Regulating crosstalk between anoikis and survival signaling pathways is crucial to regulating tissue processes and mitigating diseases like cancer. Previously, we showed that anoikis activates a CD95/Fas-mediated signaling pathway regulated by receptor-interacting protein (RIP), a kinase that shuttles between Fas-mediated cell death and integrin/FAK-mediated survival pathways. Since sirtuin-3 (SIRT3), an NAD-dependent deacetylase, is known to regulate cell survival, metabolism, and tumorigenesis, we hypothesized that SIRT3 might engage in crosstalk with Fas/RIP/integrin/FAK survival-death pathways in cancer cell systems.
Using immunohistochemical staining, immunoblotting, human tissue microarrays, and overexpression and suppression approaches in vitro and in vivo we examined the roles of RIP and SIRT3 in oral squamous cell carcinoma (OSCC) anoikis resistance and tumorigenesis.
RIP and SIRT3 have an opposite expression profile in OSCC cells and tissues. Stable suppression of RIP enhances SIRT3 levels, whereas, stable suppression of SIRT3 does not impact RIP levels in OSCC cells. As OSCC cells become anoikis-resistant they form multicellular aggregates or oraspheres in suspension conditions, and their expression of SIRT3 increases as their RIP expression decreases. Also, anoikis-resistant OSCC cells with higher SIRT3 and low RIP expression induce an increased tumor burden and incidence in mice unlike their adherent OSCC cell counterparts. Furthermore, stable suppression of SIRT3 inhibits anoikis resistance and reduces tumor incidence.
RIP is a likely upstream negative regulator of SIRT3 in anoikis resistance, and an anoikis-resistant orasphere phenotype defined by higher SIRT3 and low RIP expression contributes to a more aggressive phenotype in OSCC development.
Toll-like receptors (TLRs) are crucial for recognition of pathogen-associated molecular patterns by cells of the innate immune system. TLRs are present and functional in CD4+ T cells. Memory CD4+ T cells, predominantly central memory cells (TCM), constitute the main reservoir of latent HIV-1. However, how TLR ligands affect the quiescence of latent HIV within central memory CD4+ T cells has not been studied.
We evaluated the ability of a broad panel of TLR agonists to reactivate latent HIV-1. The TLR-1/2 agonist Pam3CSK4 leads to viral reactivation of quiescent HIV in a model of latency based on cultured TCM and in resting CD4+ T cells isolated from aviremic patients. In addition, we investigated the signaling pathway associated with Pam3CSK4 involved in HIV-1 reactivation. We show that the transcription factors NFκB, NFAT and AP-1 cooperate to induce viral reactivation downstream of TLR-1/2 stimulation. Furthermore, increasing levels of cyclin T1 is not required for TLR-mediated viral reactivation, but induction of viral expression requires activated pTEFb. Finally, Pam3CSK4 reactivates latent HIV-1 in the absence of T cell activation or proliferation, in contrast to antigen stimulation.
Our findings suggest that the signaling through TLR-1/2 pathway via Pam3CSK4 or other reagents should be explored as an anti-latency strategy either alone or in combination with other anti-latency drugs.
Age-related loss of muscle mass and function, sarcopenia, has a major impact on the quality of life in the elderly. Among the proposed causes of sarcopenia are mitochondrial dysfunction and accumulated oxidative damage during aging. Dietary restriction (DR), a robust dietary intervention that extends lifespan and modulates age-related pathology in a variety of species has been shown to protect from sarcopenia in rodents. Although the mechanism(s) by which DR modulates aging are still not defined, one potential mechanism is through modulation of oxidative stress and mitochondrial dysfunction. To directly test the protective effect of DR against oxidative stress induced muscle atrophy in vivo, we subjected mice lacking a key antioxidant enzyme, CuZnSOD (Sod1) to DR (40% of ad libitum fed diet). We have previously shown that the Sod1−/− mice exhibit an acceleration of sarcopenia associated with high oxidative stress, mitochondrial dysfunction, and severe neuromuscular innervation defects. Despite the dramatic atrophy phenotype in the Sod1−/− mice, DR led to a reversal or attenuation of reduced muscle function, loss of innervation and muscle atrophy in these mice. DR improves mitochondrial function as evidenced by enhanced Ca2+ regulation and reduction of mitochondrial reactive oxygen species (ROS). Furthermore, we show upregulation of SIRT3 and MnSOD in DR animals, consistent with reduced mitochondrial oxidative stress and reduced oxidative damage in muscle tissue measured as F2- isoprostanes. Collectively, our results demonstrate that DR is a powerful mediator of mitochondrial function, mitochondrial ROS production, and oxidative damage, providing a solid protection against oxidative stress induced neuromuscular defects and muscle atrophy in vivo even under conditions of high oxidative stress.
