Heterochromatin lossening by Oct4 in somatic cell reprogramming
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ABSTRACT:
PROVIDER: PRJNA532597 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8889400_1.fastq.gz | Fastqsanger.gz | |||
| SRR8889400_2.fastq.gz | Fastqsanger.gz | |||
| SRR8889410_1.fastq.gz | Fastqsanger.gz | |||
| SRR8889410_2.fastq.gz | Fastqsanger.gz | |||
| SRR8889391_1.fastq.gz | Fastqsanger.gz |
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Project description:The success of Yamanaka factor reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem cells (iPSCs) suggests that some factor(s) must remodel the nuclei from a condensed state to a relaxed state to facilitate factor binding to target genes. We asked how the Yamanaka factor-dependent chromatin opening occurs. We found Oct4, but neither Sox2 nor Klf4, is responsible for the loosening of the condensed state. Oct4 acts as a pioneer factor that facilitates the binding of Klf4 and the expression of epithelial genes in the early stage of reprogramming.
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Project description:OCT4 is expressed in extraembryonic endoderm stem (XEN) cell progenitors during somatic cell reprogramming
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Project description:Forced expression of four transcription factors Oct4,Sox2, Klf4 and Myc (OSKM) induces somatic cell reprogramming towards pluripotency. Major efforts have been made to characterize the molecular events involved in this process. Yet, it remains elusive how gene expression change, epigenetic landscape remodelling and cell fate conversion are triggered by expression of these Yamanaka factors.To address this gap,we utilized a secondary inducible reprogramming system and performed genome-wide profilings of Oct4 binding, histone modification(H3K4me3/H3K27me3/H3K4me1/H3K27ac), and gene expression analysis during this process. Through integrative analysis, we revealed stage-specific Oct4 binding and enhancer signatures in consistence with gene expression changes,in which the initial regression of somatic program is followed by the gradual acquisition of pluripotent program. Oct4 preferatially binds to H3K4me1 marked enhancer regions and Oct4 binding is positively correlated with active mark H3K27ac. Moreover,we observed significant enhancer activation of epigenetic related genes, especially acetylation associated genes, prior to pluripotency network activation, suggesting a pivotal role of epigenetic remodelling in the process of pluripotency acquisition and maintenance. We used ChIP-seq to explore the global changes of Oct4 binding profiling during OSKM-mediated somatic cell reprogramming. The exogenous Oct4 was tagged with 3XFlag, thus we used anti-flag antibody to monitor exogenous Oct4 changes; anti-Oct4 antibody which recognizes both endogenous and exogenous Oct4 was used to monitor total Oct4 changes during iPSC induction. In addition, we also examined genome-wide H3K4me1/H3K27ac/H3K4me3/H3K27me3/RNAPII profiling during 3Flag-OSKM 2' reprogramming.
2015-12-31 | E-GEOD-67520 | biostudies-arrayexpress
Project description:During development, progenitors of embryonic stem (ES) and extraembryonic endoderm stem (XEN) cells are concomitantly specified within the inner cell mass (ICM) of the mouse blastocyst. Similarly, XEN cells are induced (iXEN cells) alongside induced pluripotent stem (iPS) cells following overexpression ofOct4,Sox2,Klf4andMyc(OSKM) during somatic cell reprogramming. It is unclear how or why this cocktail produces both stem cell types, but OCT4 has been associated with non-pluripotent outcomes. In this report, we show that, during OSKM reprogramming, many individualOct4-GFP-expressing cells are fated to become iXEN cells. Interestingly, SKM alone was also sufficient to induce iXEN cell formation, likely via activation of endogenousOct4.These observations indicate that iXEN cell formation is not strictly an artifact ofOct4overexpression. Moreover, our results suggest that a pathway to XEN may be an integral feature of establishing pluripotency during reprogramming, as in early embryo development.
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Project description:The transcription factor Oct4/Pou5f1 is a key component of the regulatory circuitry governing pluripotency. Oct4 is also widely used to generate induced pluripotent stem cells (iPSCs) from somatic cells. These observations provide compelling rationale to understand Oct4’s functions. Here we used domain swapping and mutagenesis to compare Oct4’s reprogramming activity with the paralog Oct1/Pou2f1, identifying a redox-sensitive DNA binding domain cysteine residue (Cys48) as a key determinant of both reprogramming and differentiation. In combination with the Oct4 N-terminus, mutating Oct1’s serine at this position to cysteine is sufficient to confer strong reprogramming activity. Conversely, Oct4C48S strongly reduces reprogramming potential. Oct4C48S renders the protein insensitive to oxidative inhibition of DNA binding. The same mutation prevents oxidation-mediated protein ubiquitylation. An engineered Pou5f1C48S point mutation has little effect on undifferentiated cells embryonic stem cells (ESCs), but upon retinoic acid (RA)-mediated differentiation causes retention of Oct4 expression, decreased proliferation, and increased apoptosis. Transcriptional profiling reveals that the retention of pluripotency also manifests at the RNA level. Pou5f1C48S ESCs also contribute poorly to adult somatic tissues and form less differentiated teratomas. Collectively, the data support a model in which Oct4 redox sensing is a positive reprogramming determinant during one or more reprogramming steps, and mediates Oct4 degradation during differentiation.
