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Molecular mechanisms of global DNA demethylation to naïve pluripotency in ESC

ABSTRACT: Global demethylation is part of a conserved program of epigenetic reprogramming to naïve pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naïve hypomethylated ones (serum-to-2i) is a valuable model system for epigenetic reprogramming. We present a mathematical model, which accurately predicts global DNA demethylation kinetics. Experimentally we show that the main drivers of global demethylation are neither active mechanisms (Aicda, Tdg, Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transit to 2i and so is recruitment of the maintenance methylation machinery to replication foci. Concurrently there is global loss of H3K9me2, which is needed for chromatin binding of UHRF1. These mechanisms synergistically enforce global DNA hypomethylation in a replication-coupled fashion. Our observations establish the molecular mechanism for global demethylation in naïve ESCs, which has key parallels with those operating in primordial germ cells and early embryos.

ORGANISM(S): Mus musculus

PROVIDER: GSE77420 | GEO | 2016/05/26

SECONDARY ACCESSION(S): PRJNA310246

REPOSITORIES: GEO

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Project description:Pluripotency can be maintained in the naïve state through manipulation of ERK and WNT signalling (2i), shielding embryonic stem cells (ESCs) from inductive cues. Alternatively, inhibiting CDK8/19 (CDK8/19i), a repressor of the Mediator co-activator complex, directly stimulates super-enhancer activity, and was recently shown to stabilize cells in a functional state that resembles naïve pluripotency. Naïve ESCs exhibit important epigenetic, transcriptional and metabolic features. However, our understanding on how these regulatory layers are inter-connected to promote the naive state is in progress. To fill this gap, here we used mass spectrometry to describe the dynamic molecular events (i.e. phosphoproteome, proteome and metabolome) executed by 2i and CDK8/19i, as they transition cell identity into naïve pluripotency. We observed rapid proteomic reprogramming, revealing widespread commonalities, and some important differences, between these two approaches, suggesting a largely over-lapping mechanism. CDK8/19i acts directly on the control of the transcriptional machinery, which elicits a rapid and direct activation of key identity genes including those that maintain the naïve program. Additional molecular changes in 2i are achieved by phosphorylation of critical downstream effectors that reinforce the naïve transcriptional circuitry while repressing factors from the more-differentiated formative and primed states. Comparing transcriptomic and proteomic changes, we found that post-transcriptional de-repression is a major feature of naïve pluripotency conferred by both 2i and CDK8/19i, and this may support the enhanced mitochondrial capacity of naive cells. Furthermore, at the level of metabolome, while 2i- and CDK8/19i-treated cells share similar aspects in one-carbon metabolism and beta-oxidation, in other regards they are divergent, a feature which may explain their differences in DNA methylation. These datasets provide a valuable resource for exploring the molecular mechanisms underlying pluripotency and cell identity transitions.

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Molecular mechanisms of global DNA demethylation to naïve pluripotency in ESC

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