Project description:X chromosome reactivation (XCR) represents a paradigm to study epigenetic regulation and the reversal of chromatin silencing, although how it is linked to the pluripotency gene regulatory network, pluripotency transcription factors (TFs) and chromatin remodelling processes remains largely unexplained. Several pluripotency TFs have been linked to XCR through binding to the long non-coding RNA gene Xist, but whether other regulatory elements are involved is unclear. Here, we show that reprogramming to iPSCs induces gradual acquisition of chromatin accessibility at specific sites on the inactive X chromosome (Xi) which is subsequently propagated to other regulatory elements including enhancers and promoters. Regions that are genomically closer to day 0 accessible regions become biallelically accessible earlier, than the more distant ones. For distinct categories of chromatin regions that become biallelically accessible at different times, different sets of transcription factor (TF) motifs are enriched, including the pluripotency TFs KLF4 and cMYC. In addition, regions that become bialleliacally accessible earlier also show increased TF pluripotency binding, supporting the link between pluripotency TFs in XCR. To further explore the relationship between pluripotency TFs and XCR, we construct the active Gene Regulatory Networks (GRNs) during iPSC reprogramming using scRNA-seq and computational analyses. We reveal gene targets of 311 TFs expressed across reprogramming to iPSCs. Using this single-cell regulatory atlas, we show that X-linked genes are preferentially targeted by several pluripotency TFs. Our results suggest a new model where XCR may be coordinated by direct targeting of regulatory elements on the X chromosome by pluripotency TFs, concomitant with step-wise acquisition of chromatin accessibility. Altogether, our results demonstrate how gradual acquisition of a new GRN during reprogramming of cellular identity is linked with dynamic induction of chromatin accessibility and overcomes stable chromatin silencing on the Xi.

Project description:Pluripotency can be maintained in the naïve state through manipulation of ERK and WNT signalling (2i), shielding embryonic stem cells (ESCs) from inductive cues. Alternatively, inhibiting CDK8/19 (CDK8/19i), a repressor of the Mediator co-activator complex, directly stimulates super-enhancer activity, and was recently shown to stabilize cells in a functional state that resembles naïve pluripotency. Naïve ESCs exhibit important epigenetic, transcriptional and metabolic features. However, our understanding on how these regulatory layers are inter-connected to promote the naive state is in progress. To fill this gap, here we used mass spectrometry to describe the dynamic molecular events (i.e. phosphoproteome, proteome and metabolome) executed by 2i and CDK8/19i, as they transition cell identity into naïve pluripotency. We observed rapid proteomic reprogramming, revealing widespread commonalities, and some important differences, between these two approaches, suggesting a largely over-lapping mechanism. CDK8/19i acts directly on the control of the transcriptional machinery, which elicits a rapid and direct activation of key identity genes including those that maintain the naïve program. Additional molecular changes in 2i are achieved by phosphorylation of critical downstream effectors that reinforce the naïve transcriptional circuitry while repressing factors from the more-differentiated formative and primed states. Comparing transcriptomic and proteomic changes, we found that post-transcriptional de-repression is a major feature of naïve pluripotency conferred by both 2i and CDK8/19i, and this may support the enhanced mitochondrial capacity of naive cells. Furthermore, at the level of metabolome, while 2i- and CDK8/19i-treated cells share similar aspects in one-carbon metabolism and beta-oxidation, in other regards they are divergent, a feature which may explain their differences in DNA methylation. These datasets provide a valuable resource for exploring the molecular mechanisms underlying pluripotency and cell identity transitions.

			Action	DRS
	SRR16117547.fastq.gz	Fastqsanger.gz
	SRR16117548.fastq.gz	Fastqsanger.gz
	SRR16117549.fastq.gz	Fastqsanger.gz
	SRR16117550.fastq.gz	Fastqsanger.gz
	SRR16117551.fastq.gz	Fastqsanger.gz

Dataset Information

Landscape of Monoallelic DNA Accessibility and Gene Regulatory Networks during Reprogramming to Naive Pluripotency and X Chromosome Reactivation [allele-specific analysis]

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