Role of Factor Acetylation in the Regulation of HIV Transcription

摘要 因子乙酰化是一个公认的控制HIV潜伏期的表观遗传过程，但沉默机制 潜伏的HIV启动子上的乙酰化因子没有定义。这一点的中心假设是 建议乙酰赖氨酸阅读器蛋白，如含有溴和ET结构域的BRD4，以及 含核前信使核糖核酸结构域1(RPRD1)蛋白的调控是潜伏HIV的关键调节因子 感染。这一假说是基于我们最近的数据显示BRD4蛋白的短亚型(SBET)， 与BAF染色质重塑复合体一起，抑制HIV和内源性HIV的转录 逆转录病毒，支持Sbet-BAF复合体感知和沉默基因组入侵的模型 逆转录病毒(1)。这一模型得到了研究结果的支持，这些发现暗示了另一种溴域和 染色质重塑蛋白，ZYMND8-NuRD复合体，在DNA损伤过程中的基因沉默(2-4)。我们 进一步表明，RPRD1蛋白--没有溴结构域--与RNA中的乙酰化赖氨酸(K7ac)结合 聚合酶II，一个在潜伏的HIV LTR(5，6)高度浓缩的标记。核心假说将在三年内得到检验 具体目标：1)确定Sbet-BAF复合体在基因组监测中的作用。工作假说 Sbet-BAF，类似于ZMYND8-NuRD，感知整合引起的双链DNA断裂 逆转录病毒通过主动定位抑制性核小体(nuc-1)来沉默基因表达。我们将测试 用HIV双荧光克隆分析Sbet-BAF参与潜伏期的假说 建立，通过使用CRISPR/Cas9来测试Sbet-BAF招募到靶向DNA断裂的动力学，以及 用双末端测序法研究内源性逆转录病毒对Sbet-BAF的应答 失活。2)表征含Sbet的络合物的组成和募集。在工作中 假设Sbet与多个染色质重塑复合体相互作用，以沉默传入的逆转录病毒 并通过锌指蛋白被招募到HIV LTR中。我们特别关注的是核小体改建 以及脱乙酰酶复合体和ZNF592，因为已知与Sbet和ZMYND8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的假说 实验与击倒选定因素相结合。3)确定RPRD1蛋白如何调节HIV 抄写。工作假说是，RPRD1蛋白读取在潜伏的HIV上丰富的K7ac标记 促进并阻止暂停的RNA聚合酶II的成功延长。这一假说将在 HIV启动子和条件性CRISPRI中CTD修饰的染色质免疫沉淀 RPRD1A/B蛋白。由于初步结果表明RPRD1蛋白与脱乙酰酶的招募有关，我们将 鉴定并鉴定该脱乙酰基酶(6)的功能。我们预计拟议的工作将揭示根本 艾滋病毒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.