The transcriptional repressor REST determines the cell-specific expression of the human MAPK8IP1 gene encoding IB1 (JIP-1).

Islet-brain 1 (IB1) is the human and rat homologue of JIP-1, a scaffold protein interacting with the c-Jun amino-terminal kinase (JNK). IB1 expression is mostly restricted to the endocrine pancreas and to the central nervous system. Herein, we explored the transcriptional mechanism responsible for this preferential islet and neuronal expression of IB1. A 731-bp fragment of the 5' regulatory region of the human MAPK8IP1 gene was isolated from a human BAC library and cloned upstream of a luciferase reporter gene. This construct drove high transcriptional activity in both insulin-secreting and neuron-like cells but not in unrelated cell lines. Sequence analysis of this promoter region revealed the presence of a neuron-restrictive silencer element (NRSE) known to bind repressor zinc finger protein REST. This factor is not expressed in insulin-secreting and neuron-like cells. By mobility shift assay, we confirmed that REST binds to the NRSE present in the IB1 promoter. Once transiently transfected in beta-cell lines, the expression vector encoding REST repressed IB1 transcriptional activity. The introduction of a mutated NRSE in the 5' regulating region of the IB1 gene abolished the repression activity driven by REST in insulin-secreting beta cells and relieved the low transcriptional activity of IB1 observed in unrelated cells. Moreover, transfection in non-beta and nonneuronal cell lines of an expression vector encoding REST lacking its transcriptional repression domain relieved IB1 promoter activity. Last, the REST-mediated repression of IB1 could be abolished by trichostatin A, indicating that deacetylase activity is required to allow REST repression. Taken together, these data establish a critical role for REST in the control of the tissue-specific expression of the human IB1 gene.

Dietary obesity-associated Hif1α activation in adipocytes restricts fatty acid oxidation and energy expenditure via suppression of the Sirt2-NAD+ system.

Dietary obesity is a major factor in the development of type 2 diabetes and is associated with intra-adipose tissue hypoxia and activation of hypoxia-inducible factor 1α (HIF1α). Here we report that, in mice, Hif1α activation in visceral white adipocytes is critical to maintain dietary obesity and associated pathologies, including glucose intolerance, insulin resistance, and cardiomyopathy. This function of Hif1α is linked to its capacity to suppress β-oxidation, in part, through transcriptional repression of sirtuin 2 (Sirt2) NAD(+)-dependent deacetylase. Reduced Sirt2 function directly translates into diminished deacetylation of PPARγ coactivator 1α (Pgc1α) and expression of β-oxidation and mitochondrial genes. Importantly, visceral adipose tissue from human obese subjects is characterized by high levels of HIF1α and low levels of SIRT2. Thus, by negatively regulating the Sirt2-Pgc1α regulatory axis, Hif1α negates adipocyte-intrinsic pathways of fatty acid catabolism, thereby creating a metabolic state supporting the development of obesity.

Identification of a key lysine residue in heat shock protein 90 required for azole and echinocandin resistance in Aspergillus fumigatus.

Heat shock protein 90 (Hsp90) is an essential chaperone involved in the fungal stress response that can be harnessed as a novel antifungal target for the treatment of invasive aspergillosis. We previously showed that genetic repression of Hsp90 reduced Aspergillus fumigatus virulence and potentiated the effect of the echinocandin caspofungin. In this study, we sought to identify sites of posttranslational modifications (phosphorylation or acetylation) that are important for Hsp90 function in A. fumigatus. Phosphopeptide enrichment and tandem mass spectrometry revealed phosphorylation of three residues in Hsp90 (S49, S288, and T681), but their mutation did not compromise Hsp90 function. Acetylation of lysine residues of Hsp90 was recovered after treatment with deacetylase inhibitors, and acetylation-mimetic mutations (K27A and K271A) resulted in reduced virulence in a murine model of invasive aspergillosis, supporting their role in Hsp90 function. A single deletion of lysine K27 or an acetylation-mimetic mutation (K27A) resulted in increased susceptibility to voriconazole and caspofungin. This effect was attenuated following a deacetylation-mimetic mutation (K27R), suggesting that this site is crucial and should be deacetylated for proper Hsp90 function in antifungal resistance pathways. In contrast to previous reports in Candida albicans, the lysine deacetylase inhibitor trichostatin A (TSA) was active alone against A. fumigatus and potentiated the effect of caspofungin against both the wild type and an echinocandin-resistant strain. Our results indicate that the Hsp90 K27 residue is required for azole and echinocandin resistance in A. fumigatus and that deacetylase inhibition may represent an adjunctive anti-Aspergillus strategy.

