Person:

Charlton, Jocelyn

In normal mammalian development cytosine methylation is essential and is directed to specific regions of the genome. Despite notable advances through mapping its genome-wide distribution, studying the direct contribution of DNA methylation to gene and genome regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of the CRISPR–Cas9 system with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9 (dCas9) methyltransferase fusion protein is challenging within a highly methylated genome. Here, we report the generation and use of an engineered, methylation depleted but maintenance competent mouse ES cell line and find surprisingly ubiquitous nuclear activity of dCas9-methyltransferases. Subsequent experiments in human somatic cells refine these observations and point to an important difference between genetic and epigenetic editing tools that require unique experimental considerations.

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.