Genomic binding by an artificially evolved POU (ePOU)
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA412047 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6071690_1.fastq.gz | Fastqsanger.gz | |||
| SRR6071690_2.fastq.gz | Fastqsanger.gz | |||
| SRR6071691_1.fastq.gz | Fastqsanger.gz | |||
| SRR6071691_2.fastq.gz | Fastqsanger.gz | |||
| SRR6071692_1.fastq.gz | Fastqsanger.gz |
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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 chromatin immunoprecipitation sequencing (ChIPseq) for both factors and Sox2. Two cocktails Oct4 (O), Sox2 (S), Klf4 (K), c-Myc (M) and ePOU/S/K/M were transduced into OG2 MEF cells which is MEF cells with Oct4 promotor.
2021-02-07 | GSE104220 | 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: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:Gene expression program directed by an artificially evolved POU (ePOU)
| PRJNA412048 | ENA
Project description:Chromatin binding and regulation by Oct4 and artificially evolved POU (ePOU) in pluripotency reprogramming
| PRJNA678622 | ENA
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:Interaction of the Kaposi’s sarcoma-associated herpesvirus (KSHV) Rta protein with the cellular Notch signaling effector, Recombination Signaling Protein (RBP)-Jk (aka CSL and CBF-1), is essential for viral reactivation from latency. We previously showed that Rta binds to a DNA motif repeated in the viral Mta promoter (called “CANT” or Rta-c) to stimulate RBP-Jk DNA binding, and distinguished Rta from the activated Notch-1 protein. To determine whether Rta’s mechanism would apply generally to other viral promoters, we employed chromatin immunoprecipitation/deep sequencing (ChIP/Seq) to identify Rta and RBP-Jk binding sites across the KSHV genome. We show that RBP-Jk binds nearly exclusively to unique genome sites during latency and reactivation. Many, but not all, reactivation-specific RBP-Jk peaks were associated with Rta bound to single Rta-c motifs. Other motifs that were over-represented with stimulated RBP-Jk DNA binding including those for the cellular DNA binding proteins BCL11A, MNT, MAF B, and TCF12. Four of the top seven motifs that were most over-represented with inhibition of RBP-Jk DNA binding were putative binding sites for the Pit/Oct/Unc (POU) family of proteins, including POU3F3 (Oct-8) and POU5F1 (Oct-4). Interestingly, two other POU motifs, including one for the POU2F1 (Oct-1) protein, are associated with inhibition of RBP-Jk DNA binding unless Rta bound to a nearby Rta-c motif. The relative distances between the Rta-c, POU, and RBP-Jk motifs were conserved at reactivation-specific RBP-Jk peaks in three promoters that Rta transactivated, and the Rta-c and Oct-1 motifs overlapped in two of those. The proximity of the Rta-c/Oct-1 motif to an RBP-Jk motif is critical for Rta transactivation of the ORF50AS/KbZIP promoter, and knockdown of Oct-1 protein debilitated reactivation and production of infectious virus. Our data suggest a broad role for POU proteins in regulating DNA binding of RBP-Jk and its associated transactivators.
2019-07-14 | GSE122752 | GEO
Project description:Pluripotent stem cells are a hallmark of animal multicellularity. Sox and POU family transcription factors are pivotal for stemness and were believed to be animal innovations as they were reported absent from the genomes of their unicellular relatives. Here we describe new unicellular holozoan orthologues to Sox and POU families, indicating that they emerged before the appearance of animals. We show that choanoflagellate and filasterean Sox genes have DNA binding specificity similar to Sox2. Choanoflagellate Sox can partner with the POU member Oct4 on DNA elements found in pluripotency enhancers. Consistently, choanoflagellate – but not filasterean – Sox genes can replace Sox2 to reprogram mouse somatic cells into induced pluripotent stem cells (iPSC). In contrast, choanoflagellate POU harbors a unique DNA-binding profile that differs from Oct4 and cannot generate iPSCs. Pluripotency reprogramming with reconstructed ancestral Sox genes shows that their molecular ability to induce stemness was already present in the last common ancestor of animals and their unicellular relatives. Our findings imply that the evolution of stem cells exploited a pre-existing set of transcription factors, where the critical innovation involved an initial change in DNA specificity of POU and the exaptation of the ancestral capacity to interact with Sox transcription factors.
2024-10-23 | GSE253887 | GEO
Project description:Pluripotent stem cells are a hallmark of animal multicellularity. Sox and POU family transcription factors are pivotal for stemness and were believed to be animal innovations as they were reported absent from the genomes of their unicellular relatives. Here we describe new unicellular holozoan orthologues to Sox and POU families, indicating that they emerged before the appearance of animals. We show that choanoflagellate and filasterean Sox genes have DNA binding specificity similar to Sox2. Choanoflagellate Sox can partner with the POU member Oct4 on DNA elements found in pluripotency enhancers. Consistently, choanoflagellate – but not filasterean – Sox genes can replace Sox2 to reprogram mouse somatic cells into induced pluripotent stem cells (iPSC). In contrast, choanoflagellate POU harbors a unique DNA-binding profile that differs from Oct4 and cannot generate iPSCs. Pluripotency reprogramming with reconstructed ancestral Sox genes shows that their molecular ability to induce stemness was already present in the last common ancestor of animals and their unicellular relatives. Our findings imply that the evolution of stem cells exploited a pre-existing set of transcription factors, where the critical innovation involved an initial change in DNA specificity of POU and the exaptation of the ancestral capacity to interact with Sox transcription factors.
2024-10-23 | GSE253886 | GEO
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