International Mammalian Genome Society

logo18th International Mouse Genome Conference

17-22 October 2004, Seattle, USA


POSTER 6 - EXPERIMENTAL CONFIRMATION OF ENHANCER ACTIVITY AT LVIS1, A CONSERVED GENE-DISTANT ANCIENT REGULATORY SITE.

Weiser K, Justice MJ

Baylor College of Medicine, Houston, United States

The recent availability of genome sequence from many species has opened myriad possibilities for comparative genomics.  This allows us not only to chronicle genomic evolution with a focus on genes, but also to more effectively identify sequences distant from genes that are important for the proper development and function of the organism.  Amid an ever-changing sea of “junk” DNA sequences, there are segments that are conserved through millions of years of evolution, yet are distant from genes. These sequences are likely to be enhancers, repressors, locus control regions, matrix attachment regions, insulators, and some may have functions yet to be recognized.  We have identified one of these conserved gene-distant sites through a viral insertional mutagenesis screen for leukemia in AKXD mice.  This site was termed Lymphoid Viral Insertion Site 1 (Lvis1).  Lvis1 is the most frequent site of insertion leading to leukemia in the AKXD lymphoid tumors.  Analysis of mRNA of flanking genes by Northern blot and quantitative real-time PCR revealed two genes that are up-regulated in Lvis1 tumors, Hex (an early expressed divergent homeobox gene) and Eg5 (a kinesin-related motor protein).  These genes are 50-100kb upstream of Lvis1 and transcription from genes equivalent distances downstream is unaffected.  Sequence comparisons of Lvis1 reveal a striking evolutionary conservation with 92.5% sequence identity between Human and chicken over  226 basepairs. DNaseI hypersensitivity assays and mobility shift assays reveal factor binding within Lvis1. Luciferase assays in cell culture demonstrate enhancer activity of Lvis1.  These data imply a mechanism of gene regulation with strict sequence requirements and a mechanism for targeting specific genes for regulation at a distance.  Recent work has identified 156 intergenic sites longer than 199bp with 100% evolutionary conservation between human, rat, and mouse with enrichment of these sites near early developmental genes (Haussler, D. Science 304:1321. 2004).  The striking conservation of numerous sites distant from target genes and the discovery of enhancer activity at Lvis may indicate a common mode of gene regulation involving distant enhancer sites.

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