Elongation of Yeast Pol II Through Nucleosomes

描述（由申请人提供）：RNA聚合酶II（pol II）通过核小体的转录是真核基因表达中最基本的过程之一。酵母S.酿酒酵母提供了许多关于体内pol II延伸的见解。尽管有大量的文献，但对实际机制的理解仍然很差，因为没有实验室使用染色质模板的体外延伸系统以系统的方式解决这个问题。共转录组蛋白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其在所有真核生物中的保守性而特别令人感兴趣。然而，即使是对该机制的初步理解，也需要在具有纯化蛋白质的定义的转录系统中重新创建和分析这些事件。我最近在Stowers研究所的Jerry Workman实验室完成了为期一年的休假，在那里我建立了一个单核细胞体外转录延伸系统。我发现乙酰化和ATP依赖性重塑协同工作，使pol II通过组蛋白八聚体。我建议显着扩展这些研究，以解决几个方面的伸长机制。我的小组将使用串联亲和纯化分离相关的酵母蛋白，并将其用于染色质结合和转录测定。一个特别强大的技术，将形成基石的建议是固定化模板测定。该测定使用附着于链霉亲和素包被珠的生物素化、染色质化模板来捕获延伸复合物。然后对复合物进行功能和成分分析。这种技术和更传统的方法，如电泳迁移率偏移将用于解决以下三个目标。目的#1将检查pol II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（佐贺和NuA 4）和ATP依赖性重塑机器（RSC和SWI/SNF）。目标#2将探索H3 K4和H3 K36三甲基化功能的现有模型，以及识别甲基化组蛋白（包括佐贺，Rpd 3S和Chd 1）的机器的详细机制。目标#3将研究组蛋白伴侣，包括Spt 6，Asf 1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。我们的研究将利用来自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中pol II延伸的基本信息。B。基因调控最重要的方面之一是了解染色质，即保护真核基因组的核蛋白结构，如何在基因开启时被移除，并在基因关闭时被替换。这一步骤对所有生物体都是至关重要的，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S。酿酒酵母作为模型生物来理解这个过程。

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.