Regulation of HIV latency for Chromatin

项目1.2的目标是研究染色质重塑复合体BAF在HIV转录抑制和潜伏期建立中的作用。我们已经观察到，染色质重塑复合体BAF对于定位HIV转录起始位点下游的单个抑制核小体（nuc1）是必要的。通过选择性敲除BAF亚基来抑制BAF可诱导J-LAT系统中潜伏HIV的表达。Project 1.2的假设是，BAF染色质重塑复合体在维持HIV潜伏期中起着关键作用，干扰BAF功能是重新激活潜伏HIV的新治疗靶点。我们在这个建议中的目标是：1。确定BAF染色质重塑复合物在HIV启动子中的作用。BAF复合物与PBAF复合物的区别在于其独特的亚基（BAF250），在抑制HIV基础转录中发挥重要作用。我们将使用染色质免疫沉淀来验证BAF亚基在被抑制的HIV启动子上的存在，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。目的：研究BAF复合物在原发性淋巴样细胞HIV潜伏期中的作用。我们将与合作实验室的其他成员（Planelles, Karn, Greene）合作，确定BAF复合物在原发性淋巴样细胞HIV潜伏期中的作用。我们还将通过(1)检测其亚基（mRNA和蛋白质）在静息和活化T细胞中的表达，(2)敲除特定亚基并检测静息和活化T细胞中全局和HIV基因表达模式，以及(3)分析BAF在T细胞中的作用位点（与Genomics Core， Core D合作的chp - seq）来探索原发性淋巴细胞中BAF复合物的生物学特性。目标3。验证BAF复合物作为l/E治疗的治疗靶点。我们将探索BAF作为l/E治疗的靶点。我们将使用shRNA或slRNA对抗不同的BAF亚基，并测试它们在HIV潜伏期的原发性淋巴模型系统中重新激活潜伏HIV的能力，并探索l/fe药物与选择性BAF亚基敲除之间可能的协同作用。我们将重点分析现有的药物，如SAHA、prostratin和5-azadeoxycyfidine，以及本实验室开发的新l/E药物。

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.