Heterogenous Global Demethylation in Naïve Embryonic Stem Cells Results from Differences in DNA Methylation Maintenance in Single Cells

Epigenetic remodeling, including genome wide erasure of 5-methylcytosine (5mC) is associated with the acquisition of pluripotency. To achieve this, cells can exhibit passive demethylation, where 5mC is not faithfully copied to newly synthesized DNA during replication. Recently, we have shown that the genome wide erasure of 5mC in early mouse and human embryogenesis is heterogenous, with a subset of cells experiencing global passive demethylation¹. These findings indicate that tools to investigate passive demethylation of 5mC are critical in understanding pluripotency and development. Here we describe CpG-Dyad-sequencing (CpG-Dyad-seq) which combines enzymatic detection of modified cytosines and traditional nucleobase conversion techniques to identify the presence of hemimethylation or hemihydroxymethylation at the resolution of a single CpG dyad site. We then scale this technique down and allow for the simultaneous detection of RNA transcripts all from the same single cell. By applying single-cell CpG-Dyad-seq to different time points of mouse embryonic stem cells transitioning from a primed to a naïve state of pluripotency, we observe extreme demethylation dominated by passive processes and discover this process is highly heterogenous and delayed in some cells. By connecting RNA expression from the same cells, we detect a small set of genes directly linked to 5mC levels during this transition. Finally, we determine that regions of the genome which escape 5mC reprograming do so by retaining high levels of 5mC maintenance and are associated with specific histone modifications.

1. Sen, M. et al. Strand-specific single-cell methylomics reveals distinct modes of DNA demethylation dynamics during early mammalian development. Nat. Commun. 12, 1286 (2021).