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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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AIDS Molecular and Cellular Biology Study Section (AMCB)
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Flicker, Paula F
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J. David Gladstone Institutes
San Francisco
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Rafati, Haleh; Parra, Maribel; Hakre, Shweta et al. (2011) Repressive LTR nucleosome positioning by the BAF complex is required for HIV latency. PLoS Biol 9:e1001206
Mahmoudi, Tokameh; Parra, Maribel; Vries, Robert G J et al. (2006) The SWI/SNF chromatin-remodeling complex is a cofactor for Tat transactivation of the HIV promoter. J Biol Chem 281:19960-8