The major transcription initiation site of the SV40 late promoter is a potent thyroid hormone response element.

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor superfamily, which act as transcription factors upon binding to specific DNA sequences called thyroid hormone (T3) response elements (TREs). Such elements are found in the upstream regulatory region of promoters as well as in intragenic sequences of T3-responsive genes. In this report, we demonstrate that SV40 late (SVL) promoter activity is strongly down-regulated by TR in the absence of ligand. Addition of T3 releases this repression, but does not further induce SVL promoter activity. Electrophoretic mobility shift analyses reveal a TR binding element that overlaps with the SV40 major late transcription initiation site. This element closely fits the consensus TRE, formed of two hexanucleotides organized in a tandem repeat separated by 4 nt, and is able to confer T3 responsiveness on a heterologous promoter. We further show that, although the presence of TR leads to quantitatively modified expression of an SVL-driven reporter gene, neither displacement of the site of transcription initiation nor modification of the splicing pattern of the primary transcripts occur.

Germ cell-specific targeting of DICER or DGCR8 reveals a novel role for endo-siRNAs in the progression of mammalian spermatogenesis and male fertility.

Small non-coding RNAs act as critical regulators of gene expression and are essential for male germ cell development and spermatogenesis. Previously, we showed that germ cell-specific inactivation of Dicer1, an endonuclease essential for the biogenesis of micro-RNAs (miRNAs) and endogenous small interfering RNAs (endo-siRNAs), led to complete male infertility due to alterations in meiotic progression, increased spermatocyte apoptosis and defects in the maturation of spermatozoa. To dissect the distinct physiological roles of miRNAs and endo-siRNAs in spermatogenesis, we compared the testicular phenotype of mice with Dicer1 or Dgcr8 depletion in male germ cells. Dgcr8 mutant mice, which have a defective miRNA pathway while retaining an intact endo-siRNA pathway, were also infertile and displayed similar defects, although less severe, to Dicer1 mutant mice. These included cumulative defects in meiotic and haploid phases of spermatogenesis, resulting in oligo-, terato-, and azoospermia. In addition, we found by RNA sequencing of purified spermatocytes that inactivation of Dicer1 and the resulting absence of miRNAs affected the fine tuning of protein-coding gene expression by increasing low level gene expression. Overall, these results emphasize the essential role of miRNAs in the progression of spermatogenesis, but also indicate a role for endo-siRNAs in this process.

Characterization of PhlG, a hydrolase that specifically degrades the antifungal compound 2,4-diacetylphloroglucinol in the biocontrol agent Pseudomonas fluorescens CHA0.

The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative DeltaphlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a DeltaphlA DeltaphlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a kcat of 33 s(-1) and a Km of 140 microM at 30 degrees C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH.

Molecular clock is involved in predictive circadian adjustment of renal function.

Renal excretion of water and major electrolytes exhibits a significant circadian rhythm. This functional periodicity is believed to result, at least in part, from circadian changes in secretion/reabsorption capacities of the distal nephron and collecting ducts. Here, we studied the molecular mechanisms underlying circadian rhythms in the distal nephron segments, i.e., distal convoluted tubule (DCT) and connecting tubule (CNT) and the cortical collecting duct (CCD). Temporal expression analysis performed on microdissected mouse DCT/CNT or CCD revealed a marked circadian rhythmicity in the expression of a large number of genes crucially involved in various homeostatic functions of the kidney. This analysis also revealed that both DCT/CNT and CCD possess an intrinsic circadian timing system characterized by robust oscillations in the expression of circadian core clock genes (clock, bma11, npas2, per, cry, nr1d1) and clock-controlled Par bZip transcriptional factors dbp, hlf, and tef. The clock knockout mice or mice devoid of dbp/hlf/tef (triple knockout) exhibit significant changes in renal expression of several key regulators of water or sodium balance (vasopressin V2 receptor, aquaporin-2, aquaporin-4, alphaENaC). Functionally, the loss of clock leads to a complex phenotype characterized by partial diabetes insipidus, dysregulation of sodium excretion rhythms, and a significant decrease in blood pressure. Collectively, this study uncovers a major role of molecular clock in renal function.

Molecular insights into T cell activation : study of the TRAF, Bcl10 and Carma 1 proteins

