Roles of Protein Factors in Regulation of Imprinted Gene Expression

Genomic imprinting is an important epigenetic phenomenon in which only one parental allele is expressed. Allele-specific DNA methylation often exists in imprinted control regions (ICRs) and is required for properly imprinted expression. Imprinting control in mammals involves an insulator mechanism that requires CTCF or a long ncRNA silencing mechanism. In this dissertation, I studied functions of protein factors in genomic imprinting. First, methylated DNA binding proteins are involved in maintenance of DNA methylation at imprinted loci and required for selective silencing of one specific allele. We showed that MBD3 was localized to paternal H19 ICR. By RNA interference experiments in preimplantation embryos, we showed that MBD3 and its NuRD complex cofactor MTA2 were required for maintenance of the paternal methylation at the H19 ICR, and for silencing of the paternal H19. MTA2 is also required for Peg3 allelic expression. These results demonstrate new roles of the NuRD complex in genomic imprinting. Moreover, I showed allele-specific associations of MBD1 and Kaiso with imprinted loci, implicating functional requirements of these proteins in imprinted regulation. Second, I demonstrated colocalization of CTCF and the cohesin complex at three imprinted loci. CTCF and cohesins preferentially bind to the unmethylated allele of H19 ICR and Gtl2 DMR, and both alleles of KvDMR1 in mouse embryonic fibroblast cells (MEFs). To determine the functional importance of CTCF and cohesins, CTCF and cohesins were depleted in MEFs. Monoallelic expression of imprinted genes was maintained. However, mRNA levels for imprinted genes were typically increased; for H19 and Igf2 the increased expression was independent of the CTCF binding sites in the ICR. These experiments demonstrate an unappreciated role for CTCF and cohesins in the repression of imprinted genes in somatic cells. Finally, I have established F1 hybrid trophoblast stem (TS) cell lines that can be used as a model system to further study functions of protein factors in genomic imprinting and extraembryonic development. Overall, this dissertation has provided new and important insights into roles of protein factors in regulation of imprinted gene expression