The E3-Ubiquitin Ligase TRIM50 Interacts with HDAC6 and p62, and Promotes the Sequestration and Clearance of Ubiquitinated Proteins into the Aggresome.

In this study we report that, in response to proteasome inhibition, the E3-Ubiquitin ligase TRIM50 localizes to and promotes the recruitment and aggregation of polyubiquitinated proteins to the aggresome. Using Hdac6-deficient mouse embryo fibroblasts (MEF) we show that this localization is mediated by the histone deacetylase 6, HDAC6. Whereas Trim50-deficient MEFs allow pinpointing that the TRIM50 ubiquitin-ligase regulates the clearance of polyubiquitinated proteins localized to the aggresome. Finally we demonstrate that TRIM50 colocalizes, interacts with and increases the level of p62, a multifunctional adaptor protein implicated in various cellular processes including the autophagy clearance of polyubiquitinated protein aggregates. We speculate that when the proteasome activity is impaired, TRIM50 fails to drive its substrates to the proteasome-mediated degradation, and promotes their storage in the aggresome for successive clearance.

Genomic roles of the HCF transcriptional regulator in "Drosophila"

Abstract : Transcriptional regulation is the result of a combination of positive and negative effectors, such as transcription factors, cofactors and chromatin modifiers. During my thesis project I studied chromatin association, and transcriptional and cell cycle regulatory functions of dHCF, the Drosophila homologue of the human protein HCF-1 (host cell factor-1). The human and Drosophila HCF proteins are synthesized as large polypeptides that are cleaved into two subunits (HCFN and HCFC), which remain associated with one another by non covalent interactions. Studies in mammalian cells over the past 20 years have been devoted to understanding the cellular functions of HCF-1 and have revealed that it is a key regulator of transcription and cell cycle regulation. In human cells, HCF-1 interacts with the histone methyltransferase Set1/Ash2 and MLL/Ash2 complexes and the histone deacetylase Sin3 complex, which are involved in transcriptional activation and repression, respectively. HCF-1 is also recruited to promoters to regulate G1 -to-S phase progression during the cell cycle by the activator transcription factors E2F1 and E2F3, and by the repressor transcription factor E2F4. HCF-1 protein structure and these interactions between HCP-1 and E2F transcriptional regulator proteins are also conserved in Drosophila. In this doctoral thesis, I use proliferating Drosophila SL2 cells to study both the genomic-binding sites of dHCF, using a combination of chromatin immunoprecipitation and ultra high throughput sequencing (ChIP-seq) analysis, and dHCF regulated genes, employing RNAi and microarray expression analysis. I show that dHCF is bound to over 7500 chromosomal sites in proliferating SL2 cells, and is located at +-200 bp relative to the transcriptional start sites of about 30% of Drosophila genes. There is also a direct relationship between dHCF promoter association and promoter- associated transcriptional activity. Thus, dHCF binding levels at promoters correlated directly with transcriptional activity. In contrast, expression studies showed that dHCF appears to be involved in both transcriptional activation and repression. Analysis of dHCF-binding sites identified nine dHCF-associated motifs, four of them linked dHCF to (i) two insulator proteins, GAGA and BEAF, (ii) the E-box motif, and (iii) a degenerated TATA-box. The dHCF-associated motifs allowed the organization of the dHCF-bound genes into five biological processes: differentiation, cell cycle and gene expression, regulation of endocytosis, and cellular localization. I further show that different mechanisms regulate dHCF association with chromatin. Despite that after dHCF cleavage the dHCFN and dHCFC subunits remain associated, the two subunits showed different affinities for chromatin and differential binding to a set of tested promoters, suggesting that dHCF could target specific promoters through each of the two subunits. Moreover, in addition to the interaction between dHCF and E2F transcription factors, the dHCF binding pattern is correlated with dE2F2 genomic 4 distribution. I show that dE2F factors are necessary for recruitment of dHCF to the promoter of a set of dHCF regulated genes. Therefore dHCF, as in mammals, is involved in regulation of G1 to S phase progression in collaboration with the dE2Fs transcription factors. In addition, gene expression arrays reveal that dHCF could indirectly regulate cell cycle progression by promoting expression