Role of Factor Acetylation in the Regulation of HIV Transcription

因子乙酰化在 HIV 转录调控中的作用

• 批准号: 9303872
• 负责人: Melanie Maria Ott
• 金额: $ 47.75万
• 依托单位: J. DAVID GLADSTONE INSTITUTES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-15 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9303872
• 关键词: Acetylation; Affect; Binding; Binding Proteins; Bromodomain; C-terminal; CD4 Positive T Lymphocytes; Cell Line; Cells; Characteristics; Chromatin Remodeling Factor; Clinical; Clinical Trials; Co-Immunoprecipitations; Complement Factor B; Complex; Data; Disease; Drug Targeting; Ensure; Enzymes; Event; Funding; GLTSCR1 protein; Genes; Genetic Transcription; Goals; HIV; HIV Infections; HIV tat Protein; HIV-1; Histone Deacetylase; Histone Deacetylase Inhibitor; Histones; Ii-Key; Individual; Infection; Inflammatory; Intervention; Jurkat Cells; Laboratories; Link; Lysine; Malignant Neoplasms; Manuscripts; Mass Screening; Mass Spectrum Analysis; Mediating; Modification; Molecular; Monitor; Mutate; PCAF gene; Pharmacology; Polymerase; Positive Transcriptional Elongation Factor B; Protein Acetylation; Proteins; Publishing; RNA Polymerase II; Recruitment Activity; Regulation; Research; Reverse Transcription; Role; Site; T memory cell; T-Lymphocyte; Technology; Testing; Therapeutic; Trans-Activators; Transcript; Transcription Elongation; Transcriptional Activation; Viral; Virus Latency; Work; base; brass; cofactor; cyclin T1; endonuclease; epigenetic drug; experimental study; histone acetyltransferase; inhibitor/antagonist; insight; interest; knock-down; member; new therapeutic target; novel; programs; promoter; public health relevance; purge; small hairpin RNA; small molecule inhibitor; targeted treatment; therapeutic target; transcription activator-like effector nucleases; transcription factor; treatment strategy

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.

描述：我们寻求识别和表征控制HIV-1转录的新的调控机制，这些机制可以被用作新的治疗靶点。可逆的蛋白质乙酰化已成为控制HIV转录的关键调控机制。组蛋白脱乙酰酶抑制剂(HDAC)被临床测试为HIV转录的激活剂 以克服潜伏感染T细胞储存库中存在的转录障碍。长期以来，我的实验室一直对因子乙酰化在HI转录调控中的作用感兴趣。过去的主要贡献包括证明HIV Tat蛋白及其关键辅因子Cyclin T1是乙酰化的，并且这些乙酰化事件调节与组蛋白乙酰基转移酶PCAF和BET蛋白Brd4中存在的溴域的相互作用。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，HIV转录中的关键酶RNA聚合酶II是乙酰化的。我们目前的建议建立在这些已发表和新的未发表结果的基础上，旨在定义新的乙酰化依赖机制作为逆转HIV潜伏期的治疗靶点。我们的具体目标有三个：目标1将确定BET抑制剂如何从潜伏期重新激活艾滋病毒的机制。我们的初步数据表明，这一机制涉及BET蛋白Brd2和Brd4，并与含有BAF250的SWI/SNF重塑复合体的抑制作用有关。这种新的BET-BAF相互作用将通过芯片、免疫共沉淀和shRNA介导的J-Lat细胞系和原代CD4+T细胞的基因敲除实验来进一步表征。目的2将描述Brd4在抑制和激活转录功能之间的潜在分子开关。在未发表的质谱学结果中，我们确定了Brd4 C末端的三个乙酰化位点，它们促进了与正转录延伸因子b(P-TEFb)的相互作用。为了监测原代T细胞中Brd4的乙酰化，我们将开发新的基于精确包涵体质量筛选(AIMS)的质谱学。我们还将研究新发现的乙酰化位点与相邻的P-TEFb相互作用结构域之间的功能相互作用及其在HIV转录中的作用。AIM3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在一起，聚合酶暂停是艾滋病毒转录的一个标志。我们将使用转录激活物样效应核酸酶(TALENS)的基因编辑来建立表达突变(8KR)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在的HIV感染。我们还将基于初步结果，显示Brd4与乙酰化的CTD特异性相互作用，并测试在没有TAT的情况下，这种相互作用有助于将P-TEFb招募到HIV启动子上暂停的聚合酶的假设。我们预计，这些研究将定义因子乙酰化和含有溴域的蛋白质如何调控HIV转录的新范式。