Emmanuelle Adam

HIV-1 tat transcriptional activity is regulated by acetylation.

The human immunodeficiency virus (HIV) trans‐ activator protein, Tat, stimulates transcription from the viral long‐terminal repeats (LTR) through an RNA hairpin element, trans‐activation responsive region (TAR). We and others have shown that trans‐activator protein (Tat)‐associated histone acetyltransferases (TAHs), p300 and p300/CBP‐associating factor (PCAF), assist functionally in the activation of chromosomally integrated HIV‐1 LTR. Here, we show that p300 and PCAF also directly acetylate Tat. We defined two sites of acetylation located in different functional domains of Tat. p300 acetylated Lys50 in the TAR RNA binding domain, while PCAF acetylated Lys28 in the activation domain of Tat. In support of a functional role for acetylation in vivo, histone deacetylase inhibitor (trichostatin A) synergized with Tat in transcriptional activation of the HIV‐1 LTR. Synergism was TAR‐dependent and required the intact presence of both Lys28 and Lys50. Mechanistically, acetylation at Lys28 by PCAF enhanced Tat binding to the Tat‐associated kinase, CDK9/P‐TEFb, while acetylation by p300 at Lys50 of Tat promoted the dissociation of Tat from TAR RNA that occurs during early transcription elongation. These data suggest that acetylation of Tat regulates two discrete and functionally critical steps in transcription, binding to an RNAP II CTD‐kinase and release of Tat from TAR RNA.

The House Dust Mite Allergen Der p 1, Unlike Der p 3, Stimulates the Expression of Interleukin-8 in Human Airway Epithelial Cells via a Proteinase-activated Receptor-2-independent Mechanism

We investigated and compared the mechanisms by which two dust mite proteolytic allergens, Der p 1 and Der p 3, and a peptide agonist of proteinase-activated receptor 2 (PAR2AP) trigger interleukin (IL)-8 release from human pulmonary epithelial cells (A549). Although all three stimuli tested induced the up-regulation of IL-8 (mRNA and protein), the Der p 1-mediated signaling events did not exactly match those induced by PAR2AP and Der p 3. First, Der p 1 was less effective in stimulating IL-8 gene transcriptional activity than PAR2AP and Der p 3. Second, Der p 1-mediated IL-8 expression was mainly dependent on NF-κB, whereas Der p 3 and PAR2AP regulated IL-8 expression through the activation of both NF-κB and AP-1. Third, although all three MAP kinases, ERK1/2, p38, and JNK, were activated, Der p 1 induced IL-8 release exclusively via the ERK1/2 signaling pathway, whereas PAR2AP and Der p 3 also involved the other kinases. Fourth, in HeLa cells, Der p 1 was able to up-regulate IL-8 secretion independent of PAR2 expression, and in contrast with PAR2AP and Der p 3, Der p 1 was unable to affect calcium signaling via PAR2 in PAR2-expressing KNRK cells. Finally, cleavage by Der p 1 of a synthetic peptide representing the N-terminal activation-cleavage site of PAR2 did not release a high potency activator of PAR2 as does Der p 3. We conclude that Der p 1 (but not Der p 3)-induced IL-8 production in A549 epithelial cells is independent of PAR2 activation.

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.

Potentiation of tumor necrosis factor-induced NF-kappa B activation by deacetylase inhibitors is associated with a delayed cytoplasmic reappearance of I kappa B alpha.

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

Inositol 1,3,4,5-tetrakisphosphate is essential for T lymphocyte development

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) is phosphorylated by Ins(1,4,5)P3 3-kinase, generating inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4). The physiological function of Ins(1,3,4,5)P4 is still unclear, but it has been reported to be a potential modulator of calcium mobilization. Disruption of the gene encoding the ubiquitously expressed Ins(1,4,5)P3 3-kinase isoform B (Itpkb) in mice caused a severe T cell deficiency due to major alterations in thymocyte responsiveness and selection. However, we were unable to detect substantial defects in Ins(1,4,5)P3 amounts or calcium mobilization in Itpkb−/− thymocytes. These data indicate that Itpkb and Ins(1,3,4,5)P4 define an essential signaling pathway for T cell precursor responsiveness and development.

