基态多能干细胞的转录延伸调控

Embryonic stem cells (ESCs) have the ability to self-renew without loosing their pluripotent characteristics and differentiate into multiple lineages upon treatment with specific signaling clues. Mouse ESCs cultured with serum+LIF are considered similar to the preimplantation embryo, and are therefore termed naïve. Yet, when cultured in medium with 2 inhibitors (2i, ERK inhibitor and GSK3 inhibitor) +LIF, they behave more similarly to the early blastocyst. This is termed ground state pluripotency. Notably, the recent derivation of human ESCs in naïve conditions is supposed to mimic the mouse ESC ground state. This is important because ESCs in ground state are thought to have improved differentiation potential and also represent a better model to understand development. We want to uncover the mechanisms underlying the different transcriptional network in mouse ESCs cultured in 2i naive conditions compared to serum+LIF, and also study whether these same mechanisms participate in the maintenance of naïve human ESCs. Our specific hypothesis is that changes in the basal transcriptional machinery mediated by repurposing of the bromodomain protein BRD4 are partly responsible for the observed differences in gene regulatory networks between the 2 systems. Our preliminary data showed that BRD4 was less necessary in mouse ESCs cultured in 2i+LIF compared to serum+LIF, and that this is caused by BRD4 redistribution along the genome. We want to explore the mechanisms involved in BRD4 redistribution, with emphasis on alterations in histone acetylation (BRD4 binds acetylated histones) mediated by changes in lipid metabolism (as a source of acetyl groups) in mouse ESCs cultured with 2i+LIF. We will also aim to understand the consequences of BRD4 redistribution in the reshaping of the transcriptional landscape, with particular emphasis on changes in transcriptional pause release by RNA polymerase II (a phenomenon triggered by BRD4) at key loci. Altogether, we expect to provide an explanation to why culture of mouse ESCs in 2i+LIF induces a more naïve state that mimics the early blastocyst. In addition, we will study whether these same mechanism apply to human naïve ESCs and if not whether manipulation of BRD4 can help produce human naïve ESCs with optimal characteristics.

胚胎干细胞（Embryonic stem cell，ESC）具有自我更新和分化的能力。Serum+lif培养条件下的小鼠ESC（mESC）与植入前的胚胎相似，被称为原始态mESC；而2i+lif（即ERK抑制剂和GSK3抑制剂)条件下的mESC被称为基态ESC。后者更接近早期囊胚细胞，具有更强的分化潜能，更有助于发育研究。我们前期研究发现，2i+lif条件下的mESC对BRD4依赖更少，且BRD4在两个系统下的全基因组分布不同。因此我们假设BRD4介导的基本转录装置的差异导致了两种不同的多能性状态。为了进一步阐明这一假说，解释2i+lif条件为什么能使mESC更接近早期胚胎细胞，本项目拟研究：1.BRD4在基因组上差异分布的机制，特别是与代谢改变表观修饰的关系；2. BRD4重新分布对转录，特别是转录延伸的影响；3. BRD4在mESC中的机制是否适用于人基态ESC。

添加MEK和GSK3抑制剂（PD0325901和CHIR99021）的无血清培养基条件（2i+Lif, 2iL）下的小鼠胚胎多能性干细胞（mESCs）比血清条件(serum+Lif, SL)下的mESCs更接近着床前胚胎的内细胞团，因此2iL条件下的mESCs又被称为基态多能性干细胞。两种条件下mESCs尽管整体的多能性非常接近，但也在多个水平存在显著差异。转录起始，及BRD4和CDK9介导的转录暂停释放对维持SL条件下的mESCs的多能性和自我更新很重要，但基态多能性干细胞的转录调控机制尚不清楚。本项目主要探究2iL条件下mESCs的转录调控机制。通过shRNA敲降，抑制剂抑制，及CRISPR/CAS9敲除，我们发现SL-mESCs多能性的维持需要BRD4和CDK9，但2iL-mESCs的多能性对BRD4或CDK9的抑制有更强的抗性。但2iL-mESCs的增殖跟SL条件下的mESCs类似，仍需要BRD4和CDK9。通过在SL中加PD0325901或者CHIR99021，我们发现2iL条件下这种独特的调控机制是由WNT/β-catenin信号通路介导的，我们利用一些激活或者抑制该信号通路的基因编辑mESCs进一步证实了这个结论。抑制GSK3能保护 β-catenin不被降解，入核的β-catenin可以在多能性基因位点招募Mediator，Cohesin等co-regulators，从而促进这些基因的转录起始。这使得2iL条件下的mESCs对BRD4和CDK9介导的Pol2暂停释放不敏感，但对转录起始的抑制更敏感。相反的，细胞周期相关的基因不被β-catenin结合，其转录仍旧受BRD4和CDK9介导的调控转录暂停释放的调控。此外，我们还用白血病细胞系验证了2iL-mESCs的转录调控模型。总的来说，我们的发现从转录调控层面解释了基态多能性干细胞的多能性维持机制，并为包括癌细胞在内的其他细胞类型提供转录网络被破坏后的适应机制。