Concentrations of acetyl–coenzyme A and nicotinamide adenine dinucleotide (NAD+) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-β-hydroxybutyrate (βOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous βOHB, or fasting or calorie restriction, two conditions associated with increased βOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by βOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with βOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with βOHB conferred substantial protection against oxidative stress.
Improving the control of energy homeostasis can lower cardiovascular risk in metabolically compromised individuals. To identify new regulators of whole-body energy control, we conducted a high-throughput screen in transgenic reporter zebrafish for small molecules that modulate the expression of the fasting-inducible gluconeogenic gene pck1. We show that this in vivo strategy identified several drugs that impact gluconeogenesis in humans, as well as metabolically uncharacterized compounds. Most notably, we find that the Translocator Protein (TSPO) ligands PK 11195 and Ro5-4864 are glucose lowering agents despite a strong inductive effect on pck1 expression. We show that these drugs are activators of a fasting-like energy state, and importantly that they protect high-fat diet induced obese mice from hepatosteatosis and glucose intolerance, two pathological manifestations of metabolic dysregulation. Thus, using a whole-organism screening strategy, this study has identified new small molecule activators of fasting metabolism.
Current antiretroviral therapy for HIV-1 infection effectively suppresses but does not eradicate HIV-1. Patients on Highly Active Anti-Retroviral Therapy (HAART) maintain a persistent low-level viremia requiring lifelong adherence to antiretroviral therapies. This viremia may arise from latently infected reservoirs such as resting memory CD4+ T-cells or sanctuary sites where drug penetration is suboptimal. Understanding the mechanisms of HIV latency will help efforts to eradicate the infection. This review examines the dynamics of persistent viremia, viral reservoirs, the mechanisms behind viral latency, and methods to purge the viral reservoirs. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development.
Heterochromatin; repression; latency; transcription; transcriptional interference; chromatin; primary latency models; virus; epigenetics; histone
The Foxp3 transcription factor is the master regulator of regulatory T cell (Treg) differentiation and function. Its activity is regulated by reversible acetylation. Using mass spectrometry of immunoprecipitated proteins, we identify three novel acetylation sites in murine Foxp3 (K31, K262, and K267) and the corresponding sites in human FoxP3 proteins. Newly raised modification-specific antibodies against acetylated K31 and K267 confirm acetylation of these residues in murine Tregs. Mutant Foxp3 proteins carrying arginine substitutions at the three acetylation sites (3KR) accumulate in T cells to higher levels than wildtype Foxp3 and exert better suppressive activity in co-culture experiments. Acetylation and stability of wildtype, but not mutant, Foxp3 is enhanced when cells are treated with Ex-527, an inhibitor of the NAD+-dependent deacetylase SIRT1. Treatment with Ex-527 promotes Foxp3 expression during induced Treg differentiation, enhances Foxp3 levels in natural Tregs, and prevents loss of Foxp3 expression in adoptively transferred Tregs in mice. Our data identify SIRT1 as a negative regulator of Treg function via deacetylation of three novel target sites in Foxp3. SIRT1 inhibitors strengthen the suppressive activity of Tregs and may be useful in enhancing Treg-based therapeutic approaches to autoimmune diseases or graft rejections.
