Structural and functional analysis of gene silencing

 描述（由申请人提供）：基因沉默过程从酵母到人类都是保守的，在建立、维持和繁殖不同的基因表达模式中发挥着关键作用。基因沉默在发育、干细胞自我更新和分化中起着关键作用，其失调可导致发育性疾病、神经退行性疾病、炎症以及癌症。在所有的真核生物中，基因活性的空间和时间调节是由DNA包装到染色质中来指导的。染色质的基本重复单位是核小体，其包含包裹在组蛋白八聚体周围的146个碱基对的DNA。核小体是蛋白质和蛋白质复合物组装以调节染色体事务（例如基因转录）的平台。我们特别感兴趣的是蛋白质和蛋白质复合物，结合到核小体，以创建表观遗传沉默的染色质结构域，其调节组蛋白的翻译后修饰和它们对高阶染色质结构的影响。酵母在研究沉默染色质的建立和维持方面发挥了重要作用。酵母中基因沉默的两个被广泛研究的基本组成部分是S.酿酒酵母和异染色质蛋白1（HP 1）。粟酒这些蛋白质可以结合核小体-一个部分由组蛋白的翻译后修饰调节的过程-并以不依赖于序列的方式分布在染色质中，建立一个对转录不敏感的染色质结构。这些过程中涉及的详细机制在很大程度上是未知的。为了解决基因沉默领域的这一关键空白，我们将使用结构和功能方法。在AIM 1中，我们将确定与核小体和染色质阵列复合的Sir 3（SIR复合物的核心组分）的三维结构。在AIM 2中，我们将确定裂殖酵母HP 1（Swi 6）与带有组蛋白H3赖氨酸9甲基化的染色质底物复合的结构。我们将补充这些结构的功能在体外和体内实验。Sir 3和Swi 6与核小体复合物的结构和功能研究将揭示这些蛋白质在染色质上组装和扩散的一般原理。此外，我们将研究是否以及如何结合和传播的结果，在更高层次的折叠或染色质的压实。我们提出的全面研究将为染色质致密化和最终基因抑制的基本生物学过程提供重要见解，并将为这些复合物和染色质结构的失调如何导致疾病提供宝贵的见解。

 DESCRIPTION (provided by applicant): The process of gene silencing is conserved from yeast to humans, playing a crucial function in establishment, maintenance and propagation of distinct patterns of gene expression. Gene silencing plays a pivotal role in development, stem cell self-renewal and differentiation, and its dysregulation can cause developmental diseases, neurodegenerative disorders, inflammation as well as cancer. In all eukaryotes, spatial and temporal regulation of gene activity is directed by packaging of DNA into chromatin. The fundamental repeating unit of chromatin is the nucleosome that comprises 146 base pairs of DNA wrapped around an octamer of histone proteins. The nucleosome is the platform upon which proteins and protein complexes assemble to regulate chromosomal transactions such as gene transcription. Of particular interest to us are proteins and protein complexes that bind to nucleosomes to create epigenetically silent chromatin domains, their regulation by posttranslational modifications of histones and their effect on higher-order chromatin structure. Yeast has been instrumental in studying the establishment and maintenance of silent chromatin. Two widely studied and essential components of gene silencing in yeast are the Silent Information Regulator (SIR) complex in S. cerevisiae and Heterochromatin Protein 1 (HP1) in S. pombe. These proteins can bind nucleosomes - a process regulated in part by posttranslational modifications of histones - and spread across chromatin in a sequence independent fashion, establishing a chromatin structure that is refractory to transcription. The detailed mechanisms involved in these processes are largely unknown. To address this critical gap in the field of gene silencing, we will use structural and functional approaches. In AIM 1 we will determine three- dimensional structures of Sir3 (the core component of the SIR complex) in complex with the nucleosome and with the chromatin array. In AIM 2 we will determine the structure of fission yeast HP1 (Swi6) in complex with the chromatin substrate bearing histone H3 lysine 9 methylation. We will complement these structures with functional in vitro and in vivo experiments. The structural and functional studies of Sir3 and Swi6 in complex with nucleosomes will uncover the general principles underlying the assembly and spreading of these proteins on chromatin. Additionally we will investigate whether and how binding and spreading results in higher-order folding or compaction of chromatin. Our proposed comprehensive studies will provide crucial insights into the fundamental biological process of chromatin compaction and ultimately gene repression, and will provide invaluable insights into how deregulation of these complexes and chromatin structure contributes to disease.