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

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE56312 | GEO | 2015/08/05

SECONDARY ACCESSION(S): PRJNA242892

REPOSITORIES: GEO

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TEAD2 mediates the ground-state pluripotency by chromatin looping

Project description:Mouse embryonic stem cells (mESCs) cultured in 2i (MEK and GSK3 kinase inhibitor)/LIF and serum/LIF that we called 2i-ESCs and serum-ESCs represent ground and confused pluripotent states, respectively. However, the transcription factors that regulate ground pluripotency through chromatin-associated characteristics are not yet fully understood. By mapping chromatin accessibility and transcription factor regulatory networks during the interconversion of 2i-ESCs and serum-ESCs, we have identified TEAD2 as highly enriched in 2i-specific peaks. While Tead2 knockout did not affect the pluripotency or differentiation ability of either 2i-ESCs or serum-ESCs, it did prevent the establishment of the 2i-specific state and the exit from the serum-specific state. TEAD2 binds to active regions in 2i-specific genes and activates their expression by regulating enhancer-promoter (EP) interactions during serum-to-2i transition. Remarkably, TEAD2-mediated EP interactions were independent of chromatin architecture proteins YY1 and CTCF, but instead appear to be facilitated by TEAD2 homodimer formation.

2024-03-03 | GSE226316 | GEO

Continuous suppression of Mek1/2 impairs the developmental potential of mouse embryonic stem cells

Project description:Co-suppression of Gsk3 and Mek1/2 signaling (“2i culture”) in mouse embryonic stem cells (ESCs) induces a naïve state that more closely resembles the inner cell mass (ICM) of the pre-implantation embryo, including global DNA hypomethylation. While the ICM exists only transiently in vivo, it remains unclear how continuous suppression of Mek1/2 and Gsk3 signaling and associated hypomethylation affect the stability and functionality of ESCs in vitro. Here we show that, compared to ESCs maintained in conventional serum/LIF media, culturing male ESCs in 2i/LIF for >6 passages results in the erosion of genomic imprints and a concomitant impairment in the ability to generate adult, entirely ESC- derived mice (all-ESC mice). We further demonstrate that female ESCs cultured in serum/LIF phenocopy male ESCs cultured in 2i/LIF, including global hypomethylation, imprint erosion and impaired developmental potential. Unexpectedly, culture of female ESCs in serum/LIF and male ESCs in 2i/LIF for >16 passages causes recurrent chromosomal aberration that have been associated with a selective growth advantage. We provide evidence that inhibition of Mek1/2, but not Gsk3, is responsible for hypomethylation, imprint loss and chromosomal instability in ESCs, and identify alternative conditions that preserve epigenetic integrity. Taken together, our data suggest that while short-term suppression of Mek1/2 in ESCs captures an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise developmental potential.

2017-07-27 | GSE97995 | GEO

Genome-wide analysis of gene expression in LIF and LIF plus 2I in C57BL/6 mouse embryonic stem cells (B6 mESCs)

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2013-04-26 | E-GEOD-46369 | biostudies-arrayexpress

Derivation of ground-state female ESCs maintaining gamete-derived DNA methylation [gene expression]

Project description:Preimplantation embryos undergo a transient wave of genome-wide demethylation with the exception of imprinted genes that are critical for fetal development. Here we show that the derivation of female mouse embryonic stem cells (ESCs) in the presence of inhibitors of MEK1/2 and Gsk3 (2i-ESCs), known as ‘2i’ or “ground-state” culture conditions, results in a widespread loss of DNA methylation including a massive erasure of genomic imprints. In this study, we analyzed global gene expression profile and global DNA methylation status in 2i-ESCs and 2i-ESCs derived differentiated cells. S-ESCs are ESCs established under serum-containing medium. 2i_S_ESCs are ESCs established in 2i-containing medium, followed by maintenance in serum-containing medium.

2017-07-27 | GSE82313 | GEO

β-catenin safeguards the ground state of pluripotency by strengthening the robustness of the transcriptional apparatus [GRO-seq]

Project description:Mouse embryonic stem cells (ESCs) cultured with MEK and GSK3 inhibitors (2i) more closely resemble the inner cell mass of pre-implantation blastocysts than those cultured with serum/LIF (SL). The transcriptional mechanisms governing this pluripotent ground state are yet unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. Here, we show that β-catenin, stabilized by GSK3 inhibition in 2i, supplies transcriptional co-regulators including BRD4, CDK9, Mediator, Cohesin, and p300 at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by β-catenin and, thus, proliferation/self-renewal are still tightly controlled by the Pol2 pause release mechanism under 2i conditions. Our findings help to explain how pluripotency is preserved in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other biological contexts.