Mitochondrial protein hyperacetylation is a known consequence of sustained ethanol consumption and has been proposed to play a role in the pathogenesis of alcoholic liver disease (ALD). The mechanisms underlying this altered acetylome, however, remain unknown. The mitochondrial deacetylase sirtuin 3 (SIRT3) is reported to be the major regulator of mitochondrial protein deacetylation and remains a central focus for studies on protein acetylation. To investigate the mechanisms underlying ethanol-induced mitochondrial acetylation, we employed a model for ALD in both wild-type (WT) and SIRT3 knockout (KO) mice using a proteomics and bioinformatics approach. Here, WT and SIRT3 KO groups were compared in a mouse model of chronic ethanol consumption, revealing pathways relevant to ALD, including lipid and fatty acid metabolism, antioxidant response, amino acid biosynthesis and the electron-transport chain, each displaying proteins with altered acetylation. Interestingly, protein hyperacetylation resulting from ethanol consumption and SIRT3 ablation suggests ethanol-induced hyperacetylation targets numerous biological processes within the mitochondria, the majority of which are known to be acetylated through SIRT3-dependent mechanisms. These findings reveal overall increases in 91 mitochondrial targets for protein acetylation, identifying numerous critical metabolic and antioxidant pathways associated with ALD, suggesting an important role for mitochondrial protein acetylation in the pathogenesis of ALD.
Reactive oxygen species (ROS) are a family of compounds that can oxidatively damage cellular macromolecules and may influence lifespan. Sirtuins are a conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases that regulate lifespan in many model organisms including yeast and mice. Recent work suggests that sirtuins can modulate ROS levels notably during a dietary regimen known as calorie restriction which enhances lifespan for several organisms. Although both sirtuins and ROS have been implicated in the aging process, their precise roles remain unknown. In this review, we summarize current thinking about the oxidative stress theory of aging, discuss some of the compelling data linking the sirtuins to ROS and aging, and propose a conceptual model placing the sirtuins into an ROS-driven mitochondria-mediated hormetic response.
sirtuins; SIRT1; SIRT3; oxidative stress; mitohormesis; acetylation
Sirtuins (SIRT1-SIRT7), the mammalian homologs of the silent information regulator 2 (Sir2) in Saccharomyces cerevisiae, have been a major focus of study in the scientific community this past decade because of their emerging role in cancer biology and other age-related diseases. Emerging functions for this unique family of enzymes include roles in genomic stability, angiogenesis, metabolism, and anoikis. Here, we review recent developments on the role of sirtuins in cancer with a particular focus on SIRT3 and its role in the hallmarks of cancer and as a potential drug target for cancer treatment.
sirtuin-3; SIRT3; sirtuins; cancer; tumor promoter; tumor suppressor; anoikis
HIV-1 latency is a barrier to overcome in the effort to fully eradicate the virus from infected individuals using highly active anti-retroviral therapy (HAART). Therefore, the study of the mechanisms underlying the establishment and maintenance of HIV-1 latency are vital to achieving a cure. Transcriptional repression of the viral promoter is the major cause of HIV-1 latency. DNA methylation of genomic regions known as CpG islands (CpGIs) is a well-established transcriptional regulatory mechanism, and the HIV-1 provirus contains several conserved CpGIs including two that are located within the viral promoter region. The study of these CpGIs in both in vitro and in vivo models of HIV-1 latency using the technique of bisulfite-mediated methylcytosine mapping has led to their identification as factors that contribute to the maintenance of HIV-1 latency. Here, we discuss the identification of CpGIs within the HIV-1 provirus and the study of their differential methylation patterns in several HIV-1 latency models using bisulfite-mediated methylcytosine mapping.