of genes involved in gene expression and protein synthesis, and inhibiting expression of genes involved in cell-cell adhesion. Therefore, dHCF is an evolutionary conserved protein, which binds to many specific sites of the Drosophila genome via interaction with DNA of chromatin-binding proteins to regulate the expression of genes involved in many different cellular functions. Résumé : La regulation de la transcription est le résultat des effets positifs et négatifs des facteurs de transcription, cofacteurs et protéines effectrices qui modifient la chromatine. Pendant mon projet de thèse, j'ai étudié l'association a la chromatine, ainsi que la régulation de la transcription et du cycle cellulaire par dHCF, l'homologue chez la drosophile de la protéine humaine HCF-1 (host cell factor-1). Chez 1'humain et la V drosophile, les deux protéines HCF sont synthétisées sous la forme d'un long polypeptide, qui est ensuite coupé en deux sous-unités au centre de la protéine. Les deux sous-unités restent associées ensemble grâce a des interactions non-covalentes. Des études réalisées pendant les 20 dernières années ont permit d'établir que HCF-l et un facteur clé dans la régulation de la transcription et du cycle cellulaire. Dans les cellules humaines, HCF-1 active et réprime la transcription en interagissant avec des complexes de protéines qui activent la transcription en méthylant les histones (HMT), comme par Set1/Ash2 et MLL/Ash2, et d'autres complexes qui répriment la transcription et sont responsables de la déacétylation des histones (HDAC) comme la protéine Sin3. HCF-l est aussi recruté aux promoteurs par les activateurs de la transcription E2F l et E2F3a, et par le répresseur de la transcription E2F4 pour réguler la transition entre les phases G1 et S du cycle cellulaire. La structure de HCF-1 et les interactions entre HCF-l et les régulateurs de la transcription sont conservées chez la drosophile. Pendant ma these j'ai utilisé les cellules de la drosophile, SL2 en culture, pour étudier les endroits de liaisons de HCF-l à la chromatine, grâce a immunoprecipitation de la chromatine et du séquençage de l'ADN massif ainsi que les gènes régulés par dHCF 3 grâce a la technique de RNAi et des microarrays. Mes résultats on montré que dHCF se lie à environ 7565 endroits, et estimé a 1200 paire de bases autour des sites d'initiation de la transcription de 30% des gènes de la drosophile. J 'ai observe une relation entre dHCF et le niveau de la transcription. En effet, le niveau de liaison dHCF au promoteur corrèle avec l'activité de la transcription. Cependant, mes études d'expression ont montré que dHCF est implique dans le processus d'activation et mais aussi de répression de la transcription. L'analyse des séquences d'ADN liées par dHCF a révèle neuf motifs, quatre de ces motifs ont permis d'associer dl-ICF a deux protéines isolatrices GAGA et BEAF, au motif pour les E-boxes et a une TATA-box dégénérée. Les neuf motifs associes à dHCF ont permis d'associer les gènes lies par dHCF au promoteur a cinq processus biologiques: différentiation, cycle cellulaire, expression de gènes, régulation de l'endocytosis et la localisation cellulaire, J 'ai aussi montré qu'il y a plusieurs mécanismes qui régulent l'association de dHCF a la chromatine, malgré qu'après clivage, les deux sous-unites dHCFN and dHCFC, restent associées, elles montrent différentes affinités pour la chromatine et lient différemment un group de promoteurs, les résultats suggèrent que dHCF peut se lier aux promoteurs en utilisant chacune de ses sous-unitées. En plus de l'association de dHCF avec les facteurs de transcription dE2F s, la distribution de dHCF sur le génome corrèle avec celle du facteur de transcription dE2F2. J'ai aussi montré que les dE2Fs sont nécessaires pour le recrutement de dHCF aux promoteurs d'un sous-groupe de gènes régules par dHCF. Mes résultats ont aussi montré que chez la drosophile comme chez les humains, dl-ICF est implique dans la régulation de la progression de la phase G1 a la phase S du cycle cellulaire en collaboration avec dE2Fs. D'ailleurs, les arrays d'expression ont suggéré que dHCF pourrait réguler le cycle cellulaire de façon indirecte en activant l'expression de gènes impliqués dans l'expression génique et la synthèse de protéines, et en inhibant l'expression de gènes impliqués dans l'adhésion cellulaire. En conclusion, dHCF est une protéine, conservée dans l'évolution, qui se lie spécifiquement a beaucoup d'endroits du génome de Drosophile, grâce à l'interaction avec d'autres protéines, pour réguler l'expression des gènes impliqués dans plusieurs fonctions cellulaires.

