A novel role for proline- and acid-rich basic region leucine zipper (PAR bZIP) proteins in the transcriptional regulation of a BH3-only proapoptotic gene.

Proline- and acid-rich (PAR) basic region leucine zipper (bZIP) proteins thyrotroph embryonic factor (TEF), D-site-binding protein (DBP), and hepatic leukemia factor have been involved in neurotransmitter homeostasis and amino acid metabolism. Here we demonstrate a novel role for these proteins in the transcriptional control of a BH3-only gene. PAR bZIP proteins are able to transactivate the promoter of bcl-gS. This promoter is particularly responsive to TEF activation and is silenced by NFIL3, a repressor that shares the consensus binding site with PAR bZIP proteins. Consistently, transfection of TEF induces the expression of endogenous bcl-gS in cancer cells, and this induction is independent of p53. A naturally occurring variant of DBP (tDBP), lacking the transactivation domain, has been identified and shown to impede the formation of active TEF dimers in a competitive manner and to reduce the TEF-dependent induction of bcl-gS. Of note, treatment of cancer cells with etoposide induces TEF activation and promotes the expression of bcl-gS. Furthermore, blockade of bcl-gS or TEF expression by a small interfering RNA strategy or transfection with tDBP significantly reduces the etoposide-mediated apoptotic cell death. These findings represent the first described role for PAR bZIP proteins in the regulation of a gene involved in the execution of apoptosis.

Regulation of the DNA-binding and transcriptional activities of Xenopus laevis NFI-X by a novel C-terminal domain.

The nuclear factor I (NFI) family consists of sequence-specific DNA-binding proteins that activate both transcription and adenovirus DNA replication. We have characterized three new members of the NFI family that belong to the Xenopus laevis NFI-X subtype and differ in their C-termini. We show that these polypeptides can activate transcription in HeLa and Drosophila Schneider line 2 cells, using an activation domain that is subdivided into adjacent variable and subtype-specific domains each having independent activation properties in chimeric proteins. Together, these two domains constitute the full NFI-X transactivation potential. In addition, we find that the X. laevis NFI-X proteins are capable of activating adenovirus DNA replication through their conserved N-terminal DNA-binding domains. Surprisingly, their in vitro DNA-binding activities are specifically inhibited by a novel repressor domain contained within the C-terminal part, while the dimerization and replication functions per se are not affected. However, inhibition of DNA-binding activity in vitro is relieved within the cell, as transcriptional activation occurs irrespective of the presence of the repressor domain. Moreover, the region comprising the repressor domain participates in transactivation. Mechanisms that may allow the relief of DNA-binding inhibition in vivo and trigger transcriptional activation are discussed.

Neuronal traits are required for glucose-induced insulin secretion.

The transcriptional repressor RE1 silencer transcription factor (REST) is an important factor that restricts some neuronal traits to neurons. Since these traits are also present in pancreatic beta-cells, we evaluated their role by generating a model of insulin-secreting cells that express REST. The presence of REST led to a decrease in expression of its known target genes, whereas insulin expression and its cellular content were conserved. As a consequence of REST expression, the capacity to secrete insulin in response to mitochondrial fuels, a particularity of mature beta-cells, was impaired. These data provide evidence that REST target genes are required for an appropriate glucose-induced insulin secretion.

Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules. RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P. aeruginosa. An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P. aeruginosa. A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P. aeruginosa. Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation. Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA.

E2F activation of S phase promoters via association with HCF-1 and the MLL family of histone H3K4 methyltransferases.

E2F transcriptional regulators control human-cell proliferation by repressing and activating the transcription of genes required for cell-cycle progression, particularly the S phase. E2F proteins repress transcription in association with retinoblastoma pocket proteins, but less is known about how they activate transcription. Here, we show that the human G1 phase regulator HCF-1 associates with both activator (E2F1 and E2F3a) and repressor (E2F4) E2F proteins, properties that are conserved in insect cells. Human HCF-1-E2F interactions are versatile: their associations and binding to E2F-responsive promoters are cell-cycle selective, and HCF-1 displays coactivator properties when bound to the E2F1 activator and corepressor properties when bound to the E2F4 repressor. During the G1-to-S phase transition, HCF-1 recruits the mixed-lineage leukemia (MLL) and Set-1 histone H3 lysine 4 methyltransferases to E2F-responsive promoters and induces histone methylation and transcriptional activation. These results suggest that HCF-1 induces cell-cycle-specific transcriptional activation by E2F proteins to promote cell proliferation.

