International Mammalian Genome Society

logo18th International Mouse Genome Conference

17-22 October 2004, Seattle, USA


ORAL PRESENTATION

WEDNESDAY OCTOBER 20

9.00am – 9.15am

USING ENU MUTAGENESIS TO IDENTIFY NEW GENES INVOLVED IN EPIGENETIC GENE REGULATION

Lossie AC, Justice MJ

Baylor College of Medicine, Houston, United States

DNA methylation is an important biological process that plays a central role in epigenetic gene regulation. Imprinted genes often demonstrate differential methylation between the maternal and paternal chromosomes. In the past few years, studies have shown that DNA Methylation, in concert with chromatin structure, can alter gene expression. Defects in the processes that establish and maintain DNA methylation imprints often cause severe birth defects, including Angelman, Prader-Willi and immunodeficiency-centromeric instability facial-anomalies syndromes. In addition, some spontaneous abortions could be caused by defects in epigenetic gene regulation.

Although many imprinted genes have been characterized, we are just beginning to learn how these genes are regulated. In order to determine the mechanisms underlying mammalian epigenetic gene regulation, we are using N-ethyl-N-nitrosourea mutagenesis to select for homozygous mutant mice that have defects in establishing and/or maintaining well-established DNA methylation profiles. We identified 22 recessive mutant lines that are excellent candidates. These mutants die early during embryogenesis, and some demonstrate a broad phenotypic spectrum. These characteristics are consistent with the disruption of one or more global DNA methylation processes.

We used sodium bisulfite to identify mutant lines that have defective DNA methylation imprints. Since sodium bisulfite converts unmethylated (U) cytosines to uracils while leaving methylated (M) cytosines intact, we designed sets of primers to amplify either the M or U allele of several well-characterized differentially methylated regions (DMR) including: p57Kip2, Kvlqt1, Snrpn, Gnas, H19, Grb10, Ube3a and U2af1-rs1. So far, three lines show abnormal methylation at one or more DMR, and complementation studies indicate that two of the lines are allelic. This complementation group has two distinct phenotypes. The most severe allele dies by E6.5 with a pronounced lack of methylation at U2af1-rs1, while the other survives beyond E8.5, and has a less obvious methylation defect. The third mutant dies at E9.5 with defects in cardiac development and a disruption in methylation at Kvlqt1. Current studies will determine if other DMRs are abnormal in these three mutants. The identification of these mutations will give us a better understanding of the mechanisms underlying DNA methylation during embryogenesis, and provide us with a model to study epigenetic gene regulation in the mammal.

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