Allele-specific non-CG DNA methylation marks domains of active chromatin in female mouse brain

DNA methylation at gene promoters in a CG context is associated with transcriptional repression, including at genes silenced on the inactive X chromosome in females. Non-CG methylation (mCH) is a distinct feature of the neuronal epigenome that is differentially distributed between males and females on the X chromosome. However, little is known about differences in mCH on the active (Xa) and inactive (Xi) X chromosomes because stochastic X-chromosome inactivation (XCI) confounds allele-specific epigenomic profiling. We used whole-genome bisulfite sequencing in a mouse model with nonrandom XCI to examine allele-specific DNA methylation in frontal cortex. Xi was largely devoid of mCH, whereas Xa contained abundant mCH similar to the male X chromosome and the autosomes. In contrast to the repressive association of DNA methylation at CG dinucleotides (mCG), mCH accumulates on Xi in domains with transcriptional activity, including the bodies of most genes that escape XCI and at the X-inactivation center, validating this epigenetic mark as a signature of transcriptional activity. Escape genes showing CH hypermethylation were the only genes with CG-hypomethylated promoters on Xi, a well-known mark of active transcription. Finally, we found extensive allele-specific mCH and mCG at autosomal imprinted regions, some with a negative correlation between methylation in the two contexts, further supporting their distinct functions. Our findings show that neuronal mCH functions independently of mCG and is a highly dynamic epigenomic correlate of allele-specific gene regulation.