Analysis of higher order chromatin structures in normal and cancer epigenomes

描述(由申请人提供)：染色质结构的病理改变通过影响表观基因组和基因组的稳定性并导致异常转录而促进肿瘤的发生。沉默的和活跃的染色质都可以在大的基因组距离上传播，以创建扩展的岛和结构域的更高级别的结构。我们发现，绝缘蛋白CTCF结合位点在人类基因组中发现的一些大的异染色质结构域上定义了精确的分子边界，包括HOXA基因座，这是一组已知在发育和疾病中都很重要的同源盒基因。Hoxa基因座的表观遗传和遗传去调控定义了白血病中常见的致癌程序。我们推测CTCF是在HOXA基因座建立和维持适当的染色质结构和高阶组织所必需的。为了研究CTCF在HOXA基因座的功能，我们将使用一系列的分子分析，包括报告分析、BAC重组工程、染色质免疫沉淀和染色体构象捕获。对HOXA基因的详细分析将使我们能够了解染色质大规模组织背后的生化和分子机制，并破译CTCF功能改变如何导致癌细胞中常见的HOXA基因表达改变。同时，我们将使用生化和蛋白质组学方法来鉴定与HOXA基因上CTCF结合位点差异和唯一相关的反式作用因子，并利用短干扰RNA介导的消融来研究它们的功能。最后，我们将表征与癌症相关的CTCF基因突变的影响，以及竞争副同源基因Boris在HOXA基因座和整个人类基因组中的异位表达。这些拟议的实验代表了一项综合努力，以确定CTCF在限制异染色质和常染色质中的功能，揭示控制基因组表达的其他机制，并扩大我们对人类基因组和表观基因组结构和功能的理解。该项目将为染色质结构致癌改变的潜在机制提供关键的见解，并将提供负责癌症表观基因组的新的药物靶点。

DESCRIPTION (provided by applicant): Pathologic alterations of chromatin structure promote tumorigenesis by affecting epigenomic and genomic stability and causing aberrant transcription. Both silent and active chromatin can spread over large genomic distances to create higher-order structures of extended islands and domains. We have found that the insulator protein CTCF binding sites define precise molecular boundaries at a number of large heterochromatin domains found in the human genome, including the HOXA locus, a cluster of homeo box genes known to be important in both development and disease. Epigenetic and genetic deregulations of the HOXA locus define a common oncogenic program in leukemia. We hypothesize that CTCF is required for establishment and maintenance of the proper chromatin structure and higher order organization at the HOXA locus. To examine CTCF function at the HOXA locus, we will employ a battery of molecular analyses, including reporter assays, BAC recombineering, chromatin immunoprecipitation, and chromosome conformation capture. Detailed analyses of the HOXA locus will enable us to understand biochemical and molecular mechanisms underlying the large-scale organization of chromatin and to decipher how alteration of CTCF function might lead to altered expression of HOXA genes which is frequently observed in cancer cells. In parallel, we will use biochemical and proteomic approaches to identify trans-acting factors that are differentially and uniquely associated with the CTCF binding site at the HOXA locus, and utilize short interfering RNA mediated ablation to study their function. Lastly, we will characterize the effect of cancer-associated mutations in the CTCF gene and ectopic expression of the competitive paralog BORIS at the HOXA locus and throughout the entire human genome. These proposed experiments represent an integrated effort to determine the function of CTCF in restricting heterochromatin and euchromatin, to uncover additional mechanisms that control genome expression, and to extend our understanding of the human genome and epigenome structure and function. This project will provide critical insights into mechanisms underlying cancer-promoting alterations of chromatin structures and will provide new drug targets responsible for the cancer epigenome.