Maintenance of Paternal Methylation and Repression of the Imprinted H19 Gene Requires MBD3

Abstract

Paternal repression of the imprinted H19 gene is mediated by a differentially methylated domain (DMD) that is essential to imprinting of both H19 and the linked and oppositely imprinted Igf2 gene. The mechanisms by which paternal-specific methylation of the DMD survive the period of genome-wide demethylation in the early embryo and are subsequently used to govern imprinted expression are not known. Methyl-CpG binding (MBD) proteins are likely candidates to explain how these DMDs are recognized to silence the locus, because they preferentially bind methylated DNA and recruit repression complexes with histone deacetylase activity. MBD RNA and protein are found in preimplantation embryos, and chromatin immunoprecipitation shows that MBD3 is bound to the H19 DMD. To test a role for MBDs in imprinting, two independent RNAi-based strategies were used to deplete MBD3 in early mouse embryos, with the same results. In RNAi-treated blastocysts, paternal H19 expression was activated, supporting the hypothesis that MBD3, which is also a member of the Mi-2/NuRD complex, is required to repress the paternal H19 allele. RNAi-treated blastocysts also have reduced levels of the Mi-2/NuRD complex protein MTA-2, which suggests a role for the Mi-2/NuRD repressive complex in paternal-specific silencing at the H19 locus. Furthermore, DNA methylation was reduced at the H19 DMD when MBD3 protein was depleted. In contrast, expression and DNA methylation were not disrupted in preimplantation embryos for other imprinted genes. These results demonstrate new roles for MBD3 in maintaining imprinting control region DNA methylation and silencing the paternal H19 allele. Finally, MBD3-depleted preimplantation embryos have reduced cell numbers, suggesting a role for MBD3 in cell division.

Author Summary

Genomic imprinting is a specialized system of gene regulation whereby only one copy of a gene is used, either the maternal or the paternal copy. Misregulation of imprinting in humans results in developmental disorders such as Beckwith-Wiedemann Syndrome, and is implicated in many cancers. Study of imprinted genes in mice can lead to a greater understanding of these diseases as well as insight into gene regulation. Many imprinted genes are associated with methylation on the silenced allele. The imprinted gene H19 is maternally expressed and paternally methylated in a region upstream of the promoter known as the differentially methylated domain. This region is required for proper imprinted expression of H19 and its upstream imprinted neighbor Igf2. Our studies have explored the requirement for methyl-CpG binding protein 3 (MBD3) in silencing of the paternal allele. MBD3 is known to be part of a repressive complex that resides at silenced genes. In our experiments, we have shown that MBD3 is required for imprinting of H19, and is also required for the maintenance of methylation on the paternal allele. Finally, the MBD3 protein can be found at the differentially methylated domain. The identification of a protein required for silencing of the paternal allele of H19 is an important step in understanding regulation of this gene.