Knowledge of how Mediator and TFIID cross-talk contributes to promoter–enhancer (P-E) communication is important for elucidating the mechanism of enhancer function. We conducted an shRNA knockdown screen in murine embryonic stem cells to identify the functional overlap between Mediator and TFIID subunits on gene expression. Auxin-inducible degrons were constructed for TAF12 and MED4, the subunits eliciting the greatest overlap. Degradation of TAF12 led to a dramatic genome-wide decrease in gene expression accompanied by destruction of TFIID, loss of Pol II preinitiation complex (PIC) at promoters, and significantly decreased Mediator binding to promoters and enhancers. Interestingly, loss of the PIC elicited only a mild effect on P-E looping by promoter capture Hi-C (PCHi-C). Degradation of MED4 had a minor effect on Mediator integrity but led to a consistent twofold loss in gene expression, decreased binding of Pol II to Mediator, and decreased recruitment of Pol II to the promoters, but had no effect on the other PIC components. PCHi-C revealed no consistent effect of MED4 degradation on P-E looping. Collectively, our data show that TAF12 and MED4 contribute mechanistically in different ways to P-E communication but neither factor appears to directly control P-E looping, thereby dissociating P-E communication from physical looping.

中文翻译：

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Pol II 预启动复合体 (PIC) 影响介体结合但不影响启动子-增强子循环

了解 Mediator 和 TFIID 串扰如何促进启动子-增强子 (PE) 通信对于阐明增强子功能的机制很重要。我们在小鼠胚胎干细胞中进行了 shRNA 敲除筛选，以确定 Mediator 和 TFIID 亚基之间在基因表达上的功能重叠。为 TAF12 和 MED4 构建了生长素诱导的降解决定子，这两个亚基引起最大的重叠。TAF12 的降解导致基因表达在全基因组范围内急剧下降，同时伴随着 TFIID 的破坏、启动子处 Pol II 预启动复合体 (PIC) 的丢失，以及显着降低 Mediator 与启动子和增强子的结合。有趣的是，PIC 的丢失仅对启动子捕获 Hi-C (PCHi-C) 的 PE 循环产生轻微影响。MED4 的降解对 Mediator 的完整性影响很小，但会导致基因表达的双重损失，减少 Pol II 与 Mediator 的结合，并减少 Pol II 向启动子的募集，但对其他 PIC 组件没有影响。PCHi-C 显示 MED4 降解对 PE 循环没有一致的影响。总的来说，我们的数据显示 TAF12 和 MED4 以不同的方式对 PE 通信做出机械贡献，但似乎没有一个因素直接控制 PE 循环，从而将 PE 通信与物理循环分离。