Fig. 6. Effect of rs7225881 and rs4803219 on IFN-λ3 promoter activity.

A) Schematic of the p1.4kbIFNL3 construct used in the study showing the location of the SNPs rs4803219 and rs28416813 and the TA dinucleotide repeat polymorphism rs7225881 in the context of the different IRF7 and NF-κB TF binding sites. The exact location of each binding site from the ATG start site is shown above them. B) Effect of TA repeat-length on the IFN-λ3 promoter activity when tested against p65 or IRF7. The TA repeats were cloned at the site rs7225881 as shown in (A) in the p1.4IFNL3 construct carrying the C allele at rs28416813; this construct did not include the IFN-λ3 3’UTR. The data was drawn from 9 to 10 separate experiments representing three different plasmid preparations transfected in to HEK293T cells grown in 96-well plates. The histograms show the mean value of all the 9–10 experiments, while error bars are depicting SD. The grey line below the histograms shows the trend in mean Luciferase expression values for the different construct transfected with different TFs. C) The ancestral alleles of the three SNPs rs4803219, rs28416813 and rs4803217 significantly decrease transcription from the IFN-λ3 promoter. All four TFs as shown were transfected in to HEK293T cells grown in 12-well plates and further stimulated by polyI:C as described in the methods section. p1.4kbIFNL3-3’ UTR construct carrying the different haplotypes at the three SNP positions were used. The data is from six separate experiments representing three different plasmid preparations. The histograms show mean values from the six experiments with error bars showing SD. For B and C one tailed student t-test for independent means was used for calculating statistical significance; *p < 0.05, **p < 0.01; ***p < 0.001. All the Luciferase ratio values from each individual experiment for B and C are shown in Suppl. Table 1.