FIG. 4.

The SREBP-1a peptide inhibits Tax-activated transcription on chromatin templates. (A) SREBP-1a inhibits Tax/CREB/p300-mediated transcription. Transcription reaction mixtures contained the chromatin-assembled p4TxRE-G-less template, CEM nuclear extract, and Tax/CREB or Tax/CREB/p300, as indicated. GST-SREBP-1a and GST alone were added to the transcription reactions at equimolar concentrations (+) or fivefold molar excess (++) relative to the Tax/CREB complex, as indicated. The recovery standard, size markers, and position of the full-length transcript are indicated. (B) SREBP-1a does not affect Tax/CREB transcriptional activation on naked DNA templates. Transcription reaction mixtures contained the unassembled p4TxRE-G-less template, CEM nuclear extract, Tax/CREB, SREBP-1a, or GST, as indicated. (C) SREBP inhibits p300 binding to the Tax/CREB/viral CRE DNA complex. Streptavidin-agarose DNA pull-down reactions were performed as described in Fig. 2D. GST-SREBP-1a or GST was added at equimolar concentrations (+) or fivefold molar excess (++) relative to the Tax/CREB complex, as indicated. Reactions were analyzed by Western blotting. (D) Analysis of purified proteins used in the transcription and DNA binding reactions. Purified recombinant GST-SREBP-1a and GST were analyzed by SDS-PAGE and staining with Coomassie brilliant blue. The sizes of molecular mass markers (in kilodaltons) are indicated. (E) GST-SREBP-1a interacts with p300. The GST pull-down assay contained purified recombinant p300 and GST or GST-SREBP-1a bound to glutathione-agarose, as indicated. p300 input (10%) is shown in lane 1. Reactions were analyzed by Western blotting.