We have demonstrated a specific interaction between the acetylated Tat protein of HIV and the bromodomain of the coactivator PCAF. The structure of acetylated Tat bound to the PCAF bromodomain has been solved and mutations in both Tat and PCAF that abrogated this interaction inhibited the transactivation of the HIV promoter by Tat. These experiments define the Tat/PCAF interaction as a critical step in the transactivation of the HIV promoter and a possible target for drug intervention. In this proposal, we will use a variety of experimental approaches to further establish the role of PCAF in Tat-mediated transactivation of the HIV promoter. We will also examine the role of PCAF-B and the chromatin remodeling factors BRGl and hSNF2, three transcription factors containing a bromodomain closely related to the bromodomain of PCAF. To further define the role of PCAF in HIV transcription, we will conduct experiments to distinguish between the following three models: 1. PCAF is being recruited by acetylated Tat to the transcription start site by the TAR element; 2. PCAF and its associated cofactors are recruited to the HIV promoter and deposited at the transcriptional start site in a histone-like manner leading to chromatin remodeling of the HIV promoter; 3. Acetylated Tat serves as a bridge to recruit PCAF and its associated factors onto the elongating RNA polymerase 11. We will design and implement quantitative assays aimed at measuring the Tat/PCAF interaction and its possible inhibition by compounds derived from the structural analysis of the Tat/PCAF complex. This process will be reiterated and should lead to a further understanding of the molecular forces underpinning the Tat/PCAF interaction and to the rational design of better inhibitors. To examine the full range of biological effects occurring in response to small molecule inhibitors of the PCAF bromodomain, we will conduct a genome-wide analysis of gene expression using microarrays in response to these inhibitors. We anticipate that the combination of strong structural and biological programs will lead to the identification of specific and active inhibitors of HIV transcription and to a better understanding of HIV transcriptional regulatory mechanisms.
