Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Systems analyses reveal shared and diverse attributes of Oct4 regulation in pluripotent cells

ABSTRACT: Epiblast stem cells (EpiSCs) are pluripotent cells that can be isolated and cultured from post implantation embryos. In contrast to embryonic stem cells (ESCs), systematic studies to investigate the genes that maintain pluripotency in EpiSCs have not been reported. Here we combine a genome-wide RNAi screen with genetic interaction, protein localization and protein-level dependency studies to delineate connectivity between factors that control Oct4 expression in EpiSCs and compare the role of these factors to their function in ES cells. We demonstrate the power of this integrative approach by the identification of Tox4 as an interactor of PP1 (Protein Phosphatase 1) and Paf1C, a complex that acts in multiple aspects of RNAPII regulation. Our results indicate that Tox4 cooperates with Paf1C and PP1 to influence the phosphorylation status of the RNAPII CTD tail during transcription and that this function is vital for maintenance of pluripotent cell identity. RNA-seq of Tox4 knockdown in mouse EpiSCs

ORGANISM(S): Mus musculus

SUBMITTER: Maciej Paszkowski-Rogacz

PROVIDER: E-GEOD-62357 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Recently, (in vitro) pluripotent EpiSCs were derived from the post-implantation egg cylinder stage epiblasts of mouse and rat. These EpiSCs resemble and correspond very closely to the conventional human embryonic stem cells (hESCs) in the colony morphology and culture/signaling requirements for maintaining pluripotency, but exhibit a range of significant phenotypic and signaling response differences from the conventional mouse ES cells (mESCs). These observations strongly support the notion that EpiSCs and hESCs are intrinsically similar, and raise an attractive hypothesis: as mESCs and EpiSCs/hESCs represent two distinct pluripotency states: the mESC-like state representing the ICM of pre-implantation blastcyst and the EpiSC-like state representing the post-implantation epiblasts, whether the epiblast state (including conventional hESCs) can be converted back to the ICM state. Despite studies providing evidence that epiblast-like cells exist and transition back and forth within colony of conventional mESCs; mESCs and EpiSCs share substantial set of pluripotency transcriptional factors, including Oct4, Sox2 and Nanog; and mESCs are more stable in culture, in the present study we found that EpiSCs differentiated rapidly under mESC culture conditions and no âspontaneouslyâ converted mESC could be readily identified and isolated over serial passages at the population or clonal level. Remarkably, we found that blockage of the TGFÎ² pathway or inhibition of the H3K4 demethylase LSD1 with small molecule inhibitors induced dramatic morphological changes of EpiSCs towards mESC phenotypes with activation of some ICM-specific gene expression. However, full conversion of EpiSCs to a mESC-like state with competence to chimeric contribution can only be readily generated with a combination of inhibitors of LSD1 and ALK. These observations underscore a powerful and direct induction of reprogramming from the developmentally later-stage EpiSCs to a mESC-like stage by a synergy of signaling and direct epigenetic modulations. It also highlights a significant role for TGFÎ² pathway inhibition in promoting reprogramming to and sustaining true pluripotency, which further supports our recent studies in generating chimerism-competent rat pluripotent cells. Collectively, our studies provide a proof-of-concept demonstration that pluripotency-restricted EpiSCs can be readily converted to a mESC-like state in the absence of any genetic manipulation by precise pharmacological control of signaling pathways that distinguish the two pluripotency states and an epigenetic target simultaneously, and offer a convenient experimental system to further study the mechanism. Such method and concept may also provide an avenue for generating a new type of mESC-like human pluripotent cell. Global gene-expression analyses of the parnate/mAMFGi cells

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Systems analyses reveal shared and diverse attributes of Oct4 regulation in pluripotent cells

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