Genomic Enhancers at 8q24 and Prostate Cancer

描述（由申请人提供）：8 q24处的基因组增强子和前列腺癌在过去三年中，对前列腺癌（PCa）遗传易感性的理解和危险等位基因的鉴定经历了一场革命，这主要是由于高通量基因组技术的利用。例如，在生殖系变异和PCa风险的综合性多种族研究中，我们在染色体8 q24上跨越~500 kb的3个区域中建立了多个独立的风险等位基因。这些等位基因在随后的许多研究中得到了验证。所有的变体都位于非蛋白质编码序列中，并且与任何已知基因相距>200-kb。一个5-Mb的染色质片段，包括所有的风险区域的RNA表达，组蛋白修饰和RNA聚合酶II和雄激素受体占据的位置进行了分析。这导致了转录增强子的鉴定，其使用报告基因测定进行验证。在其中两个单核苷酸多态性（SNPs）影响TCF 7 L2和FoxA 1结合，分别在后者雄激素依赖性增强子活性。我们的总体假设是：8 q24处的PCa易感性受增强子中的SNP控制，其影响转录因子结合和远端基因表达。在具体目标#1中，将在体外表征含有风险SNP的增强子，重点是遗传变异如何影响增强子活性的组合调节（子目标1.1）。将特别关注已经确定的TCF 7 L2-、FoxA 1-和雄激素受体介导的风险机制。此外，将在携带由候选增强子元件控制的报告基因的小鼠中研究体内增强子活性（子目的1.2）。报道活性的空间和时间模式将揭示含风险SNP的增强子最大程度起作用的细胞类型和发育阶段，以及这些模式如何被风险SNP修饰。在具体目标#2中，我们将鉴定增强子的靶基因。我们打算采用三种方法：（i）在细胞培养物和小鼠中在不同生理条件下测试等位基因的大型细菌人工染色体（子目标2.1），（ii）测试候选靶基因（如Myc）的染色质成环试验（子目标2.2）和（iii）在全基因组范围内鉴定新靶基因的无偏成环筛选（子目标2.3）。这些目标的成功完成将导致对与PCa风险相关的遗传学机制的更全面的理解，正如在8 q24的非蛋白质编码序列中观察到的那样。此外，我们的方法可以提供一种新的范式来研究与其他疾病状态相关的非蛋白质编码区中发现的许多其他遗传基因座。 公共卫生相关性：8 q24基因组增强子与前列腺癌该项目旨在揭示前列腺癌风险的基本机制，前列腺癌是美国和世界上最广泛传播的癌症之一;据估计，仅在美国，每年约有20万男性被诊断患有前列腺癌，每年约有27，000人死于这种疾病。来自非洲血统的男性患前列腺癌的风险几乎是来自其他种族的男性的两倍，遗传因素可能显着导致这种增加。我们之前已经表明，人类8号染色体上500，000个碱基对（连续的DNA“字母”）的区域与前列腺癌易感性/风险密切相关，并且在本申请中将确切地揭示这是如何产生的（机制）。

DESCRIPTION (provided by applicant): Genomic Enhancers at 8q24 and Prostate Cancer The understanding of genetic predisposition to prostate cancer (PCa) and the identification of at-risk alleles have undergone a revolution during the past three years, mainly due to the utilization of high-throughput genomic technologies. For example, in a comprehensive multi-ethnic study of germline variation and PCa risk, we established multiple independent risk alleles in 3 regions that span ~500-kb on chromosome 8q24. The alleles were verified in many other subsequent studies. All the variants are located in non-protein coding sequences and are >200-kb from any known gene. A 5-Mb chromatin segment encompassing all the risk regions was profiled for RNA expression, histone modifications and locations occupied by RNA polymerase II and the androgen receptor. This led to the identification of transcriptional enhancers, which were verified using reporter assays. In two of them single nucleotide polymorphisms (SNPs) affected TCF7L2 and FoxA1 binding, respectively and in the latter androgen-dependent enhancer activity. Our overall hypothesis is: PCa predisposition at 8q24 is governed by SNPs in enhancers, which affect transcription factor binding and distant gene expression. In specific aim #1, the enhancers containing risk SNPs will be characterized in vitro with emphasis on how genetic variations affect the combinatorial regulation of enhancer activities (subaim 1.1). Special attention will be directed towards already-identified, TCF7L2-, FoxA1- and androgen receptor-mediated risk mechanisms. Additionally, in vivo enhancer activities will be investigated in mice carrying reporter genes controlled by candidate enhancer elements (subaim 1.2). The spatial and temporal pattern of reporter activity will disclose the cell types and developmental stages at which the risk SNP-containing enhancers are maximally operative and how these patterns are modified by the risk SNPs. In specific aim #2, we will identify the target genes of the enhancers. We intend to employ three approaches: (i) large bacterial artificial chromosomes to test alleles under different physiologic conditions in cell culture and in mice (subaim 2.1), (ii) chromatin looping assays to test candidate target genes (such as Myc) (submain 2.2) and (iii) an unbiased looping screen to identify novel target genes genome-wide (subaim 2.3). Successful completion of the aims will lead to a more complete understanding of the biological mechanisms underlying genetic associations with PCa risk, as observed with variants located in non-protein coding sequences at 8q24. Additionally, our approach may provide a novel paradigm to study many other genetic loci found in non-protein coding areas associated with other disease states. PUBLIC HEALTH RELEVANCE: Genomic Enhancers at 8q24 and Prostate Cancer This project seeks to unravel fundamental mechanisms of prostate cancer risk, one of the most wide spread cancers in the U.S and the world; it is estimated that in the U.S. alone ~200,000 men are diagnosed annually with prostate cancer and ~27,000 die each year from the disease. Men from African ancestry have nearly twice the risk for prostate cancer development than men from other racial-ethnic groups and genetic factors probably significantly contribute to this increase. We have previously shown that an area of 500,000 base pairs (consecutive DNA 'letters') on human chromosome 8 is robustly involved in prostate cancer predisposition/risk and in this application will expose exactly how (mechanism) this is brought about.