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FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency.

ABSTRACT: Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3β signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency.

SUBMITTER: Ficz G

PROVIDER: S-EPMC3765959 | biostudies-literature |

REPOSITORIES: biostudies-literature

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FGF Signaling Inhibition in ESCs Drives Rapid Genome-wide Demethylation to the Epigenetic Ground State of Pluripotency

Project description:Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos but the signalling mechanisms that induce reprogramming are unknown. Here we show that inhibition of Erk1/2 and Gsk3bM-BM- signalling in mouse embryonic stem cells (ESCs) by small molecule inhibitors (PD0325901 and CHIR99021, hereafter called 2i)M-BM- induces genome-wide demethylation on a scale similar to that in PGCs and early embryos, with only major satellites, intracisternal A particles (IAPs) and imprinted genes relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) in part by Tet1 together with repression of theM-BM- de novoM-BM- methyltransferases (Dnmt3a, Dnmt3b) and their regulator Dnmt3L and we identify aM-BM- cis-acting regulatory region inM-BM- Dnmt3bM-BM- that is highly responsive to signalling. Notably, this epigenetic and transcriptional ground state of pluripotency resembles closely that of inner cell mass (ICM) cells of the blastocyst. These insights provide a novel framework for understanding how signalling pathways regulate epigenetic reprogramming. mRNA and methylation profiles of ES cells passaged with or without 2i inhibitors were generated by deep sequencing, using Illumina GAIIx and HiSeq.

2013-06-27 | E-GEOD-42923 | biostudies-arrayexpress

FGF Signaling Inhibition in ESCs Drives Rapid Genome-wide Demethylation to the Epigenetic Ground State of Pluripotency

Project description:Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos but the signalling mechanisms that induce reprogramming are unknown. Here we show that inhibition of Erk1/2 and Gsk3b signalling in mouse embryonic stem cells (ESCs) by small molecule inhibitors (PD0325901 and CHIR99021, hereafter called 2i) induces genome-wide demethylation on a scale similar to that in PGCs and early embryos, with only major satellites, intracisternal A particles (IAPs) and imprinted genes relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) in part by Tet1 together with repression of the de novo methyltransferases (Dnmt3a, Dnmt3b) and their regulator Dnmt3L and we identify a cis-acting regulatory region in Dnmt3b that is highly responsive to signalling. Notably, this epigenetic and transcriptional ground state of pluripotency resembles closely that of inner cell mass (ICM) cells of the blastocyst. These insights provide a novel framework for understanding how signalling pathways regulate epigenetic reprogramming.

2013-06-27 | GSE42923 | GEO

Synergistic mechanisms of DNA demethylation during transition to ground-state pluripotency.

Project description:Pluripotent stem cells (PSCs) occupy a spectrum of reversible molecular states ranging from a naive ground-state in 2i, to metastable embryonic stem cells (ESCs) in serum, to lineage-primed epiblast stem cells (EpiSCs). To investigate the role of DNA methylation (5mC) across distinct pluripotent states, we mapped genome-wide 5mC and 5-hydroxymethycytosine (5hmC) in multiple PSCs. Ground-state ESCs exhibit an altered distribution of 5mC and 5hmC at regulatory elements and dramatically lower absolute levels relative to ESCs in serum. By contrast, EpiSCs exhibit increased promoter 5mC coupled with reduced 5hmC, which contributes to their developmental restriction. Switch to 2i triggers rapid onset of both the ground-state gene expression program and global DNA demethylation. Mechanistically, repression of de novo methylases by PRDM14 drives DNA demethylation at slow kinetics, whereas TET1/TET2-mediated 5hmC conversion enhances both the rate and extent of hypomethylation. These processes thus act synergistically during transition to ground-state pluripotency to promote a robust hypomethylated state.

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Ground State Conditions Induce Rapid Reorganization of Core Pluripotency Factor Binding before Global Epigenetic Reprogramming.

Project description:Mouse embryonic stem cells (mESCs) cultured under serum/LIF conditions exhibit heterogeneous expression of pluripotency-associated factors that can be overcome by two inhibitors (2i) of the MEK and GSK3 pathways. Several studies have shown that the "ground state" induced by 2i is characterized by global hypomethylation and specific transcriptional profiles, but little is known about the contributing effectors. Here we show that 2i conditions rapidly alter the global binding landscape of OCT4, SOX2, and NANOG. The dynamic binding influences enhancer activity and shows enrichment for regulators linked to Wnt and Erk signaling. Epigenomic characterization provided limited insights to the immediate transcriptional dynamics, suggesting that these are likely more secondary effects. Likewise, loss of the PRC2 component EED to prevent H3K27me3 deposition had minimal effect on the transcriptome, implying that it is largely dispensable for continued repression of bivalent genes and de novo silencing in 2i.

