Genomic Enhancers at 8q24 and Prostate Cancer

描述(申请人提供)：基因组增强剂8q24与前列腺癌在过去三年中，人们对前列腺癌遗传易感性的理解和高危等位基因的识别经历了一场革命，这主要是由于高通量基因组技术的使用。例如，在一项关于生殖系变异和PCA风险的综合多种族研究中，我们在染色体8q24上跨越~500-kb的3个区域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其他研究中得到了验证。所有的变异体都位于非蛋白质编码序列中，并且都是任何已知基因的200-kb。包括所有危险区域的5-Mb染色质片段被描述为RNA表达、组蛋白修饰以及RNA聚合酶II和雄激素受体所占据的位置。这导致了转录增强子的鉴定，并使用报告分析进行了验证。在其中两个基因中，单核苷酸多态(SNPs)分别影响TCF7L2和FoxA1的结合，在后者中影响雄激素依赖的增强子活性。我们的总体假设是：8q24的Pca易感性是由增强子中的SNPs控制的，它影响转录因子结合和远距离基因表达。在具体目标#1中，将在体外表征含有风险SNPs的增强子，重点是基因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TCF7L2、FoxA1和雄激素受体介导的风险机制。此外，体内增强子活性将在携带由候选增强子元件控制的报告基因的小鼠身上进行研究(SUAIM 1.2)。报告活性的空间和时间模式将揭示含有风险SNP的增强子发挥最大作用的细胞类型和发育阶段，以及这些模式是如何被风险SNP改变的。在特定的目标2中，我们将确定增强子的靶基因。我们打算使用三种方法：(I)大的细菌人工染色体在细胞培养和小鼠中测试不同生理条件下的等位基因(SubAim 2.1)，(Ii)染色质环状试验来测试候选目标基因(如Myc)(Submain 2.2)和(Iii)无偏环筛选以识别全基因组范围的新目标基因(SubAim 2.3)。成功完成这些目标将导致更全面地理解遗传与前列腺癌风险相关的生物学机制，如8q24处非蛋白质编码序列中的变体所观察到的那样。此外，我们的方法可能提供一种新的范例来研究在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位点。 公共卫生相关性：基因组增强剂与前列腺癌这个项目寻求揭示前列腺癌风险的基本机制，前列腺癌是美国和世界上传播最广泛的癌症之一；据估计，仅在美国每年就有约20万名男性被诊断患有前列腺癌，每年约有2.7万人死于前列腺癌。非洲血统的男性患前列腺癌的风险几乎是其他种族男性的两倍，遗传因素可能是这种增加的重要原因。我们之前已经证明，人类8号染色体上500,000个碱基对(连续的DNA‘字母’)区域与前列腺癌易感性/风险密切相关，在本应用中将揭示这一点是如何产生的(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.