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 Low-complexity domains of eukaryotic transcription factors form hubs via dynamic, multivalent, sequence-specific interactions. INTRODUCTION DNA binding transcription factors (TFs) are quintessential regulators of eukaryotic gene expression. Early studies of TFs revealed their well-structured DNA binding domains (DBDs) and identified functionally critical activation domains (ADs) required for transcription. It later became evident that many ADs contain intrinsically disordered low-complexity sequence domains (LCDs), but how LCDs activate transcription has remained unclear. Although it is known that transcriptional activation by LCDs requires selective interaction with binding partners, it has been challenging to directly measure selective LCD-LCD recognition in vivo and unravel its mechanism of action. RATIONALE Traditional biochemical reconstitution and genetics studies have identified most of the molecular players central to transcription regulation. However, the mechanism by which weak, dynamic protein-protein interactions drive gene activation in living cells has remained unknown. Advances in live-cell single-molecule imaging have opened a new frontier for studying transcription in vivo. In this study, we used synthetic LacO (Lac operator) arrays as well as endogenous GGAA microsatellite loci to study LCD-LCD interactions of TFs such as EWS/FLI1, TAF15, and Sp1 in live cells. To probe the dynamic behavior of TF LCDs at target genomic loci, we have combined CRISPR-Cas9 genome editing, mutagenesis, gene activation, cell transformation assays, and various high-resolution imaging approaches including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, lattice light-sheet microscopy, three-dimensional DNA fluorescence in situ hybridization, and live-cell single-particle tracking. RESULTS Live-cell single-molecule imaging revealed that TF LCDs interact to form local high-concentration hubs at both synthetic DNA arrays and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic (seconds to minutes), selective for binding partners, and differentially sensitive to disruption by hexanediols. These findings suggest that under physiological conditions, rapid, reversible, and selective multivalent LCD-LCD interactions occur between TFs and the RNA Pol II machinery to activate transcription. We observed formation of functional TF LCD hubs at a wide range of intranuclear TF concentrations. Although we detected apparent liquid-liquid phase separation with gross overexpression of LCDs, transcriptionally competent TF LCD hubs were observed at physiological TF levels at endogenous chromosomal loci in the absence of detectable phase separation. In addition, mutagenesis, gene expression, and cell transformation assays in Ewing’s sarcoma cells revealed a functional link between LCD-LCD interactions, transactivation capacity, and oncogenic potential. CONCLUSION The use of various imaging methods in live cells powerfully complements in vitro studies and provides new insights into the nature of LCD interactions and their role in gene regulation. We propose that transactivation domains function by forming local high-concentration hubs of TFs via dynamic, multivalent, and specific LCD-LCD interactions. It also seems likely that weak, dynamic, and transient contacts between TFs play a role in disease-causing dysregulation of gene expression (i.e., EWS/FLI1 in Ewing’s sarcoma), suggesting that LCD-LCD interactions may represent a new class of viable drug targets. Although we examined a small subset of TF LCDs, the principles uncovered regarding the dynamics and mechanisms driving LCD-LCD interactions may be applicable to other classes of proteins and biomolecular interactions occurring in many cell types. From hubs to phase separation: Activation occurs in a wide range of TF concentrations. In vivo LCD-dependent transactivation occurs in hubs formed over a broad range of TF concentrations (100 nM to 100 μM) and time scales (<1 s to minutes). At endogenous concentrations, TF LCDs form transactivation hubs at native genomic loci without undergoing evident phase separation. Upon TF LCD overexpression, phase separation is observed at synthetic TF binding site arrays. Many eukaryotic transcription factors (TFs) contain intrinsically disordered low-complexity sequence domains (LCDs), but how these LCDs drive transactivation remains unclear. We used live-cell single-molecule imaging to reveal that TF LCDs form local high-concentration interaction hubs at synthetic and endogenous genomic loci. TF LCD hubs stabilize DNA binding, recruit RNA polymerase II (RNA Pol II), and activate transcription. LCD-LCD interactions within hubs are highly dynamic, display selectivity with binding partners, and are differentially sensitive to disruption by hexanediols. Under physiological conditions, rapid and reversible LCD-LCD interactions occur between TFs and the RNA Pol II machinery without detectable phase separation. Our findings reveal fundamental mechanisms underpinning transcriptional control and suggest a framework for developing single-molecule imaging screens for drugs targeting gene regulatory interactions implicated in disease.