CpG Island; DNA methylation; HIV-1 latency; Bisulfite-mediated methylcytosine mapping
Acetylation is increasingly recognized as an important metabolic regulatory post-translational protein modification, yet the metabolic consequence of mitochondrial protein hyperacetylation is unknown. We find that high-fat diet (HFD) feeding induces hepatic mitochondrial protein hyperacetylation in mice and downregulation of the major mitochondrial protein deacetylase SIRT3. Mice lacking SIRT3 (SIRT3KO) placed on a HFD show accelerated obesity, insulin resistance, hyperlipidemia, and steatohepatitis compared to wild-type (wt) mice. The lipogenic enzyme stearoyl-CoA desaturase 1 is highly induced in SIRT3KO mice, and its deletion rescues both wt and SIRT3KO mice from HFD-induced hepatic steatosis and insulin resistance. We further identify a single nucleotide polymorphism in the human SIRT3 gene that is suggestive of a genetic association with the metabolic syndrome. This polymorphism encodes a point-mutation in the SIRT3 protein, which reduces its overall enzymatic efficiency. Our findings show loss of SIRT3 and dysregulation of mitochondrial protein acetylation contribute to the metabolic syndrome.
Progressive mitochondrial dysfunction contributes to neuronal degeneration in age-mediated disease. An essential regulator of mitochondrial function is the deacetylase, sirtuin 3 (SIRT3). Here we investigate a role for CNS Sirt3 in mitochondrial responses to reactive oxygen species (ROS)- and Alzheimer’s disease (AD)-mediated stress. Pharmacological augmentation of mitochondrial ROS increases Sirt3 expression in primary hippocampal culture with SIRT3 over-expression being neuroprotective. Furthermore, Sirt3 expression mirrors spatiotemporal deposition of β-amyloid in an AD mouse model and is also upregulated in AD patient temporal neocortex. Thus, our data suggest a role for SIRT3 in mechanisms sensing and tackling ROS- and AD-mediated mitochondrial stress.
Although highly active antiretroviral therapy (HAART) has converted HIV into a chronic disease, a reservoir of HIV latently infected resting T cells prevents the eradication of the virus from patients. To achieve eradication, HAART must be combined with drugs that reactivate the dormant viruses. We examined this problem in an established model of HIV postintegration latency by screening a library of small molecules. Initially, we identified eight molecules that reactivated latent HIV. Using them as templates, additional hits were identified by means of similarity-based virtual screening. One of those hits, 8-methoxy-6-methylquinolin-4-ol (MMQO), proved to be useful to reactivate HIV-1 in different cellular models, especially in combination with other known reactivating agents, without causing T-cell activation and with lower toxicity than that of the initial hits. Interestingly, we have established that MMQO produces Jun N-terminal protein kinase (JNK) activation and enhances the T-cell receptor (TCR)/CD3 stimulation of HIV-1 reactivation from latency but inhibits CD3-induced interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α) gene transcription. Moreover, MMQO prevents TCR-induced cell cycle progression and proliferation in primary T cells. The present study documents that the combination of biological screening in a cellular model of viral latency with virtual screening is useful for the identification of novel agents able to reactivate HIV-1. Moreover, we set the bases for a hypothetical therapy to reactivate latent HIV by combining MMQO with physiological or pharmacological TCR/CD3 stimulation.
Taxanes are potent inhibitors of cell motility, a property implicated in their antiangiogenic and antimetastatic activity and unrelated to their antiproliferative effect. The molecular mechanism of this anti-motility activity is poorly understood. In this study, we found that paclitaxel induced tubulin acetylation in endothelial and tumor cells, at concentrations that affected cell motility but not proliferation (10-8 to 10-9 M, for 4 hours). Induction of tubulin acetylation correlated with inhibition of motility but not proliferation based on a comparison of highly and poorly cytotoxic taxanes (paclitaxel and IDN5390) and tumor cell lines sensitive and resistant to paclitaxel (1A9 and 1A9 PTX22). Consistent with the hypothesis that tubulin deacetylase activity might affect cell response to the anti-motility activity of taxanes, we found that overexpression of the tubulin deacetylase SIRT2 increased cell motility and reduced cell response to the anti-motility activity of paclitaxel. Conversely, the SIRT2 inhibitor splitomicin reduced cell motility and potentiated the anti-motility activity of paclitaxel. The inhibitory effect was further potentiated by the addition of the HDAC6 inhibitor trichostatin A. Paclitaxel and splitomicin promoted translocation into the nucleus—and hence activation—of FOXO3a, a negative regulator of cell motility. This study indicates a role for SIRT2 in the regulation of cell motility and suggests that therapies combining sirtuin inhibitors and taxanes could be used to treat cell motility-based pathologic processes such as tumor angiogenesis, invasion, and metastasis.
