Functional cooperativity between Myc and PI3K signaling for indefinite self-renewal property of embryonic stem cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE60344 | GEO | 2014/10/30
SECONDARY ACCESSION(S): PRJNA258069
REPOSITORIES: GEO
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Project description:We explored the relationship between Myc activity and PI3K signaling in ESCs. Our data demonstrate that Myc and PI3K signaling function cooperatively for supporting pluripotent property of ESCs. Moreover, our data demonstrate that exposure of ESCs to 2i condition render both Myc and PI3K dispensable for preserving ESC status. Effect of PI3K inhibitor, LY4294002 on EBRTcH3 ESCs or their derivatives overexpressing c-Myc (wild-type or T58A mutant) was examined. Effect of LY4294002 on EBRTcH3 ESCs under 2i conditions was also examined. Furthermore, effect of Max expression ablation was compared between ESCs and those overexpressing p110 alpha.
2014-10-30 | E-GEOD-60344 | biostudies-arrayexpress
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
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
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
Project description:c-Myc is one of key players that are crucially involved in maintaining the undifferentiated state and the self-renewal of ESCs. To understand the mechanism by which c-Myc helps preserve these prominent characteristics of ESCs, we generated null-ES cells for the Max gene, which encodes the best characterized partner protein for all Myc family proteins. Although Myc/Max complexes have been widely regarded as crucial regulators of the ESC status, our data reveal that ESCs do not absolutely require these complexes in so-called ground state or related conditons and that this requirement is restricted to conventional ES culture conditions without using a MAPK inhibitor. Simply Dox-treated or Nanog (WT or D67G mutant)-rescued Max-null ESCs which were cultured under conventional culture condition and 2i/Nam-treated completely Max-null ES cells from which Dox-regulatable cDNA was removed were used for RNA source. Samples from Dox-untreated Max-null ESCs cultured under conventional culture condition were used as reference samples for Dox-treated cells and Nanog-rescued cells, while a sample from wild-type ESCs cultured under 2i/Nam condition was used as a reference sample for completely Max-null ES cells cultured with 2i/Nam.
2011-07-19 | E-GEOD-27881 | biostudies-arrayexpress
Project description:Mechanisitc model of PI3K and ERK signal integration by Myc. ERK and PI3K regulated Myc satbility by phosphorylating the same. (PMID:18463697)
2019-09-19 | BIOMD0000000818 | BioModels
Project description:Embryonic stem cells (ESCs) can be maintained in the naïve state through inhibition of Mek1/2 and Gsk3 (2i). A relevant effect of 2i is the inhibition of Cdk8/19, which are negative regulators of the Mediator complex, responsible for the activity of enhancers. Inhibition of Cdk8/19 (Cdk8/19i) stimulates enhancers and, similar to 2i, stabilizes ESCs in the naïve state. Here, we use mass spectrometry to describe the molecular events (phosphoproteome, proteome, and metabolome) triggered by 2i and Cdk8/19i on ESCs. Our data reveal widespread commonalities between these two treatments, suggesting overlapping processes. We find that post-transcriptional de-repression by both 2i and Cdk8/19i might support the mitochondrial capacity of naive cells. However, proteome reprogramming in each treatment is achieved by different mechanisms. Cdk8/19i acts directly on the transcriptional machinery, activating key identity genes to promote the naïve program. In contrast, 2i stabilizes the naïve circuitry through, in part, de-phosphorylation of downstream transcriptional effectors.
2021-02-10 | MTBLS301 | MetaboLights
Project description:Mouse embryonic stem cells can be maintained in a naive state of pluripotency by culture conditions containing inhibitors of Mek and Gsk3. Myc activity is essential for efficient cellular reprogramming. We genetically addressed the role of c-myc and N-myc in naive ESCs cultured in 2i by inducing the deletion of both genes using CRE-mediated recombination. Our findings show that myc activity controls the biosynthetic and proliferative machines of ESCs without significantly affecting the pluripotency network.
2016-02-11 | E-MTAB-3386 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE25170: MYC drives resistance to PI3K/mTOR targeted inhibition (Sty SNP array) GSE25172: MYC drives resistance to PI3K/mTOR targeted inhibition (gene expression) Refer to individual Series
2011-09-13 | E-GEOD-25173 | biostudies-arrayexpress
Project description:Simultaneous inhibition of Gsk3α/β and Mek1/2 activity in the presence of LIF (2i/L) induces a naïve state in mouse embryonic stem cells (ESCs) that resembles the inner cell mass (ICM) of the pre-implantation embryo. Since the ICM exists only transiently in vivo, it remains unclear how extended propagation of naïve ESCs in vitro affects their stability and functionality. Here we show that extended culture of male ESCs in 2i/L results in the progressive erosion of genomic imprints, loss of H2A.X binding, and accumulation of chromosomal aberrations. Consistent with these observations, we find that the developmental potential of ESCs cultured in 2i/L is impaired. Mechanistically, we demonstrate that Mek1/2 inhibition is predominantly responsible for these effects, in part through downregulation of DNA methyltransferases. Additionally, we demonstrate that female ESCs cultured in conventional serum/LIF media phenocopy male ESCs cultured in 2i/L, including the aforementioned epigenetic and developmental abnormalities. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as developmental potential of ESCs. Taken together, our data suggest that, while short-term suppression of Mek1/2 in ESCs maintains an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential.
2017-07-27 | GSE97954 | GEO