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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Scientist Development Award - Research & Training (K01)
Project #
1K01CA115906-01
Application #
6960114
Study Section
Subcommittee G - Education (NCI)
Program Officer
Ojeifo, John O
Project Start
2005-07-01
Project End
2010-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
1
Fiscal Year
2005
Total Cost
$111,947
Indirect Cost
Name
University of Pennsylvania
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
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