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

ABSTRACT: We combine a genome-scale RNAi screen in mouse epiblast stem cells (EpiSCs) with genetic interaction, protein localization and “protein-level dependency” studies – a systematic technique that uncovers post-transcriptional regulation – to delineate the network of factors that control the expression of Oct4, a key regulator of pluripotency. Our data signify that there are similarities, but also fundamental differences in Oct4 regulation in EpiSCs vs. embryonic stem cells (ESCs). Through multiparametric data analyses we predict that Tox4 is associating with the Paf1C complex, which maintains cell identity in both cell types and validate that this protein-protein interaction exists in ESCs and EpiSCs. We also identify numerous knockdowns that increase Oct4 expression in EpiSCs, indicating that, in stark contrast to ESCs, Oct4 is under active repressive control in EpiSCs. These studies provide a framework for better understanding pluripotency and for dissecting the molecular events that govern the transition from the pre-implantation to the post-implantation state.

ORGANISM(S): Mus musculus

PROVIDER: GSE62357 | GEO | 2015/08/07

SECONDARY ACCESSION(S): PRJNA263915

REPOSITORIES: GEO

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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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