HM-DyadCap – Capture and Mapping of Asymmetric 5-Hydroxymethylcytosine/5-Methylcytosine CpG Dyads in Mammalian DNA
5-Methylcytosine (mC) and 5-hydroxymethylcytosine (hmC) are the two main epigenetic modifications of mammalian DNA, and play crucial roles in cell differentiation, development, and tumorigenesis. Both modifications co-exist with unmodified cytosine in palindromic CpG dyads in different strand-symmetric and -asymmetric combinations, each having unique regulatory potential. To facilitate investigating the individual functions of such dyad modifications, we here report HM-DyadCap. This method employs MECP2 HM – an evolved methyl-CpG-binding domain of the mC reader MECP2 – for the simple capture and sequencing of DNA fragments containing the CpG dyad hmC/mC that frequently occurs in mouse embryonic stem cell and brain genomes. In vitro binding studies reveal a high discrimination of MECP2 HM against diverse off-target dinucleotides in vitro. We conduct comparative mapping experiments for mESC genomes with HM-DyadCap, standard MethylCap based on wild type MECP2, as well as the antibody-based MeDIP and hMeDIP protocols. We find that MECP2 HM is blocked by hmC glucosylation, and conduct control enrichments with glucosylated genomes that indicate highly selective enrichment of hmC/mC dyads by MECP2 HM. Metagene profiles correlate hmC/mC with actively transcribed genes, and reveal global enrichment in gene bodies as well as depletion at transcription start sites. We anticipate that HM-DyadCap will enable effective mapping of hmC/mC with implications for biomarker discovery and unravelling the function of this dyad in diverse aspects of chromatin biology.