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Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency. (RNA-seq)

ABSTRACT: The mechanisms underlying enhancer activation and the extent to which enhancer-promoter rewiring contributes to spatiotemporal gene expression are not well understood. Using integrative and time resolved analyses we show that the extensive transcriptome and epigenome resetting during the conversion between ‘serum-’ and ‘2i’-states of mouse embryonic stem cells (ESCs) takes place with minimal enhancer-promoter rewiring that becomes more evident in primed-state pluripotency. Instead, differential gene expression is strongly linked to enhancer activation via H3K27ac. Conditional depletion of TFs and allele-specific enhancer analysis reveals an essential role for Esrrb in H3K27-acetylation and activation of 2i-specific enhancers. Restoration of a polymorphic ESRRB motif using CRISPR/Cas9 in a hybrid ESC-line, restores ESRRB binding and enhancer H3K27ac in an allele-specific manner but has no effect on chromatin interactions. Our study shows that enhancer activation in serum- and 2i-ESCs is largely driven by TF-binding and epigenetic marking in a hardwired network of chromatin interactions.

ORGANISM(S): Mus musculus

PROVIDER: GSE92403 | GEO | 2019/04/30

REPOSITORIES: GEO

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Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency. (ATAC-seq)

Project description:The mechanisms underlying enhancer activation and the extent to which enhancer-promoter rewiring contributes to spatiotemporal gene expression are not well understood. Using integrative and time resolved analyses we show that the extensive transcriptome and epigenome resetting during the conversion between ‘serum-’ and ‘2i’-states of mouse embryonic stem cells (ESCs) takes place with minimal enhancer-promoter rewiring that becomes more evident in primed-state pluripotency. Instead, differential gene expression is strongly linked to enhancer activation via H3K27ac. Conditional depletion of TFs and allele-specific enhancer analysis reveals an essential role for Esrrb in H3K27-acetylation and activation of 2i-specific enhancers. Restoration of a polymorphic ESRRB motif using CRISPR/Cas9 in a hybrid ESC-line, restores ESRRB binding and enhancer H3K27ac in an allele-specific manner but has no effect on chromatin interactions. Our study shows that enhancer activation in serum- and 2i-ESCs is largely driven by TF-binding and epigenetic marking in a hardwired network of chromatin interactions.

2019-04-30 | GSE92411 | GEO

Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency. (ChIP-seq)

2019-04-30 | GSE92407 | GEO

Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency. (4C-seq)

2019-04-30 | GSE113750 | GEO

Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency.

2019-04-30 | GSE92412 | GEO

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

Quantitative subcellular proteomics using SILAC reveals enhanced metabolic buffering in ground state pluripotency

Project description:The ground state of pluripotency is defined as a minimal unrestricted state as present in the Inner Cell Mass (ICM). Mouse embryonic stem cells (ESCs) grown in a defined serum-free medium with two kinase inhibitors (‘2i’) reflect this state, whereas ESCs grown in the presence of serum (‘serum’) share more similarities with post implantation epiblast cells. Pluripotency results from an intricate interplay between cytoplasmic, nuclear and chromatin-associated proteins. Therefore, quantitative information on the (sub)cellular proteome is essential to gain insight in the molecular mechanisms driving different pluripotent states. Here, we describe a full SILAC workflow and quality controls for proteomic comparison of 2i and serum ESCs. We demonstrate that this workflow is applicable for subcellular proteomics of the cytoplasm, nuclear and chromatin. The obtained quantitative information revealed increased levels of naïve pluripotency factors on the chromatin of 2i ESCs. Further, we demonstrate that these pluripotent states are supported by distinct metabolic programs, which include upregulation of free radical buffering by the glutathione pathway in 2i ESCs. Through induction of intracellular radicals, we show that the altered metabolic environment renders 2i ESCs less sensitive to oxidative stress. Altogether, this work provides novel insights into the proteome landscape underlying ground state pluripotency.

2018-11-07 | PXD009217 | Pride

Integrative proteomics reveals a protective role of PRC2 for the ground state pluripotent epigenome

Project description:The ground state of pluripotency is defined as a basal proliferative state free of epigenetic restriction, represented by mouse embryonic stem cells (ESCs) cultured with two kinase inhibitors (so-called “2i”). Through comparison with serum-grown ESCs, we identify epigenetic features characterizing 2i ESCs by proteome profiling of chromatin including post-translational histone modifications. The most prominent difference is H3K27me3 and its enzymatic writer complex PRC2 that are highly abundant on eu- and heterochromatin in 2i ESCs, with H3K27me3 redistributing outside canonical PRC2 targets in a CpG-dependent fashion. Using PRC2-deficient 2i ESCs, we identify epigenetic crosstalk with H3K27me3, including significant increases in H4 acetylation and DNA methylation. This suggests that the unique H3K27me3 configuration protects 2i ESCs from preparation to lineage priming. Interestingly, removal of DNA methylation in PRC2-deficient 2i ESCs lacking H3K27me3 using 5-azacytidine hardly affected ESC viability and transcriptome, showing that ESCs are independent of both major repressive epigenetic marks.

2018-11-21 | PXD007154 | Pride

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

Transcriptome profiles of mouse embryonic stem cells cultured in different conditions by RNA-seq analysis

Project description:Pluripotent mouse embryonic stem cells (ESCs) were originally derived and stably maintained on feeder cells such as inactivated mouse embryo fibroblasts, and can generate complete ESC-pups by tetraploid embryo complementation (TEC), the most stringent functional test of naive pluripotency. Remarkably, 2i (inhibitors of Mek and Gsk3β signaling) medium with LIF in the absence of serum and feeders was developed to achieve ground state of mouse ESCs, and also has been successfully used for derivation of germline competent ESCs in other species such as rat. Notably, 2i-culture gives rise to transcriptional profiles and epigenetic landscapes quite distinct from serum-based ESCs. To better understand transcriptional landscape and signal transductions, we performed RNA-seq analysis of ESCs cultured in four different conditions: serum without feeder, serum with feeder, serum with feeder and 2i, and N2B27+2i.

2018-01-20 | GSE109418 | GEO

Derivation of ground-state female ESCs maintaining gamete-derived DNA methylation

Project description:Inhibitors of MEK1/2 and Gsk3b, known as '2i' culture conditions, enhance the derivation of embryonic stem cells (ESCs) and promote ground-state pluripotency in rodents 1,2. Here we show that the derivation of female mouse ESCs in the presence of 2i (2i-ESCs) results in a widespread loss of DNA methylation including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, the early-passage 2i-ESCs efficiently differentiate into somatic cells and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in the 2i-ESC-derived differentiated cells. Consistently, 2i-ESCs exhibit impairment of autonomous embryonic and placental development by tetraploid embryo complementation and nuclear transplantation. We identified the derivation conditions of female ESCs that display 2i-ESC-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ESCs exhibit ICR demethylation, regardless of culture conditions. Our results provide insights into derivation of female ESCs reminiscent of ICM of preimplantation embryos. 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 | GSE84164 | GEO

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