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CRISPR Screen: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

ABSTRACT: CRISPR Screen: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

PROVIDER: PRJNA989564 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR25097700.fastq.gz	Fastqsanger.gz
	SRR25097701.fastq.gz	Fastqsanger.gz
	SRR25097702.fastq.gz	Fastqsanger.gz
	SRR25097703.fastq.gz	Fastqsanger.gz
	SRR25097704.fastq.gz	Fastqsanger.gz

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CRISPR Screen: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

Project description:To investigate the genes and pathways involved in X-chromosome reactivation and pluripotency acquisition, we generated a whole-genome CRISPR knockout screen during female NPC reprogramming into iPSCs. We analyzed the gRNA abundance in NPCs and three different reprogramming populations: non-pluripotent, early pluripotent and late pluripotent. Pair-wise comparisons of gRNA frequencies in different populations were done to identify genes and pathways involved in cell survival in reprogramming conditions, colony formation, early pluripotency acquisition and late pluripotency/X-chromosome reactivation.

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Targeted Amplicon Sequencing: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

Project description:Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) requires activation of the pluripotency network and resetting of the epigenome by erasing the epigenetic memory of the somatic state. In female mouse cells, a critical epigenetic reprogramming step is the reactivation of the inactive X chromosome. Despite its importance, a systematic understanding of the regulatory networks linking pluripotency and X-reactivation is missing. Here we reveal important pathways for pluripotency acquisition and X-reactivation using a genome-wide CRISPR screen during neural precursor to iPSC reprogramming. In particular, we discover that activation of the interferon γ (IFNγ) pathway early during reprogramming accelerates pluripotency acquisition and X-reactivation. IFNγ stimulates STAT3 signaling and the pluripotency network and leads to enhanced TET-mediated DNA demethylation, which consequently boosts X-reactivation. We therefore gain a mechanistic understanding of the role of IFNγ in reprogramming and X-reactivation and provide a comprehensive resource of the molecular networks involved in these processes.

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The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

Project description:The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

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RNA-seq: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

Project description:RNA-seq: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

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DNA methylation arrays: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

Project description:DNA methylation arrays: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

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Project description:Targeted Amplicon Sequencing: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

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DNA methylation arrays: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

Project description:To investigate how IFNγ treatment during early stages of reprogramming could affect DNA methylation levels and its link with X-chromosome reactivation, we analyzed 5mC and 5hmC levels in cells undergoing reprogramming, in the presence or absence of IFNγ treatment, using mouse DNA methylation arrays.

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RNA-seq: The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

Project description:In order to identify trasncriptomic changes happening upon the activation of the IFNγ pathway during pluripotency acquisition, female NPCs were induced for reprogramming in the presence or absence of IFNγ for the first five days of the process. RNA-sequencing was performed at days 2, 5 and 7 in control and treated samples. ESCs and NPCs were also included in the study as controls.

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The Interferon γ Pathway Enhances Pluripotency and X-Chromosome Reactivation in iPSC Reprogramming

Project description:This SuperSeries is composed of the SubSeries listed below.

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X Chromosome Reactivation Dynamics Reveal Stages of Reprogramming to Pluripotency

Project description:Reprogramming to iPSCs resets the epigenome of somatic cells including the reversal of X chromosome-inactivation. We sought to gain insight into the steps underlying the reprogramming process by examining the means by which reprogramming leads to X chromosome-reactivation (XCR). Analyzing single cells in situ, we found that hallmarks of the inactive X (Xi) change sequentially, providing a direct readout of reprogramming progression. Several epigenetic changes on the Xi occur in the inverse order of developmental X-inactivation, while others are uncoupled from this sequence. Among the latter, DNA methylation has an extraordinary long persistence on the Xi during reprogramming, and, like Xist expression, is erased only after pluripotency genes are activated. Mechanistically, XCR requires both DNA demethylation and Xist silencing, ensuring that only cells undergoing faithful reprogramming initiate XCR. Our study defines the epigenetic state of multiple sequential reprogramming intermediates and establishes a paradigm for studying cell fate transitions during reprogramming. RRBS profiles were generated from female and male ES cells, female iPS cells, SSEA1+ and SSEA1- reprogramming intermediates, and male and female MEFs, for a total of 18 samples.

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