Abstract : Activation of naïve T lymphocytes is essential for the onset of an adaptive immune response against a pathogenic threat. T lymphocytes are activated through the engagement of their highly specific cell surface antigen-receptor (TCR), together with co-stimulatory receptors, by activated antigen-presenting cells that display antigenic peptide fragments from the pathogen that they have detected. Dissection of the mechanisms that modulate TCR- and co-stimulation- induced signals is therefore crucial for the understanding of the molelcular basis of adaptive immune responses. Following antigen-receptor triggering, the Carma1, Bcl10 and Malt1 (CBM) proteins assemble into an oligomeric complex, which is essential for activation of the NF-κB and JNK signaling pathways in lymphocytes. In this work, by using human epithelial and lymphocytic cell lines, we identified the TNF-receptor-associated factor (TRAF) proteins TRAF3 and TRAF7 as new binding partners of Bcl10 and Carma1, respectively. We could show that TRAF3 is required for the proper transcriptional upregulation of IL-2 in activated T cells, and that endogenous TRAF3 is recruited to Bcl10 following TCR engagement. Although the mechanisms used by TRAF3 to modulate the transcriptional activation of the IL-2 promoter are not elucidated, the stimulus-dependent association ofTRAF3 with its direct binding partner Bcl10 suggests that TRAF3 is regulating Bcl10 function in TCR-activated lymphocytes. We also demonstrated that TRAF7 acts as a negative regulator of Carma1-induced NFκB-and AP1-dependent transcription by overexpression in 293T cells. These data suggest that TRAF7 could contribute to the negative regulation of TCR-dependent Carma1 functions. Finally, we showed that Carma1 is processed upon antigen-receptor triggering in B and T cell lines, as well as in primary human CTLs, and that this processing is dependent on the proteolytic activity of Malt1. Collectively, this work contributes to describe new proteins and regulatory mechanisms that modulate CBM-dependent functions in activated lymphocytes. Furthermore, it uncovers new tracks that could lead to a better molecular understanding of the complex interplay between the activatory and inhibitory regulators associated with the CBM complex. Résumé : L'activation des lymphocytes T naifs est une étape essentielle à la mise en place d'une réponse immunitaire adaptative pour combattre une infection. Après la détection d'un pathogène, les cellules présentatrices d'antigènes exposent à leur surface des fragments peptidiques provenant du pathogène, qui activent le récepteur à antigène (TCR) spécifique des lymphocytes T, ainsi que des molécules co-stimulatrices qui contribuent à l'activation complète des lymphocytes T. La caractérisation des mécanismes qui modulent les cascades de signaux émanant du TCR et des récepteurs de co-stimulation est essentielle à la compréhension du fonctionnement moléculaire de la réponse immunitaire adaptative. La ligation du TCR induit la formation d'un complexe oligomérique comprenant les protéines Carma1, Bcl10 et Malt1, qui est essentiel à l'activation des voies de signalisation cellulaires NF-κB et JNK induisant l'activation complète des lymphorctes T. Dans cette étude, à l'aide de lignées de cellules humaines épithéliales et lymphocytaires, nous avons identifié que deux protéines de la famille des TRAF (Tumor Necrosis Factor Receptor-Associated Factor), TRAF3 et TRAF7, s'associent à Bc110 et à Carma1, respectivement. Les TRAFs sont d'importants régulateurs des voies de signalisation dans les cellules du système immunitaire inné et adaptatif. Nous avons démontré que TRAF3 était important pour permettre la transcription de l'interleukine-2 (IL-2) dans les lymphocytes T activés, et que TRAF3 s'associait à Bc110 à la suite de la stimulation du TCR Les mécanismes que TRAF3 utilise pour moduler l'activation du promoteur de l'IL-2 ne sont pas connus, mais l'association de TRAF3 à Bc110 suite à la stimulation du TCR suggère que TRAF3 régule la fonction de Bc110. Nous avons également identifié TRAF7 comme un nouveau régulateur négatif des voies NF-κB et JNK induites par surexpression de la protéine Carma1. Nos données suggèrent que TRAF7 pourrait également contribuer à la régulation négative de la fonction de Carma1 dans les lymphocytes activés. Enfin, nous avons découvert que Carma1 était clivé suite à la stimulation du TCR, et que ce clivage dépendait de l'activité protéolytique de Malt1. Cette étude contribue ainsi à la description de nouvelles protéines et de nouveaux mécanismes qui modulent l'activité du complexe CBM dans les lymphocytes activés, et ouvre la voie à la caractérisation moléculaire de ces nouveaux mécanismes importants pour la régulation de la réponse immunitaire adaptative.

Spatio-temporal transcript profiling of rice roots and shoots in response to phosphate starvation and recovery.

Using rice (Oryza sativa) as a model crop species, we performed an in-depth temporal transcriptome analysis, covering the early and late stages of Pi deprivation as well as Pi recovery in roots and shoots, using next-generation sequencing. Analyses of 126 paired-end RNA sequencing libraries, spanning nine time points, provided a comprehensive overview of the dynamic responses of rice to Pi stress. Differentially expressed genes were grouped into eight sets based on their responses to Pi starvation and recovery, enabling the complex signaling pathways involved in Pi homeostasis to be untangled. A reference annotation-based transcript assembly was also generated, identifying 438 unannotated loci that were differentially expressed under Pi starvation. Several genes also showed induction of unannotated splice isoforms under Pi starvation. Among these, PHOSPHATE2 (PHO2), a key regulator of Pi homeostasis, displayed a Pi starvation-induced isoform, which was associated with increased translation activity. In addition, microRNA (miRNA) expression profiles after long-term Pi starvation in roots and shoots were assessed, identifying 20 miRNA families that were not previously associated with Pi starvation, such as miR6250. In this article, we present a comprehensive spatio-temporal transcriptome analysis of plant responses to Pi stress, revealing a large number of potential key regulators of Pi homeostasis in plants.

