Role of Chromatin in HIV Transcriptional Regulation

描述(由申请人提供)：本提案的长期目标是了解染色质背景下艾滋病毒转录调控的机制。我们以前已经证明，整合的HIV启动子是由一系列精确定位的核小体和两个转录因子结合的无核小体区域组成的。单个核小体(nuc-1)位于转录起始点之后，在TAT转录激活HIV启动子的过程中被干扰。我们最近发现人类SWI/SNF复合体是NUC-1重塑的关键。我们建议进一步研究TAT介导的HIV启动子反式激活所必需的不同辅助因子的有序招募。我们的具体目标是：1.研究HuSWI/SNF染色质重塑复合体PBAF在nuc-1重塑和Tat激活HIV转录中的作用和作用机制。2.目的2.研究BAF染色质重塑复合体在HIV转录抑制中的作用。我们最近已经证明，BAF复合体特定亚基的敲除与HIV启动子的转录下调有关。我们计划扩大这些观察，并研究TAT蛋白如何在HIV转录激活过程中介导BAF和PBAF复合体之间的切换。3.研究体内转录调控蛋白在HIV启动子上的募集顺序。我们已经证明了染色质调节因子p300、huSWI/SNF和PCAF参与了染色质背景下HIV转录的转录调控。我们建议使用染色质免疫沉淀试验和RNA干扰来研究这些因子与pTEFb复合体一起对HIV启动子的招募顺序。我们将检验这样的假设，即TAT乙酰化在这些因子有序招募到HIV启动子的过程中起着协调作用。我们预计这些实验将进一步加深我们对染色质中整合的HIV转录的理解。这些实验有可能确定介导艾滋病毒转录调控的新因素，这些因素可能在未来成为治疗的靶点。

DESCRIPTION (provided by applicant): The long term goal of this proposal is to understand the mechanism of HIVtranscriptional regulation in the context of chromatin. We have previously shown that the integrated HIV promoter is organized in an array of precisely positioned nucleosomes and two nucleosome-free regions where transcription factors bind. A single nucleosome (nuc-1), is positioned after the transcription start site and is disrupted during transcriptional activation of the HIV promoter by Tat. We have recently identified the human SWI/SNF complex as critical for nuc-1 remodeling. We propose to further study the ordered recruitment of distinct cofactors that are necessary for Tat-mediated transactivation of the HIV promoter. Our specific aims are to: 1. To characterize the role and the mechanism of action of the huSWI/SNF chromatin remodeling complex called PBAF in nuc-1 remodeling and in HIV transcriptional activation by Tat. 2. Aim 2. To characterize the role of the BAF chromatin remodeling complex in HIV transcriptional repression. We have recently demonstrated that knockdown of specific subunits of the BAF complex are associated with transcriptional derepression of the HIV promoter. We plan to expand these observations and to study how the Tat protein mediates the switch between the BAF and PBAF complexes during HIV transcriptional activation. 3. To study the order of recruitment of transcriptional regulatory proteins to the HIV promoter in vivo. We have demonstrated that the chromatin regulators p300, huSWI/SNF, and PCAF participate to the transcriptional regulation of HIV transcription in the context of chromatin. We propose to use chromatin immunoprecipitation assays and RNA interference to study the order of recruitment of these factors along with the pTEFb complex, to the HIV promoter. We will test the hypothesis that Tat acetylation plays a coordinating role in the ordered recruitment of these factors to the HIV promoter. We anticipate that these experiments will further our understanding of HIV transcription integrated into chromatin. These experiments have the potential of identifying novel factors mediating HIV transcriptional regulation that could be targeted therapeutically in the future.