Role of Factor Acetylation in the Regulation of HIV Transcription

摘要 因子乙酰化是公认的控制HIV潜伏期的表观遗传过程，但沉默机制 与潜伏HIV启动子处的乙酰化因子相关的蛋白质没有定义。这个问题的核心假设是 建议是乙酰基-赖氨酸阅读器蛋白，如含溴和ET结构域的BRD 4，和 含核前mRNA结构域1（RPRD 1）蛋白是潜伏HIV的关键调节因子 感染这一假设是基于我们最近的数据，显示BRD 4蛋白的短同种型（sBET）， 与BAF染色质重塑复合物一起，抑制HIV和内源性 逆转录病毒，支持sBET-BAF复合物感知和沉默基因组入侵的模型。 逆转录病毒（1）。这一模型得到了涉及溴结构域的另一种复合物的研究结果的支持， 染色质重塑蛋白，ZYMND 8-NuRD复合物，在DNA损伤过程中的基因沉默中（2-4）。我们 进一步表明，RPRD 1蛋白-无溴结构域-结合RNA内的乙酰化赖氨酸（K7 ac 聚合酶II，一种在潜伏HIV LTR高度富集的标记（5，6）。中心假设将在三个测试 具体目的：1）明确sBET-BAF复合物在基因组监测中的作用。工作假设 sBET-BAF与ZMYND 8-BHD类似，能感知由整合引起的双链DNA断裂， 逆转录病毒并通过主动定位抑制性核小体（nuc-1）使基因表达沉默。我们将测试 通过使用HIV的双荧光克隆来分析sBET-BAF参与潜伏期的假设 通过使用CRISPR/Cas9来测试sBET-BAF募集到靶向DNA断裂的动力学， 通过使用配对末端测序来表征内源性逆转录病毒对sBET-BAF的反应 失活2)表征含sBET复合物的组成和募集。工作 假设sBET与多种染色质重塑复合物相互作用以沉默进入的逆转录病毒 并通过锌指蛋白募集到HIV LTR。我们的具体重点是NuRD核小体重塑 和脱乙酰酶复合物和ZNF 592，因为已知与sBET和ZMYND 8的相互作用。我们将测试 综合诱变、免疫共沉淀和染色质免疫沉淀假说 实验结合选择因子的敲除。3)为了确定RPRD 1蛋白如何调节HIV 转录。工作假设是RPRD 1蛋白读取在潜伏的HIV中富集的K7 ac标记。 启动子，并防止成功延长暂停的RNA聚合酶II。这一假设将在 HIV启动子处CTD修饰和条件性CRISPRi的详细染色质免疫沉淀 RPRD 1A/B蛋白。由于初步结果表明RPRD 1蛋白参与了脱乙酰酶的募集，我们将 鉴定和功能性表征该脱乙酰酶（6）。我们希望这项工作能够揭示基本的 HIV潜伏期的新生物学，可能会为未来的药物开发提供信息。

ABSTRACT Factor acetylation is a recognized epigenetic process governing HIV latency, but the silencing mechanisms associated with acetylated factors at the latent HIV promoter are not defined. The central hypothesis of this proposal is that acetyl-lysine reader proteins, such as the bromo- and ET domain-containing BRD4, and the Regulation of Nuclear Pre-MRNA Domain Containing 1 (RPRD1) proteins are critical regulators of latent HIV infection. This hypothesis is based on our recent data showing that the short isoform of the BRD4 protein (sBET), together with the BAF chromatin-remodeling complex, suppresses transcription of HIV and of endogenous retroviruses, supporting a model in which the sBET-BAF complex senses and silences genome-invading retroviruses (1). This model is supported by findings implicating another complex of bromodomain and chromatin-remodeling proteins, the ZYMND8-NuRD complex, in gene silencing during DNA damage (2-4). We further showed that RPRD1 proteins—without bromodomains—bind acetylated lysines (K7ac) within the RNA polymerase II, a mark highly enriched at the latent HIV LTR (5, 6). The central hypothesis will be tested in three specific aims: 1) To define the role of the sBET-BAF complex in genome surveillance. The working hypothesis is that sBET-BAF, similar to ZMYND8-NURD, senses double-strand DNA breaks caused by integrating retroviruses and silences gene expression by actively positioning a repressive nucleosome (nuc-1). We will test the hypothesis by using a dual-fluorescent clone of HIV to analyze sBET-BAF involvement in latency establishment, by using CRISPR/Cas9 to test dynamics of sBET-BAF recruitment to targeted DNA breaks, and by using paired-end sequencing to characterize the response of endogenous retroviruses to sBET-BAF inactivation. 2) To characterize composition and recruitment of sBET-containing complexes. The working hypothesis is that sBET interacts with multiple chromatin-remodeling complexes to silence incoming retroviruses and is recruited to the HIV LTR via zinc-finger proteins. Our specific focus is the NuRD nucleosome-remodeling and deacetylase complex and ZNF592 because of known interactions with sBET and ZMYND8. We will test the hypothesis in comprehensive mutagenesis, co-immunoprecipitation and chromatin immunoprecipitation experiments combined with knockdown of select factors. 3) To determine how RPRD1 proteins regulate HIV transcription. The working hypothesis is that RPRD1 proteins read K7ac marks enriched at the latent HIV promoter and prevent successful elongation of the paused RNA polymerase II. This hypothesis will be tested in detailed chromatin immunoprecipitations of CTD modifications at the HIV promoter and with conditional CRISPRi for RPRD1A/B proteins. As preliminary results implicate RPRD1 proteins in deacetylase recruitment, we will identify and functionally characterize this deacetylase (6). We expect the proposed work to reveal fundamental new biology of HIV latency that may inform future drug development.