FIG. 3.

The KIX peptide inhibits Tax-activated transcription on chromatin templates. (A) KIX inhibits Tax/CREB/p300-mediated transcription. The p4TxRE/G-less cassette template was assembled into chromatin by using recombinant Xenopus histones. Transcription reaction mixtures contained CEM nuclear extract and purified recombinant Tax/CREB or Tax/CREB/p300, as indicated. GST-KIX (aa 588 to 683) and GST-KIXmut (aa 597 to 719) were added to the transcription reaction mixtures at equimolar concentrations (+) or fivefold molar excess (++) relative to the Tax/CREB complex (see Materials and Methods). Recovery standard, size markers, and the position of the full-length transcript are indicated. (B) KIX inhibits Tax/CREB transcriptional activation in the absence of exogenous p300. Transcription reactions were performed as described in panel A, except in the absence of exogenous p300. (C) KIX does not affect Tax/CREB transcriptional activation on naked DNA templates. In vitro transcription reactions were performed on the unassembled p4TxRE/G-less template. Transcription reaction mixtures contained CEM nuclear extract nd purified Tax/CREB, Tax/CREB/p300, GST-KIX, or GST-KIXmut, as indicated. (D) KIX disrupts p300 binding to the Tax/CREB/viral CRE DNA complex. Binding reaction mixtures contained a DNA fragment carrying a single viral CRE linked to a streptavidin-agarose bead. CREB, Tax, and p300 were added to the immobilized viral CRE DNA as indicated. GST-KIX or GST-KIXmut was added at 0.5× molar (+) or equimolar (++) amounts relative to the Tax/CREB complex. Reactions were analyzed by Western blotting. For this experiment, Ser133-phosphorylated CREB was used. (E) Analysis of purified proteins used in the transcription and DNA binding reactions. Purified recombinant GST-KIX and GST-KIXmut were analyzed by SDS-PAGE and staining with Coomassie brilliant blue. The sizes of molecular mass markers (in kilodaltons) are indicated.