Long range inter-chromosomal interaction of Oct4 distal enhancer loci regulates ESCs pluripotency [DNA-seq]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE122748 | GEO | 2019/11/30
REPOSITORIES: GEO
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Project description:Nuclear architecture underlies the transcriptional programs within the cell to establish cell identity. As previously demonstrated, long-range chromatin interactions of the Oct4 distal enhancer (DE) are correlated with active transcription in naïve state embryonic stem cells. Here, we identify and characterize extreme long range interactions of the Oct4 DE through a novel CRISPR labeling technique we developed and chromosome conformation capture to identify lethal giant larvae 2 (Llgl2) and growth factor receptor-bound protein 7 (Grb7) as putative functional interacting target genes in different chromosomes. We show that the Oct4 DE directly regulates expression of Llgl2 and Grb7 in addition to Oct4. Expression of Llgl2 and Grb7 closely correlates with the pluripotent state, where knock down of either result in loss of pluripotency, and overexpression enhances somatic cell reprogramming. We demonstrated that biologically important interactions of the Oct4 DE can occur at extreme distances that are necessary for the maintenance of the pluripotent state.
2019-11-30 | GSE122753 | GEO
Project description:Chavez2009 - a core regulatory network of OCT4 in human embryonic stem cells
A core OCT4-regulated network has been identified as a test case, to analyase stem cell characteristics and cellular differentiation.
This model is described in the article:
In silico identification of a core regulatory network of OCT4 in human embryonic stem cells using an integrated approach.
Chavez L, Bais AS, Vingron M, Lehrach H, Adjaye J, Herwig R
BMC Genomics, 2009, 10:314
Abstract:
BACKGROUND: The transcription factor OCT4 is highly expressed in pluripotent embryonic stem cells which are derived from the inner cell mass of mammalian blastocysts. Pluripotency and self renewal are controlled by a transcription regulatory network governed by the transcription factors OCT4, SOX2 and NANOG. Recent studies on reprogramming somatic cells to induced pluripotent stem cells highlight OCT4 as a key regulator of pluripotency.
RESULTS: We have carried out an integrated analysis of high-throughput data (ChIP-on-chip and RNAi experiments along with promoter sequence analysis of putative target genes) and identified a core OCT4 regulatory network in human embryonic stem cells consisting of 33 target genes. Enrichment analysis with these target genes revealed that this integrative analysis increases the functional information content by factors of 1.3 - 4.7 compared to the individual studies. In order to identify potential regulatory co-factors of OCT4, we performed a de novo motif analysis. In addition to known validated OCT4 motifs we obtained binding sites similar to motifs recognized by further regulators of pluripotency and development; e.g. the heterodimer of the transcription factors C-MYC and MAX, a prerequisite for C-MYC transcriptional activity that leads to cell growth and proliferation.
CONCLUSION: Our analysis shows how heterogeneous functional information can be integrated in order to reconstruct gene regulatory networks. As a test case we identified a core OCT4-regulated network that is important for the analysis of stem cell characteristics and cellular differentiation. Functional information is largely enriched using different experimental results. The de novo motif discovery identified well-known regulators closely connected to the OCT4 network as well as potential new regulators of pluripotency and differentiation. These results provide the basis for further targeted functional studies.
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2005-01-01 | MODEL1305010000 | BioModels
Project description:Oct4 is considered a master transcription factor for pluripotent cell self-renewal and embryo development. It primarily collaborates with other transcriptional factors or coregulators to maintain pluripotency. However, it is still unclear how Oct4 interacts with its partners. Here, we show that the Oct4 linker interface mediates competing and balanced Oct4 protein interactions which are crucial for maintaining pluripotency. Linker mutant ESCs maintain the key pluripotency genes expression, but show decreased expression of self-renewal genes and increased expression of differentiation genes which result in impaired ESCs self-renewal and early embryonic lethality. Linker mutation dose not affect Oct4 genomic binding and transactivation potential, but breaks the balanced Oct4 interactome. In mutant ESCs, the interaction between Oct4 and Klf5 was decreased, but interactions between Oct4 and Cbx1, Ctr9, Cdc73 were increased which disrupt the epigenetic state of ESCs. Overexpression of Klf5 or knockdown Cbx1, Cdc73 rescue the cellular phenotype of linker mutant ESCs by rebalancing Oct4 interactome indicating that different partners interact with Oct4 competitively. Thus, by showing how Oct4 interacts with different partners, we provide novel molecular insights to explain how Oct4 contributes to the maintenance of pluripotency.
