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Probing the signaling requirements for naïve human pluripotency by high-throughput chemical screening

ABSTRACT: Probing the signaling requirements for naïve human pluripotency by high-throughput chemical screening

PROVIDER: PRJNA641786 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12088421_1.fastq.gz	Fastqsanger.gz
	SRR12088421_2.fastq.gz	Fastqsanger.gz
	SRR12088422_1.fastq.gz	Fastqsanger.gz
	SRR12088422_2.fastq.gz	Fastqsanger.gz
	SRR12088423_1.fastq.gz	Fastqsanger.gz

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Probing the signaling requirements for naïve human pluripotency by high-throughput chemical screening [WGBS]

Project description:Probing the signaling requirements for naïve human pluripotency by high-throughput chemical screening [WGBS]

| PRJNA641790 | ENA

Probing the signaling requirements for naïve human pluripotency by high-throughput chemical screening [WGBS]

Project description:Background: Naïve human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional “primed” hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Methods:Here we performed high-throughput screening using a library of >3,000 well-annotated compounds to identify essential signaling requirements for naïve human pluripotency. Results:We report that MEK1/2 inhibitors can be replaced during maintenance of naïve human pluripotency by inhibitors targeting either upstream (FGFR, RAF1) or downstream (ERK1/2) kinases. Naïve hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naïve resetting in combination with PKC, ROCK, and TNKS inhibitors and Activin A. Conclusions: This work demonstrates that induction and maintenance of naïve human pluripotency are governed by distinct signaling requirements.

2021-06-15 | GSE153213 | GEO

Probing the signaling requirements for naïve human pluripotency by high-throughput chemical screening [RNA-seq]

Project description:Probing the signaling requirements for naïve human pluripotency by high-throughput chemical screening [RNA-seq]

| PRJNA641789 | ENA

Probing the signaling requirements for naïve human pluripotency by high-throughput chemical screening [RNA-seq]

2021-06-15 | GSE153212 | GEO

Probing the signaling requirements for naïve human pluripotency by high-throughput chemical screening

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

2021-06-15 | GSE153215 | GEO

Probing the signaling requirements for naive human pluripotency by high-throughput chemical screening.

Project description:Naive human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional "primed" hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Here, we describe the results from a high-throughput screen using ∼3,000 well-annotated compounds to identify essential signaling requirements for naive human pluripotency. We report that MEK1/2 inhibitors can be replaced during maintenance of naive human pluripotency by inhibitors targeting either upstream (FGFR, RAF) or downstream (ERK1/2) kinases. Naive hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naive resetting in combination with PKC, ROCK, and TNKS inhibitors and activin A. This work demonstrates that induction and maintenance of naive human pluripotency are governed by distinct signaling requirements.

| S-EPMC8272458 | biostudies-literature

Interpreting reverse transcriptase termination and mutation events for greater insight into the chemical probing of RNA

Project description:Chemical probing has the power to provide insight into RNA conformation in vivo and in vitro, but interpreting the results depends on methods to detect the chemically modified nucleotides. Traditionally, the presence of modified bases was inferred from their ability to halt reverse transcriptase during primer extension and the locations of termination sites observed by electrophoresis or sequencing. More recently, modification-induced mutations have been used as a readout for chemical probing data. Given variable propensity for mismatch incorporation and read-through with different reverse transcriptases, we examined how termination and mutation events compare to each other in the same chemical probing experiments. We found that mutations and terminations induced by dimethyl sulfate probing are both specific for methylated bases, but these two measures have surprisingly little correlation and represent largely non-overlapping indicators of chemical modification data. We also show that specific biases for modified bases depend partly on local sequence context, and that different reverse transcriptases show different biases toward reading a modification as a stop or a mutation. These results support approaches that incorporate analysis of both termination and mutation events into RNA probing experiments.

2017-09-26 | GSE97609 | GEO

Carbodiimide reagents for the chemical probing of RNA structure in cells

Project description:Deciphering the conformations of RNAs in their cellular environment allows identification of RNA elements with potentially functional roles within biological contexts. Insight into the conformation of RNA in cells has been achieved using chemical probes that were developed to react specifically with flexible RNA nucleotides, or the Watson-Crick face of single-stranded nucleotides. The most widely used probes are either selective SHAPE (2'-hydroxyl acylation and primer extension) reagents that probe nucleotide flexibility, or dimethyl sulfate (DMS), which probes the base-pairing at adenine and cytosine but is unable to interrogate guanine or uracil. The constitutively charged carbodiimide N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) is widely used for probing G and U nucleotides, but has not been established for probing RNA in cells. Here, we report the use of a smaller and conditionally charged reagent, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), as a chemical probe of RNA conformation, and the first reagent validated for structure probing of unpaired G and U nucleotides in intact cells. We showed that EDC demonstrates similar reactivity to CMC when probing transcripts in vitro. We found that EDC specifically reacted with accessible nucleotides in the 7SK noncoding RNA in intact cells. We probed structured regions within the Xist lncRNA with EDC and integrated these data with DMS probing data. Together, EDC and DMS allowed us to refine predicted structure models for the 3’ extension of repeat C within Xist. These results highlight how complementing DMS probing experiments with EDC allows the analysis of Watson-Crick base-pairing at all four nucleotides of RNAs in their cellular context.

2018-12-26 | GSE118309 | GEO

AMPK signaling for naïve pluripotency [Hs]

Project description:We show activation of AMP kinase (AMPK) with single chemical compounds can endow naïve pluripotency. AMPK activators reverted early-stage differentiating cells or epi-stem cells to naïve state. Moreover, AMPK activators reverted human ESCs to naïve state with pluripotent gene expression profiles and X-chromosome reactivation.

2019-03-30 | GSE92725 | GEO

AMPK signaling for naïve pluripotency [Mm]

2019-03-30 | GSE92441 | GEO