Oct4, a master pluripotency regulator, is actively repressed in epiblast stem cells
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RNAi screen of EpiSCs reveals Oct4 regulators not found in embryonic stem cells
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Combining four large-scale techniques predicts that Tox4 interacts with Paf1C
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Binding between Tox4 and Paf1C is confirmed in ESCs and EpiSCs
Summary
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 versus 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.
