Mechanisms of RSC recruitment and its role in transcription

描述（由申请人提供）： 染色质结构的动态调节是真核生物基因表达所必需的，部分是通过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调地调节转录。RSC（重塑染色质结构）是一种染色质重塑复合物，从酵母到人类都是保守的，是生存所必需的。虽然已知RSC调节参与重要生物过程（如细胞分裂和对DNA损伤和应激的反应）的基因的转录，但RSC如何被募集到其靶基因以及它如何发挥作用以调节其表达仍有待确定。RSC复合物具有可以识别和结合乙酰化组蛋白的多个亚基，这支持了乙酰化组蛋白残基的识别对于RSC募集和功能很重要的流行观点。我们最近发现编码序列中的乙酰化与组蛋白占有率呈负相关，这进一步表明RSC识别并结合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，这表明聚合酶可能将RSC靶向转录基因。为了支持这一假设，我们的初步数据显示，RSC与Pol II的相互作用在kin 28-ts（C-末端结构域（CTD）激酶）突变体中丢失，表明Kin 28对Pol II CTD的磷酸化可能促进RSC在转录延伸过程中向编码序列的募集。因此，我们提出了一个两步模型，其中RSC最初通过延长聚合酶被招募到编码序列，随后识别特定的组蛋白乙酰化模式，以靶向核小体进行重塑或驱逐。 为了了解靶向RSC转录编码区的机制，我们将在野生型细胞和Pol II CTD激酶和组蛋白乙酰转移酶突变体细胞中进行全基因组定位测定。使用一种新的分析，我们最近开发的，我们将确定和表征的关键组蛋白赖氨酸残基的相互作用的RSC与染色质。然后，我们将分析耗尽RSC对转录延伸的影响，以确定RSC能够促进Pol II通过编码区移动的机制。这些贡献将是重要的，因为它们有望深入了解RSC如何被招募到染色质的机制，以及它如何在健康生物体中调节其靶基因的转录。这些信息将有助于我们了解RSC复合物中的突变如何导致癌症等疾病的发生和进展，并可能有助于确定新的治疗靶点。 公共卫生相关性： 组蛋白修饰模式的改变和染色质重塑复合物的突变与发育缺陷和疾病有关，包括各种癌症的发生。这项研究有望为了解一种重要的染色质重塑复合物是如何被招募到其靶基因中以调节其表达提供有价值的机制见解。了解这些复合物如何在健康生物体的生长和发育过程中调节染色质结构的精确变化，最终将使我们能够确定这些复合物中的突变如何导致疾病状态。

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. PUBLIC HEALTH RELEVANCE: PUBLIC HEALTH RELEVANCE Alterations in histone modification patterns and mutations in chromatin remodeling complexes are linked to developmental defects and disease, including the onset of various cancers. The proposed study is expected to provide valuable mechanistic insight as to how an essential chromatin remodeling complex is recruited to its target genes to regulate their expression. An understanding of how such complexes regulate precise changes in chromatin structure during the growth and development of a healthy organism will ultimately allow us to determine how mutations in those complexes lead to a disease state.