RNAseq of ground state mouse embryonic stem cells.
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE87632 | GEO | 2017/12/30
SECONDARY ACCESSION(S): PRJNA345440
REPOSITORIES: GEO
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Project description:We apply deep small-RNA sequencing technology for high-throughput profiling of microRNAs in ground state embryonic stem cells (ESCs). We provide global expression signatures of microRNAs in ESCs cultured under serum, 2i, and R2i conditions. We report that microRNAs are significantly differentially expressed when ESCs are cultured under different conditions, and that ground state pluripotency features a uniqure microRNA signature which is mainly encoded by microRNA-coding sequences within the developmentally important DLK1-Dio3 locus. Finally, we indicate that microRNA upregulated in ground state pluripotent cells (i.e. 2i/R2i) contribute to the maintenace of ground state pluripotency through stimulating self-renewal and inhibiting multi-lineague differentiation.
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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.
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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.
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Project description:Derivation of naive state of mouse embryonic stem cells (mESCs) in LIF+serum (LS) culture condition is strain dependent, whereas derivation of ground state mESCs is readily possible from all strains tested so far in “2i” culture condition. ESCs can be derived from the post-implantation stage mouse embryos (EpiSCs), showing primed characteristics. In the present study, we characterized and compared the transcriptional profile of naïve, primed and ground state mESCs. Considering the importance of genetic background of mouse model for ESCs derivation in conventional culture conditions, all ESCs lines used in the study were derived from the same strain of mice. We found distinct transcriptional profiles between naive, primed and ground state mESCs. Primed state mESCs exhibit lower expression of pluripotency markers along with higher expression of lineage specific markers compared to naive and ground state mESCs. We also demonstrate that the differentiation propensity of ESCs to specific germ layer varies depending on the pluripotency state of ESCs.
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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.
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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.
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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.
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Project description:Mouse Embryonic Stem Cells (ESCs) grown in serum-supplemented conditions are characterized by an extremely short G1-phase due to the lack of G1-phase control. Concordantly, the G1-phase-specific P53-P21 pathway is compromised in serum ESCs. Here we provide evidence that P53 is activated upon transition of serum ESCs to their pluripotent ground state using serum-free 2i conditions and modulates G1-phase progression. Our data shows that the elongated G1-phase characteristic of ground state ESCs is dependent on P53. RNA-seq and ChIP-seq analyses reveal that P53 directly regulates the expression of the Retinoblastoma (RB) protein and that the hypo-phosphorylated, active RB protein plays a key role in G1-phase control. Our findings suggest that the P53-P21 pathway is active in ground state 2i ESCs and that its role in the G1-checkpoint is abolished in serum ESCs. Taken together, the data reveals a mechanism by which inactivation of P53 can lead to loss of RB and uncontrolled cell proliferation.
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