Elongation of Yeast Pol II Through Nucleosomes

描述（由申请人提供）：RNA聚合酶II （pol II）通过核小体转录是真核生物基因表达中最基本的过程之一。遗传学、染色质免疫沉淀和分子生物学的研究为酿酒酵母体内pol II延长提供了许多见解。尽管有大量的文献，但对实际机制的理解很差，因为没有实验室以系统的方式使用体外延伸系统与染色质模板来解决这个问题。组蛋白的共转录乙酰化和快速去乙酰化现象，以及相关的共转录甲基化和去甲基化过程，由于它们在所有真核生物中都是守恒的，因此特别令人感兴趣。然而，即使是对机制的初步了解，也需要在一个明确的转录系统中，用纯化的蛋白质来重现和分析这些事件。我最近在斯托斯研究所杰瑞·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atp依赖性重塑协同工作，允许pol II通过组蛋白八聚体。我建议显著扩展这些研究，以解决延伸机制的几个方面。我的团队将使用串联亲和纯化分离相关酵母蛋白，并将其用于染色质结合和转录分析。一个特别强大的技术，将形成该建议的基石是固定化模板测定。该分析使用生物素化，染色质化的模板连接到链霉亲和素涂层珠捕获延伸复合物。然后对配合物进行功能和成分分析。这项技术和更传统的方法，如电泳迁移率转移将用于解决以下三个目标。目的1将研究pol II通过核小体的机制以及单核小体和多核小体模板上组蛋白八聚体的命运。该目的将利用纯化的组蛋白乙酰转移酶（SAGA和NuA4）和ATP依赖性重塑机器（RSC和SWI/SNF）。Aim #2将探索H3K4和H3K36三甲基化功能的当前模型，以及识别甲基化组蛋白（包括SAGA， Rpd3S和Chd1）的机器的详细机制。Aim #3将研究组蛋白伴侣蛋白（包括Spt6、Asf1和FACT）如何组装和拆卸核小体，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作和测试pol II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的假设。这些知识将提供适用于所有真核生物pol II延伸的基本信息。基因调控最重要的方面之一是了解染色质（保护真核生物基因组的核蛋白结构）是如何在基因开启时被移除，在基因关闭时被替换的。这一步骤是所有生物体的基础，对这一过程的了解是理解人类疾病、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酿酒酵母作为模型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.