhiro Fujiwara*1,2, Risako Nishino2,3, Tanmoy Bhattacharyya1, Petko M Petkov1, Tetsuo Kunieda4, and Mary Ann Handel1
1The Jackson Laboratory, Bar Harbor, ME 04609
2Graduate School of Natural Science and Technology, Okayama University, Okayama University, Okayama, Okayama 700-8530, Japan
3The Institute of Environmental Toxicology, Joso, Ibaraki, 303-0043, Japan
4Graduate School of Environmental and Life Science, Okayama University, Okayama, Okayama 700-8530, Japan
Organisms that belong to different subspecies within the same species sometimes exhibit reproductive isolation owing to failure in gametogenesis, and hybrid sterility (HS) of F1 offspring. Here, we investigate the basis of HS mechanism found in intersubspecific F1 hybrid mice produced by matings between Mus m. domesticus (domesticus) classical laboratory mouse strains and Mus m. molossinus (molossinus) Asian mouse strains. We found that F1 (designated CJFF1) males from a mating between female BALB/cJ mouse (domesticus) and male Japanese fancy mouse JF1/Ms (molossinus) were sterile, although the F1 females were fertile, and the reciprocal F1 males (designated JFCF1) were sub-fertile. Sterility of the CJFF1 males was due to high rate of arrest during the first meiotic division phase. Pachytene spermatocytes of the CJFF1 males exhibited a high rate of dissociation of the XY Chromosomes, and metaphase spermatocytes showed dissociation of the XY Chromosomes as well as premature separation of autosomal chromosomes. Although many aspects of recombination-related DSB formation and repair were similar between sterile and sub-fertile F1 males, patterns of crossing over were atypical in the CJFF1 males. We therefore infer that incompatibilities between the BALB/cJ and JF1/Ms subspecies, manifest asymmetrically in their F1 males, affect multiple aspects of meiosis, culminating in arrest of the division phase. Supported in part by NIH HD33816 to MAH.
P-031: ENU mutation cataloging in the RINEN ENU mutant mouse library using whole exome analysis with Ion Proton sequencer.
Ryutaro Fukumura*, Hayato Kotaki, Shigeru Makino, Yuichi Ishitsuka, Yuji Nakai, and Yoichi Gondo
Mutagenesis and Genomics Team,RIKEN BRC
We have been providing allelic series of mutant mice carrying a single point mutation in the target gene as a public resource named “RIKEN ENU-based gene-driven mutagenesis system (RGDMS). Since we find the base substitution first, it is very easy to phenotype not only the heterozygotes but also homozygotes. The KO mice become available from the IKMC or by the genome editing. The CRISPR/Cas9 system is also able to provide targeted base substitutions if we know which base pair to be substituted before hands. Thus, when target base-pair(s) are not known, the RGDMS is more effective because it provide about 10 allelic series of base-substitutions in the target gene. In addition, each G1 mouse carries ~5,000 base substitutions that provide a resource to detect and analyze the genetic interactions and polygenic functions among them.
We have started the whole exome sequencing (WES) for G1 genome in order to comprehensively detect SNV induced by ENU. We have so far discovered more than 3000 SNVs by using SOLiD4 and HiSEQ2000. Here, we report the WES study using Ion Proton sequencer. We obtained ~80 million reads from each G1 mouse per PI chip. The realignment BAM files mapped on GRCm38/mm10 were made by Torrent server. Using the MassARRAY or the amplicon sequencing, we validated the SNV candidates that may have a significant effect on the protein sequences, such as missense or nonsense mutations. As a result, we newly extracted 1207 SNV candidates on the 1124 genes from 27 G1 genomes. From OMIM database, 237 genes were related to the 525 diseases. We experimentally validated the 246 SNV candidates from 5 G1 genomes and found that 246 (100%) were true mutations. By making the catalog of the ENU mutations, our system should contribute to understand the mechanism of the genome functions with polygenic disorders.
P-032: Correlation of Trp53cor1 and Trp53 expression in the Trp53cor1 gene trap mouse line
Riki Furuhata*1, Mai Nakahara1, Haruka Ito1, Masatake Araki2, Kumiko Yoshinobu2, Ken‑ichi Yamamura3, and Kimi Araki1
1Division of Developmental Genetics, IRDA, Kumamoto University
2Division of Bioinfomatics, IRDA, Kumamoto University
3Yamamura Project Laboratory, IRDA, Kumamoto University
Trp53cor1 (LincRNA-p21) was recently identified as a long intergenic non-coding RNA (lincRNA) regulated by TRP53, and reported to function in TRP53-dependent repression of target genes and induction of apoptosis (Huarte et al, Cell 142, 409-419). Many other interesting functions of Trp53cor1 have been reported, such as regulation of Warburg effect by promoting glycolysis under hypoxia, transcriptional activation of the neighboring Cdkn1a (p21) gene in cis. Most studies of Trp53cor1 were performed using culture cells, therefore, the in vivo function of Trp53cor1 is unclear. We found one of our exchangeable gene trap clones, Ayu21-B186, has trapped the Trp53cor1 gene and established the Trp53cor1 gene trap mouse line B6.Cg-Trp53cor1Gt(Ayu21-B186)Imeg to analyze its functions in mice.
The trap vector was inserted in the intron at 12kb downstream of the 1st exon, and the fusion transcript of the 1st exon and the lacZ gene was produced. To examine the expression pattern of Trp53cor1 in mice, RT-PCR and X-gal staining of adult tissues were performed. Although RT-PCR analysis revealed that Trp53cor1 was weakly expressed in almost all mouse tissues examined, relatively strong X-gal staining was detected in the cerebellum and pancreas. In order to know whether Trp53cor1 is induced by TRP53 in vivo, we crossed B6.Cg-Trp53cor1Gt(Ayu21-B186)Imeg gene trap line to TRP53-deficient (B6.Cg-Trp53tm1Sia) mice. In B6.Cg-Trp53tm1Sia/Trp53tm1Sia Trp53cor1Gt(Ayu21-B186)Imeg/+ mice, X-gal staining in the cerebellum and pancreas completely disappeared. Next, we induced TRP53 expression through high-fat diet and experimental pancreatitis and examined the Trp53cor1 expression. In the TRP53 up-regulated tissues, the Trp53cor1 was also highly expressed. These results indicate that Trp53cor1 is regulated by TRP53 in vivo.
P-033: Maternal malnutrition alters gene expression, genomic methylation and behavioral phenotypes of progeny
Tamio Furuse*1, Takashi Kohda2, Kunio Miyake3, Takae Hirasawa4, Tomoko Kushida1, Ikuko Yamada1, Misho Kashimura1, Hideki Kaneda1, Kimio Kobayashi1, Fumitoshi Ishino2, Takeo Kubota3, and Shigeharu Wakana1
1Japan mouse clinic, RIKEN BRC, Japan
2Dept. of Epigenetics, Tokyo Med. & Dent. Univ., Japan
3Dept. of Epigenetic Med., Univ. of Yamanashi, Japan
4Dept. of Bioscience., Teikyo University, Japan
The developmental origins of health and disease paradigm (DOHaD) is a concept that fetal environmental factors affect the adult phenotypes. We have been performing study that validates the DOHaD theory in the pathogenesis of psychiatric disorders and developmental disorders using mouse models. In this study, in vitro fertilization (IVF) and embryo transfer (ET) technics were used for mouse reproduction. ICR female mice were used as recipient and foster mothers. The embryos were prepared from eggs and sperms of C57BL/6J. We provided following diets as experimental diets to pre-pregnant and pregnant females, AIN93G (control diet, CD), low-protein (LP), and low-protein diet that contains folate supplement (LP + FA). The embryos obtained from C57BL/6J were transferred to the recipient mothers. The offspring which were exposed to malnutrition in utero exhibited increased activity in the home cage, decreased contact to novel object, and decreased social investigation. The adult offspring of LP group and LP+FA group exhibited different pattern of mRNA expression and genomic methylation in the brain. In addition, we are developing new DOHaD model by using maternal mice which carry mutations in genes that relate to nutrient transport. In this meeting, we will report about the summary of past study and progress of the development of new model.
P-034: Transcriptional control of hibernation: first insights on comparative genomics of dormice
(See abstract TS-03 in the Trainee Symposium)
P-035: Conservative miRNA target analysis: are we limiting our discoveries of neuronal miRNA function?
(See abstract TS-06 in the Trainee Symposium)
P-036: Needs of fundamental revision of mouse genome reference sequences.
Yoichi Gondo*1, Kazuki Mori2, Ryutaro Fukumura1, Atsushi Toyoda3, Satoshi Oota1, Toyoyuki Takada4, Ryutaro Himeno5, Toshihiko Shiroishi4, Satoru Kuhara2, and Asao Fujiyama3
1RIKEN BioResource Ctr
2Grad. Sch. Agr., Kyushu Univ.
3Comp. Genome, Natnl. Inst. Genet.
4Mamm. Genet., Natnl. Inst. Genet.
5RIKEN Adv. Ctr. Comp. Comm.
Chaisson et al.  and Pendleton et al.  have conducted re-assembly of whole human genome reference sequences by using long-read WGS this year. The gaps and whole structural variations (SVs) have been significantly closed and revised, respectively. The mouse genome reference sequences have been considered to be more precise than human’s, since they are from C57BL/6J inbred genomic DNA. Fairfield et al.  reported that ˜50% of Mendelian traits in the mutant mouse strains were identified by WES, the efficiency of which was very equivalent to those in human. It implies that the mouse genome reference sequences also have some unexpected issues in addition to those found in human genome reference sequences. We have conducted WGS of C57BL/6JJcl with >500-fold depth and found peculiar SNVs as well as SNPs, suggesting that not only the unfinished gaps but also the SV issues must be solved in the mouse genome reference sequences as well. The re-assessment and re-assembly of mouse genome reference sequences seems to be urgent and necessary, since the designing of PCR primers and genome editings are all relied on the genome reference sequences. Not only the re-sequencing to find de novo mutations and SNPs but also RNAseq, ChIPseq, Egigenome-seq, etc. are also depending on the genome reference sequences.
1. Chaisson et al. Nature 517: 608-611, 2015.
2. Pendleton et al. Nat Method 12: 780-786, 2015.
3. Fairfield et al. Genome Res 25: 948-957, 2015.
P-037: Mapping SARS-Coronavirus susceptibility alleles using the Collaborative Cross
(See abstract TS-02 in the Trainee Symposium)
P-038: SINEUPs: a new class of natural and synthetic antisense long non-coding RNAs that activate translation.
Silvia Zucchelli1,2, Diego Cotella2, Hazuki Takahashi3, Francesca Fasolo1, Michael Jones4, Francesca Persichetti2, Remo Sanges5, Claudio Santoro2, Piero Carninci3,6, and Stefano Gustincich*1,6,7
1Area of Neuroscience, SISSA, Trieste, Italy
2Dipartimento di Scienze della Salute, Universita del Piemonte Orientale, Novara, Italy
3RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Japan
4Cell Guidance Systems, Cambridge, United Kingdom
5Animal Physiology and Evolution Laboratory, Stazione Zoologica Anton Dorhn, Napoli, Italy
6TransSINE Technologies, Yokohama, Japan
7Department of Neuroscience and Brain Technologies, Italian Institute of Technology (IIT), Genova, Italy.
We present the discovery of a new functional class of natural and synthetic antisense lncRNAs that stimulate translation of sense mRNAs. These molecules have been named SINEUPs since their function requires the activity of an embedded inverted SINEB2 sequence to UP-regulate translation. Natural SINEUPs suggest that embedded Transposable Elements may represent functional domains in long non-coding RNAs. Synthetic SINEUPs may be designed by targeting the antisense sequence to the mRNA of choice representing the first scalable tool to increase protein synthesis of potentially any gene of interest.
We will discuss potential applications of SINEUP technology in the field of molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.
P-039: Genome-wide mapping of hyper-acetylated chromatin with a novel antibody in lung cancer cells
Lusy Handoko*1, Marina Lizio1, Wakamori Masatoshi2, Michiel De Hoon1, Aki Minoda1, and Takashi Umehara2,3
1Division of Genomic Technologies, RIKEN CLST
2Epigenetic Drug Discovery Unit, Division of Structural and Synthetic Biology, RIKEN CLST
Changes in acetylation of histone H4 are a common hallmark of cancer cells. In leukemia cells, histone H4 is characterized by a loss of K16 mono-acetylation. Bromodomain proteins, implicated in cancer, specifically recognize acetylated lysines. Recently, inhibitors of DNER(BET)-bromodomain (JQ-1 and I-BET) have been developed as promising anti-cancer agents. These indicate prominent roles of H4-acetylation in transcriptional regulation in cancer cells. Less study, however, has been focused on histone H4-acetylation at a genome-wide level. Here, we aim to profile histone H4-acetylation sites by ChIP-seq in lung cancer cells. With a novel monoclonal antibody we generated, we found around 70% of hyper-acetylated H4 is associated with active enhancers. Furthermore, a small subset of hyper-acetylated H4 is associated with super-enhancers. Incorporating the genome-wide H4-acetylation patterns with other epigenetic state marks (enhancers, repressors and transcription factors BRDs) will give us insight into the role of histone H4 acetylation in the epigenetic regulation of lung cancer cells
P-040: Up-regulation of non-coding RNAs in adult and pediatric liver cancers
Kosuke Hashimoto*1, Ana Maria Suzuki1, Alexandre Dos Santos2, Christophe Desterke2, Emilie Braun2, Alessandro Bonetti1, Alexandre Fort1, Xian‑Yang Qin1, Bogumil Kaczkowski1, Alistair Forrest1, Soichi Kojima1, Didier Samuel2, Marie Annick Buendia2, Jamila Faivre2, and Piero Carninci1
1RIKEN Center for Life Science Technologies
2INSERM U785, Centre Hepatobiliaire
An increasing number of non-coding RNAs (ncRNAs) are implicated in various human diseases including cancer, however ncRNA transcriptome of liver cancer remains largely unexplored. We use CAGE to comprehensively map transcription start sites (TSSs) and measure their expression in two types of liver cancers, human hepatocellular carcinoma and hepatoblastoma with special emphasis on ncRNAs. We found thousands of significantly up-regulated ncRNAs in both types of liver cancers compared to their matched non-tumors. In HCC, many LTR retroviral promoters are activated in a subfamily-specific manner. Intriguingly, the expression of highly up-regulated LTRs tends to be limited in reproductive tissues, such as testis and placenta. 3' RACE for 15 highly up-regulated LTRs revealed that the transcripts are multi-exon ncRNAs typically 0.5-2kb in length. On the other hand, hepatoblastoma didn’t show strong activation of LTR promoters. Instead, we identified activated ncRNAs that have TCF/LEF binding motifs, which are likely to be targets of the wnt/β-catenin pathway. Together, this study sheds light on ncRNA transcriptome of liver cancers, which might play important roles in tumor progression.
P-041: Dissecting the contribution of genes from the 17q21.31 region in the Koolen-deVries deletion syndrome using the mouse
Thomas Arbogast1, Hamid Meziane2, Giovanni Iacono3, Arnaud Duchon1, Mohammed Selloum2, Marie‑Christine Birling2, Tania Sorg2, Henk G. Stunnenberg3, the Gencodys Network, Hans van Bokhoven3, David Koolen3, Bert de Vries3, and Yann Herault*1,2
1Institut Clinique de la Souris, PHENOMIN-ICS, CNRS, INSERM, Universite de Strasbourg, 1 rue Laurent Fries, 67404 Illkirch, France
2Institut de Genetique Biologie Moleculaire et Cellulaire, IGBMC, CNRS, INSERM, Universite de Strasbourg, UMR7104, UMR964, 1 rue Laurent Fries, 67404 Illkirch, France
3Department of Human Genetics, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, Nijmegen, The Netherlands
Genetic diseases with intellectual disability (ID) can occur during the developmental period and is defined by an intellectual quotient below 70. Several genetic causes, including Down syndrome, deletion or duplication of genomic regions, and more than 500 genes, have been associated with ID. To better understand the physiopathology of the 17q21.31 Koolen deVries syndromes (KdVS), characterized by a deletion of a short genomic region, we generated a series of mouse mutants including the deletion and the duplication of the homologous region, and the inactivation of candidate genes. Here we will report the characterization of mouse models using standardized behavioural and cognitive paradigms. Based on the new series of models and further studies, we identified one candidate gene as the main contributor to the syndrome having a smaller contribution to the disease. In addition we went further and we were able to identify gene networks and chromatin changes in the models. The data generated are challenging our current knowledge and offer new perspectives for a better understanding of the KdV syndrome and its future treatment.
P-042: Effects of early-life exposure to Western diet on adult activity levels and associated behavioral and physiological traits in mice
Layla Hiramatsu*, Ralph Albuquerque, Gerald Claghorn, Jarren Kay, Jennifer Singleton, Zoe Thompson, James Colbath, Brett Ho, Brittany Ho, Gabriela Sanchez‑Andrade, Danny Thai, and Theodore Garland Jr.
University of California Riverside, Riverside, CA 92521
Lack of physical activity contributes to many human diseases, as well overweight and obesity. Numerous factors that influence activity levels, genetic or environmental, may occur during development and early life of an individual. Using an ongoing selection experiment with 4 replicate lines of mice bred for high voluntary wheel running (and 4 replicate, non-selected control lines), 100 female mice were given a “Western diet” (WD) with increased fat and added sucrose, while an additional 100 female mice were given standard diet (SD), from 2 weeks prior to mating until their pups could feed on solid food (~14 days of age). Of the resulting pups, 100 males (50 WD and 50 SD) were considered focal mice and received various tests: behavioral spot checks from birth, elevated plus maze, VO2max during forced treadmill exercise, 6 days of wheel access (with additional mice), home-cage activity, body fat composition, and organ masses. Nested ANCOVA was used with line nested within linetype (high-runner or control), with body mass and/or age as covariates. Exposure to WD during early life had a significant positive effect on the adult body mass of males for both HR and C lines, and caused a diet-by-line interaction in HR females. Early-life WD increased wheel running in C males, and caused a diet-by-line interaction in HR females. As expected from previous studies, HR mice had higher VO2max than C, but VO2max was unaffected by WD. Heart ventricle mass was increased by early-life WD in both HR and C mice. Interestingly, early-life WD did not increase fat pad masses. Analyses of additional behavioral and physiological traits are ongoing. Supported by US NSF grant IOS-1121273 to TG.
P-043: An atlas of 5’ complete transcripts reveals the genomic origins and expression landscape of human long non-coding RNAs
Chung‑Chau Hon*1, Jordan A Ramilowski1, The FANTOM Consortium1, Yoshihide Hayashizaki2, Piero Carninci1, and Alistair Forrest1,3
1RIKEN Center for Life Science Technologies (Division of Genomic Technologies),1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045 Japan
2RIKEN Preventive Medicine and Diagnosis Innovation Program, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan
3Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, the University of Western Australia, Nedlands, Western Australia, Australia
There is a strong controversy over the fraction of long non-coding RNA (lncRNAs) that are products of regulated and functional, versus spurious and non-functional, transcription events. One aspect hampering their study is the incomplete transcript models assembled from short reads, leading to misidentification of their 5’ends and hence their regulatory regions. Herein we applied Cap Analysis of Gene Expression (CAGE) to identify the 5’ ends of lncRNA transcript models from various sources. Overlaying these 5’ complete models onto DNase I hypersensitivity sites (DHS), we defined the putative regulatory regions (e.g. promoter or enhancer) controlling their transcription. Epigenetic marks indicate that the majority of intergenic lncRNAs are derived from enhancer-like regions (e-lncRNAs). These regulatory regions, and sometimes the RNA stretches outside of the DHS, showed evidence of non-neutral selection, and were significantly enriched in GWAS and eQTL associated SNPs. Using the expression profiles of lncRNAs measured by the FANTOM5 atlas, we show that those that overlap an eQTL associated SNP are significantly more correlated with the expression of their eQTL associated mRNA partner than random pairs of lncRNAs and mRNAs at similar distances, providing multiple layers of evidence supporting coordinated regulation of lncRNA and mRNA loci. Lastly, using sample ontology enrichment and differential expression analyses, we annotate these transcripts with the cell types in which they are enriched in and the physiological conditions in which they are dynamically regulated, summarizing these annotations in a web resource. This forms the foundation for a new effort to functionally characterize lncRNAs in the genome.
P-044: CAGE revealed novel biomarker of periodontitis-associated fibroblasts
Masafumi Horie*1,2,3, Yoko Yamaguchi4, Akira Saito1,2, Takahide Nagase1, Masayoshi Itoh3,5, Hideya Kawaji3,5, Timo Lassmann3,5, Piero Carninci3,5, Yoshihide Hayashizaki6, Alistair Forrest3,5, The FANTOM consortium, Marina Lizio3,5, Masahiro Kondo7, Tatsuo Suzutani8, Patrick Micke9, and Mitsuhiro Ohshima10
1Department of Respiratory Medicine, Graduate School of Medicine, The University of Tokyo
2Division for Health Service Promotion, The University of Tokyo
3Division of Genomic Technologies (DGT), RIKEN Center for Life Science Technologies
4Department of Biochemistry, Nihon University School of Dentistry
5Omics Science Center, RIKEN Yokohama Institute,
6RIKEN Preventive Medicine and Diagnosis Innovation Program
7Department of Physiology, Nihon University School of Dentistry
8Department of Microbiology, Fukushima Medical University School of Medicine
9Department of Immunology, Genetics and Pathology, Uppsala University
10Department of Biochemistry, Ohu University School of Pharmaceutical Sciences.
Bacterial infection has long been regarded as the main cause of periodontitis, and antibiotics therapy including plaque control has been widely used as treatment measures. However, their effects are limited leading to a new concept that causative of periodontitis might involve other than infection. Therefore, elucidation of pathogenesis of periodontitis by novel approach, and the establishment of novel therapy is desirable. We have previously established periodontitis-associated fibroblasts (PAFs) which degrade collagen matrix when co-cultured with gingival epithelial cells in 3D culture. Because teeth are supported by collagen fibers in alveolar bone, PAFs might be mainly involved in the pathogenesis of periodontitis. In this research, to characterize PAFs, we performed transcriptome analysis by CAGE-seq of PAFs.
From the FANTOM5 database, we firstly characterized normal gingival fibroblast (GF), which has low ability of collagen degradation, by comparing CAGE data of GF and fibroblasts from various organs. By the characteristic reference of normal GF, comparative analysis of CAGE profiles of 3 pairs of PAFs and non-PAFs, 2 from aggressive periodontitis and 1 from chronic periodontitis, were performed.
In normal GF, homeobox transcriptional factors, such as BARX1, LHX8, and DLX5/6, which play an important role in the mesenchyme during tooth development, are specifically expressed. Furthermore, CAGE revealed the peaks of RUNX2 long variant are specifically expressed in normal GF. By comparing CAGE profile of PAFs and non-PAFs, the peaks of both DLX5, GF-specific homeobox transcriptional factor, and RUNX2 long variant, which is regulated by DLX5, are lost in PAFs. In contrast, short variant of RUNX2 is ubiquitously expressed. These results are confirmed by qRT-PCR.
CAGE revealed DLX5 - RUNX2 long variant signaling is specifically inactivated in PAF. These results might lead to the elucidation of the mechanism of development of PAFs, and establishment of novel therapy of periodontitis targeting PAFs.
P-045: Evidence of a direct role for endothelial cells in the development of acute leukemias
Indhu Subramanian1,2,3, Emily Fuller1,3, Katie Powell2,3, Anthony Ashton2,3,4, and Viive Howell*1,3,4
1Bill Walsh Translational Cancer Research Laboratory
2Perinatal Research Laboratory
3Kolling Institute of Medical Research, University of Sydney, St Leonards NSW 2065 Australia
Pronounced increases in angiogenesis occur in the bone marrow of patients with acute leukemia, supportive of an endothelial contribution to leukemia. However, the molecular basis of how the endothelium propagates /initiates leukemia remains poorly understood.
The aim of this study was to determine the role of the endothelium in leukemia. Mice with altered endothelial cell behavior via Sleeping Beauty (SB) random insertional mutagenesis system were generated by breeding homozygous floxed SB (STOCK TgTn(sb-T2/Onc2)6113Njen/Nci Gt(ROSA)26Sortm2(sb11)Njen) female mice with hemizygous Tie2-Cre (B6.Cg-Tg(Tek-cre)12Flv/J) male mice. Litters were genotyped for the presence of the Cre allele indicating endothelial-specific activation of the SB system (Cre+), and tissues collected for analysis at ethically defined endpoints or at 1 year of age. Colony forming unit (CFU) assays were performed on magnetically selected CD45-/CD31+ endothelial cells (EC) and CD45-/CD31-/Sca1+ haematopoietic stem cells (HSC) isolated from bone marrow.
All Cre+ (n=106) and no Cre- (n=35) mice became moribund between 6 and 52 weeks of age. Cre+ mice displayed peripheral leucocytosis and gross anatomical/histopathological changes in spleens, livers and thymii. Flow cytometry and immunohistochemistry of Cre+ spleens and bone marrow revealed amplifications of T cell (in 49% of mice) or myeloid (28%) populations or both (23%). In vitro CFU assays identified elevations in CD3+ and CD14+ populations arising from the Cre+ EC cells but neither HSCs nor Cre- ECs. Amongst the SB-mutated genes identified by targeted deep sequencing were known mediators of haematological lineage specification.
Targeting SB to the endothelium resulted in acute forms of leukaemia signifying direct control over both myeloid and lymphoid differentiation by endothelial cells. Identification of genes known to be involved in leukemia validate our model. These data, for the first time, provide evidence for endothelial regulation of post-natal haematopoiesis and preliminary justification for the use of anti-angiogenic therapy in treatment of leukaemia.
P-046: Identification of key regulators contributing to the different responses of transforming growth factor beta in A549 cells by single-cell transcriptome
Yi Huang*, Masaaki Furuno, Harukazu Suzuki, and Erik Arner
RIKEN Center for Life Science Technologies, Division of Genomic Technologies 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan
The cellular heterogeneity within cell populations have been revealed in recent studies due to the advantages of single-cell analytic platforms. Both of single-cell genomic and transcriptomic data have been used to investigate the single nucleotide variation or gene expression-signatures associated to drug responses. However, there are still lacking a robust approach that identify subpopulations and design specific therapeutic targets for different sub-groups of cell. Our ultimate goal is to establish an integrative framework to combine drug responses of cells and single-cell transcriptomic data to identify key regulators contributing to the different responses of small molecules treatment. Here, we used transforming growth factor beta stimulation on A549 cells to induce cell migration and collect samples after 0 hour, 6 hours and 24 hours treatment to perform single-cell RNA sequencing. We could identify differentially expressed transcripts between subpopulations in each time-point and discover expression-signatures for insensitive cells. Besides, the key regulatory networks associated to TGF-beta response can be established to identify key regulators that will be manipulated in vitro to validate our findings. Our framework will provide a robust method to investigate new targets for developing new drug as combinatorial or alternative therapies for insensitive subpopulations of original treatment.
P-047: Dissecting the regulation of olfactory receptor expression in the mouse.
(See abstract TS-07 in the Trainee Symposium)
P-048: KRAS mutation specific alkylating pyrrole-imidazole polyamide, KR12 showed significant anti-tumor efficacy and preferential localization in KRAS mutant xenografts without adverse effects
(See abstract TS-13 in the Trainee Symposium)
P-049: Optimization of a Mathematical Model for Explanation of Biological Functions Using Monte-Carlo Methods and Parallel Computing
Kazuo Ishii*, and Toshinori Kozaki
Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183-8509, Japan
In the clinical diagnosis and genomic plant breeding, it is very effective to create mathematical models for prediction of phenotype using big data obtained from NGS and microarray by supervised learning, such as linear discriminant analysis and support vector machine. For example, differential expression genes from RNA-Seq and differential methylated CpG sites from WGBS (Whole Genome Bisulfite Sequence) can be used as explanatory variables of mathematical models to explain biological phenomena. Normally, hundreds or thousands of factors are selected from these analyses. A mathematical model is created from the combination of these factors like the following expression, and the phenotype can be predicted by the value f(x):
f(x) = a1x1 + a3x2 + a3x3 + …. + anxn + C,
where x1, x2, x3, …. , xn are explanatory valuables (values of differential expression genes or differential methylated CpG sites); a1, a2, a3, …. , an are coefficients for each valuables; C is a constant term. But because there are vast numbers of combinations of explanatory valuables, it is often difficult to discover the optimal combinations of factors.
To solve the problem, random selection of mathematical models from vast numbers of combinations of explanatory variable and large numbers of calculations, such as LDA, by parallel computing were performed. Fitting accuracy of models was evaluated and optimized based on sensitivity, specificity and Wilks' lambda. The optimized model with RNA-Seq data from colon cancers (18 colon cancer, 18 normal colon tissues) from public databases (GSM1228184 - GSM1228237; SRA) showed significant high sensitivity and specificity (100 % in both indices). It is showed that optimization of mathematical models using Monte-Carlo methods and parallel computing is very powerful to obtain a mathematical model for explanation of biological functions.
P-050: Molecular and functional characterization of Angelman syndrome patient-derived iPSCs and neurons
Mitsuru Ishikawa*1, Hironobu Okuno1,3, Shoma Tanaka1, Nakatake Yuki2, Hajime Komano1, Wado Akamatsu1,4, Minoru Ko2, Kenjiro Kosaki3, Shinji Saitoh5, and Hideyuki Okano1
1Dept Physiol, Keio Univ, Sch Med
2Dept System Med, Keio Univ, Sch Med
3Cent Med Genet, Keio Univ, Sch Med
4Cent Genomics and Regenerative Med, Juntendo Univ, Sch Med
5Dept Pediat and Neonatol, Nagoya City Univ, Grad Sch Med Sci
Genomic imprinting is a form of epigenetic regulation that results in parent‒of‒origin differential gene expression. Angelman syndrome (AS) is one of the most famous imprinted disorders which shows sever neurodevelopmental deficit, speech impairments, intellectual disability, epilepsy, prognathism, abnormal sleep patterns, and hyperactivity. In most cases, AS is caused by large deletion of maternally inherited human Chromosome 15q11‒q13, whose region contains UBE3A (ubiquitin protein ligase E3A) gene. In this study, using the episomal plasmids carrying the reprogramming and the tumor suppressor factors, POU5F1, KLF4, SOX2, MYCL, LIN28A, EBNA, and dominant negative TP53, we established novel iPSCs from T lymphocytes of seven different AS individuals, including not only a large genomic deletion in 15q11-13, but also uniparental disomy, or imprinting defect or epimutation in the imprinting center region in 15q11-13. To investigate the AS‒specific neuronal phenotypes, we perform the neuronal induction of iPSCs using not only neurosphere culture method but the rapid single‒step induction method. All the AS‒specific iPSC lines were successfully differentiated into mature neurons which expressed the marker genes of glutamatergic cortical neurons. As expected, UBE3A expression levels in the neurons are different between each AS patients and control iPSCs. We are now investigating neuronal functions of healthy control and AS iPSC clones, including analysis of the expression of synapse‒related genes, subcellular localization of glutamate receptor subunits, and neuronal morphology.
P-051: Recurrent Transcriptome Alterations Across Multiple Cancer Types
Bogumil Kaczkowski*1, Yuji Tanaka1,3, Hideya Kawaji1,2,3, Albin Sandelin4, Robin Andersson4, Masayoshi Itoh2, Timo Lassmann1,5, FANTOM5 consortium1, Yoshihide Hayashizaki2, Piero Carninci1, and Alistair Forrest1,6
1RIKEN Center for Life Science Technologies, Division of Genomic Technologies,1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan
2RIKEN Preventive Medicine & Diagnosis Innovation Program, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan
3RIKEN Advanced Center for Computing and Communication, Preventive Medicine and Applied Genomics unit, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan
4The Bioinformatics Centre, Department of Biology and Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark
5Telethon Kids Institute, the University of Western Australia
6Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, the University of Western Australia, Nedlands, Western Australia, Australia.
