FIG. 1.

Synergistic IE62-USF activation of a model minimal promoter. (A) Schematic of the model luciferase reporter vector, pUSFTAluc, indicating the relative positions and sequences of the USF site and TATA element. Consensus binding motifs are shown in boldface. Mutations are underlined. (B) Results of luciferase assays. One microgram of each reporter plasmid including the basic pTALuc plasmid, which lacks the USF binding site, was cotransfected with or without 0.02 μg of the IE62-expressing plasmid, pCMV62, into MeWo cells. The luciferase activity expressed from the pTALuc reporter in the absence of IE62 was normalized to 1. The promoter activities resulting from the presence of USF, IE62, or both are reported as induction (n-fold) of the luciferase activity over the pTALuc level. The open and solid bars represent promoter activity in the absence and presence of IE62, respectively. (C) Results of EMSAs confirming that recombinant USFΔN binds to the consensus binding site inserted into pUSFTALuc and that the complex is supershifted by anti-USF1 antibody. Recombinant Sp1 and anti-Sp1 antibody were used as negative controls. (D) Control transfection assays showing the requirement of the TATA element for both USF and IE62 activation. These results were normalized to the activity observed with the pUSFTALuc reporter in the absence of IE62. Luciferase assay data in panels B and D represent the averages of triplicate transfections. The average values are shown above each bar, and the error bars represent standard deviations.