β-catenin safeguards the ground state of pluripotency by strengthening the robustness of the transcriptional apparatus [RNA-seq]

Mouse embryonic stem cells (ESCs) cultured with inhibitors of MEK and GSK3 (2iL) more closely resemble the pre-implantation embryo inner cell mass than cultures in serum/LIF (SL). Unveiling the differences between both systems is important for understanding development and could also assist in isolating an ESC equivalent for other mammalian species. In SL, pluripotency and cell cycle gene transcription is a multistep process requiring release of promoter-proximal paused RNA polymerase II (Pol2) by the histone acetylation reader BRD4 and the Pol2 kinase CDK9. Here, we show that recruitment of co-regulators including Mediator and Cohesin by β-catenin changes the mode of transcriptional regulation at BRD4/CDK9-bound loci in 2iL. This switch renders pluripotency genes more reliant on transcriptional initiation and less on Pol2 pause release for effective gene body elongation. Conversely, cell cycle genes are not bound by β-catenin and are still dependent on Pol2 pause release. Thus, pluripotency is more resistant to BRD4/CDK9 suppression in 2iL while self-renewal remains highly sensitive. Our findings help to explain how pluripotency is shielded in the ground state and provide insight into transcriptional adaptation upon network perturbation in other contexts. RNA-seq of mouse embryonic stem cells (ESCs) cultured either with inhibitors of MEK and GSK3 (2iL) or cultured in serum/LIF (SL), and treated with DMSO or JQ1 (100nM).

采用丝裂原活化蛋白激酶激酶（MEK）与糖原合成酶激酶3（GSK3）抑制剂培养的小鼠胚胎干细胞（ESCs），即2iL培养体系，相较血清/白血病抑制因子（SL）培养体系的细胞，其表型更接近于植入前胚胎的内细胞团。阐明这两种培养体系间的差异，不仅有助于理解胚胎发育进程，还可辅助分离其他哺乳动物的等效胚胎干细胞。在SL培养体系中，多能性基因与细胞周期基因的转录属于多步骤过程，需由组蛋白乙酰化阅读器BRD4以及RNA聚合酶II（Pol2）激酶CDK9介导，释放启动子近端暂停的RNA聚合酶II（Pol2）。本研究显示，在2iL培养体系中，β-连环蛋白（β-catenin）招募包括中介体复合物（Mediator）与黏连蛋白（Cohesin）在内的共调控因子，会改变BRD4/CDK9结合位点的转录调控模式。这种调控模式转变使得多能性基因更依赖转录起始过程，而在高效完成基因体延伸时，对Pol2暂停释放的依赖程度显著降低。与之相反，细胞周期基因并未受到β-连环蛋白的结合调控，仍依赖Pol2暂停释放过程。因此，在2iL培养体系中，多能性对BRD4/CDK9抑制的抗性更强，而干细胞自我更新过程仍对该抑制作用高度敏感。本研究结果有助于解释基态多能性的维持机制，并为其他场景下基因调控网络扰动后的转录适应过程提供了全新见解。本数据集包含分别采用MEK与GSK3抑制剂（2iL）或血清/白血病抑制因子（SL）培养的小鼠胚胎干细胞（ESCs），并经二甲基亚砜（DMSO）或JQ1（100nM）处理后的RNA测序（RNA-seq）数据。