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对真核基因的表达至关重要，部分是通过组蛋白修饰和染色质重塑复合体的联合作用实现的。我们的长期目标是了解这些复合体如何协调作用来调节转录。RSC(重塑染色质的结构)是一种从酵母到人类保存的染色质重塑复合体，是生存所必需的。虽然已知RSC调节细胞分裂、DNA损伤和应激反应等重要生物学过程中涉及的基因的转录，但RSC如何被招募到其靶基因以及它如何调节其表达仍有待确定。RSC复合体具有多个亚基，可以识别和结合乙酰化组蛋白，这支持了流行的观点，即识别乙酰化组蛋白残基对RSC的招募和功能至关重要。我们最近发现编码序列中的乙酰化与组蛋白的占有率呈负相关，这进一步表明RSC识别并结合乙酰化组蛋白来移除它们。有趣的是，RSC还与RNA聚合酶II(Pol II)相互作用，这表明该聚合酶可能针对RSC转录基因。为了支持这一假说，我们的初步数据显示，在一个kin28-ts(C末端结构域(CTD)激酶)突变体中，RSC与Pol II的相互作用丢失，表明Kin28对Pol II CTD的磷酸化可能促进RSC在转录延伸过程中招募到编码序列中。因此，我们提出了一个两步模型，在这个模型中，RSC最初通过延长聚合酶来招募编码序列，然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突变细胞中进行POL II CTD激酶和组蛋白乙酰转移酶的全基因组定位分析。使用我们最近开发的一种新的分析方法，我们将识别和表征对RSC和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，因为他们有望深入了解RSC是如何被招募到染色质的机制，以及它如何在健康的生物体中调节其目标基因的转录。这些信息将有助于我们了解RSC复合体的突变是如何导致癌症等疾病的发生和发展的，并可能有助于确定新的治疗靶点。

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.