Auxin-degron system identifies immediate mechanisms of Oct4 [Nanog ChIP-seq]

The pluripotency factor Oct4 is essential for the maintenance of naïve pluripotent stem cells in vitro and in vivo. However, the specific role of Oct4 in this process remains unknown. Here, we developed a rapid protein-level Oct4 depletion system that demonstrates that the immediate downstream response to loss of Oct4 is reduced expression of key pluripotency factors. Our data show a requirement for Oct4 for the efficient transcription of several key pluripotency factors, and suggest that expression of trophectoderm markers is a subsequent event. Additionally, we find that Nanog is competent to bind to the genome in the absence of Oct4, and this binding is in fact enhanced. Globally, however, active enhancer associated histone mark H3K27ac is depleted. Our work establishes that while Oct4 is required for the maintenance of the naïve transcription factor network, at a normal ESC level it antagonises this network through inhibition of Nanog binding Overall design: Mouse ESCs and iPSCs in which Oct4 could be depleted were analysed at different times following removal of Oct4 for changes in gene expression by RNA sequencing, in their epigenetic landscape by H3K27ac ChIP sequencing, and in Nanog binding by Nanog ChIP sequencing

多能性因子Oct4对于在体外及体内维持初始态多能干细胞（naïve pluripotent stem cells）至关重要。然而，Oct4在该过程中的具体功能仍未明确。本研究构建了一套快速蛋白水平Oct4耗竭系统，该系统证实，Oct4缺失后即刻的下游响应表现为关键多能性因子的表达水平下调。我们的数据表明，Oct4对于多个关键多能性因子的高效转录是必需的，同时提示滋养外胚层标志物的表达是后续发生的事件。此外，我们发现在Oct4缺失的情况下，Nanog仍可结合基因组，且该结合实际上得到了增强。但在全基因组水平上，与活性增强子相关的组蛋白修饰标记H3K27ac出现了耗竭。本研究证实，尽管Oct4对于维持初始态转录因子网络是必需的，但在正常胚胎干细胞（ESC）水平下，Oct4可通过抑制Nanog的结合来拮抗该网络。整体实验设计：将可诱导Oct4耗竭的小鼠胚胎干细胞（ESC）与诱导多能干细胞（iPSCs）在移除Oct4后的不同时间点收集，分别通过RNA测序检测基因表达变化、通过H3K27ac染色质免疫共沉淀测序（ChIP sequencing）分析表观遗传图谱，以及通过Nanog染色质免疫共沉淀测序检测Nanog的基因组结合情况。