Fig. 2

IRF6 directly promotes the expression of KLF4 through binding to super-enhancers of KLF4. (A and B) Representative image of anti-KLF4 immunofluorescence (green) and nuclear DNA (DAPI, blue) in coronal section of mouse E14.5 head of (A) wild-type (Irf6^+/+) and (B) Irf6-null mice (Irf6^-/-). Arrowheads, immunoreactivity in oral periderm in wild-types, absent from mutants. P, palate shelf; T, tongue. Bar, 50 µm. (C) Quantitation of KLF4 mRNA in oral epithelial cell line without and with over-expressed human IRF6. Asterisks indicate statistical significance compared with control plasmid-transfected group: **P < 0.01. (D) Quantitative chromosome configuration capture (3C) experiment with the anchoring (i.e. bait) primer in the promoter of KLF4. Upper panel: UCSC Genome Browser view of hKLF4 E1-E5, peaks of IRF6 binding according to ChIP-seq and positions of two NHEK super-enhancers 1000 kb downstream of the KLF4 TSS. Lower panel: results of chromatin conformation capture experiment in human oral epithelial cell line confirming physical crosstalk between KLF4-E1 and -E5 with KLF4 promoter. Grids in x-axis represent each EcoRI restriction site. (E) Dual luciferase reporter assays for KLF4 activity in 293FT cells validate that IRF6 can bind to the putative IRF6 binding sites in hKLF4-E1 and -E5 and increase their activities. Asterisks indicate statistical significance compared with the luciferase activity of empty luciferase reporter co-transfected with IRF6 plasmid: **P < 0.01. (F-H) Lateral or (I) ventral epifluorescence views of live, wild-type zebrafish embryos at the indicated stage, previously injected with the indicated reporter construct. Arrowheads, GFP expression in internal, oral epithelium cells.