Mus musculus
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ABSTRACT:
PROVIDER: PRJNA288679 | ENA |
REPOSITORIES: ENA
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Project description:The self-renewing pluripotent state was first captured in mouse embryonic stem cells (mESCs) over two decades ago. The standard condition requires the presence of serum and LIF, which provide growth promoting signals for cell expansion. However, there are pro-differentiation signals which destabilize the undifferentiated state of mESCs. The dual inhibition (2i) of the pro-differentiation Mek/Erk and Gsk3/Tcf3 pathways in mESCs is sufficient to establish an enhanced pluripotent “ground state” which bears features resembling the pre-implantation mouse epiblast. Gsk3 inhibition alleviates the repression of Esrrb, a transcription factor that can substitute for Nanog function in mESCs. The molecular mechanism that is mediated by Mek inhibition is however not clear. In this study, we investigate the pathway through which Mek inhibition operates to maintain ground state pluripotency. We have found that in mESCs, Kruppel-like factor 2 (Klf2) is a protein target of the Mek/Erk pathway; and that Klf2 protein is phosphorylated by Erk2 and subsequently degraded through the proteosome. It is therefore by Mek-inhibition through PD0325901 or 2i that enables the stabilization and accumulation of Klf2 to sustain ground state pluripotency. Importantly, we found that Klf2-null mESCs, while viable under LIF/Serum conditions, cannot be maintained and eventually gradually die within a few passages. Our result thus demonstrates that Klf2 is an essential factor of ground state pluripotency. Collectively, our study defines the Mek/Klf2 axis that cooperates with the Gsk3/Esrrb pathway in mediating ground state pluripotency.
2014-06-04 | E-MTAB-2365 | biostudies-arrayexpress
Project description:The self-renewing pluripotent state was first captured in mouse embryonic stem cells (mESCs) over two decades ago. The standard condition requires the presence of serum and LIF, which provide growth promoting signals for cell expansion. However, there are pro-differentiation signals which destabilize the undifferentiated state of mESCs. The dual inhibition (2i) of the pro-differentiation Mek/Erk and Gsk3/Tcf3 pathways in mESCs is sufficient to establish an enhanced pluripotent “ground state” which bears features resembling the pre-implantation mouse epiblast. Gsk3 inhibition alleviates the repression of Esrrb, a transcription factor that can substitute for Nanog function in mESCs. The molecular mechanism that is mediated by Mek inhibition is however not clear. In this study, we investigate the pathway through which Mek inhibition operates to maintain ground state pluripotency. We have found that in mESCs, Kruppel-like factor 2 (Klf2) is a protein target of the Mek/Erk pathway; and that Klf2 protein is phosphorylated by Erk2 and subsequently degraded through the proteosome. It is therefore by Mek-inhibition through PD0325901 or 2i that enables the stabilization and accumulation of Klf2 to sustain ground state pluripotency. Importantly, we found that Klf2-null mESCs, while viable under LIF/Serum conditions, cannot be maintained and eventually gradually die within a few passages. Our result thus demonstrates that Klf2 is an essential factor of ground state pluripotency. Collectively, our study defines the Mek/Klf2 axis that cooperates with the Gsk3/Esrrb pathway in mediating ground state pluripotency.
2014-06-04 | E-MTAB-2366 | biostudies-arrayexpress
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
Project description:Human pluripotent cells were reset to ground state pluripotency by transient overexpression of NANOG and KLF2 and subsequent inhibition of ERK and protein kinase C. Transcriptional profiling of reset cells and conventional pluripotent stem cell cultures was carried out by RNA-seq, in tandem with mouse embryonic stem cells propagated under similar conditions to assess the combinatorial effects of MEK inhibitor PD0325901, GSK3 inhibitor CHIR99021 and PKC inhibitor Go6983.
2014-09-10 | E-MTAB-2857 | biostudies-arrayexpress
Project description:Understanding mechanisms of epigenetic regulation in embryonic stem cells (ESCs) is of fundamental importance for stem cell and developmental biology. Here we identify Spic, a member of the ETS family of transcription factors, as a specific marker of ground state pluripotency. We show that Spic is rapidly induced in ESCs cultured with GSK3-, MEK-inhibitors and LIF (2iL), and in response to MEK/ERK inhibition. ChIP-seq analysis demonstrated that Spic binds to enhancer elements that are associated with pluripotency genes. Interaction proteomics and genomic profiling confirmed that SPIC interacts with NANOG and stabilizes its binding to chromatin in 2iL-ESCs. Additional gain of function and loss of function experiments revealed that Spic controls genes involved in one carbon (1C) metabolism, Bhmt, Bhmt2, and Dmgdh, and the flux of SAM-to-SAH in 2iL-ESCs. By maintaining low levels of SAM, Spic controls the level of H3K4me3 and H3R17me2 histone methylation in ground state ESCs. Our data highlight the role of uncharacterized axillary transcription factors that link cellular metabolism to epigenetic regulation in ground state pluripotency.
