Factor acetylation is a recognized epigenetic process governing HIV latency, but the silencing mechanisms associated with acetylated factors at the latent HIV promoter are not defined. The central hypothesis of this proposal is that acetyl-lysine reader proteins, such as the bromo- and ET domain-containing BRD4, and the Regulation of Nuclear Pre-MRNA Domain Containing 1 (RPRD1) proteins are critical regulators of latent HIV infection. This hypothesis is based on our recent data showing that the short isoform of the BRD4 protein (sBET), together with the BAF chromatin-remodeling complex, suppresses transcription of HIV and of endogenous retroviruses, supporting a model in which the sBET-BAF complex senses and silences genome-invading retroviruses (1). This model is supported by findings implicating another complex of bromodomain and chromatin-remodeling proteins, the ZYMND8-NuRD complex, in gene silencing during DNA damage (2-4). We further showed that RPRD1 proteins?without bromodomains?bind acetylated lysines (K7ac) within the RNA polymerase II, a mark highly enriched at the latent HIV LTR (5, 6). The central hypothesis will be tested in three specific aims: 1) To define the role of the sBET-BAF complex in genome surveillance. The working hypothesis is that sBET-BAF, similar to ZMYND8-NURD, senses double-strand DNA breaks caused by integrating retroviruses and silences gene expression by actively positioning a repressive nucleosome (nuc-1). We will test the hypothesis by using a dual-fluorescent clone of HIV to analyze sBET-BAF involvement in latency establishment, by using CRISPR/Cas9 to test dynamics of sBET-BAF recruitment to targeted DNA breaks, and by using paired-end sequencing to characterize the response of endogenous retroviruses to sBET-BAF inactivation. 2) To characterize composition and recruitment of sBET-containing complexes. The working hypothesis is that sBET interacts with multiple chromatin-remodeling complexes to silence incoming retroviruses and is recruited to the HIV LTR via zinc-finger proteins. Our specific focus is the NuRD nucleosome-remodeling and deacetylase complex and ZNF592 because of known interactions with sBET and ZMYND8. We will test the hypothesis in comprehensive mutagenesis, co-immunoprecipitation and chromatin immunoprecipitation experiments combined with knockdown of select factors. 3) To determine how RPRD1 proteins regulate HIV transcription. The working hypothesis is that RPRD1 proteins read K7ac marks enriched at the latent HIV promoter and prevent successful elongation of the paused RNA polymerase II. This hypothesis will be tested in detailed chromatin immunoprecipitations of CTD modifications at the HIV promoter and with conditional CRISPRi for RPRD1A/B proteins. As preliminary results implicate RPRD1 proteins in deacetylase recruitment, we will identify and functionally characterize this deacetylase (6). We expect the proposed work to reveal fundamental new biology of HIV latency that may inform future drug development.

Public Health Relevance

The proposed research is relevant to public health because it will determine if and how new acetyl-lysine reader proteins sBET and RPRD1 control HIV latency. These results will improve the understanding of the basic biology of HIV latency and will, ultimately, inform the development of new therapies to reverse HIV latency in infected individuals. This project supports the NIH's mission and aligns with one of its high-priority topics of research that awards AIDS-designated funds to support research toward a cure for HIV.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI083139-11
Application #
9977900
Study Section
HIV Molecular Virology, Cell Biology, and Drug Development Study Section (HVCD)
Program Officer
Mcdonald, David Joseph
Project Start
2009-07-15
Project End
2024-06-30
Budget Start
2020-07-01
Budget End
2021-06-30
Support Year
11
Fiscal Year
2020
Total Cost
Indirect Cost
Name
J. David Gladstone Institutes
Department
Type
DUNS #
099992430
City
San Francisco
State
CA
Country
United States
Zip Code
94158