Figure 8  GATA3 Represses the Human FOXP3 Promoter
(A) Jurkat, Th2 cells, and human primary CD4 cells were transfected with an empty vector (pGL3 basic) or a vector containing the putative FOXP3 promoter region fused to the luciferase reporter gene. Bars show the mean ± SD of arbitrary light units normalized for renilla luciferase of four independent experiments; samples were measured in triplicates.
(B) Naive CD4 T cells were transfected with the FOXP3 promoter reporter construct together with a GATA3 expression vector or an empty vector. Bars show the mean ± SD of three independent experiments.
(C) Naive (left panel) or memory (right panel) CD4 T cells were transfected with wild-type or a GATA3 mutated 511-FOXP3 promoter reporter construct and activated with PMA and ionomycin. Bars show the mean ± SD of arbitrary light units normalized for renilla luciferase of eight independent experiments; samples were measured in triplicates.
(D) Nuclear extracts were prepared from HEK cells transfected with GATA3 or an empty vector, (E) Th1, Th2, or iTreg cells and binding factors precipitated using biotinylated oligonucleotides. The oligonucleotides–transcription factor complexes were separated on a SDS-PAGE gel. The amounts of GATA3 protein in the precipitates were assessed by immunoblotting with anti-GATA3 mAb. Total nuclear extracts were also run as controls. Data are representative of three different experiments.
(F) iTreg or Th2 cells were analyzed by ChIP for GATA3 binding to the FOXP3 promoter. The “input” represents PCR amplification of the total sample, which was not subjected to any precipitation. Results are representative of three independent experiments.