Elongation of Yeast Pol II Through Nucleosomes

描述(申请人提供)：RNA聚合酶II(PolII)通过核小体转录是真核基因表达中最基本的过程之一。酿酒酵母的遗传学、染色质免疫沉淀和分子研究提供了许多关于PolII在体内延伸的见解。尽管有大量的文献，但对实际机制的了解很少，因为还没有实验室以系统的方式使用染色质模板的体外延长系统来解决这个问题。组蛋白共转录组蛋白乙酰化和快速去乙酰化现象以及与之相关的共转录甲基化和去甲基化过程在所有真核生物中都是特别有趣的，因为它们在所有真核生物中都是保守的。然而，即使是对这一机制的初步了解，也需要用纯化的蛋白质在特定的转录系统中重新创建和分析这些事件。最近，我在斯托尔斯研究所曾傑瑞·沃克曼的实验室完成了为期一年的休假，在那里我建立了一个单核小体的体外转录延伸系统。我发现乙酰化和依赖三磷酸腺苷的重塑协同作用，使PolII通过组蛋白八聚体。我建议大大扩展这些研究，以解决伸长机制的几个方面。我的团队将使用串联亲和纯化技术分离相关酵母蛋白，并将其用于染色质结合和转录分析。形成该提案基石的一项特别强大的技术是固定化模板分析。这种分析方法使用附着在链霉亲和素包裹的珠子上的生物素化、显色化模板来捕获伸长复合体。然后将对这些络合物进行功能和组成分析。这项技术和更传统的方法，如电泳迁移率变化，将被用来解决以下三个目标。目的#1将研究PolII通过核小体的机制以及组蛋白八聚体在单核小体和多核小体模板上的命运。这一目标将利用纯化的组蛋白乙酰转移酶(SAGA和NuA4)和ATP依赖的重塑机器(RSC和SWI/SNF)。目的#2将探索目前H3K4和H3K36三甲基化的功能模型，以及识别包括sagA、Rpd3S和Chd1在内的甲基化的组蛋白的机器的详细机制。目的#3将研究包括Spt6、ASF1和FACT在内的组蛋白伴侣蛋白如何组装和拆解核小体，重点是共价修饰如何影响这一过程。我们的研究将利用酵母遗传学和分子生物学的大量知识来制定和测试假设，即PolII如何通过核小体，以及染色质的共价修饰如何调节这一过程。这一知识将提供适用于所有真核生物中PolII延伸的基本信息。B.项目说明基因调控最重要的方面之一是了解当基因开启时如何移除染色质，以及在基因关闭时如何替换染色质。染色质是保护真核生物基因组的核蛋白结构。这一步骤对所有生物体来说都是基本的，了解这一过程是理解人类疾病、分化和发育过程中基因调控的关键。我们的建议将使用酵母S.cerevisiae作为模式生物来理解这一过程。

DESCRIPTION (provided by applicant): RNA polymerase II (pol II) transcription through a nucleosome is one of the most fundamental processes in eukaryotic gene expression. Genetics, chromatin immunoprecipitation and molecular studies in the yeast S. cerevisiae have provided many insights into pol II elongation in vivo. Despite the extensive literature there is a poor understanding of the actual mechanism because no lab has attacked the issue in a systematic manner using in vitro elongation systems with chromatin templates. The phenomena of co-transcriptional histone acetylation and rapid deacetylation, and the related process of co-transcriptional methylation and demethylation are of particular interest due to their conservation in all eukaryotes. Yet even a rudimentary understanding of the mechanism requires that these events be recreated and analyzed in a defined transcription system with purified proteins. I recently completed a 1-year sabbatical in Jerry Workman's lab at the Stowers Institute, where I set up an in vitro transcription elongation system on mononucleosomes. I found that acetylation and ATP-dependent remodeling work in concert to permit pol II passage through the histone octamer. I propose to significantly extend these studies to address several aspects of the elongation mechanism. My group will isolate relevant yeast proteins using tandem affinity purification and employ these in chromatin binding and transcription assays. A particularly powerful technique that will form the cornerstone of the proposal is the immobilized template assay. This assay uses biotinylated, chromatinized templates attached to streptavidin-coated beads to capture the elongation complex. The complexes will then be subjected to functional and compositional analyses. This technique and more traditional methods such as electrophoretic mobility shift will be used to address the three aims below. Aim #1 will examine the mechanism by which pol II passes through a nucleosome and the fate of the histone octamer on mono and poly-nucleosomal templates. This aim will utilize purified histone acetyltransferases (SAGA and NuA4) and ATP dependent remodeling machines (RSC and SWI/SNF). Aim #2 will explore current models for the function of H3K4 and H3K36 trimethylation and the detailed mechanisms of the machines that recognize the methylated histones including SAGA, Rpd3S and Chd1. Aim #3 will examine how histone chaperones, including Spt6, Asf1 and FACT, assemble and disassemble nucleosomes with an emphasis on how covalent modifications affect the process. Our study will leverage the vast body of knowledge from yeast genetics and molecular biology to craft and test hypotheses for how pol II passes through a nucleosomes and how covalent modifications of chromatin regulate this process. The knowledge will provide fundamental information applicable to pol II elongation in all eukaryotes. B. Project Narrative One of the most important aspects of gene regulation is understanding how chromatin, the nucleoprotein structure that protects eukaryotic genome, is removed when genes are turned on and replaced when genes are turned off. This step is fundamental to all organisms and knowledge of the process is key to understanding gene regulation during disease, differentiation and development in humans. Our proposal will use the yeast S. cerevisiae as a model organism to understand this process.