Structural Biology of the Sin3 Corepressor Complex

描述(申请人提供)：真核基因转录水平的调控依赖于多种蛋白质因子的协调作用。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协同调节复合体募集到基因组上的特定位点，是调控基因转录的重要步骤。SIN3辅阻遏物复合体是迄今为止在哺乳动物细胞中发现的为数不多的主要辅阻遏物复合体之一。该复合体对基因转录的负面影响部分依赖于核组蛋白脱乙酰酶(HDACs)的作用，组蛋白脱乙酰酶是一组催化乙酰化染色质底物上的乙酰基水解的酶。~150 kDa的Sin3蛋白既可作为辅阻遏子复合体组装的分子支架，又可直接或间接地通过复合体的其他成分与大量的DNA结合转录因子和染色质结合蛋白相互作用，这些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着重要作用。我们在下一个资助期的研究将广泛地解决与以下问题有关的问题：一方面，不同的启动子结合的转录因子如何招募Sin3辅阻遏子复合体的成分；另一方面，翻译后修饰的组蛋白的特定模式，也被称为“染色质标记”。另一个主要问题涉及该综合体是如何组装的，以及它如何促进其功能多样性。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核磁共振波谱，因为它非常适合于转录因子的研究，因为它们经常含有自然展开的片段，而且被研究的系统相对较小和易于处理。此外，该技术非常适合研究弱相互作用，如涉及染色质结合模块的相互作用。功能分析将依赖于各种生化和生物物理方法。该项目的目标主要集中在三个主题上：(1)启动子结合转录因子对Sin3辅阻遏子的招募；(2)辅抑制子复合体的招募和靶向特定染色质标记；(3)核心辅抑制子复合体的组装。与公共卫生相关：真核基因转录成信使RNA，随后翻译成蛋白质，由一类称为转录共调节因子的蛋白质调节。这些共调节因子被特异结合DNA的蛋白质和/或结合组蛋白的蛋白质招募到基因组的特定区域。涉及SIN3辅助调节因子的分子相互作用以及涉及与SIN3相互作用的组蛋白结合蛋白的分子相互作用是这一提议的重点，因为人们对这些蛋白质的功能知之甚少，而且这些蛋白质的缺陷和异常数量与癌症等疾病有关。这项拟议的工作将试图获得在与SIN3相互作用时捕获的蛋白质或在与组蛋白相互作用时捕获的SIN3相互作用蛋白的详细分子图像，试图合理解释这些相互作用或缺乏这些相互作用如何导致疾病状态，并为开发合适的分子治疗方法提供思路。

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.