Programme in Developmental Genetics; The Babraham Institute
Possibly the most consistent epigenetic features associated with imprinting are parental allele-specific DNA methylation and differential replication timing. Whereas allelic methylation is often confined to smaller sequence elements, differential timing of replication seems to involve larger domains and may reflect chromatin differences between the parental alleles. A more direct analysis of chromatin conformation is provided by nuclease sensitivity assays on nuclei. In the imprinted, splice factor-encoding, U2afl-rs1 gene, we found that the paternally inherited (unmethylated) chromosomes display a greater sensitivity to DNase-I and MspI than the maternally inherited (methylated) chromosomes. This differential "generalized" nuclease sensitivity involves the gene and direct flanking sequences (i.e. the domain of differential methylation) and is detected regardless of expression. It is unclear what features of chromatin give rise to the generalized sensitivity differences; however, micrococcal nuclease digestions indicate that nucleosomes are equally present on both chromosomes. One possible modification within nucleosomes that could be involved, is core histone acetylation. This suggestion is supported by our observation, that when embryonic stem cells are treated with Trichostatin-A (a specific inhibitor of histone deacetylases), the two parental alleles of U2afl-rs1 become equally sensitive to DNase-I.
In order to directly analyze the histone acetylation status of the U2afl-rs1 locus we have carried out immuno-precipitations on chromatin using affinity-purified antibodies to specific acetylated lysine residues of histone H4. To enable us to compare the relative level of acetylation on both parental alleles within the same immuno-precipitation, we developed a novel PCR-based methodology. Using this technique we have detected allele-specific histone H4 acetylation, and this appears to be confined to the domain of differential nuclease sensitivity. Interestingly, the strongest differences were detected in lysine residue 5, which had significantly higher levels of acetylation on the paternal than on the maternal chromosome. These initial findings provide the first link between allele-specific nuclease sensitivity and histone acetylation at an imprinted locus.