The Mediator complex directs signals from DNA-binding transcription factors to RNA polymerase II (Pol II). Despite this pivotal position, mechanistic understanding of Mediator in human cells remains incomplete. Here we quantified Mediator-controlled Pol II kinetics by coupling rapid subunit degradation with orthogonal experimental readouts. In agreement with a model of condensate-driven transcription initiation, large clusters of hypophosphorylated Pol II rapidly disassembled upon Mediator degradation. This was accompanied by a selective and pronounced disruption of cell-type-specifying transcriptional circuits, whose constituent genes featured exceptionally high rates of Pol II turnover. Notably, the transcriptional output of most other genes was largely unaffected by acute Mediator ablation. Maintenance of transcriptional activity at these genes was linked to an unexpected CDK9-dependent compensatory feedback loop that elevated Pol II pause release rates across the genome. Collectively, our work positions human Mediator as a globally acting coactivator that selectively safeguards the functionality of cell-type-specifying transcriptional networks.

介体复合物将来自DNA结合转录因子的信号导向RNA聚合酶II（Pol II）。尽管处于这一关键位置，但对人体细胞中介体的机理了解仍然不完整。在这里，我们通过耦合快速亚基降解与正交实验读数量化介体控制的Pol II动力学。与冷凝物驱动的转录起始模型一致，在Mediator降解后，大量的次磷酸化的Pol II簇迅速分解。这伴随着细胞类型特异性转录回路的选择性和显着破坏，其组成基因具有极高的Pol II周转率。值得注意的是，大多数其他基因的转录输出在很大程度上不受急性介体消融的影响。这些基因转录活性的维持与意想不到的依赖CDK9的补偿性反馈回路有关，该回路提高了基因组中Pol II暂停释放的速率。总的来说，我们的工作将人类介体定位为一种全球性的协同激活剂，可以选择性地保护特定于细胞类型的转录网络的功能。