Figure 3: The rs2238126 alleles affect the activity of enhancer MAX at the 12p13.2 locus.
From: Common genetic variation in ETV6 is associated with colorectal cancer susceptibility
(a) A putative enhancer region flanking rs2238126 (chr12:12,009,241-12,010,241) with A or G alleles was cloned upstream of the ETV6 promoter-luciferase reporter vector. HCT116 and SW480 cells were transiently transfected with each of these constructs and assayed for luciferase activity after 24 h. The P-value was calculated with two-sided t-test. *P<0.001. (b) EEL analysis predicted the binding affinity of MAX to the rs2238126 alleles. (c) EMSA with biotin-labelled rs2238126 A or G probes and HCT116 nuclear extracts. Lanes 1 and 5 represent negative controls with probes only. The biotin-labelled rs2238126 A allele probe (lane 2) produced a much denser band of a specific DNA–protein complex (arrow) than the G allele probe (lane 6). The specific complex with rs2238126-labelled A probe can be partly competed by 300-fold unlabelled A probe (lane 3) or G probe (lane 4). The complex with the labelled G allele probe can be completely abolished by 300-fold unlabelled A probe (lane 8), but not G probe (lane 7). (d) ChIP and quantitative RT–PCR assays confirm that rs2238126 binds to MAX in HCT116 cells. Relative enrichment was calculated as a ratio of the signals from MAX or IgG to the signals from the input DNA.