DiC14-amidine cationic liposomes stimulate myeloid dendritic cells through Toll-like receptor 4.

DiC14‐amidine cationic liposomes were recently shown to promote Th1 responses when mixed with allergen. To further define the mode of action of diC14‐amidine as potential vaccine adjuvant, we characterized its effects on mouse and human myeloid dendritic cells (DC). First, we observed that, as compared with two other cationic liposomes, only diC14‐amidine liposomes induced the production of IL‐12p40 and TNF‐α by mouse bone marrow‐derived DC. DiC14‐amidine liposomes also activated human DC, as shown by synthesis of IL‐12p40 and TNF‐α, accumulation of IL‐6, IFN‐β and CXCL10 mRNA, and up‐regulation of membrane expression of CD80 and CD86. DC stimulation by diC14‐amidine liposomes was associated with activation of NF‐κB, ERK1/2, JNK and p38 MAP kinases. Finally, we demonstrated in mouse and human cells that diC14‐amidine liposomes use Toll‐like receptor 4 to elicit both MyD88‐dependent and Toll/IL‐1R‐containing adaptor inducing interferon IFN‐β (TRIF)‐dependent responses.

Suboptimal Enhancer Sequences Are Required for Efficient Bovine Leukemia Virus Propagation In Vivo: Implications for Viral Latency

ABSTRACT Repression of viral expression is a major strategy developed by retroviruses to escape from the host immune response. The absence of viral proteins (or derived peptides) at the surface of an infected cell does not permit the establishment of an efficient immune attack. Such a strategy appears to have been adopted by animal oncoviruses such as bovine leukemia virus (BLV) and human T-cell leukemia virus (HTLV). In BLV-infected animals, only a small fraction of the infected lymphocytes (between 1 in 5,000 and 1 in 50,000) express large amounts of viral proteins; the vast majority of the proviruses are repressed at the transcriptional level. Induction of BLV transcription involves the interaction of the virus-encoded Tax protein with the CREB/ATF factors; the resulting complex is able to interact with three 21-bp Tax-responsive elements (TxRE) located in the 5′ long terminal repeat (5′ LTR). These TxRE contain cyclic AMP-responsive elements (CRE), but, remarkably, the “TGACGTCA” consensus is never strictly conserved in any viral strain (e.g.,AGACGTCA, TGACGGCA, TGACCTCA). To assess the role of these suboptimal CREs, we introduced a perfect consensus sequence within the TxRE and showed by gel retardation assays that the binding efficiency of the CREB/ATF proteins was increased. However,trans-activation of a luciferase-based reporter by Tax was not affected in transient transfection assays. Still, in the absence of Tax, the basal promoter activity of the mutated LTR was increased as much as 20-fold. In contrast, mutation of other regulatory elements within the LTR (the E box, NF-κB, and glucocorticoid- or interferon-responsive sites [GRE or IRF]) did not induce a similar alteration of the basal transcription levels. To evaluate the biological relevance of these observations made in vitro, the mutations were introduced into an infectious BLV molecular clone. After injection into sheep, it appeared that all the recombinants were infectious in vivo and did not revert into a wild-type virus. All of them, except one, propagated at wild-type levels, indicating that viral spread was not affected by the mutation. The sole exception was the CRE mutant; proviral loads were drastically reduced in sheep infected with this type of virus. We conclude that a series of sites (NF-κB, IRF, GRE, and the E box) are not required for efficient viral spread in the sheep model, although mutation of some of these motifs might induce a minor phenotype during transient transfection assays in vitro. Remarkably, a provirus (pBLV-Δ21-bp) harboring only two TxRE was infectious and propagated at wild-type levels. And, most importantly, reconstitution of a consensus CRE, within the 21-bp enhancers increases binding of CREB/ATF proteins but abrogates basal repression of LTR-directed transcription in vitro. Suboptimal CREs are, however, essential for efficient viral spread within infected sheep, although these sites are dispensable for infectivity. These results suggest an evolutionary selection of suboptimal CREs that repress viral expression with escape from the host immune response. These observations, which were obtained in an animal model for HTLV-1, are of interest for oncovirus-induced pathogenesis in humans.

