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Bromodomain-containing Proteins in HIV Transcription & Latency

  • Author(s): Conrad, Ryan J.
  • Advisor(s): Ott, Melanie
  • et al.
Abstract

HIV-1 latency is a phenomenon of transcriptional silencing wherein components of the host epigenetic machinery impose reversible restrictions to viral chromatin. Bromodomain-containing factors are emergent epigenetic effector proteins that recognize acetyl-lysine residues, and have been implicated in the regulation of HIV-1 transcription & latency. This dissertation presents a review of HIV-1 transcription and viral epigenetics, with a particular focus on the roles of protein acetylation and bromodomain-containing proteins in these processes. Original scientific work describing a novel corepressive function of the bromodomain and extraterminal domain (BET) subfamily member bromodomain-containing protein 4 (BRD4) is reported. Specifically, we establish that the understudied short isoform of BRD4 interacts with SWI/SNF chromatin remodeling machines to maintain transcriptional repression of latent HIV-1 via nucleosome positioning. Through genetic and pharmacological loss of function, chromatin biology, and immunoprecipitation experiments, we detail the physical and functional interaction between BRD4 and BRG1, a catalytic SWI/SNF subunit. Although primarily a gene-centered study surrounding the latent HIV-1 promoter, this dissertation also includes high throughput sequencing data establishing genomic co-binding of BRD4 and BRG1 at both coding and non-coding regions that is disrupted under pharmacological inhibition of BET bromodomains. These data i.) uncover SWI/SNF remodelers as critical downstream targets of BET inhibitors in the reversal of HIV-1 latency, which is a promising therapeutic strategy to achieve eradication in infected individuals, ii.) support the hypothesis that BRD4 executes isoform-specific functions, iii.) underscore the essential role of chromatin in HIV-1 latency maintenance and iv.) provide a global view of genomic regulation by BRD4 and SWI/SNF. Lastly, this dissertation includes preliminary data demonstrating that the long isoform of BRD4 is itself subject to reversible acetylation. We have mapped a cluster of acetylation-competent lysine residues within the long BRD4 C-terminus, generated modification-specific antibodies, and begun to characterize the effects of this BRD4 modification on its transactivation capacity via the P-TEFb kinase. Collectively, the work presented herein advances our knowledge of both isoforms of the bromodomain-containing protein BRD4, and further develops our understanding of the role of chromatin in HIV-1 transcription & latency.

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