Fine-mapping of multiple melanoma GWAS signals on chromosome band 2q33.1.

(Top) A view of the 2q33.1 locus (hg19) including all fine-mapped candidate causal variants nominated by log-likelihood ratio (LLR; red), Bayesian fine-mapping either unweighted (DAP-G, green) or weighted using melanocyte-specific epigenomic annotations (PAINTOR, brown), or LD to the melanoma lead SNP (rs10931936, r2 ≥ 0.625, orange). Also shown are tracks representing NCBI RefSeq genes and imputed chromatin state (ChromHMM) from two primary melanocyte cultures, two primary keratinocyte cultures, and two breast mammary cultures generated by the Roadmap Epigenome Project (Red: Active TSS; Orange-Red: Flanking Active TSS; Yellow: Enhancers; Green-Yellow: Genic enhancers; Green: Strong transcription, Dark Green: Weak transcription). (Bottom) A zoomed-in view of the same annotations for a region encompassing select candidate causal variants. Data and rendering was performed using the UCSC Genome Browser.

The melanoma risk-associated T allele of rs10931936 is correlated with lower CASP8 expression.

eQTL analyses were performed for rs10931936 combining genotype and expression level data derived from (A) 106 human primary melanocyte cultures (TT, n=7; TC, n=39; CC, n=60), (B) 349 melanoma tumors from TCGA-SKCM (TT, n=42; TC, n=126; CC, n=181), and (C) a panel of 59 early-passage melanoma cell lines (TT, n=8; TC, n=23; CC, n=25), where the risk-T allele is labeled in red. A significant eQTL effect with higher expression driven from the C-protective allele was observed for CASP8 in both melanocytes and melanoma tumors (P = 1.2 x 10-9 and P = 6.8 x 10-3, respectively), while the result was marginal but in the same direction in melanoma cell lines (P = 0.07). Significance determined by linear regression; mean with SEM are plotted along with individual data values.

CASP8 eQTL in human primary melanocyte cultures is colocalized with the melanoma GWAS.

(A) LocusZoom plots present –log10 P-values for melanoma GWAS (upper) and melanocyte CASP8 eQTL (lower) for a 1 Mb region encompassing rs10931936. The melanoma risk lead SNP rs10931936 is labeled and highlighted in purple in both panels, and LD (r2 based on 1000G EUR) of all other SNPs to the melanoma GWAS lead SNP is color-coded. (B) A LocusCompare plot compares P-values between melanoma GWAS (x-axis) and melanocyte CASP8 eQTL (y axis) for the same region. Genomic coordinates are based on hg19.

The melanoma-associated rs3769823 is a functional cis-regulatory variant and displays allele-preferential binding by E4F1 and IRF2.

(A) Individual luciferase reporter activity assays for rs3769823 were conducted using the melanoma cell line UACC903. 138 bp sequences encompassing rs3769823 construct were cloned 5’ of the pGL4.23 minimal TATA promoter and transfected. Luciferase activity was measured 24 h after transfection and was normalized against Renilla luciferase activity. One representative set from three biological replicate experiments is shown. Mean with SEM are plotted. Individual P values are shown for A-risk allele versus G-protective allele for the replicate shown (two-tailed, unpaired t-test assuming unequal variances). TATA, minimal promoter control; A, risk allele construct marked in red; G, protective allele construct. (B) EMSAs were performed using 21 bp biotin-labeled double-stranded oligonucleotides for the A-risk (red) or G-protective (black) alleles of rs3769823 and nuclear extract from UACC903 melanoma cells. 2x, 5x, or 10x molar excess of unlabeled competitor was added in specified lanes. One representative set from three replicate experiments is shown. (C) Allele-specific rs3769823 binding proteins were identified by quantitative mass spectrometry using UACC903 melanoma nuclear extracts and 21 bp biotinylated double-stranded DNA probed with A-risk or G-protective alleles. The dimethyl-labeling ratios of proteins bound to A/G or G/A probes are plotted on the x and y axes for label-swapping experiments. Red-filled circles highlight proteins enriched above the background in both experiments. (D) Antibody super-shift assay of rs3769823 EMSA in UACC903 melanoma cells using anti-E4F1 or anti-IRF2 antibody is shown, where the A-risk specific shifted band (arrows) is diminished. One representative set from three independent experiments is shown. (E) ChIP was performed using antibody to anti-E4F1, anti-IRF2 or anti-IgG and chromatin prepared from UACC903 melanoma cells, followed by qPCR. DNA quantity was normalized to input DNA for each immunoprecipitation. Mean of qPCR triplicates with SEM are plotted and P-values shown for one representative experiment from three biological replicates.

E4F1 or IRF2 and rs3769823 regulate CASP8 expression in UACC903 melanoma cells.

(A) E4F1 or IRF2 were knocked down using respective pools of four different siRNAs in UACC903 melanoma cells, and E4F1, IRF2 and CASP8 levels were measured. GAPDH- normalized E4F1, IRF2 or CASP8 mRNA levels are shown as fold change over those from non-targeting siRNA. A representative experiment from three biological replicates is shown (individual datapoints, mean, and SEM are plotted). P-values are shown from one representative set. (B) Protein levels were examined using anti-E4F1, anti-IRF2, or anti-GAPDH antibody with UACC903 cell lysates of siRNAs transfected with either non-targeting control, E4F1, or IRF2 siRNAs. GAPDH was used as a loading control. One representative set of three replicate experiments is shown. (C) Individual luciferase assays were performed by co-transfecting rs3769823 luciferase constructs with E4F1, IRF2, or non-targeting control siRNAs into UACC903 melanoma cells. Renilla-normalized relative luciferase activities are shown relative to an empty construct containing only a minimal promoter (TATA). One representative set is shown from three biological replicates. Mean with SEM is plotted, n = 6 technical replicates. A two-tailed t-test assuming unequal variances was used to calculate all P values shown against control siRNA.