Chromatin-dependent mechanisms of CBP/p300 coactivator function in HTLV-I Tax transactivation

Efficient transcription of the human T-cell leukemia virus (HTLV-I) genome requires Tax, a virally encoded oncogenic transcription factor, complexed with the cellular transcription factor CREB and the coactivators p300/CBP. To examine the role of Tax transactivation in a chromatin context, we used chromatin assembled with recombinant core histones. Using this chromatin-based transcription system, we typically saw greater than 100-fold activation of transcription in the presence of Tax, CREB and p300. We found that transcription from chromatin templates was dependent on acetyl CoA. To test the hypothesis that this effect was due to the role of acetyl CoA in histone tail acetylation, we generated chromatin templates selectively lacking amino terminal histone tails. Compared with wild type chromatin, these tailless chromatin templates demonstrated enhanced transcriptional activation by Tax and CREB, with significantly reduced dependence on acetyl CoA. However, Tax/CREB activation from tailless chromatin templates retained a substantial requirement for acetyl CoA. Additionally, Tax transactivation from tailless templates was refractory to stimulation by endogenous p300, suggesting that tail deletion and acetylation are functionally equivalent. Importantly, p300 did not stimulate transcription from unassembled DNA templates, indicating that p300 functions by a chromatin-dependent mechanism in Tax-transactivation.; We also investigated the mechanism of CBP/p300 recruitment to the HTLV-I promoter. Tax has been characterized previously to interact with the KIX domain of CBP/p300, but this interaction has not been dissected in a functional context. Using polypeptides to inhibit CBP/p300 recruitment to the DNA-bound Tax/CREB complex, we found that the KIX domain is a primary mediator of CBP/p300 coactivator function on the HTLV-I promoter. Additionally, we found that Tax/CREB transcription strongly requires CBP/p300, since the polypeptides potently inhibited the function of CBP/p300 endogenous to the nuclear extract used in these studies.; We used Lys-CoA, a selective inhibitor of CBP/p300 acetyltransferase function, to further investigate the contribution of endogenous CBP/p300 in Tax-transactivation. Our data suggest that CBP/p300 may act in concert with other acetyltransferases to acetylate histone tails during Tax-transactivation. Furthermore, we discovered that CBP/p300 acetyltransferase activity is required on tailless chromatin templates, and is targeted at an unknown substrate whose acetylation is critical to Tax-transactivation.