Intron 1 of GRHL2 Is Transcriptionally Active and PPCD4 Mutations Cause Increased Promoter Activity In Vitro
A dual-luciferase reporter assay was used to determine whether intron 1 of GRHL2 had promoter activity and the effect of PPCD4 mutations. HEK293 cells were co-transfected with pRL-CMV (Renilla luciferase) and pGL3-basic (firefly luciferase) containing 2,728 bp of the wild-type or respective GRHL2 promoter sequence mutants. Wild-type activity was normalized to 1, and the relative luciferase activity of all mutants was expressed with respect to the wild-type. All PPCD4-associated GRHL2 variants significantly increased the relative luciferase activity. Data represent a minimum of three independent biological replicates in triplicate. Error bars represent ±1 SD. p values were calculated by one-way ANOVA (∗∗∗p ≤ 0.001).