Genomic binding by an artificially evolved POU (ePOU)
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE104220 | GEO | 2021/02/07
REPOSITORIES: GEO
Similar Datasets
Project description:We generated Oct4 libraries by randomizing selected amino acids and by recombining domains of paralogous POU family genes. These libraries were subjected to iterative rounds of pooled screens to select variants that enhance pluripotency induction. We identified an artificially evolved POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of mouse embryonic fibrobast (MEF) reprogramming. To probe whether the ePOU and Oct4 differentially engage the chromatin of reprogramming cells, we performed Assay for Transposase-Accessible Chromatin with highthroughput sequencing (ATACseq) for both factors and different combination. Two cocktails Sox2 (S), Klf4 (K), c-Myc (M) plus Oct4 (O) or ePOU(e) were transduced into OG2 MEF cells which is MEF cells with Oct4 promotor.
2021-01-31 | GSE161512 | GEO
Project description:We generated Oct4 libraries by randomizing selected amino acids and by recombining domains of paralogous POU family genes. These libraries were subjected to iterative rounds of pooled screens to select variants that enhance pluripotency induction. We identified an artificially evolved POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of mouse embryonic fibrobast (MEF) reprogramming. We compared the transcriptomes of cells reprogramming under ePOU or Oct4 conditions at day 3 using RNA sequencing.
2021-01-31 | GSE104219 | GEO
Project description:Embryonic stem cell (ESCs) identity is orchestrated by co-operativity between the transcription factors (TFs) Sox2 and the class V POU-TF, Oct4 at composite Sox/Oct motifs. Neural stem cells (NSCs) lack Oct4 but express Sox2 and class III POU-TFs. This raises the question of how Sox2 interacts with POU-TFs to transcriptionally specify ESCs or NSCs. Here we show that Oct4 alone binds the Sox/Oct motif and the octamer-containing palindromic MORE equally well. Sox2 binding selectively increases the affinity of Oct4 for the Sox/Oct motif. In contrast, Oct6 binds preferentially to the MORE, and is unaffected by Sox2. ChIP-seq in NSCs shows the MORE to be the most enriched motif for class III POU-TFs, with MORE sub-types apparent, but no Sox/Oct motif enrichment. These results suggest that in NSCs, co-operativity between Sox2 and class III POU-TFs may not occur and that POU-TF driven transcription uses predominantly the MORE cis architecture. Thus, distinct interactions between Sox2 and POU-TF subclasses distinguish pluripotent ESCs from multipotent NSCs, providing molecular insight into how Oct4 alone can convert NSCs to pluripotency.
2015-08-21 | GSE69859 | GEO
Project description:Next Generation Sequencing with differdent pluripotent transcript factor overexpression in MEF Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by defined factors. The low efficiency of reprogramming limited the potential application of iPSCs. Here we found that knockdown LSD1 , which demethylates histone H3 Lys 4 or 9 , could increase iPSCs generation. It has been reported that LSD1 interaction with Oct4 which is the core factor of reprogramming. So we try to find out the different of overexpression Oct4 or Kllf4/Sox2 in MEF and LSD1 inhibitor. Retrovirus-mediated different pluripotent transcript factor overexpression in MEF cells after 4 days collect mRNA profiles processing with Illumina MiSeq; MEF, Mouse Embryonic Fibroblast O, MEF infected with Oct4 KS,MEF infected with Klf4 and Sox2 OSK, MEF infected with Oct4 Klf4 and Sox2 T20, MEF infected with Oct4 Klf4 and Sox2 and treated with 20nm LSD1 inhibitor Tranylcypromine
2015-09-17 | E-GEOD-61694 | biostudies-arrayexpress
Project description:Octamer-binding Pit-Oct-Unc (POU) family members have distinct reprogramming competences. OCT4 induces pluripotency, whereas POU III factors (OCT6, OCT7, OCT8, and OCT9) lack this ability, but are prone to inducing neural identities. However, which specific features of these proteins render the distinct reprograming competences remains unknown. Here, we present that OCT6 can also induce pluripotency. But, it works only with human cells, indicating its species-dependent reprogramming activity. Functional readouts with a series of reciprocal mutants uncover that the central role of OCT4 and its strong reprogramming competence to pluripotency arise from its C-terminal transactivation domain. Furthermore, we identify intrinsic properties of OCT7, OCT8, and OCT9 that are detrimental for inducing pluripotency. A chemical screen reveals that their persistent deficiency for inducing pluripotency can be surmounted by reducing H3K79 methylation in donor cells. Our findings delineate that intrinsic properties of POU factors and their responsive donor-cell epigenome state are tightly linked to the reprogramming competence.
