The study of epigenetic mechanisms will lead to understanding of how the genome functions as a developmental blueprint and how perturbations of gene expression patterns can lead to cancer. The goal of this proposal is to elucidate epigenetic mechanisms leading to allele-specific gene silencing at two clusters of imprinted genes. Imprinted genes have parental-specific monoallelic expression. Differential DNA methylation at specific sequences is the epigenetic modification most consistently associated with imprinted genes. Many imprinted domains also exhibit expression of non-coding RNAs. Very little is known of the molecular mechanisms involved in allele-specific silencing and the role of the non-coding RNAs in modulating this process. Using the mouse as a model, this proposal will first elucidate the physical interactions between regulatory elements at the H19/lgf2 locus through chromosome conformation capture technology, testing the hypothesis that there are allele-specific interactions. Mice with targeted mutations in the differentially methylated domain (DMD) at H19 will be compared to the wild-type mice. Second, a targeting experiment will test whether transcription of Kcnq1ot, an imprinted antisense non-coding RNA produced from exon 10 of Kcnq1, is required to maintain the imprinting at this locus by inserting a polyadenylation site in the Kcnq1ot1 gene. A third experiment will exploit transgenic RNA interference to target the Kcnq1ot1 transcript and test whether the Kcnq1ot1 RNA itself plays a role in establishing and maintaining allele-specific gene silencing. Genes in both the H19/lgf2 and the Kcnql domains are involved in Beckwith-Wiedemann syndrome, which causes prenatal overgrowth and predisposition to cancer. Loss of imprinting at both domains has also been implicated in several human neoplasias. Thus, these studies will provide a more thorough understanding of the regulatory mechanisms deployed in gene silencing and will give insight into how these mechanisms can go awry in cancerous cells.
|Engel, Nora; Raval, Anjali K; Thorvaldsen, Joanne L et al. (2008) Three-dimensional conformation at the H19/Igf2 locus supports a model of enhancer tracking. Hum Mol Genet 17:3021-9|
|Engel, Nora; Thorvaldsen, Joanne L; Bartolomei, Marisa S (2006) CTCF binding sites promote transcription initiation and prevent DNA methylation on the maternal allele at the imprinted H19/Igf2 locus. Hum Mol Genet 15:2945-54|