Abstract
SummaryGlobal demethylation is part of a conserved program of epigenetic reprogramming to naive pluripotency. The transition from primed hypermethylated embryonic stem cells (ESCs) to naive 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, and Tet1-3) nor the reduction of de novo methylation. UHRF1 protein, the essential targeting factor for DNMT1, is reduced upon transition 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 naive ESCs, which has key parallels with those operating in primordial germ cells and early embryos.
Highlights
Pluripotency describes the transient embryonic potential to form all embryonic germ layers and the germline, excluding the extraembryonic tissues (Smith, 2001)
While originally mouse embryonic stem cells (ESCs) were grown in serum/leukemia inhibitory factor (LIF)-containing media (‘‘serum ESCs’’), recently, serum-free culture conditions have been established that favor derivation and propagation of mouse ESCs in the absence of serum (Ying et al, 2008)
Demethylation Dynamics during Serum-to-2i Reprogramming DNA demethylation dynamics can be attributed to three major pathways (Wu and Zhang, 2014): (1) maintenance DNA methylation or replication dependent passive dilution, (2) de novo DNA methylation, and (3) active DNA demethylation, primarily via DNA hydroxymethylation (Figure 1A)
Summary
Pluripotency describes the transient embryonic potential to form all embryonic germ layers and the germline, excluding the extraembryonic tissues (Smith, 2001). While originally mouse ESCs were grown in serum/leukemia inhibitory factor (LIF)-containing media (‘‘serum ESCs’’), recently, serum-free culture conditions have been established that favor derivation and propagation of mouse ESCs in the absence of serum (Ying et al, 2008). These conditions rely on specific inhibition of GSK3beta and Erk1/2 and downstream signaling by two smallmolecule inhibitors (‘‘2i ESCs’’). The state of 2i ESCs has been designated the ‘‘ground- or naive-state’’ of pluripotency, and cells grown in 2i are believed to be a much better representation of the cells from the ICM, compared to ‘‘primed’’ serum ESCs (Marks et al, 2012; Martello and Smith, 2014)
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