Developmental Profile of H19 Differentially Methylated Domain (DMD) Deletion Alleles Reveals Multiple Roles of the DMD in Regulating Allelic Expression and DNA Methylation at the Imprinted H19/Igf2 Locus

Abstract

The differentially methylated domain (DMD) of the mouse H19 gene is a methylation-sensitive insulator that blocks access of the Igf2 gene to shared enhancers on the maternal allele and inactivates H19 expression on the methylated paternal allele. By analyzing H19 DMD deletion alleles H19^ΔDMD and H19^{Δ3.8kb-5′H19} in pre- and postimplantation embryos, we show that the DMD exhibits positive transcriptional activity and is required for H19 expression in blastocysts and full activation of H19 during subsequent development. We also show that the DMD is required to establish Igf2 imprinting by blocking access to shared enhancers when Igf2 monoallelic expression is initiated in postimplantation embryos and that the single remaining CTCF site of the H19^ΔDMD allele is unable to provide this function. Furthermore, our data demonstrate that sequence outside of the DMD can attract some paternal-allele-specific CpG methylation 5′ of H19 in preimplantation embryos, although this methylation is not maintained during postimplantation in the absence of the DMD. Finally, we report a conditional allele floxing the 1.6-kb sequence deleted from the H19^ΔDMD allele and demonstrate that the DMD is required to maintain repression of the maternal Igf2 allele and the full activity of the paternal Igf2 allele in neonatal liver.

We thank J. Richa and the University of Pennsylvania Transgenic Core Facility for the production of chimeric mice and Grace Yang for technical assistance. We thank Mark Magnuson for the C57BL6-TgN(AlbCre)21Mgn mice. We thank Nora Engel and Raluca Verona for their comments on the manuscript.

This work was supported by U.S. Public Service grant GM51279 and the Howard Hughes Medical Institute. A.M.F. was supported by an NIH predoctoral training grant (T32 HD-07516).