Vincent Quivy

Synergistic Activation of Human Immunodeficiency Virus Type 1 Promoter Activity by NF-κB and Inhibitors of Deacetylases: Potential Perspectives for the Development of Therapeutic Strategies

ABSTRACT The transcription factor NF-κB plays a central role in the human immunodeficiency virus type 1 (HIV-1) activation pathway. HIV-1 transcription is also regulated by protein acetylation, since treatment with deacetylase inhibitors such as trichostatin A (TSA) or sodium butyrate (NaBut) markedly induces HIV-1 transcriptional activity of the long terminal repeat (LTR) promoter. Here, we demonstrate that TSA (NaBut) synergized with both ectopically expressed p50/p65 and tumor necrosis factor alpha/SF2 (TNF)-induced NF-κB to activate the LTR. This was confirmed for LTRs from subtypes A through G of the HIV-1 major group, with a positive correlation between the number of κB sites present in the LTRs and the amplitude of the TNF-TSA synergism. Mechanistically, TSA (NaBut) delayed the cytoplasmic recovery of the inhibitory protein IκBα. This coincided with a prolonged intranuclear presence and DNA binding activity of NF-κB. The physiological relevance of the TNF-TSA (NaBut) synergism was shown on HIV-1 replication in both acutely and latently HIV-infected cell lines. Therefore, our results open new therapeutic strategies aimed at decreasing or eliminating the pool of latently HIV-infected reservoirs by forcing viral expression.

Synthetic Vpr protein activates activator protein-1, c-Jun N-terminal kinase, and NF-kappaB and stimulates HIV-1 transcription in promonocytic cells and primary macrophages.

The human immunodeficiency virus (HIV) Vpr protein plays a critical role in AIDS pathogenesis, especially by allowing viral replication within nondividing cells such as mononuclear phagocytes. Most of the data obtained so far have been in experiments with endogenous Vpr protein; therefore the effects of extracellular Vpr protein remain largely unknown. We used synthetic Vpr protein to activate nuclear transcription factors activator protein-1 (AP-1) and NF-κB in the promonocytic cell line U937 and in primary macrophages. Synthetic HIV-1 Vpr protein activated AP-1, c-Jun N-terminal kinase, and MKK7 in both U937 cells and primary macrophages. Synthetic Vpr activated NF-κB in primary macrophages and to a lesser extent in U937 cells. Because synthetic Vpr activated AP-1 and NF-κB, which bind to the HIV-1 long terminal repeat, we investigated the effect of synthetic Vpr on HIV-1 replication. We observed that synthetic Vpr stimulated HIV-1 long terminal repeat in U937 cells and enhanced viral replication in chronically infected U1 promonocytic cells. Similarly, synthetic Vpr stimulated HIV-1 replication in acutely infected primary macrophages. Activation of transcription factors and enhancement of viral replication in U937 cells and primary macrophages were mediated by both the N-terminal and the C-terminal moieties of synthetic Vpr. Therefore, our results suggest that extracellular Vpr could fuel the progression of AIDS via stimulation of HIV-1 provirus present in such cellular reservoirs as mononuclear phagocytes in HIV-infected patients.

Diversity of acetylation targets and roles in transcriptional regulation: the human immunodeficiency virus type 1 promoter as a model system.

Persuasive evidence has accumulated that reversible acetylation of proteins is key post-translational modification regulating transcription in eukaryotes. Deacetylase inhibitors (such as trichostatin A) modulate the expression of approximately 2% of all cellular genes. We and others have demonstrated a marked transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) promoter in response to deacetylase inhibitors. Deacetylation events seem to be an important mechanism of HIV-1 transcriptional repression during latency, whereas acetylation events play critical functional roles in HIV-1 reactivation from latency. These deacetylation/acetylation events are implicated in chromatin remodeling of the viral promoter region, as well as in modulating the functional properties of cellular and viral transcription factors binding to this promoter region. Thereby, the HIV-1 promoter constitutes a unique regulatory model system to study the complex relationship between acetylation processes and transcriptional activity.

Potentiation of Tumor Necrosis Factor-Induced NF-κB Activation by Deacetylase Inhibitors Is Associated with a Delayed Cytoplasmic Reappearance of IκBα

Previous studies have implicated acetylases and deacetylases in regulating the transcriptional activity of NF-B. Here, we show that inhibitors of deacetylases such as trichostatin A (TSA) and sodium butyrate (NaBut) potentiated TNF-induced expression of several natural NF-B-driven promoters. This transcriptional synergism observed between TNF and TSA (or NaBut) required intact B sites in all promoters tested and was biologically relevant as demonstrated by RNase protection on two instances of endogenous NF-B-regulated gene transcription. Importantly, TSA prolonged both TNF-induced DNA-binding activity and the presence of NF-B in the nucleus. We showed that the p65 subunit of NF-B was acetylated in vivo. However, this acetylation was weak, suggesting that other mechanisms could be implicated in the potentiated binding and transactivation activities of NF-B after TNF plus TSA versus TNF treatment. Western blot and immunofluorescence confocal microscopy experiments revealed a delay in the cytoplasmic reappearance of the IB inhibitor that correlated temporally with the prolonged intranuclear binding and presence of NF-B. This delay was due neither to a defect in IB mRNA production nor to a nuclear retention of IB but was rather due to a persistent proteasome-mediated degradation of IB. A prolongation of IB kinase activity could explain, at least partially, the delayed IB cytoplasmic reappearance observed in presence of TNF plus TSA.

