Reprogramming competence of POU factors is linked to the donor-cell epigenome
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ABSTRACT:
PROVIDER: PRJNA377635 | ENA |
REPOSITORIES: ENA
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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:Identifying molecular targets that regulate reprogramming competence of transcription factors in donor cells broadens our understanding of reprogramming process. Here, by a chemical screen targeting major epigenetic pathways in human reprogramming, we discovered that inhibiting specific epigenetic roadblocks allows iPSC generation with almost all OCT factors. Amongst these epigenetic pathways was not only DOT1L-mediated H3K79 methylation but also other modifications catalyzed by LSD1, DNMTs and HDACs, and we found that simultaneous inhibition of these pathways not only dramatically enhances reprogramming competence of most OCT factors but in fact enables to dismantle species-dependent reprograming competence of OCT6, NR5A1, NR5A2, TET1 and GATA3. Harnessing these permissive epigenetic states, we performed an additional screen with 98 transcriptional regulators. Thereby, we identified 25 novel genes that can functionally replace OCT4 in inducing pluripotency. Our findings provide a conceptional framework for understanding how transcription factors elicit reprogramming in dependency of the donor cell epigenome that differs across species.
2020-06-24 | GSE149017 | GEO
Project description:We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner-transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counter-selection of mutants in 21 different genes during the challenge. Eight of the counter-selected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants in which seven of the eight remaining genes, i.e., spr0459/spr0460, spr0777, spr0838, spr1259/spr1260, and spr1357, were deleted, displayed reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation of chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and DNA damage repair in S. pneumoniae. Keywords: GAF competence Selection for genes essential for transformation. Aliquots of a marinerT7 transposon library generated in S. pneumoniae R6 containing approximately 40,000 independent mutants were transformed with an antibiotic resistance marker and grown without (initial library) or with (challenged library) antibiotics for 25 generation until DNA extraction.
2007-09-04 | E-GEOD-8362 | biostudies-arrayexpress
Project description:We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner-transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counter-selection of mutants in 21 different genes during the challenge. Eight of the counter-selected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants in which seven of the eight remaining genes, i.e., spr0459/spr0460, spr0777, spr0838, spr1259/spr1260, and spr1357, were deleted, displayed reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation of chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and DNA damage repair in S. pneumoniae. Keywords: GAF competence
2007-09-05 | GSE8362 | GEO
Project description:OCA-B, OCA-T1, and OCA-T2 belong to a family of transcriptional coactivators that bind to POU transcription factors (TFs) to regulate gene expression in immune cells. Here, we identify IkBz (encoded by the NFKBIZ gene) as the fourth member of the OCA protein family. While originally discovered as an inducible regulator of NFkB, we show here that IkBz shares a microhomology with OCA proteins and uses this segment to simultaneously bind to POU transcription factors and octamer motif-containing DNA. Our functional reporter assays suggest that IkBz requires its interaction with POU TFs to coactivate immune-related genes. This finding is reinforced by our epigenomic analysis of MYD88 L265P-mutant lymphoma cells, which revealed colocalization of IkBz, the POU transcription factor OCT2, and NFkB:p50 at hundreds of DNA elements harboring octamer and kB motifs. These results suggest that IkBz is a transcriptional coactivator that integrates and amplifies the output of NFkB and POU transcription factors at inducible genes in immune cells.
2024-01-09 | GSE239374 | 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: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 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: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