Phase separation and gene control Many components of eukaryotic transcription machinery—such as transcription factors and cofactors including BRD4, subunits of the Mediator complex, and RNA polymerase II—contain intrinsically disordered low-complexity domains. Now a conceptual framework connecting the nature and behavior of their interactions to their functions in transcription regulation is emerging (see the Perspective by Plys and Kingston). Chong et al. found that low-complexity domains of transcription factors form concentrated hubs via functionally relevant dynamic, multivalent, and sequence-specific protein-protein interaction. These hubs have the potential to phase-separate at higher concentrations. Indeed, Sabari et al. showed that at super-enhancers, BRD4 and Mediator form liquid-like condensates that compartmentalize and concentrate the transcription apparatus to maintain expression of key cell-identity genes. Cho et al. further revealed the differential sensitivity of Mediator and RNA polymerase II condensates to selective transcription inhibitors and how their dynamic interactions might initiate transcription elongation. Science, this issue p. eaar2555, p. eaar3958, p. 412; see also p. 329 Phase-separated condensates compartmentalize the transcription apparatus at super-enhancers of key cell-identity genes. INTRODUCTION Mammalian genes that play prominent roles in healthy and diseased cellular states are often controlled by special DNA elements called super-enhancers (SEs). SEs are clusters of enhancers that are occupied by an unusually high density of interacting factors and drive higher levels of transcription than most typical enhancers. This high-density assembly at SEs has been shown to exhibit sharp transitions of formation and dissolution, forming in a single nucleation event and collapsing when chromatin factors or nucleation sites are deleted. These features led us to postulate that SEs are phase-separated multimolecular assemblies, also known as biomolecular condensates. Phase-separated condensates, such as the nucleolus and other membraneless cellular bodies, provide a means to compartmentalize and concentrate biochemical reactions within cells. RATIONALE SEs are formed by the binding of master transcription factors (TFs) at each component enhancer, and these recruit unusually high densities of coactivators, including Mediator and BRD4. Mediator is a large (~1.2 MDa) multisubunit complex that has multiple roles in transcription, including bridging interactions between TFs and RNA polymerase II (RNA Pol II). BRD4 facilitates the release of RNA Pol II molecules from the site of transcription initiation. The presence of MED1, a subunit of Mediator, and BRD4 can be used to define SEs. We reasoned that if transcriptional condensates are formed at SEs, then MED1 and BRD4 should be visualized as discrete bodies at SE elements in cell nuclei. These bodies should exhibit behaviors described for liquid-like condensates. We investigated these possibilities by using murine embryonic stem cells (mESCs), in which SEs were originally described. Because intrinsically disordered regions (IDRs) of proteins have been implicated in condensate formation, we postulated that the large IDRs present in MED1 and BRD4 might be involved. RESULTS We found that MED1 and BRD4 occupy discrete nuclear bodies that occur at SEs in mESCs. These bodies exhibit properties of other well-studied biomolecular condensates, including rapid recovery of fluorescence after photobleaching and sensitivity to 1,6-hexanediol, which disrupts liquid-like condensates. Disruption of MED1 and BRD4 bodies by 1,6-hexanediol was accompanied by a loss of chromatin-bound MED1 and BRD4 at SEs, as well as a loss of RNA Pol II at SEs and SE-driven genes. The IDRs of both MED1 and BRD4 formed phase-separated liquid droplets in vitro, and these droplets exhibited features characteristic of condensates formed by networks of weak protein-protein interactions. The MED1-IDR droplets were found to concentrate BRD4 and RNA Pol II from transcriptionally competent nuclear extracts, which may reflect their contribution to compartmentalizing and concentrating biochemical reactions associated with transcription at SEs in cells. CONCLUSION Our results show that coactivators form phase-separated condensates at SEs and that SE condensates compartmentalize and concentrate the transcription apparatus at key cell-identity genes. These results have implications for the mechanisms involved in the control of genes in healthy and diseased cell states. We suggest that SE condensates facilitate the compartmentalization and concentration of transcriptional components at specific genes through the phase-separating properties of IDRs in TFs and cofactors. SE condensates may thus ensure robust transcription of genes essential to cell-identity maintenance. These properties may also explain why cancer cells acquire large SEs at driver oncogenes and why SEs that facilitate transcriptional dysregulation in disease can be especially sensitive to transcriptional inhibitors. Phase separation of coactivators compartmentalizes and concentrates the transcription apparatus. Enhancers are gene regulatory elements bound by transcription factors that recruit coactivators and the transcription apparatus (not shown) to regulate gene expression. Super-enhancers are clusters of enhancers bound by master transcription factors that concentrate high densities of coactivators and the transcription apparatus to drive robust expression of genes that play prominent roles in cell identity. This is achieved by the phase separation of coactivators, which is driven in part by high-valency and low-affinity interactions of intrinsically disordered regions. Super-enhancers (SEs) are clusters of enhancers that cooperatively assemble a high density of the transcriptional apparatus to drive robust expression of genes with prominent roles in cell identity. Here we demonstrate that the SE-enriched transcriptional coactivators BRD4 and MED1 form nuclear puncta at SEs that exhibit properties of liquid-like condensates and are disrupted by chemicals that perturb condensates. The intrinsically disordered regions (IDRs) of BRD4 and MED1 can form phase-separated droplets, and MED1-IDR droplets can compartmentalize and concentrate the transcription apparatus from nuclear extracts. These results support the idea that coactivators form phase-separated condensates at SEs that compartmentalize and concentrate the transcription apparatus, suggest a role for coactivator IDRs in this process, and offer insights into mechanisms involved in the control of key cell-identity genes.

