Epigenetic Regulation of Imprinting in Mouse Embryo

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The imprinted H19 gene is closely linked to the oppositely imprinted Igf2 gene in both mouse and humans. The imprinted expression of these genes is mediated by a 2 kb differentially methylated region (DMR) located 5' to the H19 gene. In vitro when the DMR is situated between an enhancer and promoter, it functions as a boundary that blocks the enhancer from engaging the promoter. The boundary function is likely mediated by the methylation-sensitive transcriptional regulatory protein CTCF. According to the boundary model, CTCF binds to the DMR on the maternal allele, thereby allowing the maternal H19 gene exclusive access to the enhancers. On the paternal chromosome, the hypermethylated DMR cannot bind CTCF. This hypermethylation results in both suppressing paternal H19 promoter activity and permitting the enhancers to engage exclusively the paternal Igf2 promoter. The model thus accounts for the observed maternal H19 expression and the reciprocal paternal Igf2 expression. Specific Aim 1 will use RNA interference (RNAi) in the mouse to test the hypotheses that (1) CTCF binding on the maternal allele is responsible for the exclusive maternal H19 expression, and (2) DNA hypermethylation and the interacting DNA methyl-binding proteins on the paternal allele mediate the repression of H19 and hence permits the exclusive paternal Igf2 expression. In addition, this aim will test the hypothesis that CTCF binding to the maternal DMR in the oocyte prevents DNA methylation and thereby ensures maternal H19 expression in the embryo. Understanding the expression of imprinted genes has implications for the practice of Assisted Reproductive Technology (ART). The practice of ART has increased dramatically during the past two decades and has resulted in the birth of tens of thousands of children. Subtle changes in the culture media for preimplantation mouse embryos can result in loss-of-imprinting of the normally imprinted and maternally expressed H19 gene. The resulting biallelic expression correlates with the loss of methylation in the DMR on the paternal allele. Although it is not known if such changes occur during the course of culture of human preimplantation embryos (and if there are long-term consequences), ART is occurring in the virtual absence of any basic research using an animal model. Specific Aim 2 will use the mouse as a model system to test the hypotheses that (1) embryo culture results in the differential loss-of-imprinting of imprinted genes, (2) this loss-of- imprinting is linked with the loss of DNA methylation of specific cytosines in the DMR, and (3) following implantation the ability of the embryo to restore the correct DNA methylation pattern and imprinted gene expression is coupled with successful development to term.