Article Figures & Data
Figures
- FIG. 1.
Recruitment of p300 and CBP to a viral CRE-containing promoter requires pCREB and Tax. (A) Analysis of recombinant, purified proteins by SDS-PAGE and silver staining. Molecular weight markers are indicated. (B) Immobilized 4TxRE promoter DNA (1 pmol) was incubated with full-length p300 (2 pmol) in the presence of CREB, CREB plus Tax, pCREB, and pCREB plus Tax (8 pmol each) as indicated. Bound complexes were washed, and DNA-bound proteins were separated on a 6%-to-12%-gradient SDS-polyacrylamide gel and analyzed by Western blotting. (C) Densitometric analysis of p300 binding from three independent experiments is presented graphically. (D) Analysis of recombinant, full-length CBP recruitment to the immobilized 4TxRE template in the presence of CREB, CREB plus Tax, pCREB, and pCREB plus Tax (8 pmol each), as indicated. The experiment was performed exactly as described for panel B, except purified, recombinant full-length CBP (2 pmol) was used in the binding reaction in place of p300.
- FIG. 2.
Individual viral and cellular CRE sequences support Tax/pCREB recruitment of p300. (A) The immobilized template assay was performed with the natural HTLV-1 promoter. Binding reactions and analyses were performed as described for Fig. 1B. (B) Partial top-strand sequence of the double-stranded oligonucleotides used in the immobilized template assays shown in panels C and D (see Materials and Methods for the complete sequences). The conserved Tax-binding sequences are indicated with a dashed underline. Nucleotides conforming to the consensus CRE are underlined. (C) The immobilized template assay was performed with the individual 45-bp doubled-stranded CRE sequences shown in panel B. Binding reactions and analyses were performed as described for Fig. 1B. The upper panel is a darker and higher-contrast version of the same p300 blot shown directly below and shows weak detection of p300 in lanes 1 and 2. (D) Immobilized template assay performed in parallel with the experiment shown in panel C, except that GST-KIX was included in the binding reactions in place of p300.
- FIG. 3.
Recruitment of endogenous p300 correlates with transcriptional activation from chromatin templates. (A) Immobilized, chromatin-assembled 4TxRE promoter DNA (1 pmol) was incubated in the absence (lane 1) or presence of pCREB (2.5 pmol) (lane 2) and increasing amounts of Tax (2.5 or 10 pmol) (lane 3 and 4), as indicated. Following the preincubation step, CEM nuclear extract was added and samples were incubated for 60 min at 4°C. Immobilized complexes were washed and analyzed by using a 4-to-20% SDS-polyacrylamide gradient gel and Western blotting (upper three panels). A portion of each binding reaction was resolved on an 18% SDS-polyacrylamide gel and stained with Coomassie to detect bound histone proteins (lower panel). Western blot of p300 present in the nuclear extract input (50%) and a Coomassie-stained gel of input histone (100%) are shown. (B) Small aliquots from each binding reaction mixture above (8% of the total) were incubated with acetyl-CoA and nucleoside triphosphates and analyzed in duplicate by in vitro transcription. The positions of molecular weight size markers, recovery standard, and full-length G-less transcripts are indicated. (C) Transcription reactions were performed with the unphosphorylatable mutant Ser133→A CREB (CREBS133A) and compared with results for pCREB. (All samples were analyzed on a single gel, but lanes 7 and 8 and lanes 9 and 10 were rearranged for this figure to maintain consistency with other experiments.) (D) Protein binding and transcription reactions were performed as described for panels A and B but in the presence of increasing amounts of purified GST-KIX (2.5, 5, and 25 pmol) (lanes 4 to 6), as indicated. GST alone (25 pmol) was used as a negative control (lane 7). Results from the transcription assay are shown in the top panel, and Western blots of bound p300, Tax, GST-KIX, and pCREB are shown in the lower three panels. Input Tax, pCREB, GST-KIX (2.5 pmol purified protein), and p300 (50% of nuclear extract) are shown in lane 8. (E) HTLV-1-negative Jurkat T cells were serum starved (0.5% FBS) and then cotransfected with the vCRE-luc reporter plasmid (100 ng) (17) and a Tax expression plasmid (pSG-Tax, 100 ng) (42). Eight hours prior to harvest, cells were treated with forskolin (FSK) (10 μM) or the dimethylsulfoxide vector and then analyzed by luciferase assay. Transient transfections were performed in triplicate, and each experiment was repeated three times. Relative luciferase activity is shown on the y axis. Corresponding Western blots probed against pCREB and Tax are shown for the samples analyzed in the luciferase assay.
- FIG. 4.
Model comparing the recruitment of full-length CBP/p300 and the KIX domain by CRE-bound Tax and pCREB. (A) Full-length CBP/p300 recruitment by the DNA-bound Tax/pCREB complex. The model depicts initial KIX contact by Tax, promoting a conformational change that enables pCREB binding and stable coactivator recruitment. (B) The model depicts distinct mechanisms of KIX recruitment by pCREB and the Tax/CREB complex. Tax recruits KIX in the presence of unphosphorylated CREB (left). The pCREB binding site on KIX is accessible, allowing recruitment in the absence of Tax (right).
