Role of Factor Acetylation in the Regulation of HIV Transcription

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DESCRIPTION: We seek to identify and characterize novel regulatory mechanisms controlling HIV-1 transcription that can be exploited as new therapeutic targets. Reversible protein acetylation has emerged as a key regulatory mechanism that controls HIV transcription. Inhibitors of histone deacetylases (HDACs) are clinically tested as activators of HIV transcription in order to overcome the transcriptional block present in the reservoir of latently infected T cell. My laboratory has a longstanding interest in the role of factor acetylation in the regulation of HI transcription. Key contributions in the past include the demonstrations that the HIV Tat protein and its pivotal cofactor cyclin T1 are acetylated, and that these acetylation events regulate the interaction with bromodomains present in the histone acetyltransferase PCAF and the BET protein Brd4. We recently demonstrated that small-molecule inhibitors of BET proteins activate HIV from latency. We also showed that RNA polymerase II, the key enzyme in HIV transcription, is acetylated. Our current proposal builds on these published and new unpublished results and aims at defining novel acetylation-dependent mechanisms as therapeutic targets in the reversal of HIV latency. Our specific aims are three-fold: Aim 1 will identify the mechanisms how BET inhibitors reactivate HIV from latency. Our preliminary data show that this mechanism involves the BET proteins Brd2 and Brd4 and is linked to the suppressive action of the BAF250-containing SWI/SNF remodeling complex. This new BET-BAF interaction will be further characterized using ChIP, coimmunoprecipitation and shRNA-mediated knockdown experiments in J-Lat cell lines and primary CD4+ T cells. Aim 2 will characterize a potential molecular switch between repressive and activatory transcriptional functions of Brd4. In unpublished mass spectrometry results, we identified three acetylation sites in the C- terminus of Brd4 that promote interaction with the positive transcription elongation factor b (P-TEFb). To monitor Brd4 acetylation in primary T cells, we will develop new accurate inclusion mass screening (AIMS)-based mass spectrometry. We will also characterize the functional interaction between the newly identified acetylation sites and the adjacent P-TEFb-interacting domain (PID) and their role in HIV transcription. Aim3 will define how acetylation of RNA polymerase II regulates HIV transcription. Our recently published data links polymerase acetylation to polymerase pausing, a hallmark of HIV transcription. We will use gene editing with transcription activator-like effector nucleases (TALENs) to establish Jurkat T cells expressing mutated (8KR) RNA polymerase II to study how the modification affects active and latent HIV infection. We will also build on preliminary results showing that Brd4 specifically interacts with the acetylated CTD and test the hypothesis that this interaction serves to recruit P-TEFb to the paused polymerase at the HIV promoter in the absence of Tat. We anticipate that these studies will define new paradigms of how factor acetylation and bromodomain- containing proteins regulate HIV transcription.