FIGURE 4.

Methylated Gata3 is able to recruit JunB and Ets1 to the Il5 promoter. A, the DNA sequences of the proximal Il5 promoter region are shown. The GATA, AP-1, and Ets1 consensus binding sites are underlined. The numbers indicate positions relative to the transcriptional start site of the Il5 gene. NFAT, nuclear factor of activated T-cells. B, schematic representations of the transcriptional activation Gata3 complex bound to the Il5 promoter. C and D, 293T cells were transfected with expression plasmids encoding FLAG-tagged Gata3 and Myc-tagged JunB (C) or Ets1 (D). Two days later, the extracts were immunoprecipitated with a FLAG mAb, followed by I.B. with a Myc mAb (upper panel). The total lysates were also subjected to I.B. in parallel (lower panel). Mock, mock-transfected. E and F, naive CD4 T cells were stimulated under Th1 conditions and then infected with a retrovirus vector carrying WT or mutant Gata3 cDNAs. Four days later, the cells were restimulated with phorbol 12-myristate 13-acetate and ionomycin for 16 h, and the binding of JunB and Est1 at the indicated regions was determined by a ChIP assay with a qPCR analysis. The relative intensities (/input) are shown with standard deviations.