Dual profiling of DNA modifications with enhancer features during the exit of naive pluripotency

Cis-regulatory elements, such as enhancers, play an essential role in coordinating gene expression programs during cellular transitions. As such, substantial efforts have been made to characterize enhancer elements, e.g., chromatin accessibility, transcription factor (TF) binding sites, histone post-translational modifications (PTMs), and 5-cytosine DNA methylation and hydroxymethylation (5mC and 5hmC). Elevated 5mC levels are typically correlated with the inactive enhancer state. However, whether 5mC precludes TF binding or is simply a downstream consequence of enhancer decommissioning is difficult to determine. Bulk genomics assays from cell populations fail to fully capture cellular heterogeneity, and single-cell assays often suffer from limited read counts per cell. Here, we leveraged tagmentation-based technologies to assess chromatin features with 5mC simultaneously: Methyl-ATAC and Methyl-CUT&Tag. In addition, we modified the technique to interrogate 5hmC dynamics (hM-ATAC). We employed these techniques during the exit of naive pluripotency in mouse embryonic stem cells (mESCs), which recapitulates the embryonic DNA methylation establishment program. This system withstands the complete absence of DNA methylation and demethylation machinery, allowing us to further dissect the temporal contributions of 5mC and 5hmC. Given the affordability of these techniques, we were able to obtain robust, dynamic single-molecule 5mC/5hmC information during this cellular transition for chromatin accessibility and histone marks in wild-type and mutant conditions. The sum of these data allowed for unprecedented insight into the role that 5mC turnover plays at enhancers during a key window of mammalian embryonic development.