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Tracking and mitigating imprint erasure during induction of naïve human pluripotency at single-cell resolution [RNA-Seq]

ABSTRACT: Tracking and mitigating imprint erasure during induction of naïve human pluripotency at single-cell resolution [RNA-Seq]

PROVIDER: PRJNA1117755 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR29212702_1.fastq.gz	Fastqsanger.gz
	SRR29212702_2.fastq.gz	Fastqsanger.gz
	SRR29212703_1.fastq.gz	Fastqsanger.gz
	SRR29212703_2.fastq.gz	Fastqsanger.gz
	SRR29212704_1.fastq.gz	Fastqsanger.gz

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Tracking and mitigating imprint erasure during induction of naïve human pluripotency at single-cell resolution [Bisulfite-Seq]

Project description:Naïve human pluripotent stem cells (hPSCs) model the pre-implantation epiblast. However, parent-specific epigenetic marks (imprints) are eroded in naïve hPSCs, which represents an important deviation from the epiblast in vivo. To track the dynamics of imprint erasure during naïve resetting in real time, we established a dual-colored fluorescent reporter at both alleles of the imprinted SNRPN locus. During primed-to-naïve resetting, SNRPN expression becomes biallelic in most naïve cells and biallelic SNRPN expression is irreversible upon re-priming. We utilized this live-cell reporter to evaluate chemical and genetic strategies to minimize imprint erasure. Decreasing the level of MEK/ERK inhibition or overexpressing the KRAB zinc finger protein ZFP57 protected a subset of imprints during naïve resetting. Combining these two strategies protected imprint levels to a further extent than either strategy alone. This study offers an experimental tool to track and enhance imprint stability during transitions between human pluripotent states in vitro.

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Tracking and mitigating imprint erasure during induction of naïve human pluripotency at single-cell resolution [RNA-Seq]

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Project description:Genome-wide DNA demethylation, including the erasure of genome imprints, in primordial germ cells (PGCs), is critical as a first step for creating the totipotent epigenome in the germ line. Here, we provide evidence that contrary to the prevailing model involving active DNA demethylation, imprint erasure in mouse PGCs occurs in a manner consistent with replication-coupled passive DNA demethylation: PGCs erase imprints during their rapid proliferation with little de novo as well as maintenance DNA methylation potential and no major chromatin alterations. Our findings necessitate the re-evaluation of and provide novel insights into the mechanism of genome-wide DNA demethylation in PGCs. We performed expression analysis of primordial germ cells (PGCs) at embryonic days 10.5-13.5. Because the number of PGCs available at these stages were low, cDNAs were amplified by the method that we previously published (Kurimoto et al. 2006, NAR 34: e42 (PMID 16547197)). To include analysis of PGC gene expression at E9.5, we re-normalized the data from our E9.5 PGC samples (GSM744103, GSM744104) from our previous publication (Hayashi et al., 2011, Cell 146: 519-32 (PMD 21820164)) together with the data from this submission.

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