Genes that are recurrently deregulated in cancer are clinically attractive as both potential pan-cancer diagnostic markers and therapeutic targets. Here as part of the FANTOM5 project we compare Cap Analysis of Gene Expression (CAGE) profiles from a collection of 216 different cancer cell lines and corresponding primary cells to identify transcripts that are recurrently deregulated in a broad range of cancer types. We perform a complementary analysis using RNA-seq data from 4,055 tumors and 563 normal tissues profiled by the TCGA and present a core set of pan-cancer biomarkers that are identified in both the FANTOM5 and TCGA analyses. Additionally, we make the first report of enhancer RNAs that are up-regulated in cancer and using chromatin interaction data show that several are physically associated with the promoters of known oncogenes. Finally, we show that promoters that overlap repetitive elements (especially SINE/Alu and LTR/ERV1 elements) are more likely to be up regulated in cancer and report for the first time the activation of multiple copies of the REP522 interspersed repeat in cancer.
P-052: Analysis of mutational landscape in protein domains across a broad spectrum of pathological conditions
Anastasia Terskikh1, Anastasia Samsonova1, and Alexander Kanapin*2
1Peter the Great St.Petersburg Polytechnic University, St. Petersburg, Russia
2Department of Oncology, University of Oxford, Oxford, UK
Understanding of the disease etiology and potential treatment strategies, particularly in the areas of cancer and rare genetic disorders remains incomplete without functional interpretation of the impact of mutations on protein domains. We explored the protein domain-level landscape of genetic variants in WGS500 cohort (Taylor et al., Nat Gen, 2015) of whole genome sequencing data from patients with rare diseases and cancers to reveal potential driver pathways in pathological context.
We applied penalized logistic regression to extract disease-specific signatures of nonsynonymous variants, scored by mutation outcome, in PFAM domains. Pathway enrichment analysis on corresponding gene sets revealed metabolic and signaling pathways significantly over-represented specific to various genetic disorders and cancers, thus providing the functional context for how the variants contribute to disease. We also devised an integrated domain vulnerability score, which facilitated domain stability ranking in a context of various pathological conditions.
This study reveals a functional context of disease-specific mutations in protein domains and aid prioritization of domain target candidates for drug development.
P-053: Variation in transcriptional response to 1,25-dihydroxyvitamin D3 and bacterial lipopolysaccharide in primary human monocytes
Silvia Kariuki*, John Blischak, David Witonsky, and Anna DiRienzo
Department of Human Genetics, University of Chicago, Chicago, IL, USA
The active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), plays an important immunomodulatory role, regulating transcription of genes in the monocyte microbicidal pathway. We aimed to better characterize the complex immunomodulatory role of 1,25D by studying variation in transcriptional response to 1,25D and the bacterial lipopolysaccharide (LPS) in primary monocytes in vitro.
Monocytes were isolated from 10 African-American (AA) and 10 European-American (EA) healthy donors and treated with 1,25D, LPS, and ethanol for 24 hours. Transcript levels of RNA were measured on the Illumina HumanHT12 microarray. Genome-wide differential expression (DE) patterns were analyzed using linear mixed-effect models.
In the combined 1,25D and LPS treatment, pro-inflammatory cytokine genes such as CCL7 and CCL19 were downregulated, while genes with important antimicrobial activities such as CAMP and ITGAM were upregulated. Genes with inter-ethnic DE patterns in response to the combined 1,25D and LPS treatment were also identified, including PPAP2B, which encodes a phosphatidic acid phosphatase previously implicated in coronary artery disease risk, and AKNA, which encodes a transcription factor that induces antigen-dependent-B-cell development.
Studying the patterns of transcription response to 1,25D and LPS in primary monocytes in vitro enabled us to better characterize the immunomodulatory role of 1,25D, where it upregulates antimicrobial activity while attenuating the pro-inflammatory response. It also enabled us to detect several genes with inter-ethnic DE patterns, a finding that has not been previously reported. We speculate that these inter-ethnic differential transcription responses to 1,25D and LPS could contribute to inter-ethnic variation in susceptibility to immune-related diseases.
P-054: Versatile instrument for efficient single cell collection and deposition
Stanislav Karsten*, Zhongcai Ma, Anialac Zavala, and Lili Kudo
NeuroInDx, Inc., Signal Hill, California, 90755, USA
Single cell genome analysis demands for the development of cost-efficient instruments and approaches permitting routine collection and deposition of mammalian single cells from both cultures and tissues. We have developed a versatile capillary-based vacuum-assisted cell and tissue acquisition instrument (UnipicK+TM) with which individual fluorescently labeled and/or morphologically distinct cells can be acquired from any cultures grown in standard cell culture dishes including adherent, suspended, and 3D cultures. Collected cell(s) may be deposited in any desired well without significant impact on its viability for downstream clonal expansion or direct molecular analysis such as sequencing. The system is compatible with the collection and deposition of cells from multi-well plates. Moreover, the instrument is suitable for individual cell collection and region specific acquisition of cell clusters and subanatomical areas from tissues. Unlike LCM, UnipicK+TM is a free standing instrument that may be mounted over any inverted microscope providing exceptional flexibility in the laboratory. Because small sample volume is incremental for single cell analysis, UnipicK+TM enables the collection and dispensing of a single cell in as small as 15 nl volume. Our current work demonstrates the efficiency of UnipicK+TM for collection and population of multi-well plates by single cells from several different cell cultures including primary neural progenitor (NPC), SH-SY5Y, CHO and 3T3 cell cultures based on morphology or fluorescent label. Further, the collected cells were successfully deposited and re-cultured, demonstrating the minimal effect the process has on cellular viability. In addition, applicability of the instrument for the collection and deposition of individual cell clusters grown in three-dimensional (3D) culture system was shown using MDA-MB-435 cells. The instrument’s low cost, ease of use, and minimal impact on cell viability, makes it ideal for single cell analysis including sequencing applications.
P-055: The function of non-coding RNA in stem cell maintenance and differentiation
Kaori Kashi*1, Alessandro Bonetti1, Kosuke Hashimoto1, Alexandre Fort1,2, and Piero Carninci1
1RIKEN Center for Lice Sciences Technologies, Division of Genomics Technologies, 1-7-22 Suehiro, Tsurumi, Yokohama, Kanagawa 230-0045 Japan
2Department of Genetic Medicine and Development, University Medical Center, Geneva, Switzerland
Non-coding RNA (ncRNA) is a term referred to RNA molecules that are not translated into proteins. Although variable in size, ncRNAs that are longer than 200 nucleotides are referred as long non-coding RNAs (lncRNAs). Examples of well-studied ncRNA are tRNA and rRNA, whereas for the overwhelming majority of other ncRNAs the function and role in cells is not yet elucidated. However, in the past few years, a growing body of evidences has shown that they have role in supporting multiple cellular mechanisms.
Just like in other cells, lncRNAs are also present in stem cells. The main objective of this study is to identify lncRNAs which are important for stem cell maintenance and differentiation, using mouse ES and iPS cells. A previous study performed at RIKEN has revealed thousands of unannotated human and mouse lncRNAs whose function has not been yet identified. LncRNA expression will be altered using genetic perturbation technique and further analysis to understand their roles in molecular network and their interaction with transcriptional factors. Furthermore, as lncRNAs are involved in the regulation epigenomic machinery, association with the chromatin organization and remodeling will also be analyzed. The study of the interaction between RNA molecules and chromatin will be central to unravel the mechanisms of action for selected lncRNAs.
P-056: SEC23A functionally compensates for SEC23B deficiency in mice
Rami Khoriaty*1, Lesley Everett2, Jennifer Chase5, Guojing Zhu5, Bin Zhang7, Mark Hoenerhoff6, Ivan Maillard1,5, and David Ginsburg1,2,3,4
1Department of Internal Medicine, University of Michigan, Ann Arbor, MI
2Department of Human Genetics, University of Michigan, Ann Arbor, MI
3Department of Pediatrics, University of Michigan, Ann Arbor, MI
4Howard Hughes Medical Institute
5Life Sciences Institute, University of Michigan, Ann Arbor, MI
6In Vivo Animal Core, Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, MI
7Genomic Medicine Institute, Cleveland Clinic Lerner Research Institute, Cleveland, OH
SEC23A/SEC23B are paralogous components of COPII vesicles, which transport secretory proteins from Endoplasmic Reticulum (ER) to Golgi apparatus. SEC23B mutations in humans result in the autosomal recessive disease Congenital Dyserythropoietic Anemia type II (CDAII). We previously demonstrated that mice homozygous for a Sec23b genetrap allele die perinatally exhibiting pancreatic degeneration. To examine the impact of SEC23B-deficiency on adult murine hematopoiesis, we generated mice with erythroid specific and pan-hematopoietic SEC23B-deficiency by crossing a second, conditional allele Sec23btm1c(EUCOMM)Wtsi (exons 5-6 flanked by loxP sites) to an Eportm1(EGFP/cre)Uk and Tg(Vav1-cre)A2Kio, respectively. These mice did not exhibit any CDAII characteristic. Similarly, mice transplanted with SEC23B-deficient fetal liver cells did not exhibit CDAII. Pancreas-specific Sec23b knockout (using Ptf1atm1.1(cre)Cvw or Tg(Pdx1-cre)6Tuv) generated a phenotype indistinguishable from mice with germline Sec23bGt(AD0407)Wtsi deletion, indicating that loss of pancreatic SEC23B is sufficient to explain the perinatal-lethality of global SEC23B-deficiency. We examined the relative expression of SEC23B/SEC23A in human and mouse tissues. This ratio is higher in human bone marrow (BM) (7.8) compared to pancreas (5.5), while it was higher in mouse pancreas (12.7) compared to BM (2.6), suggesting that the tissue-specific expression of SEC23A and SEC23B has shifted during evolution. To determine if SEC23A can rescue the phenotype of SEC23B-deficient mice, we genetically engineered the Sec23a cDNA into the endogenous genomic locus of Sec23b (Sec23btm1(Sec23a)Dgi, Sec23a‑b) via recombinase mediated cassette exchange. A heterozygous Sec23a-b intercross generated the expected number of Sec23a-b/Sec23a-b mice. Sec23a-b/Sec23a-b mice exhibited normal survival and development, with normal pancreatic weights and histology and lack of dilated ER by electron microscopy. Western blot analysis confirmed the absence of SEC23B in Sec23a-b/Sec23a-b pancreata, with high levels of SEC23A expression. These data demonstrate that SEC23A and SEC23B have overlapping functions, suggesting that therapies that increase the expression of either paralog in erythroid cells might be effective in CDAII.
P-057: Developing mouse models of open angle glaucoma using large scale ENU mutagenesis
Stephen C Kneeland*1, and Simon W M John1,2
1Howard Hughes Medical Institute and The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA
2Department of Ophthalmology, Tufts University School of Medicine, Boston, MA 02115, USA
Despite recent advances in gene editing technologies, a forward-genetic screen remains a powerful tool to identify human disease causing genes. Mutagenesis screens provide an unbiased approach to create disease models that cannot be anticipated from current knowledge. In a large-scale screen utilizing N-ethyl-N-nitrosourea (ENU), we aimed to discover new mouse models of glaucoma using innovative approaches in phenotype detection combined with genetic and physiologic sensitizers on a modified DBA/2J background. The ongoing screen assesses the impact of dominant mutations on G1 mice, and has identified over 60 mutant lines exhibiting the hallmarks of open angle glaucoma: high intraocular pressure and retinal ganglion cell loss. To maintain the integrity of the phenotype, mutant lines were mapped on a pure background using a panel of incidental mutations identified during exome sequencing of the G1. At present, 8 mutant lines have been mapped to chromosomal regions that contain a range of 2 to 10 mutations identified by exome sequencing. RNA sequencing of ocular drainage tissue was used to determine functional effects of the mutations in disease relevant tissue. In several mutant lines, genes with mutations in mapped regions were differentially expressed compared to age-matched, healthy control ocular tissue, and multiple novel glaucoma genes are being investigated to elucidate disease-inducing mechanism. In addition to the new phenotypic lines generated during the screen, cryopreservation paired with exome sequencing of over 1000 G1 males has created a vast collection of potentially disease relevant point mutations that are available to the community. Our searchable database consists of 145,587 mutations affecting 22,117 genes that can be recovered and used for study in a gene targeting approach. These ENU-induced point mutations are a valuable resource of variant alleles that mimic naturally observed disease causing mutations.
P-058: Establishing new mouse resource, wild-derived heterogeneous stock, which is useful for genetic analysis of tameness and other complex traits
Tsuyoshi Koide*1,2,3, Yuki Matsumoto1,2, Hirofumi Nakaoka4, Jo Nishino5, and Tatsuhiko Go6
1Mouse Genomics Resource Laboratory, National Institute of Genetics
2Department of Genetics, SOKENDAI
3Transdisciplinary Research Integration Center, ROIS
4Division of Human Genetics, National Institute of Genetics
5Graduate School of Medicine, Nagoya University
6College of Agriculture, Ibaraki University
Tameness is a behavioral characteristic with two potential components: reluctance to avoid humans (passive tameness) and motivation to approach humans (active tameness). We quantified these two different components of tameness separately, using three novel behavioral tests: the ‘active tame’, ‘passive tame’, and ‘stay-on-hand’ tests. We subjected genetically diverse mouse strains to these tests, including ten wild strains and seven laboratory strains. They clearly showed differences in the features of tameness in domesticated and wild strains. Most of the domesticated strains showed significantly greater passive tameness than wild strains, whereas there was no significant difference in the level of active tameness between these two groups. These results suggested that domesticated strains were predominantly selected for passive tameness but not for active tameness over the course of their domestication history. To reveal genetic loci contributing to active tameness, we are trying to map loci using newly established genetic mapping resource, wild-derived heterogeneous stocks. The stocks were established from eight wild-derived strains, MSM, HMI, BLG2, PGN2, KJR, CHD, NJL, and BFM/2. In the course of establishment, mice were randomly crossed in each generation, but one group of the stocks was selected for higher level of active tameness even though the other stock was not. After the selective breeding for eight generations, we observed significant difference of active tameness in the two groups. We applied 77K genome-wide SNPs array typing for genomic DNA samples of these groups and the allelic frequencies were compared with the frequencies calculated with computer simulation. The results showed that one locus is significantly associate with active tameness. The results obtained from our study supports that the method used in this study will lead us to understand genetic basis of complex traits in mice.
P-059: Does inter-subspecific and -specific swapping of Prdm9 ZFA affect recombination and reproduction in mice?
Hiromitsu Kono*1,2, Kunihiro Ohta1, and Toshihiko Shiroishi2
1Department of Biophysics and Biochemistry, Graduate school of Science, The University of Tokyo, Japan
2Mammalian Genetics Laboratory, National Institute of Genetics, Mishima, Japan
In mice, homologous recombination is observed at specific genomic sites referred to as hotspots in a strain-dependent manner. Now, it was well established that a histone methyltransferase named PRDM9 makes histone modification at specific genomic regions, thereby serving a site-specific marker of the hotspots. Binding of zinc finger array (ZFA) of PRDM9 to specific genome sequences ensures the site-specificity of recombination. We previously conducted a large-scale population analysis of wild-captured mice, and showed extremely high level of polymorphism of the Prdm9 ZFA and its very rapid evolution (Kono et al. 2014). One of the possible explanations for driving force of its rapid evolution is that each hotspot sequences are changed at times of every recombination repair process, but individual is under pressure for securing a sufficient amount of hotspots to progress meiosis. Therefore, Prdm9 has been subjected to natural selection to accelerate the polymorphism. On the other hand, the Prdm9 polymorphism is known to act for reproduction isolation by preventing gene flow between genetically diverged populations. To reconcile the above paradoxical actions of the Prdm9 polymorphism and to understand its implication on mouse evolution, we intended to test the effect of inter-subspecific and -specific swapping of the Prdm9 ZFA on recombination rate and reproduction in mice. To do so, we generated a Prdm9 knock-in (KI) mouse strain to replace the ZFA of C57BL/6J by that of R209-wm7 haplotype, which originated from Mus musculus molossinus, flanked with HA-tag. In this KI line named Prdm9wm7HA, the last exon of Prdm9 is flanked by mutated lox sequence to facilitate swapping ZFA afterward. With this line, we are trying swapping of ZFA with different origins by pronuclear injection of ZFA-swapping constructs and a cre-recombinase vector. We report phenotype of the Prdm9wm7HA mice, and the current progress of the ZFA-swapping experiment.
P-060: C1 CAGE: Quantifying coding and non-coding RNAs at single-cell and single molecule level
Tsukasa Kouno*, Mickael Mendez, Yi Huang, Erik Arner, Sachi Kato, Imad Abugessaisa, Michael Boettcher, Efthymios Motakis, Takeya Kasukawa, Piero Carninci, Charles Plessy, and Jay W Shin
RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama, Kanagawa, 230-0045 Japan
Single-cell research is a powerful tool to reveal cellular heterogeneity and dynamics. Previously, we used single-cell qRT-PCR technology to demonstrate the temporal dynamics of gene expression during the monocyte differentiation (Kouno T., et al., Genome Biology 2013). This work led us to reconstruct co-expression networks revealing the rewiring of gene-to-gene regulation. However, single-cell qRT-PCR only allows analysis of limited number of genes. Recently we developed C1-CAGE (Single-cell Cap Analysis Gene Expression) method which enable to reveal 5’-end of transcripts at single-cell and single-molecule resolution including non-poly adenylated RNAs, non-codiing RNA with unique molecular identifier. Additionally, we developed a systematic pipeline to process single cells combined with high-throughput imaging and fluorescent quantification to sequencing data processing and visualization. Our pipeline is scalable and can elucidate how cell state switches focusing on non-coding RNA and eRNAs for drug stimulation at single cell resolution.
P-061: Targeting PIK3CA gene by Pyrrole Imidazole polyamide seco-CBI conjugates in cervical cancer
Sakthisri Krishnamurthy*, Takatori Atsushi, Yoda Hiroyuki, Hiraoka Kiriko, Inoue Takahiro, Watanabe Takayoshi, Koshikawa Nobuko, and Nagase Hiroki
Division of Cancer Genetics, Chiba Cancer Center Research Institute
Mutations of the PIK3CA gene have been reported in many human cancers including cervical cancer. Activation of PI3K caused by increased expression and/or mutations leads to a constitutively active enzyme, which results in promoting cell proliferation and reducing apoptosis. We have developed a novel DNA alkylating agent, Pyrrole-Imidazole polyamide-seco-CBI conjugates (termed P3AE5K1) to target E545K mutation, one of the hotspot mutations of PIK3CA. P3AE5K1 was designed to specifically bind to the minor groove of double strand DNA within PIK3CA coding region harbouring E545K mutation. WST assay demonstrated that PIK3CA mutated cells (ME-180 and Ca-ski) displayed higher sensitivity to P3AE5K1 compared to PIK3CA wild-type HeLa cells. The IC50 of P3AE5K1 in ME-180 and Ca-ski cells was substantially lower than that of PI3K inhibitors, BYL719 and LY294002. The expression of PIK3CA mRNA was significantly suppressed by P3AE5K1 treatment in ME-180 and Ca-ski cells. These results strongly suggest that P3AE5K1 is a promising new drug candidate for targeting cervical cancer cells with specific PIK3CA (E545K) mutation. We will present the preliminary study of antitumor activity in mouse model of human cancers.
P-062: Role of a novel asRNA in human white adipose differentiation and metabolism
Hiroko Kushige*, Yas Sasaki, Yutaka Saito, Toutai Mituyama, and Yasuyuki Kida
National Institute of Advanced Industrial Science and Technology, Japan
Obesity is associated with an increased risk of developing insulin resistance and type 2 diabetes. Indeed, elucidating the mechanisms controlling adipogenesis and cellular metabolism will contribute to the fundamental understanding of metabolic disorders. In this study, we identified a novel adipose regulator that controls adipocyte differentiation under the peroxisome proliferator-activated receptor gamma (PPARg) 's organization. Previously, we investigated gene expressions and epigenetic modifications in human adipose derived stem cells (ADSCs) and mature fat cells. Our RNA-seq and Whole Genome Bisulfite (WGBS)-seq combined with published ChIP-seq by PPARg uncovered a direct link between DNA methylations and PPARg bindings on the target promoters. Based on the results, we focused on unknown antisense non-cording RNA (named as asRNA-1) as a one of the epigenetic targets of PPARg. Our RNA-seq data, asRNA-1 is located in a coding gene and has more than 50 kb length. The RNA has no protein coding regions, by qPCR analysis using homologues regions in mouse, it was highly conserved between human and mouse genome. Moreover, fluorescent in situ hybridization in differentiated 3T3-L1 cells reveals that asRNA-1 was existed in the nucleus. During adipogenesis, expression of asRNA-1 was significantly increased soon after adipose induction, while its sense RNA/Protein expression was detected only pre-differentiation state. In addition, knockdown of asRNA-1 expression in ADSCs inhibited adipogenesis with down-regulations of PPARg common target genes that were confirmed by microarray analysis. Although the molecular mechanisms are still unknown, we will discuss the role of the novel antisense RNA in the adipose differentiation and fat remodeling.
P-063: Transcriptome analysis of controlled and therapy-resistant childhood asthma reveals distinct gene expression profiles
Helena Persson1, Andrew T Kwon*2,3, Jordan A Ramilowski2,3, Gilad Silberberg4, Cilla Soederhall1,5, Christina Orsmark‑Pietras1, Bjorn Nordlund5,6,7, Jon R Konradsen5,6,7, Michiel De Hoon2,3, Erik Melen5,8,9, Yoshihide Hayashizaki2,10, Gunilla Hedlin5,6,7, Juha Kere1,5,11,12, and Carsten O Daub1,2,3
1Dept of Biosciences and Nutrition, Karolinska Institutet (KI), Stockholm, Sweden
2Omics Science Center, RIKEN Yokohama Institute, Yokohama, Japan
3Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Japan
4Unit of Computational Medicine, Dept of Medicine, KI
5Centre for Allergy Research, KI
6Astrid Lindgren Childrens Hospital, Karolinska University Hospital, Stockholm, Sweden
7Dept of Womens and Childrens Health, KI
8Institute of Environmental Medicine, KI
9Sachs Childrens Hospital, Stockholm, Sweden
10Preventive Medicine and Diagnosis Innovation Program, RIKEN Research Cluster for Innovation, Wako, Japan
11Folkhalsan Institute of Genetics, Helsinki, Finland
12Research Programs Unit, University of Helsinki, Helsinki, Finland
Children with severe forms of asthma have poor responsiveness to the existing treatment methods, including high doses of inhaled corticosteroids. This not only leads to greater personal suffering by the patients, with early deterioration of their lung functions, but also result in significant consumption of healthcare resources. If no exacerbating factors such as smoking or allergies can be found, these children are diagnosed as having therapy-resistant (or -refractory) asthma. To better understand the inner mechanisms of this disease pathology, we analyze the gene expression levels in peripheral blood leukocytes from various patient groups, including children with therapy-resistant asthma (n = 13), controlled persistent asthma (n = 15) and age-matched healthy controls (n = 9). We generate the transcriptome data using Cap Analysis of Gene Expression (CAGE), and model the underlying regulatory transcription factor (TF) networks based on the results. CAGE sequencing detects the transcription start sites (TSSs) of known and novel messenger RNAs (mRNAs) and non-coding RNAs.
* Published in J Allergy Clin Immunol 2015 Apr 9 (doi: 10.1016/j.jaci.2015.02.026)
P-064: A mouse model of development syndrome associated with kaptin (Kptn) mutations
MO Levitin*1, SJ Sawiak3, Christopher Lelliott1, L Robenson1, EL Baple2, AH Crosby2, Darren W. Logan1, and ME Hurles1
1Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton-Cambridge, CB10 1SA, United Kingdom
2Monogenic Molecular Genetics, University of Exeter Medical School, St. Luke"s Campus, Magdalen Road, Exeter EX1 2LU, UK
3Behavioural and Clinical Neuroscience Institute
4Wolfson Brain Imaging Centre.
Windows of Hope (WOH) is a large population-based medical project, aimed at understanding the underlying genetic causes of inherited conditions, including distinct uncharacterised developmental disability, mainly focusing on Amish, Mennonite, and Hutterite populations. Modelling these mutations in animal models can provide evidence supporting a causal link between the candidate genes and the previously uncharacterised developmental disorders, shed light on their underpinning neuronal circuitry, and potentially inform treatment. One of the recent findings from the WOH project are nine patients with a developmental delay syndrome with distinct morphological characteristics. The patients all have recessive mutations in the kaptin gene and have macrocephaly and intellectual and cognitive disability as their main endophenotypes. We are now modelling this syndrome in mice, using a Kptntm1a(EUCOMM)Wtsi “knockout first” mouse model. We have combined molecular experiments with cognitive phenotyping, to dissect the molecular and neuronal pathology associated with this mutation, as well as study the main behavioural outputs of the syndrome. We have tested the mice in a series of behavioural assays and morphometric analysis and the mutants phenocopy the hyperactivity, cognitive impairment, and macrocephaly phenotypes observed in the human patients. Moreover, our preliminary data suggests that automated touch-screen technology may be used as a standardized phenotyping platform for assessment of cognitive impairment in this mouse model, analogous to approaches used for testing intellectual disability in humans.
P-065: Collateral damage: Identification and characterisation of spontaneous mutations causing deafness from a targeted knockout programme
Morag Lewis*1,2, Francesca Carlisle2, Selina Pearson2, Sohinder Rekhi1, Jing Chen1,2, Matthew Drake1, Cassandra Whelan1, Johanna Pass1,2, Thomas Keane2, David Adams2, Jacqueline White2, Neil Ingham1,2, and Karen Steel1,2
1King"s College London
2Wellcome Trust Sanger Institute
The Sanger Institute’s Mouse Genetics Project is a large-scale mutant mouse production and phenotyping programme. An unexpected side effect has been the discovery of spontaneous mutations which arose during the creation of the knockout mice. More than twenty lines have been identified as carrying a spontaneous mutation affecting hearing, with at least six distinct phenotypes. We have isolated and characterised seven lines representing four of those phenotypes; early severe progressive hearing loss, later onset progressive hearing loss, low frequency progressive hearing loss and complete deafness with vestibular dysfunction. In those seven lines, we have identified multiple mutations, some of which affect hearing and some of which appear to have no effect on the mouse, to the limit of our investigations. The mutations identified so far include new genes not previously associated with deafness and new alleles of genes known to be involved in hearing, two of which show a phenotype much reduced in severity compared to other mutant alleles of the same gene, which could be very useful for further research.
As well as providing new genes and alleles for studying hearing and deafness, this work demonstrates the unintentional consequences of ES cell manipulation, and provides interesting insights into the mutations thus induced. For example, five of the seven mutations identified so far have been single base pair substitutions, and although several of the phenotypes are shared, the causative mutations are almost all different. There is only one which appears to be widespread among the mice of the Mouse Genetics Project, having been identified in several lines displaying the low frequency hearing loss phenotype.
ES cell manipulation is a common experimental approach, and knockout mice are a highly useful resource. This study highlights the potential side effects of ES cell manipulation, which in this case have been an unexpected benefit.
P-066: The long non-coding RNA NEAT1 in cell biology, cancer and paraspeckle function
Ruohan Li*1, Stuart Hodgetts2, Alan Harvey2, and Archa Fox1
1Harry Perkins Institute of Medical Research, University of Western Australia
2School of Anatomy, Physiology, and Human Biology, University of Western Australia
This project focuses on understanding the functional significance of a novel long non-coding RNA named NEAT1, whose main role is thought to be nucleating RNA and proteins in the nucleus to form structures named paraspeckles. Over the past decade, despite increasing knowledge of the molecules associated with NEAT1 and paraspeckles, the significance of them on cellular level is still poorly understood. This PhD project addresses this problem by systematically examining the upstream and downstream pathways for NEAT1, utilizing a number of novel techniques including data mining from the ENCODE project, CRISPR genomic engineering, complex three-way transcriptomic analysis, and pathway analysis. The result of this study revealed a diverse range of pathways regulating NEAT1 expression, as well as a wide range of cellular functions being influenced by NEAT1 and paraspeckles. A novel regulatory relationship between NEAT1 and paraspeckles is also indicated by our data. This study is important for our understanding of paraspeckles and NEAT1, and the molecular mechanisms used by long non-coding RNAs. We hope these results can provide us a good indication on any therapeutic usage of NEAT1 and paraspeckles in the future.
P-067: Alkaline ceramidase 1 (Acer1) is indispensable for mammalian skin homeostasis and thermoregulation
Kifayathullah Liakath‑Ali*1,7, Valerie Vancollie2, Christopher Lelliott2, Anneliese Speak2, David Lafont2, Hayley Protheroe2, Camilla Ingvorsen2, Antonella Galli2, Angela Green2, Diane Gleeson2, Ed Ryder2, Leanne Glover6, Gema Vizcay‑Barrena6, Natasha Karp2, Mark Arends3, Thomas Brenn4, Sarah Spiegel5, David Adams2, Fiona Watt1, and Louise Van Der Weyden2
1Centre for Stem Cells and Regenerative Medicine, Kings College London, UK
2Wellcome Trust Sanger Institute, Cambridge, UK
3Division of Pathology, Edinburgh Cancer Research Centre, University of Edinburgh, Edinburgh, UK
4Department of Pathology, NHS Lothian University Hospitals Trust and The University of Edinburgh, Edinburgh, UK
5Department of Biochemistry and Molecular Biology, Virginia Commonwealth University School of Medicine, Richmond, USA
6Centre for Ultrastructural Imaging, Kings College London, London, UK
7Department of Biochemistry, University of Cambridge, Cambridge, UK
The epidermis is the outer most layer of skin that acts as a barrier to protect the body from the external environment and control water and heat loss. This barrier function is established through the multistage differentiation of keratinocytes in the epidermis, and the presence of bioactive lipids such as ceramides, the levels of which are tightly regulated by a balance of ceramide synthase and ceramidase activities. Here we reveal the essential role of alkaline ceramidase 1 (Acer1) in the skin, as Acer1-deficient (C57BL/6NTac-Acer1tm1a(EUCOMM)Wtsi/Acer1tm1a(EUCOMM)Wtsi aka Acer-/-) mice showed elevated levels of different ceramide species in the skin, aberrant hair shaft cuticle formation and cyclic alopecia. We show that Acer1 is specifically expressed in differentiated interfollicular epidermis, infundibulum and sebaceous glands and consequently Acer-/- mice showed significant alterations in infundibulum and sebaceous gland architecture. Acer-/- skin also showed perturbed hair follicle stem cell compartments. These alterations in the epidermis led to Acer-/- mice showing increased transepidermal water loss and a hypermetabolism phenotype with associated reduction of fat content with age. Our study collectively suggests that Acer1 is indispensible for mammalian skin homeostasis and thermoregulation.
P-068: Skin Megagenetics - Novel skin phenotypes revealed by a genome-wide mouse reverse genetic screen
(See abstract TS-12 in the Trainee Symposium)
P-069: Mapping histon modification marks for activated enhancers genome wide by ChIP of articular cartilage and underlying subchondral bone in human osteoartritic knees
Ye Liu*1, Yanfei Zhang1, Jen‑Chien Chang2, Mitsunori Yahata1, Akiko Minoda2, and Ming Ta Michael Lee1
1Riken Center for Integrative Medical Sciences, Laboratory for International Alliance on Genomic Research, Yokohama, Kanagawa, JAPAN
2Riken Center for Life Science Technologies, Laboratory for Omics Application Technology Research, Yokohama, Kanagawa, JAPAN
Osteoarthritis (OA), a degenerative joint disease, is a large and growing global health burden that represents one of the most common causes of disability in the Western world. Around 27 million US and 7 million Japanese adults are affected by OA. OA has a strong genetic component that is polygenic in nature and GWAS has identified several variants associated with OA. However, the small percentage of heritability explained by these variants suggests epigenetic components may be an important factor in the progression of the disease. However, very little epigenetic landscape of the disease is known. There have been some reports on the genome-wide DNA methylation studies for knee and hip OA. We have developed a unique model system for studying knee OA progression by selecting regions on the knee tissue with different stages of cartilage degradation. Expression and methylation profiling have been generated using this model system. However, changes in the expression could not be all explained by changes in methylation profiling. Our preliminary data showed that majority (69%) of the differentially methylated regions are hypo-methylated and are found at gene enhancers in late stages of OA. The enhancer activation statuses are altered in late disease stage of OA patients leading to differential RNA expression. And the ChIP-seq datasets will be identified potential enhancers that are activated (presence of both H3K27ac and H3K4me1) only in OA regions.
