A subset of genes in mammals is regulated by genomic imprinting, a process that results in unequal expression of the maternal and paternal alleles of certain genes. As a consequence, deleterious mutations or deletions in the single expressed allele of an imprinted gene will result in the absence of a functional gene product. In humans, disruptions in imprinting and imprinted genes account for the human genetic diseases Beckwith-Wiedemann Syndrome, Prader-Willi Syndrome and Angelman Syndrome and for cancers such as Wilms' tumor. The objective of this proposal is to investigate the mechanism by which parental identity of imprinted genes is established and maintained. The studies will employ the H19 gene, which is expressed from the maternally-derived allele in mice and humans. The imprinting of H19 and the linked and oppositely imprinted Igf2 gene, is mediated, at least in part, through the 2 kb differentially methylated domain (DMD) that is located 2 kb upstream from the start of H19 transcription. The DMD is hypermethylated on the repressed paternal allele, hypersensitive to nucleases on the expressed maternal allele and is hypothesized to act as a methylation-sensitive insulator. The insulator function is suggested to act through a set of 4 short repetitive elements that bind the transcriptional regulatory protein CTCF in vitro. This proposal will investigate the mechanism of imprinting at the H19/Igf2 locus through the following experiments: (1) to determine whether CTCF binding sites and the CpG dinucleotides within the binding sites are necessary for H19 and Igf2 imprinting, mutations will be introduced at the endogenous locus; (2) to determine whether conserved sequence 3' to the H19 transcription unit is important for H19 and Igf2 imprinting and expression, a 4.2 kb fragment deletion and replacement of H19 endodermal enhancers with AFP enhancers will be performed at the endogenous locus; (3) to determine the source of allelic marking leading to the allele specific remethylation of the H19 locus in the male embryonic germ cells, chromatin patterns will be examined. Elucidation of the sequences required for imprinting and the nature of imprint establishment are essential for the identification of the trans-acting factors that mediate genomic imprinting.
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