Molecular mechanisms for the transcriptional regulation of the Connexin36 gene expression in insulin-secreting cells

Résumé Régulation de l'expression de la Connexin36 dans les cellules sécrétrices d'insuline La communication intercellulaire est en partie assurée via des jonctions communicantes de type "gap". Dans la cellule ß pancréatique, plusieurs observations indiquent que le couplage assuré par des jonctions gap formées parla Connexine36 (Cx36) est impliqué dans le contrôle de la sécrétion de l'insuline. De plus, nous avons récemment démontré qu'un niveau précis d'expression de la Cx36 est nécessaire pour maintenir une bonne coordination de l'ensemble des cellules ß, et permettre ainsi une sécrétion synchrone et contrôlée d'insuline. Le développement du diabète et du syndrome métabolique est partiellement dû à une altération de la capacité des cellules ß à sécréter de l'insuline en réponse à une augmentation de la glycémie. Cette altération est en partie causée par l'augmentation prolongée des taux circulant de glucose, mais aussi de lipides, sous la forme d'acides gras libres, et de LDL (Low Density Lipoproteins), particules assurant le transport des acides gras et du cholestérol dans le sang. Nous avons étudié la régulation de l'expression de la Cx36 dans différentes conditions reflétant la physiopathologie du diabète de type 2 et du syndrome métabolique et démontré qu'une exposition prolongée à des concentrations élevées de glucose, de LDL, ainsi que de palmitate (acide gras saturé le plus abondant dans l'organisme), inhibent l'expression de la Cx36 dans les cellules ß. Cette inhibition implique l'activation de la PKA (Proteine Kinase A), qui stimule à son tour l'expression du facteur de transcription ICER-1 (Inductible cAMP Early Repressor-1). Ce puissant répresseur se fixe spécifiquement sur un motif CRE (cAMP Response Element), situé dans le promoteur du gène de la Cx36, inhibant ainsi son expression. Nous avons de plus démontré que des cytokines pro-inflammatoires, qui pourraient contribuer au développement du diabète, inhibent également l'expression de la Cx36. Cependant, les cytokines agissent indépendamment du répresseur ICER-1, mais selon un mécanisme requérant l'activation de l'AMPK (AMP dependant protein kinase). Sachant qu'un contrôle précis des niveaux d'expression de la Cx36 est un élément déterminant pour une sécrétion optimale de l'insuline, nos résultats suggèrent que la Cx36 pourrait être impliquée dans l'altération de la sécrétion de l'insuline contribuant à l'apparition du diabète de type 2. Summary A particular way by which cells communicate with each other is mediated by gap junctions, transmembrane structures providing a direct pathway for the diffusion of small molecules between adjacent cells. Gap junctional communication is required to maintain a proper functioning of insulin-secreting ß-cells. Moreover, the expression levels of connexin36 (Cx36), the sole gap junction protein expressed in ß-cells, are critical in maintaining glucose-stimulated insulin secretion. Chronic hyperglycemia and hyperlipidemia exert deleterious effects on insulin secretion and may contribute to the progressive ß-cell failure linked to the development of type 2 diabetes and metabolic syndrome. Since modulations of the Cx36 levels might impair ß-cell function, the general aim of this work was to elucidate wether elevated levels of glucose and lipids affect Cx36 expression. The first part of this work was dedicated to the study of the effect of high glucose concentrations on Cx36 expression. We demonstrated that glucose transcriptionally down-regulates the expression of Cx36 in insulin-secreting cells through activation of the protein kinase A (PKA), which in turn stimulates the expression of the inducible cAMP early repressor-1 (ICER-1). This repressor binds to a highly conserved cAMP response element (CRE) located in the Cx36 promoter, thereby inhibiting Cx36 expression. The second part of this thesis consisted in studying the effects of sustained exposure to free fatty acids (FFA) and human lipoproteins on Cx36 levels. The experiments revealed that the most abundant FFA, palmitate, as well as the atherogenic low density lipoproteins (LDL), also stimulate ICER-1 expression, resulting in Cx36 down-regulation. Finally, the third part of the work focused on the consequences of long-term exposure to proinflammatory cytokines on Cx36 content. Interleukin-1 ß (IL-1 ß) inhibits Cx36 expression and its effect is potentialized by tumor necrosis factor α (TNFα) and interferon γ (IFNγ). We further unveiled that the cytokines effect on Cx36 levels requires activation of the AMP dependent protein kinase (AMPK). Prolonged exposures to glucose, palmitate, LDL, and pro-inflammatory cytokines have all been proposed to contribute to the development of diabetes and metabolic syndrome. Since Cx36 expression levels are critical to maintain ß-cell function, Cx36 down-regulation by glucose, lipids, and cytokines might participate to the ß-cell failure associated with diabetes development.