中文翻译：

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成像控制基因转录的动态和选择性低复杂度域相互作用

相分离和基因控制真核转录机制的许多组成部分——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在转录调控中的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。发现转录因子的低复杂性域通过功能相关的动态、多价和序列特异性蛋白质-蛋白质相互作用形成集中的中枢。这些中心有可能在较高浓度下发生相分离。事实上，Sabari 等人。表明在超级增强子中，BRD4 和 Mediator 形成类似液体的冷凝物，将转录装置分隔并浓缩，以维持关键细胞身份基因的表达。赵等人。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，这个问题 p。eaar2555，页。eaar3958，页。412；另见 p. 329 真核转录因子的低复杂性域通过动态、多价、序列特异性相互作用形成中枢。引言 DNA 结合转录因子 (TF) 是真核基因表达的典型调节因子。TF 的早期研究揭示了它们结构良好的 DNA 结合域 (DBD)，并确定了转录所需的功能关键激活域 (AD)。后来很明显，许多 AD 包含本质上无序的低复杂性序列域 (LCD)，但 LCD 如何激活转录仍不清楚。虽然已知 LCD 的转录激活需要与结合伙伴的选择性相互作用，但直接测量体内选择性 LCD-LCD 识别并阐明其作用机制一直具有挑战性。基本原理 传统的生化重组和遗传学研究已经确定了大多数对转录调控至关重要的分子参与者。然而，弱的、动态的蛋白质-蛋白质相互作用驱动活细胞中基因激活的机制仍然未知。活细胞单分子成像技术的进步为研究体内转录开辟了一个新领域。在这项研究中，我们使用合成 LacO（Lac 操作符）阵列以及内源性 GGAA 微卫星位点来研究活细胞中 EWS/FLI1、TAF15 和 Sp1 等转录因子的 LCD-LCD 相互作用。为了探索 TF LCD 在目标基因组位点的动态行为，我们结合了 CRISPR-Cas9 基因组编辑、诱变、基因激活、细胞转化分析和各种高分辨率成像方法，包括荧光相关光谱、光漂白后的荧光恢复、晶格光片显微术、三维 DNA 荧光原位杂交和活细胞单粒子追踪。结果 活细胞单分子成像显示，TF LCD 相互作用，在合成 DNA 阵列和内源基因组位点形成局部高浓度中心。TF LCD 中枢稳定 DNA 结合、募集 RNA 聚合酶 II (RNA Pol II) 并激活转录。中枢内的 LCD-LCD 交互是高度动态的（秒到分钟），对结合伙伴具有选择性，并且对己二醇的破坏具有不同的敏感性。这些发现表明，在生理条件下，转录因子和 RNA Pol II 机制之间会发生快速、可逆和选择性的多价 LCD-LCD 相互作用以激活转录。我们观察到在各种核内 TF 浓度下功能性 TF LCD 中心的形成。尽管我们检测到明显的液-液相分离以及 LCD 的过度表达，在没有可检测相分离的情况下，在内源染色体位点的生理 TF 水平上观察到具有转录能力的 TF LCD 中心。此外，尤文氏肉瘤细胞的诱变、基因表达和细胞转化分析揭示了 LCD-LCD 相互作用、反式激活能力和致癌潜力之间的功能联系。结论 在活细胞中使用各种成像方法有力地补充了体外研究，并为 LCD 相互作用的性质及其在基因调控中的作用提供了新的见解。我们建议反式激活域通过动态、多价和特定的 LCD-LCD 相互作用形成局部高浓度转录因子中枢来发挥作用。似乎也可能是虚弱的，动态的，TF 之间的瞬时接触在引起疾病的基因表达失调（即尤文氏肉瘤中的 EWS/FLI1）中发挥作用，表明 LCD-LCD 相互作用可能代表一类新的可行药物靶标。尽管我们检查了一小部分 TF LCD，但揭示的有关驱动 LCD-LCD 相互作用的动力学和机制的原理可能适用于其他类别的蛋白质和发生在许多细胞类型中的生物分子相互作用。从中心到相分离：激活发生在广泛的 TF 浓度范围内。体内 LCD 依赖性反式激活发生在广泛 TF 浓度（100 nM 至 100 μM）和时间尺度（<1 秒至分钟）范围内形成的中枢中。在内源性浓度下，TF LCD 在原生基因组位点形成反式激活中心，而不会发生明显的相分离。在 TF LCD 过表达后，在合成 TF 结合位点阵列处观察到相分离。许多真核转录因子 (TF) 包含本质上无序的低复杂性序列域 (LCD)，但这些 LCD 如何驱动反式激活仍不清楚。我们使用活细胞单分子成像来揭示 TF LCD 在合成和内源基因组位点形成局部高浓度相互作用中心。TF LCD 中枢稳定 DNA 结合、募集 RNA 聚合酶 II (RNA Pol II) 并激活转录。中枢内的 LCD-LCD 相互作用是高度动态的，显示选择性与结合伙伴，并且对己二醇的破坏不同敏感。在生理条件下，TF 和 RNA Pol II 机器之间会发生快速且可逆的 LCD-LCD 相互作用，而没有可检测到的相分离。