Elongation of Yeast Pol II Through Nucleosomes

酵母 Pol II 通过核小体的延伸

• 批准号: 8076376
• 负责人: MICHAEL F CAREY
• 金额: $ 28.72万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8076376
• 关键词: ATP phosphohydrolase; Acetylation; Address; Affect; Affinity Chromatography; Animal Model; Applications Grants; Binding; Biochemical; Biological Assay; Bromodomain; Chromatin; Complex; DNA Polymerase II; Data; Deacetylation; Development; Disease; Electrophoretic Mobility Shift Assay; Elongation Factor; Enzymes; Eukaryota; Event; Excision; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genome; Histone Acetylation; Histones; Homologous Gene; Human; ISWI; In Vitro; Institutes; Knowledge; Link; Literature; Lysine; Mediating; Methods; Methylation; Modeling; Modification; Molecular; Molecular Biology; Molecular Chaperones; Molecular Genetics; Mono-S; Nucleoproteins; Nucleosomes; Organism; Pentas; Physiological; Play; Polymerase; Process; Proteins; RNA Polymerase II; Role; SAGA; Saccharomyces cerevisiae; Streptavidin; Structure; System; Techniques; Technology; Testing; Time; Transcription Elongation; Work; Yeasts; chromatin immunoprecipitation; chromatin modification; chromatin remodeling; demethylation; histone acetyltransferase; histone modification; in vivo; insight; interest; research study; yeast genetics; yeast protein

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.

描述（由申请人提供）：通过核小体的RNA聚合酶II（POL II）转录是真核基因表达中最基本的过程之一。酿酒酵母中的遗传学，染色质免疫沉淀和分子研究为体内的pol II伸长提供了许多见解。尽管文献广泛，但对实际机制的了解很差，因为没有实验室使用带有染色质模板的体外伸长系统以系统的方式攻击了该问题。共转录组蛋白乙酰化和快速脱乙酰化的现象以及共转录甲基化和脱甲基化的相关过程尤其引起了人们的关注，这是由于它们在所有真核生物中的保存。然而，即使对机制的基本理解也要求在具有纯化蛋白质的定义转录系统中重新创建和分析这些事件。我最近在Stowers Institute的Jerry Workman的实验室完成了一个1年的休假，在那里我在单核小体上建立了一个体外转录伸长系统。我发现乙酰化和ATP依赖性重塑作品协同允许POL II通过组蛋白八聚体。我建议显着扩展这些研究，以解决伸长机制的几个方面。我的小组将使用串联亲和力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。构成该提案基石的一种特别强大的技术是固定的模板分析。该测定法使用附着在链霉亲和素涂层的珠子附着的染色体模板来捕获伸长络合物。然后，将对复合物进行功能和组成分析。该技术和更传统的方法（例如电泳移动性转移）将用于解决以下三个目标。 AIM＃1将检查POL II通过核小体和单核小体模板上组蛋白蛋白酶的命运的机制。该目标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（RSC和SWI/SNF）。 AIM＃2将探索用于H3K4和H3K36三甲基化功能的当前模型，以及识别包括SAGA，RPD3S和CHD1的甲基化组蛋白的机器的详细机制。 AIM＃3将检查包括SPT6，ASF1和FACT，组装和拆卸核小体的组蛋白伴侣如何侧重于共价修改如何影响该过程。我们的研究将利用从酵母遗传学和分子生物学的广泛知识来制作和检验Pol II如何通过核小体以及染色质的共价修饰的假设。这些知识将提供适用于所有真核生物的Pol II延伸的基本信息。 B.项目叙事基因调控的最重要方面之一是了解当基因打开并替换基因时，染色质是如何去除核蛋白结构（保护真核基因组的）如何去除的。此步骤对于所有生物体都是基础的，对过程的了解是了解人类疾病，分化和发育过程中基因调节的关键。我们的建议将使用酵母菌酿酒酵母作为模型生物来理解这一过程。

项目成果

The Rpd3 core complex is a chromatin stabilization module.

• DOI: 10.1016/j.cub.2011.11.042
• 发表时间: 2012-01-10
• 期刊: CURRENT BIOLOGY
• 影响因子: 9.2
• 作者: Chen, Xiao-Fen;Kuryan, Benjamin;Kitada, Tasuku;Tran, Nancy;Li, Jing-Yu;Kurdistani, Siavash;Grunstein, Michael;Li, Bing;Carey, Michael
• 通讯作者: Carey, Michael