Role of salt-inducible kinase 1 in the activation of MEF2-dependent transcription by BDNF.

Substantial evidence supports a role for myocyte enhancer factor 2 (MEF2)-mediated transcription in neuronal survival, differentiation and synaptic function. In developing neurons, it has been shown that MEF2-dependent transcription is regulated by neurotrophins. Despite these observations, little is known about the cellular mechanisms by which neurotrophins activate MEF2 transcriptional activity. In this study, we examined the role of salt-inducible kinase 1 (SIK1), a member of the AMP-activated protein kinase (AMPK) family, in the regulation of MEF2-mediated transcription by the neurotrophin brain-derived neurotrophic factor (BDNF). We show that BDNF increases the expression of SIK1 in primary cultures of rat cortical neurons through the extracellular signal-regulated kinase 1/2 (ERK1/2)-signaling pathway. In addition to inducing SIK1 expression, BDNF triggers the phosphorylation of SIK1 at Thr182 and its translocation from the cytoplasm to the nucleus of cortical neurons. The effects of BDNF on the expression, phosphorylation and, translocation of SIK1 are followed by the phosphorylation and nuclear export of histone deacetylase 5 (HDAC5). Blockade of SIK activity with a low concentration of staurosporine abolished BDNF-induced phosphorylation and nuclear export of HDAC5 in cortical neurons. Importantly, stimulation of HDAC5 phosphorylation and nuclear export by BDNF is accompanied by the activation of MEF2-mediated transcription, an effect that is suppressed by staurosporine. Consistent with these data, BDNF induces the expression of the MEF2 target genes Arc and Nur77, in a staurosporine-sensitive manner. In further support of the role of SIK1 in the regulation of MEF2-dependent transcription by BDNF, we found that expression of wild-type SIK1 or S577A SIK1, a mutated form of SIK1 which is retained in the nucleus of transfected cells, is sufficient to enhance MEF2 transcriptional activity in cortical neurons. Together, these data identify a previously unrecognized mechanism by which SIK1 mediates the activation of MEF2-dependent transcription by BDNF.

Epigenetic regulation of histone H3 serine 10 phosphorylation status by HCF-1 proteins in C. elegans and mammalian cells.