Histone Deacetylase Inhibitor Trichostatin A Sustains Sodium Pervanadate-induced NF-κB Activation by Delaying IκBα mRNA Resynthesis COMPARISON WITH TUMOR NECROSIS FACTOR α

NF-κB is a crucial transcription factor tightly regulated by protein interactions and post-translational modifications, like phosphorylation and acetylation. A previous study has shown that trichostatin A (TSA), a histone deacetylase inhibitor, potentiates tumor necrosis factor (TNF) α-elicited NF-κB activation and delays IκBα cytoplasmic reappearance. Here, we demonstrated that TSA also prolongs NF-κB activation when induced by the insulino-mimetic pervanadate (PV), a tyrosine phosphatase inhibitor that initiates an atypical NF-κB signaling. This extension is similarly correlated with delayed IκBα cytoplasmic reappearance. However, whereas TSA causes a prolonged IKK activity when added to TNFα, it does not when added to PV. Instead, quantitative reverse transcriptase-PCR revealed a decrease of iκbα mRNA level after TSA addition to PV stimulation. This synthesis deficit of the inhibitor could explain the sustained NF-κB residence in the nucleus. In vivo analysis by chromatin immunoprecipitation assays uncovered that, for PV induction but not for TNFα, the presence of TSA provokes several impairments on the iκbα promoter: (i) diminution of RNA Pol II recruitment; (ii) reduced acetylation and phosphorylation of histone H3-Lys14 and -Ser10, respectively; (iii) decreased presence of phosphorylated p65-Ser536; and (iv) reduction of IKKα binding. The recruitment of these proteins on the icam-1 promoter, another NF-κB-regulated gene, is not equally affected, suggesting a promoter specificity of PV with TSA stimulation. Taken together, these data suggest that TSA acts differently depending on the NF-κB pathway and the targeted promoter in question. This indicates that one overall histone deacetylase role is to inhibit NF-κB activation by molecular mechanisms specific of the stimulus and the promoter.

Histone deacetylase inhibitor trichostatin a sustains sodium pervanadate-induced NF-κB activation by delaying IκBα mRNA resynthesis - comparison with TNFα

NF-κB is a crucial transcription factor tightly regulated by protein interactions and post-translational modifications, like phosphorylation and acetylation. A previous study has shown that trichostatin A (TSA), a histone deacetylase inhibitor, potentiates tumor necrosis factor (TNF) α-elicited NF-κB activation and delays IκBα cytoplasmic reappearance. Here, we demonstrated that TSA also prolongs NF-κB activation when induced by the insulino-mimetic pervanadate (PV), a tyrosine phosphatase inhibitor that initiates an atypical NF-κB signaling. This extension is similarly correlated with delayed IκBα cytoplasmic reappearance. However, whereas TSA causes a prolonged IKK activity when added to TNFα, it does not when added to PV. Instead, quantitative reverse transcriptase-PCR revealed a decrease of iκbα mRNA level after TSA addition to PV stimulation. This synthesis deficit of the inhibitor could explain the sustained NF-κB residence in the nucleus. In vivo analysis by chromatin immunoprecipitation assays uncovered that, for PV induction but not for TNFα, the presence of TSA provokes several impairments on the iκbα promoter: (i) diminution of RNA Pol II recruitment; (ii) reduced acetylation and phosphorylation of histone H3-Lys14 and -Ser10, respectively; (iii) decreased presence of phosphorylated p65-Ser536; and (iv) reduction of IKKα binding. The recruitment of these proteins on the icam-1 promoter, another NF-κB-regulated gene, is not equally affected, suggesting a promoter specificity of PV with TSA stimulation. Taken together, these data suggest that TSA acts differently depending on the NF-κB pathway and the targeted promoter in question. This indicates that one overall histone deacetylase role is to inhibit NF-κB activation by molecular mechanisms specific of the stimulus and the promoter.

