Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2

Abstract

Differentially methylated sequences associated with imprinted genes are proposed to control genomic imprinting. A 2-kb region located 5 to the imprinted mouse H19 gene is hypermethylated on the inactive paternal allele throughout development. To determine whether this differentially methylated domain (DMD) is required for imprinted expression at the endogenous locus, we have generated mice harboring a 1.6-kb targeted deletion of the DMD and assayed for allelic expression of H19 and the linked, oppositely imprinted Igf2 gene. H19 is activated and Igf2 expression is reduced when the DMD deletion is paternally inherited; conversely, upon maternal transmission of the mutation, H19 expression is reduced and Igf2 is activated. Consistent with the DMD's hypothesized role of setting up the methylation imprint, the mutation also perturbs allele-specific methylation of the remaining H19 sequences. In conclusion, these experiments show that the H19 hypermethylated 5 flanking sequences are required to silence paternally derived H19.Additionally, these experiments demonstrate a novel role for the DMD on the maternal chromosome where it is required for the maximal expression of H19 and the silencing of Igf2. Thus, the H19 differentially methylated sequences are required for both H19 and Igf2 imprinting.

Sequence and Comparative Analysis of the Mouse 1-Megabase Region Orthologous to the Human 11p15 Imprinted Domain

“…CpG islands are normally unmethylated, but allele-specific methylation of CpG islands appears to mark both the inactive X chromosome (Yen et al 1984) and many imprinted genes, for example, H19, Snrpn, and Igf2r (Brandeis et al 1993;Shemer et al 1997;Wutz et al 1997). In addition, GC-rich sequences that are not CpG islands (i.e., they meet the first, but not the second criterion above) may also be differentially methylated (termed a differentially methylated region) in the vicinity of imprinted genes, for example Igf2 (Sullivan et al 1999) and a second site 2-4 kb upstream of the H19 CpG island (Thorvaldsen et al 1998). Therefore, one of our goals was to identify conserved CpG islands and GC-rich sequences that might serve as a substrate for future experiments to investigate allelespecific methylation.…”

“…Thus it is likely that both local and regional cis-acting elements are involved in the regulation of genomic imprinting. However, almost nothing is known about the identity or location of such regulatory elements, with the notable exception of a region that has been intensively studied upstream of and downstream from the H19 gene (Thorvaldsen et al 1998;Bell and Felsenfeld 2000;Hark et al 2000;Srivastava et al 2000).…”

Epigenetic deregulation of imprinting in congenital diseases of aberrant growth

“…Particularly, deletion of the H19 ICR resulted in biallelic expression of Igf2 ('loss of imprinting'), when maternally transmitted. (16) Deletion of the KvDMR1 Imprinting control region abrogates imprinting of the paternally repressed genes in this domain, when paternally inherited. This causes growth retardation, which supports the hypothesis that abnormal silencing at CDKN1C and other genes along the domain negatively affects growth.…”

Erasure of methylation imprint at the promoter and CTCF-binding site upstream of H19 in human testicular germ cell tumors of adolescents indicate their fetal germ cell origin

“…The 5 0 region of H19 is methylated only on the paternal allele in both mouse (Bartolomei et al, 1993;Ferguson-Smith et al, 1993) and human (Jinno et al, 1996). This region is regarded as a key domain in the control of imprinting at this locus (Thorvaldsen et al, 1998). The DMR of H19 contains seven potential CTCF-binding sites.…”