2020-06-24 | GSE95608 | GEO
Project description:Chromatin binding and regulation by Oct4 and artificially evolved POU (ePOU) in pluripotency reprogramming
| PRJNA678622 | ENA
Project description:Human and mouse somatic cells can be reprogrammed by the combination of Oct4, Sox2, Klf4, and c-Myc, but the efficiency of reprogramming is low. To better understand the process of reprogramming we sought to identify factors that mediate reprogramming at higher efficiency. For this we established an assay to screen nuclear fractions from the extracts of pluripotent cells based on Oct4 reactivation. We identified components of the ATP-dependent SWI/SNF chromatin-remodeling complex, which when used along with the above four factors increase reprogramming efficiency by five-fold and improve the quality of the reprogrammed cells. These cells were found to be capable of germline transmission and exhibited pluripotency according to gene expression and in vivo and in vitro assays. SWI/SNF was found to replace c-Myc and mediate its effects by facilitating recruitment of Oct4 on target promoters during reprogramming. Thus, somatic cell reprogramming using chromatin-remodeling molecules represents an efficient method of generating reprogrammed cells. DNA-free RNA samples to be hybridized on Illumina expression BeadChips were processed using a linear amplification kit (Ambion) (generating IVT duration: 12h). cRNA samples were quality-checked on a 2100 Bioanalyzer (Agilent) and hybridized as biological triplicates onto MouseWG-6 V2 chips as recommended and using materials / reagents provided by the manufacturer (hybridization time: 18h). The Myc probe on the V2 arrays was found to be defective. The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina), background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model, variance stabilization was performed using log2 scaling, and gene expression normalization was calculated with the quantile method implemented in the lumi package of R-Bioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab. Hierarchical clusters of genes and samples were performed with a one minus correlation metric and the average (unweighted pair group) linkage method. 4 samples were analyzed, each one them by triplicate. ESC: Mouse Embryonic Stem Cells (OG2 mix female and male) MEF: Mouse Embryonic Fibroblasts (OG2 male) iPS4F: Mouse induced Pluripotent Stem cells with 4 factors (Oct4, Sox2, Klf4, c-myc) iPS6F: Mouse induced Pluripotent Stem cells with 6 factors (Oct4, Sox2, Klf4, c-myc, Brg1, Baf155)
2010-06-23 | E-GEOD-18330 | biostudies-arrayexpress
Project description:Somatic cells can be reprogrammed into pluripotent stem cells by the ectopic expression of OCT4, SOX2, KLF4, and c-MYC. Although SOX2, KLF4, and c-MYC (SKM) can be substituted by their respective family members, OCT4 is considered to not be interchangeable with any octamer-binding POU proteins. Through a screening with 102 candidate genes, here we have identified that the POU protein OCT6 (also known as SCIP, TST-1, and POU3F1), in conjunction with SKM, is capable of functionally replacing OCT4 and inducing pluripotency. OCT6-mediated reprogramming works with any human cell type, but not with mouse cells. The reprogramming process involving OCT6 is relatively inefficient and slow. This is mainly due to either inefficient formation of OCT6-SOX2 heterodimers onto canonical SOX-OCT sites or lower transactivation activity of OCT6. We demonstrate that modulating either the DNA-binding propensity or the transactivation activity of OCT6 enhances iPSC generation to the efficiency of OCT4. These results thus provide the first evidence that a POU factor other than OCT4 can induce human pluripotency.
2019-01-17 | GSE93706 | GEO
Project description:Pluripotency reprogramming by competent and incompetent POU factors uncovers temporal dependency for Oct4 and Sox2
| PRJNA407838 | ENA
Project description:Pluripotent stem cell lines derived from embryos of different stages have distinct pluripotent ground states, but similar levels of the transcription factor Oct4. Epiblast-derived pluripotent stem cells (EpiSCs), in contrast to embryonic stem (ES) cells, cannot form chimeras. We show that EpiSCs express lower levels of the transcription factors Sox2 and Klf4 than ES cells and have limited reprogramming potential, as shown by cell fusion. Sox2 overexpression dramatically increases the reprogramming potential, chimera formation, and germline contribution of EpiSCs. Therefore, although Oct4 is essential for reprogramming, the level of Sox2 defines both the reprogramming capability and the pluripotent ground states. RNA samples to be analyzed on microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Ambion), which involved synthesis of T7-linked double-stranded cDNA and 12 hrs of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was then hybridized for 18 hrs onto MouseRef-8 v2 expression BeadChips (Illumina) according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were hybridized as biological replicates. 12 sample types were analyzed, each of them in duplicate. ESCm: Mouse ESC male; ESCf: Mouse ESC OG2 female; F9 EC: F9 EC (mouse embryonic carcinoma cell); F9-Sox2: F9 EC (mouse embryonic carcinoma cell) overexpressing wild type Sox2; EpiSCf: Mouse EpiSC OG2 female; Epi-Sox2f: Mouse EpiSC Sox2 (OG2 female) overexpressing wild type Sox2; P19 EC: P19 EC (mouse embryonic carcinoma cell); P19-Sox2: P19 EC (mouse embryonic carcinoma cell) overexpressing wild type Sox2; EpiSCm: Mouse EpiSC (GOF18 male) (duplicates); EpiSox2mL2: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in condition EpiSC medium (CM); EpiSox2mE1: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in ESC medium (ESC like1); EpiSox2mE2: Mouse EpiSC Sox2 (GOF18 male) overexpressing wild type Sox2 cultured in ESC medium (ESC like2).
2010-12-31 | E-GEOD-15358 | biostudies-arrayexpress