Role of Factor Acetylation in the Regulation of HIV Transcription

产品说明：我们试图确定和表征控制HIV-1转录的新的调控机制，这些机制可以作为新的治疗靶点。可逆的蛋白质乙酰化已经成为控制HIV转录的关键调节机制。组蛋白去乙酰化酶（HDAC）抑制剂作为HIV转录激活剂进行临床试验 以克服潜伏感染的T细胞库中存在的转录阻断。我的实验室对因子乙酰化在HI转录调控中的作用有着长期的兴趣。过去的主要贡献包括证明HIV达特蛋白及其关键辅因子细胞周期蛋白T1被乙酰化，并且这些乙酰化事件调节与组蛋白乙酰转移酶PCAF和BET蛋白Brd 4中存在的溴结构域的相互作用。我们最近证明了BET蛋白的小分子抑制剂从潜伏期激活HIV。我们还发现，RNA聚合酶II，在艾滋病毒转录的关键酶，是乙酰化。我们目前的建议建立在这些已发表的和新的未发表的结果，旨在定义新的乙酰化依赖性机制作为逆转HIV潜伏期的治疗靶点。我们的具体目标有三个方面：目标1将确定BET抑制剂如何从潜伏期重新激活HIV的机制。我们的初步数据表明，这种机制涉及BET蛋白Brd 2和Brd 4，并与含BAF 250的SWI/SNF重塑复合物的抑制作用有关。将使用ChIP、免疫共沉淀和shRNA介导的敲减实验在J-Lat细胞系和原代CD 4 + T细胞中进一步表征这种新的BET-BAF相互作用。目的2将表征Brd 4的抑制和激活转录功能之间的潜在分子开关。在未发表的质谱结果中，我们鉴定了Brd 4的C末端中的三个乙酰化位点，其促进与正转录延伸因子B（P-TEF B）的相互作用。为了监测原代T细胞中Brd 4的乙酰化，我们将开发新的基于精确包涵体质量筛选（AIMS）的质谱法。我们还将表征新发现的乙酰化位点和相邻的P-TEFb相互作用结构域（PID）之间的功能相互作用及其在HIV转录中的作用。Aim 3将定义RNA聚合酶II的乙酰化如何调节HIV转录。我们最近发表的数据将聚合酶乙酰化与聚合酶暂停联系起来，聚合酶暂停是HIV转录的标志。我们将使用转录激活因子样效应核酸酶（TALEN）进行基因编辑，以建立表达突变（8 KR）RNA聚合酶II的Jurkat T细胞，以研究这种修饰如何影响活跃和潜伏的HIV感染。我们还将建立在初步结果表明，Brd 4特异性相互作用的乙酰化CTD和测试的假设，这种相互作用用于招募P-TEFb暂停聚合酶在HIV启动子的情况下，达特。我们预期这些研究将定义因子乙酰化和含溴结构域蛋白如何调节HIV转录的新范例。

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 Our current proposal builds on these published and new unpublished results and aims at defining novel acetylation-dependent mechanisms as therapeutic targets in the reversal of HIV latency.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.