BACKGROUND: The human herpes simplex virus (HSV) host cell factor HCF-1 is a transcriptional coregulator that associates with both histone methyl- and acetyltransferases, and a histone deacetylase and regulates cell proliferation and division. In HSV-infected cells, HCF-1 associates with the viral protein VP16 to promote formation of a multiprotein-DNA transcriptional activator complex. The ability of HCF proteins to stabilize this VP16-induced complex has been conserved in diverse animal species including Drosophila melanogaster and Caenorhabditis elegans suggesting that VP16 targets a conserved cellular function of HCF-1. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of HCF proteins in animal development, we have characterized the effects of loss of the HCF-1 homolog in C. elegans, called Ce HCF-1. Two large hcf-1 deletion mutants (pk924 and ok559) are viable but display reduced fertility. Loss of Ce HCF-1 protein at reduced temperatures (e.g., 12 degrees C), however, leads to a high incidence of embryonic lethality and early embryonic mitotic and cytokinetic defects reminiscent of mammalian cell-division defects upon loss of HCF-1 function. Even when viable, however, at normal temperature, mutant embryos display reduced levels of phospho-histone H3 serine 10 (H3S10P), a modification implicated in both transcriptional and mitotic regulation. Mammalian cells with defective HCF-1 also display defects in mitotic H3S10P status. CONCLUSIONS/SIGNIFICANCE: These results suggest that HCF-1 proteins possess conserved roles in the regulation of cell division and mitotic histone phosphorylation.

The function of a new regulator of epidermal differentiation, HOP, and pathomechanisms underlying epidermolytic hyperkeratosis

The process of epidermal differentiation involves proliferation, differentiation, migration and maturation of keratinocytes to form an impermeable barrier against water loss and outside environment. It is controlled by highly balanced regulatory machinery, involving many molecules that are still under investigation.Homeobox proteins are involved in body patterning and morphogenesis of organs and are studied as potentially good candidates to regulate this process. In the first project we investigated the role of a protein named HOP which belongs to a group of homeobox proteins. Even if HOP is a small protein almost completely composed of the homeodomain and without DNA binding capacity, it is considered as transcriptional regulator in different tissues. HOP interacts with serum response factor (SRF) and histone deacetylase type 2 (HDAC2). By microarray analysis we found that HOP expression increases in cultured human primary keratinocytes (NHK) which undergo calcium-induced differentiation. HOP protein was localized in granular layer of the epidermis of healthy individuals. Lack of HOP was demonstrated in psoriatic lesions, whereas a strong expression was demonstrated in the lesional skin of patients affected with lichen planus (LP). Since LP is characterized by hypergranulosis while psoriatic lesions by progressive lack of the granular layer, the obtained data indicated that HOP might have a potential function in granular layer of epidermis. To investigate HOP function, we inhibited its expression by using HOP specific StealthRNAi and we overexpressed HOP using lentiviral vectors in differentiating NHK. The conclusion of both experiments indicated that HOP positively regulates the expression of late differentiation markers, such as profilaggrin, loricrin and transglutaminase 1. The in vitro data were next confirmed in vivo using HOP knockout mouse model.The second part of my study involved analysis of mechanisms underlying the pathogenesis of epidermolytic hyperkeratosis (EHK). EHK is a genetic disorder characterized by erythema, skin blistering, keratinocyte hyperproliferation and hyperkeratosis. EHK is caused by mutations in keratin 1 or 10 (K1, K10) which are major structural proteins of differentiated keratinocytes and participate in the cellular scaffold formation. To investigate how the structural proteins carrying mutations alter cellular signaling, we established an in vitro model for EHK by overexpression of one of the most common K10 mutations reported so far (K10R156H), in primary human keratinocytes. In order to mimic the in vivo situation, mutated keratinocytes growing on silicone membranes were subjected to mechanical stretch. We observed strong collapse of KIF in K10R156H keratinocytes when subjected to stretch for 30 minutes. Our data demonstrated stronger activation of p38, a member of MAPK stress signaling pathways, in K10R156H when compared to control cells. We demonstrated also that K10R156H keratinocytes showed an induction of TNF-α and RANTES release in response to stretch.Taken together these studies characterize a novel regulator of epidermal differentiation - HOP and demonstrate new aspects implicated in the pathogenesis of EHK.