Astrocyte dense-core vesicle gliosecretion is governed by rest/nrsf

Astrocytes are the brain non-nerve cells competent for the expression of clear and dense-core vesicles (DCVs) and for their regulated exocytosis. This process, called gliosecretion, nearly resembles the neurosecretion occurring in neurons and neurosecretory cells. REST/NRSF is a transcription repressor known to orchestrate nerve-cell differentiation, governing the expression of hundreds of neuron-specific genes through their repression in the non-nerve and their fine modulation in the nerve cells. Our previous studies in neurosecretory rat PC12 cells identified REST as the critical factor for the expression not only of individual genes, but also of the whole neurosecretory process via multiple, direct and indirect mechanisms (D'Alessandro et al., J. Neurochem., 2008; Klajn et al., J. Neurosci., 2009). Therefore we wondered whether gliosecretion was governed by REST. We investigated rat astrocyte primary cultures: they exhibited high REST, which directly represses the transcription of at least one target gene, and expressed neither DCVs nor their markers (granins, peptides, membrane proteins). Transfection of a dominant-negative construct of REST (REST/ DBD-GFP) induced the appearance of DCVs filled with secretogranin2 and NPY that are distinct from other intracellular organelles. TIRF analysis of astrocytes co-transfected with REST/DBD-GFP and NPY-mRFP constructs revealed NPY-mRFP-positive DCVs undergoing Ca2þ-dependent exocytosis, largely prevented by BoNT/B. Immunohistochemistry of the I-II layers of the human temporal brain cortex showed all neurons and microglia exhibiting the expected inappreciable and high levels of REST, respectively. In contrast astrocyte RESTwas variable, going from inappreciable to high, accompanied by variable expression of DCVs. In this work it has been demonstrated that astrocyte DCV expression and gliosecretion are governed by REST (Prada et al., 2011 in press). The variable in situ REST levels may contribute to the well known structural/functional heterogeneity of astrocytes and this new observation might be of great interest for the understanding of both astrocyte physiology and pathology.

Comparative study and identification of potent eukaryotic transcriptional repressors in gene switch systems.

In mammalian cells, proper gene regulation is achieved by the complex interplay of transcription factors that activate or repress gene expression by binding to the regulatory regions of target promoters. While transcriptional activators have been extensively characterised and classified into functional groups, relatively little is known about the comparative strength and cell type-specificity of transcriptional repressors. Here, we have compared the ability of a series of eukaryotic repression domains to silence basal and activated transcription. A series of the most potent repression domains was further tested in the context of a gene therapy gene-switch system in various cell types. The results indicate that the analysed repression domains exert varying silencing activities in different promoter contexts. Furthermore, their potential for gene silencing varies also depending on the cellular context. When multimerised within one chimeric repressor protein, particular combinations of repressor domains were found to display synergistic repressing effects and efficient repression in a panel of cell lines. This approach thus allowed the identification of transcriptional repressors that are both potent and versatile in terms of cellular specificity as a basis for gene switch systems.

cAMP prevents the glucose-mediated stimulation of GLUT2 gene transcription in hepatocytes.

