To date, there has been little investigation of the extent and nature of ASHM across the human genome. In this study, we undertook a comprehensive, genome-wide investigation of ASHM in an hESC line. Of more than 1.7 million putative heterozygote sites identified in this study, more than 81,000 displayed high ChIP-sequencing coverage (35x
or more, see Methods), of which 51 displayed significant evidence of different allele ratios in the reads from two or more histone-modification datasets. The results in this study therefore highlight that although sites of ASHM are found throughout the genome, they are probably not common. Owing to this being the first genome-wide analysis of ASHM in humans, a relatively stringent approach was used in this study, which probably resulted in a higher false-negative rate. However, the data used were combined from two large international sequencing projects, so although the generation of more data and for more modifications would probably identify further sites, we can still state that ASHM does not seem to be widespread, at least in hESCs. In yeast, it has been shown that single base changes can lead to predictable changes in nucleosome positioning [41
]; however, the results from the current study argue against SNPs having a substantial widespread effect on the modifications each nucleosome carries. Changes in the underlying DNA are rarely associated with changes in the modification carried by any overlying nucleosome. This is in broad agreement with a recent study that could find no evidence for selection acting on particular base changes at DNA underlying nucleosomes carrying different histone modifications [42
]. The fact that there is little evidence that underlying DNA has a substantial affect on histone modifications is perhaps unsurprising; patterns and distributions of histone modifications vary between cell types even when from the same genetic background. Histone modifications do not seem to be strongly reliant on the DNA sequences immediately underlying the nucleosomes on which they occur. These data are in contrast to the results we obtained when investigating ASM, which was found to be relatively common and closely associated with the underlying DNA, with almost all sites of ASM identified being attributable to a neighboring SNP. These results would suggest there are probably different mechanisms controlling ASHM and ASM in embryonic stem cells, and that one does not necessarily lead to the other.
We have shown in this study that despite sites of ASHM not being widespread, they are likely to be important. ASHM sites were found to be enriched around genes displaying evidence of ASE. Although this is not evidence of a direct link, it is likely, given the known role of histone modifications in gene expression, that at least some of these sites of ASHM are directly associated with allele-specific gene expression. ASM, which is already known to be directly associated with allele-specific gene expression, was substantially less enriched around sites of ASE. Consequently, the few sites of ASHM identified in this study would seem to be having a disproportionate affect on gene expression.
Sites of ASHM were also substantially more enriched around known imprinted genes than sites of ASM. We found that 1 in 6 sites of ASHM were within 10
kb of a known imprinted locus, as opposed to 1 in 417 sites of ASM. This suggests that ASHM is a substantially stronger marker of imprinted genes in embryonic stem cells than is ASM, and will probably be more informative in any searches for novel imprinted loci. It would seem that the reason that sites of ASM display such limited enrichment around imprinted regions is because the most significant sites are overwhelmingly the result of adjacent polymorphisms. This result also suggests that strong epialleles (DNA methylation differences between alleles that are not attributable to underlying DNA polymorphisms) are likely to be relatively rare in hESCs. The lack of strong enrichment of sites of ASM around imprinted regions may also be partly a result of imprinted regions having smaller differences in methylation between chromosomes across longer stretches, compared with sites at which CpG motifs are disrupted by a heterozygous SNP. Smaller differences in methylation levels will be difficult to detect without extremely high coverage, when robustly accounting for the substantial number of sites tested genome-wide.
Interestingly, despite the highly significant enrichment of ASHM sites around known imprinted regions, most ASHM sites were not associated with such regions. This suggests that either there are a substantial number of imprinted regions yet to be defined in the human genome or that ASHM is often located distinct from imprinted loci.
Analysis of the distribution of ASHM sites relative to the position of known microdeletion syndrome loci led to the discovery that one in six sites of allele-specific histone modification were within 250
kb of a locus at which deletions have been associated with developmental disorders. The observation of sites of ASHM at the PWS genomic interval on 15q11-13 is in agreement with previous studies [43
]. Targeted studies in mice looking at the patterns of histone modification at this imprinted locus in murine embryonic brain samples have identified enrichment of H3K4me2 and H3Ac modifications along the paternal chromosome, with H4K20me3-, H3K36me3-, H3K9me3-, and H3K79me3-modified nucleosomes enriched along the silenced maternal copy [43
]. Our observation of preferential enrichment of the H3K9me3 marker along the same phased haplotype in this study is therefore in broad agreement with the observed parent-of-origin chromosome bias of this modification in mice. However, the observed bias of the H3K36me3 histone modification along the silenced maternal chromosome in the same mouse study was more surprising, given that this histone mark has previously been associated with active, transcribed regions [44
]. In the current study looking at hESCs, we found sites in this region preferentially covered by nucleosomes carrying H3K36me3 were located on both chromosomes, suggesting that this modification is not preferentially associated with either chromosome copy in hESCs. This observation of sites of H3K36me3 on both the silenced and active copies of this region in humans is in agreement with the broader observation that this marker is found at both transcriptionally active and repressed regions in mice, including mouse embryonic stem cells [43
Although rare, ASHM is consequently likely to play an important role in human development. The location of sites of ASHM at key imprinted and ASE loci and at sites associated with developmental disorders suggests that ASHM is an important but as yet undercharacterized phenomenon involved in embryonic development.