2020-07-01 | GSE140340 | GEO

RNA-seq analysis of ES cell populations with different functional potentials in serum/LIF and 2i/LIF, as well as analysis of the transcriptional effect of LIF in ground state (2i) culture conditions.

Project description:We report that ES cells cultured in ground state (2i and 2i/LIF) culture conditions are heterogeneous and show heterogeneus expression of extraembryonic markers. Using a highly sensitive reporter for the endoderm marker Hex we can sort Hex high and low populations from either serum/LIF or 2i/LIF and demonstrate that they have different functional properties. Here we explored the transcriptional basis of these functional differences and noted that Hex low (HV-) and Hex high (HV+) populations showed more distinct expression profiles in 2i/LIF than in serum/LIF. Additionally in 2i/LIF the HV+ population showed an upregulation of extraembryonic markers (such as trophoblast stem cell specific genes) and also imprinted genes compared to the HV- population, which is not observed when these populations are sorted from serum/LIF. We also analysed the transcriptional effect of LIF in 2i by analysing unsorted ES cells cultured in either 2i alone or 2i with LIF. We observed that the addition of LIF led to an upregulation of extraembryonic markers but did not effect the expression of pluripotency genes, other than Klf4. Additionally, the most significantly upregulated genes from 2i/LIF cultured ES cells compared to 2i cultured ES cells showed the greatest correlation to placental tissue when compared to the GNF tissue specific expression database. This analysis, alongside functional experiments, suggested that HV+ ES cells in 2i/LIF corresponded to an extraembryonically primed population of cells and that the addition of LIF supported this population.

2013-06-11 | GSE45182 | GEO

Transcriptome upon Mof deletion and Etomoxir treatment in ground-state mouse (m) ESCs

Project description:This study describes the transcriptome profiling of: 1) Mof fl/fl mESCs cultured in 2i/LIF medium with Ethanol (WT) or 4-OHT (Mof-/-); 2) E14 mESCs cultured in 2i/LIF medium treated with H2O (control) or Etomoxir treatment (ETO, FAO inhibition) and ETO-released (Released).

2020-07-02 | GSE137138 | GEO

RNA-seq analysis of ES cell populations with different functional potentials in serum/LIF and 2i/LIF, as well as analysis of the transcriptional effect of LIF in ground state (2i) culture conditions.

2013-06-11 | E-GEOD-45182 | biostudies-arrayexpress

Integrative Proteomic Profiling Reveals PRC2-Dependent Epigenetic Crosstalk Maintains Ground-State Pluripotency.

Project description:The pluripotent ground state is defined as a basal state free of epigenetic restrictions, which influence lineage specification. While naive embryonic stem cells (ESCs) can be maintained in a hypomethylated state with open chromatin when grown using two small-molecule inhibitors (2i)/leukemia inhibitory factor (LIF), in contrast to serum/LIF-grown ESCs that resemble early post-implantation embryos, broader features of the ground-state pluripotent epigenome are not well understood. We identified epigenetic features of mouse ESCs cultured using 2i/LIF or serum/LIF by proteomic profiling of chromatin-associated complexes and histone modifications. Polycomb-repressive complex 2 (PRC2) and its product H3K27me3 are highly abundant in 2i/LIF ESCs, and H3K27me3 is distributed genome-wide in a CpG-dependent fashion. Consistently, PRC2-deficient ESCs showed increased DNA methylation at sites normally occupied by H3K27me3 and increased H4 acetylation. Inhibiting DNA methylation in PRC2-deficient ESCs did not affect their viability or transcriptome. Our findings suggest a unique H3K27me3 configuration protects naive ESCs from lineage priming, and they reveal widespread epigenetic crosstalk in ground-state pluripotency.

2018-11-24 | GSE101675 | GEO

Ground state of pluripotency cannot be maintained in mouse embryonic stem cells upon prolonged inhibition of GSK3

Project description:We compare gene expression changes in mESCs culture in serum/LIF or 2i/LIF which favour the ground state pluripotency. Published RNASeq data where compared with newly generated data set in order to indentify transciptional signatures associated with pluripotency of mouse ES cells

2019-03-03 | GSE70436 | GEO

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