Regulation of HIV latency for Chromatin

项目1.2的目标是研究染色质重塑复合物BAF在HIV转录抑制和潜伏期建立中的作用。我们已经观察到，染色质重塑复合物BAF是定位一个单一的抑制性核小体（nuc-1）下游的HIV转录起始位点所必需的。通过选择性敲低BAF亚基来抑制BAF诱导潜伏HIV在J-LAT系统中的表达。项目1.2的假设是BAF染色质重塑复合物在维持HIV潜伏期中起关键作用，并且干扰BAF功能代表了潜伏HIV再激活的新治疗靶点。我们在这一建议中的目标是：1。确定BAF染色质重塑复合物对HIV启动子的作用。BAF复合物与PBAF复合物的独特亚基（BAF 250）不同，在抑制基础HIV转录中起重要作用。我们将使用染色质免疫沉淀来验证BAF亚基在受抑制的HIV启动子中的存在，并测试BAF亚基特异性shRNA在HIV转录中的作用。我们将研究BAF募集到HIV启动子的分子机制。目标2.研究BAF复合物在HIV潜伏于初级淋巴细胞中的作用。我们将与合作实验室的其他成员（Planelles，Karn，格林）合作，以确定BAF复合物在初级淋巴细胞中HIV潜伏期中的作用。我们还将通过以下方法探讨BAF复合物在初级淋巴细胞中的生物学特性：（1）检测其亚单位的表达（2）敲低特定亚基并检查静息和活化T细胞中的总体和HIV基因表达模式，以及（3）分析BAF在T细胞中的作用位点（ChIP-Seq与Genomics Core，Core D合作）。目标3.验证BAF复合物作为I/E治疗的治疗靶点。我们将探索BAF作为I/E治疗的靶点。我们将使用针对不同BAF亚基的shRNA或slRNA，并测试它们在初级淋巴模型系统中重新激活潜伏HIV的能力，以用于HIV潜伏，并探索I/Fe试剂和选择性BAF亚基敲低之间可能的协同作用。我们将重点分析现有的代理商，如SAHA，prostratin和5-氮脱氧胞嘧啶，并在新的I/E代理开发的合作实验室。

The goal of Project 1.2 is to study the role of the chromatin remodeling complex BAF in the transcriptional repression of HIV and in the establishment of latency. We have observed that the chromatin remodeling complex BAF is necessary for the positioning of a single repressive nucleosome (nuc-1) downstream of the HIV transcriptional start site. Inhibition of BAF by selective knockdown of its subunits induces the expression of latent HIV in the J-LAT system. The hypothesis of Project 1.2 is that the BAF chromatin remodeling complex plays a critical role in the maintenance of HIV latency and that interfering with BAF function represents a new therapeutic target for reactivation of latent HIV. Our aims in this proposal are: 1. To determine the role of BAF chromatin remodeling complexes on the HIV promoter. The BAF complex, distinguished from the PBAF complex by a unique subunit (BAF250), plays an important role in the repression of basal HIV transcription. We will use chromatin immunoprecipitation to validate the presence of BAF subunits at the repressed HIV promoter and test the role of shRNAs specific for BAF subunits in HIV transcription. We will study the molecular mechanism of recruitment of BAF to the HIV promoter. Aim 2. To study the role of the BAF complex in HIV latency in primary lymphoid cells. We will collaborate with other members of the Collaboratory (Planelles, Karn, Greene) to define the role of the BAF complex in HIV latency in primary lymphoid cells. We will also explore the biology of the BAF complex in primary lymphoid cells by (1) examining expression of its subunits (mRNA and protein) in resting and activated T cells, (2) knocking down specific subunits and examining patterns of global and HIV gene expression in resting and acti</ated T cells, and (3) analyzing the sites of action of BAF in T cells (ChlP-Seq in collaboration with the Genomics Core, Core D). Aim 3. To validate the BAF complex as a therapeutic target for l/E therapy. We will explore BAF as a target for l/E therapy. We will use shRNA or slRNA against different BAF subunits and test their ability to reactivate latent HIV in primary lymphoid model systems for HIV latency and explore possible synergies between l/fe agents and selective BAF subunit knockdown. We will focus this analysis on existing agents, such as SAHA, prostratin and 5-azadeoxycyfidine, and on newer l/E agents developed in this Collaboratory.