Massively parallel reporter assays combined with cell-type specific eQTL identified a functional melanoma risk variant in HIV-1 restriction gene, MX2 [MX2_overexpress]

Genome-wide association studies (GWAS) have identified twenty melanoma susceptibility loci. To identify susceptibility genes and variants simultaneously from multiple GWAS loci, we integrated massively-parallel reporter assays (MPRA) with melanocyte-specific expression quantitative trait locus (eQTL) profiling. We identified thirty-nine candidate functional variants displaying allelic transcriptional activity, of which nine from four loci were also correlated with local gene expression in melanocytes (CTSS, CASP8, MX2, and MAFF). Among these, we further characterized the locus in MX2 gene on chromosome band Chr21q22.3 and validated a functional variant, rs398206. The functional variant mediates allelic transcriptional activity via binding of the transcription factor, YY1. This allelic transcriptional regulation largely accounts for a significant cis-eQTL of the HIV-1 restriction gene, MX2, in primary melanocytes, where the melanoma risk-associated A allele is correlated with higher MX2 levels. Melanocyte-specific transgenic expression of human MX2 in a zebrafish model demonstrated an accelerated melanoma formation in a BRAFV600E background. Thus, using an efficient scalable approach to streamline GWAS follow-up functional studies, we uncovered a pleiotropic function of MX2 in melanoma susceptibility. To identify genes that are differentially expressed upon MX2 over-expression in primary human melanocytes, we performed RNA-seq of total of 27 samples (melanocyte cultures from 3 individuals, in 3 conditions, and 3 biological replicates). Three conditions include 1) MX2 control (with 0 doxycycline treatment on the cells transduced with MX2 cDNA under the control of tetracycline-inducible promoter), 2) MX2 over-expression (with 100ng/ml doxycycline treatment resulting in 2-10 fold increase of MX2 levels), and 3) Doxycycline control (with 100ng/ml doxycycline treatment on the cells transduced with empty pINDUCER20 vector). We identified differentially expressed genes in MX2 over-expression samples compared to MX2 control samples for pathway analysis. Same type of analysis was performed to identify doxycycline-induced genes by comparing Doxycycline control samples (100ng/ml treatment on Empty vector) to MX2 control samples (no treatment) for pathway analysis.