Sirtuins (SIRT1 7), the mammalian homologues of the Sir2 gene in yeast, have emerging roles in age-related diseases, such as cardiac hypertrophy, diabetes, obesity, and cancer. However, the role of several sirtuin family members, including SIRT1 and SIRT3, in cancer has been controversial. The aim of this review is to explore and discuss the seemingly dichotomous role of SIRT3 in cancer biology with particular emphasis on its potential role as a tumor promoter and tumor suppressor. This review will also discuss the potential role of SIRT3 as a novel therapeutic target to treat cancer.
Sirtuin-3; SIRT3; Cancer; Tumor promoter; Tumor Suppressor
Several sirtuin family members (SIRT1-7), evolutionarily conserved NAD-dependant deacetylases, play an important role in carcinogenesis. However, their role in oral cancer has not yet been investigated. Therefore, the aim of this study was to investigate whether sirtuins play a role in oral cancer carcinogenesis.
We examined the expression levels of all sirtuins in several oral squamous cell carcinoma (OSCC) cell lines compared to normal human oral keratinocytes, and found SIRT3 was highly expressed. Therefore, tissue microarrays were used to evaluate the clinical relevance of this overexpression. SIRT3 downregulation in OSCC cell proliferation and survival was then investigated and analyzed by cell proliferation and cell viability assays. Ionizing radiation and cisplatin were used to investigate whether SIRT3 downregulation can increase the sensitivity of OSCC to both treatments. To further assess the in vivo role of SIRT3 in OSCC carcinogenesis, we used a floor-of-mouth oral cancer murine model to study the effect of SIRT3 downregulation on OSCC tumor growth in immunodeficient mice.
We show for the first time that SIRT3 is overexpressed in OSCC in vitro and in vivo, compared to other sirtuins. Downregulation of SIRT3 inhibited OSCC cell growth and proliferation, and increased its sensitivity to radiation and cisplatin treatments in vitro. SIRT3 downregulation reduced tumor burden in vivo.
Our findings reveal a novel role for SIRT3 in oral cancer carcinogenesis as a promoter of cell proliferation and survival, thus implicating SIRT3 as a new potential therapeutic target to treat oral cancer.
SIRT3; Sirtuins; Carcinogenesis; Oral cancer; Squamous cell carcinoma
Despite advances in metabolic and postmetabolic labeling methods for quantitative proteomics, there remains a need for improved label-free approaches. This need is particularly pressing for workflows that incorporate affinity enrichment at the peptide level, where isobaric chemical labels such as isobaric tags for relative and absolute quantitation and tandem mass tags may prove problematic or where stable isotope labeling with amino acids in cell culture labeling cannot be readily applied. Skyline is a freely available, open source software tool for quantitative data processing and proteomic analysis. We expanded the capabilities of Skyline to process ion intensity chromatograms of peptide analytes from full scan mass spectral data (MS1) acquired during HPLC MS/MS proteomic experiments. Moreover, unlike existing programs, Skyline MS1 filtering can be used with mass spectrometers from four major vendors, which allows results to be compared directly across laboratories. The new quantitative and graphical tools now available in Skyline specifically support interrogation of multiple acquisitions for MS1 filtering, including visual inspection of peak picking and both automated and manual integration, key features often lacking in existing software. In addition, Skyline MS1 filtering displays retention time indicators from underlying MS/MS data contained within the spectral library to ensure proper peak selection. The modular structure of Skyline also provides well defined, customizable data reports and thus allows users to directly connect to existing statistical programs for post hoc data analysis. To demonstrate the utility of the MS1 filtering approach, we have carried out experiments on several MS platforms and have specifically examined the performance of this method to quantify two important post-translational modifications: acetylation and phosphorylation, in peptide-centric affinity workflows of increasing complexity using mouse and human models.
