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An RNAi screen probing signalling control of human embryonic stem cells establishes the cell cycle-regulated restriction of the exit from pluripotency (DNA damage checkpoint)

ABSTRACT: Analysis of effect of S-phase arrest and replication checkpoint activation on differentiating hESCs at the gene expression level. The hypothesis tested in the present study was that replication checkpoint activation prevents the exit from pluripotency in hESCs. Results provide important information of the response of hESCs to replication arrest, such as upregulation of genes involved in the TGF-beta signaling pathway, and subsequent maintenance of pluripotency marker expression.

ORGANISM(S): Homo sapiens

PROVIDER: GSE62062 | GEO | 2015/07/28

SECONDARY ACCESSION(S): PRJNA263140

REPOSITORIES: GEO

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An RNAi screen probing signalling control of human embryonic stem cells establishes the cell cycle-regulated restriction of the exit from pluripotency (DNA damage checkpoint)

Project description:Analysis of effect of S-phase arrest and replication checkpoint activation on differentiating hESCs at the gene expression level. The hypothesis tested in the present study was that replication checkpoint activation prevents the exit from pluripotency in hESCs. Results provide important information of the response of hESCs to replication arrest, such as upregulation of genes involved in the TGF-beta signaling pathway, and subsequent maintenance of pluripotency marker expression. Total RNA obtained from hESCs incubated in medium without bFGF and TGF-beta supplemented with either DMSO, Aphidicolin or Aphidicolin+AZD7762 for 0, 48 and 96 hours.

2015-07-28 | E-GEOD-62062 | biostudies-arrayexpress

An RNAi screen probing signaling control of human embryonic stem cells establishes the cell cycle-regulated restriction of the exit from pluripotency (CCNB1 overexpression)

Project description:Analysis of effect of Cyclin B1 overexpression on differentiating hESCs at the gene expression level. The hypothesis tested in the present study was that Cyclin B1 overexpression prevents the exit from pluripotency in hESCs. Results provide important information on the effect of Cyclin B1 on hESCs, such as changes in expression of TGFb-related genes and pluripotency markers, as well as mesodermic and neuroectodermic genes. Total RNA obtained from CCNB1 overexpressing hESCs incubated in medium without bFGF and TGF-beta for 0, 48 and 96 hours.

2015-07-28 | E-GEOD-63215 | biostudies-arrayexpress

An RNAi screen probing signaling control of human embryonic stem cells establishes the cell cycle-regulated restriction of the exit from pluripotency (CCNB1 overexpression)

2015-07-28 | GSE63215 | GEO

Probing the signaling requirements for naïve human pluripotency by high-throughput chemical screening [WGBS]

Project description:Background: Naïve human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional “primed” hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Methods:Here we performed high-throughput screening using a library of >3,000 well-annotated compounds to identify essential signaling requirements for naïve human pluripotency. Results:We report that MEK1/2 inhibitors can be replaced during maintenance of naïve human pluripotency by inhibitors targeting either upstream (FGFR, RAF1) or downstream (ERK1/2) kinases. Naïve hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naïve resetting in combination with PKC, ROCK, and TNKS inhibitors and Activin A. Conclusions: This work demonstrates that induction and maintenance of naïve human pluripotency are governed by distinct signaling requirements.

2021-06-15 | GSE153213 | GEO

Probing the signaling requirements for naïve human pluripotency by high-throughput chemical screening [RNA-seq]

2021-06-15 | GSE153212 | GEO

The functional role of OGDH for maintaining mitochondrial respiration and identity of primed human embryonic stem cells

Project description:Human embryonic stem cells (hESCs) can self-renew infinitely or differentiate into the 3 germ layer lineages in vitro with certain cues, holding great promise to model early human embryo development ex vivo. It is wildly accepted that hESCs take advantage of both glycolysis and mitochondrial respiration to favor the naïve pluripotency, while prefer glycolysis to support the primed pluripotency. Thus, the function of mitochondrial respiration in primed hESCs has been underestimated for a long time, and has not been fully understood yet. Herein, we report that the adenosine triphosphate (ATP) production rate is comparable between mitochondrial respiration and glycolysis, suggesting that mitoATP may serve as one of the major sources of the ATP pool in primed hESCs. To further reveal the function of mitochondrial respiration, we deployed inducible CRISPRi method to inhibit OGDH expression with high efficiency, resulting in the TCA cycle blockade as well as the diminishment of mitochondrial respiration activity and total ATP level. Of note, OGDH deficiency led to the exit of primed pluripotency accompanied by cell death. Furthermore, the treatment with small molecule inhibitors to block electron transport chain (ETC) can phenocopy the loss-of-function of OGDH in hESCs. Therefore, genetic and pharmacological perturbations on mitochondrial respiration can impair the stemness of primed hESCs. Collectively, the current study unveils that OGDH acts as a key regulator to fine-tune the abundance of TCA cycle metabolites to ensure the mitochondrial respiration activity in primed hESCs, and highlights the pivotal roles of mitochondrial respiration in terms of ATP production and the maintenance of primed pluripotency. Our findings may provide insights into the linkage between pluripotent states and energy metabolism in hESCs.

