Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region.

Abstract

ABSTRACTExpression of imprinted genes is classically asso-ciated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus isconsidered a paradigm for epigenetic regulation. Inmice, as in humans, the essential H19 DMR—targetof the CTCF insulator—is located between the twogenes. Here, we performed a pyrosequencing-basedquantitative analysis of its CpG methylation innormal human tissues. The quantitative analysis ofthe methylation level in the H19 DMR revealed threeunexpected discrete, individual-specific methylationstates. This epigenetic polymorphism was confinedto the sixth CTCF binding site while a uniquemedian-methylated profile was found at the thirdCTCF binding site as well as in the H19 promoter.Monoallelic expression of H19 and IGF2 was main-tained independently of the methylation status atthe sixth CTCF binding site and the IGF2 DMR2displayed a median-methylated profile in all indi-viduals and tissues analyzed. Interestingly, themethylation profile was genetically transmitted.Transgenerational inheritance of the H19 methyla-tion profile was compatible with a simple modelinvolving one gene with three alleles. The existenceof three individual-specific epigenotypes in theH19 DMR in a non-pathological situation means itis important to reconsider the diagnostic valueand functional importance of the sixth CTCFbinding site.INTRODUCTIONImprinted genes are expressed from only one of the parentalchromosomes (1,2). Generally they are located in clusters andepigenetically marked by DNA methylation, histoneacetylation/deacetylation and histone methylation and oftenassociated with antisense RNAs (3,4). In mice, extensivestudies of the paradigmatic imprinted H19/Igf2 regionrevealed that the physical contacts between differentiallymethylated regions (DMRs), containing insulators, silencersand activators, lead to transcriptional regulation of bothH19 and Igf2 genes (5,6). Methylation of the paternallyderived allele at an imprinting control region (ICR) located2 kb upstream of H19 (H19 DMR) is required to silenceH19 and to activate Igf2 on the chromosome of paternal ori-gin. Reciprocally, absence of methylation on the maternalallele in the H19 DMR leads to expression of the maternalH19 allele and the silencing of Igf2 throughout development(7). Mechanistically, the H19 DMR is the biological target for