Systematic Functional Interrogation of Genes in GWAS Loci Identified ATF1 as a Key Driver in Colorectal Cancer Modulated by a Promoter-Enhancer Interaction

Abstract

Genome-wide association studies (GWASs) have identified approximately 100 colorectal cancer (CRC) risk loci. However, the causal genes in these loci have not been systematically interrogated. We conducted a high-throughput RNA-interference functional screen to identify the genes essential for proliferation in the CRC risk loci of Asian populations. We found that ATF1, located in the 12q13.12 region, functions as an oncogene that facilitates cell proliferation; ATF1 has the most significant effect of the identified genes and promotes CRC xenograft growth by affecting cell apoptosis. Next, by integrating a fine-mapping analysis, a two-stage affectedcontrol study consisting of 6,213 affected individuals and 10,388 controls, and multipronged experiments, we elucidated that two risk variants, dbSNP: rs61926301 and dbSNP: rs7959129, that located in the ATF1 promoter and first intron, respectively, facilitate a promoter-enhancer interaction, mediated by the synergy of SP1 and GATA3, to upregulate ATF1 expression, thus synergistically predisposing to CRC risk (OR ¼ 1.77, 95% CI ¼ 1.42-2.21, p ¼ 3.16 3 10 À7 ; P multiplicative-interaction ¼ 1.20 3 10 À22 ; P additive-interaction ¼ 6.50 3 10 À3 ). Finally, we performed RNA-seq and ChIP-seq assays in CRC cells treated with ATF1 overexpression in order to dissect the target programs of ATF1. Results showed that ATF1 activates a subset of genes, including BRAF, NRAS, MYC, BIRC2, DAAM1, MAML2, STAT1, ID1, and NKD2, related to apoptosis, Wnt, TGF-b, and MAPK pathways, and these effects could cooperatively increase the risk of CRC. These findings reveal the clinical potential of ATF1 in CRC development and illuminate a promoter-enhancer interaction module between the ATF1 regulatory elements dbSNP: rs61926301 and dbSNP: rs7959129, and they bring us closer to understanding the molecular drivers of cancer.We selected candidate genes on the basis of CRC GWASs, which identified 15 loci associated with CRC risk (2016.12, Table S1) in Asian (ASN) populations. To select candidate genes in each region for functional screening, we performed fine mapping by extending 1 Mb upstream and downstream of the tag SNPs. After we excluded microRNAs, noncoding RNAs, and pseudogenes on the basis of their functional annotation in the National Center for Biotechnology Information database, we ultimately selected a total of 157 protein-coding genes (Table S2) for a proliferation measurement of CRC cells by a large-scale RNAi interrogation. The siRNA library was provided by ViewSolid Biotech, and the repression efficiencies were guaranteed by the provider. Both p < 0.05 and an n-fold change >1.1 or <0.9 were selected as the threshold of significance.

miR-34b/crs4938723 T>C Decreases Neuroblastoma Risk: A Replication Study in the Hunan Children

“…Such protective role could also be seen in other cancer types, such as colorectal cancer [34], gastric cancer [54], and esophageal cancer [55]. Other genetic, environmental factors, and gene-environment interaction may cooperatively determine the protective role of miR-34b/c rs4938723 T>C in neuroblastoma risk [56, 57].…”

Systematic analysis of genetic variants in cancer-testis genes identified two novel lung cancer susceptibility loci in Chinese population

“…To gain insight into the potential functions of our identified SNPs and their related variants (r 2 ≥ 0.6), several public-available databases, including Haplo Reg v4.1 (https://pubs.broadinstitute.org/mammals /haploreg/), ENCODE, rSNPBase (http://rsnp.psych .ac.cn/index.do) [17,18], SNP2TFBS (https://ccg.epfl. ch/snp2tfbs/) [19], PINES (http://genetics.bwh.harv ard.edu/pines/index.html) and RegulomeDB (http: //regulome.stanford.edu/) [20], were used for functional annotations.…”

ANKLE1 N6‐Methyladenosine‐related variant is associated with colorectal cancer risk by maintaining the genomic stability

“…The genotyping in the discovery stage was using the Illumina HumanExome Bead Chip and was reported before. 18 We phased the haplotypes with segmented haplotype estimation and imputation tool 20 and performed a genome-wide imputation with IMPUTE2 software, 21 based on the linkage disequilibrium and haplotypes information from the 1,000 Genomes Project Phase 3 Asian samples as references. A total of 69 poorly imputed variants with an information measure <0.40 were excluded from subsequent analyses (Fig.…”

“…The principal components of genetic ancestry have been analyzed in the previous study and the results showed that there was no significant population stratification for the participants in the discovery stage. 18 Written informed consent was obtained from each subject at recruitment, and our study was approved by the Chinese Academy of Medical Sciences Cancer Institute and Institutional Review Board of Tongji Medical College, Huazhong University of Science and Technology.…”