PROJECT SUMMARY (See instructions): Fully exploiting the promise of factor acetylation as a relevant drug target in HIV infection has the potential to significantly advance progress towards effective functional cure strategies. The overall objective of this proposal is to define the unique role of factor acetylation in latency establishment, maintenance and reactivation. The central hypothesis is that acetyl-lysine readers proteins (BRD4 and RPRD1) are critical regulators of latent HIV infection. The short isoform of the BET protein BRD4 (sBET) and the BAF chromatin-remodeling complex silence transcription of HIV and endogenous retroviruses, consistent with the model that the complex senses and silences genome-invading retroviruses. RPRD1 proteins are a novel class of acetyl-lysine reader proteins that, without bromodomains, bind acetyl-lysine residues (K7ac) within the RNA polymerase II C-terminal domain, a mark that is found highly enriched at the latent HIV LTR. A model is proposed of a cascade of latency-establishing events during or shortly after proviral integration including the methyltransferase SMYD5, the KAT8 acetyltransferase (MOF complex) and the sBET/BAF complexes. To test this model, the following experiments are planned: Aim 1: To define the role of the sBET-BAF-KAT8- SMYD5 factors in genome surveillance. The working hypothesis is that a cascade of SMYD5, KAT8 and sBET/BAF activity senses double-stranded DNA breaks caused by integrating retroviruses and silences gene expression by positioning a repressive nucleosome at the transcription start site. Dual-fluorescent HIV, Cas9- induced DSBs and knockdown studies of sBET, KAT8 and SMYD5 will be used to test this hypothesis. Aim 2: To characterize recruitment of sBET-containing complexes to the integrated provirus. The working hypothesis is that a cascade of H3K36me3 and H4K16ac modifications recruits sBET-BAF to the integrating HIV promoter. Comprehensive mutagenesis, co-immunoprecipitation and ChIP experiments combined with factor knockdown are planned to test this hypothesis. Aim 3: To determine how RPRD1 proteins regulate HIV transcription via R-loops. The working hypothesis is that RPRD1 proteins read K7ac marks at the latent HIV promoter and prevent transcription elongation of the paused RNA polymerase through R-Loop formation (DNA-RNA hybrids). ChIP, DRIP (DNA-RNA IPs) and CRISPR-targeted nuclease recruitment to dissolve R-loops are planned to test this hypothesis. Important insights into the roles of H3K36me3, H4K16ac and R-loops in HIV latency establishment and maintenance are expected, enabling new cure therapies.