The full potential of genome wide association studies (GWAS) will only be realized once we fully understandthe biological consequences of genetic risk associations. The goal of the proposed study is to identify genetargets of validated colorectal cancer (CRC) GWAS risk enhancers using a series of complementaryapproaches and to begin to establish the biological role of risk enhancers in normal crypt development andCRC etiology using a novel in vivo murine-based method. This study builds upon our previous successes inidentifying 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 ofthese CRC risk enhancers by conducting genome wide eQTL analyses using RNA-Seq data from >1000normal colon epithelial biopsies and by CRISPR/Cas9-mediated knock out of the risk enhancers in CRC celllines 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 physicalinteraction 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 befurther 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 bedeveloped that harbor selected human BACs corresponding to 3 risk enhancer GWAS regions (including themultiple 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 intomouse ES cells and CRISPR/Cas9 technology will be used to introduce either risk or non-risk variants withinrisk enhancers. The modified ES cells will be combined with wild type tetraploid embryos to generate chimericmice in which the entire embryo-proper was derived from the modified ES cells. The effects of the risk andnon-risk SNPs on target gene transcript levels using transcriptome profiling (RNA-Seq) will be determined inthese mice in intestinal crypts and non-colon cells (e.g. liver, spleen). Histological studies will be conducted toexamine the effects of risk enhancer SNPs on normal crypt and intestine polyp/tumor development. Theseexperiments will be carried out in transgenic mice that are wild-type for Apc, as well as mice that carry aheterozygous-null mutation in the Apc gene. The proposed research will provide insight into the biological roleof risk enhancers in the intestinal crypt and CRC etiology and the discovery of risk enhancer target genes willprovide tools for future early surveillance and prevention studies of CRC.
Our work is motivated by our success in identifying a growing number of risk enhancers for colorectal cancer(CRC) identified through post-genome wide association studies (post GWAS). The goal of this proposal is toidentify target genes of known CRC risk enhancers for CRC using complementary but independentapproaches and to model a number of risk enhancers in mice to determine their potential role in intestinedevelopment and polyp/cancer risk.