P-070: Gateways to the FANTOM5 promoter level mammalian expression atlas
Marina Lizio*1, Jayson Harshbarger1, Alistair Forrest3, and Hideya Kawaji1,2
3University of Western Australia
The FANTOM5 project investigates transcription initiation activities in more than 1,000 human and mouse primary cells, cell lines and tissues using CAGE. Based on manual curation of sample information and development of an ontology for sample classification, we assemble the resulting data into a centralized data resource. This resource contains web-based tools and data-access points for the research community to search and extract data related to samples, genes, promoter activities, transcription factors and enhancers across the FANTOM5 atlas.
P-071: Diversity Outbred Mice Indicate Idiosyncratic Drug-Induced Liver Injury Potential
LE Lyn‑Cook*1, Daniel M Gatti2, S Luo1, Gary A Churchill2, and AH Harrill1
1University of Arkansas for Medical Sciences, Little Rock, AR
2The Jackson Laboratory, Bar Harbor, ME
Hepatotoxicity is a major cause of attrition during pharmaceutical development. While newer models have offered improvements in predicting incidence of common (high frequency) hepatotoxic events, the ability to detect idiosyncratic (low frequency) drug-induced liver injury (DILI) has remained elusive. While rodent models involving external or internal manipulation have enabled mechanistic study of certain drugs, there remains a need for an animal model that can detect liver liabilities where the mode of action is unknown. A critical issue is that conventional models lack genetic diversity, which in several instances has been shown to play a role in adverse drug reactions. The Diversity Outbred (DO) mice comprise a genetically diverse population with variability that surpasses that of the human population, but in which the minor allele frequency is greater in the DO (12.5% on average). We hypothesized that the DO could provide a model for low frequency DILI in patient populations. In this study, female DO mice (N=50/group) were administered orally one of three drugs associated with rare liver toxicity that are still used clinically (diclofenac, zileuton, isoniazid) or 0.5% methylcellulose vehicle. Mice were dosed (i.g.) daily up to 14 days and blood samples were taken before dosing and at necropsy. As a group, diclofenac and zileuton both caused significant elevations in alanine aminotransferase (ALT) from the pre-dose (baseline) values at necropsy (P<0.05). ALT was not elevated by 0.5% methylcellulose (P>0.05). Fold elevations in ALT ranged from 0.2-8.3 fold for diclofenac and from 0.2-13.6 fold for zileuton, and group mean±SEM for diclofenac and zileuton post-dosing were 82.8±7.3 U/L and 123.8±10.0 U/L, respectively, compared to 32.39±5.4 U/L in the vehicle group. While preliminary, the data provide an important first step to qualifying the DO mouse population as a tool for improved prediction of rare safety liabilities that may call for personalized prescribing strategies.
P-072: Age-related retinal abnormalities and Bloom Syndrome
Erica Macke*1, Hitoshi Higuchi1, Sakae Ikeda1,2, and Akihiro Ikeda1,2
1Department of Medical Genetics, University of Wisconsin-Madison, Madison, WI, 53706, USA
2McPherson Eye Research Institute, University of Wisconsin-Madison, Madison, WI 53706, USA
The normal aging retina goes through a series of distinct pathological changes, including ectopic localization of photoreceptor cell synapses, photoreceptor cell degeneration, and an increase in inflammation. Similar retinal abnormalities have been observed in retinal degenerative diseases such as age-related macular degeneration (AMD), suggesting a link between the molecular mechanisms of normal aging and age-dependent retinal diseases. By utilizing recombinant A/J and C57BL/6 (B6) inbred strains we have genetically mapped two loci affecting the severity of retinal abnormalities in A/J mice, termed rta1 and rta2. Our purpose is to identify genes within the rta1 and rta2 regions that affect the severity of retinal abnormalities.
One candidate for rta1 is the DNA repair enzyme Bloom syndrome, RecQ helicase-like (Blm). A/J mice exhibit a non-synonymous SNP in the 7th exon of Blm. To test Blm as a candidate, we obtained a mutant line, Blmtm3Brd/Blmtm3Brd (Blm mutant) that exhibits a truncation of the protein. Immunohistochemical analysis of 8 month retinal sections from Blm mutant and 129S1/SvImJ (129) controls was performed to examine age related phenotypes. Subcellular localization of BLM was investigated using immunocytochemistry on fibroblast cells isolated from B6 mice.
Blm mutants display ectopic localization of photoreceptor cell synapses at 8 months of age, while wild-type controls do not. This indicates that BLM plays a role in retinal maintenance and is likely the rta1 causative gene. BLM is known to play a role in double strand break repair, and is important for maintaining nuclear DNA integrity. Immunocytochemistry performed using fibroblast cells demonstrates that BLM also localizes to the mitochondria, which was not previously described. Additionally, we have shown that BLM co-localizes with mitochondrial DNA. We hypothesize that BLM may play a role in maintaining or repairing the mitochondrial genome.
P-073: Homeostasis of motifs for transcription factors binding withstanding cancer somatic mutations
Ilya Vorontsov1, Grigory Khimulya1, Darya Nikolaeva3, Elena Lukianova5, Ivan Kulakovskiy1,2, and Vsevolod Makeev*1,2,4
1Vavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
2Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
3Department of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow, Russia
4Department of Medical and Biological Physics, Moscow Institute of Physics and Technology, Moscow Region, Russia
5Human Stem Cells Institute PJSC, Moscow, Russia
We used an extensive collection of transcription factor binding site models to predict transcription factor binding sites (TFBS) overlapping somatic mutations in a large set of cancer samples. By careful statistical assessment taking into account biased cancer mutations signatures we demonstrated that TFBS for many factors had a tendency to overlap or avoid somatic mutations. Moreover, for many TFs somatic mutations were predominantly found at or strongly avoided specific crucial positions in the binding motifs. Similarly, TFBS for some TFs tended to emerge owing to somatic mutations in relevant contexts more or less often than expected. We have demonstrated that TFBS for a number of TFs were protected from disruption by somatic mutations and simultaneously rarely emerged in the mutated regulatory regions. This protection from disruption/suppressed emergence of TFBS resulted in homeostasis of TF binding motifs for a number of TF; the process opposite to the TFBS turnover in the regulatory regions found in inter-species comparison. Surprisingly, we observed comparatively small number of cases of the turnover, or the increased tendency of mutations to disrupt TFBS simultaneously with the increased rate of TFBS emergence for the same TF. Comparison of different type of cancers demonstrated that the list of TF displaying homeostasis was mostly cancer type specific, but several TFs, notably zinc fingers or those belonging to HOX family demonstrated a clear homeostasis in a number cancer samples with different mutation signatures. We believe this provides an important evidence for negative selection pressure acting in cancer cells population and allows one to identify transcription factors substantial for cancer progression.
P-074: Production of a truncated protein from the Gli3 gene with a frameshift mutation, which is introduced by the CRISPR/Cas9 system
Shigeru Makino*, and Yoichi Gondo
RIKEN BioResource Center
Genome-editing technology is a powerful tool to analyze gene functions. Recent works have pointed out that the endonuclease used in the CRISPR/Cas9 system frequently induces off-target mutations as well. In this study, we demonstrate the importance of surveying not only off-target mutations but also the effect of the on-target mutation on translation of the target gene.
GLI transcription factors are critical mediators of Hedgehog signaling in fetal development and cancer. To uncover functions of GLI3 protein, we plan to investigate subcellular localization and activity of exogenous tagged-GLI3 in a Gli3-deficient cell line. Thus, we firstly knocked down the Gli3 gene in mouse embryonic fibroblast cells (3T3) by the CRISPR/Cas9 system.
We established several cell lines with frameshift mutations in the either second or third exon. To analyze expression of GLI3 protein, we performed Western analysis with an anti-GLI3 antibody against the N-terminal fragment of GLI3, which corresponds to the region from exon2 to exon10. As a result, we observed partial expression of GLI3 protein in the “homozygous” mutant cell lines carrying two independently targeted frame-shift mutations. The GLI3 protein in the mutant cells showed a slightly lower molecular weight than that in original 3T3, suggesting that nonsense mutation near the 5’ region in the Gli3 mRNA allowed reinitiation of translation from the second inframe ATG codon. Because of the presence of the DNA binding domain in the presumed truncated GLI3, it might still function even in the “homozygous” mutant cells. Thus, expression of the protein product of interest needs to be carefully analyzed, even if a frame-shift mutation is introduced to the target gene by the CRISPR/Cas9 system.
P-075: Development of Semantic Web/RDF based integrated database of experimental animals
Hiroshi Masuya*1, Terue Takatsuki1, Mikako Saito1, Kai Lents2, Eiki Takayama1, Kazuya Ohshima1, Nobuhiko Tanaka1, Kiyoshi Naruse3, Toyoyuki Takada4, Takashi Kuramoto5, Shigeharu Wakana1, and Norio Kobayashi2
1RIKEN BioResource Center
2Adv Cent for Comput and Commun, RIKEN
3Laboratory of BioResources, Natl Inst Basic Biology
4Mammalian Genetics Laboratory, Natl Inst Genetics
5Graduate School of Medicine, Kyoto Univ
Resource Description Framework (RDF) is a standardized technology recommended by World Wide Web Consortium (W3C) enables cross database data integration in the Web. RDF provides common fundamental procedures for data linking using unique resource identifier (URI), description of knowledge with ontologies and query language can be used across databases (RDF endpoints). For better dissemination and utility of experimental animal-related information, we worked out development of fully RDF-based integrated database for phenotypes of experimental animals developed in Japan including mouse, rat, mammalian cell lines, zebrafish and medaka.
Currently we established RDF-based “meta”-databases for mouse strains in RIKEN BRC (http://metadb.riken.jp/metadb/db/rikenbrc_mouse), RIKEN BRC Cell Bank (http://metadb.riken.jp/metadb/db/rikenbrc_cell), and NBRP Medaka (http://metadb.riken.jp/metadb/db/NBRP_medaka). Phenotype data in these databases are described in common procedure and easily integrated. Furthermore, using importing or generating RDF data from public data resources such as MGI and RGD, data are easily linked with genomic data. All data are open for download for use in other RDF-based (and also non-RDF based) databases. We expect that these efforts will contribute to global sharing and improvement of utilities of bio-resources.
P-076: Combination of selective breeding and genome-wide SNP analysis revealed the genetic loci associated with tame behavior in mice
(See abstract TS-04 in the Trainee Symposium)
P-077: Conservation and evolution of splicing patterns during postnatal development of prefrontal cortex in primates
(See abstract TS-05 in the Trainee Symposium)
P-078: Site-Directed Endonuclease Mutagenesis: Naming Mutations
Monica McAndrews*, and The International Committee on Standardized Genetic Nomenclature for Mice
Mouse Genome Informatics, The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA
The Mouse Genome Informatics (MGI, www.informatics.jax.org) Database serves as the authoritative repository of official symbols and names for mouse genes, alleles, and strains, implementing the rules and guidelines established by the International Committee on Standardized Genetic Nomenclature for Mice. MGI hosts the website, http://www.informatics.jax.org/nomen, where the most current version of the guidelines for mouse and rat nomenclature for genes, alleles and strains can be found.
The application of powerful new site-directed endonuclease mutagenesis systems, such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeat, CRISPR associated protein 9 (CRISPR/Cas9), is resulting in a flood of publications reporting the creation of new mutant alleles and strains of mice.
The current guidelines established by the International Committee to designate these endonuclease-mediated mutant alleles use the following format:
Geneem#Labcode where "Gene" is the gene symbol, “em” designates the allele as endonuclease-mediated, "#" is a serial number from the laboratory of origin, and "Labcode" is the ILAR-registered laboratory code of the investigator or institution where the mutation was produced. For example, Scn8aem1Mm is the first endonuclease-mediated mutation of the Scn8a gene produced in the laboratory of Miriam Meisler.
Not every endonuclease-mediated mutation created requires official nomenclature. Similar to transgenes, many endonuclease-mutations are often made in an experiment, but only one or a few of the mutations are actually studied or maintained. Those that are published, phenotyped, and maintained are the most important for establishing official allele symbols and names. You can reserve nomenclature for your endonuclease-mediated mutations by submitting to MGI at http://www.informatics.jax.org/mgihome/submissions/amsp_submission.cgi.
P-079: EpiFACTORS: a comprehensive database of human epigenetic factors and complexes
Yulia Medvedeva*1,2, Andreas Lennartsson3, Rezvan Ehsani4, Ivan Kulakovskiy2,5, Ilya Vorontsov2, Pouda Panahandeh4, Grigory Khimulya2, Takeya Kasukawa6, FANTOM Consortium6, and Finn Drablos4
1Institute of Personal and Predictive Medicine of Cancer, 08916 Badalona Spain
2Vavilov Institute of General Genetics, Russian Academy of Sciences, 119991, Moscow, Russia
3Department of Biosciences and Nutrition, Karolinska Institutet, 14183 Huddinge, Sweden
4Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, NO-7489 Trondheim, Norway
5Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
6Division of Genomic Technologies (DGT), RIKEN Center for Life Science Technologies, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Kanagawa, Japan
Epigenetics refers to stable and long-term alterations of cellular traits that are not caused by changes in the DNA sequence per se. Rather, covalent modifications of DNA and histones affect gene expression and genome stability via proteins that recognize and act upon such modifications. Many enzymes that catalyze epigenetic modifications or are critical for enzymatic complexes have been discovered, encouraging investigators to study their role in diverse normal and pathological processes. Rapidly growing knowledge in the area has resulted in the need for a resource that compiles, organizes and presents curated information to the researchers in an easily accessible and user friendly form. Here we present EpiFactors, a manually curated database providing information about epigenetic regulators, their complexes, targets and products. EpiFactors contains information on 815 proteins, including 95 histones and protamines. For all genes we include expressions values across a collection of 573 human primary cell samples (200 cell types from up to 3 donors), covering most mammalian cell steady states, 250 different cancer cell lines (representing 154 distinct cancer subtypes) and 152 human post-mortem tissues, obtained by FANTOM5 consortium using CAGE (Cap Analysis of Gene Expression) technique. EpiFactors also contains information on 69 protein complexes that are involved in epigenetic regulation. The resource is practical for a wide range of users, including biologists, pharmacologists and clinicians.
P-080: Cell-cycle classification at the single-cell level with Random Forest
Mickael Mendez*1, Efthymios Motakis1, Michael Bottcher1, Tsukasa Kouno1, Elo Madissoon2, Piero Carninci1, Timo Lassmann3, Jay W Shin1, and Charles Plessy1
1RIKEN Center for Life Sciences Technologies, Division of Genomics Technologies
2Karolinska Institutet, Department of Biosciences and Nutrition, Molecular genetics and biology of complex diseases group
3Present address: Telethon Kids Institute, West Perth Western Australia 6872, Australia
Over the last two decades there have been considerable developments towards the elucidation of the molecular mechanisms that control cell cycle progression. On the one hand, recent sequencing technologies have enabled the study of the transcriptome at the single-cell level. On the other hand Fluorescent ubiquitination-based cell cycle indicator (Fucci) technologies have provided valuable information on the spatiotemporal dynamics governing multicellular cell cycle progression. Both technologies have opened up new perspectives for the analysis of the cell cycle mechanism. In this work we follow a multi-platform approach that combines the merits of both technologies. Fucci unravels the cell cycle transition dynamics by the expression of phase specific protein markers. Its results are subsequently combined with the single cell transcriptome data to obtain a machine learning (Random Forests) predictive model of the cell cycle. Our goal is to provide a predictive model able to identify in which specific cell cycle phase a single cell is. The raw data for this study have been collected through a series of experimental and analytical steps with detailed quality control, image processing and in-house developed methodologies.
P-081: Updates about the Collaborative Cross and Features of the Systems Genetics Core Facility at UNC
Darla R Miller*1,2,3, Sarah E Cates1,2,3, James M Holt4, Chen‑Ping Fu4, Chia‑Yu Kao4, Kenneth F Manly1, Nashiya N Robinson1,2,3, Ginger D Shaw1,2,3, Catherine E Welsh4, Leonard McMillan4, and Fernando Pardo‑Manuel&nbnbsp;de Villena1,2,3
1Department of Genetics, UNC at Chapel Hill, Chapel Hill, NC
2Lineberger Comprehensive Cancer Center, UNC at Chapel Hill, Chapel Hill, NC
3Carolina Center for Genome Sciences, UNC at Chapel Hill, Chapel Hill, NC
4Department of Computer Science, UNC at Chapel Hill, Chapel Hill, NC
The Collaborative Cross is a genetic reference population derived from eight inbred strains by an international consortium of researchers. In 2011and 2012 a series of proof of principle experiments and the initial description of the population were reported. The Systems Genetics Core Facility (SGCF; http://csbio.unc.edu/CCstatus/index.py?run=AvailableLines) at UNC distributes CC lines that have reached a defined minimum level of inbreeding. The SGCF has also reconstructed the founder mosaic of each CC line. We have combined these reconstructions with the whole genome sequence published by the Sanger Institute to generate pseudogenomes from each CC line that incorporate all high quality SNP and indel variants while retaining the extensive annotation of the mouse reference genome (http://csbio.unc.edu/CCstatus/index.py?run=Pseudo).
The SGCF has distributed CC mice to >30 laboratories. CC projects fall into four categories: Strain surveys to determine the genetics of a wide variety of traits; Follow up experiments in smaller sets of CC strains; Development of models of human disease and Identification and genetic and molecular dissection of novel biological phenomena. Manuscripts using the CC have been published recently including a strain survey for susceptibility of Ebola virus infection, a new mouse model for spontaneous colitis, the discovery of a meiotic drive system and a new parent of origin effect on gene expression. We will present summaries on the status of the CC population (number of lines, inbreeding and breeding performance); use of the CC and publications. We will also discuss recent efforts by funders to broaden the use of the CC.
P-082: CRISPR/Cas9-based generation of knockdown mice using long single-stranded DNA
Hiromi Miura*1, Channabasavaiah Gurumurthy2, Sato Masahiro3, and Masato Ohtsuka1
1Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine
2Mouse Genome Engineering Core Facility, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center
3Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University
RNA interference (RNAi)-mediated gene silencing (knockdown) serves as a tool to study gene function. It is used as an alternate tool to complete knockout models and also in situations where hypomorhic expression of the gene is preferred. Typically, knockdown mice are generated using transgenic models by injecting artificial miRNA (amiRNA) expression cassettes containing several kilobase pair of sequence of various elements; a promoter, amiRNA sequence often coupled with a reporter gene and a polyA signal.
In this study, we applied CRISPR/Cas9 system for generation of knockdown mice by targeted insertion of amiRNA sequences. Considering that endogenous miRNAs are often contained within introns of protein-coding genes, we reasoned that targeting the amiRNAs to introns would yield better knockdown models and we tried an intron of Eef2 gene as a candidate target site. For this purpose, long single-stranded DNAs (ssDNAs) containing amiRNA sequences against eGFP gene and Otx2 gene were synthesized by in vitro Transcription and Reverse Transcription (we termed this strategy as ivTRT) of a double-stranded DNA template; a simple molecular biology technique. The amiRNA-eGFP-containing ssDNA was injected into the fertilized eggs derived from the eGFP transgenic mouse, together with Cas9 mRNA and sgRNA targeted to the Eef2 intron. The E14.5 embryos from this experiment were analyzed that showed reduced eGFP fluorescence indicative of amiRNA insertion. The genotyping analyses detected targeted insertion of amiRNA sequences with an efficiency of 83%. Next we tried an amiRNA ssDNA against an endogenous gene (Otx2) using this approach. We observed putative Otx2 knockdown phenotypes in some embryos with an overall insertion efficiency of of 66.7%. We named this new method as (Isi)-CRISPR: ivTRT-ssDNA insertion CRISPR (pronounced Easy-CRISPR). Taken together, Easy-CRISPR offers a simple, fast, and efficient method to generate knockdown models using longer ssDNA.
P-083: The Integrated Transcriptome Analysis of Adipocyte and Osteoblast Differentiation
Yosuke Mizuno*1, Yutaka Nakachi1,2, Yukiko Yatsuka1, Yoshimi Tokuzawa1, and Yasushi Okazaki1,2
1Division of Functional Genomics & Systems Medicine, Research Center for Genomic Medicine, Saitama Medical University
2Division of Translational Research, Research Center for Genomic Medicine, Saitama Medical University
Mesenchymal stem cells can differentiate into various cell types. Particularly the regulatory mechanisms of the balance between differentiation to adipocytes and osteoblasts are closely linked to the pathogenesis of metabolic syndrome and osteoporosis. We have displayed the gene regulatory networks for osteoblastogenesis and adipogenesis of mesenchymal cells, and several genes and microRNAs (miRNAs) were identified as key regulators for those differentiation. Meanwhile, several RNA variants, produced by such as different transcription sites or exon usage, are known to be expressed from a gene locus, and they are functionally discriminated among body tissues and cell types. In addition, it is revealed that various types of noncoding RNAs (ncRNAs) as well as miRNAs are crucially involved in several biological functions. Discovery of specific regulatory RNA variants and ncRNAs could lead to the development of medical therapy with reduced side effects. Therefore, to identify the precise RNA variants and ncRNAs that are involved in regulation of osteoblastogenesis and adipogenesis, we performed detailed RNA expression analysis using differentiating cells. Differential expression of each exon and ncRNAs are comprehensively analyzed using exon array, miRNA array and RNA-sequencing. As a result, we successfully detected specific increase in PPARG2 expression, which is a crucial variant of PPARG gene for adipogenesis using exon array. We also detected various RNA variants of other genes and ncRNAs that are differentially expressed between both types of differentiation. We estimate that specific RNA variants and ncRNAs which regulate the differentiation are involved in those differentially expressed RNA variants, and currently the experiments for their validation are underway.
P-084: Reliable and efficient analysis of non-coding DNA elements in vivo at the mouse Tyr locus using CRISPR-Cas9 mutagenesis
D Seruggia1, A Fernandez1, M Cantero1, P Pelczar2, and L Montoliu*1
1Department of Molecular and Cellular Biology, National Centre for Biotechnology (CNB-CSIC) and CIBERER-ISCIII, Madrid, Spain
2Center for Transgenic Models, University of Basel, Basel, Switzerland
Studies of gene function in mice have been supported during the last decade by a nearly exhaustive collection of mutants, systematically obtained by homologous recombination in murine ES cells. The study of non-coding fraction of the genome, however, does not benefit of the same valuable resources. First, regulatory elements are usually found in the non-coding fraction of the genome, accounting for 98% of our genome and constitute a very large list of DNA sequences to be individually ablated genetically and, second, they are technically hard to target due to the nature of intergenic DNA, often populated by repeated sequences. We have applied CRISPR-Cas9 mutagenesis to efficiently inactivate a non-coding regulatory element found in the 5’ upstream region of the mouse tyrosinase (Tyr) locus, which we had been studying extensively in transgenes, hence in ectopic sites, although it was refractory to homologous recombination in mouse ES cell, to assess its function at the endogenous location. Mice lacking this element show a striking reduction of coat colour pigmentation, confirmed by histological analyses and by melanin measurements, thus highlighting the relevance, in vivo, for the first time, of this DNA regulatory element, evolutionary conserved in the human genome, and hence suggesting its potential pathogenic role in some oculocutaneous albinism type I cases. Melanin loss is also observed in choroidal melanocytes, but not in the retinal pigment epithelium layer. Comparative analyses of a series of distinct deletion alleles generated allowed a fine genetic mapping of the key nucleotides relevant for achieving proper gene expression. Systematic Sanger sequencing of multiple potential off-target sites in all founder mice showed that efficient mutagenesis can be achieved in the absence of undesired, off-target mutations. Together with ENCODE and EPIGENOME datasets, this study illustrates how CRISPR-Cas9 mutagenesis can be used to rapidly crack the code of the non-coding genome.
P-085: Identification of novel chimeric transcripts associated with human-specific retroposed gene copies
Saori Mori*1, Takuji Oshima1, Masaaki Hayashi1, Shun Inagaki1, Ken Tateishi1, and Shunsuke Suzuki1,2
1Shinshu University Faculty of Agriculture
2Shinshu University Institute for Biomedical Sciences
Duplicate gene copies potentially act as genetic resources that evolve novel genomic functions. To investigate contribution of human-specific retrocopies that specifically retrotransposed during the human evolution after the divergence of chimpanzee to produce changes of genomic regulation in the human lineage, we identified and analyzed several human-specific retroposed loci in which novel CpG islands have emerged with the insertion of retrocopies.
DNA methylation analysis of the CpG islands located in 5’ region of the human-specific retrocopies showed various levels of methylation, not all loci were highly methylated. In some loci, highly tissue-specific undermethylation and differential methylation level between normal tissues and cancer cell lines were observed. These data suggest the expression potential of these human-specific retrocopies as mRNA or protein, considering that ORF of the retrocopy is still intact with several amino acid substitutions in many cases.
We next performed retrocopy-specific RT-PCR specifically amplifying the target retrocopies distinguishing from their parent genes that have highly similar sequence to check if the retrocopies were transcribed. Most retrocopies were transcribed in multiple normal tissues and cancer cell lines and cancer-specific expression was observed in some loci. Analysis of transcript structure by RACE experiment revealed that some human-specific retrocopies were transcribed as chimeric transcripts associated with neighboring genes. In this meeting, we would like to discuss relevance of these novel chimeric transcripts in regulation or dysregulation of the human genome based on the function of the associated genes.
P-086: Gene-trap mutagenesis is useful for analysis of long intergenic non-coding RNA genes.
Mai Nakahara*1, Kumiko Yoshinobu2, Haruka Ito1, Riki Furuhata1, Ken‑ichi Yamamura3, Masatake Araki2, and Kimi Araki1
1Division of Developmental Genetics, IRDA, University of Kumamoto
2Division of Bioinformatics, IRDA, University of Kumamoto
3Yamamura Project Laboratory, IRDA, University of Kumamoto
Recent studies imply that many thousands of long non-coding RNA genes are contained in mammalian genomes. Long intergenic non-coding RNAs (lincRNA) have been identified through genome-wide screening for epigenetic marks of histone H3 with trimethylation of lysine4 in promoter region and lysine 36 in gene body (K4-K36 domain) (Guttman et al., Nature 458, 223-227, 2009). Although several lincRNA genes have been disrupted, most of them remained to be analyzed for their in vivo function.
Gene trapping in ES cells is a proven and versatile method to induce insertional mutations and analyze gene function in mice. Previously, we developed an exchangeable gene-trap vector and constructed a gene-trap library [Exchangeable Gene-Trap Clones (EGTC), http://egtc.jp/]. We selected 13 trap clones in which their 5’RACE products mapped within or close to K4-K36 domains and established mouse lines. Since our trap vector carries the beta-geo gene to monitor expression patterns of trapped genes, we first performed X-gal staining of adult and embryo tissues. Eleven out of 13 lines showed wide and/or specific expression patterns, suggesting their functionality in vivo. We next performed heterozygous cross and observed phenotypes of 3 trap lines in which whole cDNA sequences are available from Mouse Genome Informatics database. In two lines, homozygotes were born at the expected Mendelian ratio and have no apparent abnormality. One line showed lower birth rate of homozygotes, suggesting a crucial role in early developmental stages. In 5 lincRNA trap lines, there was no cDNA sequence information in genome browser. We tried 3’RACE using ES RNA and succeeded to identify their transcripts. Thus, gene-trap can efficiently mutate lincRNA genes even if the transcripts were not identified. Since many number of trap clones are available from the International Gene Trap Consortium, they should be a useful tool for analysis of unknown lincRNA genes in vivo.
P-087: Exploring new gene integration sites for gene knock-in by gene-trapping strategy.
Isamu Nanchi*1,2, Yuki Yoshimura1, Kazuomi Nakamura3, Yusaku Masago2, Tetsuya Ohbayashi3, and Tomohiko Okuda2
1Division of Molecular Genetics and Biofunction, Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Japan
2Discovery Research Laboratory for Core Therapeutic Areas, Shionogi Pharmaceutical Research Center, Toyonaka, Osaka, 561-0825, Japan
3Division of Laboratory Animal Science, Research Center for Bioscience and Technology, Tottori University, Yonago, Japan
The knock-in mouse is a powerful tool for biological research, but the stability of expression of an integrated gene strongly depends on where it is integrated in the mouse genome. At present, there are an insufficient number of loci suitable for gene knock-in, such as the Gt(ROSA)26Sor locus. Therefore, in this study, we developed an efficient strategy for identifying genome loci suitable for gene knock-in and characterized the properties of such loci for gene integration. For efficient discovery and characterization, we constructed a new gene-trapping vector that enables monitoring of the expression of both trapped and integrated genes using fluorescence. We successfully obtained fluorescent–positive mouse embryonic stem cell (mESC) clones with the vector. Thorough analysis of the expression of fluorescent proteins in chimera embryos generated with the obtained mESC clones, some of the gene-trapped chimera embryos showed stable and ubiquitous expression of the integrated gene. Furthermore, adult mice derived from one of the gene-trapped mESC clones showed ubiquitous expression of the integrated gene in various tissues without any unusual phenotype. This indicated that the identified locus possesses high potential for foreign gene integration. Our strategy allows for efficient discovery and characterization of mouse genome loci for gene integration.
P-088: Identification of Genetic Susceptibility Loci to Alveoler Bone Loss Affected by Type 2 Diabetes Induced by High-Fat-Food Using Collaborative Cross Mice
Aysar Nashef*1,4,5, Hanifa Abu‑Toamih‑Atamni2,4,5, Ervin Weiss3, Yael Houri‑Haddad1,4,5, and Fuad Iraqi2,4,5
1Department of Prosthodontics,School of Dental mediceine, Haddasa medical center ,Jerusalem Israel
2Department of Clinical Microbiology and Immunology ,Medical school, Tel-Aviv University Tel Aviv Israel
3School of Dental Medicine Tel-Aviv University Tel Aviv Israel
4Have Equal Contributions
5Have Equal Contributions
Background: In previous studies, it was shown that oral microbiome, obesity and Type 2 diabetes (T2D) have an association with alveolar bone loss in periodontal patients.
Aims of study: To explore the power of collaborative cross (CC) mouse population for dissecting the host genetic susceptibility loci underlying alveolar bone loss affected by T2D development induced by High Fat Food (HFD).
Methods: 301 mice from 35 different CC lines were assessed in this study. The mice were divided into two groups; 1) 192 mice were maintained on chow and used as control group; and 2) 109 mice were maintained on - 42% fat HFD. Mice were mainted on the respected diet for three months, and subsequently intraperitoneal glucose tolerance test (IPGTT) was conducted at the terminal point. Following the IPGTT assessment, the maxillary jaws were harvested and bone volume around molar teeth were quantified by micro-CT technique.
Results: The level of alveolar bone varied between the different CC lines both in the control and challenge groups. Some CC lines showed a significant alveolar bone loss following dietary challenge, while others did not (One-Way Anova). Person correlation analysis has shown a significant (P<0.01) negative correlation (-0.267) between IPGTT values and bone volume level among dietary challenge group . Broad sense heritability of the bone loss trait following high fat food diet was 0.49 and genetic coefficient of variation of 0.25.
Conclusion: Our results have shown, for the first time a conclusive association analysis between alveolar bone loss and T2D traits. These results strongly support of conducting the QTL analysis and identify host genes associated with these complex traits, which may provide a novel basis for the development of alternative therapeutic targets to control periodontitis among T2D patients.
