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Genome-wide analysis of gene expression of IWR1/Y27632-maintained epiblast stem cells (EpiSCs)

ABSTRACT: Analysis of specific genes regulated by IWR1 and Y27632 (Y27), respectivly.

ORGANISM(S): Mus musculus

PROVIDER: GSE77476 | GEO | 2016/06/01

SECONDARY ACCESSION(S): PRJNA310607

REPOSITORIES: GEO

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Genome-wide analysis of gene expression of IWR1/Y27632-maintained epiblast stem cells (EpiSCs)

Project description:Analysis of specific genes regulated by IWR1 and Y27632 (Y27), respectivly. Total RNA obtained from No Treatment (NT) control EpiSCs and EpiSCs treated by 2 ÂµM IWR1 or 0.5 ÂµM Y27.

2016-06-01 | E-GEOD-77476 | biostudies-arrayexpress

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Project description:Epiblast stem cells (EpiSCs) were derived from the epiblast or the ectoderm (epi/ect) of pre-gastrula stage to late-bud stage mouse embryos. To identify if the EpiSCs retain any original stage specific characteristics or which developmental stage of epi/ect they most closely related to, we performed microarray analysis to compare the gene expression profile of multiple EpiSC lines with that of epi/ect of 7 different stages. Eighteen EpiSC lines established from R1-129 embryos of different stages and 1 line (EpiSC9) imported were harvested in triplicate cultured separately for 3 days. The * marks the subline thawed at a different time and harvested but originated from the same embryo. EpiSC9_T is an RNA sample provided by Dr Tesar. ESCs, iPSCs and MEFs were prepared accordingly. For the epiblast samples, in order to avoid the averaging effect by pooling samples, we linearly amplified the RNA starting from a single epiblast or ectoderm.

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A genome-wide screen in EpiSCs identifies Nr5a nuclear receptors as potent inducers of ground state pluripotency.

Project description:In rodents, the naïve early epiblast undergoes profound morphogenetic, transcriptional and epigenetic changes after implantation. These differences are maintained between blastocyst-derived embryonic stem (ES) cells and egg cylinder-derived epiblast stem cells (EpiSCs). Notably, ES cells robustly colonise chimaeras, whereas EpiSCs show little or no contribution. ES cells self-renew independently of mitogenic growth factors, whereas EpiSCs require fibroblast growth factor. However, EpiSCs retain the core pluripotency factors Oct4 and Sox2 and the developmental barrier dividing them from unrestricted pluripotency can be surmounted by a single reprogramming factor. This provides an opportunity to identify molecules that can reset the naïve state. We undertook a forward genetic screen for effectors of EpiSC reprogramming, employing piggyBac transposition to activate endogenous gene expression at random and selecting for undifferentiated colonies in the absence of growth factor signalling. Three recovered clones harboured integrations that activate the closely related orphan nuclear receptor genes Nr5a1 and Nr5a2. Activity of Nr5a1 and Nr5a2 was confirmed by direct transfection. Reprogrammed colonies were obtained without transgene integration and at 10-fold higher frequency than with other single factors. Converted cells exhibited the diagnostic self-renewal characteristics, gene expression profile and X chromosome activation signature of ground state pluripotency. They efficiently produced adult chimaeras and gave germline transmission. Nr5a receptors regulate Oct4 transcription but this is insufficient for reprogramming. Intriguingly, unlike previously identified reprogramming molecules, Nr5a receptors play no evident role in ES cell self-renewal. This implies a different foundation for their capacity to reset pluripotency and suggests that further factors remain to be identified.

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Mus musculus

Project description:Genome-wide analysis of gene expression of IWR1/Y27632-maintained epiblast stem cells (EpiSCs)

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Genome-wide maps of chromatin state in epiblast stem cells and nodal inhibited ebiblast stem cells

Project description:We reported H3K4me3, H3K27ac and H3K27me3 histone modification in EpiSCs and Nodal inhibited EpiSCs(EpiSCS/F). Using ChIP sequencing, we demonstrate that the active modification of H3K4me3 and H3K27ac level are higher in EpiSCS/F, suggesting EpiSCsS/F were in an active chromatin state. Interestingly, H3K27me3, the supressive modification enriched more in EpiSCS/F down regulated genes, suggesting H3K27me3 plays pivital roles in regulating gene expression.

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Genome wide analysis of SMAD1 binding in mouse mesodermal cells treated or not with the WNT inhibitor IWR1

Project description:Using chromatin-immunoprecipitation followed by next-generation sequencing, we studied the effect of WNT pathway inhibition of the binding of SMAD1 in mouse mesodermal cells from Embryoid Bodies (EB) cultures. The WNT inhibitor IWR1 (or the vehicle control DMSO) were added in day 3 EB cultures and cells were collected 24h later for ChIP assay.

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Genome-wide maps of five major transcription factors in epiblast stem cells

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Genome-wide maps of five major transcription factors in epiblast stem cells

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MLL1 Inhibition Reprograms Epiblast Stem Cells to Naive Pluripotency.

Project description:The interconversion between naive and primed pluripotent states is accompanied by drastic epigenetic rearrangements. However, it is unclear whether intrinsic epigenetic events can drive reprogramming to naive pluripotency or if distinct chromatin states are instead simply a reflection of discrete pluripotent states. Here, we show that blocking histone H3K4 methyltransferase MLL1 activity with the small-molecule inhibitor MM-401 reprograms mouse epiblast stem cells (EpiSCs) to naive pluripotency. This reversion is highly efficient and synchronized, with more than 50% of treated EpiSCs exhibiting features of naive embryonic stem cells (ESCs) within 3 days. Reverted ESCs reactivate the silenced X chromosome and contribute to embryos following blastocyst injection, generating germline-competent chimeras. Importantly, blocking MLL1 leads to global redistribution of H3K4me1 at enhancers and represses lineage determinant factors and EpiSC markers, which indirectly regulate ESC transcription circuitry. These findings show that discrete perturbation of H3K4 methylation is sufficient to drive reprogramming to naive pluripotency.

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