Figure 2.

Allele-specific transcriptional regulation and AHR binding via rs117132860

(A) motifbreakR analysis identified an AHR::ARNT binding motif as strongly altered, where the melanoma-protective rs117132860-G allele matches the motif, while rs117132860-A is predicted to weaken AHR binding.

(B–D) Luciferase reporter activity of a 375 bp fragment encompassing either allele of rs117132860 is measured in (B) the human melanoma cell line UACC903 and (C and D) two primary melanocyte cultures (C197 and C23; labeled). pGL4.23 is the control vector containing a minimal TATA promoter; risk (labeled in red) and protective alleles of rs117132860 are assayed in both forward and reverse directions relative to the promoter. Three biological replicates were combined for each cell (three technical replicates of each, mean, and SEM are plotted).

(E) rs117132860 displays allele-preferential binding to AHR in C87, melanocytes (heterozygous for rs117132860) by chromatin immunoprecipitation assay with anti-AHR antibody or normal IgG control. Relative quantities are shown as fold over input DNA (one representative experiment from three biological replicates is shown; individual datapoints, mean, and SEM are plotted).

(F) Taqman genotyping of rs117132860 with AHR ChIP DNA from the same experiment was normalized to input DNA (one representative set from three biological replicates is shown; mean of PCR triplicates with SEM is plotted).