P-089: Efficient State of the Art generating of mutant mouse models under full cost accounting conditions in relation to the 3 R's and personnel management
MPI-CBG Dresden Germany
Today, the mouse is a widely used animal model in the scientific community. The Transgenic Core Facility (TCF) provides a centralized resource and state-of-the-art technology in production of Knock-Out (-IN) mice by injection or aggregation of embryonic stem cells into mouse embryos, and transgenic mice by injection of DNA or CRISPR into the cytoplasm or pronuclear of mouse zygotes. Our transgenic service facility generated about 70 mutant mouse lines per year. We work very successful and efficient in full cost accounting. I would like inform about management, 3R's animal welfare, budget, animal and personal resources and the generating of mutant mice in a high health level (SPF). We show that using of fresh or frozen mouse embryos from breeding companies as donor for ES-cell injection is a flexible tool to organise customised projects. All the CRISPR, plasmid or BAC injections into zygotes will done in specific user background to reduce the numbers of recrossing generations.
P-090: Rat models for complex human disease: utilizing phenotypes and genotypes to identify the desired strains
Rajni Nigam*, Stan Laulederkind, G Thomas Hayman, Victoria Petri, Shur‑Jen Wang, Jennifer R Smith, Jeff De Pons, Marek Tutaj, Melinda Dwinell, and Mary Shimoyama
Rat Genome Database, Medical College of Wisconsin, Milwaukee, WI, USA
The rat is an important and versatile model for studying various aspects of complex human diseases in a genetically traceable environment, and great strides have been made in the understanding of hypertension, alcoholism, cancers, diabetes, autoimmune diseases and many other debilitating human conditions. To capitalize on the strength of the rat as a physiological and disease model, we annotate all rat strains and QTLs with ontologies to standardize traits (Vertebrate Trait Ontology - VTO), strains used (Rat Strain Ontology – RS), and the experimental parameters for phenotype identification including ‘what was measured’ (Clinical Measurement Ontology – CMO), ‘how it was measured’ (Measurement Method Ontology - MMO), and ‘the stressors or conditions imposed’ (Experimental Condition Ontology – XCO). These annotations allow users to query and filter results by multiple experimental components using similar data from multiple strains and return those of the most interest to users and the results can be retrieved, downloaded and visualized in our user-friendly tool, PhenoMiner.
Strain Variation tracks for selected strains are available on the GBrowse/JBrowse. SNPs and SNVs related to a particular strain can also be compared in GBrowse/JBrowse and SNPlotyper. Moreover, the details of certain strains that are models for complex human diseases are included in the disease portals.
Additionally, RGD is the hub for the strain and QTL data registration. We urge all rat researchers to submit their strain and QTL data to RGD. Direct data submission to RGD is an extremely simple process and with multiple advantages like ensuring correct official nomenclature and data availability at RGD when paper is published or whenever the submitter desires. These prime directives continue to meet RGD’s goal of providing continuous support for ongoing rat physiological, genetic and genomic research.
All strain and QTL data is readily available from the RGD ftp site for free downloads.
P-091: Single-cell data integration platform
Shuhei Noguchi*, Imad Abugessaisa, Michael Boettcher, Tsukasa Kouno, Timo Lassmann, Piero Carninci, Jay W Shin, Charles Plessy, and Takeya Kasukawa
RIKEN Center for Life Science Technologies(CLST)
Single-cell omics recently emerged as improvements in sequencing, microscopy, mass spectrometry and microfluidic technologies to investigate heterogeneity of large populations of cells in single cellular resolution. Since experiments of single cells use several kinds of equipment, these results let to a rapid increase of complex datasets with single-cell resolution. However, due to a lack of available platforms to easily share and integrate complex single-cell datasets, the accessibility of such datasets can be a barrier to efficient usage.
To address the issue of efficient single-cell data management, we developed a single-cell database integration platform (http://single-cell.clst.riken.jp/). Currently, we provide a dataset of transcriptome analysis with fluorescent, ubiquitination-based cell cycle indicator (Fucci) cells. In addition to their single-cell RNA-Seq data, the database also includes their fluorescence intensities, cDNA concentrations, and images at the capture chambers of Fluidigm’s C1 capture arrays for providing QC and phenotypic information of single-cells. The system is web-based with a graphical user-interface that enables data exploration and retrieval in an intuitive way.
The system provides several functionalities that includes but not limited to data search and retrieval, bulk download and data export. Important feature is the search for gene(s) that are expressed in any of the cell samples. The user is able to search for gene(s) in a list of more the 36,100 genes and their expression values [TPM] in 472 single-cell by ENCODE gene id, gene symbol or gene description.
Moreover, the system also provides accesses to experimental metadata and sequence data of published third party single-cell datasets. The database is designed to be able to store various types of data such as epigenetic and proteomics data, and we will expand our database to store various types of omics and image data. We hope the database can provide useful and easy-to-use resources in the single-cell omics research field.
P-092: rRNA depletion for low quantity RNA-seq involving coding and non-coding RNA
Shohei Noma*1, Chitose Takahashi1, and Masayoshi Itoh1,2
1RIKEN Center for Life Science Technologies(CLST)
2RIKEN Preventive Medicine and Diagnosis Innovation Program(PMI)
RNA-seq is an useful tool for transcriptome analysis. Several kinds of RNA-seq library preparation kits for Illumina platform are available, but are not compatible on the expression profiles among different kits. And some kits can not detect non-polyA RNA because of polyA(+) selection by using oligo dT beads or dT primer based procedure. Here, we optimized TruSeq RNA library prep procedure and combined with NEBNext rRNA depletion kit which can deplete the rRNA sequence selectively. Thereby, we are able to analyze comprehensive transcriptome including non-cording RNAs start with 10 ng total RNA input.
P-093: Partition Heritability of Variants in Gene Regulatory Regions for Complex Traits in Mice
Hiroko Ohmiya*, and Gota Morota
University of Nebraska-Lincoln
Many variants in the genome have been revealed to be associated with different traits by genome-wide association studies (GWASs); however, how those variants contribute to phenotypes is unclear. Especially, since the variants in regulatory regions affect expressions of target genes, it is crucial to assess the contribution to traits in terms of understanding gene regulatory networks and signal pathways.
We applied the variance-component method to imputed genotype data for 20 quantitative traits associated with anxiety, type II diabetes, and asthma in the heterogeneous stock mouse population to partition the heritability explained by input SNPs into nine functional categories. Many of 3,948,884 imputed SNPs were classified into protein coding regions (21.7%) other than transcription factors (TFs) and long non-coding RNAs (lncRNAs). Interestingly, transcription factor binding sites (TFBSs) have more variants (2.8%) than TFs (1.3%) as well as microRNA (miRNA) target regions (1.5%) and miRNAs (0.0%). On average, 11.3% of imputed SNPs explained the phenotypes, and most of those SNPs were categorized as coding genes (60.8%). SNPs in the TFBS and miRNA target regions explain larger proportions of the heritability than TFs and miRNAs, implying that TF and miRNA binding to sites regulates transcriptional pathways rather than TF and miRNA expression levels.
Our result suggests the importance of the regulation by miRNAs and TFs for quantitative traits associated with diseases. Those findings would enable us to reveal gene regulatory pathways for complex traits.
P-094: A Spontaneous and Novel Pax3 Mutant Mouse That Models Waardenburg Syndrome and Neural Tube Defects
Tetsuo Ohnishi*1, Ikuo Miura2, Hisako Ohba1, Yoshimi Iwayama1, Shigeharu Wakana2, and Takeo Yoshikawa1
1Laboratory for Molecular Psychiatry, RIKEN Brain Science Institute, Wako, Saitama, Japan
2Technology and Development Team for Mouse Phenotype Analysis, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan
Genes responsible for reduced pigmentation phenotypes in rodents are associated with human developmental defects, such as Waardenburg syndrome, where patients display congenital deafness along with various abnormalities related to craniofacial development. In this study, we identified a spontaneous mutant mouse line Rwa, which displays variable white spots on mouse bellies and white digits and tail, on a C57BL/6N genetic background. Curly tail and spina bifida were also observed, although at a lower penetrance. These phenotypes were dominantly inherited by offspring. Using a rapid mouse gene mapping system newly developed in our laboratories, we identified a region within Chromosome 1 as a probable locus for the causal mutation. Dense mapping using interval markers narrowed the locus down to a 670-kbp region, containing four genes including Pax3, a gene known to be implicated in the types I and III Waardenburg syndrome. Extensive mutation screening of Pax3 detected an 841-bp deletion, spanning the promoter region and intron 1 of the gene. The defective allele of Pax3, named Pax3Rwa, lacked the first coding exon and co-segregated perfectly with the phenotypes, confirming its causal nature. The genetic background of Rwa mice is almost identical to that of inbred C57BL/6N. These results highlight Pax3Rwa mice as a beneficial tool for analyzing biological processes involving Pax3, in particular the development and migration of neural crest cells and melanocytes.
P-095: GONAD: a novel CRISPR/Cas9 genome editing method that does not require ex vivo handling of fertilized eggs
Masato Ohtsuka*1, Gou Takahashi1,2, Kenta Wada2, Hiromi Miura1, Channabasavaiah Gurumurthy3, and Masahiro Sato4
1Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan
2Department of Bioproduction, Tokyo University of Agriculture, 196 Yasaka, Abashiri, Hokkaido, 099-2493, Japan
3Mouse Genome Engineering Core Facility, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, 68198, USA
4Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8544, Japan.
The traditional methods to generate genetically modified mice involve three critical and complex steps: 1) isolation of fertilized eggs, 2) microinjection of nucleic acids into them, 3) transfer of injected eggs to recipient females, all of which demand sophisticated equipment and highly skilled personnel to perform it. In this study, we developed novel method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) that allowed us to bypass all the three critical steps of animal transgenesis. Unlike the traditional methods, the GONAD system does not require specialized microinjection equipment but it uses an electroporator to deliver nucleic acids (NAs) to the zygotes within the intact mouse oviduct in situ.
To test the system, we first instilled eGFP mRNA into the oviductal lumen of super-ovulated pregnant females (corresponding to 2-cell stage) and then electroporated the oviducts. The embryos flushed and collected next day exhibited eGFP fluorescence, indicating that mRNAs can be delivered directly to 2-cell staged embryos. We next delivered CRISPR/Cas9 genome editing components (e.g., Cas9 mRNA and sgRNAs) through the GONAD method and showed that they too can be effectively delivered to pre-implantation embryos within the intact mouse oviduct and result in the desired genetic modification in the target genes (e.g. eGFP or Hprt). We also confirmed that the GONAD-treated embryos could implant and develop further. These results demonstrate that NAs can be successfully delivered to pre-implantation embryos through in situ electroporation without isolating them out from the female reproductive tract and that targeted gene modification can be achieved when CRISPR/Cas9 tools were delivered with this method. The GONAD method can also be potentially applied to generate genetically engineered models in other species.
P-096: A cancer modifier role for Parathyroid Hormone in mouse skin carcinogenesis
Kazuhiro Okumura*1, Megumi Saito1, Yasuhiro Yoshizawa1, Eriko Isogai1, Ikuo Miura2, Shigeharu Wakana2, Midori Shimanuki3, Hiroshi Shitara3, Choji Taya3, Ryo Kominami4, and Yuichi Wakabayashi1
1Department of Carcinogenesis Research, Division of Experimental Animal Research, Chiba Cancer Center Research Institute, Chiba, Chiba, Japan
2Technology and Development Team for Mouse Phenotype Analysis, Japan Mouse Clinic, Riken Bioresource Center, Tsukuba, Ibaraki, Japan
3Laboratory for Transgenic Technology, the Animal Research Division, Tokyo Metropolitan Institute Medical Science, Setagaya, Tokyo, Japan
4Department of Molecular Genetics, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Niigata, Japan
Using a forward genetics approach to map several loci in a mouse skin cancer model, we previously identified strong genetic loci skin tumor modifier of MSM 1 (Stmm1) on Chromosome 7 with a large number of [(FVB/N x MSM/Ms) x FVB/N] F1 backcross mice. The parathyroid hormone (Pth) gene was found in the vicinity of Stmm1b. Here, we report a genetic polymorphism located in Pth gene, which produces Val/Met pro-PTH variants. Skin carcinogenesis experiments with MSM-BAC transgenic mouse lines FVB/N-Tg(MSMg01-047A16) (FVB-PthMet Tg) showed PthMet alleles conferred resistance to tumors. In addition, we investigated the amount of serum intact-PTH of FVB-PthMet Tg and wild type littermate mice (FVB-PthVal). As a result, the concentration of serum intact-PTH was more than two times higher in FVB-PthMet Tg than in FVB-PthVal mice. Moreover, transfection of NIH-3T3 cells with PthMet-GFP resulted in higher expression of intracellular PTH-GFP in the protein level than in PthVal-GFP-transfected cells. These results suggested that Pth might be a good candidate gene for Stmm1 and the amount of serum PTH could be a marker of skin cancer risk.
P-097: A new framework to analyze a mouse aberrant gait pattern by using a neuro-musculoskeletal model
Satoshi Oota*1, Yosuke Ikegami2, Koh Ayusawa3, Kazunarii Takeich3, Akihiko Murai3, Nobunori Kakusho4, Atushi Yoshiki1, Yuko Okamura‑Oho5, Hideo Yokota5, and Yoshihiko Nakamura2
1RIKEN BioResource Center
2The Department of Mechano-Informatics, University of Tokyo
3Intelligent Systems Research Institute, National Institute of Advanced Industrial Science and Technology
4Japan Neutron Optics Inc.
5Image Processing Research Team, Extreme Photonics Research Group, Center for Advanced Photonics, RIKEN
Traditionally, mouse neuro-behavioral phenotypes are analyzed by a battery of various kinds of tests, each of which depends on a distinctive paradigm. While this approach is useful for conceptually known phenomena, it is generally hard to reach fundamental understanding of mechanisms of novel and subtle neuro-motor functions, which can be often crucial clues to elucidate early-onset neurological diseases. We applied a biomechanics framework to overcome this limitation, by which we can conduct physics-based analysis, rather than context-dependent analysis that requres prior knowledge. To analyze an abnormal gait pattern of a mutant mouse, we developed a detailed yet feasible mouse neuro-musculoskeletal physics model by using high-resolution X-ray CT scanning data. We also performed motion capture on the mutant mouse (hugger: C57BL/6JJcl-hugRbrc) hindlimb with the elaborate neuro-musculoskeletal model, by which detailed motor function data were obtained. Considering that its responsible gene Rorb is exclusively expressed in the central nervous system, the mutant mouse is thought to encode aberrant motor commands that innvervate certain motor units involved in the mutant-specific gait pattern. We elucidated the mechanism of the neuro-motor functions in the biomechanics framework: i.e., we performed both inverse-kinematics and dynamics by using the mouse hindlimb model associated with a simple neural model to compensate unobserved parameters. While the laboratory mouse is an excellent genetic tool (i.e., a genetically modifiable mammal), there was no reliable physics model for mouse neuro-motor function analyses. We show how neuro-biomechanics framework can be applied to laboratory mice, putatively too small to be analyzed at individual level. With the mouse neuro-biomechanics framework, it is potentially possible to create a novel domain of the neuroscience: simulation-based neuroinformatics that covers broad-spectrum phenomena from the neural circuitry-level to the macroscopic motor function-level, which will potentially contribute to totally different disciplines, like neuro-robotics.
P-098: Genetic dissection of Rift Valley fever pathogenesis: Rfvs2 on mouse Chromosome 11 impacts tolerance to early onset hepatitis
Leandro Batista1,2,3, Gregory Jouvion4, Dominique Simon‑Chazottes1,2, Satoko Tokuda1,2, Tania Zaverucha do Valle1,2,5, Marie Flamand6, Xavier Montagutelli1,2, and Jean‑Jacques Panthier*1,2
1Institut Pasteur, Development and Stem Cell Biology, Mouse functional Genetics,75015, Paris, France
2Centre National de la Recherche Scientifique, URA 2578, 75015 Paris, France
3Sorbonne Universites, UPMC Univ Paris 06, IFD, 75006 Paris, France
4Institut Pasteur, Human Histopathology and Animal Models, 75015, Paris, France
5Instituto Oswaldo Cruz, Laboratorio de Imunomodulacao e Protozoologia. FIOCRUZ - RJ, Brasil
6Institut Pasteur, Structural Virology, 75015 Paris, France.
Rift Valley fever (RVF) is an emerging zoonosis, caused by an arbovirus, the Rift Valley fever virus (RVFV). The disease affects mainly livestock, but it can also have a severe impact on human health. In humans, RVF may progress into hepatitis or encephalitis leading to a fatal outcome. No human vaccine or specific treatment is available. Recent genetic studies emphasize the influence of host genetic background on the outcome of the disease, but the identity of genes involved remains unknown. The systemic inoculation of mice with RVFV reproduces major pathological features of severe human disease, notably the rapid onset hepatitis and delayed onset encephalitis. We study the genetic factors determining the susceptibility of MBT inbred strain to RVF. Previous QTL analysis identified a genetic interval on Chromosome 11 (Rvfs2) linked to susceptibility to infection with RVFV. On the BALB/c genetic background, Rvfs2 by itself is sufficient to reduce the survival time of RVFV-infected mice of both sexes. This study investigates the mechanisms by which Rvfs2 confers increased susceptibility to BALB/c mice that are congenic for Rvfs2 (C.MBT‑Rvfs2) after infection with the virulent strain ZH548. Detailed studies showed that Rvfs2 has no effect on clinical parameters and RNA viral loads in the peripheral blood and liver. In addition, C.MBT‑Rvfs2 and BALB/c mice exhibit similar liver damage. However, C.MBT‑Rvfs2 mice die between days 3 and 5 post infection with hepatitis, whereas BALB/c mice recover from this condition. This suggests that Rvfs2 impacts the host tolerance to the acute early-onset hepatitis. An increased hepatocyte proliferation in BALB/c livers accounts for their full recovery from hepatitis and the tolerance phenotype. We propose that Rvfs2 affects the regenerative capacity of the liver tissue in response to RVFV infection.
P-099: Myeloid cell specific interferon hyporesponse contributes to Rift Valley fever susceptibility and virus induced sepsis
Rashida Lathan1,2, Gregory Jouvion3, Marie Flamand4, and Jean‑Jacques Panthier*1,2
1Institut Pasteur, Development and Stem Cell Biology, Mouse functional Genetics, 75015, Paris, France
2Centre National de la Recherche Scientifique, URA 2578, 75015 Paris, France
3Institut Pasteur, Human Histopathology and Animal Models, 75015, Paris, France
4Institut Pasteur, Structural Virology, 75015, Paris, France.
Rift Valley Fever virus (RVFV) an arbovirus causes a wide range of severity and morbidity in humans and in mice. We identified a host genetic basis for RVFV resistance and susceptibility in the resistant BALB/cByJ (BALB) and in highly susceptible, wild-derived MBT/Pas (MBT) mouse models. Since susceptible MBT mice die within only 3 days post RVFV infection with a high viral titer, and since innate immune myeloid response has been shown to be key in limiting early viral replication, we investigated the role of innate cells and their ability to generate interferon and activation responses during the course of infection in both strains. Daily cell suspensions derived from blood, liver, and spleen of mice infected with 102 PFU of virulent ZH548 RVFV, were labeled and FACS analyzed. MBT immune cell profile was a hyperresponse (characterized by high DCs, NKs, and neutrophil peripheral recruitment and high TNF production) at the onset of infection on day 1 p.i. and reverted to a hypo-responsive profile (characterized by low DCs, NKs, and neutrophil peripheral recruitment and low IFN α/β receptor upregulation) by day 3 p.i.. Neutrophils were the most incapacitated MBT cell type, failing to proliferate in sufficient numbers from the bone marrow and failing to upregulate IFN α/β receptor in all organs. However, neutrophil activation status appears vital as an increase in neutrophil number alone by GCSF treatment failed to extend MBT survival. Study also revealed a day 3 p.i. defect in MBT apoptosis, lower spleen and liver cell viability, and blood spikes in pro-inflammatory cytokines which are hallmark symptoms of septicemia. The precise genetic controls of BALB protection and MBT susceptibility require further investigation, however, this study on the mechanism of innate control will hopefully serve as a bridge in a comprehensive understanding of RVFV pathogenesis as controlled by complex genetic traits.
P-100: Epigenetic Studies Reveal the Role of Genetic Background in Corticosteroid Response in Mouse Hepatocytes
Catrina Spruce, Robyn Ball, Wendy Pitman, Narayanan Raghupathy, Anna Tyler, Michael Walker, Gary A Churchill, Kenneth Paigen, Gregory W Carter, and Petko M Petkov*
Center for Genome Dynamics, The Jackson Laboratory, Bar Harbor, ME 04609, USA
Epigenetic changes such as DNA methylation and histone modifications can modulate gene expression at the level of transcription by affecting chromatin access to transcription factors and RNA polymerase. Recent human and mouse genetic studies have shown the existence of many cis-regulating quantitative trait loci, many of them likely due to variation in regulatory regions such as promoters and enhancers. However, the effects of genetic variation on regulatory regions and the extent to which alterations in DNA methylation and local chromatin configuration play a role in them have not been extensively studied. To address this gap in our knowledge, we initiated a study of genetics of epigenetic marks in norm and after drug stimulation, using hepatocytes as a model, in a set of diverse mouse strains – C57BL/6J, A/J, DBA/2J, 129S1/SvImJ, NZO/HiLtJ, NOD/ShiLtJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ. Three months old female mice of each strain were injected three times, 24 hours apart, with 5 mg dexamethasone/kg body weight. Livers were perfused 24 hours after the last injection, and pure hepatocytes were isolated from three dexamethasone-treated and three control animals. We then performed RNA-seq, and ChIP-seq for H3K4me1, H3K4me3, and the glucocorticoid receptor Nr3c1, to assess the impact of genetic variation on gene expression and epigenetic states at promoters and enhancers. We will present the initial analysis of these studies.
P-101: Transcriptome analysis of FACS-sorted single cells with nanoCAGE
Stephane Poulain*, Mickael Mendez, Ophelie Arnaud, Sachi Kato, and Charles Plessy
Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama
NanoCAGE is a powerful method that allows for characterizing cell’s transcriptome and identifying promoter regions by capturing the 5'-ends of capped RNA molecules. The standard nanoCAGE reaction typically requires 50 ng of bulk total RNA as a starting material. Here, we adapted the protocol in order to study the transcriptome of single cells sorted in 96-well plates by flow cytometry. On this poster are shown the results of single cell nanoCAGE experiments designed in order to compare the efficiency of the initial reverse transcription step of the protocol when using either standard template switching oligonucleotides or new template switching oligonucleotides containing a fingerprint sequence. The fingerprint is composed of a unique combination of 8 random bases that is specific of each template switching oligonucleotide molecule, which further enable to count the number of transcripts assigned to each gene expressed in each single cell. We show that, with libraries sequenced at small scale on Illumina's MiSeq platform, an average number of 1,500 genes can be detected per single HeLa cell with the standard template switching oligonucleotides, while around 1,000 genes are detected per cell with the oligonucleotides containing a fingerprint. We found that the sequencing profiles and promoter detection rates are consistent with both kinds of template switching oligonucleotides, and that the number of genes and transcripts detected per cell are highly dependent on the amount of RNA molecules expressed in the type of cell analyzed. We also illustrate how the fingerprint sequence is used in order to count and visualize the different transcripts expressed in each cell. With this methodology, we further expect to identify sub-populations of cells within a sample based on their transcriptome profiles and thereby identify potential biomarkers and therapeutic targets.
P-102: Cognitive Endophenotypes of Modern and Extinct Hominins Associated With NTNG Gene Paralogs
(See abstract TS-11 in the Trainee Symposium)
P-103: A draft network of ligand-receptor mediated multicellular signaling in human
Jordan A Ramilowski*1, Tatyana Goldberg2,3, Jayson Harshbarger1, Edda Kloppman2,4, Marina Lizio1, Venkata Satagopam5, Masayoshi Itoh1,6, Hideya Kawaji1,6, Piero Carninci1, Burkhard Rost2,3,4, and Alistair Forrest1,7
1RIKEN Center for Life Science Technologies (Division of Genomic Technologies), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045 Japan
2Informatics, Department of Bioinformatics and Computational Biology, Technische Universitat Munchen, Boltzmannstr 3, 85748 Garching, Germany
3TUM Graduate School, Center of Doctoral Studies in Informatics and its Applications (CeDoSIA), Boltzmannstr. 11, 85748 Garching, Germany
4New York Consortium on Membrane Protein Structure, New York Structural Biology Center, 89 Convent Avenue, New York, NY 10027, USA
5Luxembourg Centre for Systems Biomedicine, Campus Belval, 7 Avenue des Hauts Fourneaux, L-4362, Belval, Luxembourg
6RIKEN Preventive Medicine and Diagnosis Innovation Program, 2-1 Hirosawa, Wako, Saitama, 351-0198, Japan
7Harry Perkins Institute of Medical Research, QEII Medical Centre and Centre for Medical Research, the University of Western Australia, Nedlands, Western Australia, Australia. Postal address: PO Box 7214, Shenton Park, WA, 6008
Cell-to-cell communication across multiple cell types and tissues strictly governs proper functioning of metazoans and it extensively relies on interactions between secreted ligands and cell-surface receptors. Herein, we present the first large-scale map of cell-to-cell communication between 144 human primary cell types. We reveal that most cells express tens to hundreds of ligands and receptors to create a highly connected signaling network through multiple ligand-receptor paths. We also observe extensive autocrine signaling with approximately two-thirds of partners possibly interacting on the same cell type. We find that plasma membrane and secreted proteins have the highest cell-type-specificity, they are evolutionarily younger than intracellular proteins, and that most receptors had evolved before their ligands. We provide an online tool to interactively query and visualize our networks and demonstrate how this tool can reveal novel cell-to-cell interactions with the prediction that mast cells signal to monoblastic lineages via the CSF1-CSF1R interacting-pair.
This work has just been publsihed in Nat. Commun. (doi:10.1038/ncomms8866)
P-104: Insights into aberrant methylation enhancer regions in hepatocellular carcinoma
Claire Renard‑Guillet*1, Genta Nagae1, Shingo Tsuji1, Shogo Yamamoto1, Kenji Tatsuno1, Hiroki Ueda1, Yutaka Midorikawa1, Yasushi Totoki2, Hidewaki Nakagawa1, Tatsuhiro Shibata2, and Hiroyuki Aburatani1
1Genome Science Division, Research Center for Advanced Science and Technology, The University of Tokyo, Tokyo, Japan
2Division of Cancer Genomics, National Cancer Center Research Institute, Tokyo, Japan
Hepatocellular carcinoma (HCC) is a cancer with a high prevalence worldwide associated to a poor prognosis, and it has then been extensively studied over the past years.
It is now well known that, as in other cancers, tumor cells conceal deregulation of expression of key genes, accompanied by aberrant mutations or changes in epigenetic regulation. Amongst epigenetic mechanisms, DNA methylation alteration has appeared to be an early event of cancer, and has been mainly described as hyper-methylation of (CpG island) gene promoters, associated with repressed transcription of the associated gene.
However, DNA methylation alteration at other regulatory regions, such as distal or proximal enhancer, may also be of great interest since they play key roles in gene expression pattern within a given tissue. Here, using a dataset of methylation data (450K Infinium Illumina arrays) from over 400 patients suffering from HCC, we focus specifically on the methylation changes within enhancer regions. We annotate probes as “enhancer” based on recently defined epi-genomic annotations, and subsequently characterize them with additional information such as CpG island and gene positions, methylation level in liver and chromatin states in other cell lines.
We also compare methylation changes within these probe sets to changes in cancer-related mutations. We focus mainly on the CTNNB1 hotspot mutation, carried by over 1/3 of patients, and affecting WNT-signaling pathway by subsequent alteration of AXIN2 expression. We describe the sites and regions specifically demethylated in those patients, and the changes in expression for the associated genes, as well as the pathways affected.
P-105: Serotonin receptor HTR3A in the development of sacral autonomic and sensory ganglia
K Elaine Ritter*1,2, Dennis P Buehler1, Hsiao‑Huei Wu3, and E Michelle Southard‑Smith1
1Division of Genetic Medicine, Department of Medicine, Vanderbilt University, Nashville TN, USA
2Neuroscience Graduate Program, Vanderbilt Brain Institute, Vanderbilt University, Nashville TN, USA
3Department of Pediatrics, Keck School of Medicine, University of Southern California, Los Angeles CA, USA
Decades of research demonstrate the importance of serotonin (5-HT) signaling in the fetal development and postnatal maturation of various neuronal populations. While most of these studies focused on the brain and enteric nervous system of the gut, very few have inspected the role of 5-HT in development of other peripheral nervous system components. In a gene expression analysis conducted in our lab, we noted expression of the 5-hydroxytryptamine (serotonin) receptor 3A (HTR3A) in fetal pelvic ganglia that provide autonomic innervation to the bladder and urethra. This finding, along with reports of HTR3A in adult urinary function and visceral pain, led us to pursue the role of this receptor in the development of bladder autonomic and sensory innervation. We aim to define the temporal, spatial, and cell-type specific expression patterns of the HTR3A receptor throughout lower urinary tract development as a basis for interrogating gene function in this system. By employing the Tg(Htr3a-EGFP)DH30Gsat transgenic reporter mouse line and immunohistochemistry, we have characterized expression of the HTR3A receptor in a variety of both autonomic and sensory neuronal subtypes within the pelvic and dorsal root ganglia. Expression of HTR3A begins early in development of bladder innervation at 12 days post coitus and becomes more restricted in fetal and postnatal stages. HTR3A co-localizes with both sympathetic and parasympathetic markers in the pelvic ganglia throughout fetal development, as well as several markers of nociceptive neurons in the dorsal root ganglia and bladder urothelium. Ongoing experiments are focused on determining the effects of HTR3A loss on urinary function and neural development. Our studies are relevant for elucidating underlying neurologic causes of urinary incontinence and chronic pelvic pain syndromes.
P-106: Cell-cell-communication in cancer
(See abstract TS-15 in the Trainee Symposium)
P-107: Computer Simulation of Scoliosis-like Phenotypes: Skeletal Analysis of Unbalanced Vertebral Bone Growth in ENU-induced KTA41 Mutant Mice
Nobuho Sagawa*1, Satoshi Ota2, Helmut Fuchs3, Sibylle Sabrautzki3, Martin Hrabe de Angelis3, and Koichiro Abe1
1Tokai University School of Medicine
2Bioresource Center, RIKEN
3Helmholtz Zentrum Muenchen,Institute of Experimental Genetics
In humans, the spine, together with vertebral discs, provides structural strength and flexibility to the body. Thus, abnormal positioning of vertebrae renders physical activity defective. Scoliosis is clinically defined as lateral curvature and convolution of the spine and is not a life-threatening disease. In severe cases, however, locomotive and respiratory functions are impaired. Although scoliosis is classified as either congenital or adolescent, most of patients are judged around the age of puberty by radiograph. The molecular mechanisms to cause vertebral anomaly in scoliosis are almost unknown, and lack of animal models for scoliosis hampers development of new medical treatment. In this study, to establish a novel animal model for scoliosis, we focused on a novel ENU-induced recessive mutant mouse strain, KTA41, derived from the Munich mutagenesis screen. Because adult homozygous KTA41 mice show mild kinky tails and thoracic vertebral fusion, we performed detailed skeletal analyses in various developmental stages. In the newborn stage, hyperossification was detected in vertebrae and limbs of KTA41 mice. Further, KTA41 mice showed abnormal synostosis and curvature of spine around 7 weeks of age. Thus we attempt to simulate how curvature of the spine during vertebral growth using micro CT image data from 7 to 9 weeks old KTA41 homozygous mice. We set 20 landmarks on the L4 vertebra, and angles of each three landmark combination were compared between wild type and KTA41 mice. Interestingly, the angle among spinous process and posterior vertebral body landmarks was increased according to the age. Thus, hyperossification and abnormal bone growth in KTA41 mice resulted in curvature of spine. Our results indicate that KTA41 mice could be a mouse model for unbalanced vertebral growth of adolescent scoliosis. Various types of animal models are useful to develop the early stage diagnosis and prevention medicine of scoliosis.