Analyse des modifications de la chromatine impliquées dans l'effet barrière de CTF1 aux télomères humains

Eukaryotic genomes are compartmentalized in different structural domains that can affect positively or negatively gene expression. These regions of euchromatin and heterochromatin are characterized by distinct histones marks which can facilitate or repress gene transcription. The chromatin environment represents thus one of the main problems to control gene expression in biotechnological applications or gene therapy, since its expression is affected by the chromatin neighboring its locus of insertion. Some chromatin regions like telomeres are composed of constitutive heterochromatin which leads to the telomeric position effect (TPE) that silences genes adjacent to the telomere. TPE is known to spread by the selfrecruitment of the SIR histone deacetylase complex from the telomere in S.cerevisiae, but the histone marks that are associated to telomeric chromatin in mammalian cells remain mostly unknown. The transcription factor CTF1 has shown antisilencing properties in mammalian cells and also a boundary activity against TPE in yeast cells when fused to the yeast Gal4 DNA binding domain. In the work presented here, we describe a dual-reporter system to assess the boundary activity of proteins such as CTF1 at human telomeres. When located between the two reporter genes, CTF1 shields the telomere distal gene from TPE, while the telomereproximal gene remains silenced by telomeric heterochromatin. The boundary activity of CTF1 is shown to act regardless its function of transcriptional activator, by opposition to the transcriptional activator VP16 which activates indifferently both transgenes. Moreover, this study shows that CTF1 boundary activity is linked to its H3 binding function, as expected from a chromatin remodeler. ChIP experiments showed that histone deacetylation is the main histone modification involved in gene silencing at mammalian cell telomeres. Distinctly to yeast cells, the histone deacetylation signal in human cells extented over a short range along the chromosome. CTF1 may help to block this propagation and therefore to restore histones acetylation level on telomere protected locus. Surprisingly, other histone marks such as trimethyl-H3K9 or trimethyl-H4K20 were found on telomere protected locus, while in another clone, unsilencing of telomere distal transgene was associated with recruitment of the histone variant H2A.Z. Thus, I conclude that CTF1 displays a chromatin boundary function which is independent of its transcriptional activity and therefore exhibit features required for use as chromatin insulator in biotechnological applications. RESUME Les génomes eucaryotes sont compartementalisés en domaines structurels qui peuvent affecter positivement ou négativement l'expression des gènes avoisinants. Ces régions dites d'euchromatine ou d'hétérochromatine sont caractérisées par des modifications posttraductionnelles des histones qui peuvent faciliter ou au contraire inhiber la transcription des gènes qui s'y trouvent. Ainsi, isoler un gène de son environnement chromatinien est problème fréquent lorsqu'il s'agit de contrôler son expression dans le cadre d'applications en biotechnologie ou encore en thérapie génique. Certaines régions de chromatine telles que les télomères sont composées d'hétérochromatine constitutive qui mène au silençage des gènes avoisinants. Cet effet de position télomérique (TPE) est connu dans la levure S.cerevisiae comme se propageant par auto-recrutement du complexe de déacétylation d'histone SIR, alors que peu de modifications de chromatine ont pu être associées à ce phénomène dans les cellules de mammifères. Le facteur de transcription CTF1 a montré des propriétés d'anti-silençage dans les cellules de mammifères, ainsi qu'une activité barrière contre le silençage télomérique dans les cellules de levures lorsqu'il est fusionné au domaine de liaison à l'ADN de la protéine de levure Gal4. Dans le travail présenté ci-après est décrit un système à deux gènes rapporteurs permettant de mesurer l'activité barrière de protéines telles que CTF1 aux télomères humains, et les modifications de chromatine qui y sont associées. Lorsque CTF1 est placé entre les deux gènes rapporteurs, le gène distant du télomère est protégé du silençage qui lui est associé, alors que le gène proche du télomère reste soumis à ce silençage induit par l'hétérochromatine télomérique. L'activité barrière de CTF1 est montrée ici comme agissant indépendamment de son activité transcriptionnelle, par opposition à l'activateur transcriptionnel VP16 qui active indifféremment les deux transgènes. En outre, cette étude appuie l'hypothèse stipulant que CTF1 agisse comme remodeleur chromatinien puisqu'elle démontre que son activité barrière est directement dépendante de son activité de liaison avec l'histone H3. De plus, des expériences d'immuno-précipitation de la chromatine démontrent que la déacétylation des histones est le majeur phénomène intervenant dans le silençage télomérique. Par opposition à la levure, ce signal de déacétylation ne se propage dans les cellules humaines que sur une courte distance le long du chromosome. CTF1 agit ainsi en bloquant cette propagation et en restaurant le niveau d'acétylation des histones sur le locus protégé du télomère. De manière surprenante et inattendue, d'autres modifications d'histones telles que 4 les H3K9 et H4K20 triméthylées sont aussi observées à ce locus, tandis le recrutement du variant H2A.Z peut aussi être suffisant à restaurer l'expression du gène distant du télomère. En terme de cette analyse, CTF1 exhibe ainsi une fonction de barrière chromatinienne qui exclue une activité transcriptionnelle non désirée - propriété qui est requise dans l'établissement des isolateurs visant à permettre le contrôle d'un transgène dans le cadre d'applications en biotechnologies.

