Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE5895 | GEO | 2007/05/23
SECONDARY ACCESSION(S): PRJNA97323
REPOSITORIES: GEO
Similar Datasets
Project description:To characterize the differentiation by Sox2 KO, we performed microarray analyses of mouse ES cell line 2TS22C during the time-course being induced of Sox2 KO Inducible Sox2 KO cell line 2TS22C were assigned to eight experimental groups and cultured in GMEM medium (SIGMA) with LIF at 37°C in an atmosphere of 5% CO2 until the specified time point before the harvest: (1)for controls, cultured in the absence of Tc; (2) for 3h after the administration of Tc; (3) for 6h; (4) for 12h; (5) for 24h; (6) for 48h; (7) for 72h; (8) for 96h.
2010-06-11 | E-GEOD-5895 | biostudies-arrayexpress
Project description:Protein-protein proximity of core pluripotency transcription factors plays an important role during cell reprogramming. Pluripotent embryonic stem (ES) cell identity is controlled by a trio of transcription factors: Sox2, Oct4, and Nanog. These proteins often bind to closely localized genomic sites. The precise mode by which Sox2, Oct4, and Nanog interact with DNA is likely to make a crucial contribution to their function. Here, a detailed protocol for in vivo detection and quantitative analysis of protein-protein proximity of Sox2 and Oct4 using Proximity Utilizing Biotinylation (PUB) method based on the use of the BAP/BirA (target/enzyme) system is described. The method includes design and cloning of DNA plasmid construct, transient transfection of HEK293T cells, Western blot analysis of nuclei fraction and LC-MS/MS analysis. Experiments with coexpression of BAP-X+BirA-Y (X, Y=Sox2, Oct4 and GFP as control) revealed strong biotinylation level of target proteins when X and Y were pluripotency transcription factors compared with control when X=GFP. Since mass spectrometry provides both high sensitivity and more accurate quantification of data a modified workflow was used, in which SDS-PAGE step was eliminated and His-tagged BAP-fused proteins from cell lysate were purified in 6M guanidine HCl buffer, washed, propionylated, digested directly on the Ni sepharose beads using trypsin and analysed on Q-TOF Impact II instrument. Using mass spectrometry allows making quantitative estimation of in vivo interaction of BAP-Sox2 and BirA-Oct4 which was demonstrated by measuring ratios of biotinylation levels of BAP fused either with Sox2 or GFP at different biotin pulse times. After vector preparation this protocol can be completed in seven working days.
2020-01-21 | PXD015756 | Pride
Project description:To characterize Klf4 downstream genes, we performed microarray analyses of the ES cells knocked down by siRNA of Klf4 gene. Keywords: individual genetic characteristic design
2006-09-30 | GSE5930 | GEO
Project description:POU transcription factor Pou5f1 (Oct3/4) is required to maintain ES cells in an undifferentiated state. Here we show that global expression profiling of Oct3/4-manipulated ES cells delineates the downstream target genes of Oct3/4. Combined with data from genome-wide chromatin-immunoprecipitation assays, this analysis identifies not only primary downstream targets of Oct3/4, but also secondary or tertiary targets. Furthermore, the analysis also reveals that downstream target genes are regulated either positively or negatively by Oct3/4. Identification of a group of genes that show both activation and repression depending on Oct3/4 expression levels provides a possible mechanism for the requirement of appropriate Oct3/4 expression to maintain undifferentiated ES cells. As a proof-of-principle study, one of the downstream genes, Tcl1, has been analyzed in detail. We show that Oct3/4 binds to the promoter region of Tcl1 and activates its transcription. We also show that Tcl1 is involved in the regulation of proliferation, but not differentiation, in ES cells. These findings suggest that the global expression profiling of gene-manipulated ES cells can help to delineate the structure and dynamics of gene regulatory networks. Keywords: development or differentiation design,time series design
2006-12-21 | GSE5936 | GEO
Project description:SOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. How temporal fluctuations in their expression levels bias lineage commitment is unknown. Here we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cells towards both neuroectodermal and mesendodermal fates. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.
2019-09-23 | GSE126554 | GEO
Project description:We report the effect on genome-wide gene expression after deletion of an enhancer region downstream of Sox2 in F1 ES cells. The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Reporter assays reveal novel enhancers, including two enhancers over 100 kb downstream (SRR107 and SRR111) which, through the formation of chromatin loops, localise to the Sox2 promoter in ES cells. Using CRISPR/Cas9 we deleted a region containing these two enhancers, which we term the Sox2 control region (SCR). This deletion revealed that the SCR is required for Sox2 transcription in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together these data identify a distal control region essential for Sox2 transcription in ES cells.
