Identification and characterization of functional risk variants for colorectal cancer mapping to chromosome 11q23.1

Abstract

Genome-wide association studies of colorectal cancer (CRC) have identified a number of common variants associated with modest risk, including rs3802842 at chromosome 11q23.1. Several genes map to this region but rs3802842 does not map to any known transcribed or regulatory sequences. We reasoned, therefore, that rs3802842 is not the functional single-nucleotide polymorphism (SNP), but is in linkage disequilibrium (LD) with a functional SNP(s). We performed ChIP-seq for histone modifications in SW480 and HCT-116 CRC cells, and incorporated ChIP-seq and DNase I hypersensitivity data available through ENCODE within a 137-kb genomic region containing rs3802842 on 11q23.1. We identified SNP rs10891246 in LD with rs3802842 that mapped within a bidirectional promoter region of genes C11orf92 and C11orf93. Following mutagenesis to the risk allele, the promoter demonstrated lower levels of reporter gene expression. A second SNP rs7130173 was identified in LD with rs3802842 that mapped to a candidate enhancer region, which showed strong unidirectional activity in both HCT-116 and SW480 CRC cells. The risk allele of rs7130173 demonstrated reduced enhancer activity compared with the common allele, and reduced nuclear protein binding affinity in electromobility shift assays compared with the common allele suggesting differential transcription factor (TF) binding. SNPs rs10891246 and rs7130173 are on the same haplotype, and expression quantitative trait loci (eQTL) analyses of neighboring genes implicate C11orf53, C11orf92 and C11orf93 as candidate target genes. These data imply that rs10891246 and rs7130173 are functional SNPs mapping to 11q23.1 and that C11orf53, C11orf92 and C11orf93 represent novel candidate target genes involved in CRC etiology.

Figure 1.

Chromatin features and LD structure for the 11q23.1 CRC GWAS locus. (A) Coordinates for the LD block (r² ≥ 0.2, CEU population) surrounding tagSNP rs3802842 are noted above coding transcripts from Genome Browser. SNPs in LD r² ≥ 0.2 with rs3802842 are noted. ChIP-seq tracks for H3K4me1 and H3K4me3 in HCT-116 and SW480 are presented along with DNAse hypersensitivity tracks from UW ENCODE dataset for HCT-116 and the CaCo-2 cell lines (29,30). The blue stripe highlights the promoter fragment described in Figure 2. The green stripe highlights the enhancer fragment described in Figure 3. (B) In line with the above panel, a linkage disequilibrium plot for SNPs in the region is shown. Arrows denote the tagSNP rs3802842, and putative functional SNPs rs7130173 and rs10891246. The LD plot was created using 1000 Genomes Project data and created with Haploview. r² = 0—white, 0 < r² < 1—shades of gray, r² = 1—black.

Figure 2.

Bidirectional promoter activity for DNA fragment surrounding rs10891246. (A) A 2 kb DNA fragment centered over the H3K4me3 peak on 11q23.1 (forward and reverse) along with a negative control sequence from chromosome 10 were tested for promoter activity in HCT-116 and SW480 CRC cells lines in transient assays. Bidirectional promoter activity is seen in both cell lines. (B) Two SNPs within the fragment highly correlated with rs3802842 (rs7105857, r² = 0.83 and rs10891246, r² = 0.96) were mutated from their common alleles, T and G, to the risk alleles, C and A, respectively. Fold change in activity values are presented as mean ± SD of at least four-independent transfections. A statistically significant reduction in promoter activity was observed bidirectionally for rs10891246 in both cell lines.

Figure 3.

Unidirectional enhancer activity for DNA fragment surrounding rs7130173. (A) 2 kb DNA fragment centered over H3K4me1 peak on 11q23.1, chr11:111,152,290–111,154,290, (forward, F, and reverse, R) along with a negative control sequence (CT) from the chromosome 8q24 region (13) were tested for enhancer activity in HCT-116 and SW480 CRC cells lines in transient assays along with pRL-TK Renilla luciferase plasmid. Relative luciferase activity is presented as mean ± SD of at least four independent transfections and reveals unidirectional enhancer activity in both HCT-116 and SW480 CRC cells. The locations of two SNPs within the fragment highly correlated with rs3802842 (rs1987128, r² = 0.77 and rs7130173, r² = 0.96) are depicted in the inset. The 2 kb fragment was sub-cloned into two 1 kb fragments, ‘1 kb-A’ and ‘1 kb-B’, and a 300-bp DNA fragment encompassing only the DNase I hypersensitive site, ‘300 bp’. Enhancer activity was seen only in those fragments containing the common SNP rs7130173 in the forward orientation. (B) The common (C) and risk (A) alleles of rs7130173 were tested in enhancer activity assays following site directed mutagenesis of fragments ‘1 kb-B’ and ‘300 bp’ in both HCT-116 and SW480. The risk allele A reduces enhancer activity for the 1 kb fragment, while it increases enhancer activity for the 300 bp fragment.

Figure 4.

Minor allele of the rs7130173 variant was associated with reduced gene expression levels of three putative genes (C11orf53, C11orf92 and C11orf93) mapping to 11q23.1. Relative gene expression quantities (RQs) in C11orf53 (Hs00736614_m1), C11orf92 (Hs04186919_m1), C11orf93 (Hs00416978_m1) and POU2AF1 (Hs01573371_m1) were measured in normal colon epithelial tissue samples from surveillance colonoscopies. Linear regression was used to estimate the effect of each extra minor allele on log-RQs (additive model). Two-sided P-values were obtained from a likelihood ratio test. Levels of log-RQs were plotted as a function of genotypes using a box plot—dot plot overlay. The positions of SNPs rs7130173 and rs10891246 are shown relative to the genes tested.

Figure 5.

DNA fragments containing the A (risk) allele of rs7130173 show reduced DNA-protein binding compared with fragments containing the C (common) allele. EMSA were performed with IRDye-labeled oligos: C (major) allele (700/red), A (risk) allele (800/green). (A)–(C) The same gel and image with (A) showing the dual color scan, (B) showing the 700 nm channel (red above) in black and white and (C) showing the 800 nm channel (green above) in black and white for clarity of reproduction. Lanes 1 and 2 are probes incubated without nuclear extract. Lanes 3–7 show the probe shift following incubation with 5 μg SW480 nuclear extract. Lanes 8–12 show the probe shift following incubation with 5 μg HCT-116 nuclear extract. Lanes 6 and 11 and Lanes 7 and 12 show the effect of 200-fold excess competition with unlabeled C and A oligos, respectively. Bands that show protein/DNA complexes specific to rs7130173 alleles are noted with arrows.