Structural Biology of the Sin3 Corepressor Complex

描述（由申请人提供）：真核基因在转录水平的表达调控依赖于多种蛋白因子的协调作用。序列特异性DNA结合因子和染色质结合蛋白将含有染色质修饰活性的多亚基辅助调节因子复合物募集到基因组上的特定位点是调节基因转录的重要步骤。Sin 3辅阻遏物复合物是迄今为止在哺乳动物细胞中鉴定的少数主要辅阻遏物复合物之一。这种复合物对基因转录的负面影响部分依赖于核组蛋白脱乙酰酶（HDAC）的作用，HDAC是一组催化乙酰化染色质底物中乙酰基水解的酶。约150 kDa的Sin 3蛋白作为辅阻遏物复合物组装的分子支架发挥作用，并直接或间接通过复合物的其他组分与DNA结合转录因子和染色质结合蛋白（在正常生长、发育和细胞内稳态中发挥重要作用）的惊人的大而多样的组相互作用。我们在下一个资助期的研究将解决与Sin 3辅阻遏物复合物的组成部分如何一方面被不同的启动子结合的转录因子招募，另一方面被特定模式的转录后修饰的组蛋白（也称为“染色质标记”）招募有关的问题。另一个主要问题涉及复合体是如何组装的，以及它如何有助于其功能的多样性。这些问题的答案依赖于结构-功能分析。结构研究的主要工具将是溶液状态NMR光谱，因为它非常适合于转录因子的研究，因为它们经常含有天然未折叠的片段，并且还因为所研究的系统相对较小且易于处理。此外，该技术非常适合弱相互作用的研究，如涉及染色质结合模块。功能分析将依赖于各种生物化学和生物物理方法。该项目的目标主要集中在三个主题：（1）通过启动子结合的转录因子募集Sin 3辅阻遏物，（2）募集辅阻遏物复合物并将其靶向特定的染色质标记，以及（3）组装核心辅阻遏物复合物。公共卫生相关性：真核生物基因转录成信使RNA，随后翻译产生蛋白质，这是由一类称为转录辅调节因子的蛋白质调节的。这些辅助调节因子被特异性结合DNA的蛋白质和/或结合组蛋白的蛋白质募集到基因组的特定区域。涉及Sin 3辅助调节因子的分子相互作用和涉及与Sin 3相互作用的组蛋白结合蛋白的分子相互作用是该提议的焦点，因为对这些蛋白质的功能知之甚少，并且因为这些蛋白质的缺陷和异常量与癌症等疾病有关。拟议的工作将试图获得详细的分子图像中捕获的蛋白质的行为与Sin 3或那些Sin 3相互作用的蛋白质捕获的行为与组蛋白相互作用，试图合理化这些相互作用或缺乏可能导致疾病的状态，并提供思路开发合适的分子疗法。

DESCRIPTION (provided by applicant): Regulation of eukaryotic gene expression at the transcriptional level relies on the coordinated action of multiple protein factors. The recruitment of multi-subunit coregulator complexes containing chromatin-modifying activities by sequence-specific DNA binding factors and chromatin-binding proteins to specific loci on the genome is an important step in regulating gene transcription. The Sin3 corepressor complex is one of only a handful of major corepressor complexes identified thus far in mammalian cells. The negative influence on gene transcription exerted by this complex relies in part on the action of nuclear histone deacetylases (HDACs), a group of enzymes that catalyze the hydrolysis of acetyl groups from acetylated chromatin substrates. The ~150 kDa Sin3 protein functions both as a molecular scaffold for corepressor complex assembly and interacts directly, or indirectly through other components of the complex, with a surprisingly large and diverse group of DNA-binding transcription factors and chromatin-binding proteins that play important roles in normal growth, development, and cellular homeostasis. Our studies for the next funding period will address questions broadly relating to how components of the Sin3 corepressor complex are recruited by diverse promoter-bound transcription factors on the one hand and by specific patterns of post-translationally modified histones, also referred to as "chromatin marks," on the other. Another major question relates to how the complex is assembled and how it contributes to its functional diversity. Answers to these questions rely on structure-function analyses. The primary tool for the structural investigations will be solution-state NMR spectroscopy because it is ideally suited for studies of transcription factors as they frequently harbor natively unfolded segments and also because of the relatively small size and tractable nature of the systems being studied. In addition, the technique is well-suited for studies of weak interactions such as those involving chromatin-binding modules. Functional analyses will rely on a variety of biochemical and biophysical approaches. The aims of the project broadly focus on three themes: (1) recruitment of the Sin3 corepressor by promoter-bound transcription factors, (2) recruitment and targeting of the corepressor complex to specific chromatin marks and (3) assembly of the core corepressor complex. PUBLIC HEALTH RELEVANCE: The transcription of eukaryotic genes into messenger RNAs for subsequent translation to produce proteins is regulated by a class of proteins known as transcriptional coregulators. These coregulators are recruited to specific regions of the genome by proteins that bind DNA specifically and/or by proteins that bind histones. The molecular interactions involving the Sin3 coregulator and those involving histone-binding proteins that interact with Sin3 are the focus of this proposal because little is known about how these proteins function and because defects in and abnormal amounts of these proteins are associated with diseases such as cancer. The proposed work will attempt to derive detailed molecular pictures of proteins captured in the act of interacting with Sin3 or those of Sin3-interacting proteins captured in the act of interacting with histones, attempt to rationalize how these interactions or lack thereof could lead to diseased states, and provide ideas for developing suitable molecular therapies.