Interrelated roles of repressive chromatin modifiers in genomic imprinting

Genomic imprinting is an epigenetic process in which genes are monoallelically expressed according to their parent of origin. This process requires an imprinting mark to designate the chromosomes\u27 parental identity. The best–studied candidate mark is DNA methylation. Imprinted genes are found in clusters, where their expression is controlled by regulatory elements that are methylated on only one allele. Mutations in the maintenance DNA methyltransferase gene, Dnmt1, cause loss of imprinting—biallelic expression or silencing—of nearly all imprinted genes. However, not all imprinted genes lose imprinting in these mutants, suggesting that other mechanisms are also involved. I have investigated the ways in which three different chromatin-modifying factors regulate imprinting. First, I performed a systematic examination of the effects of Dnmt1 mutations on imprinting, comparing the effects of two different mutant alleles on expression in the embryo and placenta. I identified genes that do not rely entirely on DNA methylation for proper expression. The extent to which genes depended on DNMT1 varied between imprinted domains, providing insight into the different regulatory mechanisms employed by these domains. Second, I investigated the individual contributions of maternal and zygotic Embryonic Ectoderm Development (EED) to imprinting. EED is a member of the Polycomb Repressive Complex 2, a histone H3 lysine 27-specific methyltransferase and an important repressor of gene expression. I found that maternal EED is required in the oocyte for establishment of DNA methylation at the Snrpn domain; maternal EED is also required following fertilization to repress the paternal allele of H19. Finally, I have been part of a collaborative effort to investigate the roles of the Histone Deacetylases 1 and 2 in the establishment of DNA methylation in oocytes. We found that these genes are also required for deposition of methylation in the Snrpn domain. Furthermore, the role of HDAC1 and –2 in de novo methylation appears to be specific to imprinted genes, and may be mediated by subcellular localization of the methyltransferase DNMT3A. These results provide important insights into the relative contributions of these chromatin modifiers to the regulation of imprinting and the ways in which they cooperate to ensure proper gene expression