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2i Maintains a Naive Ground State in ESCs through Two Distinct Epigenetic Mechanisms.

Project description:Mouse embryonic stem cells (ESCs) are maintained in serum with leukemia inhibitory factor (LIF) to maintain self-renewal and pluripotency. Recently, a 2i culture method was reported using a combination of MEK inhibition (MEKi) and GSK3 inhibition (GSK3i) with LIF to maintain ESCs in a naive ground state. How 2i maintains a ground state of ESCs remains elusive. Here we show that MEKi and GSK3i maintain the ESC ground state by downregulating global DNA methylation through two distinct mechanisms. MEK1 phosphorylates JMJD2C for ubiquitin-mediated protein degradation. Therefore, MEKi increased JMJD2C protein levels but decreased DNMT3 expression. JMJD2C promotes TET1 activity to increase 5-hydroxymethylcytosine (5hmC) levels. GSK3i suppressed DNMT3 expression, thereby decreasing DNA methylation without affecting 5hmC levels. Furthermore, 2i increased PRDM14 expression to inhibit DNMT3A/B protein expression by promoting G9a-mediated DNMT3A/B protein degradation. Collectively, 2i allows ESCs to maintain a naive ground state through JMJD2C-dependent TET1 activation and PRDM14/G9a-mediated DNMT3A/B protein degradation.

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Proteome Analysis of Ground State Pluripotency.

Project description:The differentiation potential of pluripotent embryonic stem cells (ESCs) can be manipulated via serum and medium conditions for direct cellular development or to maintain a naïve ground state. The self-renewal state of ESCs can thus be induced by adding inhibitors of mitogen activated protein kinase (MAPK) and glycogen synthase kinase-3 (Gsk3), known as 2 inhibitors (2i) treatment. We have used a shotgun proteomics approach to investigate differences in protein expressions between 2i- and serum-grown mESCs. The results indicated that 164 proteins were significantly upregulated and 107 proteins downregulated in 2i-grown cells compared to serum. Protein pathways in 2i-grown cells with the highest enrichment were associated with glycolysis and gluconeogenesis. Protein pathways related to organ development were downregulated in 2i-grown cells. In serum-grown ESCs, protein pathways involved in integrin and focal adhesion, and signaling proteins involved in the actin cytoskeleton regulation were enriched. We observed a number of nuclear proteins which were mostly involved in self-renewal maintenance and were expressed at higher levels in 2i compared to serum - Dnmt1, Map2k1, Parp1, Xpo4, Eif3g, Smarca4/Brg1 and Smarcc1/Baf155. Collectively, the results provided an insight into the key protein pathways used by ESCs in the ground state or metastable conditions through 2i or serum culture medium, respectively.

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Ground state conditions induce rapid reorganization of core pluripotency factor binding that precede global epigenetic reprogramming

Project description:Mouse embryonic stem cells (mESCs) cultured under serum/LIF conditions exhibit heterogeneous expression of pluripotency-associated factors that can be overcome by two inhibitors (2i) of the MEK and GSK3 pathway, respectively. Several studies have shown that the “ground state” induced by 2i is characterized by global hypomethylation and specific transcriptional profiles, but little is known about the contributing effectors. Here we show that 2i conditions rapidly alter the global binding landscape of OCT4, SOX2 and NANOG. The dynamic binding influences enhancer activity and shows enrichment for regulators linked to Wnt and Erk signaling. Epigenomic characterization provided limited insights to the immediate transcriptional dynamics suggesting these are likely more secondary effects. Likewise, knock-out of the PRC2 component EED to prevent H3K27me3 deposition had minimal effect on the transcriptome, rendering it largely dispensable for continued repression of bivalent genes and de novo silencing in 2i.

2015-08-05 | GSE56312 | GEO

The translational landscape of ground state pluripotency.

Project description:Translational control plays a central role in regulation of gene expression and can lead to significant divergence between mRNA- and protein-abundance. Here, we used genome-wide approaches combined with time-course analysis to measure the mRNA-abundance, mRNA-translation rate and protein expression during the transition of naïve-to-primed mouse embryonic stem cells (ESCs). We find that the ground state ESCs cultured with GSK3-, MEK-inhibitors and LIF (2iL) display higher ribosome density on a selective set of mRNAs. This set of mRNAs undergo strong translational buffering to maintain stable protein expression levels in 2iL-ESCs. Importantly, we show that the global alteration of cellular proteome during the transition of naïve-to-primed pluripotency is largely accompanied by transcriptional rewiring. Thus, we provide a comprehensive and detailed overview of the global changes in gene expression in different states of ESCs and dissect the relative contributions of mRNA-transcription, translation and regulation of protein stability in controlling protein abundance.

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Klf4 reverts developmentally programmed restriction of ground state pluripotency.

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