Biology of Colorectal Cancer Risk Enhancers

 描述(由申请人提供)：只有当我们充分了解遗传风险关联的生物学后果时，全基因组关联研究(GWAS)的全部潜力才能实现。这项研究的目的是利用一系列互补的方法确定已验证的结直肠癌(CRC)GWA型风险增强剂的基因靶点，并开始使用一种新的体内小鼠方法确定风险增强剂在正常隐窝发育和结直肠癌病因学中的生物学作用。这项研究建立在我们之前在染色体1q41、3p14.1、8q24.21、11q23.1、15q13.3(3个风险增强子)、18q21.1、19q13.11、19q21和20p12.3基因座上成功识别CRC风险增强子的基础上。在目标1中，我们将通过使用来自&gt；1000正常结肠上皮活组织的RNA-Seq数据进行全基因组eQTL分析，以及通过CRISPR/Cas9介导的敲除CRC细胞系中的风险增强子，然后进行RNA-Seq eQTL分析，来识别这些CRC风险增强子的新的靶基因。在目标2中，我们将使用染色体构象捕获方法来识别和验证风险增强子-靶基因(S)的相互作用。我们将使用循环染色体构象捕获(4C)方法，使用HCT116和SW480大肠癌细胞株来鉴定和验证风险增强剂和靶基因之间的物理相互作用。利用染色质构象捕捉(3C)和荧光原位杂交(FISH)进一步验证特定增强子-靶基因的相互作用。在目标3中，我们将使用一种新的小鼠模型系统来测试结直肠癌风险增强剂的生物学效应。小鼠将发育成含有与3个风险增强子GWA区(包括15q13.3上的多个增强子区域)相对应的精选人BAC的小鼠，这些区域具有已知的局部靶基因(8q24.21/cMYC/CCAT2、11q23.1/C11orf53/C11orf92/C11orf93和15q13.3/GREM1/FMN1/ax747968)。BACs将被插入小鼠ES细胞，CRISPR/Cas9技术将被用于在风险增强剂中引入风险或非风险变体。改良的胚胎干细胞将与野生型四倍体胚胎相结合，产生嵌合体小鼠，在嵌合鼠中，整个胚胎本身都来自改良的胚胎干细胞。风险和非风险SNPs对转录组分析(RNA-Seq)靶基因转录水平的影响将在这些小鼠的肠腺和非结肠细胞(如肝、脾)中确定。将进行组织学研究，以检查风险增强剂SNPs对正常隐窝和肠息肉/肿瘤发展的影响。这些实验将在野生型APC转基因小鼠以及携带APC基因杂合零突变的小鼠身上进行。这项拟议的研究将深入了解风险增强剂在肠道隐窝和结直肠癌病因学中的生物学作用。 风险增强子靶基因的发现将为今后结直肠癌的早期监测和预防研究提供工具。

 DESCRIPTION (provided by applicant): The full potential of genome wide association studies (GWAS) will only be realized once we fully understand the biological consequences of genetic risk associations. The goal of the proposed study is to identify gene targets of validated colorectal cancer (CRC) GWAS risk enhancers using a series of complementary approaches and to begin to establish the biological role of risk enhancers in normal crypt development and CRC etiology using a novel in vivo murine-based method. This study builds upon our previous successes in identifying CRC risk enhancers within GWAS loci on chromosomes 1q41, 3p14.1, 8q24.21, 11q23.1, 15q13.3 (3 risk enhancers), 18q21.1, 19q13.11, 19q21 and 20p12.3. In Aim 1 we will identify novel target genes of these CRC risk enhancers by conducting genome wide eQTL analyses using RNA-Seq data from >1000 normal colon epithelial biopsies and by CRISPR/Cas9-mediated knock out of the risk enhancers in CRC cell lines followed by RNA-Seq eQTL analysis. In Aim 2 we will identify and validate risk enhancer-target gene(s) interactions using chromosome conformation capture methods. We will identify and validate the physical interaction between risk enhancers and target genes using the circularized chromosome conformation capture (4C) method using HCT116 and SW480 CRC cell lines. Specific enhancer-target gene interactions will be further validated using chromatin conformation capture (3C) and fluorescence in situ hybridization (FISH). In Aim 3 we will test the biological effect of CRC risk enhancers using a novel mouse model system. Mice will be developed that harbor selected human BACs corresponding to 3 risk enhancer GWAS regions (including the multiple enhancer region on 15q13.3) with known local target genes (8q24.21/cMYC/ CCAT2, 11q23.1/C11orf53/ C11orf92/ C11orf93 and 15q13.3/GREM1/ FMN1/ ax747968). BACs will be inserted into mouse ES cells and CRISPR/Cas9 technology will be used to introduce either risk or non-risk variants within risk enhancers. The modified ES cells will be combined with wild type tetraploid embryos to generate chimeric mice in which the entire embryo-proper was derived from the modified ES cells. The effects of the risk and non-risk SNPs on target gene transcript levels using transcriptome profiling (RNA-Seq) will be determined in these mice in intestinal crypts and non-colon cells (e.g. liver, spleen). Histological studies will be conducted to examine the effects of risk enhancer SNPs on normal crypt and intestine polyp/tumor development. These experiments will be carried out in transgenic mice that are wild-type for Apc, as well as mice that carry a heterozygous-null mutation in the Apc gene. The proposed research will provide insight into the biological role of risk enhancers in the intestinal crypt and CRC etiology and the discovery of risk enhancer target genes will provide tools for future early surveillance and prevention studies of CRC.