P-108: Genetic analysis of Stmm3 locus controlling tumor progression in a Japanese wild-derived mouse strain, MSM/Ms
Megumi Saito*1, Kazuhiro Okumura1, Eriko Isogai1, Shigeharu Wakana2, Ryo Kominami3, and Yuichi Wakabayashi1
1Department of Carcinogenesis Research, Division of Experimental Animal Research, Chiba Cancer Center Research Institute
2Technology and Development Team for Mouse Phenotype Analysis: Japan Mouse Clinic, Riken Bioresource Center
3Department of Molecular Genetics, Graduate School of Medical and Dental Sciences, Niigata University
In this study, we showed that MSM/Ms mice exhibit dominant resistance when crossed with susceptible FVB/N mice and subjected to the two-stage skin carcinogenesis protocol using DMBA/ TPA. A series of F1 backcross mice were generated by crossing Trp53+/+ or Trp53tm1Sia/+ (FVB/N × MSM/Ms) F1 males with FVB/N female mice. In recent study, we successfully mapped highly significant QTLs for late stage papillomas (>6 mm) and identified Stmm3 (skin tumor modifier of MSM 3) locus on Chromosome 4. To narrow down Stmm3 region, we generated three congenic lines (Trp53+/+ or Trp53tm1Sia/+) and subjected these mice to the two-stage skin carcinogenesis protocol using DMBA/TPA. As a result, we narrowed down Stmm3 region to 88-93 Mb (5 Mb; 2 cM) on Chromosome 4. Furthermore, Trp53tm1Sia/+ congenic mice developed a significantly increased number of late stage papillomas compared with Trp53+/+ congenic mice. These data suggested that Stmm3 locus, which maps near the Cdkn2a (p19Arf), was entirely Trp53-dependent. A previous study demonstrated that the size of each papilloma was larger in Cdkn2a-deficient mice, compared with wild-type mice using the DMBA/TPA skin carcinogenesis experiment. These results suggest that Cdkn2a may play a major role in the transition to large papillomas. And there are non-synonymous polymorphisms in Cdkn2a between FVB/N and MSM/Ms mice. Therefore, to determine the relative biological activities of the MSM/Ms and FVB/N p19Arf proteins, we compared their protein stability and cell cycle arrest. In these experiments, MSMp19Arf stability was higher than FVBp19Arf by TPA treatment. And MSMp19Arf was more effective in inducing G1 arrest. These date suggested that Cdkn2a might be expected to be a candidate gene controlling tumor progression in MSM/Ms.
P-109: Nuclease-Mediated Conditional Allele in a Gene Refractory to Gene Targeting in ES Cells
Elizabeth Hughes1, Wanda Filipiak1, Michael Zeidler1, Galina Gavrilina1, and Thomas Saunders*1,2
1University of Michigan Medical School, Transgenic Animal Model Core
2University of Michigan Medical School, Department of Internal Medicine, Division of Molecular Medicine and Genetics, 1150 W. Medical Center Dr., Ann Arbor, MI 48109
Muscle phosphofructokinase is an essential enzyme for glucose homeostasis. Patients with phosphofructokinase deficiency present with Tarui disease and exercise intolerance in the clinic. In order to generate a mouse model for this disease, exon 3 in the mouse Pfkm gene was selected for gene targeting in mouse embryonic stem (ES) cells. Mutation of exon 3 is expected to disrupt both isoforms of Pfkm by nonsense-mediated decay of mRNA encoding a premature termination codon. A gene-targeting vector designed to replace exon 3 with a sequence flanked by loxP sites was introduced into mouse ES cells. Genetic screening of 480 drug-resistant ES cell clones showed that none of them had undergone homologous recombination with the targeting vector. Subsequent efforts to generate a mutant mouse model turned to the direct manipulation of the mouse genome in fertilized eggs by the microinjection of nucleases targeted to exon 3. Four transcription activator-like effector nucleases (TALENs), one zinc finger nuclease (ZFN), and one CRISPR- associated Cas9 nuclease were designed to target Pfkm exon 3 and prepared for microinjection. Nucleases were obtained from vendors or prepared in-house from publicly available resources. Microinjection of plasmids expressing TALENs did not produce mutant mice although this method has been reported to be effective for other genes. The microinjection of mRNA coding for TALENs also failed to generate mouse mutants, independent of the origin of the TALENs reagents. The use of ZFN mRNA for microinjection produced multiple mouse mutants. Co-injection of Cas9 mRNA and guide RNA produced numerous mice that are undergoing genetic analysis. Active Cas9 reagents were then used to generate animals with a floxed exon 3 using a novel single chromosome break. The phenotype of homozygous mutant animals will be discussed. Factors affecting the success of nuclease-mediated gene targeting include nuclease activity and toxicity of microinjected reagents.
P-110: A natural antisense RNA to the protein phosphatase 1 regulatory subunit 12A (PPP1R12A) functions as a SINEUP in human cells
Aleks Schein*1,2, Silvia Zucchelli3, Sakari Kauppinen2, Stefano Gustincich3, and Piero Carninci1
1Division of Genomic Technologies, RIKEN Center for Life Science Technologies, Yokohama, Japan
2Center for RNA Medicine, Department of Clinical Medicine, Aalborg University Campus Copenhagen, Copenhagen, Denmark
3Scuola Internazionale Superiore di Studi Avanzati, Area of Neuroscience, Trieste, Italy
Recent data imply that up to 80% of mammalian genome is transcribed and that at least 20% of the expressed genomic loci produce RNAs from both DNA strands, giving rise to a broad range of sense/antisense (S/AS) RNA interactions. In fact, more than 50% of all mammalian RNAs may overlap an opposite-strand transcript in a divergent, convergent or a full-length configuration. Natural antisense transcripts (NATs) have been shown to regulate gene expression by affecting transcription and mRNA stability. Recently, a NAT to mouse ubiquitin carboxyl-terminal esterase L1 (Uchl1), was reported to increase UCHL1 protein synthesis, representing a new functional class of lncRNAs, named SINEUPs. However, no SINEUP homologs have been yet described in other species.
Here, we show that a NAT to the protein phosphatase 1 regulatory subunit 12A (PPP1R12A), designated as PPP1R12A-AS1, functions as a SINEUP in human cells, increasing PPP1R12A protein synthesis at the post-transcriptional level. The ~2kb PPP1R12A-AS1 overlaps with the 5’ UTR and first coding exon of the PPP1R12A mRNA, thereby creating an overlap of 236-337 nucleotides, depending on the mRNA variant and PPP1R12A-AS1 transcription start site. The SINEUP activity depends on the presence of the aforementioned sense-antisense interaction and a free right Alu monomer repeat element (FRAM) at the 3’ end of PPP1R12A-AS1. Furthermore, we show that a synthetic SINEUP ncRNA, containing FRAM repeat also increases translation of recombinant PPP1R12A and up-regulates green fluorescent protein (GFP) more effectively compared to a previously described SINEUP, containing the mouse SINEB2 element.
Our results demonstrate for the first time that a NAT can up-regulate protein translation in human cells, suggesting that SINEUP ncRNAs may be widespread and present in many mammalian species. The short FRAM sequence provides a basis for uncovering the molecular mechanism of SINEUP-mediated up-regulation of protein synthesis, and an attractive opportunity for many biotechnological applications.
P-111: Water-soluble fullerene [C60] derivative causes myogenic differentiation of human tissue-derived mesenchymal stem cells.
Vasilina A. Sergeeva*, Svetlana V. Kostyuk, Elisaveta S. Ershova, Larisa P. Kamenava, and Natalya N. Veiko
Federal State Budgetary Institution, Research Centre For Medical Genetics, Moscow, Russian Federation
Recently it was shown that [C60] fullerenes and their derivatives possess anti-viral activity, antioxidant properties, moreover, they are under studies as potential anticancer medicine and nanovectors for the drug delivery across biological barriers. But the affect of such derivatives on human cells is to be further investigated. We assessed the affect of 3 water-soluble [C60] derivatives (polyphosphonated, polycarboxylated and polypiperazined) on human adipose-derived mesenchymal stem cells. Level of mRNA of myogenic, adipogenic and osteogenic differentiation genes was estimated using RT-PCR. It has been shown that in the absence of fullerene derivatives level of adipogenic differentiation genes PPARG, eEPPb, FABP4 rises. After 14 days of exposure to polyphosphonated [C60] fullerene derivative in concentration 195 µM level of gene expression of Myod, Myog, Myf5 increases in 7.90, 1.60 and 5.0 times, respectively. Furthermore, level of corresponding proteins also gets higher (shown by means of flow cytometry). Fluorescent microscopy revealed increase of myogenin, myf5 and myf6, whereas alkaline phosphatase and oil red staining barely change compared to control. No increase in levels of adipogenic or osteogenic differentiation genes was statistically distinguishable. Other fullerene derivatives did not lead to increase in levels of myogenic differentiation genes under the same conditions.
P-112: Visualization and Analysis of Cell and Tissue Omics data with ZENBU genome browser system
Jessica Severin*1, Marina Lizio1, Jayson Harshbarger1, Hideya Kawaji1,2, Michiel De Hoon1, Carsten O Daub1,4, Yoshihide Hayashizaki2, Piero Carninci1, Nicolas Bertin3, and Alistair Forrest5
1RIKEN Center for Life Science Technologies(CLST)(DGT)
2RIKEN Preventive Medicine and Diagnosis Innovation Program
3Cancer Science Institute Singapore
4Department of Biosciences and Nutrition and Science for Life Laboratory, Karolinska Institutet
5Harry Perkins Institute of Medical Research
Recent genome-wide compendium studies, such as FANTOM5, Encode, Epigenome Roadmap, TCGA, and more recently single-cell studies are changing the nature of biological science due to their unprecedented breadth and scale. With this new mode of research requires novel tools to visualize, analyze, and manage such data.
To address this need, we developed the ZENBU system (Severin et al., /Nature Biotechnology/, 2014). ZENBU extends the genome browser concept by integrating advanced, on-demand data processing and analysis with visualization optimized for the comparison across 1000s of experimental samples. A key feature of ZENBU is the data security and collaboration system, allowing users to upload 100s-1000s of experimental data sets and share them with a selected group of collaborators. ZENBU thus enables anyone from individual researchers to large consortia to use the system with their unpublished data while maintaining confidentiality.
ZENBU can easily work with both transcriptomics and epigenomics data sets providing raw data visualization, data clustering, gene expression analysis and comparative analysis as some examples. Recently, we have expanded ZENBU’s statistical toolbox to enable sample enrichment and grouping analysis.
Future areas of development include expanding the statistical analysis capabilities of ZENBU in addition to enhancing the collaboration tools and interfaces.
ZENBU is currently installed at several mirrors around the world and has been averaging over 200,000 hits per month.
P-113: Live Imaging Analysis of Mouse Development during DVE migration
Go Shioi*1, Hideharu Hoshino2, Kazuki Nakao1,3, Toshihiko Fujimori1,4, Yasuhide Furuta1, and Shinichi Aizawa2
1Genetic Engineering Team, RIKEN CLST, Japan
2Laboratory for Vertebrate Body Plan, RIKEN CDB, Japan
3Laboratory of Animal Resources, CDBIM, University of Tokyo, Japan
4Division of Embryology, National Institute for Basic Biology (NIBB), Japan
In mouse embryos the anterior-posterior axis is initially formed along the proximal-distal axis at E5.25, with the emergence of a unique population of cells that express a series of head organizer markers in the distal visceral endoderm (DVE). Subsequently, prior to gastrulation, the DVE cells migrate to the proximal future anterior unidirectionally, generating the anterior visceral endoderm (AVE). We examined the behavior of each embryonic VE cell during this axis rotation by labeling the cell nucleus with histone H2B-tagged fluorescent protein and found that: 1) daughter and grand-daughter cells divide at the same time and are closely positioned to each other, 2) DVE cells divide during migration, and 3) the cells located posterior to the DVE do not migrate but stay in this region. DVE movement is apparently not the result of the overall movement of the VE cells or of the forward pushing by the posterior cells; rather the DVE cells migrate actively toward the future anterior region. We also examined the shape of VE cells during DVE migration. Analysis of the behaviors of each VE cell will be discussed in the context of the overall cell movement.
P-114: The Genetic Regulation of Serpine1, Plasminogen Activator Inhibitor-1, in the LEWES/EiJ Mouse Strain
Amy E Siebert*1, Stephanie Verbeek1, Marisa A Brake1, Alexander J Johnston1, Lena Mishack1, Fernando Pardo‑Manuel de Villena3,4,5, Andrew P Morgan3,4,5, Amy Banes‑Berceli1, Guojing Zhu2, Jill M Johnsen6,7, and Randal J Westrick1
1Department of Biological Sciences,Oakland University, Rochester, Michigan, USA
2Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, USA
3Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
4Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
5Carolina Center for Genome Sciences, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
6Bloodworks NW, Seattle, Washington, USA
7Department of Internal Medicine, University of Washington, Seattle, Washington, USA
Cardiovascular disease is one of the world’s leading causes of mortality. A final event in cardiovascular disease is the formation of intravascular blood clots (thrombi) consisting of platelets and fibrin. As the fibrinolytic system is a potent endogenous regulator of intravascular thrombi, decreased fibrinolytic activity greatly contributes to cardiovascular disease progression. Serpine1 (plasminogen activator inhibitor 1, PAI-1) is an essential fibrinolysis inhibitor produced primarily by platelets. PAI-1 is consistently elevated in plasma from cardiovascular patients but mechanisms driving this increase are unknown. In order to elucidate genetic regulators of Serpine1 expression, we surveyed the following mouse strains for platelet PAI-1 antigen: RIIIS/J, C57BL/6J, WSB/EiJ, SWR/J, KK/HiJ, CD, PERC/EiJ, PERA/EiJ, RF/J, SF/CamEiJ, CF-1, and LEWES/EiJ. We found that while the inbred strains RIIIS/J, C57BL/6J, WSB/EiJ, SWR/J, KK/HiJ each had <0.2 ± 0.2 pg/μg total protein, the LEWES/EiJ strain had a striking increase in platelet PAI-1 antigen with 1.8 ± 0.8 pg/μg total protein (q = 0.004). LEWES/EiJ x C57BL/6J crossbreeding experiments produced F1 mice with average platelet PAI-1 levels of 0.8 ± 0.1 pg/μg total protein (q = 0.01), suggesting a semidominant Serpine1 regulatory effect. To identify regulatory genes, we produced 48 F2 mice, measured platelet PAI-1 levels and genotyped 12 with <0.4 pg/μg total protein and 12 with >1.0 pg/μg total protein using the second generation Mouse Universal Genotyping Array (MegaMUGA). QTL mapping revealed several candidate regions including a significant 14.4 megabase interval (128.2-142.6 megabases) on Chromosome 5 (LOD score = 4.81). The Serpine1 gene resides within this candidate interval, strongly suggesting that a Serpine1 cis-eQTL is responsible for expression differences between LEWES/EiJ and C57BL/6J. Additional studies are underway to identify this element. The identification of elements controlling platelet Serpine1 expression will provide insight into platelet-specific gene expression and identification of novel therapeutic targets for modulating thrombotic disease.
P-115: Using RGD Genome and Phenotype Resources to Find and Assess a Model for Human Disease
Jennifer R Smith*, Jeff De Pons, Stan Laulederkind, G Thomas Hayman, Rajni Nigam, Victoria Petri, Shur‑Jen Wang, Marek Tutaj, Melinda Dwinell, and Mary Shimoyama
Rat Genome Database, Medical College of Wisconsin, Milwaukee, WI, USA
The Rat Genome Database (RGD, http://rgd.mcw.edu), the premier online resource for rat genetic, genomic and phenotypic data, offers a large body of cross-species functional, phenotype and disease data and multiple innovative software tools which facilitate the search for appropriate models for human diseases. As an example workflow, we used the OLGA Object List Generator and Analysis tool to generate the list of rat genes annotated to the RGD Pathway Ontology (PW) term for the type 2 diabetes mellitus (T2DM) pathway. To ascertain whether any of these genes are known to have single nucleotide variants (SNVs) or indels that might adversely affect function of the associated protein product in any sequenced rat strain, the gene list was used as input for the Variant Visualizer tool. Selecting all available strains and limiting the results to variants predicted to be "probably damaging" by Polyphen, a list of gene-strain-variant triples was generated. Among these is a C to T transition in the Slc2a2 gene in each of three substrains of WKY (WKY/Gcrc, WKY/N and WKY/NCrl). The variant causes a change from threonine to methionine in one of the cytoplasmic regions of the protein. Using the RGD PhenoMiner tool to assess diabetes-related phenotypes for all of the strains with probably damaging SNVs in T2DM pathway genes, we found that the WKY/N strain shows significantly elevated blood glucose levels after glucose injection. In addition, RGD's imported ClinVar dataset includes a variant in the human SLC2A2 gene associated with T2DM. Taken together, these results suggest that WKY/N could be an appropriate model for T2DM. As a confirmation, both the literature and the information in the RGD Phenotypes and Models portal list the WKY strain as a model for glucose intolerance, hyperglycemia and hyperinsulinemia.
P-116: A Mutation in Greb1l Results in Multiple Organogenesis Defects
Olivia Sommers*1,2, Jabier Gallego1,2, and David Beier1,2
1Seattle Children"s Research Institute
2University of Washington
In a forward genetic screen for recessive mutations affecting organogenesis in mice treated with the chemical mutagen N-ethylnitrosourea, a line with a wide variety of developmental defects was identified. A positional cloning analysis revealed that this line carried two missense mutations in a highly conserved region of the growth regulation by estrogen in breast cancer-like gene (Greb1l). As little is known about this gene the aim of the current research is to understand its function. Characterization of Greb1l E18.5 mutant embryos by micro-CT and histology revealed a spectrum of phenotypes with variable penetrance. These included increased or decreased digit number, many skeletal defects including a decrease in the number of costal ribs and malformation of the sternum, agenesis or underdevelopment of one or both kidneys, cleft palate often associated with cleft lip, heart defects, brain malformations, and a neural tube closure defect (exencephaly). Notably, a malformation of the eye including disorganization of neural retina and pigmented epithelium with occasional lack of lens was highly penetrant. For this reason, and because the process of eye development has been well characterized, we are focusing our analysis on this defect to understand the role of Greb1l in organogenesis. We are analyzing the morphology of the eye defect by histology at different stages in development. Additionally, in situ hybridizations are being used to assess the level and pattern of expression of genes responsible for regulation of eye formation including Pax6, Otx2, and Mitf. We are also generating knockouts in zebrafish by CRISPR/Cas9, and knock-downs in cells using shRNA. Given the multiple developmental anomalies, we hypothesize this poorly annotated gene plays a modulating role in many different developmental signaling pathways.
P-117: CARDIOGENE : deciphering the genetic mechanisms of cardiovascular diseases
Tania Sorg*1, Ghina Bou‑About1, Marie‑France Champy1, Winfried Maerz2, Heiko Runz3, Mohammed Selloum1, Francois Spitz4, Amandine Velt1, and Yann Herault1
1Institut Clinique de la Souris-ICS-MCI, PHENOMIN, CNRS UMR7104, INSERM U964, Universite de Strasbourg, 1 rue Laurent Fries BP 10142 Parc d Innovation 67404 Illkirch, France
2University of Heidelberg, Mannheim
3University Clinic of Heidelberg
Cardiovascular diseases (CVD) and their complications are a leading cause of death. Our main objective is to gain a better understanding of the genetic mechanisms that influence the incidence of cardiovascular diseases in humans, particularly in order to better understand the factors contributing to their development and to identify markers allowing to improve the prevention and appropriate treatments of CVDs.
Our approach combines a detailed genetic analysis of a large cohort of patients (~ 3800) from Baden-Wuerttemberg and Rhineland-Palatinate (Germany) followed longitudinally for many clinical and metabolic parameters (LURIC project) to identify genetic variants associated to CVDs. To study more precisely the effects of these variants, we generated several mouse models carrying the genomic changes found in these loci and characterized their effects on multiple molecular, metabolic and physiological parameters.
One of the generated mouse models carries a duplication of a target genomic region and was characterized for its physiological and metabolic traits upon challenge with a diet enriched in cholesterol. This model showed an important decrease in fat mass and in body weight, but with a higher food intake, and a significant decrease in blood lipids. In addition, no changes could be observed in aortic cholesterol levels, while atherosclerotic plaques of various sizes were induced in the aorta, the carotid, and the abdominal and renal arteries.
Other mouse models have been generated and are currently under investigation and will be presented, one of them carrying either a duplication or a deletion of another genomic region, showing conversely an obesity or a leanness phenotype.
All these results taken together, associated to molecular analysis and combined to the genetic variants observed in the human population will allow to better understand the pathophysiological mechanisms of CVDs and to identify markers of risk factors to improve the prevention and treatment of these diseases.
P-118: A combinatorial approach for targeted therapy of triple negative breast cancers: interference peptides against transcription factors, chemotherapy and nanoparticles
Anabel Sorolla*1, Diwei Ho2, Edina Wang1, Callum Ormonde2, Cameron Evans2, K. Swaminathan Iyer2, and Pilar Blancafort1
1Cancer Epigenetics, Harry Perkins Institute of Medical Research, The University of Western Australia, Crawley, Western Australia, Australia
2Bio-nano, School of Chemistry & Biochemistry, The University of Western Australia, Crawley, Western Australia, Australia
Triple negative breast cancers have the worst prognosis among all breast cancers. These cancers are very difficult to treat and highly resistant to standard chemotherapy in the mestastatic setting. Thus, there is an urgent need to develop novel and more specific therapies to treat this lethal disease. Triple negative breast cancers show selective overepression of the transcription factor engrailed homobox 1 (EN1) which promotes chemotherapy resistance. To overcome this situation, here we propose a novel therapeutic strategy that combines interference peptides against EN1 (EN1-iPeps) and Docetaxel (DTX), both integrated into a poly(glycidyl methacrilate) polymer nanoparticle (PGMA) decorated with poly(acrylic acid) (PAA) to create an electrostatically favourable surface that allows for the binding of EN1-iPeps. The nanoparticles (NPs) will serve for the delivery of the interference peptides.
The EN1-iPeps that we engineered are truncated forms of the native transcription factor EN1 and include a highly conserved hexapeptide which is essential for their binding with DNA and the binding partners. The EN1-iPeps act as a dominant negative, blocking and preventing protein-protein and protein-DNA interactions which leads to the disruption of EN1 function. These iPeps are highly selective as they induce apoptotic cell death in the triple negative breast cancer cell lines T11 and SUM149 while having a null efect in the nonneoplastic human mammary epithelial cell line MCF10A. The assembling of the EN1-iPeps on the surface of the (NPs) clearly facilitates their internalisation. Moreover, EN1-iPeps sensitise T11 cells to the commonly used chemotherapeutic drug Docetaxel and the incorporation of these two agents in the nanoparticle significantly enhances the antitumoral effects in vitro. Interestingly, NPs loaded with Docetaxel and coated with EN1-iPeps increase mice survival in 45% and slowed down tumour growth in an advanced model of the disease.
In conclusion, our results demonstrate that targeting transcription factors using interference peptides constitutes a promising therapy for triple negative breast cancers.
P-119: Identification of novel hypomorphic and null mutations in Nrf1 and Klf1 derived from a genetic screen for modifiers of alpha-globin transgene variegation
Anabel Sorolla*1, Michael R Tallack2, Harald M Oey3, Sarah K Harten4, Lucia Daxinger5, Andrew C Perkins2, and Emma Whitelaw3
1Cancer Epigenetics, Harry Perkins institute of Medical Research, The University of Western Australia, Crawley, Western Australia, Australia
2Mater-UQ Research Institute, The University of Queensland, Translational Research Institute, Woolloongabba, Queensland, Australia
3La Trobe Institute for Molecular Science, Department of Genetics, La Trobe University, Bundoora, Victoria, Australia
4Epigenetics Laboratory, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia
5Center for Human and Clinical Genetics, Leiden University Medical Center, Leiden, The Netherlands
Position-effect variegation of transgene expression is sensitive to chromatin state. We previously reported a forward genetic screen in mice carrying a variegated alpha-globin GFP transgene (Tg(HBA1-Gfp)1Ew) to find novel genes encoding epigenetic regulators. We named the phenovariant strains "Mommes" for modifiers of murine metastable epialleles.
Here we report positional cloning of mutations in three FVB/NJ-Momme strains. One Momme (dominant 44; MommeD44) is an enhancer of variegation while the other two (MommeD11 and MommeD45) are suppressors of variegation, as seen by the positive or the negative shift on the percentage of erythrocytes expressing the GFP transgene. We have identified the chromosomal location of the mutations by SNP-Chip and the putative causative point mutations in these Mommes by exome sequencing. MommeD44 carries a point mutation in the nuclear respiratory factor 1 (Nrf1) which consists of a substitution of a leucine for a proline at the position 71, just before the predicted NRF1 DNA binding domain. The other two strains harbour point mutations in the erythroid transcription factor, Klf1. One (MommeD11) generates a stop codon in the zinc finger domain and the other (MommeD45) generates an amino acid transversion (H350R) within a conserved linker between zinc fingers two and three. The mutation in Nrf1MommeD44 is homozygous lethal and associated with an increase in DNA methylation. The mutation in Klf1MommeD11 generates a homozygous null phenotype. Finally, homozygous Klf1MommeD45 mice have chronic microcytic anaemia which models the phenotype in a recently described family.
This is the first evidence of Nrf1 as being a player in the epigenetic control of gene expression. Also, that the linkers between the zinc fingers of transcription factors have a function beyond that of a simple spacer.
P-120: GUDMAP – GenitoUrinary Development Molecular Anatomy Project, an open resource.
E Michelle Southard‑Smith*1, Frances Wong2, Jamie Davies2, Richard Baldock3, and GUDMAP Consortium4
1Vanderbilt University Medical Center, USA
2University of Edinburgh, UK
3Institute of Molecular Medicine, UK
4GUDMAP Project, International
The GenitoUrinary Development Molecular Anatomy Project (GUDMAP) is an open access online resource developed by an international consortium of laboratories working to provide the scientific and medical community with gene expression data, transgenic mice and tools to facilitate research and teaching in genitourinary (GU) development. The goal is to identify novel cell types and expression domains in the kidney, ureter, lower urinary tract and reproductive tract. The GUDMAP database includes data from large-scale in-situ hybridization screens, 3D Optical Projection Tomography (OPT) data, microarray gene expression data and sequencing data of the developing mouse GU system. Expression data are annotated using a high-resolution ontology specific to the developing murine GU system. More specifically, gene expression is annotated by describing both the presence and strength of expression in different sub-compartments. The database is searchable based on gene name, function or by anatomical structure. An advanced Boolean query allows users to further refine their search according to the stage, organ, or expression pattern. Initially, GUDMAP focused on the murine GU system. More recently, GUDMAP has extended its focus to two new projects: Nociceptive GUDMAP (nGUDMAP) and Human GUDMAP (hGUDMAP). nGUDMAP focuses on nociceptors and associated cell types in pain processing of the murine urinary tract and pelvic region. hGUDMAP extends the gene expression data to include Human studies in bladder, urethra and kidney. GUDMAP data are curated and freely accessible at www.gudmap.org
P-121: Identification of Active Signaling pathways in Neural Crest Derived Progenitors that Innervate the Lower Urinary Tract
Dennis P Buehler, Sara Ireland, and E Michelle Southard‑Smith*
Vanderbilt University Medical Center
The neural crest (NC) gives rise to the peripheral nervous system including the innervation of the lower urogenital tract (LUT) which is essential for proper function of the bladder and genitalia. However, remarkably little is known about the molecular mechanisms that control development of sacral NC-derived cells that give rise to pelvic innervation. We utilized transgenic mice that express a Histone2BVenus (H2BVenus) reporter driven from Sox10 regulatory regions, to visualize migration of sacral NC and capture these progenitors for analysis of gene expression in the developing bladder and pelvic ganglia. We imaged fetal tissues fromTg(Sox10-HIST2H2BE/YFP*)1Sout transgenic mice, hereafter referred to as Sox10-H2BVenus, from 12-16 days post coitus (dpc) to determine migration routes and timing of NC cell entry into the LUT. NC progenitors labeled by H2BVenus expression form the anlagen of the pelvic ganglia by 12 dpc, enter the bladder by 13dpc, and reach the bladder dome by 15dpc. Using robust flow sorting methods, we isolated H2BVenus+ progenitors from bladder and pelvic ganglia of transgenic embryos to obtain stage and sub-domain specific transcriptional profiles. Gene expression profiles of sacral NC-derived progenitors were compared as a means to identify pathways that regulate development of pelvic innervation. We specifically focused on gene expression changes within the pelvic ganglia from 13-15dpc because our prior work has shown that neurogenesis within pelvic ganglia is ongoing at 15dpc. This analysis identified significant up-regulation of serotonin receptors that are known to play roles in neurogenesis and neural connectivity in the brain. In vitro explants of pelvic ganglia cultured in the presence of serotonin receptor specific agonists and antagonists further supported a functional role for serotonin receptors in development of pelvic innervation. Our identification of signaling pathways that function in pelvic innervation provides a basis for understanding normal development and pathological processes that contribute to bladder dysfunction.
P-122: WNT inhibition facilitates the establishment of stable and homogeneous EpiSCs
Michihiko Sugimoto*1,2, Masayo Kondo2, Yumiko Koga2, Rieko Ikeda2, Susana M. Chuva de Sousa Lopes3, and Kuniya Abe2
1Division of Developmental Genetics, Institute of Resource Development and Analysis, Kumamoto University
2BioResource Center, RIKEN
3Department of Anatomy and Embryology, Leiden University Medical Center
Epiblast stem cells (EpiSCs) are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos, representing a useful model for studying “primed” pluripotent states. Here we devised a simple and robust technique to derive high quality EpiSCs using an inhibitor of WNT secretion. Using this method, EpiSC lines were readily established with high efficiency; whole embryonic portions could be used without separation of epiblast from visceral endoderm (VE). Expression analyses revealed that these EpiSCs maintained a homogeneous, undifferentiated status, yet showed high potential for differentiation both in vitro and in teratomas. Unlike EpiSCs derived by the original protocol, new EpiSC lines required continuous treatment with the WNT inhibitor, suggesting some intrinsic differences from the existing EpiSCs. The homogeneous properties of this new version of EpiSCs should facilitate studies on the establishment and maintenance of a “primed” pluripotent state, and on directed differentiation from the primed state.
P-123: Analysis of long non-coding RNAs functions in the human genome
Supat Thongjuea*, Jordan Ramilowski, Jay W Shin, Masayoshi Itoh, Takeya Kasukawa, Naoto Kondo, Harukazu Suzuki, Michiel De Hoon, and Piero Carninci
RIKEN Center for Life Science Technologies (Division of Genomic Technologies) (CLST (DGT)), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
In recent years, transcriptome analysis derived from various cell types generated by a series of FANTOM, ENCODE, and other large-scale projects has revealed a large number of long non-coding RNAs (lncRNAs). However, only a small fraction of these RNAs have been functionally characterized, whereas the majority of them are largely unknown. To characterize biological functions of lncRNAs, we have developed the large-scale perturbation system and workflow to perturb a large number of lncRNAs in human cell types (e.g. dermal fibroblasts). This project is part of the pilot phase of FANTOM6, a worldwide collaborative project hosted by RIKEN aiming to identify all functional elements in mammalian genomes. We use CAGE technology to profile the genome-wide transcriptome of each perturbed lncRNA. Large-scale bioinformatics analyses identify a set of differentially expressed protein- and non-coding RNAs per each targeted lncRNA. Gene ontology, pathway, and gene set enrichment analyses have been then used to computationally identify the significance in associated biological functions. Gene network analysis has been performed to discover the pattern of lncRNAs networks. Finally, a subset of the most interesting lncRNAs from bioinformatics analyses will be selected to functionally characterize and to validate the findings using other complementary technologies.
