Sequencing DNA methylation and hydroxymethylation at co-occurring chromatin features

Epigenetic modifications govern chromatin dynamics and cell state. However, current methods cannot simultaneously resolve the presence of multiple DNA modifications at co-occurring chromatin-associated features. It is thus not clear how these features are physically coupled and how their combinations regulate genome function. To address this key question, we report 6-base-CUT&Tag, a method for simultaneous 6-base DNA sequencing at target chromatin features. Using 6-base-CUT&Tag to profile 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at co-occurring histone modifications in mouse embryonic stem cells (mESCs), we identify feature-dependent 5mC/5hmC signatures previously unresolvable with untargeted or bisulfite-based workflows. We show that DNA methylation and hydroxymethylation are specifically coupled with the H3K4me1 mark in mESC enhancers and that H3K4me1-derived signatures robustly distinguish different enhancer functional states.