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

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. Overall design: RNA-seq of Tox4 knockdown in mouse EpiSCs

本研究将小鼠上胚层干细胞（epiblast stem cells, EpiSCs）中的全基因组RNA干扰（RNAi）筛选，与遗传互作、蛋白质定位以及"蛋白质水平依赖性"研究——一种用于揭示转录后调控的系统性技术——相结合，以绘制调控多能性关键调控因子Oct4表达的因子网络。本研究数据表明，在EpiSCs与胚胎干细胞（embryonic stem cells, ESCs）中，Oct4的调控机制既存在相似性，也存在根本性差异。通过多参数数据分析，我们预测Tox4与在两种细胞类型中均维持细胞身份的Paf1复合物（Paf1C）存在相互作用，并验证了该蛋白质互作在ESCs和EpiSCs中均真实存在。我们还鉴定出多个可上调EpiSCs中Oct4表达的基因敲低靶点，这表明与ESCs截然不同的是，Oct4在EpiSCs中处于活跃的抑制性调控之下。这些研究为深入理解多能性以及解析调控从植入前到植入后状态转变的分子事件提供了研究框架。整体实验设计：小鼠EpiSCs中Tox4基因敲低的RNA测序（RNA-seq）