Crosstalk between RNA Pol II C-Terminal Domain Acetylation and Phosphorylation via RPRD Proteins.

Post-translational modifications of the RNA polymerase II C-terminal domain (CTD) coordinate the transcription cycle. Crosstalk between different modifications is poorly understood. Here, we show how acetylation of lysine residues at position 7 of characteristic heptad repeats (K7ac)-only found in higher eukaryotes-regulates phosphorylation of serines at position 5 (S5p), a conserved mark of polymerases initiating transcription. We identified the regulator of pre-mRNA-domain-containing (RPRD) proteins as reader proteins of K7ac. K7ac enhanced CTD peptide binding to the CTD-interacting domain (CID) of RPRD1A and RPRD1B proteins in isothermal calorimetry and molecular modeling experiments. Deacetylase inhibitors increased K7ac- and decreased S5-phosphorylated polymerases, consistent with acetylation-dependent S5 dephosphorylation by an RPRD-associated S5 phosphatase. Consistent with this model, RPRD1B knockdown increased S5p but enhanced K7ac, indicating that RPRD proteins recruit K7 deacetylases, including HDAC1. We also report autoregulatory crosstalk between K7ac and S5p via RPRD proteins and their interactions with acetyl- and phospho-eraser proteins.

Targeted degradation via direct 26S proteasome recruitment.

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.

The Short Isoform of BRD4 Promotes HIV-1 Latency by Engaging Repressive SWI/SNF Chromatin-Remodeling Complexes.

BET proteins commonly activate cellular gene expression, yet inhibiting their recruitment paradoxically reactivates latent HIV-1 transcription. Here we identify the short isoform of BET family member BRD4 (BRD4S) as a corepressor of HIV-1 transcription. We found that BRD4S was enriched in chromatin fractions of latently infected T cells, and it was more rapidly displaced from chromatin upon BET inhibition than the long isoform. BET inhibition induced marked nucleosome remodeling at the latent HIV-1 promoter, which was dependent on the activity of BRG1-associated factors (BAF), an SWI/SNF chromatin-remodeling complex with known repressive functions in HIV-1 transcription. BRD4S directly bound BRG1, a catalytic subunit of BAF, via its bromodomain and extraterminal (ET) domain, and this isoform was necessary for BRG1 recruitment to latent HIV-1 chromatin. Using chromatin immunoprecipitation sequencing (ChIP-seq) combined with assay for transposase-accessible chromatin coupled to high-throughput sequencing (ATAC-seq) data, we found that the latent HIV-1 promoter phenotypically resembles endogenous long terminal repeat (LTR) sequences, pointing to a select role of BRD4S-BRG1 complexes in genomic silencing of invasive retroelements.

Lysine Acetylation Goes Global: From Epigenetics to Metabolism and Therapeutics.

Post-translational acetylation of lysine residues has emerged as a key regulatory mechanism in all eukaryotic organisms. Originally discovered in 1963 as a unique modification of histones, acetylation marks are now found on thousands of nonhistone proteins located in virtually every cellular compartment. Here we summarize key findings in the field of protein acetylation over the past 20 years with a focus on recent discoveries in nuclear, cytoplasmic, and mitochondrial compartments. Collectively, these findings have elevated protein acetylation as a major post-translational modification, underscoring its physiological relevance in gene regulation, cell signaling, metabolism, and disease.

Therapeutics Targeting Protein Acetylation Perturb Latency of Human Viruses.

Persistent viral infections are widespread and represent significant public health burdens. Some viruses endure in a latent state by co-opting the host epigenetic machinery to manipulate viral gene expression. Small molecules targeting epigenetic pathways are now in the clinic for certain cancers and are considered as potential treatment strategies to reverse latency in HIV-infected individuals. In this review, we discuss how drugs interfering with one epigenetic pathway, protein acetylation, perturb latency of three families of pathogenic human viruses-retroviruses, herpesviruses, and papillomaviruses.

Retrovirus Integration: Some Assembly Required?

Integration is a key feature of the retroviral life cycle. This process involves packaging of the viral genome into chromatin, which is often assumed to occur as a post-integration step. In this issue of Cell Host & Microbe, Wang and colleagues (Wang et al., 2016) show that chromatinization occurs before integration, raising new questions about the role of histones in retroviral integration and transcription.

The mTOR Complex Controls HIV Latency.

A population of CD4 T lymphocytes harboring latent HIV genomes can persist in patients on antiretroviral therapy, posing a barrier to HIV eradication. To examine cellular complexes controlling HIV latency, we conducted a genome-wide screen with a pooled ultracomplex shRNA library and in vitro system modeling HIV latency and identified the mTOR complex as a modulator of HIV latency. Knockdown of mTOR complex subunits or pharmacological inhibition of mTOR activity suppresses reversal of latency in various HIV-1 latency models and HIV-infected patient cells. mTOR inhibitors suppress HIV transcription both through the viral transactivator Tat and via Tat-independent mechanisms. This inhibition occurs at least in part via blocking the phosphorylation of CDK9, a p-TEFb complex member that serves as a cofactor for Tat-mediated transcription. The control of HIV latency by mTOR signaling identifies a pathway that may have significant therapeutic opportunities.

Bromodomain proteins in HIV infection.

Bromodomains are conserved protein modules of ~110 amino acids that bind acetylated lysine residues in histone and non-histone proteins. Bromodomains are present in many chromatin-associated transcriptional regulators and have been linked to diverse aspects of the HIV life cycle, including transcription and integration. Here, we review the role of bromodomain-containing proteins in HIV infection. We begin with a focus on acetylated viral factors, followed by a discussion of structural and biological studies defining the involvement of bromodomain proteins in the HIV life cycle. We end with an overview of promising new studies of bromodomain inhibitory compounds for the treatment of HIV latency.