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hiv转录调控的机制。我们之前已经证明，整合的HIV启动子被组织在一系列精确定位的核小体和转录因子结合的两个核小体无区中。单个核小体（nuc1）位于转录起始位点之后，在HIV启动子的转录激活过程中被Tat破坏。我们最近发现人类SWI/SNF复合体对nuc1重塑至关重要。我们建议进一步研究tat介导的HIV启动子转激活所必需的不同辅助因子的有序募集。我们的具体目标是：1。研究huSWI/SNF染色质重塑复合物PBAF在nuc-1重塑和HIV转录激活中的作用及其机制。目标2。表征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复合物的特定亚基的敲低与HIV启动子的转录抑制有关。我们计划扩展这些观察结果，并研究Tat蛋白如何在HIV转录激活过程中介导BAF和PBAF复合物之间的转换。3. 研究体内HIV启动子转录调控蛋白的募集顺序。我们已经证明了染色质调控因子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法和RNA干扰来研究这些因子与pTEFb复合物一起募集到HIV启动子的顺序。我们将检验Tat乙酰化在这些因子有序募集到HIV启动子中起协调作用的假设。我们预计这些实验将进一步加深我们对HIV转录整合到染色质的理解。这些实验有可能确定介导HIV转录调控的新因子，这些因子可能在未来用于靶向治疗。

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.