中文翻译：

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超级增强剂处的共激活剂凝聚将相分离和基因控制联系起来

相分离和基因控制真核转录机制的许多组件（例如转录因子和辅助因子，包括 BRD4、介体复合物的亚基和 RNA 聚合酶 II）包含本质上无序的低复杂性结构域。现在，一个将它们相互作用的性质和行为与其转录调控功能联系起来的概念框架正在出现（参见 Plys 和 Kingston 的观点）。冲等人。发现转录因子的低复杂性结构域通过功能相关的动态、多价和序列特异性的蛋白质-蛋白质相互作用形成集中的枢纽。这些中心有可能在较高浓度下发生相分离。事实上，萨巴里等人。研究表明，在超级增强子处，BRD4 和 Mediator 形成液体状冷凝物，将转录装置分隔并集中，以维持关键细胞识别基因的表达。曹等人。进一步揭示了Mediator和RNA聚合酶II缩合物对选择性转录抑制剂的不同敏感性，以及它们的动态相互作用如何启动转录延伸。科学，本期第 14 页。eaar2555，p。eaar3958，p。412；另见 p. 329 相分离的凝聚物将转录装置划分在关键细胞识别基因的超级增强子处。简介 在健康和患病细胞状态中发挥重要作用的哺乳动物基因通常由称为超级增强子 (SE) 的特殊 DNA 元件控制。SE 是增强子簇，被异常高密度的相互作用因子占据，并且比大多数典型增强子驱动更高水平的转录。SE 上的这种高密度组装已被证明表现出形成和溶解的急剧转变，在单个成核事件中形成，并在染色质因子或成核位点被删除时崩溃。这些特征使我们假设 SE 是相分离的多分子组装体，也称为生物分子缩合物。相分离的凝聚物，例如核仁和其他无膜细胞体，提供了一种划分和集中细胞内生化反应的方法。基本原理 SE 是由主转录因子 (TF) 在每个增强子成分上结合而形成的，它们会招募异常高密度的共激活子，包括 Mediator 和 BRD4。介体是一种大型 (~1.2 MDa) 多亚基复合物，在转录中具有多种作用，包括桥接 TF 和 RNA 聚合酶 II (RNA Pol II) 之间的相互作用。BRD4 促进 RNA Pol II 分子从转录起始位点的释放。MED1（Mediator 的一个子单元）和 BRD4 的存在可用于定义 SE。我们推断，如果转录缩合物在 SE 处形成，那么 MED1 和 BRD4 应在细胞核中的 SE 元件处可视化为离散体。这些物体应该表现出类液体凝结物所描述的行为。我们通过使用小鼠胚胎干细胞（mESC）研究了这些可能性，其中 SE 最初被描述。由于蛋白质的本质无序区域 (IDR) 与冷凝物的形成有关，因此我们推测 MED1 和 BRD4 中存在的大 IDR 可能参与其中。结果我们发现 MED1 和 BRD4 占据 mESC 中 SE 处的离散核体。这些物体表现出其他经过充分研究的生物分子凝聚体的特性，包括光漂白后荧光的快速恢复以及对 1,6-己二醇的敏感性，1,6-己二醇会破坏液体状凝聚体。1,6-己二醇对 MED1 和 BRD4 体的破坏伴随着 SE 处染色质结合的 MED1 和 BRD4 的丢失，以及 SE 处 RNA Pol II 和 SE 驱动基因的丢失。MED1和BRD4的IDR在体外形成相分离的液滴，并且这些液滴表现出由弱蛋白质-蛋白质相互作用网络形成的冷凝物的特征。发现 MED1-IDR 液滴从具有转录能力的核提取物中浓缩 BRD4 和 RNA Pol II，这可能反映了它们对划分和浓缩与细胞 SE 转录相关的生化反应的贡献。结论 我们的结果表明，共激活剂在 SE 处形成相分离的凝聚物，并且 SE 凝聚物将转录装置划分并集中在关键的细胞识别基因处。这些结果对健康和患病细胞状态中基因控制所涉及的机制具有影响。我们认为，SE 缩合物通过 TF 和辅因子中 IDR 的相分离特性，促进特定基因上转录成分的区室化和浓缩。因此，SE 凝聚物可以确保细胞身份维持所必需的基因的稳健转录。这些特性也可以解释为什么癌细胞在驱动癌基因上获得大量 SE，以及为什么促进疾病中转录失调的 SE 对转录抑制剂特别敏感。共激活剂的相分离分隔并集中了转录装置。增强子是由转录因子结合的基因调控元件，转录因子招募共激活子和转录装置（未显示）来调节基因表达。超级增强子是由主转录因子结合的增强子簇，它们集中了高密度的共激活子和转录装置，以驱动在细胞身份中发挥重要作用的基因的稳健表达。这是通过共激活剂的相分离来实现的，这部分是由本质上无序区域的高价和低亲和力相互作用驱动的。超级增强子（SE）是增强子簇，它们协作组装高密度的转录装置，以驱动在细胞身份中发挥重要作用的基因的稳健表达。在这里，我们证明富含 SE 的转录共激活因子 BRD4 和 MED1 在 SE 处形成核点，这些核点表现出液体状凝聚物的特性，并被扰乱凝聚物的化学物质破坏。BRD4 和 MED1 的本质无序区域 (IDR) 可以形成相分离的液滴，MED1-IDR 液滴可以分隔和浓缩核提取物中的转录装置。这些结果支持这样的观点，即共激活剂在 SE 上形成相分离的凝聚物，从而分隔和集中转录装置，表明共激活剂 IDR 在此过程中的作用，并提供对关键细胞识别基因控制所涉及机制的见解。