Genome-wide deciphering the translation ratio of hESCs under OCT4 siRNA treatment and hypoxia by RNC-seq
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE186550 | GEO | 2025/03/05
REPOSITORIES: GEO
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Project description:Genome-wide deciphering the translation ratio of hESCs under OCT4 siRNA treatment and hypoxia by RNC-seq
| PRJNA774358 | ENA
Project description:We set out to systematically decipher the OCT4-RNA interactome by HITS-CLIP.
2024-07-24 | GSE154655 | GEO
Project description:We set out to develop an unbiased, genome-wide method to screen for proteins that locate to the OCT4 proximal enhancer (PE) in hESCs; for this purpose, we developed an optimized locus-specific proteomics approach in hESCs. First, we designed TALEN (Transcription Activator-Like Effector Nuclease) constructs to target the sequences that are near the OCT4 PE, located in a region of DNaseI hypersensitivity. TALEN constructs with the highest cutting efficiency were chosen for locus-specific proteomics. We then made modifications to the original TALEN protein to transform it into a catalytically-dead TALE (dTALE) protein that is optimized for locus-specific proteomics in hESCs via three steps: 1) The nuclease-domain FokI at the C-terminus was replaced by a GFP (green fluorescence protein); 2) a 3X FLAG tag at the N-terminus was included for following pull-down analysis; 3) the existing CMV promoter was replaced with an EF1alpha promoter that has robust expression in hESCs. This dTALE protein could then be chemically crosslinked to the OCT4 locus together with all the other proteins that bind to the locus. We verified that dTALE protein binds to the targeted locus by chromatin immunoprecipitation (ChIP)-qPCR. Following crosslinking, chromatin was sheared, and all the associated proteins were immunoprecipitated using an anti-FLAG antibody. Immunoprecipitation pulled down the dTALE protein as well as other proteins and complexes that are also attached to that region. Crosslinking was then reversed, and the samples were subjected to mass spectrometry to enable generation of a list of proteins that potentially bind to OCT4 PE locus.
2019-01-07 | PXD010963 | Pride
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
Project description:This study is directed to systematically map out phosphorylation sites in OCT4 protein that are differentially phosphorylated between naïve hESCs and their primed counterparts, and to further identify the upstream kinases that phosphorylate the sites in naïve hESC-specific manner
2025-05-06 | PXD033619 | Pride
Project description:RNC-seq experiment of the simulated acetylation mutant S1K247Q of S1 protein
2024-09-15 | GSE254167 | GEO
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
Project description:Here, we performed RNA-interactome capture (RIC) on nuclear fractions from human embryonic stem cells (hESCs). The poly(A)+ RNA-bound proteome was determined by UV light-mediated cross-linking (CL) of RNAs to proteins in living cells, followed by nuclei isolation, oligo(dT) purification of poly(A)-RNA-protein complexes, and mass spectrometry analysis of captured proteins. As a control, we applied a similar strategy to non-cross-linked (non-CL) samples. RIC was performed in four independent 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 | PXD025262 | Pride
Project description:The transcription factors Nanog, Oct4 and Sox2 are the master regulators of pluripotency in mouse embryonic stem cells (mESCs), however, their functions in human ESCs (hESCs) have not been rigorously defined. Here we show that the requirements for NANOG, OCT4 and SOX2 in hESCs differ from those in mESCs. Both NANOG and OCT4 are required for self-renewal and repress differentiation. OCT4 controls both extraembryonic and epiblast-derived cell fates in a BMP4-dependent manner. OCT4-depleted hESCs commit to trophectoderm and primitive endoderm in the presence of BMP4, but undergo neuroectoderm differentiation in the absence of BMP4. NANOG represses neuroectoderm and neural crest commitment, but has little or no effect on the other lineages. We find that SOX2 is not required for self-renewal because it is redundant with SOX3, which is induced in SOX2-depleted hESCs. Simultaneous depletion of both SOX2 and SOX3 induces differentiation into the primitive streak. Unexpectedly, we identify significant variability in the usage of pluripotency factors by individual hESC lines, suggesting that the pluripotency network is remodelled to support a continuum of developmental states. Our study revises the general view of how NANOG, OCT4 and SOX2 orchestrate self-renewal in hESCs.
2012-04-06 | GSE34920 | GEO
Project description:The transcription factors Nanog, Oct4 and Sox2 are the master regulators of pluripotency in mouse embryonic stem cells (mESCs), however, their functions in human ESCs (hESCs) have not been rigorously defined. Here we show that the requirements for NANOG, OCT4 and SOX2 in hESCs differ from those in mESCs. Both NANOG and OCT4 are required for self-renewal and repress differentiation. OCT4 controls both extraembryonic and epiblast-derived cell fates in a BMP4-dependent manner. OCT4-depleted hESCs commit to trophectoderm and primitive endoderm in the presence of BMP4, but undergo neuroectoderm differentiation in the absence of BMP4. NANOG represses neuroectoderm and neural crest commitment, but has little or no effect on the other lineages. We find that SOX2 is not required for self-renewal because it is redundant with SOX3, which is induced in SOX2-depleted hESCs. Simultaneous depletion of both SOX2 and SOX3 induces differentiation into the primitive streak. Unexpectedly, we identify significant variability in the usage of pluripotency factors by individual hESC lines, suggesting that the pluripotency network is remodelled to support a continuum of developmental states. Our study revises the general view of how NANOG, OCT4 and SOX2 orchestrate self-renewal in hESCs.
2012-04-06 | GSE34918 | GEO