2021-12-10 | PXD017456 | Pride
Project description:The impact of signal dependent transcription factors, such as glucocorticoid receptor (GR) and NFκB on the three-dimensional organization of chromatin remains a topic of discussion. The possible scenarios range from remodeling of higher order chromatin architecture by activated transcription factors to recruitment of activated transcription factors to pre-established long-range interactions. Using 4C-seq and high-resolution ChIA-PET analysis of P300 we observed agonist-induced changes in long-range chromatin interactions, and uncovered interconnected enhancer-enhancer hubs spanning up to one megabase. The vast majority of activated GR and NFκB appears to join pre-existing P300 enhancer hubs without affecting the chromatin conformation. In contrast, binding of the activated transcription factors to loci with their consensus response elements leads to increased formation of an active epigenetic state of enhancers and a significant increase in long-range interactions within pre-existing enhancer networks. De novo enhancers or ligand-responsive enhancer hubs preferentially interact with ligand-induced genes. We demonstrate that, at a subset of genomic loci, ligand-mediated induction leads to active enhancer formation and an increase in long-range interactions, facilitating efficient regulation of target genes. Therefore, our data suggest an active role of signal dependent transcription factors in chromatin and long-range interaction remodeling.
2015-09-30 | GSE61911 | GEO
Project description:Long-Range Enhancer Interactions Are Prevalent in Mouse Embryonic Stem Cells and Are Reorganized upon Pluripotent State Transition
| PRJNA400069 | ENA
Project description:Brief expression of pluripotency-associated factors such as OCT4, KLF4, SOX2 and c-MYC (OKSM), in combination with differentiation-inducing signals, was reported to trigger transdifferentiation of fibroblasts into alternative cell types. Here, we show that OKSM expression gives rise to both induced pluripotent stem cells (iPSCs) and iNSCs under conditions that were previously shown to induce only NSC transdifferentiation. Fibroblast-derived iNSC colonies silenced retroviral transgenes and reactivated silenced X chromosomes, both hallmarks of pluripotent stem cells. Moreover, lineage tracing via an Oct4-CreER labeling system demonstrated that virtually all iNSC colonies originate from cells transiently expressing Oct4, whereas ablation of Oct4-positive cells prevented iNSC formation. Lastly, use of an alternative transdifferentiation cocktail that lacks OCT4 and was reportedly unable to support induced pluripotency, yielded iPSCs and iNSCs carrying the Oct4-CreER-derived lineage label. Together, these data suggest that iNSC generation using OKSM and related reprogramming factors requires passage through a transient iPSC state. 5 samples were anlyzed in total, 2 induced pluripotent stem cells (iPSCs), 1 neural stem cells (NSCs) and 2 induced NSCs (iNSCs)
2015-04-30 | E-GEOD-65951 | biostudies-arrayexpress
Project description:The transcription factors OCT4 and SOX2 are required for generating induced pluripotent stem cells (iPSCs) and for maintaining embryonic stem cells (ESCs). To this end, OCT4 and SOX2 associate and bind to DNA in different configurations depending on the composite DNA element present in their target genes. Here, we have investigated the role of the different OCT4-SOX2 conformational arrangements in regulating and inducing pluripotency. To this end, we have generated SOX2 mutants that interfere with specific OCT4-SOX2 heterodimer configurations and we have assessed their abilities to generate iPSCs and to rescue ESC self-renewal. Our results demonstrate that the OCT4-SOX2 configuration that dimerizes on a Hoxb1-like composite, a canonical element with juxtaposed individual binding sites, plays a more critical role in the induction and maintenance of pluripotency than other OCT4-SOX2 configurations. Overall, the results of this study provide a new insight into the protein interactions required to establish a de novo pluripotent network and to maintain a true pluripotent cell fate.