Overlapping CRE and E Box Motifs in the Enhancer Sequences of the Bovine Leukemia Virus 5′ Long Terminal Repeat Are Critical for Basal and Acetylation-Dependent Transcriptional Activity of the Viral Promoter: Implications for Viral Latency

ABSTRACT Bovine leukemia virus (BLV) infection is characterized by viral latency in a large proportion of cells containing an integrated provirus. In this study, we postulated that mechanisms directing the recruitment of deacetylases to the BLV 5′ long terminal repeat (LTR) could explain the transcriptional repression of viral expression in vivo. Accordingly, we showed that BLV promoter activity was induced by several deacetylase inhibitors (such as trichostatin A [TSA]) in the context of episomal LTR constructs and in the context of an integrated BLV provirus. Moreover, treatment of BLV-infected cells with TSA increased H4 acetylation at the viral promoter, showing a close correlation between the level of histone acetylation and transcriptional activation of the BLV LTR. Among the known cis-regulatory DNA elements located in the 5′ LTR, three E box motifs overlapping cyclic AMP responsive elements (CREs) in U3 were shown to be involved in transcriptional repression of BLV basal gene expression. Importantly, the combined mutations of these three E box motifs markedly reduced the inducibility of the BLV promoter by TSA. E boxes are susceptible to recognition by transcriptional repressors such as Max-Mad-mSin3 complexes that repress transcription by recruiting deacetylases. However, our in vitro binding studies failed to reveal the presence of Mad-Max proteins in the BLV LTR E box-specific complexes. Remarkably, TSA increased the occupancy of the CREs by CREB/ATF. Therefore, we postulated that the E box-specific complexes exerted their negative cooperative effect on BLV transcription by steric hindrance with the activators CREB/ATF and/or their transcriptional coactivators possessing acetyltransferase activities. Our results thus suggest that the overlapping CRE and E box elements in the BLV LTR were selected during evolution as a novel strategy for BLV to allow better silencing of viral transcription and to escape from the host immune response.

Probiotic Escherichia coli Nissle 1917 activates DC and prevents house dust mite allergy through a TLR4-dependent pathway

Experimental animal and human studies have demonstrated that probiotic strains have beneficial effects on allergy. Here we report that the probiotic Escherichia coli Nissle 1917 strain (EcN) is able to activate DC, as shown by important cytokine synthesis together with up‐regulation of membrane expression of CD40, CD80 and CD86. This EcN‐induced DC activation was strictly dependent on the TLR4 signaling pathway and was also associated with stimulation of NF‐κB and MAPK. We next investigated the prophylactic potential of i.n. co‐administration of EcN with a recombinant form of Der p 1 (ProDer p 1) in a murine model of mite allergy. I.n. vaccinations with EcN plus ProDer p 1 prevented the subsequent allergic response following Der p 1 sensitization and airway challenge with aerosolized mite extracts through the induction of an allergen‐specific IgG2a response, the prevention of specific IgE production and a strong reduction of IL‐5 secretion by allergen‐restimulated splenocytes. EcN alone or in combination with ProDer p 1 inhibited the development of airway eosinophilia and neutrophilia. This in vivo protective effect of EcN was, in part, mediated by TLR4 signaling. Our results suggest that EcN represents an efficient adjuvant to prevent allergic responses.

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.