A 5′ 2-Kilobase-Pair Region of the Imprinted Mouse H19 Gene Exhibits Exclusive Paternal Methylation throughout Development

Abstract

The imprinted mouse H19 gene is hypermethylated on the inactive paternal allele in somatic tissues and sperm. Previous observations from a limited analysis have suggested that methylation of a few CpG dinucleotides in the region upstream from the start of transcription may be the mark that confers parental identity to the H19 alleles. Here we exploit bisulfite mutagenesis coupled with genomic sequencing to derive the methylation status of 68 CpGs that reside in a 4-kb region 5 to the start of transcription. This method reveals a 2-kb region positioned between 2 and 4 kb upstream from the start of transcription that is strikingly differentially methylated in midgestation embryos. At least 12 of the cytosine residues in this region are exclusively methylated on the paternal allele in blastocysts. In contrast, a 350-bp promoter-proximal region is less differentially methylated in midgestation embryos and, like most of the genome, is largely devoid of methylation on both alleles in blastocysts. We also demonstrate exclusive expression of the maternal H19 allele in the embryos that exhibit paternal methylation of the upstream 2-kb region. These data suggest that the 2-kb differentially methylated region acts as a key regulatory domain for imprinted H19 expression.

Role of CTCF Binding Sites in the Igf2/H19 Imprinting Control Region

“…4A; see also Fig. 5B), as is the case in normal fetuses (45,46). The ϩ/Ϫ(P) fetuses showed similar hypermethylation of the paternal chromosome around the G-rich repeats, at the pro- (Fig.…”

“…Bisulfite sequencing was performed on cells embedded into agarose beads (25). DNA from 20,000 and 25,000 cells was amplified using primers described earlier (45). One PCR was performed on DNA from each of two conversion reactions.…”

“…On the paternal chromosome, the ICR is methylated (45,46). CpG methylation inhibits CTCF binding (3,10,12,16,28,38); therefore, the methylated paternal ICR lacks insulator activity and the paternal Igf2 promoter interacts with the downstream enhancers.…”

“…Differential methylation of the Igf2-H19 ICR, the establishment of which could be viewed as the imprinting mechanism itself, is laid down in the male and female germ lines (6,(45)(46)(47). It was shown that establishment of differential methylation is a function autonomous to the ICR (15), but the germ line-specific processes that establish this differential ICR methylation are undefined.…”

Parental Allele-Specific Chromatin Configuration in a Boundary–Imprinting-Control Element Upstream of the Mouse H19 Gene

“…During prenatal and early-postnatal development, H19 is expressed in tissues of mesodermal and endodermal origin, in which the gene and several kilobases of upstream sequences are hypermethylated on the inactive paternal allele (4,7,14,18,44). Detailed methylation studies have pinpointed a core region, localized at approximately 2 to 4 kb upstream of the gene, in which CpG dinucleotides are paternally methylated throughout development (38,52,53,57). Furthermore, a study with transgenic mice has suggested that the H19 upstream region is necessary for the imprinted expression of H19 (13).…”