Analysis of higher order chromatin structures in normal and cancer epigenomes

描述（由申请方提供）：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并引起异常转录促进肿瘤发生。沉默和活跃的染色质都可以在大的基因组距离上传播，以创建扩展的岛和结构域的高级结构。我们已经发现，绝缘子蛋白CTCF结合位点在人类基因组中发现的许多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失调定义了白血病中常见的致癌程序。我们假设CTCF是建立和维持HOXA基因座上正确的染色质结构和更高层次组织所必需的。为了研究CTCF在HOXA位点的功能，我们将采用一系列分子分析，包括报告基因测定、BAC重组工程、染色质免疫沉淀和染色体构象捕获。HOXA基因座的详细分析将使我们能够了解染色质大规模组织的生化和分子机制，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，我们将使用生物化学和蛋白质组学的方法来确定的反式作用因子的差异和独特的CTCF结合位点在HOXA基因座，并利用短干扰RNA介导的消融研究其功能。最后，我们将描述CTCF基因中的癌症相关突变和HOXA基因座和整个人类基因组中竞争性旁腺BORIS的异位表达的影响。这些拟议的实验代表了一个综合的努力，以确定CTCF在限制异染色质和常染色质的功能，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的促癌改变机制提供重要见解，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.