2014-11-19 | GSE58339 | GEO
Project description:We report the effect on genome-wide gene expression after deletion of an enhancer region downstream of Sox2 in F1 ES cells. The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency. Reporter assays reveal novel enhancers, including two enhancers over 100 kb downstream (SRR107 and SRR111) which, through the formation of chromatin loops, localise to the Sox2 promoter in ES cells. Using CRISPR/Cas9 we deleted a region containing these two enhancers, which we term the Sox2 control region (SCR). This deletion revealed that the SCR is required for Sox2 transcription in ES cells. Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together these data identify a distal control region essential for Sox2 transcription in ES cells. Examination of PolA+ RNA after heterozygous and homozygous enhancer deletion in F1 ES cells (M. musculus129 x M. castaneus).
2014-11-19 | E-GEOD-58339 | biostudies-arrayexpress
Project description:Previous studies have demonstrated that pluripotency-associated transcription factors, such as Oct3/4, Nanog and Sox2, play a crucial role in the development and malignant progression of various types of tumors. Breast cancer is the most frequent cancer among females, being a heterogeneous disease, with distinct morphologies, metastatic behavior and therapeutic responses. The expression of Oct3/4, Nanog and Sox2 in 3 human breast cancer cell lines, MCF7, T-47D and MDA-MB-231, was determined. The expression of Oct3/4, Nanog and Sox2 mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR) and protein expression was detected by immunocytohistochemistry. RT-PCR revealed that all three human breast cancer cell lines tested expressed evident Oct3/4, Nanog and Sox-2 mRNA at various levels. Higher levels of Oct3/4 were identified in MCF7 and MDA-MB-231 cells compared with T-47D cells. Higher levels of Nanog were observed in MCF7 and T-47D cells compared with MDA-MB-231 cells and the highest expression of Sox-2 was detected in MCF7 cells. The nuclear localization of Oct3/4, Nanog and Sox-2 was confirmed by immunostaining. Oct3/4, Nanog and Sox2 were expressed in human breast cancer cell lines. Further studies are required to characterize the role of Oct3/4, Nanog and Sox2 in human breast cancer.
| S-EPMC3506717 | biostudies-literature
Project description:Previous studies have demonstrated that pluripotency-associated transcription factors, such as Oct3/4, Nanog and Sox2, play a crucial role in the development and malignant progression of various types of tumors. Breast cancer is the most frequent cancer among females, being a heterogeneous disease, with distinct morphologies, metastatic behavior and therapeutic responses. The expression of Oct3/4, Nanog and Sox2 in 3 human breast cancer cell lines, MCF7, T-47D and MDA-MB-231, was determined. The expression of Oct3/4, Nanog and Sox2 mRNA was determined by reverse transcription polymerase chain reaction (RT-PCR) and protein expression was detected by immunocytohistochemistry. RT-PCR revealed that all three human breast cancer cell lines tested expressed evident Oct3/4, Nanog and Sox-2 mRNA at various levels. Higher levels of Oct3/4 were identified in MCF7 and MDA-MB-231 cells compared with T-47D cells. Higher levels of Nanog were observed in MCF7 and T-47D cells compared with MDA-MB-231 cells and the highest expression of Sox-2 was detected in MCF7 cells. The nuclear localization of Oct3/4, Nanog and Sox-2 was confirmed by immunostaining. Oct3/4, Nanog and Sox2 were expressed in human breast cancer cell lines. Further studies are required to characterize the role of Oct3/4, Nanog and Sox2 in human breast cancer.
| S-EPMC3506717 | biostudies-literature
Project description:We present a microarray experiment with the same tet-inducible system but with multiple time points within 24 hr to capture early responses to Oct4 suppression. These data were analyzed together with published genome-wide ChIP data. We found that most tentative target genes (TTG) of Oct4 in ES cells were activated by Oct4 and only a few genes were suppressed. The same method was then applied to find target genes of Sox2 and Nanog based mostly on previously published data. Because the interaction between Oct4, Sox2, and Nanog is supported by immunoprecipitation, functional analysis, and co-localization of binding sites, we explored the relationships between their target genes. Keywords: time series design
2008-04-24 | GSE8617 | GEO