SIRT1 Activates MAO-A in the Brain to Mediate Anxiety and Exploratory Drive.

SIRT1 is a NAD(+)-dependent deacetylase that governs a number of genetic programs to cope with changes in the nutritional status of cells and organisms. Behavioral responses to food abundance are important for the survival of higher animals. Here we used mice with increased or decreased brain SIRT1 to show that this sirtuin regulates anxiety and exploratory drive by activating transcription of the gene encoding the monoamine oxidase A (MAO-A) to reduce serotonin levels in the brain. Indeed, treating animals with MAO-A inhibitors or selective serotonin reuptake inhibitors (SSRIs) normalized anxiety differences between wild-type and mutant animals. SIRT1 deacetylates the brain-specific helix-loop-helix transcription factor NHLH2 on lysine 49 to increase its activation of the MAO-A promoter. Both common and rare variations in the SIRT1 gene were shown to be associated with risk of anxiety in human population samples. Together these data indicate that SIRT1 mediates levels of anxiety, and this regulation may be adaptive in a changing environment of food availability.

Extracellular-signal-regulated kinase 5 modulates the antioxidant response by transcriptionally controlling Sirtuin 1 expression in leukemic cells.

Cancer cell metabolism differs from that of non-transformed cells in the same tissue. This specific metabolism gives tumor cells growing advantages besides the effect in increasing anabolism. One of these advantages is immune evasion mediated by a lower expression of the mayor histocompatibility complex class I molecules. The extracellular-signal-regulated kinase-5 regulates both mayor histocompatibility complex class I expression and metabolic activity. However, the mechanisms underlying are largely unknown. We show here that extracellular-signal-regulated kinase-5 regulates the transcription of the NADH(+)-dependent histone deacetylase silent mating type information regulation 2 homolog 1 (Sirtuin 1) in leukemic Jurkat T cells. This involves the activation of the transcription factor myocyte enhancer factor-2 and its binding to the sirt1 promoter. In addition, extracellular-signal-regulated kinase-5 is required for T cell receptor-induced and oxidative stress-induced full Sirtuin 1 expression. Extracellular-signal-regulated kinase-5 induces the expression of promoters containing the antioxidant response elements through a Sirtuin 1-dependent pathway. On the other hand, down modulation of extracellular-signal-regulated kinase-5 expression impairs the anti-oxidant response. Notably, the extracellular-signal-regulated kinase-5 inhibitor BIX02189 induces apoptosis in acute myeloid leukemia tumor cells without affecting T cells from healthy donors. Our results unveil a new pathway that modulates metabolism in tumor cells. This pathway represents a promising therapeutic target in cancers with deep metabolic layouts such as acute myeloid leukemia.

Mice generated by in vitro fertilization exhibit vascular dysfunction and shortened life span.