Glucose homoeostasis necessitates the presence in the liver of the high Km glucose transporter GLUT2. In hepatocytes, we and others have demonstrated that glucose stimulates GLUT2 gene expression in vivo and in vitro. This effect is transcriptionally regulated and requires glucose metabolism within the hepatocytes. In this report, we further characterized the cis-elements of the murine GLUT2 promoter, which confers glucose responsiveness on a reporter gene coding the chloramphenicol acetyl transferase (CAT) gene. 5'-Deletions of the murine GLUT2 promoter linked to the CAT reporter gene were transfected into a GLUT2 expressing hepatoma cell line (mhAT3F) and into primary cultured rat hepatocytes, and subsequently incubated at low and high glucose concentrations. Glucose stimulates gene transcription in a manner similar to that observed for the endogenous GLUT2 mRNA in both cell types; the -1308 to -212 bp region of the promoter contains the glucose-responsive elements. Furthermore, the -1308 to -338 bp region of the promoter contains repressor elements when tested in an heterologous thymidine kinase promoter. The glucose-induced GLUT2 mRNA accumulation was decreased by dibutyryl-cAMP both in mhAT3F cells and in primary hepatocytes. A putative cAMP-responsive element (CRE) is localized at the -1074/-1068 bp region of the promoter. The inhibitory effect of cAMP on GLUT2 gene expression was observed in hepatocytes transfected with constructs containing this CRE (-1308/+49 bp fragment), as well as with constructs not containing the consensus CRE (-312/+49 bp fragment). This suggests that the inhibitory effect of cAMP is not mediated by the putative binding site located in the repressor fragment of the GLUT2 promoter. Taken together, these data demonstrate that the elements conferring glucose and cAMP responsiveness on the GLUT2 gene are located within the -312/+49 region of the promoter.

The Quorum-Sensing Molecules Farnesol/Homoserine Lactone and Dodecanol Operate via Distinct Modes of Action in Candida albicans.

Living as a commensal, Candida albicans must adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects on C. albicans, with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C(12)-homoserine lactone, a quorum-sensing molecule secreted by Pseudomonas aeruginosa, block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of the Candida adenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving the C. albicans hyphal repressor, Sfl1p. Deletion of SFL1 did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing in C. albicans is mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that the Burkholderia cenocepacia diffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C(12)-homoserine lactone, may be used by other quorum-sensing molecules.

Role of the transcriptional factor C/EBPbeta in free fatty acid-elicited beta-cell failure.

Fatty acids can favour the development of Type 2 diabetes by reducing insulin secretion and inducing apoptosis of pancreatic beta-cells. Here, we show that sustained exposure of the beta-cell line MIN6 or of isolated pancreatic islets to the most abundant circulating fatty acid palmitate increases the level of C/EBPbeta, an insulin transcriptional repressor. In contrast, two unsaturated fatty acids, oleate and linoleate were without effect. The induction of C/EBPbeta elicited by palmitate was prevented by inhibiting the ERK1/2 MAP kinase pathway or by reducing mitochondrial fatty acid oxidation with an inhibitor of Carnitine Palmitoyl Transferase-1. Overexpression of C/EBPbeta mimicked the detrimental effects of palmitate and resulted in a drastic reduction in insulin promoter activity, impairment in the capacity to respond to secretory stimuli and an increase in apoptosis. Our data suggest a potential involvement of C/EBPbeta as mediator of the deleterious effects of unsaturated free fatty acids on beta-cell function.

Multifaceted roles of peroxisome proliferator-activated receptors (PPARs) at the cellular and whole organism levels.

Chronic disorders, such as obesity, diabetes, inflammation, non-alcoholic fatty liver disease and atherosclerosis, are related to alterations in lipid and glucose metabolism, in which peroxisome proliferator-activated receptors (PPAR)α, PPARβ/δ and PPARγ are involved. These receptors form a subgroup of ligand-activated transcription factors that belong to the nuclear hormone receptor family. This review discusses a selection of novel PPAR functions identified during the last few years. The PPARs regulate processes that are essential for the maintenance of pregnancy and embryonic development. Newly found hepatic functions of PPARα are the mediation of female-specific gene repression and the protection of the liver from oestrogen induced toxicity. PPARα also controls lipid catabolism and is the target of hypolipidaemic drugs, whereas PPARγ controls adipocyte differentiation and regulates lipid storage; it is the target for the insulin sensitising thiazolidinediones used to treat type 2 diabetes. Activation of PPARβ/δ increases lipid catabolism in skeletal muscle, the heart and adipose tissue. In addition, PPARβ/δ ligands prevent weight gain and suppress macrophage derived inflammation. In fact, therapeutic benefits of PPAR ligands have been confirmed in inflammatory and autoimmune diseases, such as encephalomyelitis and inflammatory bowel disease. Furthermore, PPARs promote skin wound repair. PPARα favours skin healing during the inflammatory phase that follows injury, whilst PPARβ/δ enhances keratinocyte survival and migration. Due to their collective functions in skin, PPARs represent a major research target for our understanding of many skin diseases. Taken altogether, these functions suggest that PPARs serve as physiological sensors in different stress situations and remain valuable targets for innovative therapies.