2017-02-13 | GSE65345 | GEO
Project description:Serum-to-2i interconversion of mouse Embryonic Stem Cells (mESCs) is a valuable in vitro model for early embryonic development. To assess whether 3D chromatin organization changes during this transition, we established Capture Hi-C with target-sequence enrichment of DNase I hypersensitive sites. We detected extremely long-range intra- and inter-chromosomal interactions between a small subset of H3K27me3 marked bivalent promoters involving the Hox clusters in serum grown cells. Notably, these promoter-mediated interactions are not present in 2i ground-state pluripotent mESCs but appear upon further development into primed-like serum mESCs. Reverting serum mESCs to ground-state 2i mESCs removes these promoter-promoter interactions in a spatiotemporal manner. H3K27me3, which is largely absent at bivalent promoters in ground-state 2i mESCs, is necessary but not sufficient to establish these interactions, as confirmed by Capture Hi-C on Eed-/- serum mESCs. Our results implicate H3K27me3 and PRC2 as critical players in chromatin alteration during priming of ESCs for differentiation. To study dynamics in chromatin architecture and to characterize long-range interaction, we performed Hi-C using DpnII as the restriction enzyme, potentially reaching a genome-wide coverage at a less than 1Kb resolution. We subsequently performed enrichment of interaction by a target capture similar to the exome sequencing approach. We enriched for DNaseI hyper-sensitive sites (DHSâs) in chromatin from mESCs. Probes were designed against the union of all DHSâs of Serum and 2i mESCs. Capture Hi-C reveals Extremely Long-Range Interactions (ELRI) in Serum but not in 2i ESCs. We observed H3K27me3 as a prominent characteristic, but not exclusive feature of ELRI loci in Serum mESCs. To further elucidate the involvement of constituents of PRC1 and PRC2 in ELRI, we performed ChIP-seq experiment on Suz12 and Ring1B during serum-to-2i transition. In addition, RNA-seq was performed to compare the expression levels of genes.
2015-12-07 | E-GEOD-72164 | biostudies-arrayexpress
Project description:Naïve and primed pluripotent stem cells (PSCs) and germ cells express the Pou5f1 (Oct4) gene. The Oct4 gene contains two cis-regulatory elements, the distal (DE) and proximal enhancer (PE), which differentially control Oct4 expression in a cell-type- and stage-specific manner. Here, we generated double transgenic mice carrying both Oct4-ΔPE-GFP and Oct4-ΔDE-tdTomato (RFP), enabling us to simultaneously monitor the activity of DE and PE. Oct4 expression is stage-specifically regulated by DE and PE during embryonic and germ cell development. Using this dual reporter system, we successfully cultured pure populations of naïve (GFP+RFP-) and primed (GFP-RFP+) PSCs. We found that GFP+RFP- cells were metastable in serum-containing medium; stable naïve pluripotent cells were observed in medium containing two inhibitors (Meki and GSKi) but lacking serum. Finally, we suggest that the activity of Oct4 DE and PE is regulated by the repressive histone marks H3K9me3 and DNA methylation.
2015-03-19 | GSE67031 | GEO
Project description:Human endogenous retrovirus subfamily H (HERVH) is a class of transposable elements whose members are expressed preferentially in human pluripotent stem cells. Here, we report that the long-terminal repeat regions of HERVH function as enhancers which are regulated by OCT4. Strikingly, we found that HERVH is a nuclear-localized long non-coding RNA that is required to maintain the undifferentiated state of human embryonic stem cells. Furthermore, we showed that HERVH is associated with OCT4 and co-activators such as P300, CBP and mediator subunits. Together, these results uncover a new and unexpected role of species-specific transposable elements in specifying the pluripotency of human cells.
2014-03-30 | E-MTAB-2072 | biostudies-arrayexpress