Children conceived by assisted reproductive technologies (ART) display a level of vascular dysfunction similar to that seen in children of mothers with preeclamspia. The long-term consequences of ART-associated vascular disorders are unknown and difficult to investigate in healthy children. Here, we found that vasculature from mice generated by ART display endothelial dysfunction and increased stiffness, which translated into arterial hypertension in vivo. Progeny of male ART mice also exhibited vascular dysfunction, suggesting underlying epigenetic modifications. ART mice had altered methylation at the promoter of the gene encoding eNOS in the aorta, which correlated with decreased vascular eNOS expression and NO synthesis. Administration of a deacetylase inhibitor to ART mice normalized vascular gene methylation and function and resulted in progeny without vascular dysfunction. The induction of ART-associated vascular and epigenetic alterations appeared to be related to the embryo environment; these alterations were possibly facilitated by the hormonally stimulated ovulation accompanying ART. Finally, ART mice challenged with a high-fat diet had roughly a 25% shorter life span compared with control animals. This study highlights the potential of ART to induce vascular dysfunction and shorten life span and suggests that epigenetic alterations contribute to these problems.

Fetal programming of pulmonary vascular dysfunction in mice: role of epigenetic mechanisms.

Insults during the fetal period predispose the offspring to systemic cardiovascular disease, but little is known about the pulmonary circulation and the underlying mechanisms. Maternal undernutrition during pregnancy may represent a model to investigate underlying mechanisms, because it is associated with systemic vascular dysfunction in the offspring in animals and humans. In rats, restrictive diet during pregnancy (RDP) increases oxidative stress in the placenta. Oxygen species are known to induce epigenetic alterations and may cross the placental barrier. We hypothesized that RDP in mice induces pulmonary vascular dysfunction in the offspring that is related to an epigenetic mechanism. To test this hypothesis, we assessed pulmonary vascular function and lung DNA methylation in offspring of RDP and in control mice at the end of a 2-wk exposure to hypoxia. We found that endothelium-dependent pulmonary artery vasodilation in vitro was impaired and hypoxia-induced pulmonary hypertension and right ventricular hypertrophy in vivo were exaggerated in offspring of RDP. This pulmonary vascular dysfunction was associated with altered lung DNA methylation. Administration of the histone deacetylase inhibitors butyrate and trichostatin A to offspring of RDP normalized pulmonary DNA methylation and vascular function. Finally, administration of the nitroxide Tempol to the mother during RDP prevented vascular dysfunction and dysmethylation in the offspring. These findings demonstrate that in mice undernutrition during gestation induces pulmonary vascular dysfunction in the offspring by an epigenetic mechanism. A similar mechanism may be involved in the fetal programming of vascular dysfunction in humans.

Antifungal activity of compounds targeting the Hsp90-calcineurin pathway against various mould species.

OBJECTIVES: Invasive mould infections are associated with a high mortality rate and the emergence of MDR moulds is of particular concern. Calcineurin and its chaperone, the heat shock protein 90 (Hsp90), represent an important pathway for fungal virulence that can be targeted at different levels. We investigated the antifungal activity of compounds directly or indirectly targeting the Hsp90-calcineurin axis against different mould species. METHODS: The in vitro antifungal activity of the anticalcineurin drug FK506 (tacrolimus), the Hsp90 inhibitor geldanamycin, the lysine deacetylase inhibitor trichostatin A and the Hsp70 inhibitor pifithrin-μ was assessed by the standard broth dilution method against 62 clinical isolates of Aspergillus spp. and non-Aspergillus moulds (Mucoromycotina, Fusarium spp., Scedosporium spp., Purpureocillium/Paecilomyces spp. and Scopulariopsis spp.) RESULTS: FK506 had variable antifungal activity against different Aspergillus spp. and was particularly active against Mucor spp. Geldanamycin had moderate antifungal activity against Fusarium spp. and Paecilomyces variotii. Importantly, trichostatin A had good activity against the triazole-resistant Aspergillus ustus and the amphotericin B-resistant Aspergillus terreus as well as the MDR Scedosporium prolificans. Moreover, trichostatin A exhibited synergistic interactions with caspofungin against A. ustus and with geldanamycin against Rhizopus spp. for which none of the other agents showed activity. Pifithrin-μ exhibited little antifungal activity. CONCLUSIONS: Targeting the Hsp90-calcineurin axis at different levels resulted in distinct patterns of susceptibility among different fungal species. Lysine deacetylase inhibition may represent a promising novel antifungal strategy against emerging resistant moulds.