Mechanism involved in Zta-mediated IL-13 gene expression. (A) Schematic illustration of the IL-13 promoter that drives the luciferase gene in the reporter plasmid; putative ZRE sequences are indicated. The IL-13 promoter contains 34 putative ZRE regions in the sequence from −1159 to +65. (B) HEK293T cells were transfected with Zta-expressing plasmid or vector in combination with serial 5′-deleted pIL-13 reporter plasmids and pEGFP-C1 reporter control. After 72 hours, luciferase activities from each transfectant were normalized with the GFP intensities. Zta-driven fold activation for each reporter was calculated by normalizing luciferase activities for the Zta transfectant versus that for the pGL2-basic vector control. (C) DNA sequences of the probes used in EMSA experiments. Bold lines indicate ZRE or ZREmt within the IL-13 promoter sequences from −90 to −110. Nuclear extracts were harvested at 72 hours after transfection of pRC vector or pRC-Zta in HEK293T cells. The binding of Zta with ³²P-labeled ZRE (lanes 3 and 7) or ZREmt (lane 4) probes was examined by EMSA. The supershift signal represents the complex of Zta and IL-13 promoter DNA and was detected by the addition of specific anti-Zta (lane 8) or anti-GST control (lane 9) antibodies. For the competition EMSA, a 30-fold excess of wild-type ZRE (lane 10) or ZREmt (lane 11) oligonucleotides was added to the reaction mixture. (D) A ChIP experiment was carried out as described previously. L428 cells were infected with pSIN-Zta or pSIN-vector lentivirus for 5 days. DNA-protein complexes were immunoprecipitated with anti-Zta antibody or mouse IgG (negative control). IL-13 promoter DNA (pIL-13, from nt −232 to +66) and control GAPDH promoter DNA (pGAPDH, from nt −93 to +64) were detected in the immunoprecipitates by PCR. Total DNA was harvested from L428 cells and used as the input control.