Active Transcription of the Human FASL/CD95L/TNFSF6 Promoter Region in T Lymphocytes Involves Chromatin Remodeling ROLE OF DNA METHYLATION AND PROTEIN ACETYLATION SUGGEST DISTINCT MECHANISMS OF TRANSCRIPTIONAL REPRESSION

Fas ligand (FasL/CD95L/TNFSF6), a member of the tumor necrosis factor family, initiates apoptosis in lymphoid and nonlymphoid tissues by binding to its receptor Fas (CD95/TNFRSF6). Although the transcriptional control of TNFSF6 gene expression is subjected to intense study, the role of its chromatin organization and accessibility to the transcriptional machinery is not known. Here, we determined the chromatin organization of TNFSF6 gene 5′ regulatory regions. Using the indirect end-labeling technique, a unique region named HSS1 and encompassing nucleotides –189 to +185 according to the transcriptional start site, was identified throughout a 20-kilobase nucleosomal DNA domain surrounding the promoter. The HSS1 region displayed hypersensitivity to in vivo DNase I digestion in TNFSF6-expressing cells only, including upon T cell activation. Hypersensitivity to micrococcal nuclease digestion and to specific restriction enzyme digestion suggested the precise positioning of two nucleosomes across the transcription start site and minimal promoter region, likely interfering with TNFSF6 active transcription in T lymphocytes. Indeed, HSS1 hypersensitivity to nuclease digestion strictly correlated with TNFSF6 transcription, including in primary and leukemia T cells. HSS1 chromatin remodeling preceded detectable TNFSF6 mRNA accumulation and was blocked by cycloheximide that also prevented TNFSF6 transcription. However, DNA methylation levels of the TNFSF6 HSS1 region did not correlate with transcriptional activation. Induction of global protein acetylation by treatment with histone deacetylase inhibitors was not accompanied by HSS1 chromatin remodeling and/or TNFSF6 transcription. We conclude that chromatin remodeling is a primary event in the activation of TNFSF6 expression in primary and leukemia T cells and that mechanisms independent of protein deacetylation and of DNA methylation of the TNFSF6 promoter region are involved in the repression of TNFSF6 gene expression.

Gene activation and gene silencing: a subtle equilibrium.

The genetic make-up of a cell resides entirely in its DNA. Now that the nucleotide sequence of several genomes has been determined, the major challenging problem is to understand how cell differentiation, proliferation or death are controlled. Major steps include analysis of the determinants of the cell cycle, the unravelling of RNAs and proteins involved in the control of gene expression and the dissection of the protein-destruction machinery. The successive steps to be considered are transcription of RNA on the DNA template, mRNA stabilization or degradation, and mRNA translation and protein localization in the right cell compartment. Gene expression or gene silencing is the result of many DNA-RNA-protein interactions and chromatin is among the key regulators of gene expression. Open chromatin (euchromatin) allows expression of the DNA message. This chromatin structure is generally characterized by the presence on the gene promoters of transcription complexes associated with histone acetyltransferases (HATs). On the contrary, closed chromatin (heterochromatin) is poorly acetylated and more condensed. It contains histone deacetylases (HDACs), potentially associated with DNA methyltransferases (DNMTs). DNMT activity leads to methylation and silencing of the DNA. Thus, a major problem in the field of gene regulation resides in understanding chromatin structure at each promoter, a formidable task for the years to come.

Chromatin-Associated Regulation Of Hiv-1 Transcription

Human Immunodeficiency Virus type 1 (HIV-1) infection can now be treated effectively in many patients in the developed world, using combinations of antiretroviral therapeutics, called Highly Active Anti-Retroviral Therapy (HAART). However, despite prolonged treatment with HAART, the persistence of latently HIV-1-infected cellular reservoirs harboring transcriptionally silent but replication-competent proviruses represents the major hurdle to virus eradication. These latently infected cells are a permanent source for virus reactivation and lead to a rebound of the viral load after interruption of HAART. Therefore, a greater understanding of the molecular mechanisms regulating proviral latency and reactivation should lead to rational strategies aimed at purging these cellular reservoirs of HIV-1. This review summarizes our current knowledge and understanding of the elements involved in HIV-1 transcriptional reactivation: (1) the site of integration; (2) the transcription factor NF-kappaB, which is induced by proinflammatory cytokines (such as TNFalpha) and binds to two kappaB sites in the HIV-1 promoter region; (3) the specific remodeling of a single nucleosome (called nuc-1 and located immediately downstream of the HIV-1 transcription start site under latency conditions) upon activation of the HIV-1 promoter; (4) post-translational acetylation of histones and of non-histone proteins (following treatment with deacetylases inhibitors, which induce viral transcription and nuc-1 remodeling); and (5) the viral trans-activator Tat, which promotes transcription by mediating the recruitment to the HIV-1 promoter of histone-modifying enzymes and ATP-dependent chromatin remodeling complexes required for nucleosome disruption and transcriptional processivity. Finally, this review highlights experimental therapies aimed at administrating HIV-1 gene expression activators (such as HDAC inhibitors) combined with an effective HAART in order to reactivate and decrease/eliminate the pool of latently HIV-1-infected cellular reservoirs