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稳定性以及引起异常转录来促进肿瘤发生。沉默和活跃的染色质都可以在大的基因组距离上传播，以创建扩展岛和结构域的高阶结构。我们发现绝缘体蛋白CTCF结合位点在人类基因组中发现的许多大异染色质结构域定义精确的分子边界，包括HOXA位点，已知在发育和疾病中都很重要的一组同源盒基因。HOXA基因座的表观遗传和遗传失调定义了白血病中一种常见的致癌程序。我们假设CTCF是HOXA位点建立和维持适当的染色质结构和高阶组织所必需的。为了检查CTCF在HOXA位点的功能，我们将采用一系列分子分析，包括报告基因分析、BAC重组、染色质免疫沉淀和染色体构象捕获。对HOXA基因座的详细分析将使我们能够理解大规模染色质组织背后的生化和分子机制，并破译CTCF功能的改变如何导致HOXA基因表达的改变，这在癌细胞中经常观察到。同时，我们将使用生化和蛋白质组学方法来识别与HOXA位点上CTCF结合位点差异和独特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，我们将描述CTCF基因中癌症相关突变的影响，以及HOXA位点和整个人类基因组中竞争平行物BORIS的异位表达。这些拟议的实验代表了确定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，并扩展我们对人类基因组和表观基因组结构和功能的理解的综合努力。该项目将为促进癌症的染色质结构改变的机制提供关键见解，并将提供负责癌症表观基因组的新药物靶点。

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.