Person:

Mattei, Alexandra

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.

AbstractDNA methylation plays a critical role during development, particularly in repressing retrotransposons. The mammalian methylation landscape is dependent on the combined activities of the canonical maintenance enzyme Dnmt1 and the de novo Dnmts, 3a and 3b. Here, we demonstrate that Dnmt1 displays de novo methylation activity in vitro and in vivo with specific retrotransposon targeting. We used whole-genome bisulfite and long-read Nanopore sequencing in genetically engineered methylation-depleted mouse embryonic stem cells to provide an in-depth assessment and quantification of this activity. Utilizing additional knockout lines and molecular characterization, we show that the de novo methylation activity of Dnmt1 depends on Uhrf1, and its genomic recruitment overlaps with regions that enrich for Uhrf1, Trim28 and H3K9 trimethylation. Our data demonstrate that Dnmt1 can catalyze DNA methylation in both a de novo and maintenance context, especially at retrotransposons, where this mechanism may provide additional stability for long-term repression and epigenetic propagation throughout development.