TETs compete with DNMT3 activity in pluripotent cells at thousands of methylated somatic enhancers

Abstract

Mammalian cells maintain a remarkably stable, highly methylated global DNA methylation landscape despite expressing both positive (DNMT3A/B) and negative (TET1-3) regulators. Notably, although embryonic stem cells (ESCs) can tolerate loss of DNMT3 or TET activity, whether their interplay contributes to viability or developmental competence remains unknown. Here, we used wildtype (WT) and TET triple knockout human ESCs, generated DNMT3 as well as DNMT3 and TET pentuple knockout (PKO: DNMT3A-/-;DNMT3B-/-;TET1-/-;TET2-/-;TET3-/-) lines, and compared methylation patterns using whole genome bisulfite sequencing. Interestingly, we observe the greatest impact on global methylation levels in DNMT3-deficient cells, including highly reproducible and rapid focal demethylation of thousands of normally methylated loci. This demethylation depends upon TET expression and only occurs when DNMT3 activity is absent. Dynamic loci are enriched for hydroxymethylcytosine and overlap with subsets of putative somatic enhancers that are methylated in ESCs and can be activated upon differentiation. We observe similar dynamics in mouse ESCs that were less frequenct in EpiSCs and scarce in somatic tissues, suggesting a conserved pluripotency-linked mechanism. Taken together, our data reveal tightly regulated competition between DNMT3s and TETs at thousands of somatic regulatory sequences within pluripotent cells.