AML1 is involved in the transcriptional regulation of IEX-1. (A) Activation of the IEX-1 promoter in UT7-Mpl cells. UT7-Mpl were transiently transfected with the pLucIEXp plasmid. The cells were treated with TPO (10 nM, ▪) or with EPO (2 U/mL, □) 5 hours after transfection and were assessed for luciferase activities at the indicated times. Transfection efficiencies were normalized to the Renilla luciferase activity from the cotransfected internal control plasmid (phRL-Null). The results are from one representative experiment. (B) Schematic diagram of the wild-type IEX-1 promoter-luciferase reporter. (C) Site-directed mutagenesis of the binding sites for AML1 (–1068), GATA-1 (–1322), or ETS (–1318) in the human IEX-1 promoter affects its transcriptional activation. The site-directed mutants and wild-type promoter luciferase activities were compared after transient transfection and 20 hours of TPO stimulation of UT7-Mpl cells. (D) Effect of AML1 dominant-negative (DN) mutants on IEX-1 promoter induction. The pLucIEXp was cotransfected with wild-type (AML1wt) or DN (AML1a and AML1/ETO) forms of AML1. Cells were harvested and luciferase assays were performed after 20 hours of TPO treatment. Data represent mean ± SD of at least 3 independent experiments. *P < .01.