P-124: Redefining the transcriptional regulatory dynamics of classically and alternatively activated macrophages by deepCAGE transcriptomics
Sugata Roy1,2, Sebastian Schmeier3, Erik Arner1,2, Tanvir Alam4, Suraj P Parihar5,6, Mumin Ozturk5,6, Ousman Tamgue5,6, Hideya Kawaji1,2,7, Michiel De Hoon1,2, Masayoshi Itoh1,2,7, Timo Lassmann1,2, Piero Carninci1,2, Yoshihide Hayashizaki2,7, Alistair Forrest1,2, Vladimir B Bajic4, Reto Guler5,6, Frank Brombacher5,6, and Harukazu Suzuki*1,2
1Division of Genomic Technologies, RIKEN Center for Life Science Technologies (CLST), Yokohama, Japan
2RIKEN Omics Science Center (OSC), Yokohama, Japan
3Massey University, Auckland, New Zealand
4King Abdullah University of Science and Technology (KAUST), Thuwal, Saudi Arabia
5International Centre for Genetic Engineering and Biotechnology (ICGEB), Cape Town, South Africa
6University of Cape Town, Cape Town,, South Africa
7RIKEN Preventive Medicine and Diagnosis Innovation Program (PMI), Yokohama, Japan
Classically and alternatively activated macrophages (M1 and M2, respectively) play distinct and important roles for microbiocidal activity, regulation of inflammation and tissue homeostasis. Despite this, their transcriptional regulatory dynamics are poorly understood. Using promoter-level expression profiling by non-biased deepCAGE technology, we have studied the transcriptional dynamics of IFNG- (M1) or IL4/IL13-activated (M2) macrophages. Surprisingly, transcription factor (TF) binding motif activity analysis revealed that four out of five top motifs, NFKB1_REL_RELA, IRF1,2, IRF7 and TBP, are commonly activated but have distinct activity dynamics in M1 and M2 activation. We observe matching changes in the expression profiles of the corresponding TFs and show that only a restricted set of TFs change expression. There is an overall drastic and transient up-regulation in M1 and a weaker and more sustainable up-regulation in M2. Novel TFs, such as Thap6, Maff, (M1) and Hivep1, Nfil3, Prdm1, (M2) among others, were suggested to be involved in the activation processes. Further, we find 52 (M1) and 67 (M2) genes are drastically differentially expressed including several long non-coding RNAs (lncRNAs). In conclusion, the finding of novel motifs, TFs and protein-coding and lncRNA genes is an important step forward to fully understand the transcriptional machinery of macrophage activation. We will also report transcriptional regulatory dynamics of macrophages in Mycobacterium tuberculosis infection.
P-125: Genetic mapping of metabolic traits using the Diversity Outbred mouse population: Are we there yet?
Karen L Svenson*, Daniel M Gatti, and Gary A Churchill
The Jackson Laboratory, Bar Harbor, Maine, USA
Metabolic disorders such as obesity, diabetes and lipidopathies emerge from a complex and variable interplay of genetics and environment. We used the heterogeneous Diversity Outbred mouse population to model diversity in metabolic phenotypes and identify genetic loci driving them.
Using cohorts of 150-200 mice, in alignment with production of 3 generations per year, we analyzed a total of 850 DO animals from generations 4-11 for more than 100 phenotypic traits. Female and male mice entered the study at wean age and followed a series of high-throughput noninvasive clinical assessments through 26 weeks of age. Half of the mice consumed standard chow and half consumed a high fat, high sucrose diet throughout the study.
A considerable challenge to using outbred mice with greater than two founder strains for genetically mapping metabolic traits has been in developing appropriate tools for correlation of genotype to phenotype. As our experiment progressed, we developed and refined a genetic mapping algorithm to manage 36 possible genotypes in the DO towards accurately identifying genetic loci responsible for trait outcomes. Power analyses are now supported by empirical data to inform study sample size. Our most recent analysis identified 42 unique quantitative trait loci with genome-wide significance of p ≤ 0.05. These include 22 for hematologic traits, 14 for metabolic traits (plasma lipids, glucose, liver enzymes, urinalysis, body fat content, body weight), 4 for bone density, 1 for immune function and 1 for heart rate. Plausible candidate genes underlying select metabolic traits in this study will be presented.
P-126: Genome resequencing of wild mice-derived inbred strains originated from four subspecies of Mus musculus
Toyoyuki Takada*1, Kentaro Fukuta2, Hideki Noguchi2, Atsushi Toyoda2, Asao Fujiyama2, and Toshihiko Shiroishi1
1Mammalian Genetics Laboratory,
2Comparative Genomics Laboratory, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan
Commonly used classical inbred mouse strains have mosaic genomes originated from different subspecies, namely west European subspecies Mus musculus domesticus, east European subspecies M. m. musculus and southeastern Asian subspecies M. m. castaneus. Japanese subspecies M. m. molossinus is established through hybridization between M. m. musculus and M. m. castaneus. Our recent genome-resequencing project of M. m. molossinus-derived two inbred strains, MSM/Ms and JF1/Ms, revealed over ten millions of SNPs due to large genetic distance between M. m. molossinus and M. m. domesticus (Takada et al. 2013).
To expand our knowledge of genome polymorphisms across the every subspecies of M. musculus, we have conducted more comprehensive genome-resequencing of wild mice-derived strains originated from the four subspecies. We expected that information of the genome polymorphisms could be applied not only to study the natural history of this species but also to perform functional genomics based on inter-subspecific genome diversity. Because founders of these strains had been exposed to their own specific environments for a long time, the strains should still retain many of unique quantitative traits. For instance, they have obtained characteristic energy metabolic systems that are adapted to different nutritional conditions. The strains used in this project are musculus-derived BLG2/Ms, NJL/Ms, CHD/Ms, SWN/Ms and KJR/Ms, domesticus-derived PGN2/Ms and BFM/Ms, and castaneus-derived HMI/Ms, all of which were established as inbred strains at NIG by Prof. Moriwaki since 1970's. On the HiSeq platform, we resequenced genomes of these strains with 27 to 41x coverage, and that of MSM/Ms with additional 79x coverage. In this talk, we introduce current progress in this project.
P-127: The recessive congenital cataract in nat mice caused by mislocalization of the MIP
Gou Takahashi*1, Sayaka Hasegawa1, Yoshiaki Kikkawa2, and Kenta Wada1,2
1Graduate School of Bioindustry, Tokyo University of Agriculture
2Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science
The major intrinsic protein of lens fiber (MIP) is a member of the aquaporin superfamily, and has important roles in the formation of normal lens structure and in the maintenance of lens transparency, and has been reported that its mutations lead to autosomal dominant cataract in humans and mice. Recently, we isolated a recessive cataract mutation, Nodai cataract (nat), from SJL/J colony, and identified that its responsible mutation is a missense mutation which leads to glycine-to-arginine substitution on Mip gene (c.631G>A, p.211G>R). In order to verify recessive inheritance of nat mutation, phenotype of aged heterozygous nat individuals was observed, because of all MIP/Mip mutation in humans and mice leads to dominant cataract. At 9 month after birth (9M), although the eyes of homozygous nat showed obvious opacity, that of heterozygous nat was remained normal as well as wild-type. Histological analysis also exhibited that the lens fiber cells of heterozygous nat were not disorganized, and that were similar histology to that of wild-type. Next, we performed immunohistochemical analysis using anti-MIP and anti-CTNNB1 antibody to estimate effect of p.211G>R for MIP localization on lens cells. Although MIP and CTNNB1 showed co-localization on plasma membrane of lens fiber cells in wild-type, the expression of p.211R mutant MIP was detected to nuclear membrane in homozygous nat, despite CTNNB1 expressed was similar to that of wild-type. On the other hand, MIP and CTNNB expression of heterozygous nat were identical to that of wild-type. Therefore, cataract of nat mouse is caused by mislocalization of mutant MIP protein on nuclear membrane resulting from its missense mutation, and the normal lens phenotype of heterozygous nat might be attributed to formation of complex between wild-type and mutant MIP proteins. This novel missense mutation found in nat is the first case in mouse recessive cataract by Mip mutation.
P-128: Development of HTS system to optimize SINEUPs, antisense long-noncoding RNAs that increase translation of target mRNAs
(See abstract TS-14 in the Trainee Symposium)
P-129: Site-directed DNA demethylation by transcription factor and Manipulation of DNA demethylation
Takahiro Suzuki*1,2, Yuri Nakanishi1, Erina Furuhata1, Shiori Maeda1, Mami Kishima1, Hajime Nishimura1, Yoshihide Hayashizaki3, and Harukazu Suzuki1
1RIKEN Center for Life Science Technologies (CLST), Division of Genomic Technologies
2Graduate School of Medical Life Science, Yokohama City University
3RIKEN Preventive Medical and Diagnosis Innovation Program (PMI)
DNA demethylation is the pivotal step in gene activation. DNA methylation at the cytosine of CpG dinucleotides in promoter regions is an important epigenetic mark of gene silencing that prevents TFs from binding to gene regulatory regions. Therefore, to activate genes, the regulatory regions must be initially demethylated. Although the mechanism of DNA methylation is well characterized, that of DNA demethylation is still incompletely understood. Here, we present our activities which are to understand biological impact of DNA demethylation. 1) How site-specificity of DNA demethylation is regulated. Certain TFs such as NANOG and EBF1 were recently proposed to determine site-specificity of DNA demethylation. However the number of reported TF is still limited. Therefore, we are developing an analysis pipeline to systematically identify the TFs which can determine site-specificity of DNA demethylation based on genome-wide methylome data. By using this pipeline, I identified novel candidates of the determinant TFs in several biological process. 2) Establishment of site-directed DNA demethylation method. DNA methylation/demethylation is involved in many biological phenomena such as differentiation, reprogramming or tumor genesis. Therefore, site-directed epigenetic manipulation methods are highly expected to contribute to understand link between DNA methylation of specific cite and biological process. Here, we introduce our recent progress of this project.
P-130: Targeting the mutation site of oncogenic KRAS by KR12 identifies synthetic lethal interactions in colon cancer cells
Atsushi Takatori*1, Kiriko Hiraoka1,2, Sakthisri Krishnamurthy1,2, Hiroyuki Yoda1,2, Takayoshi Watanabe1, Nobuko Koshikawa1, and Hiroki Nagase1,2
1Division of Cancer Genetics, Chiba Cancer Center Research Institute
2Graduate School of Medical and Pharmaceutical Sciences, Chiba University
Developing novel tools to detect specific sites of DNA would help to address unmet medical needs, especially in cancer therapy. Recently, we have reported that a synthetic alkylating agent, pyrrole-imidazole polyamide indole-seco-CBI conjugate (KR12), selectively recognized genomic sequence of oncogenic mutated KRAS and induced significant tumor growth inhibition. However, potential off-target binding of KR12 in genomic DNA remains to be elucidated. In the present study, microarray profiles of KR12-treated KRAS mutant cells revealed down-regulation of the genes that are predicted to have putative KR12 target sequences. Among them, we focused on previously reported genes selectively required for cell viability of KRAS mutant cells. To examine the direct involvement of the genes including BCL2 and VPRBP in tumor survival, siRNA knockdown experiments were performed and identified the cell growth inhibition effects in KRAS mutant cells and synergistic/additive cell growth inhibition with an alkylating agent. These data suggest that KR12 exerts its anti-tumor effects through targeting not only mutant KRAS suppression but also synthetic lethal interactions with mutant KRAS oncogene in colon cancer cells.
P-131: Exploring of novel mouse models for human disease with comprehensive mouse phenotyping data
Nobuhiko Tanaka*, and Hiroshi Masuya
Technology and Development Unit for Knowledge Base of Mouse Phenotype, BRC, RIKEN, Japan
The mouse (Mus musculus) has become the principal animal model for investigating and understanding the mechanisms underlying human disease. The International Mouse Phenotyping Consortium (IMPC) was launched to provide the scientific community with a complete collection of mammalian gene functional information by conducting high-throughput phenotyping of about 20,000 knockout (KO) mice lines by 2020. More than 1,800 gene-to-phenotype associations were analyzed and can be downloaded for free from IMPC web portal so far (10/Aug/2015). Here, we aimed to develop a workflow to discover novel knowledge on biological phenomena and to explore novel disease models by analyzing comprehensive mouse phenotyping data from the IMPC.
To ensure comparability between measured parameters, we first standardized the phenotypic data, namely, defined a novel index of “degree of mutation” as Mutation Index (MI). The MI is defined as the negative common logarithm of a p value (－log10(p value)) from a significance test between a mutant group and an internal control group. Also, to clearly show relationships both between measured parameters and between mutant strains, we used methods for visualization such as “heatmap with dendrogram” and “network diagram”. The following two kinds of information were mapped on the visualization of relationships mentioned above. One is disease information of each mutant strain downloaded from the ftp site of Mouse Genome Informatics (MGI). Another information is functional related gene groups from functional annotation sources such as GO and KEGG pathway for about the 1,800 genes with a gene functional annotation tool (DAVID). As a result, we demonstrated both the larger trends and the details that exist within the comprehensive phenotyping results. Future, based on this workflow, we plan to develop a supporting tool for searching novel disease models as well as generating hypotheses for further progress in user’s own study.
P-132: Higher expression of Adcyap1 gene is associated with altered behavioral and prolonged physiological responses to stress in wild-derived MSM mice
(See abstract TS-09 in the Trainee Symposium)
P-133: Valproic acid-induced vertebral malformations correlate with global expression changes in developmental regulators
Sho Tanimoto*1, Masami Yaguchi1, Joji Mochida2, and Koichiro Abe1
1Department of Molecular Life Science, Tokai University School of Medicine
2Department of Orthopaedic Surgery, Tokai University School of Medicine
During embryogenesis, there are certain sensitive periods to exogenous agents, which differ from one organ to another. Thus, environmental factors influence fetal organogenesis, but the mechanisms including interacting genetic factors are almost unknown. To elucidate gene expression changes by environmental cues during embryogenesis, we employed valproic acid (VPA) induced-vertebral malformations in mice as a model. VPA is a antiepileptic drug and also acts as a teratogen, which induces fetal vertebral malformations, when administered to pregnant females. It also acts as an inhibitor of histone deacetylase. We administered VPA (500 mg/kg) intraperitoneally to ICR mice once at the 9th day of pregnancy, and the embryos were used to analyze skeletal morphology and gene expression afterwards. In skeletal analysis, all E18.5 embryos showed vertebral abnormalities and some of them showed anterior homeotic transformation. We employed microarray, real-time quantitative PCR (qPCR), and whole mount in situ hybridization (WISH) for gene expression analyses using E10.5 embryos. First, we analyzed expression patterns of somite markers and NOTCH target genes by real-time qPCR. Interestingly, Pax1 expression levels were increased, and Tbx6 and Mesp2 expression levels were decreased. Further, we found gene expression changes in many of Hox genes by microarray analysis. Interestingly, expression of the genes positioned in telomeric side of Hox clusters, which involved in thoracic, lumbar and sacral vertebrae formation, were decreased, but the genes positioned on the centromeric side were not changed. WISH analysis confirmed these expression changes in the target tissues. Our results indicate that VPA-induced vertebral malformations are caused by global expression changes of developmental regulators. This may shed light on molecular dissection of sporadic vertebral malformations caused by the complex interrelationships between genetic and environmental factors in humans.
P-134: SATB1 orchestrates expression of lineage specifying genes during positive selection of thymocytes
Laboratory for Transcriptional Regulation, IMS, RIKEN
Thymocytes have to pass a T-cell antigen receptor (TCR) mediated selection process, known as positive selection, to become professional immune soldiers with distinct function. Several genes are involved in lineage specification during positive selection, those includes transcription factors (TFs) such as Zbtb7b, Runx3 and Foxp3 and Cd4 and Cd8a and Cd8b co-receptor genes. Although several regulatory regions are identified in these loci, it remains how TCR signals are linked with mechanisms that control their activity for appropriate expression of these genes.
Here we identified that SATB1, a chromatin organizer protein, plays multiple roles to activate above genes during positive selection. For instances, insufficient activation of Zbtb7b in the absence of SATB1 caused a redirection of MHC class II selected cells into CD8+ cells. In addition, both stably inheritable active statuses were not established in both Cd4 and Cd8 loci in part due to failure of DNA de-methylation. Collectively, our results unraveled novel function of SATB1 in orchestrating expression of several lineage-specifying genes during T cell development in the thymus.
P-135: Engineering the mouse genome using CRISPR/Cas9
Lydia Teboul*, Joffrey Mianne, Gemma Codner, Adam Caulder, MLC Microinjection and Husbandry Services, Debora Bogani, Martin Fray, and Sara Wells
The Mary Lyon Centre, MRC Harwell, HSIC, Oxon, OX11 0RD, UK
The Mary Lyon Centre is a national facility for the generation, study, archiving and dissemination of mice for biomedical research. As such, it is also a partner of the International Mouse Phenotyping Consortium, that aims to generate, characterise and distribute 20,000 mouse line worldwide in a 10-year programme.
The recently developed CRISPR/Cas system as genome-engineering tool has brought new perspectives for the generation of mouse models in a more precise fashion, at reduced price, all within a shorter time scale. We will report the use of this technology to engineer the genetic background of the C57BL/6N mouse strain. We will present data acquired from optimisation experiments of our CRISPR/Cas9 mutagenesis pipeline to enable the application of the technology to produce tailored mouse mutant lines (deletions, point mutations, floxed alleles) at large-scale at MRC Harwell. We will also discuss methods for screening both (often complex) founders and F1 animals and controlling their quality.
Finally, we will introduce the phenotyping and distribution programme for which these mouse lines are produced.
P-136: Analysis of three human genomic loci associated with Tetralogy of Fallot
(See abstract TS-10 in the Trainee Symposium)
P-137: PRDM14 promotes epigenetic changes that lead to driver mutations at Notch1 in inducible mouse models of T-ALL
Lauren J Tracey*1,2, Travis Brooke‑Bisschop2, Brandi Carofino3, and Monica J Justice1,2,3
1Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
2Program in Genetics and Genome Biology, The Hospital for Sick Children, Toronto, Ontario, Canada
3Department of Molecular and Human Genetics and Program in Translational Biology and Molecular Medicine, Baylor College of Medicine, Houston, Texas, USA
Evidence from various forms of malignancy, including leukemia, suggests that cancer is organized hierarchically, with self-renewing cancer stem cells at the apex. These cancer stem cells give rise to the tumour bulk, and enable disease relapse after treatment. Our lab showed for the first time that PRDM14, an epigenetic regulator of pluripotency in embryonic stem cells and primordial germ cells, is an oncogene in lymphoid leukemias. PRDM14 has also been implicated in breast, testicular, cervical, germ cell, and non-small cell lung cancers. To study the mechanism for PRDM14-induced tumorigenesis, we developed a Gt(ROSA)26Sortm1Jus inducible mouse model, which results in a rapid and completely penetrant T-cell acute lymphoblastic leukemia (T-ALL) when Prdm14 expression is induced in hematopoietic stem cells (HSCs). The development of T-ALL is preceded by the expansion of HSC-like cells, and is driven by constitutive activation of Notch1. Gt(ROSA)26Sortm2Jus (a FLAG-tagged version of this model) presents disease phenotypes indistinguishable to the non-FLAG-tagged mice. Using the FLAG-tagged model to isolate pre-leukemia cells, we found that PRDM14 binds a region within Notch1 prior to leukemia development, suggesting a direct role for PRDM14 in epigenetic changes at this locus. PRDM14-binding is followed by the accumulation of an H3K4me3 mark, which allows for RAG recombinases to access cryptic recombination signal sequences at Notch1, causing driver mutations. Additional RNA expression, protein/DNA interaction, and DNA methylation analysis will help to determine how and where PRDM14 modifies chromatin to cause cancer. We hypothesize that PRDM14’s potency functions allow it to maintain a cancer-initiating cell pool in our mouse models, while aberrant histone modification and DNA methylation at its binding sites promote a cell environment permissive for accumulation of DNA damage that causes driver mutations. Since PRDM14 overexpression and amplification is seen in multiple human malignancies, our findings will have implications across a wide range of cancers.
P-138: ENU mutagenesis identifies a novel molecular pathomechanism of severe immunodeficiency
(See abstract TS-16 in the Trainee Symposium)
P-139: Aromatase inhibitors counteract tamoxifen-induced activation of breast cancer stem cells
Jie‑Heng Tsai*1,2, Li‑Sung Hsu1, and Wei‑Jen Chen2
1Istitute of Biochemistry, Microbiology and Immunology, College of Medicine, Chung Shan Medical University, Taichung, Taiwan
2Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan
The cancer stem cell (CSC) model postulates the existence of a small proportion of cancer cells capable of sustaining tumor formation, self-renewal and differentiation.Elevated expression of aromatase (estrogen synthase) in ER positive breast cancer cells increases local estradiol concentrations and is associated with breast stem cancer development, but the causal relationship between aromatase and breast cancer has not been identified. In this study, we demonstrate that CDK10 is an important determinant of ER positive breast cancer cells and show that aromatase inhibitor letrozole decrease cancer-stem cell sphere formation. In monolayer or Tamoxifen of cultured MCF-7 cells, Tamoxifen sensitivity in MCF-7 cells were found to possess up-regulated ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2) and down-regulated CDK10. Interestingly, Letrozole is recover CDK10 expression and help block cells growth in inhibition by tamoxifen is associated with inhibition of ETS2. Letrozole silencing decreases ETS2-driven transcription of RAF1 (c-RAF), resulting in MAPK pathway activation and loss of tumor cell reliance upon estrogen signaling, which confers breast cancer cells cytotoxicity and sphere formation . The results suggested that CDK10 may play an important role in enhancement of tamoxifen efficiency and its expression may have a synergistic effect on letrozol treatments. Preliminary data show effective suppression of aromatase levels in ER positive breast cancer cells and in combination with tamoxifen. Letrozol was more effective in controlling tumor growth and significant reduction in mammosphere formation was observed. Hence, we suggest that the CSC population play a significant role during endocrine resistance through loss CDK10 and activity of the ETS2 , elevating ERK/MAPK kinase pathway activity. CDK10 is thus a potential biomarker for sensitivity in prospective clinical trials of patients treated with endocrine therapies.
P-140: Germline mutation rates and mutation accumulation lines in mice
Arikuni Uchimura*1, Mayumi Higuchi1, Yohei Minakuchi2, Atsushi Toyoda2, Asao Fujiyama2, Shigeharu Wakana3, Jo Nishino4, and Takeshi Yagi1
1Graduate School of Frontier Biosciences, Osaka University
2Comparative Genomics Laboratory, National Institute of Genetics
3Technology and Development Team for Mouse Phenotype Analysis, Japan Mouse Clinic, RIKEN BioResource Center
4Department of Biostatistics, Nagoya University Graduate School of Medicine
Germline mutation is the ultimate resource of all variation. However, the rate of germline mutations in laboratory mice remains unclear. Here we studied genome-wide mutation rates and their long-term effects on phenotype in more than 20 generations (9 years of breeding) of wild-type C57BL/6 mice and C57BL/6-Pold1tm1.1Uchi (mutator) mice, which have high DNA replication error rates due to the targeted disruption of 3’-5’ exonuclease activity in DNA polymerase delta. We estimated the base-substitution mutation rate to be 5.4×10-9 (95% confidence interval = 4.6×10-9 – 6.5×10-9) per nucleotide per generation in C57BL/6 laboratory mice, about half the rate reported in humans. The mutation rate in mutator mice was 17 times that in wild-type mice; the 20-generations of inbreeding of the mutator mice accumulated more than 6,300 mutations in an individual, which is much more than in an ENU (chemical mutagen) treated mouse. Abnormal phenotypes, including a number of disease phenotypes, were 4.1-fold more frequent in the mutator lines than in the wild-type lines. Quantitative traits were also more diverse in the mutator lines after several generations. In addition, the mutator mice reproduced at substantially lower rates than the controls. These results provide fundamental information about mutation rates in laboratory mice and suggest that mutation accumulation lines made by mutator mice would be a useful mutagenized resource for mammalian genetics research.
P-141: Granulosa Cell-Specific or Global PTEN Mutations in Combination with Transgenic FSH Expression Fails to Induce Ovarian Tumors
Dannielle H Upton*, Kirsty A Walters, Mark Jimenez, David J Handelsman, and Charles M Allan
Andrology Laboratory, ANZAC Research Institute, University of Sydney, Concord Hospital, Concord, NSW 2139
PTEN mutations occur frequently in ovarian cancer but in previous experimental studies granulosa cell (GC)-specific Pten inactivation alone was insufficient to cause murine tumors. Follicle stimulating hormone (FSH) is vital for ovarian function with elevated circulating levels associated with reproductive ageing and ovarian tumorigenesis in women. Transgenic FSH (B6.Cg-Tg(Ins2-CGA,-FSHB)6Cmal aka TgFSH) mice exhibit progressively rising FSH levels with ageing causing ovarian dysfunction and premature infertility, but no tumours. We hypothesized that high FSH when combined with ovarian Pten mutations may promote ovarian tumorigenesis in mice.
We used Plekha5Tg(AMH-cre)1Flor (Tg.AMH.Cre) to target Pten (Ptentm1Hwu) disruption to GCs (PtenGC) and Tg(Sox2-cre)1Amc (Tg.Sox2.Cre) for global heterozygous Pten (Pten+/Pten-) mutation combining them with TgFSH overexpression as a multi-hit strategy to use genetic and hormonal means to induce ovarian tumors. STOCK-TgFSH ± PtenGC females displayed similar ovary and uterine weights versus controls (±TgFSH) with no detectable tumours at 12 months. STOCK-TgFSH ± Pten+/Pten- ovary weights remained unchanged (p = 0.5), whereas uterine weights increased due to tumors vs controls (p < 0.001). TgFSH ± Pten+/Pten- females developed tumours in various organs (pituitary, skin, kidneys) but not in the ovary. Estrous cycling and stage lengths were not significantly altered by ovary-specific PtenGC status whereas global Pten+/Pten- mice had reduced estrous cycling (p < 0.001). Corpora lutea (CL) numbers remained unchanged among PtenGC mice vs control (p = 0.99), however Pten+/Pten- significantly increase CL numbers (p < 0.05), indicating increased ovulation rates or persisting CLs by global Pten mutation.
We conclude that specific follicular or global Pten mutations, alone, or combined with TgFSH, were not sufficient to cause ovarian tumors. These findings that the ovary is remarkably resistant to oncogenesis support the newer extra-gonadal origin hypothesis for ovarian tumorigenesis.
P-143: Loss of MECP2 results in lung defects in a mouse model for Rett syndrome
Neeti Vashi*1,2, Martin Post3, and Monica J Justice1,2
1Department of Molecular Genetics, University of Toronto, Ontario, Canada
2Genetics and Genome Biology, The Hospital for Sick Children, Ontario, Canada
3Physiology and Experimental Medicine, The Hospital for Sick Children, Ontario, Canada
Mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2) are the primary cause of Rett syndrome, a neurodevelopmental disorder that presents almost exclusively in females. While respiratory impairment is a common feature of Rett syndrome, respiratory symptoms have historically been credited to neurochemical dysregulation in patients. However, various neurotransmitter therapy experiments in rodents have only recovered a subset of respiratory symptoms, suggesting a potential non-neuronal cause. Interestingly, previous studies in humans and mice have demonstrated that MECP2 is highly expressed not only in the brain, but in the lung as well. Thus, we hypothesized that the global loss of MECP2 causes a local pathology in the respiratory system. Here, we use a Mecp2 null mouse model (B6.129P2(C)-Mecp2tm1.1Bird/J), which recapitulates many disease symptoms, to study the lung. Histology observations show thickened septae and reduced number of alveoli in the lung early in postnatal development, suggesting alveolarization defects in the Mecp2 null mice. In early adulthood, the alveolar walls thin out and start to disintegrate. Gene expression analyses reveal that genes involved in the alveolarization process including Vegfa, Foxa2, Pdgfa, and Mdk, are down-regulated in the lung during postnatal development. Altogether, our preliminary data suggest that the loss of MECP2 affects lung structure beginning early in development, and prior to the onset of neurological symptoms in our mouse model. To further investigate this pathology, we plan to develop a lung-specific Mecp2 knockout mouse line to characterize respiratory symptoms separate from any neurological influence. Our ongoing studies are designed to guide the development of therapeutics to alleviate respiratory impairments in Rett syndrome patients.
P-144: Horses: an underutilized animal model
(See abstract TS-08 in the Trainee Symposium)
P-145: IsoformSwitchAnalyzeR: Enabling Identification and Analysis of Isoform Switches, with Functional Consequences, from RNA-sequencing data
Kristoffer Vitting‑Seerup*, and Albin Sandelin
Bioinformatics Centre, Biotech Research & Innovation Centre (BRIC), Department of Biology, University of Copenhagen, Ole Maaloes Vej 5 DK-2200, Copenhagen N, Denmark
RNA-sequencing data is currently under utilized, in part because it is difficult to predict the functional consequences of changes in alternative transcription. The recent software improvements in full-length transcript deconvolution came with the promise of better understanding of these events, but unfortunately such analyses are still hard to obtain and only rarely done.
To solve this problem we developed IsoformSwitchAnalyzeR. IsoformSwitchAnalyzeR is an easy to use R package that enables annotation of full-length RNA-seq derived transcripts with protein domains, signal peptides, coding potential as well as NMD sensitivity. Furthermore IsoformSwitchAnalyzeR supports identification and visualization of isoform switches, which together with the obtained annotation, allows for prediction of functional consequences due to alternative transcription events.
During this presentation I will introduce IsoformSwitchAnalyzeR, show how easy it is to use as well as demonstrate how IsoformSwitchAnalyzeR supports hypothesis generating data exploration by identifying isoform switches with functional consequences and thereby facilitating better utilization of RNA-seq data.
P-146: Coexpression networks identify brain region-specific enhancer RNAs in the human brain
Pu Yao, Peijie Lin, Akira Gokoolparsadh, Amelia Assareh, and Irina Voineagu*
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales, Australia.
Despite major progress in identifying enhancer regions on a genome-wide scale, the majority of available data are limited to model organisms and human transformed cell lines. Using the FANTOM5 CAGE data, we have identified a robust set of enhancer RNAs (eRNAs) expressed in the human brain and constructed networks assessing eRNA-gene coexpression interactions across human fetal brain and multiple adult brain regions. Our data identify brain region-specific eRNAs and show that enhancer regions expressing eRNAs are enriched for genetic variants associated with autism spectrum disorders.
P-147: Challenging to RDoC matrix in behavior phenotype of mouse models for human psychiatric disorders
Shigeharu Wakana*, Ikuko Yamada, Misho Kashimura, Tomoko Kushida, and Tamio Furuse
The Japan Mouse Clinic, RIKEN BioResource Center
A mouse is useful for human disease models in many fields of medical science. However, there is a problem in its application to the results of mouse phenotype in mouse mutants in the field of psychiatric disorders. The Japan Mouse Clinic has analyzed the behavioral screens for several mutant mice in our pipeline. However, I wonder whether or not the results of the behavior phenotype have clarified the relationship between mouse models and human psychiatric disorders. Recently, the diagnostic criteria of human psychiatric disorders have been slightly changed to align with the DSM-5 (DSM: American Psychiatric Association 2013). The categories contained in the DSM does not have sufficient validity as biological functional constructs of psychopathology. Additionally, the Research Domain Criteria Project (RDoC) was established under a proposal by NIMH which includes the development for research purposes of new ways of classifying mental disorders based on dimensions of observable behavior and neurobiological measures. We have rearranged the mouse behavioral screens of the mutants, and we will also challenge to apply the results to classify the RDoC.
