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RNA-Seq and ATAC-Seq of HEK239T cell line carrying different genotypes at rs4420550 by CRISPR/Cas9 editing

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE152177 | GEO | 2020/10/01

REPOSITORIES: GEO

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Project description:Genome-wide association studies (GWAS) have successfully identified 145 genomic regions that contribute to schizophrenia risk, but linkage disequilibrium (LD) makes it challenging to discern causal variants. Computational finemapping prioritized thousands of credible variants, ~98% of which lie within poorly characterized non-coding regions. To functionally validate their regulatory effects, we performed a massively parallel reporter assay (MPRA) on 5,173 finemapped schizophrenia GWAS variants in primary human neural progenitors (HNPs). We identified 440 variants with allelic regulatory effects (MPRA-positive variants), with 72% of GWAS loci containing at least one MPRA-positive variant. Transcription factor binding had modest predictive power for predicting the allelic activity of MPRA-positive variants, while GWAS association, finemap posterior probability, enhancer overlap, and evolutionary conservation failed to predict MPRA-positive variants. Furthermore, 64% of MPRA-positive variants did not exhibit eQTL signature, suggesting that MPRA could identify yet unexplored variants with regulatory potentials. MPRA-positive variants differed from eQTLs, as they were more frequently located in distal neuronal enhancers. Therefore, we leveraged neuronal 3D chromatin architecture to identify 273 genes that physically interact with MPRA-positive variants. These genes annotated by chromatin interactome displayed higher mutational constraints and regulatory complexity than genes annotated by eQTLs, recapitulating a recent finding that eQTL- and GWAS-detected variants map to genes with different properties. Finally, we propose a model in which allelic activity of multiple variants within a GWAS locus can be aggregated to predict gene expression by taking chromatin contact frequency and accessibility into account. In conclusion, we demonstrate that MPRA can effectively identify functional regulatory variants and delineate previously unknown regulatory principles of schizophrenia.

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RNA-Seq and ATAC-Seq of HEK239T cell line carrying different genotypes at rs4420550 by CRISPR/Cas9 editing

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