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.
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.
Imprinting of the linked and oppositely expressed mouse H19 and Igf2 genes requires a 2-kb differentially methylated domain (DMD) that is located 2 kb upstream of H19. This element is postulated to function as a methylation-sensitive insulator. Here we test whether an additional sequence 5 of H19 is required for H19 and Igf2 imprinting. Because repetitive elements have been suggested to be important for genomic imprinting, the requirement of a G-rich repetitive element that is located immediately 3 to the DMD was first tested in two targeted deletions: a 2.9-kb deletion (⌬DMD⌬G) that removes the DMD and G-rich repeat and a 1.3-kb deletion (⌬G) removing only the latter. There are also four 21-bp GC-rich repetitive elements within the DMD that bind the insulator-associated CTCF (CCCTC-binding factor) protein and are implicated in mediating methylation-sensitive insulator activity. As three of the four repeats of the 2-kb DMD were deleted in the initial 1.6-kb ⌬DMD allele, we analyzed a 3.8-kb targeted allele (⌬3.8kb-5H19), which deletes the entire DMD, to test the function of the fourth repeat. Comparative analysis of the 5 deletion alleles reveals that (i) the G-rich repeat element is dispensable for imprinting, (ii) the ⌬DMD and ⌬DMD⌬G alleles exhibit slightly more methylation upon paternal transmission, (iii) removal of the 5 CTCF site does not further perturb H19 and Igf2 imprinting, suggesting that one CTCF-binding site is insufficient to generate insulator activity in vivo, (iv) the DMD sequence is required for full activation of H19 and Igf2, and (v) deletion of the DMD disrupts H19 and Igf2 expression in a tissue-specific manner.Genomic imprinting is an epigenetic modification to differentiate the maternal and paternal alleles of a gene, the consequence of which is parent-specific gene expression. Differential DNA methylation, allele-specific nuclease hypersensitivity, and repetitive elements have been proposed to be important for the regulation of imprinted gene expression (5, 6, 43). The imprinted mouse H19 gene, which is expressed from the maternal allele, possesses each of these characteristics. A 2-kb sequence between Ϫ4 and Ϫ2 kb relative to the H19 transcription start site (designated as the differentially methylated domain [DMD] or the imprinting control region) is exclusively methylated on the paternal allele throughout development (40,52,54). Maternally specific hypersensitive sites have been mapped to this 2-kb region in embryonic and neonatal tissues (28,34). Coincident with these hypersensitive sites are four 21-bp GC-rich repeats that are conserved in mouse, rat, and human tissue (25, 48). Finally, a 461-bp sequence harboring 32 copies of a G-rich element (GGGGTATA) resides immediately 3Ј to the DMD (48, 53).The mouse H19 gene is located 90 kb 3Ј to the oppositely imprinted insulin-like growth factor 2 (Igf2) gene. The expression of the H19 and Igf2 genes is mediated through shared enhancers that are located 3Ј to H19 (1,3,32,37). Perhaps the most critical part of the joint regulation of H19...
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