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Project description:Cellular differentiation involves profound changes in the chromatic landscape, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNAi screens targeting chromatin factors during transcription factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPSCs). Remarkably, subunits of the chromatin assembly factor-1 (CAF-1) complex emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Suppression of CAF-1 increased reprogramming efficiency by several orders of magnitude and facilitated iPSC formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 as a novel regulator of somatic cell identity during transcription factor-induced cell fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting. Gene expression analysis in CAF-1 knockdown and Renilla control during early OKSM-induced reprogramming by microarray
2015-12-10 | E-GEOD-72741 | biostudies-arrayexpress
Project description:The evolutionary origins of the gene network underlying cellular pluripotency, a central theme in developmental biology, have yet to be elucidated. In mammals, Oct4 is a factor crucial in the reprogramming of differentiated cells into induced pluripotent stem cells. The Oct4 and Pou2 genes evolved from a POU class V gene ancestor, but it is unknown whether pluripotency induced by Oct4 gene activity is a feature specific to mammals or was already present in ancestral vertebrates. Here we report that different vertebrate Pou2 and Oct4 homologues can induce pluripotency in mouse and human fibroblasts and that the inability of zebrafish Pou2 to establish pluripotency is not representative of all Pou2 genes, as medaka Pou2 and axolotl Pou2 are able to reprogram somatic cells into pluripotent cells. Therefore, our results indicate that induction of pluripotency is not a feature specific to mammals, but existed in the Oct4/Pou2 common ancestral vertebrate. 16 samples were analyzed Notation: O: stands for OCT4 reprogramming factor from human; o: stands for Oct4 reprogramming factor from Axolotl S: stands for SOX2 reprogramming factor from human; s: stands for SOX2 reprogramming factor from Axolotl K: stands for KLF4 reprogramming factor from human
2014-03-23 | E-GEOD-21302 | biostudies-arrayexpress
Project description:Forced expression of four transcription factors Oct4,Sox2, Klf4 and Myc (OSKM) induces somatic cell reprogramming towards pluripotency. Major efforts have been made to characterize the molecular events involved in this process. Yet, it remains elusive how gene expression change, epigenetic landscape remodelling and cell fate conversion are triggered by expression of these Yamanaka factors. To address this gap,we utilized a secondary inducible reprogramming system and performed genome-wide profilings of Oct4 binding, histone modification (H3K4me3/H3K27me3/H3K4me1/H3K27ac), and gene expression analysis during this process. Through integrative analysis, we revealed stage-specific Oct4 binding and enhancer signatures in consistence with gene expression changes, in which the initial regression of somatic program is followed by the gradual acquisition of pluripotent program. Oct4 preferatially binds to H3K4me1 marked enhancer regions and Oct4 binding is positively correlated with active mark H3K27ac. Moreover, we observed significant enhancer activation of epigenetic related genes, especially acetylation associated genes, prior to pluripotency network activation, suggesting a pivotal role of epigenetic remodelling in the process of pluripotency acquisition and maintenance.
