RNA-Seq and ATAC-Seq of HEK239T cell line carrying different genotypes at rs4420550 by CRISPR/Cas9 editing
Background: Genome-wide association studies (GWAS) have reported hundreds of genomic loci associated with schizophrenia, yet identifying the functional risk variations is a key step in elucidating the underlying mechanisms. Methods: We applied multiple bioinformatics and molecular approaches, including expression quantitative trait loci (eQTL) analyses, epigenome signature identification, luciferase reporter assay, chromatin conformation capture (3C), homology-directed genome editing by CRISPR/Cas9, RNA-sequencing (RNA-Seq) and assay for transposase-accessible chromatin using sequencing (ATAC-Seq). Results: We found that the schizophrenia GWAS risk variations at 16p11.2 were significantly associated with mRNA levels of multiple genes in human brain, and one of the leading eQTL genes, MAPK3, located ~200-kb away from these risk variations in the genome. Further analyses based on the epigenome marks in human brain and cell lines suggested that a noncoding single nucleotide polymorphism (SNP) rs4420550 (p=2.36×10–9 in schizophrenia GWAS) was within a DNA enhancer region, which was validated via in vitro luciferase reporter assays. The 3C experiment showed that the rs4420550 region physically interacted with the MAPK3 promoter and TAOK2 promoter. Precise CRISPR/Cas9 editing of single base pair in cells followed by RNA-Seq further confirmed the regulatory effects of rs4420550 on the transcription of 16p11.2 genes, and ATAC-Seq demonstrated that rs4420550 affected chromatin accessibility at the 16p11.2 region. The rs4420550-[A/A] cells showed significantly higher proliferation rates compared with rs4420550-[G/G] cells. Conclusions: These results together suggest that rs4420550 is a functional risk variation, and this study illustrates an example of comprehensive functional characterization of schizophrenia GWAS risk loci. Overall design: To understand if the different alleles of rs4420550 could affect nearby gene expressions and chromatin accessibility of the 16p11.2 region, we performed RNA-Seq and ATAC-Seq in four colonies carried the [A/A] genotype and the other four carried the [G/G] genotype at rs4420550.