2022-03-03 | MTBLS4223 | MetaboLights

ESRG regulates alternative splicing of TCF3 to maintain hESCs self-renewal and pluripotency

Project description:Elucidating the mechanism of self-renewal and pluripotency maintenance of human embryonic stem cells (hESCs) is of great significance in basic research and clinical applications. Long non-coding RNAs (lncRNAs) have been shown to play a key role in the self-renewal and pluripotency maintenance of hESCs. We previously reported that the lncRNA ESRG, which is highly expressed in undifferentiated hESCs, can interact with the replication licensing factor MCM2 and inhibit the p53 pathway to maintain the self-renewal and pluripotency of hPSCs. In addition to MCM2, RNA pull-down mass spectrometry showed that ESRG could also bind to other proteins, among which heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1) attracted our attention. In this study, we show that HNRNPA1 can maintain self-renewal and pluripotency of hESCs. ESRG binds to and stabilizes HNRNPA1 protein through the ubiquitin-proteasome pathway. In addition, knockdown of ESRG or HNRNPA1 resulted in alternative splicing of TCF3, which originally and primarily encodes E12, to mainly encode E47 and inhibit CDH1 expression. HNRNPA1 could rescue the biological function changes of hESCs caused by ESRG knockdown or overexpression. Our results suggest that ESRG regulates the alternative splicing of TCF3 to affect CDH1 expression and maintain hESCs self-renewal and pluripotency by binding and stabilizing HNRNPA1 protein. This study lays a good foundation for exploring the new molecular regulatory mechanism by which ESRG maintains hESCs self-renewal and pluripotency.

2024-03-05 | GSE236745 | GEO

Deep proteomic profiling of TE03 (I3) human embryonic stem cells (hESCs)

Project description:The proteome of undifferentiated human embryonic stem cells (hESCs) was profiled by deep mass spectrometry-based proteomics of whole-cell extracts from suspension cultures of TE03 cells, in four biological replicates. This data accompanies the manuscript: "Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells". Abstract: "Embryonic stem cell (ESC) self-renewal and cell-fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carry out a proteome-wide screen and identify 810 proteins that directly bind RNA in hESCs. We reveal that RBPs are preferentially expressed in hESCs and dynamically regulated during exit from pluripotency and early lineage specification. Moreover, we show that nearly 200 RBPs are affected by knockdown of OCT4, a master regulator of pluripotency, several dozen of which are directly bound by this factor. Intriguingly, over 20 percent of the proteins detected in our study are putative DNA- and RNA-binding proteins (DRBPs), among them key transcription factors (TFs). Using fluorescently labeled RNA and seCLIP (single-end enhanced crosslinking and immunoprecipitation) experiments, we discover that the pluripotency-associated STAT3 and OCT4 TFs interact with RNA in hESCs and confirm the direct binding of STAT3 to the conserved NORAD long-noncoding RNA. Taken together, our findings indicate that RBPs have a more widespread role in human pluripotency than previously appreciated, reinforcing the importance of post-transcriptional regulation in stem cell biology".

2023-05-05 | PXD025278 | Pride

OCT4 IP-MS in Naive and Primed Human ESCs

Project description:Understanding the molecular underpinnings of pluripotency is a prerequisite for optimal maintenance and application of embryonic stem cells (ESCs). While the protein-protein interactions of core pluripotency factors have been identified in mouse ESCs, their interactome in human ESCs (hESCs) has not to date been explored. Here we mapped the OCT4 interactomes in naïve and primed hESCs, revealing extensive connections to mammalian ATP-dependent nucleosome remodeling complexes.

2021-09-12 | PXD026556 | Pride

Mass spectrometry based-proteomic analysis comparing the abundance of RNA-binding proteins (RBPs) between TE03 (I3) human embryonic stem cells (hESCs) and human foreskin fibroblasts (HFFs)

Project description:Here, we used high-resolution mass spectrometry to identify differentially expressed RNA-binding proteins (RBPs) between TE03 (I3) human embryonic stem cells (hESCs) and human foreskin fibroblasts (HFFs). 2102Ep embryonal carcinoma (EC) cells were used as a reference for pluripotent cells. This data accompanies the manuscript: "Uncovering the RNA-binding protein landscape in the pluripotency network of human embryonic stem cells". Abstract: "Embryonic stem cell (ESC) self-renewal and cell-fate decisions are driven by a broad array of molecular signals. While transcriptional regulators have been extensively studied in human ESCs (hESCs), the extent to which RNA-binding proteins (RBPs) contribute to human pluripotency remains unclear. Here, we carry out a proteome-wide screen and identify 810 proteins that directly bind RNA in hESCs. We reveal that RBPs are preferentially expressed in hESCs and dynamically regulated during exit from pluripotency and early lineage specification. Moreover, we show that nearly 200 RBPs are affected by knockdown of OCT4, a master regulator of pluripotency, several dozen of which are directly bound by this factor. Intriguingly, over 20 percent of the proteins detected in our study are putative DNA- and RNA-binding proteins (DRBPs), among them key transcription factors (TFs). Using fluorescently labeled RNA and seCLIP (single-end enhanced crosslinking and immunoprecipitation) experiments, we discover that the pluripotency-associated STAT3 and OCT4 TFs interact with RNA in hESCs and confirm the direct binding of STAT3 to the conserved NORAD long-noncoding RNA. Taken together, our findings indicate that RBPs have a more widespread role in human pluripotency than previously appreciated, reinforcing the importance of post-transcriptional regulation in stem cell biology".

2023-05-05 | PXD025284 | Pride

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