2023-09-06 | GSE222053 | GEO
Project description:Understanding mechanisms of epigenetic regulation in embryonic stem cells (ESCs) is of fundamental importance for stem cell and developmental biology. Here we identify Spic, a member of the ETS family of transcription factors, as a specific marker of ground state pluripotency. We show that Spic is rapidly induced in ESCs cultured with GSK3-, MEK-inhibitors and LIF (2iL), and in response to MEK/ERK inhibition. ChIP-seq analysis demonstrated that Spic binds to enhancer elements that are associated with pluripotency genes. Interaction proteomics and genomic profiling confirmed that SPIC interacts with NANOG and stabilizes its binding to chromatin in 2iL-ESCs. Additional gain of function and loss of function experiments revealed that Spic controls genes involved in one carbon (1C) metabolism, Bhmt, Bhmt2, and Dmgdh, and the flux of SAM-to-SAH in 2iL-ESCs. By maintaining low levels of SAM, Spic controls the level of H3K4me3 and H3R17me2 histone methylation in ground state ESCs. Our data highlight the role of uncharacterized axillary transcription factors that link cellular metabolism to epigenetic regulation in ground state pluripotency.
2023-09-06 | GSE222057 | GEO
Project description:Mouse embryonic stem cells were maintained in minimal and chemically-defined culture conditions supporting naive pluripotency. Inhibitors of the Gsk3 (CHIR99021) and Mek/Erk (PD0325901) pathways were withdrawn, cultures maintained for 24 hours, and subsequently sorted by flow cytometry based on fluorescence of a short-half-life Rex1(Zfp42)::GFP reporter into two populations: Rex1-high cells, functionally capable of reversion to naive pluripotency, and Rex1-low cells that have exited the naive state.
2017-02-22 | E-MTAB-5305 | biostudies-arrayexpress
Project description:Mouse embryonic stem cells were maintained in minimal and chemically-defined culture conditions supporting naive pluripotency. Inhibitors of the Gsk3 (CHIR99021) and Mek/Erk (PD0325901) pathways were withdrawn, cultures maintained for 24 hours, and subsequently sorted by flow cytometry based on fluorescence of a short-half-life Rex1(Zfp42)::GFP reporter into two populations: Rex1-high cells, functionally capable of reversion to naive pluripotency, and Rex1-low cells that have exited the naive state.
2017-02-22 | E-MTAB-5304 | biostudies-arrayexpress
Project description:Deciphering the mechanisms that control the pluripotent ground state is key for understanding embryonic development. Nonetheless, the epigenetic regulation of ground-state mouse embryonic stem cells (mESCs) is not fully understood. Here, we identify the epigenetic protein MPP8 as being essential for ground-state pluripotency. Its depletion leads to cell cycle arrest and spontaneous differentiation. MPP8 has been suggested to repress LINE1 elements by recruiting the human silencing hub (HUSH) complex to H3K9me3-rich regions. Unexpectedly, we find that LINE1 elements are efficiently repressed by MPP8 lacking the chromodomain, while the unannotated C-terminus is essential for its function. Moreover, we show that SETDB1 recruits MPP8 to its genomic target loci, whereas transcriptional repression of LINE1 elements is maintained without retaining H3K9me3 levels. Taken together our findings demonstrate that MPP8 protects the DNA-hypomethylated pluripotent ground state through its association with the HUSH core complex, however, independently of detectable chromatin binding and maintenance of H3K9me3.
2021-03-21 | PXD019345 | Pride
Project description:Mouse embryonic stem (ES) cells cultured in defined medium with MEK and GSK3 inhibitors (2i) resemble the pre-implantation epiblast in the ground state, with full development capacity including the somatic lineages and the germline. Although β-catenin is known to be crucial for naive pluripotency of ES cells, the mechanism is not fully understood. Here we showed that β-catenin interacted with a repressive protein complex to maintain the ground state of ES cells by fine-tuning their lineage development potential. Absence of β-catenin impaired ES cell self-renewal without affecting the core self-renewal circuitry of Oct4, Sox2 and Nanog as well as other pluripotency factors. However, β-catenin-deficient cells showed a primed state transcriptional signature with perturbed expression of germline and neuronal lineage genes. Knockdown of Tcf7l1, the repressor in canonical Wnt signaling pathway, did not completely rescue the β-catenin-deficient phenotype of ES cells. Mechanistically, β-catenin formed a novel biochemical complex with E2F6, HP1γ and HMGA2 to restrain ES cells from differentiation by co-occupying the promoters of germline and neuronal lineage regulators independent of TCF7L1. Overall, out work showed that β-catenin maintained ground state ES cells by orchestrating their development plasticity through a repressive protein complex with E2F6, HP1γ and HMGA2.
2020-08-20 | GSE108620 | GEO