PML4 stimulates cooperation of GATA-1 and p300. (A) PML4 increased in vivo DNA binding ability of GATA-1. ChIP analysis was performed to detect GATA-1 occupancy at HS2 and β-major promoter in G1E-ER4 cells expressing PML4 and vector control before and after 18-hour β-estradiol induction for GATA-1 activity recovery (*P < .05, **P < .01). (B-C) 293T cells were transfected with p300 and GATA-1 expression vectors in the presence or absence of PML4 expression vector. Cell lysates were prepared and immunoprecipitated by anti-p300 (B) or anti–GATA-1 (C), followed by immunoblotting with indicated antibodies. (D) PML4 enhanced cooperation of GATA-1 and p300. 293T cells were cotransfected with indicated reporter constructs and pRL-TK in the presence or absence of expression vectors for GATA-1, PML4, and p300. Transfected cells were cultured for 24 hours and lysed for measurement of luciferase activities. Data are representative of 3 independent experiments. Error bars represent standard error of the mean. (E) Occupancy of p300 at GATA-1 binding sites in PML knockdown and control K562 cells. GATA-1 binding sites at distal regions or within intron sequences of GATA-1 target genes (as indicated) were detected. glyceraldehyde 3-phosphaate dehydrogenase–promoter region was used as negative control. Right panel shows the efficiency of PML interference. Primer sequences are available on request.