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 i n 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 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.
Understanding of HIV latency in molecular details represents the first and critical step in generating novel therapies to eliminate the pool of latently-infected cells from HIV-infected patients. We have identified a previously unrecognized cellular complex, called BAF, that plays a critical role in HIV transcriptional repression. These experiments will explore the biological role of the BAF complex in normal lymphocyte biology and in HIV latency and validate the BAF complex as a possible drug target in l/E therapy.
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