Allele-Specific H3K79 Di- versus Trimethylation Distinguishes Opposite Parental Alleles at Imprinted Regions

Abstract

Imprinted gene expression corresponds to parental allele-specific DNA CpG methylation and chromatin composition. Histone tail covalent modifications have been extensively studied, but it is not known whether modifications in the histone globular domains can also discriminate between the parental alleles. Using multiplex chromatin immunoprecipitation-single nucleotide primer extension (ChIP-SNuPE) assays, we measured the allele-specific enrichment of H3K79 methylation and H4K91 acetylation along the H19/Igf2 imprinted domain. Whereas H3K79me1, H3K79me2, and H4K91ac displayed a paternal-specific enrichment at the paternally expressed Igf2 locus, H3K79me3 was paternally biased at the maternally expressed H19 locus, including the paternally methylated imprinting control region (ICR). We found that these allele-specific differences depended on CTCF binding in the maternal ICR allele. We analyzed an additional 11 differentially methylated regions (DMRs) and found that, in general, H3K79me3 was associated with the CpG-methylated alleles, whereas H3K79me1, H3K79me2, and H4K91ac enrichment was specific to the unmethylated alleles. Our data suggest that allele-specific differences in the globular histone domains may constitute a layer of the "histone code" at imprinted genes.

MIRA-SNuPE, a quantitative, multiplex method for measuring allele-specific DNA methylation

“…7 and not shown). The U2af1 SNP in the 16-plex 44 assay lies at the outside boundary of the U2af1 DMR, and although it is suitable to discern allele-specific chromatin features associated with the U2af1 DMR, 44 it does not map to an area of the actual differential CpG methylation. Other known SNPs that map to the DMR are embedded in repeat elements, 12 and cannot be used in SNuPE assays.…”

“…7), we used MIRA in combination with our recently developed 16-plex SNuPE assays. 44 The MIRA-SNuPE Figure 5. MIRA assays to quantify CpG methylation levels in normal cells.…”

“…We where the CTCF site mutations resided in the maternally inherited 129-type ICR allele. A previously validated 7-plex Sequenom assay 44 distinguished between 129 and CS alleles in the MIRA fraction. MIRA-SNuPE results (Fig.…”

“…44 Alternative SNPs were included for the H19/Igf2 ICR (-3 kb and -2 kb from the TSS of H19). We found that each DMR, except for the U2af1 DMR, exhibited the expected parental-specific CpG methylation bias in the reciprocal crosses ( Fig.…”

“…DNA methylation was determined near single nucleotide polymorphisms (SNPs) within maternally methylated (Zac1, Snrpn, Igf2r, DMRs and at the KvDMR1) and paternally methylated DMRs (H19/Igf2 and Rasgrf1 DMRs and IG-DMR) by quantitative 16-plex assays. 44 Alternative SNPs were included for the H19/Igf2 ICR (-3 kb and -4 kb from the transcription start site of H19). MIRA was performed in three independent fetuses.…”