Mechanisms of RSC recruitment and its role in transcription

RSC 招募机制及其在转录中的作用

• 批准号: 8303263
• 负责人: Chhabi K Govind
• 金额: $ 27.38万
• 依托单位: OAKLAND UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8303263
• 关键词: Acetylation; Affinity; Binding; Biological Assay; Biological Process; Bromodomain; C-terminal; Calorimetry; Cell Cycle Progression; Cell division; Cell physiology; Cells; Chromatin; Chromatin Remodeling Factor; Chromatin Structure; Chromosome Segregation; Code; Complex; DNA Damage; Data; Defect; Disease; Disease Progression; Eukaryota; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Genome; Goals; Histone Acetylation; Histones; Human; Individual; Lead; Lysine; Malignant Neoplasms; Mammals; Maps; Measures; Mediating; Modeling; Movement; Mutation; Nucleosomes; Organism; Pattern; Peptides; Phosphorylation; Phosphotransferases; Physiological; Polymerase; Process; RNA Polymerase II; Recruitment Activity; Regulation; Relative (related person); Resolution; Role; Saccharomyces cerevisiae; Saccharomycetales; Stress; Tail; Titrations; Transcription Elongation; Yeasts; cell type; chromatin immunoprecipitation; design; genome-wide; histone acetyltransferase; histone modification; in vivo; insight; mutant; new therapeutic target; novel; research study; response

DESCRIPTION (provided by applicant): The dynamic regulation of chromatin structure, essential for the expression of eukaryotic genes, is achieved in part by the combined activity of histone modifying and chromatin remodeling complexes. Our long term goal is to understand how these complexes act coordinately to regulate transcription. RSC (Remodels the Structure of Chromatin) is a chromatin remodeling complex conserved from yeast to human that is required for viability. Although RSC is known to regulate the transcription of genes involved in important biological processes such as cell division and the responses to DNA damage and stress, how RSC is recruited to its target genes as well as how it functions to regulate their expression remains to be defined. The RSC complex possesses multiple subunits that can recognize and bind acetylated histones, which supports the prevailing view that the recognition of acetylated histone residues is important for RSC recruitment and function. We recently showed that acetylation in coding sequences is inversely correlated with histone occupancy, which further suggests that RSC recognizes and binds acetylated histones to remove them. Interestingly, RSC also interacts with RNA Polymerase II (Pol II), which suggests that the polymerase may target RSC to transcribed genes. In support of this hypothesis, our preliminary data shows that the interaction of RSC with Pol II is lost in a kin28-ts (C-terminal domain (CTD) kinase) mutant, indicating that phosphorylation of the Pol II CTD by Kin28 may promote the recruitment of RSC to coding sequences during transcription elongation. We thus propose a two-step model in which RSC is initially recruited to coding sequences by elongating polymerases, and subsequently recognizes particular patterns of histone acetylation to target nucleosomes for remodeling or eviction. To understand the mechanism involved in targeting RSC to transcribed coding regions, we will perform genome-wide localization assays in wild type cells and cells mutant for Pol II CTD kinases and histone acetyltransferases. Using a novel assay that we have recently developed, we will identify and characterize critical histone lysine residues important for the interaction of RSC with chromatin. We will then analyze the effect of depleting RSC on transcription elongation to define the mechanism by which RSC is able to facilitate Pol II movement through coding regions. These contributions will be significant in that they are expected to lend insight into the mechanism of how RSC is recruited to chromatin, as well as how it functions to regulate the transcription of its target genes in a healthy organism. This information will be valuable to our understanding of how mutations in the RSC complex lead to the initiation and progression of diseases such as cancer, and could potentially aid in identifying new therapeutic targets.

描述（由申请人提供）： 染色质结构的动态调节，对于真核基因的表达必不可少的，部分是由组蛋白修饰和染色质重塑复合物的组合活性来实现的。我们的长期目标是了解这些复合物如何协同作用以调节转录。 RSC（重塑染色质的结构）是一种从酵母到人类保守的染色质重塑复合物，这是生存力所必需的。尽管已知RSC调节与重要生物学过程有关的基因的转录，例如细胞分裂以及对DNA损伤和压力的反应，但如何募集RSC对其靶基因以及如何调节其表达的功能仍有待定义。 RSC复合物具有多个可以识别和结合乙酰化组蛋白的亚基，这支持了普遍的观点，即对乙酰化组蛋白残基的识别对于RSC募集和功能很重要。我们最近表明，编码序列中的乙酰化与组蛋白占用率成反比，这进一步表明RSC识别并结合了乙酰化组蛋白以去除它们。有趣的是，RSC还与RNA聚合酶II（POL II）相互作用，这表明聚合酶可以将RSC靶向转录基因。为了支持这一假设，我们的初步数据表明，RSC与POL II的相互作用在KIN28-TS（C-末端结构域（CTD）激酶）突变体中丢失，这表明KIN28通过KIN28对POL II CTD的磷酸化可能会促进RSC募集到转录过程中RSC的编码序列的募集。因此，我们提出了一个两步模型，其中最初通过拉长聚合酶将RSC募集到编码序列中，并随后识别组蛋白乙酰化的特定模式以进行重塑或驱逐。 为了理解针对RSC转录编码区域涉及的机制，我们将对POL II CTD激酶和组蛋白乙酰基转移酶进行野生型细胞和细胞突变体中的全基因组定位测定。使用我们最近开发的新测定法，我们将识别并表征对RSC与染色质相互作用重要的关键组蛋白赖氨酸残基。然后，我们将分析耗尽RSC对转录伸长的影响，以定义RSC能够促进Pol II通过编码区域的机制。这些贡献将具有重要意义，因为它们有望深入了解RSC如何募集到染色质的机制，以及它如何功能来调节健康生物体中其靶基因的转录。这些信息对于我们了解RSC复合物中的突变如何导致癌症等疾病的开始和进展将很有价值，并有可能有助于识别新的治疗靶标。