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. 2008 May 30;283(22):14955–14962. doi: 10.1074/jbc.M801123200

FIGURE 3.

FIGURE 3.

Identification of the IL-4/STAT6-dependent silencer region of the Foxp3 promoter. A, naïve T cells were cultured under the Th0 or iTreg conditions in the absence or presence of IL-4. 48 h later, cells were subjected to the ChIP assay with anti-rabbit IgG or anti-acetylated histone H4 Ab. The histone acetylation levels were determined by real-time PCR. Each plot represents the normalized value to the input. Data are representative of at least two independent experiments with similar results. Data of the ChIP assay for PCR 10 and 11 are shown as a graph in the lower panels. B, DNA affinity precipitation assay. T cells expanded with anti-TCR Ab and IL-2 cells were restimulated with (+) or without (–) IL-4 for 1 h. Extracts of cells were incubated with the biotinylated oligonucleotide corresponding to the putative STAT6 binding site, and then the isolated DNA-protein complexes were analyzed by immunoblotting with anti-STAT6 Ab. C, EL4 cells were transfected with the empty vector or FLAG-tagged STAT6VT (an active form of STAT6). Chromatins from transfected cells were subjected to ChIP assay using anti-FLAG Ab. The binding of STAT6VT to the putative STAT6 binding site located in the Foxp3 promoter region was determined by real-time PCR using the final DNA extractions. PCR product 11 was electrophoresed and visualized in the upper panel. D, induction of Foxp3 mRNA in EL4 cells. EL4 cells cultured with plate-bound anti-CD3 Ab in the presence or absence of TGF-β1 and IL-4 for 12 h. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. E, promoter assay in EL4 cells with the Foxp3 promoter reporter constructs. After transfection, cells were stimulated with plate-bound anti-CD3 Ab and TGF-β1 for 12 h. The luciferase activities normalized by the β-galactosidase activity and expressed as the -fold change to control cultures defined as 1.0 are shown. F, effects of point mutations introduced into the putative STAT6 binding site on the Foxp3 promoter reporter activity. EL4 cells transfected with WT or mutant pFoxp3 luciferase plasmids were stimulated with TGF-β1 with or without IL-4, and luciferase activity was measured after 10 h. The luciferase activities normalized by the β-galactosidase activity are shown as the mean ± S.D. of three independent samples. One representative experiment of three independent experiments is shown.