Genomic Enhancers at 8q24 and Prostate Cancer

描述（由申请人提供）：8q24基因组增强子与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鉴定经历了一场革命，主要是由于高通量基因组技术的使用。例如，在一项关于种系变异和PCa风险的综合多民族研究中，我们在染色体8q24上的3个区域建立了多个独立的风险等位基因，跨度约为500-kb。这些等位基因在随后的许多研究中得到了证实。所有的变异都位于非蛋白质编码序列中，与任何已知基因的距离都在100 - 200-kb之间。包含所有危险区域的5mb染色质片段被描绘为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。这导致了转录增强子的鉴定，并通过报告者试验进行了验证。其中，单核苷酸多态性（snp）分别影响TCF7L2和FoxA1的结合，并影响后者雄激素依赖性增强子的活性。我们的总体假设是：8q24位点的PCa易感性受增强子中的snp控制，增强子影响转录因子结合和远端基因表达。在具体目标#1中，包含风险snp的增强子将在体外进行表征，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的活性（见第1.2条）。报告基因活动的时空模式将揭示含风险snp增强子发挥最大作用的细胞类型和发育阶段，以及这些模式如何被风险snp修改。在具体目标#2中，我们将确定增强子的靶基因。我们打算采用三种方法：(i)在细胞培养和小鼠中测试不同生理条件下的大细菌人工染色体的等位基因（subaim 2.1）， （ii）染色质环连接测定以测试候选靶基因（如Myc） （submain 2.2）和（iii）无偏环连接筛选以识别全基因组的新靶基因（subaim 2.3）。成功完成这些目标将导致对PCa风险遗传关联的生物学机制有更全面的了解，正如在8q24非蛋白质编码序列中所观察到的那样。此外，我们的方法可能为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位点提供新的范例。

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.