The mitochondrial sirtuin SIRT3 regulates metabolic homeostasis during fasting and calorie restriction. We identified mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) as an acetylated protein and a possible target of SIRT3 in a proteomics survey in hepatic mitochondria from Sirt3−/− (SIRT3KO) mice. HMGCS2 is the rate-limiting step in β-hydroxybutyrate synthesis and is hyperacetylated at lysines 310, 447, and 473 in the absence of SIRT3. HMGCS2 is deacetylated by SIRT3 in response to fasting in wild-type mice, but not in SIRT3KO mice. HMGCS2 is deacetylated in vitro when incubated with SIRT3 and in vivo by overexpression of SIRT3. Deacetylation of HMGCS2 lysines 310, 447, and 473 by incubation with wild-type SIRT3 or by mutation to arginine enhances its enzymatic activity. Molecular dynamics simulations show that in silico deacetylation of these three lysines causes conformational changes of HMGCS2 near the active site. Mice lacking SIRT3 show decreased β-hydroxybutyrate levels during fasting. Our findings show SIRT3 regulates ketone body production during fasting and provide molecular insight into how protein acetylation can regulate enzymatic activity.
Calorie restriction results in leanness, which is linked to metabolic conditions that favor longevity. We show here that deficiency of the triglyceride synthesis enzyme acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), which promotes leanness, also extends longevity without limiting food intake. Female DGAT1-deficient mice were protected from age-related increases in body fat, tissue triglycerides, and inflammation in white adipose tissue. This protection was accompanied by increased mean and maximal life spans of ~25% and ~10%, respectively. Middle-aged Dgat1−/− mice exhibited several features associated with longevity, including decreased levels of circulating insulin growth factor 1 (IGF1) and reduced fecundity. Thus, deletion of DGAT1 in mice provides a model of leanness and extended lifespan that is independent of calorie restriction.
DGAT1; adipose tissue; longevity; triglycerides; calorie restriction
Sirtuins are a highly conserved family of proteins whose activity can extend lifespan in model organisms such as yeast, worms, and flies. Mammals contain seven sirtuins (SIRT1-7) that modulate distinct metabolic and stress response pathways. Three sirtuins, SIRT3, SIRT4 and SIRT5, are located in the mitochondrion, a dynamic organelle that functions as the primary site of oxidative metabolism and plays critical roles in apoptosis and intracellular signaling. Recent findings have shed light on how the mitochondrial sirtuins function in the control of basic mitochondrial biology, including energy production, metabolism, apoptosis, and intracellular signaling.
The SWI/SNF BAF chromatin remodeling complex generates a repressive nucleosome structure at the HIV LTR conducive to establishment and maintenance of HIV latency, while PBAF augments HIV transcription.
Persistence of a reservoir of latently infected memory T cells provides a barrier to HIV eradication in treated patients. Several reports have implicated the involvement of SWI/SNF chromatin remodeling complexes in restricting early steps in HIV infection, in coupling the processes of integration and remodeling, and in promoter/LTR transcription activation and repression. However, the mechanism behind the seemingly contradictory involvement of SWI/SNF in the HIV life cycle remains unclear. Here we addressed the role of SWI/SNF in regulation of the latent HIV LTR before and after transcriptional activation. We determined the predicted nucleosome affinity of the LTR sequence and found a striking reverse correlation when compared to the strictly positioned in vivo LTR nucleosomal structure; sequences encompassing the DNase hypersensitive regions displayed the highest nucleosome affinity, while the strictly positioned nucleosomes displayed lower affinity for nucleosome formation. To examine the mechanism behind this reverse correlation, we used a combinatorial approach to determine DNA accessibility, histone occupancy, and the unique recruitment and requirement of BAF and PBAF, two functionally distinct subclasses of SWI/SNF at the LTR of HIV-infected cells before and after activation. We find that establishment and maintenance of HIV latency requires BAF, which removes a preferred nucleosome from DHS1 to position the repressive nucleosome-1 over energetically sub-optimal sequences. Depletion of BAF resulted in de-repression of HIV latency concomitant with a dramatic alteration in the LTR nucleosome profile as determined by high resolution MNase nucleosomal mapping. Upon activation, BAF was lost from the HIV promoter, while PBAF was selectively recruited by acetylated Tat to facilitate LTR transcription. Thus BAF and PBAF, recruited during different stages of the HIV life cycle, display opposing function on the HIV promoter. Our data point to the ATP-dependent BRG1 component of BAF as a putative therapeutic target to deplete the latent reservoir in patients.