P-148: Targeting genes via architecture rather than recognition motifs: The FOS/NFY case
Martin Haubrock, Fabian Hartmann, and Edgar Wingender*
Institute of Bioinformatics, University Medical Center Goettingen, Goettingen, Germany
Large parts of the mammalian genome exert important regulatory functions as, e.g., promoters or enhancers. They are made up of arrays of regulatory sequence signals, cis-regulatory elements, which serve as docking sites for transcription factors (TFs). These TF binding sites (TFBSs) can be described with, e.g., consensus sequences or positional weight matrices (PWMs).
We launched a systematic attempt to check all matrices of the TRANSFAC PWM library for recognizing the corresponding TFBSs in chromatin-immunoprecipitated (ChIPped) fragments collected in the Encode data repository. We noticed that especially c-Fos bound regions are highly diverse with regard to (a) the cell line examined and (b) proximal or distal localization of these regions (enriched either in promoters or enhancers). The conventional binding mode of FOS to DNA is as heterodimer with a JUN-component that binds to an AP-1-motif (TGACTCA). Applying both motif matching and motif discovery approaches, we identified subsets of regulatory regions that are bound by FOS, but do not contain any significant AP-1 motif. They rather are characterized by CCAAT boxes, serving as a recognition sites for TF NFY, a heterotrimer of NFYA, B and C. Consequently, these CCAAT-marked, FOS-targeted regions also appear in collections of genomic fragments ChIPped with an NFYB antibody, where they constitute a distinct subset. When focusing on the proximal regions of this subset, we could identify as distinctive trait the presence of direct repeats of two CCAAT boxes, with a defined distance distribution peaking at 31 bp. This pattern is absent from enhancer regions and can hardly be found in promoters that have not been found yet to be bound by FOS. Since we found more ChIP datasets lacking the expected consensus, our results prompt us to support the idea that TFs may also target promoters through certain architectures rather than a specific consensus sequence.
P-149: microRNA expression during erythroid differentiation
Louise Winteringham*1, Peter Klinken1, Patrick Candy1, Erik Arner2, Hiroshi Tarui2, Shohei Noma2, Maxwell Burroughs2, Alistair Forrest1,2, Piero Carninci2, Yoshihide Hayashizaki2, and FANTOM5 Consortium2
1Harry Perkins Institute of Medical Research, QEII Medical Centre, Nedlands and Centre for Medical Research, The University of Western Australia, Crawley, Western Australia, 6009, Australia
2RIKEN Center for Life Science Technologies (Division of Genomic Technologies)(CLST (DGT)), 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
MicroRNAs are short ribonucleic acids which can influence gene expression and consequently affect diverse cellular functions. A single miRNA may regulate several target mRNAs and therefore has the potential to influence multiple signalling pathways and even complex networks. miRNA expression is commonly deregulated in many disorders therefore manipulating aberrant miRNA expression may provide a new approach to treat complex diseases such as cancer. Importantly, to understand the role miRNAs play in disease we need to understand how they affect normal cellular processes. In collaboration with the FANTOM5 consortium, we undertook a transcriptomic analysis of an in vitro differentiation model of erythropoiesis. The in vitro model is murine fetal liver cells transformed with the J2 virus. These J2E cells are able to be grown in the laboratory and when treated with erythropoietin (Epo) a proportion of them will differentiate morphologically and biochemically to produce haemoglobin. If injected into mice they cause a rapid and fatal erythroleukemia.
Differentially expressed miRNA and mRNA were identified at 6hrs, 12hrs, 24hrs and 48hrs post Epo induction. A list of candidate molecules was generated using the following criteria i) a logFC > 1.5 for both miRNA and target mRNA, ii) a FDR <0.05 for both miRNA and mRNA iii) miRNAs previously associated with myelo-erythroid differentiation or leukemia. A short list of candidate miRNAs was identified to be further validated in the laboratory. Corresponding mRNA targets were then predicted using TargetScan and intersected with our mRNA data to generate a list of predicted targets dynamic in the opposite direction to the miRNA.
To assess the biological significance of these miRNAs we are systematically up or down regulating them in J2E and murine bone marrow cells. Initial data indicate that a number of these miRNAs are required for normal erythroid differentiation and their deregulation contributes to altered haemopoiesis.
P-150: Phenoview: A tool for comparative visualisation of genotype-phenotype relationships
Gagarine Yaikhom*, Hugh Morgan, Duncan Sneddon, Andrew Blake, James M Brown, Armida Di Fenza, Tanja Fiegel, Neil Horner, Natalie Ring, Luis Santos, Henrik Westerberg, Ahmad Retha, Julian Atienza‑Herrero, Steve DM Brown, and Ann‑Marie Mallon
MRC Harwell, Medical Research Council, Harwell, UK
The International Mouse Phenotyping Consortium (IMPC) currently provides access to over 28 million data-points concerning the knockout effects of 2,145 mouse genes on 58 classes of well-defined phenotypic traits. This data was collected at 10 phenotyping centres world-wide using standardised operating procedures. To enable systematic analysis of this data, the IMPC has developed specialised visualisation and analysis tools.
Phenoview is a publicly accessible web-based tool (https://www.mousephenotype.org/phenoview) for comparative visualisation of genotype-phenotype relationships. It allows users to visualise the raw measurements, and to download the data for further analysis. The defining feature of Phenoview is its ability to visualise multiple gene-phenotype combinations simultaneously. Users select the genes they are interested in and the phenotypes they wish to investigate, and Phenoview automatically chooses the appropriate visualisation methodology to display the measurements.
Phenoview currently has the facility to display measurements as scatter, beeswarm, segmented-column and time-series plots. It also allows users to interact with the visualisations in real-time. For instance, filtering out measurements by zygosity or gender, or to overlay descriptive statistics. Users can then save the visualisations as image files. The embedded media viewer within Phenoview handles display of media, such as LacZ expression and X-ray images. This viewer is seamlessly integrated with the rest of the visualisations, thus providing a link between related phenotypes. For instance, LacZ images are linked to tissue and organ phenotypes. Finally, a heatmap interface displays statistical annotations enabling exploration of mutant gene-phenotype combinations that deviate from the wildtype controls. The heatmap displays sexual dimorphism, statistical analysis results and standardised mammalian phenotype terms. This allows users to quickly identify gene-phenotype combinations that could shed light to novel biological processes.
By providing unrestricted access to the rich and complex data-set of genotype-phenotype relationships, the IMPC aims to create a comprehensive functional catalog of the mammalian genome.
P-151: Development of innovative therapeutic strategy using DNA minor groove binder-drug conjugate in MYCN-driven neuroblastoma
(See abstract TS-01 in the Trainee Symposium)
P-152: Peroxiredoxin 2 Promotes HRAS*G12V-Induced Hepatic Tumorigenesis Through Reciprocal Regulation With Forkhead Box M1
Young‑Ho Park, Sun‑Uk Kim, Tae‑Ho Kwon, and Dae‑Yeul Yu*
Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea
Peroxiredoxin 2 (PRDX2), an antioxidant enzyme, is elevated in several human cancers, including hepatocellular carcinoma (HCC). However, the molecular mechanisms of PRDX2 in HCC remain unclear. Here, we investigated whether PRDX2 plays an important role in oncogenic HRAS*G12V-induced hepatic tumorigenesis. Expression of PRDX2 was elevated in HRAS*G12V transformed HCC cells and tumor tissues from B6-Tg(Alb-HRAS*G12V)28Yu transgenic (HRAS*G12V) mice. B6-Prdx2tm1Yu/Prdx2tm1Yu Tg(Alb-HRAS*G12V)28Yu (HRAS*G12V Prdx2-/-) mice exhibited a significantly reduced number and size of tumors. Conversely, overexpression of PRDX2 promoted colony formation in soft agar and tumor growth in nude mice. Moreover, PRDX2 expression was correlated with that of forkhead box M1 (FOXM1) in HCC patients and HRAS*G12V mice. We identified that FOXM1 is a direct transcription factor of Prdx2 induced by HRAS*G12V. Interestingly, PRDX2 modulated FOXM1 expression through MAPK1(ERK) phosphorylation. This mutual regulation between PRDX2 and FOXM1 contributes to sustain the RAF/MAP2K7/MAPK pathway and cyclin D1 activation. Conclusion: PRDX2 is an important downstream target of FOXM1 in HRAS*G12V transformed HCC cells and this novel PRDX2/MAPK1/FOXM1 signaling axis promotes hepatocarcinogenesis.
P-153: A mutation in TBC/LysM associated domain containing TLDC1 causes craniofacial abnormalities in mice
Rong Zeng*1, Victoria Keener2, Brian Dawson2, Brendan Lee2, Carlos Bacino2, Jay Shendure3, and Monica J Justice2
1The Hospital for Sick Children
2Baylor College of Medicine
3University of Washington
Craniofacial 3 (crf3) is a highly penetrant recessive trait that was previously isolated in a series of large-scale mouse N-ethyl-N-nitrosourea (ENU) mutagenesis screens on a mixed strain background. Crf3 homozygotes are small in size with visible craniofacial phenotypes including short snout, microcephaly and upright standing ears. We used a novel soft-tissue scanning technique to provide a non-invasive, reliable and quick method to accurately distinguish, quantify and classify the craniofacial abnormalities in mutant mice, and link them to craniofacial defects of human syndromes. The crf3 locus has been mapped to mouse Chromosome 8, linked to D8Mit92 (Z=5.7). To identify causal DNA variants, we sequenced one homozygous crf3 sample by using whole-exome sequencing. Short reads were aligned to the mouse reference genome by BWA and variants were called by GATK. Hard-filtering for the variants was based on a minimum SNP quality of 20, read depth of 5 and variant confidence of 1.5. Variants were filtered by removing common polymorphisms, which were present in sequence from our in-house samples and in sequence from mouse SNP database. After filtering, only one homozygous variant remained that was located on mouse Chromosome 8: a T to C nucleotide change in a splice donor site of Tldc1 (NM_028883: exon4: c.378+2T>C). Eight DNA samples presumed to be homozygotes based on phenotype were homozygous for this mutation, whereas none of seven DNA samples from unaffected, presumed heterozygous or wild type littermates were homozygous for this allele. To date, the function of human TLDC1 on HSA 16q24.1 is unknown. TLDC1 lies near the FOXF1 cluster, and is deleted in some cases of the 16q24.1 microdeletion syndrome, and lies within human duplications associated with disease. Mice with mutations in Tldc1 may prove useful as models to determine if craniofacial defects are a part of the syndrome in such patients.
P-154: Diversification of Behavior and Postsynaptic Properties by Netrin G Presynaptic Adhesion Family Proteins
Qi Zhang*, Hiromichi Goto, Sachiko Akiyoshi‑Nishimura, Pavel Prosselkov, Chei Sano, Hiroshi Matsukawa, Kunio Yaguchi, Toshiaki Nakashiba, and Shigeyoshi Itohara
RIKEN Brain Science Institute
Vertebrate-specific neuronal genes are expected to play a critical role in the diversification and evolution of higher brain functions. Among them, the GPI (glycosyl-phosphatidylinositol)-anchored netrin G sub-family members in the UNC6/netrin family are unique in their differential expression patterns in many neuronal circuits. To gain insights on the role of these genes in higher brain functions, we examined netrin G KO mice with comprehensive behavioral batteries. Two netrin G paralogs that recently diverged in evolution, netrin G1 and netrin G2 (gene symbols Ntng1 and Ntng2, respectively), were responsible for complementary behavioral functions. Netrin G2, but not netrin G1 encoded demanding sensorimotor functions. Both paralogs were responsible for complex vertebrate-specific cognitive functions and the fine-scale regulation of basic adaptive behaviors conserved between invertebrates and vertebrates. Remarkably, netrin G1 and netrin G2 encoded a genetic “division of labor” in behavioral regulation, selectively mediating different tasks or even different details of the same task. At the cellular level, netrin G1 and netrin G2 differentially regulated the sub-synaptic localization of their cognate receptors and differentiated the properties of postsynaptic scaffold proteins in complementary neural pathways. Our findings constitute the first genetic analysis of behavioral and synaptic diversification roles for a vertebrate GPI protein and presynaptic adhesion molecule family.
P-155: Genome-wide DNA methylation profile implicates potential cartilage regeneration at the late stage of knee osteoarthritis
Yanfei Zhang*1, Naoshi Fukui2, Mitsunori Yahata1,3, Yozo Katsuragawa4, Toshiyuki Tashiro5, Shiro Ikegawa6, and Ming Ta Michael Lee1
1Laboratory for International Alliance on Genomic Research, Center for Integrated Medical Sciences, RIKEN, Yokohama, Japan
2Clinical Research Center, National Hospital Organization Sagamihara Hospital, Kanagawa, Japan
3Laboratory for Pharmacogenomics, Center for Integrated Medical Sciences, RIKEN, Yokohama, Japan
4Department of Orthopaedic Surgery, National Center for Global Health and Medicine Center Hospital, Tokyo, Japan
5Department of Orthopaedic Surgery, Tokyo Yamate Medical Center, Tokyo, Japan
6Laboratory for Bone and Joint Diseases, Center for Integrated Medical Sciences, RIKEN, Tokyo, Japan
The aim of this work was to characterize the genome-wide DNA methylation profile of cartilage from three regions of tibial plateau isolated from patients with primary knee osteoarthritis (OA), providing the first DNA methylation study that reflects OA progression.
The unique model system we developed previously was used to section three regions of tibial plateau: the outer lateral tibial plateau (oLT), the inner lateral tibial plateau (iLT) and the inner medial tibial plateau (iMT) regions which represented the early, intermediate and late stages of OA, respectively. Genome-wide DNA methylation profile was examined using Illumina Infinium HumanMethylation450 BeadChip array. Comparisons of the iLT/oLT and iMT/oLT groups were carried out to identify differential methylated (DM) probes (DMPs) associated with OA progression. DM genes were analyzed to identify the gene ontologies (GO), pathways, upstream regulators and networks.
No significant DMPs were identified in iLT/oLT group, while 519 DMPs were identified in iMT/oLT group. Majority of them (68.2%) were hypo-methylated and enriched in enhancers and N-shores. Upstream regulator analysis identified many microRNAs. DM genes were enriched in transcription factors, especially homeobox genes and in Wnt/β-catenin signaling pathway. These genes also showed changes in expression when analyzed with expression profiles generated from a previously study.
Our data suggested the changes in DNA methylation occurred at the late stage of OA. Pathways and networks enriched in identified DM genes highlighted potential etiologic mechanism and implicated the potential cartilage regeneration in the late stage of knee OA.
P-156: Genetic resistance to congenital hypothyroidism rescues neurosensory and conductive hearing impairment
SA Camper*, AH Mortensen, MT Fleming, and Q Fang
University of Michigan, Dept. of Human Genetics, Ann Arbor, MI, USA
Maternal and fetal sources of thyroid hormone (TH) are important for the development of many organ systems. TH deficiencies cause variable intellectual disability and hearing impairment and in mouse and man, and the genetic basis for resistance to congenital hypothyroidism is not clear. To explore the source of this variability we studied two TH-deficient mouse strains, DF-Prop1df/df and DW-Pou1f1dw/dw that have mild elevations in hearing thresholds and profound deafness, respectively. The different hearing abilities of Prop1 and Pou1f1 mice are due to genetic variations in the backgrounds of the strains and are intrinsic to the developing fetus. Many aspects of inner ear neurosensory and middle ear development are affected in Pou1f1 mutants. We identified a major modifier locus Chromosome 2 that regulates the sensitivity to hypothyroidism induced hearing impairment, Mdwh. Thus, one chromosomal region can rescue multiple developing structures in TH-deficient animals. This region contains modifiers of thyroid gland development that could be causal. We hypothesized that Prop1 mutants are protected against hearing impairment by better thyroid gland development and residual TH production in the absence of pituitary TSH stimulation. To test this idea we measured free T4 levels in serum mice of all four genotypes. Normal mice from the Prop1 and Pou1f1 strains have significantly different T4 levels; 11.7 and 7.5 ug/dL, respectively, and mutant levels are near the limit of detection, but Prop1 mutants appear to have a higher residual level. Both mutants have arrested thyroid gland development, but Pou1f1 mutant thyroid glands appear more severely affected. These results support our hypothesis that, in the absence of TSH, strain differences in thyroid development and function underlie the difference in hearing abilities. Identification of the genetic modifiers that cause variation in thyroid development in mice could help explain the different consequences of congenital hypothyroidism in humans.
P-157: Polycomb PRC1 and PRC2 complexes contribution to MYC-mediated Hepatocarcinoma
J Gimenez*1, V Bianchi2, K Hashimoto3, T Kress2, P Nicoli2, B Amati2, P Carninci3, and V Orlando1,4
1IRCCS Santa Lucia Foundation, Rome, Italy
2Italian Institute of Technology, Campus IFOM-IEO, Milan, Italy
3RIKEN Center for Life Science Technologies, Yokohama, Japan
4KAUST Environmental Epigenetics Program, Thuwal, Kingdom of Saudi Arabia
Hepatocellular carcinoma (HCC) is one of the most common malignancies and remains largely incurable. Past decade research demonstrated that cancer is not just a “genetic” disease, but that epigenetic alterations control the pathogenic behavior of cancerous cells. Polycomb group proteins (PcGs) are epigenetic regulators fundamental for the maintenance of cell-specific gene expression pattern, thereby being essential for cell identity, development, pluripotency and differentiation. Not surprisingly, deregulation and/or mutations of PcG components have being reported to be involved in tumorigenesis, including in HCC.
PcG forms two major multiprotein complexes, Polycomb Repressive Complex 1 (PRC1) and PRC2. PRC2 is responsible for the repressive histone modification H3K27me3 onto promoters and subsequent spreading over large domains. PRC1 recognition of H3K27me3, and mono-ubiquitination of H2AK119 further enhances transcriptional silencing and chromatin compaction. This traditional model of PcG-mediated repression however is changing towards a more complicated picture. PRC1 and PRC2 are indeed far more varied than previously thought, in both their composition and their relative contributions to chromatin state and transcription. Notably BMI1 (PCGF4) and MEL18 (PCGF2) proteins which are present in similar but mutually exclusive PRC1 complexes, have been reported to have opposite consequences on tumor agressivity. Similarly, EZH2 and EZH1, the histone-methyltransferases (HMTase) of PRC2, are associated with different cell differentiation state. EZH2, the more potent HMTase, marks proliferative cells and is often upregulated in cancer. In HCC, EZH2 was recently reported to physically interact with the MYC oncogene. Finally, non-canonical PRC2 and PRC1 complexes can occupy active chromatin, and positively regulate gene expression.
By using a mouse model for MYC-induced hepatocellular carcinoma, we have been investigating how PcG complexes and diversity contribute in a MYC dependent and independent manner to hepatocarcinogenesis. Understanding better the abnormal PcG-mediated epigenetic reprogramming and interplays occurring in HCC, will help identifying the events at the basis of the disease.
|Abe, Koichiro||P-001, P-107, P-133||Ayusawa, Koh||P-097|
|Abe, Kuniya||P-002, P-122||Babarinde, Isaac Adeyemi||O-37|
|Abe, Takaya||O-09||Babina, Magda||O-45|
|Abugessaisa, Imad||P-003, P-012, P-060, P-091||Bacino, Carlos||P-153|
|Aburatani, Hiroyuki||P-104||Bagger Terkelsen, Thilde||O-43|
|Abu-Toamih-Atamni, Hanifa||P-088||Bagger, Frederik Otzen||P-007|
|Abyzov, Alexej||P-004||Bajic, Vladimir B||P-124|
|Ackerman, Kate||O-22||Baker, Christopher||P-008|
|Adams, David||O-50, P-065, P-067||Baker, Richard||O-15|
|Adams, Ian||O-33||Balcova, Maria||P-029|
|Adler, Thure||TS-16/P-138||Baldock, Richard||P-120|
|Ahmed, Jehangir||P-005||Balick, DJ||O-32|
|Airhart, Susan||O-01||Ball, Robyn||P-100|
|Aizawa, Shinichi||P-113||Balling, Rudi||O-14|
|Akamatsu, Wado||P-050||Bando, Toshikazu||TS-13/P-048|
|Akiyoshi-Nishimura, Sachiko||P-154||Banes-Berceli, Amy||P-114|
|Alam, Tanvir||O-45, P-124||Baple, EL||P-064|
|Alanis-Lobato, Gregorio||O-46||Baric, Ralph||TS-02/P-037|
|Albuquerque, Ralph||P-042||Barratt, Kristen||P-005|
|Allan, Charles M||P-141||Barrington, William||P-009|
|Alzahrani, Alaa||P-005||Bartunek, Jan||O-20|
|Amati, B||P-157||Batista, Leandro||P-098|
|Amenduni, Mariangela||P-004||Bauer, Denis||O-02|
|Amiri, Anahita||P-004||Bazaga, David||P-010|
|Andersson, Leif||TS-08/P-144||Beall, Anne||TS-02/P-037|
|Andersson, Lisa||TS-08/P-144||Beamer, Gillian||O-15|
|Andersson, Robin||O-35, O-43, O-44, P-007, P-051||Beckers, Johannes||O-26|
|Andre, Philippe||O-29||Beguin, Pascal||O-13|
|Andreas, Jonathan||O-19||Beier, David||O-32, P-116|
|Andres, Christian||TS-16/P-138||Bell, Timothy A||O-34|
|Angel, Joe||P-010||Benavides, Fernando||P-010|
|Araki, Kimi||P-032, P-086||Bennett, Brian||P-009|
|Araki, Masatake||P-032, P-086||Bergstrom, David||O-31|
|Arbogast, Thomas||P-041||Berletch, Joel B||O-24|
|Arends, Mark||P-067||Bertin, Nicolas||O-13, O-45, P-112|
|Arkell, Ruth||P-005||Bhattacharyya, Tanmoy||P-029, P-030|
|Arnaud, Ophelie||P-006, P-101||Bianchi, V||P-157|
|Arner, Erik||O-44, O-45, P-046, P-060, P-124, P-149||Bickmore, Wendy||O-33, O-35|
|Arner, Peter||O-45||Billings, Timothy||P-016|
|Ashoor, Haitham||O-45||Birling, Marie-Christine||O-29, P-041|
|Ashton, Anthony||P-045||Bjerrum, Jacob||O-43|
|Assareh, Amelia||P-146||Blake, Andrew||P-150|
|Astrom, Gaby||O-45||Blake, Judith||P-011|
|Atienza-Herrero, Julian||P-150||Blancafort, Pilar||P-118|
|Atsushi, Takatori||P-061||Blau, Carl Anthony||O-24|
|Ay, Ferhat||O-24||Blauwendraat, Cornelis||O-08|
|Blischak, John||P-053||Carninci, Piero||O-13, O-35, O-44, O-45, O-49, P-012, P-038, P-040, P-043, P-044, P-051, P-055, P-060, P-080, P-091, P-103, P-110, P-112, P-123, P-124, P-149, TS-14/P-128|
|Blott, Sarah||TS-08/P-144||Carofino, Brandi||P-137|
|Bodea, Gabriela-Oana||O-07||Carouge, Delphine||O-25|
|Bodega, Beatrice B||O-46||Carter, Gregory W||P-016, P-100|
|Boettcher, Michael||P-012, P-060, P-091||Cassa, CA||O-32|
|Bogani, Debora||P-135||Castillo-Lizardo, Melissa||O-08|
|Bondareva, Olga||TS-03/P-034||Cates, Sarah E||P-081|
|Bonetti, Alessandro||P-040, P-055||Caulder, Adam||P-135|
|Bornholdt, Jette||O-43||Cazzella, Valentina||O-46|
|Bottcher, Michael||P-080||Champy, Marie-France||P-117|
|Bou-About, Ghina||P-117||Chang, Jen-Chien||P-017, P-069|
|Boyd, Mette||O-43||Chang-Chien, Ju||P-018|
|Bozzoni, Irene||O-46||Chase, Jennifer||P-056|
|Brake, Marisa A||P-013, P-114||Chen, Chun-Yu||P-019|
|Braun, Emilie||P-040||Chen, Jing||P-065|
|Brenn, Thomas||P-067||Chen, Wei-Jen||P-018, P-139|
|Britton, Steven L||O-03||Cheng, Kai||O-31|
|Brombacher, Frank||P-124||Chick, Joel||O-53|
|Brommage, Robert||O-40||Chimura, Takahiko||O-13|
|Brooke-Bisschop, Travis||P-137||Choi, Kwangbom||O-53|
|Brown, James M||P-014, P-150||Christie, Karen||P-011|
|Brown, Steve DM||P-150||Chung, Elaine KY||P-020|
|Buehler, Dennis P||P-105, P-121||Churchill, Gary A||O-19, O-53, P-024, P-071, P-100, P-125|
|Buendia, Marie Annick||P-040||Chuva de Sousa Lopes, Susana M.||P-122|
|Bult, Carol J||O-01, P-015||Claghorn, Gerald||P-042|
|Bupp, Sujata||O-19||Clark, Grace||O-34|
|Burant, Charles F||O-03||Clayshulte, Amelia M-F||O-34|
|Burgio, Gaetan||O-02||Cleak, James||P-014|
|Burroughs, Maxwell||O-45, P-149||Codner, Gemma||P-135|
