Genomic Enhancers at 8q24 and Prostate Cancer

描述（由申请人提供）：8q24 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高危等位基因的识别发生了一场革命，这主要归功于高通量基因组技术的利用。例如，在一项关于种系变异和 PCa 风险的综合多种族研究中，我们在染色体 8q24 上跨越约 500 kb 的 3 个区域中建立了多个独立的风险等位基因。这些等位基因在许多其他后续研究中得到了验证。所有变体都位于非蛋白质编码序列中，并且距离任何已知基因均>200-kb。对包含所有风险区域的 5 Mb 染色质片段进行了 RNA 表达、组蛋白修饰以及 RNA 聚合酶 II 和雄激素受体占据的位置的分析。这导致了转录增强子的鉴定，并使用报告基因检测进行了验证。其中两个单核苷酸多态性 (SNP) 分别影响 TCF7L2 和 FoxA1 结合，并影响后者雄激素依赖性增强子活性。我们的总体假设是：8q24 的 PCa 易感性受增强子中的 SNP 控制，影响转录因子结合和远端基因表达。在具体目标#1中，将在体外对含有风险SNP的增强子进行表征，重点关注遗传变异如何影响增强子活性的组合调节（subaim 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，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（subaim 1.2）。报告基因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，以及风险 SNP 如何修改这些模式。在具体目标#2中，我们将确定增强子的目标基因。我们打算采用三种方法：（i）大型细菌人工染色体来测试细胞培养物和小鼠中不同生理条件下的等位基因（subaim 2.1），（ii）染色质循环测定来测试候选靶基因（例如Myc）（submain 2.2）和（iii）无偏循环筛选来识别全基因组范围内的新靶基因（submain 2.3）。成功完成这些目标将有助于更全面地了解与 PCa 风险遗传相关的生物学机制，如观察到的位于 8q24 非蛋白质编码序列的变异。此外，我们的方法可能提供一种新的范例来研究与其他疾病状态相关的非蛋白质编码区域中发现的许多其他遗传位点。 公共健康相关性：8q24 的基因组增强剂和前列腺癌 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传播最广泛的癌症之一；据估计，仅在美国，每年就有约 200,000 名男性被诊断患有前列腺癌，每年约有 27,000 人死于该疾病。非洲血统的男性患前列腺癌的风险几乎是其他种族群体男性的两倍，遗传因素可能对这种增加有显着影响。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”）的区域与前列腺癌易感性/风险密切相关，在本应用中，我们将准确揭示这是如何（机制）发生的。

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.