Excluding Oct4 from Yamanaka cocktail unleashes the developmental potential of iPSCs
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ABSTRACT:
PROVIDER: PRJNA564252 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10078400_1.fastq.gz | Fastqsanger.gz | |||
| SRR10078400_2.fastq.gz | Fastqsanger.gz | |||
| SRR10078401_1.fastq.gz | Fastqsanger.gz | |||
| SRR10078401_2.fastq.gz | Fastqsanger.gz | |||
| SRR10078402_1.fastq.gz | Fastqsanger.gz |
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Project description:Oct4 is widely considered the most important among the four Yamanaka reprogramming factors. Here we show that the combination of Sox2, Klf4, and cMyc (SKM) suffices for reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs). Simultaneous induction of Sox2 and cMyc in fibroblasts triggers immediate retroviral silencing, which explains the discrepancy with previous studies that attempted but failed to generate iPSCs without Oct4 using retroviral vectors. SKM induction could partially activate the pluripotency network even in Oct4-knockout fibroblasts. Importantly, reprogramming in the absence of exogenous Oct4 results in greatly improved developmental potential of iPSCs, determined by their ability to give rise to all-iPSC mice in the tetraploid complementation assay. Our data suggest that overexpression of Oct4 during reprogramming leads to off-target gene activation during reprogramming and epigenetic aberrations in resulting iPSCs, and thereby bear major implications for further development and application of iPSC technology.
2019-11-06 | GSE137001 | GEO
Project description:Oct4 is widely considered the most important among the four Yamanaka reprogramming factors. Here, we show that the combination of Sox2, Klf4, and cMyc (SKM) suffices for reprogramming mouse somatic cells to induced pluripotent stem cells (iPSCs). Simultaneous induction of Sox2 and cMyc in fibroblasts triggers immediate retroviral silencing, which explains the discrepancy with previous studies that attempted but failed to generate iPSCs without Oct4 using retroviral vectors. SKM induction could partially activate the pluripotency network, even in Oct4-knockout fibroblasts. Importantly, reprogramming in the absence of exogenous Oct4 results in greatly improved developmental potential of iPSCs, determined by their ability to give rise to all-iPSC mice in the tetraploid complementation assay. Our data suggest that overexpression of Oct4 during reprogramming leads to off-target gene activation during reprogramming and epigenetic aberrations in resulting iPSCs and thereby bear major implications for further development and application of iPSC technology.
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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:Mouse induced pluripotent stem cells (iPSCs) were derived from embryonic fibroblasts by overexpressing the Yamanaka factors Oct4, Sox2, Klf4 and c-Myc for 32 consecutive days. iPSCs were isolated by RNA FACS (endogenous Sox2) from the heterogeneous reprogramming culture and transcriptionally compared with (1) iPSCs stabilized in 2i medium (iPS-2i) and (2) iPSCs stabilized in co-culture with mouse embryonic fibroblasts (iPS-MEF) n = 2 replicates for each sample/condition
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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.
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