Deregulated MHC class II transactivator expression leads to a strong Th2 bias in CD4+ T lymphocytes.

The MHC class II (MHC-II) transactivator (CIITA) is the master transcriptional regulator of genes involved in MHC-II-restricted Ag presentation. Fine tuning of CIITA gene expression determines the cell type-specific expression of MHC-II genes. This regulation is achieved by the selective usage of multiple CIITA promoters. It has recently been suggested that CIITA also contributes to Th cell differentiation by suppressing IL-4 expression in Th1 cells. In this study, we show that endogenous CIITA is expressed at low levels in activated mouse T cells. Importantly CIITA is not regulated differentially in murine and human Th1 and Th2 cells. Ectopic expression of a CIITA transgene in multiple mouse cell types including T cells, does not interfere with normal development of CD4(+) T cells. However, upon TCR activation the CIITA transgenic CD4(+) T cells preferentially differentiate into IL-4-secreting Th2-type cells. These results imply that CIITA is not a direct Th1-specific repressor of the IL-4 gene and that tight control over the expression of CIITA and MHC-II is required to maintain the normal balance between Th1 and Th2 responses.

Control and host-dependent activation of insect toxin expression in a root-associated biocontrol pseudomonad.

Pseudomonas fluorescens CHA0 is a root-associated biocontrol agent that suppresses soil-borne fungal diseases of crops. Remarkably, the pseudomonad is also endowed with systemic and oral activity against pest insects which depends on the production of the insecticidal Fit toxin. The toxin gene (fitD) is part of a virulence cassette encoding three regulators (FitF, FitG, FitH) and a type I secretion system (FitABC-E). Immunoassays with a toxin-specific antibody and transcriptional analyses involving fitG and fitH deletion and overexpression mutants identified LysR family regulator FitG and response regulator FitH as activator and repressor, respectively, of Fit toxin and transporter expression. To visualize and quantify toxin expression in single live cells by fluorescence microscopy, we developed reporters which in lieu of the native toxin protein express a fusion of the Fit toxin with red fluorescent mCherry. In a wild-type background, expression of the mCherry-tagged Fit toxin was activated at high levels in insect hosts, i.e. when needed, yet not on plant roots or in batch culture. By contrast, a derepressed fitH mutant expressed the toxin in all conditions. P. fluorescens hence can actively induce insect toxin production in response to the host environment, and FitH and FitG are key regulators in this mechanism.

Negative control of quorum sensing by RpoN (sigma54) in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa PAO1, the expression of several virulence factors such as elastase, rhamnolipids, and hydrogen cyanide depends on quorum-sensing regulation, which involves the lasRI and rhlRI systems controlled by N-(3-oxododecanoyl)-L-homoserine lactone and N-butyryl-L-homoserine lactone, respectively, as signal molecules. In rpoN mutants lacking the transcription factor sigma(54), the expression of the lasR and lasI genes was elevated at low cell densities, whereas expression of the rhlR and rhlI genes was markedly enhanced throughout growth by comparison with the wild type and the complemented mutant strains. As a consequence, the rpoN mutants had elevated levels of both signal molecules and overexpressed the biosynthetic genes for elastase, rhamnolipids, and hydrogen cyanide. The quorum-sensing regulatory protein QscR was not involved in the negative control exerted by RpoN. By contrast, in an rpoN mutant, the expression of the gacA global regulatory gene was significantly increased during the entire growth cycle, whereas another global regulatory gene, vfr, was downregulated at high cell densities. In conclusion, it appears that GacA levels play an important role, probably indirectly, in the RpoN-dependent modulation of the quorum-sensing machinery of P. aeruginosa.