|Busch, Dirk H.||TS-16/P-138||Colbath, James||P-042|
|Buys, Nadine||TS-08/P-144||Consalvi, Sara||O-46|
|Byers, Candice||O-06||Coppee, Jean-Yves||O-04|
|Cairns, Murray J||TS-06/P-035||Cormier, RT||P-022|
|Camper, SA||P-156||Coskun, Mehmet||O-43|
|Candy, Patrick||P-149||Cotella, Diego||P-038, TS-14/P-128|
|Cantero, M||P-084||Crawford, Nigel||O-19|
|Carlisle, Francesca||P-065||Crosby, AH||P-064|
|Carniel, Elisabeth||O-04||Cunningham, Julie||P-004|
|Carninci, P||P-157||Czechanski, Anne||O-06|
|Dalby, Maria||P-007||Eriksson, Susanne||TS-08/P-144|
|Daub, Carsten O||O-44, O-45, P-063, P-112||Ershova, Elisaveta S.||P-111|
|Davies, Jamie||P-120||Estabel, Jeanne||TS-12/P-068|
|Dawson, Brian||P-153||Evans, Cameron||P-118|
|Daxinger, Lucia||P-119||Everett, Lesley||P-056|
|De Hoon, Michiel||O-45, O-49, P-039, P-063, P-112, P-123, P-124||Faivre, Jamila||P-040|
|De Pons, Jeff||P-090, P-115||Faltusova, Barbora||P-029|
|De Rie, Derek||O-45||Fan, Meng-Ni||P-019|
|de Souza, TA||P-023||Fang, Q||P-156|
|de Vries, Bert||P-041||Fasolo, Francesca||P-038|
|Della Valle, Francesco||O-46||Faulkner, Geoffrey J.||O-07|
|Demant, Peter||O-20||Fegraeus, Kim||TS-08/P-144|
|Demeure, Christian||O-04||Fernandez, A||P-084|
|den Uil, SH||P-022||Fernandez, J||O-27|
|Denda, Atsumi||O-09||Ferris, Martin T||O-15, P-027, TS-02/P-037|
|Deng, Xinxian||O-24||Fiegel, Tanja||P-150|
|Dengler, Leonie||O-14||Fijneman, RJA||P-022|
|Desterke, Christophe||P-040||Filipiak, Wanda||P-109|
|Detmar, Michael||O-45||Fine, Alexander||P-016|
|Devailly, Guillaume||O-11||Flachs, Petr||P-008|
|Dhingra, Ashutosh||O-08||Flamand, Marie||P-098, P-099|
|Di Fenza, Armida||P-150||Fleming, MT||P-156|
|Diamand, Koula||P-005||Flores, E"Lissa||P-028|
|Didion, John P||P-024||Foote, Simon||O-02|
|Dillies, Marie-Agnes||O-04||Forejt, Jiri||P-029|
|Ding, James||O-33||Forrest, Alistair||O-35, O-44, O-45, P-040, P-043, P-044, P-051, P-070, P-103, P-112, P-124, P-149, TS-15/P-106|
|Dinh, TH Tra||P-025||Fort, Alexandre||O-45, P-040, P-055|
|DiRienzo, Anna||P-053||Fotopulosova, Vladana||P-029|
|Disteche, Christine M||O-24||Fox, Archa||P-066|
|DiTommaso, Tia||TS-12/P-068||Francescatto, Margherita||O-08|
|Drablos, Finn||P-079||Francois, Liesbeth||TS-08/P-144|
|Drake, Matthew||P-065||Fray, Martin||P-135|
|Duan, Zhijun||O-24||Frith, Martin||O-51|
|Duchon, Arnaud||P-041||Fu, Chen-Ping||P-081|
|Ducro, Bart||TS-08/P-144||Fuchs, Helmut||O-40, P-001, P-107, TS-16/P-138|
|Dwinell, Melinda||P-090, P-115||Fujimori, Toshihiko||P-113|
|Dyckhoff, Daniela||O-26||Fujita, Keisuke||P-017|
|Ebrahimi-Rad, Mina||P-026||Fujiwara, Yasuhiro||P-030|
|Ehsani, Rezvan||P-079||Fujiyama, Asao||P-036, P-126, P-140|
|Emam, Mohammadmehdi||P-026||Fukamizu, Akiyoshi||P-025|
|Eppig, Janan||O-38, P-011||Fukui, Naoshi||P-155|
|Fukumura, Ryutaro||O-52, P-031, P-036||Green, Richard R||P-027|
|Fukuta, Kentaro||P-126||Greenaway, S||O-27|
|Fuller, Emily||P-045||Gregorova, Sona||P-029|
|Furuhata, Erina||P-129||Grekov, Igor||O-20|
|Furuhata, Riki||P-032, P-086||Greth, Andreas||O-02|
|Furuno, Masaaki||P-046||Groll, Michael||TS-16/P-138|
|Furuse, Tamio||P-033, P-147||Guhl, Sven||O-45|
|Furuta, Yasuhide||O-09, P-113||Guler, Reto||P-124|
|Gailus-Durner, Valerie||O-40, TS-16/P-138||Gurumurthy, Channabasavaiah||P-082, P-095|
|Gallego, Jabier||P-116||Gusev, Oleg||TS-03/P-034|
|Galli, Antonella||P-067||Gustincich, Stefano||P-038, P-110, TS-14/P-128|
|Garland Jr., Theodore||P-042||Gygi, Steven||O-53|
|Gatti, Daniel M||O-19, O-53, P-071, P-125||Haagen Nielsen, Ole||O-43|
|Gavrilina, Galina||P-109||Hadsell, Darryl||O-54|
|Gazizova, Guzel||TS-03/P-034||Hadsell, Louise||O-54|
|Geffers, Robert||O-14||Hale, Paul||P-015|
|Gelfand, Mikhail||TS-05/P-077||Handel, Mary Ann||P-030|
|Gerdes, Patricia||O-07||Handelsman, David J||P-141|
|Gergelits, Vaclav||P-029||Handoko, Lusy||P-039|
|Gerhardt, Daniel J.||O-07||Harrill, AH||P-071|
|Gerstein, Mark||P-004||Harrow, Jennifer||O-48, O-50|
|Gimenez, J||P-157||Harshbarger, Jayson||O-45, P-070, P-103, P-112|
|Ginsburg, David||O-16, P-056||Harten, Sarah K||P-119|
|Gleeson, Diane||P-067||Hartmann, Fabian||P-148|
|Glover, Leanne||P-067||Harvey, Alan||P-066|
|Go, Tatsuhiko||P-058||Hasegawa, Akira||O-45, P-003|
|Goedert, Michel||O-10||Hasegawa, Sayaka||P-127|
|Gokoolparsadh, Akira||P-146||Hashimoto, K||P-157|
|Goldberg, Tatyana||P-103||Hashimoto, Kosuke||O-45, P-040, P-055|
|Goldie, Belinda J||TS-06/P-035||Hashimoto, Ryota||TS-11/P-102|
|Goldowitz, Dan||O-45||Haubrock, Martin||P-148|
|Gondo, Yoichi||O-52, P-031, P-036, P-074||Havelkova, Helena||O-20|
|Gonzales, Natalia M.||O-41||Hayashi, Masaaki||P-085|
|Goodwin, Leslie||O-31||Hayashizaki, Yoshihide||O-35, O-44, O-45, P-012, P-043, P-044, P-051, P-063, P-112, P-124, P-129, P-149|
|Goos, JACM||P-022||Hayman, G Thomas||P-090, P-115|
|Gopalakrishnan, Shyam||O-41||Heath, Emma||TS-12/P-068|
|Gosheh, Yanal||O-46||Hedlin, Gunilla||P-063|
|Goto, Hiromichi||P-154||Heetveld, Sasja||O-08|
|Goto, Tatsuhiko||TS-04/P-076||Heinemeyer, Wolfgang||TS-16/P-138|
|Graber, Joel H||O-01||Heise, Mark T||P-027|
|Gralinski, Lisa||TS-02/P-037||Herault, Yann||O-29, P-041, P-117|
|Green, Angela||P-067||Heutink, Peter||O-08, O-17|
|Higuchi, Hitoshi||P-072||Ikegawa, Shiro||P-155|
|Higuchi, Mayumi||P-140||Imoto, Seiya||O-36|
|Hill, Andrew||O-24||Inagaki, Shun||P-085|
|Himeno, Ryutaro||P-036||Ingham, Neil||P-065|
|Hiramatsu, Layla||P-042||Ingvorsen, Camilla||P-067|
|Hiraoka, Kiriko||O-36, P-130, TS-13/P-048||Inoue, Takahiro||O-36, TS-01/P-151, TS-13/P-048|
|Hirasawa, Takae||P-033||Iraqi, Fuad||P-088|
|Hiroki, Nagase||P-061||Ireland, Sara||P-121|
|Hiroyuki, Yoda||P-061||Iseki, Hiroyoshi||P-025|
|Ho, Brett||P-042||Ishida, Junji||P-025|
|Ho, Brittany||P-042||Ishihara, Akihiko||TS-03/P-034|
|Ho, Diwei||P-118||Ishii, Kazuo||P-049|
|Hodges, C||P-022||Ishikawa, Mitsuru||P-050|
|Hodgetts, Stuart||P-066||Ishino, Fumitoshi||P-033|
|Hoenerhoff, Mark||P-056||Ishioka, Noriaki||TS-03/P-034|
|Hohenauer, Tobias||P-020||Ishitsuka, Yuichi||P-031|
|Holan, Vladimir||O-20||Isogai, Eriko||P-096, P-108|
|Holt, James M||O-34, P-081||Ito, Haruka||P-032, P-086|
|Hon, Chung-Chau||P-043||Itoh, Masayoshi||O-35, P-044, P-051, P-092, P-103, P-123, P-124|
|Horie, Masafumi||P-044||Itohara, Shigeyoshi||P-154, TS-11/P-102|
|Horner, Neil||P-014, P-150||Iwayama, Yoshimi||P-094|
|Hoshino, Hideharu||P-113||Iyer, K. Swaminathan||P-118|
|Houri-Haddad, Yael||P-088||Jackson, Andrew||O-33|
|Howe, Kerstin||O-50||Jacquot, Sylvie||O-29|
|Howell, Viive||P-045||Jagla, Bernd||O-04|
|Hrabe de Angelis, Martin||O-26, O-40, P-001, P-107, TS-16/P-138||Jarosikova, Tatana||O-20|
|Hsu, Li-Sung||P-139||Jaubert, Jean||O-04|
|Hsu, Yu-Chen||P-019||Javorkova, Eliska||O-20|
|Hsuc, Tsai-Ching||P-018||Jen, Jin||P-004|
|Hu, Ying||O-19||Jesuadian, J. Samuel||O-07|
|Huang, Hong Ming||O-02||Jimenez, Mark||P-141|
|Huang, Yi||P-046, P-060||John, Simon W M||P-057|
|Huber, Eva M.||TS-16/P-138||Johnsen, Jill M||P-114|
|Hughes, Elizabeth||P-109||Johnson, Sara||P-014|
|Hume, David A||O-44||Johnston, Alexander J||P-114|
|Hurles, ME||P-064||Jones, Michael||P-038|
|Huttlin, Edward||O-53||Jordan, DM||O-32|
|Huypens, Peter||O-26||Joshi, Anagha||O-11|
|Iacono, Giovanni||P-041||Jouvion, Gregory||P-098, P-099|
|Ibarra-Soria, Ximena||TS-07/P-047||Justice, Monica J||O-21, P-137, P-143, P-153|
|Ienne, S||P-023||Kaczkowski, Bogumil||P-040, P-051|
|Ikeda, Akihiro||P-072||Kakusho, Nobunori||P-097|
|Ikeda, Rieko||P-122||Kamenava, Larisa P.||P-111|
|Ikeda, Sakae||P-072||Kanapin, Alexander||P-052|
|Ikegami, Yosuke||P-097||Kaneda, Hideki||P-033|
|Kaneko, Mari||O-09||Knopf, Corrina||P-029|
|Kaneko, Megumi||O-13||Ko, Minoru||P-050|
|Kaneko, Takehito||O-30||Kobayashi, Kimio||P-033|
|Kao, Chia-Yu||P-081||Kobayashi, Norio||P-075|
|Kariuki, Silvia||P-053||Kobets, Tatyana||O-20|
|Karp, Natasha||P-067||Koch, Lauren G||O-03|
|Karsten, Stanislav||P-054||Kocher, Jacob||TS-02/P-037|
|Kashi, Kaori||P-055||Koga, Yumiko||P-122|
|Kashimura, Misho||P-033, P-147||Kohda, Takashi||P-033|
|Kasukawa, Takeya||P-003, P-012, P-060, P-079, P-091, P-123||Koide, Tsuyoshi||O-30, P-058, TS-04/P-076, TS-09/P-132|
|Kato, Sachi||O-13, P-006, P-012, P-060, P-101||Kojima, Soichi||P-040|
|Katsuragawa, Yozo||P-155||Kollmus, Heike||O-14|
|Kauppinen, Sakari||P-110||Komano, Hajime||P-050|
|Kawaji, Hideya||O-35, O-45, P-003, P-044, P-051, P-070, P-103, P-112, P-124||Kominami, Ryo||P-096, P-108|
|Kay, Jarren||P-042||Kondo, Masahiro||P-044|
|Kazutaka, Ohi||TS-11/P-102||Kondo, Masayo||P-122|
|Keane, Thomas||O-50, P-065||Kondo, Naoto||P-123|
|Keavney, Bernard||TS-10/P-136||Kono, Hiromitsu||P-059|
|Keck, James||O-01||Konradsen, Jon R||P-063|
|Keener, Victoria||P-153||Koolen, David||P-041|
|Kemp, Harriet||O-33||Kosaki, Kenjiro||P-050|
|Kempen, Marie-Jeanne H.C.||O-07||Koshikawa, Nobuko||O-36, P-130, TS-01/P-151, TS-13/P-048|
|Kere, Juha||P-063||Kostyuk, Svetlana V.||P-111|
|Keum, Sehoon||O-18||Kotaki, Hayato||P-031|
|Khaitovich, Philipp||TS-05/P-077||Kouno, Tsukasa||P-012, P-060, P-080, P-091|
|Khalife, Manal||O-04||Kozaki, Toshinori||P-049|
|Khimulya, Grigory||P-073, P-079||Kozhuharova, Ana||TS-14/P-128|
|Khoriaty, Rami||P-056||Kratz, Anton||O-13|
|Kida, Yasuyuki||P-062||Kress, T||P-157|
|Kikkawa, Yoshiaki||P-127||Krishnamurthy, Sakthisri||O-36, P-061, P-130|
|Kim, Jun-Dal||P-025||Kubota, Takeo||P-033|
|Kim, Sun-Uk||P-152||Kudo, Lili||P-054|
|Kiriko, Hiraoka||P-061||Kuhara, Satoru||P-036|
|Kishima, Mami||P-129||Kulakovskiy, Ivan||P-073, P-079|
|Kitaeva, Kristina||TS-03/P-034||Kumar, S||O-27|
|Kiyonari, Hiroshi||O-09||Kundra, Rishika||O-10|
|Klein-Rodewald, Tanja||TS-16/P-138||Kunieda, Tetsuo||P-030|
|Klingenspor, Martin||O-40||Kunita, Satoshi||P-025|
|Klinken, Peter||O-45, P-149||Kuramoto, Takashi||P-075|
|Kloppman, Edda||P-103||Kushida, Tomoko||P-033, P-147|
|Kneeland, Stephen C||P-057||Kushige, Hiroko||P-062|
|Kwon, Andrew T||P-063||Madissoon, Elo||P-012, P-080|
|Kwon, Tae-Ho||P-152||Maeda, Shiori||P-129|
|Kyle, Stephanie M||O-21||Maerz, Winfried||P-117|
|Lafont, David||P-067||Maillard, Ivan||P-056|
|Lampkin, Shelley||O-02||Makeev, Vsevolod||O-45, P-073|
|Largaespada, DA||P-022||Makino, Shigeru||P-031, P-074|
|Lassmann, Timo||O-13, O-35, O-45, P-012, P-044, P-051, P-080, P-091, P-124||Mallon, Ann-Marie||O-27, P-014, P-150|
|Lathan, Rashida||P-099||Manly, Kenneth F||P-081|
|Laulederkind, Stan||P-090, P-115||Mantsoki, Anna||O-11|
|Launey, Thomas||O-13||Marasca, Federica||O-46|
|Lecellier, Charles||O-45||Marchuk, Douglas||O-18|
|Lee, Brendan||P-153||Mariani, Jessica||P-004|
|Lee, Han Kyu||O-18||Marion de Proce, Sophie||O-33|
|Lee, Ming Ta Michael||P-069, P-155||Marioni, John C.||TS-07/P-047|
|Lee, Weonju||O-45||Marschall, Susan||O-26|
|Leidy-Davis, Tiffany||O-31||Martin, Whitney||O-06|
|Leist, Sarah||O-14||Martone, Jenny||O-46|
|Lelliott, Christopher||P-064, P-067||Masago, Yusaku||P-087|
|Lelliott, Patrick||O-02||Masahiro, Sato||P-082|
|Lennartsson, Andreas||P-079||Masatoshi, Wakamori||P-039|
|Lents, Kai||P-075||Mashimo, Tomoji||O-30|
|Levitin, MO||P-064||Massironi, SG||P-023|
|Lewis, Morag||P-065||Masuya, Hiroshi||P-075, P-131|
|Li, Jun Z||O-03||Mathelier, Anthony||O-45|
|Li, Ruohan||P-066||Matsukawa, Hiroshi||P-154|
|Liakath-Ali, Kifayathullah||P-067, TS-12/P-068||Matsumoto, Yuki||P-058, TS-04/P-076|
|Lin, Jason||O-36||Matsunaga, Atsuko||O-13|
|Lin, Peijie||P-146||Mattick, John||O-42|
|Lin, Shu-Wha||P-019||Mazin, Pavel||TS-05/P-077|
|Lindgren, Gabriella||TS-08/P-144||McAndrews, Monica||P-078|
|Linnekamp, JF||P-022||McCoy, Kathy D.||TS-16/P-138|
|Lipoldova, Marie||O-20||McHugh, Thomas J.||TS-11/P-102|
|Liu, Ye||P-069||McMillan, Leonard||O-34, P-081|
|Lizio, Marina||O-45, P-039, P-044, P-070, P-103, P-112||McMorran, Brendan||O-02|
|Lo, Donald||O-18||McMullan, Rachel C||O-34|
|Logacheva, Maria||TS-03/P-034||Meadows, Jennifer||TS-08/P-144|
|Logan, Darren W.||O-10, P-064, TS-07/P-047||Medema, JP||P-022|
|Long, Jarukit||O-15||Medvedeva, Yulia||O-45, P-079|
|Lukianova, Elena||P-073||Meehan, Terrence F||O-39|
|Luo, S||P-071||Meijer, GA||P-022|
|Lyn-Cook, LE||P-071||Melen, Erik||P-063|
|Lynes, Emily M||O-08||Menachery, Vineet||TS-02/P-037|
|Ma, Wenxiu||O-24||Menck, CF||P-023|
|Ma, Zhongcai||P-054||Mendez, Mickael||P-060, P-080, P-101|
|Macke, Erica||P-072||Meziane, Hamid||P-041|
|Macpherson, Andrew J.||TS-16/P-138||Mianne, Joffrey||P-135|
|Micke, Patrick||P-044||Nakahara, Mai||P-032, P-086|
|Midorikawa, Yutaka||P-104||Nakai, Yuji||P-031|
|Mihola, Ondrej||P-008, P-029||Nakamura, Kazuomi||P-087|
|Mikko, Sofia||TS-08/P-144||Nakamura, Yoshihiko||P-097|
|Miller, Darla R||P-081||Nakanishi, Yuri||P-129|
|Minakuchi, Yohei||P-140||Nakao, Kazuki||P-113|
|Minoda, Aki||P-039||Nakaoka, Hirofumi||P-058, TS-04/P-076|
|Minoda, Akiko||P-017, P-069||Nakashiba, Toshiaki||P-154|
|Mirkhanim, Fatemeh||P-026||Nakouzi, Ghunwa||O-25|
|Mishack, Lena||P-114||Nanchi, Isamu||P-087|
|Mishra, Bibhuti||O-15||Naruse, Kiyoshi||P-075|
|Mitchell, Rod||O-33||Nashef, Aysar||P-088|
|Mituyama, Toutai||P-062||Naumann, Ronald||P-089|
|Miura, Hiromi||P-082, P-095||Neff, Frauke||TS-16/P-138|
|Miura, Ikuo||P-094, P-096||Network, the Gencodys||P-041|
|Miyakawa, Tsuyoshi||O-23||Nicoli, P||P-157|
|Miyake, Kunio||P-033||Nigam, Rajni||P-090, P-115|
|Miyano, Satoru||O-36||Nikolaeva, Darya||P-073|
|Mizuno, Seiya||P-025||Nishimura, Hajime||P-129|
|Mizuno, Yosuke||P-083||Nishino, Jo||P-058, P-140, TS-04/P-076|
|Mizuno-Iijima, Saori||P-025||Nishino, Risako||P-030|
|Mochida, Joji||P-133||Noble, William S||O-24|
|Montagutelli, Xavier||P-098||Nobuko, Koshikawa||P-061|
|Montoliu, L||P-084||Noguchi, Hideki||P-126|
|Moore, Adrian W||P-020||Noguchi, Shuhei||P-003, P-091|
|Mora, Marina||O-46||Noll, Kelsey E||P-027|
|Morgan, Andrew P||O-34, P-024, P-114||Noma, Shohei||O-45, P-092, P-149|
|Morgan, Hugh||P-150||Nordlund, Bjorn||P-063|
|Morgan, Judy||O-31||Nunomura, Satochi||P-001|
|Morgante, Michele||O-46||Nurullin, Leniz||TS-03/P-034|
|Mori, Kazuki||P-036||Nusinow, DP||O-32|
|Mori, Saori||P-085||Oestereicher, Manuela||O-40|
|Morota, Gota||P-093||Oey, Harald M||P-119|
|Morrison, Clayton R||P-027||Ohba, Hisako||P-094|
|Mortensen, AH||P-156||Ohbayashi, Tetsuya||P-087|
|Motakis, Efthymios||P-012, P-060, P-080||Ohmiya, Hiroko||P-093|
|Mukumoto, Yoshiko||O-09||Ohnishi, Tetsuo||P-094|
|Mungall, Chris||O-45||Ohshima, Kazuya||P-075|
|Munger, Steven||O-04, O-53||Ohshima, Mitsuhiro||O-45, P-044|
|Murai, Akihiko||P-097||Ohta, Kunihiro||P-059|
|Nadeau, Joseph||O-25||Ohtsuka, Masato||P-082, P-095|
|Nagae, Genta||P-104||Okamura-Oho, Yuko||P-097|
|Nagase, Hiroki||O-36, P-130, TS-01/P-151, TS-13/P-048||Okano, Hideyuki||O-47, P-050|
|Nagase, Takahide||P-044||Okazaki, Yasushi||P-083|
|Nakachi, Yutaka||P-083||Okuda, Tomohiko||P-087|
|Nakagawa, Hidewaki||P-104||Okumura, Kazuhiro||P-096, P-108|
|Okuno, Hironobu||P-050||Pomp, Daniel||P-009, P-024|
|Olea, Walter||O-54||Porse, Bo Torben||P-007|
|Ollert, Markus||TS-16/P-138||Post, Martin||P-143|
|Onada, Hiroaki||P-011||Poulain, Stephane||O-13, P-006, P-101|
|Oota, Satoshi||O-52, P-036, P-097||Powell, Katie||P-045|
|Orlando, V||P-157||Prosselkov, Pavel||P-154, TS-11/P-102|
|Orlando, Valerio||O-46||Protheroe, Hayley||P-067|
|Ormonde, Callum||P-118||Proulx, Megan||O-15|
|Orsmark-Pietras, Christina||P-063||Puri, Per Lorenzo||O-46|
|Oshima, Takuji||P-085||Pyz, Elwira||O-08|
|O'Sullivan, MG||P-022||Qi, Nathan R||O-03|
|Ota, Satoshi||P-107||Qin, Xian-Yang||P-040|
|Ozaki, Toshinori||TS-13/P-048||Ra, Chisei||P-001|
|Ozturk, Mumin||P-124||Raghupathy, Narayanan||O-53, P-100|
|Paigen, Kenneth||P-008, P-016, P-100||Ramani, Vijay||O-24|
|Palmer, Abraham A.||O-41||Ramilowski, Jordan||P-123|
|Panahandeh, Pouda||P-079||Ramilowski, Jordan A||P-043, P-063, P-103, TS-15/P-106|
|Panthier, Jean-Jacques||O-04, P-098, P-099||Ramirez-Solis, Ramiro||TS-12/P-068|
|Pardo-Manuel de Villena, Fernando||O-05, O-34, P-024, P-081, P-114, TS-02/P-037||Rapin, Nicolas||P-007|
|Parihar, Suraj P||P-124||Rathkolb, Birgit||O-40, TS-16/P-138|
|Paris, Nicole||O-22||Ravasi, Timothy||O-46|
|Park, Young-Ho||P-152||Rehli, Michael||O-45|
|Pass, Johanna||P-065||Reijns, Martin||O-33|
|Paten, Benedict||O-50||Reinholdt, Laura G.||O-06, O-50|
|Pavlovic, Guillaume||O-29||Rekhi, Sohinder||P-065|
|Pearson, Selina||P-065||Ren, Yu-yu||O-03|
|Pelczar, P||P-084||Renard-Guillet, Claire||P-104|
|Perez, Carlos||P-010||Retha, Ahmad||P-150|
|Perkins, Andrew C||P-119||Richardson, Joel||P-011, P-015|
|Persichetti, Francesca||P-038||Richardson, Sandra R.||O-07|
|Persson, Helena||O-45, P-063||Ring, Natalie||P-150|
|Petkov, Petko M||P-008, P-016, P-030, P-100||Ritter, K Elaine||P-105|
|Petkova, Pavlina||P-008||Rizzu, Patrizia||O-08|
|Petri, Victoria||P-090, P-115||Robenson, L||P-064|
|Pham, Son||O-50||Robinson, Nashiya N||P-081|
|Phipps, Richard||P-028||Rocha de Carvalho, MAC||P-023|
|Phuah, Jia Yao||O-15||Rost, Burkhard||P-103|
|Pilzner, Carolin||O-14||Roudnicky, Filip||O-45|
|Pinosio, Sara||O-46||Roy, Riti||TS-15/P-106|
|Pitman, Wendy||P-100||Roy, Sugata||P-124|
|Plante, Jessica||TS-02/P-037||Rozman, Jan||O-40|
|Plessy, Charles||O-12, O-13, P-006, P-012, P-060, P-080, P-091, P-101||Rubin, Carl-Johan||TS-08/P-144|
|Pokorna, Tereza||O-20||Runz, Heiko||P-117|
|Polygalov, Denis||TS-11/P-102||Ryder, Ed||P-067|
|Sabrautzki, Sibylle||P-107||Sethi, S||O-27|
|Saccone, Valentina||O-46||Severin, Jessica||O-45, P-112|
|Saetrom, Pal||O-45||Sharma, Harshita||TS-14/P-128|
|Sagawa, Nobuho||P-107||Shaw, Ginger D||P-081|
|Saghiri, Reza||P-026||Shendure, Jay||O-24, P-153|
|Saito, Akira||P-044||Sheppard, Keith||P-015|
|Saito, Megumi||P-096, P-108||Shi, Jiayuan||O-31|
|Saito, Mikako||P-075||Shibata, Tatsuhiro||P-104|
|Saito, Yutaka||P-062||Shimanuki, Midori||P-096|
|Saitoh, Shinji||P-050||Shimoyama, Mary||P-090, P-115|
|Saitou, Naruya||O-37, O-52||Shin, Jay W||O-49, P-012, P-060, P-080, P-091, P-123|
|Samsonova, Anastasia||P-052||Shioi, Go||P-113|
|Samuel, Didier||P-040||Shiroishi, Toshihiko||P-036, P-059, P-126|
|Sanchez-Andrade, Gabriela||O-10, P-042||Shitara, Hiroshi||P-096|
|Sanchez-Luque, Francisco J.||O-07||Shiura, Hirosuke||P-002|
|Sandelin, Albin||O-35, O-43, O-44, O-45, P-051, P-145||Shrestha, Merina||TS-08/P-144|
|Sanges, Remo||P-038||Siebert, Amy E||P-013, P-114|
|Sano, Chei||P-154||Siemieniak, David R||O-16, P-013|
|Santoro, Claudio||P-038, TS-14/P-128||Silberberg, Gilad||P-063|
|Santos, Alexandre Dos||P-040||Sima, Matyas||O-20|
|Santos, Luis||P-150||Simecek, Petr||O-53|
|Sasaki, Yas||P-062||Simon, M||O-27|
|Sasivarevic, Damir||P-007||Simon-Chazottes, Dominique||P-098|
|Sass, Steffen||O-26||Singleton, Jennifer||P-042|
|Sassetti, Christopher||O-15||Sismeiro, Odile||O-04|
|Satagopam, Venkata||P-103||Slapnickova, Martina||O-20|
|Sato, Masahiro||P-095||Smedley, Damian||TS-12/P-068|
|Saunders, Thomas||O-16, P-109||Smith, Clare||O-15|
|Sawiak, SJ||P-064||Smith, Cynthia||P-011|
|Schaefer, Alexandra||TS-02/P-037||Smith, Jennifer R||P-090, P-115|
|Schaefer, Sabine||O-25||Smyth, Ian||TS-12/P-068|
|Schaeffer, Laurence||O-29||Sneddon, Duncan||P-150|
|Schein, Aleks||P-110||Soederhall, Cilla||P-063|
|Schmeier, Sebastian||P-124||Sohi, Sina Hadi||P-007|
|Schughart, Klaus||O-14||Sohrabi, Yahya||O-20|
|Schultz, Meghan||O-31||Sommers, Olivia||P-116|
|Schumann, A||P-022||Sorg, Tania||P-041, P-117|
|Schurink, Anouk||TS-08/P-144||Sorolla, Anabel||P-118, P-119|
|Scott, PM||P-022||Southard-Smith, E Michelle||P-105, P-120, P-121|
|Selloum, Mohammed||P-041, P-117||Speak, Anneliese||P-067|
|Seo, Jungkyun||O-41||Spiegel, Sarah||P-067|
|Sergeeva, Vasilina A.||P-111||Spillantini, Maria Grazia||O-10|
|Seridi, Loqmane||O-46||Spitz, Francois||P-117|
|Seruggia, D||P-084||Spruce, Catrina||P-100|
|Stanke, Mario||O-50||Tanimoto, Sho||P-133|
|Starr, TK||P-022||Taniuchi, Ichiro||P-134|
|Steel, Karen||P-065, TS-12/P-068||Tarui, Hiroshi||O-45, P-149|
|Stinckens, Anneleen||TS-08/P-144||Tashiro, Toshiyuki||P-155|
|Strom, Tim M.||TS-16/P-138||Tateishi, Ken||P-085|
|Stunnenberg, Henk G.||P-041||Tatsuno, Kenji||P-104|
|Subramanian, Indhu||P-045||Taya, Choji||P-096|
|Sugimoto, Michihiko||P-122||Taylor, Martin||O-33, O-35|
|Sugiyama, Fumihiro||P-025||Teboul, Lydia||P-014, P-135|
|Sugiyama, Hiroshi||O-36, TS-13/P-048||Tee, Chee Wee||P-020|
|Sumiyama, Kenta||TS-09/P-132||Tenin, Gennadiy||TS-10/P-136|
|Summers, Kim M||O-44, O-45||Terskikh, Anastasia||P-052|
|Sunter, David||TS-12/P-068||Thai, Danny||P-042|
|Sunyaev, SR||O-32||Than, BLN||P-022|
|Suzuki, AM||P-157||Theis, Fabian||O-26|
|Suzuki, Ana Maria||O-13, P-040||Thimma, Manjula||O-46|
|Suzuki, Harukazu||P-012, P-046, P-123, P-124, P-129||Thodberg, Malte||O-43|
|Suzuki, Shunsuke||P-085||Thomas, Mark||O-48|
|Suzuki, Takahiro||P-129||Thompson, Zoe||P-042|
|Suzutani, Tatsuo||P-044||Thomson, Peter||O-54|
|Svenson, Karen L||O-53, P-125||Thongjuea, Supat||P-123|
|Svobodova, Milena||O-20||Threadgill, David||P-009|
|Swinburne, June||TS-08/P-144||Thybert, David||O-50|
|Synofzik, Matthis||O-08||Tokuda, Satoko||P-098|
|Szoke-Kovacs, Zsombor||P-014||Tokuzawa, Yoshimi||P-083|
|Tajima, Atsushi||P-001||Tomasini, Livia||P-004|
|Takada, Toyoyuki||P-036, P-075, P-126||Tomberg, Kart||O-16, P-013|
|Takahashi, Aki||TS-09/P-132||Totoki, Yasushi||P-104|
|Takahashi, Chitose||P-092||Toyoda, Atsushi||P-036, P-126, P-140|
|Takahashi, Gou||P-095, P-127||Tracey, Lauren J||P-137|
|Takahashi, Hazuki||P-038, TS-14/P-128||Trachtulec, Zdenek||P-008, P-029|
|Takahashi, Masayo||O-28||Treise, Irina||TS-16/P-138|
|Takahiro, Inoue||P-061||Trempenau, Mette Louise||P-007|
|Takatori, Atsushi||O-36, P-130, TS-01/P-151, TS-13/P-048||Treutelaar, Mary K.||O-03|
|Takatsuki, Terue||P-075||Troelsen, Jesper||O-43|
|Takayama, Eiki||P-075||Tsai, Jie-Heng||P-139|
|Takayoshi, Watanabe||P-061||Tsai, Ming-Shian||P-019|
|Takeda, Masatoshi||TS-11/P-102||Tsuji, Shingo||P-104|
|Takeich, Kazunarii||P-097||Tutaj, Marek||P-090, P-115|
|Tallack, Michael R||P-119||Tyapkina, Oksana||TS-03/P-034|
|Talmane, Lana||O-33||Tyler, Anna||P-100|
|Tamgue, Ousman||P-124||Uchimura, Arikuni||P-140|
|Tanaka, Nobuhiko||P-075, P-131||Ueda, Hiroki||P-104|
|Tanaka, Shoma||P-050||Umehara, Takashi||P-017, P-039|
|Tanaka, Yuji||P-051||Upton, Dannielle H||P-141|
|Tanave, Akira||TS-09/P-132||Urban, Alexander||P-004|
|Taniguchi, Yuichi||P-017||Urun, Fatma||P-020|
|Vaccarino, Flora||P-004||West, Ande||P-027|
|van Bokhoven, Hans||P-041||Westerberg, Carl Henrik||P-014|
|Van Den Broek, E||P-022||Westerberg, Henrik||P-150|
|Van Der Weyden, Louise||P-067||Westrick, Randal J||O-16, P-013, P-114|
|Vancollie, Valerie||P-067, TS-12/P-068||Whelan, Cassandra||P-065|
|Vashi, Neeti||P-143||White, Jacqueline||P-065, TS-12/P-068|
|Vasmatzis, Nikolaos||P-004||Whitelaw, Emma||P-119|
|Veiko, Natalya N.||P-111||Whitmore, Alan C||P-027|
|Velie, Brandon||TS-08/P-144||Wieland, Thomas||TS-16/P-138|
|Velt, Amandine||P-117||Williams, Kendra||O-19|
|Vendruscolo, Michele||O-10||Williams, Robert||O-15|
|Verbeek, Stephanie||P-114||Williamson, Peter||O-54|
|Vermeulen, L||P-022||Wilson, Michael||P-004|
|Vigot, Rejan||O-13||Wingender, Edgar||P-148|
|Vikhlyantsev, Ivan||TS-03/P-034||Winteringham, Louise||O-45, P-149|
|Vitezic, Morana||O-43||Winther, Ole||P-007|
|Vitting-Seerup, Kristoffer||O-43, O-44, O-45, P-145||Witonsky, David||P-053|
|Vizcay-Barrena, Gema||P-067||Woeller, Collynn||P-028|
|Voineagu, Irina||P-146||Wolf, Eckhard||O-40, TS-16/P-138|
|Vojtiskova, Jarmila||O-20||Wong, Frances||P-120|
|Volkova, Valeriya||O-20||Wu, Hsiao-Huei||P-105|
|Vorontsov, Ilya||P-073, P-079||Wu, Moya||O-26|
|Wada, Kenta||P-095, P-127||Wu, Yanhong||P-004|
|Wakabayashi, Yuichi||P-096, P-108||Xiao, Mei||P-015|
|Wakana, Shigeharu||P-033, P-075, P-094, P-096, P-108, P-140, P-147, Panel, TS-13/P-048||Yadgary, Liran||P-024|
|Walker, Denise||P-004||Yagami, Ken-ichi||P-025|
|Walker, Michael||P-008, P-016, P-100||Yagi, Takeshi||P-140|
|Walters, Kirsty A||P-141||Yaguchi, Kunio||P-154|
|Wang, Dan O||TS-06/P-035||Yaguchi, Masami||P-133|
|Wang, Edina||P-118||Yahata, Mitsunori||P-069, P-155|
|Wang, Shur-Jen||P-090, P-115||Yaikhom, Gagarine||P-150|
|Wang, Yu||O-03||Yamada, Ikuko||P-033, P-147|
|Watanabe, Takayoshi||O-36, P-130, TS-01/P-151, TS-13/P-048||Yamaguchi, Yoko||O-45, P-044|
|Watase, Megumi||O-09||Yamamoto, Shogo||P-104|
|Watt, Fiona||P-067, TS-12/P-068||Yamamura, Ken-ichi||P-032, P-086|
|Wattenhofer-Donze, Marie||O-29||Yamashita, Yui||O-09|
|Wei, Jerry||O-54||Yanagida, Toshio||P-017|
|Weiss, Ervin||P-088||Yang, Fan||O-24|
|Weissman, Sherman||P-004||Yang, Jiann-Jou||P-018|
|Wells, Christine||O-44, O-45||Yang, Sujeong||O-10|
|Wells, Sara||P-135||Yao, Pu||P-146|
|Welsh, Catherine E||P-081||Yatsuka, Yukiko||P-083|
|Yoda, Hiroyuki||O-36, P-130, TS-01/P-151, TS-13/P-048|
|Yoshinobu, Kumiko||P-032, P-086|
|Young, Robert||O-33, O-35|
|Zaverucha do Valle, Tania||P-098|
|Zhang, Qi||P-154, TS-11/P-102|
|Zhang, Yanfei||P-069, P-155|
|Zhu, Guojing||O-16, P-013, P-056, P-114|
|Zucchelli, Silvia||P-038, P-110, TS-14/P-128|