Signaling through NFAT and NF-κB is required for induction of twist1 expression. (A) Comparison of the genomic sequence of the murine and the human proximal twist1 promoter (−150 to −100). The murine sequence is displayed with conserved bases in capital letters. Selected putative DNA-binding motifs are indicated. ISRE, IFN-stimulated response element. (B) Twist1 mRNA in 24-d-old Th1 cells restimulated for 3 h with PMA/ionomycin and IL-2 in the presence of serial dilutions of the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC; circles; starting concentration 50 μM), the NFAT inhibitor BTP1 (squares; starting concentration 100 nM), or the NF-κB and NFAT inhibitor cyclosporine A (CsA; triangles; starting concentration 30 nM). (C) 24-d-old Th1 cells were restimulated in the presence of IL-2 for 3 h with IL-2 alone (unstimulated) or 10 ng/ml TNF-α, PMA, or PMA/ionomycin. The binding of NFAT1 (D) and the NF-κB subunit p65 (E) to the proximal promoter of twist1 was analyzed by ChIP. 18–24-d-old Th1 and Th2 cells either in the resting state (-) or after restimulation with PMA/ionomycin and IL-2 for 1 h (+) were used. The immunoprecipitated DNA was quantified by RT-PCR using primers specific for the proximal promoter. The precipitated DNA was normalized to the amount of input DNA. The amount of twist1 transcripts precipitated in activated Th1 cells was set to 1. Data represent mean ± SD of 3 experiments.