FIG. 3.

Brd4 CTD inhibits E2-dependent activation of transcription from the native BPV1 promoter. (A) Dual luciferase reporter assay with different cell lines. Cells were transfected with the following plasmids: pGL3P2, which expresses firefly luciferase under the control of early viral promoters in the BPV1 URR (C127, 1 μg; CHO, 250 ng; C33A, 1 μg); pRL-TK, which expresses Renilla luciferase under the control of the thymidine kinase promoter (C127, 100 ng; CHO, 25 ng; C33A, 500 ng); BPV1 wt E2 plasmid pCGE2 (+E2 columns) (C127, 500 ng; CHO, 25 ng; C33A, 500 ng); and either CTD expression plasmid pCGCTD (+CTD) or control vector pCGdXS (−CTD) (C127, 3 μg; CHO, 1 μg; C33A, 3 μg). The cells were lysed 2 days after transfection of cells by electroporation and processed for luciferase analysis. The results of firefly luciferase expression were normalized to Renilla luciferase data for every sample and are shown relative to the values from transfections with E2 without CTD in all series (+E2 −CTD). The data from three different series are summarized in the case of C127 and CHO and two series in the case of C33A cells; error bars show the average deviation. “basal” corresponds to control transfections with luciferase reporters only. The control transfection with CTD expression only (+CTD) was not included in the C33A series. (B) Analysis of luciferase expression with different CTD concentrations in C127 and CHO cells and (C) parallel Western blotting analysis of the samples. One microgram of pGL3P2 and 100 ng of pRL-TK were transfected into C127 cells; 250 ng of pGL3P2 and 25 ng of pRL-TK were transfected into CHO cells. In addition, either E2 expression vector pCGE2 (shaded columns) or VP16E2 expression vector pCGVP16E2 (open columns) (14) was cotransfected into cells (C127, 200 ng; CHO, 50 ng), together with 0 to 3,000 ng (C127) or 0 to 1,000 ng (CHO) of CTD expression vector pCGCTD as indicated. pCGdXS control vector was added to all transfections marked as +CTD to keep the total amount of pCGdXS plus pCGCTD constant in the series (3,000 ng for C127 and 1,000 ng for CHO). Anti-E2Tag (which recognizes E2 and epitope-tagged CTD) and 1E4 (which recognizes E2 and VP16E2) (10) antibodies were used for Western blotting (C) in the upper blots. For the wt E2 series in C127 cells (lanes 1 to 6), the blot with anti-E2Tag antibody alone is shown at the bottom, to give an idea of the relative molar ratio of E2 to CTD in the series. Equal amounts of the total protein were loaded on the gel in each series.