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Project description:Differentiated cells can be reprogrammed into induced pluripotent stem cells (iPSCs) following the forced overexpression of a specific combination of transcription factors, of which Oct4 is essential. To better understand the mechanism underlying iPSC reprogramming, we investigated the immediate transcriptional response to ectopic Oct4 expression in somatic cells. To this end, we derived different somatic cell types from Oct4-inducible transgenic mice and induced Oct4 expression for different time periods. Using microarray analysis, we have identified early transcriptional Oct4 targets, including genes not previously known to interact with Oct4. Comparison of the sets of Oct4-regulated genes revealed pronounced differences among the individual cell types. Furthermore, a notable up-regulation of pluripotent markers could not be detected. In contrast, we observed a down-regulation of somatic markers specific to each cellular population. This finding suggests that Oct4 interferes with the somatic transcriptional program in a cell type-specific manner, thereby promoting an unstable epigenetic state that facilitates reprogramming rather than the direct initiation of a pluripotent gene expression network. Finally, we show that Mgarp, a gene expressed in pluripotent cells, is commonly up-regulated in all cell types after Oct4 induction. Unexpectedly, Mgarp expression decreases during the first steps of reprogramming due to a Klf4-dependent inhibition. Our data show that Oct4 counteracts Klf4 repressive activity and enhances Mgarp expression at the late stages of reprogramming. This temporal pattern of Mgarp expression is crucial for the efficient generation of iPSCs. In summary, our study provides new insights into the specific role that Oct4 plays during the early stages of reprogramming. Different somatic cell types were derived from transgenic mice containing both a tetracycline-inducible transactivator (rtTA-M2) and a tetracycline operon-controlled Oct4 expression cassette (TO) (Hochedlinger 2005, Cell 121:465-477) that were mated with GOF18-GFP mice in which the green fluorescent protein (GFP) is driven by 18 kb of the Oct4 regulatory region (Oct4-GFP) (Yoshimizu 1999, Dev Growth Differ 41:675-684), thus indicating endogenous Oct4 expression. In addition, a control neural stem cell (CtrlNSC) line was generated from transgenic mice that entail a tetracycline-inducible transactivator (irtTA-VP16-GBD) (Anastassiadis 2002, Gene 298:159-172) plus an OG2 Oct4-GFP reporter transgene (Yoshimizu 1999),but lack a TO cassette. A second control consisted of wild type (C57BL/6) mouse embryonic fibroblasts (CtrlMEF). Oct4-inducible mouse embryonic fibroblasts (TO-MEFs) and neural stem cell (TO-NSCs) were derived from 14.5 days post coitum (d.p.c.) embryos and cultured as previously described (Conti 2005, PLoS Biol 3:e283). Primary bone marrow cells (TO-BMCs) were isolated from 6-week old mice after flushing femora and tibiae and plated onto dishes with a combined coating of gelatin (PAA, Pasching, Austria), laminin (Santa Cruz Biotechnology, Santa Cruz, CA), and poly-L-lysine (Sigma, St. Louis, MO). Adherent cells were then cultivated in DMEM Low Glucose (Gibco, Carlsbad, CA) with 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM/L glutamine (all PAA). The transgenic Oct4 expression was induced by either 6 µg/ml doxycycline (Sigma) or by 2 µg/ml doxycycline (Dox) plus 10–7 M dexamethasone (Sigma), depending on the respectively contained tetracycline transactivator. 5-azacytidine (10 nM, Sigma) and cycloheximide (30 µg/ml, Sigma) were applied as indicated. Generation, culture, and differentiation of ESCs and iPSCs were performed as previously described (Tiemann 2011, Cytometry A 79:426-435). Microarray Gene Expression Analysis Samples from Oct4-inducible cell types and controls were collected at different time points (0, 6, 12, and 24 hours after induction). Biotin-labeled cRNA was prepared with the linear TotalPrep RNA Amplification Kit (Ambion, Austin, TX) from 500 ng original RNA and hybridized onto MouseRef 8 v2.0 Expression BeadChips (Illumina, San Diego, CA). The chips were stained with streptavidin-Cy3 (GE Healthcare, Little Chalfont, UK) and scanned using the iScan reader (Illumina). Bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina) and background correction was performed using the Affymetrix robust multiarray analysis model. Variance stabilization was performed by log2 scaling and the R-Bioconductor lumi package was used for normalization. 17 samples were analyzed. ESC: Mouse Embryonic Stem Cell (ESC) TO_MEF_0: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 0h TO_MEF-6: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 6h TO_MEF-12: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 12h TO_MEF-24: Tetracycline Oct4 (TO) Mouse Embryonic Fibroblast (MEF), time 24h TO_BMC-0: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 0h TO_BMC-6: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 6h TO_BMC-12: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 12h TO_BMC-24: Tetracycline Oct4 (TO) Mouse Bone Marrow Cell (BMC), time 24h TO_NSC-0: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 0h TO_NSC-6: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 6h TO_NSC-12: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 12h TO_NSC-24: Tetracycline Oct4 (TO) Mouse Neural Stem Cell (NSC), time 24h Ctrl_MEF_0: Control wild type Mouse Embryonic Fibroblast (MEF) untreated Ctrl_MEF_24: Control wild type Mouse Embryonic Fibroblast (MEF) 24h treated Ctrl_NSC_0: Control wild type Mouse Neural Stem Cell (NSC) untreated Ctrl_NSC_24: Control wild type Mouse Neural Stem Cell (NSC) 24h treated
2014-05-13 | E-GEOD-47061 | biostudies-arrayexpress
Project description:The goal of this study is to identify the role of OCT4 in combination with small molecules as the critical cue governing the reprogramming of human somatic cells into OPCs using ChIP-seq
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