Department of Anatomy; University of Cambridge
Parental-origin specific regulation of imprinted genes is controlled by modifications to the DNA and chromatin. In the mouse Igf2/H19 domain on chromosome 7, many of the key regulatory sites have been identified and parental specific regions of DNA methylation have been mapped. Indirect evidence has suggested that chromatin modification may play a role in the regulation of imprinting. This is through the identification of parent-specific nuclease hypersensitive sites and the finding that the maternal and paternal chromosomes show asynchronous replication. Bearing this in mind we have directly assessed whether histone acetylation may be implicated in gene regulation at this domain.
Analysis of H4 acetylation on the two parental chromosomes was compared using a panel of antisera generated against the four different acetylated isoforms of histone H4. Chromatin was compared from fibroblasts made from normal embryos and embryos containing maternal duplication/paternal deficiency of the imprinted region on distal chromosome 7. Chromatin was immunoprecipitated and DNA isolated from bound and unbound fractions, then hybridized with probes from key areas in the imprinted domain.
The CpG-island H19 promoter which shows parent-specific methylation and nuclease accessibility exhibited hyperacetylation on the unmethylated active maternal allele with all four antibodies. The methylated inactive paternal promoter was deacetylated. Interestingly, the Igf2 promoter which is also a CpG island but is not methylated on either the maternal or paternal chromosome, also shows hyperacetylation of the active allele. In contrast to the H19 promoter, the inactive maternal allele does not show hypoacetylation, however, the acetylation level is considerably lower than that on the active allele. These results show a correlation between acetylation and allelic activity and are consistent with a recently described role for methylation in the recruitment of deacetylase activity.