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, a number of cases of Silver-Russell 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 imprinting control region (ICR) that is located 2 kb upstream from the start of H19 transcription. The ICR, which is also designated the differentially methylated domain (DMD), is hypermethylated on the repressed paternal allele and acts as a methylation-sensitive insulator through CTCF-binding on the maternal allele. This proposal will investigate the mechanism of imprinting at the H19/Igf2 locus through the following experiments: (1) to determine whether ICR/DMD sequence in addition to CTCF binding sites and the proper spacing of the CTCF binding sites are critical for H19/Igf2 imprinting;(2) to determine if CpG mutations outside of the CTCF-binding sites disrupt imprinting;(3) To examine the chromatin structure of the H19 locus in germ cells and embryonic cells and assess the role of these epigenetic modifications in imprinting. In addition to elucidating the mechanism of imprint establishment and maintenance at this locus during development, these experiments will model newly identified mutations in individuals with Beckwith-Wiedemann Syndrome and Silver-Russell Syndrome, providing a better understanding of the etiology of the disease.
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