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

Mouse embryonic stem cells (ESCs) cultured with MEK and GSK3 inhibitors (2i) more closely resemble the inner cell mass of pre-implantation blastocysts than those cultured with serum/LIF (SL). The transcriptional mechanisms governing this pluripotent ground state are yet unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. Here, we show that β-catenin, stabilized by GSK3 inhibition in 2i, supplies transcriptional co-regulators including BRD4, CDK9, Mediator, Cohesin, and p300 at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by β-catenin and, thus, proliferation/self-renewal are still tightly controlled by the Pol2 pause release mechanism under 2i conditions. Our findings help to explain how pluripotency is preserved in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other biological contexts. GRO-seq of mouse ESCs cultured in different conditions.

与采用血清/白血病抑制因子（serum/LIF, SL）培养的小鼠胚胎干细胞（mouse embryonic stem cells, ESCs）相比，使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（2i）培养的小鼠胚胎干细胞，其表型更接近于植入前囊胚的内细胞团（inner cell mass）。目前，调控该多能基态（pluripotent ground state）的转录机制尚未阐明。启动子近端暂停的RNA聚合酶II（promoter-proximal paused RNA polymerase II, Pol2）的释放是一个多步骤过程，对于SL培养条件下的多能性维持及细胞周期基因转录至关重要。本研究发现，在2i条件下被GSK3抑制剂稳定的β-连环蛋白（β-catenin），可向多能性基因座募集包括溴结构域蛋白4（BRD4）、细胞周期蛋白依赖性激酶9（CDK9）、中介体复合物（Mediator）、黏连蛋白复合物（Cohesin）以及p300组蛋白乙酰转移酶（p300）在内的转录共调控因子（transcriptional co-regulators）。该过程选择性强化了多能性基因座，使其依赖转录起始以实现有效的基因体延伸，反而以牺牲Pol2暂停释放为代价。与之相反，细胞周期基因并不会结合β-连环蛋白，因此在2i培养条件下，细胞增殖与自我更新仍受Pol2暂停释放机制的严格调控。本研究结果不仅阐明了多能基态下多能性的维持机制，还为其他生物场景中基因调控网络扰动后的转录韧性与适应性提供了通用模型。本数据集包含不同培养条件下小鼠胚胎干细胞的全局运行测序（GRO-seq）数据。