Figure 6.

Runx3 directly binds to the promoter and intron 8 of il12rb2 (A) Graph of the il12r2 gene and location of Runx3 binding sites. (B) Luciferase activity in HEK293 cells transfected with various combinations (below plot) of vector alone (pGL3) or with 100, 200, or 500 ng of vector containing the wild-type promoter of il12rb2 (il12rb2-p) or with mutations in the Runx3-binding site of the promoter (il12rb2-p-MUT), together with empty vector (EV) or vector expressing Runx3 (Runx3). The results are presented relative to cells transfected with pGL3 and empty vector (far left), set to one. (C) Luciferase activity in HEK293 cells transfected with various combinations (below plot) of vector alone (pGL3) or with 100, 200 ng, or 500 ng of vector containing the wild-type intron 8 of il12rb2 (il12rb2-I8) or with mutations in the Runx3-binding site of the promoter (il12rb2-I8-MUT), together with empty vector (EV) or vector expressing Runx3 (Runx3). The results are presented relative to cells transfected with pGL3 and empty vector (far left), set to one. (D) Runx3 binding at the il12rb2 locus in EL4 stable cell lines expressing either a triple flag peptide (TFTC) or triple-flag-tagged Runx3 (TFTC-RORγt) was monitored using a flag ChIP assay. The fold enrichment of Runx3 binding at each locus was normalized to TFTC-empty EL4 cells. The actin and ifng loci were used as negative and positive controls, respectively. (E) ILC1s were sorted from wild type and cKO mice and infected with retrovirus containing the indicated vector. ELISA for IFNγ secretion with the indicated vector overexpressed in ILC1s after stimulation with IL12 and IL18 for 36 h (mean ± SD of three samples in (B–E); ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 by Student's t test). Data are from one experiment representative of three independent experiments with similar results in (A,C,D) and two independent experiments with similar results in (E).