Despite the effectiveness of antiretroviral medication, the HIV virus persists in resting memory T cells of infected patients in a latent state, providing the main impediment to eradication of the virus. In this article, we examined the molecular mechanism responsible for the establishment and maintenance of HIV latency and its re-activation, and uncovered the role played in this process by the SWI/SNF class of chromatin remodeling complexes, which use energy from ATP to alter the structure of chromatin. We show that two distinct sub-classes of SWI/SNF, BAF and PBAF, play functionally opposing roles in distinct steps of the HIV promoter (or long terminal repeat, LTR) transcription cycle. The PBAF complex augments transcription of the LTR by the viral transactivator Tat. In contrast, the distinct BAF complex generates a chromatin structure at the LTR that is energetically unfavorable with respect to the intrinsic histone-DNA sequence preferences. Specifically, we find that BAF positions a repressive nucleosome immediately downstream of the HIV transcription start site, abrogating transcription, and in this way contributes to the establishment and maintenance of HIV latency. Our data describe a novel molecular mechanism for the establishment and maintenance of HIV latency, and we identify the catalytic subunit of BAF, the enzyme BRG1, as a putative molecular target to deplete the latent reservoir in infected patients.
SIRT1 and SIRT3 are NAD+-dependent protein deacetylases that are evolutionarily conserved across mammals. These proteins are located in the cytoplasm/nucleus and mitochondria, respectively. Previous reports demonstrated that human SIRT1 deacetylates Acetyl-CoA Synthase 1 (AceCS1) in the cytoplasm, whereas SIRT3 deacetylates the homologous Acetyl-CoA Synthase 2 (AceCS2) in the mitochondria. We recently showed that 3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) is deacetylated by SIRT3 in mitochondria, and we demonstrate here that SIRT1 deacetylates the homologous 3-hydroxy-3-methylglutaryl CoA synthase 1 (HMGCS1) in the cytoplasm. This novel pattern of substrate homology between cytoplasmic SIRT1 and mitochondrial SIRT3 suggests that considering evolutionary relationships between the sirtuins and their substrates may help to identify and understand the functions and interactions of this gene family. In this perspective, we take a first step by characterizing the evolutionary history of the sirtuins and these substrate families.
sirtuins; evolution; deacetylases; aging
Posttranslational modifications of the HIV-1 Tat protein have emerged as critical regulatory mechanisms that fine-tune interactions of the viral transactivator with TAR RNA and cellular cofactors. Here, we identify the lysine methyltransferase Set7/9 (renamed KMT7) as a novel co-activator of HIV transcription. Set7/9-KMT7 associates with the HIV promoter in vivo and monomethylates lysine 51, a highly conserved residue located in the RNA-binding domain of Tat. Knockdown of Set7/9-KMT7 suppresses Tat transactivation of the HIV promoter, but does not affect the transcriptional activity of methylation-deficient Tat (K51A). Set7/9-KMT7 itself binds TAR RNA and forms a complex with Tat and the positive transcription elongation factor P-TEFb. Our findings uncover novel RNA-binding properties of Set7/9-KMT7 and demonstrate a positive role of Tat methylation in early steps of the Tat transactivation cycle.