Mechanisms of HCF protein proteolytic maturation

Abstract : Post-translational modifications such as proteolytic processing, phosphorylation, and glycosylation, add extra layers of complexity to proteomes and allow a finely tuned regulation of the activity of many proteins. The evolutionarily conserved cell-cycle and transcriptional regulator HCP-] is regulated by proteolytic maturation via which a stable heterodirneric complex of two cleaved subunits is formed from a single precursor protein. The human HCF-1 precursor is cleaved at six nearly identical 26 amino acid sequence repeats, called HCF-1pro repeats, which represent uncommon protease recognition sites dedicated to human HCF-1 proteolysis. This proteolytic maturation process is conserved in vertebrate HCF-1 homologues and is essential for the functions of the human protein in cell-cycle regulation; the mechanisms that execute and control HCF-1 proteolysis, however, remain poorly understood. In this dissertation I investigate the mechanisms of proteolytic maturation of HCF-1 proteins in different species. I show that the Drosophila homolog of human HCF-1, called dHCP, is proteolytically cleaved via a different mechanism than human HCF-1. dHCP is processed by the same protease, called Taspase], which cleaves one of the key developmental regulators in flies, the Trithorax protein. Maturation of HCP proteins via Taspase] cleavage is probably not particular to dHCP as many invertebrate HCP proteins, particularly insects and flatworms, possess Taspase] recognition sites. In contrast, the vertebrate HCF-1 proteins lack Taspase] recognition sites and the HCF-1pro repeats are not Taspase1 substrates, suggesting that multiple mechanisms for HCF-1 proteolytic maturation have appeared during evolution. I also show that the proteolytic activity responsible for the cleavage of the HCP- 1pro repeats is very difficult to characterize, being resistant to most protease inhibitors and very sensitive to biochemical fractionation. Moreover, the HCF-1pro repeats represent complex protease recognition sites and I demonstrate that, in addition to be the HCF-1 cleavage sites, these repeated sequences, also recruit the OG1cNAc transferase OGT. The OGT protein and the OG1cNAc modification of HCF-1 are both important for HCF-1pro repeat proteolysis. Interestingly, a human recombinant OGT purified from insect cells is able to induce cleavage of a HCF-1pro-repeat precursor in vitro, indicating that OGT either (i) induces HCF-1 autoproteolysis,(ii) is the HCF-1pro- repeat proteolytic activity itself, or (iii) physically associates with a proteolytic activity that is conserved in insect cells. In any case, OGT plays an important role in HCF-1 proteolytic maturation and perhaps a broader role in HCF-1 biological function. Résumé : Les modifications post-traductionelles pomme le clivage protéolytique, la phosphorylation, et la glycosylation, augmentent significativement la complexité des protéomes et permettent une régulation fine de l'activité de beaucoup de protéines. La protéine HCF-1, qui est un régulateur du cycle cellulaire et de la transcription, est elle- même régulée par clivage protéolytique. La protéine HCF-1 est en effet coupée en deux sous-unités qui s'associent l'une a l'autre pour former la protéine mature. Le précurseur de la protéine HCF-1 humaine est clivé à six sites correspondant à six séquences répétées nommées les HCF-1pro repeats, chacune composée de 26 acide aminés. Les HCF-1pro- repeats ne ressemblent ai aucune séquence de clivage protéolytique connue et sont présentes seulement dans les protéines HCF-1 chez les vertébrés. Bien que la maturation protéolytique d'HCF-1 soit essentielle pour les activités de cette protéine pendant le cycle cellulaire, les mécanismes qui la contrôlent restent inconnus. Au cours de mon travail de thèse, j'ai analysé les mécanismes de clivage protéolytique des protéines HCF dans différentes espèces. J'ai montré que la protéine de Drosophile homologue d'HCF-1 humaine nommée dHCF est clivée par une protéase nommée Taspase1. Ainsi, dHCF est clivé par la même protéase que celle qui induit la maturation protéolytique d'un des principaux facteurs du développement chez la mouche, la protéine Trithorax. La maturation de dHCF via le clivage par la Taspase1 n'est pas spécifique à la mouche, mais est probablement étendu à plusieurs protéines HCF chez les invertébrés, surtout dans les familles des insectes et des plathehninthes, car ces protéines HCF présentent des sites de reconnaissance pour la Taspasel. Par contre, les protéines HCF-1 chez les vertébrés n'ont pas de sites de reconnaissance pour la Taspasel et cela suggère que différents mécanismes de maturation des protéines HCF- ls ont apparu au cours de l'évolution. J'ai montré aussi que les HCF-1pro-repeats sont clivés par une activité protéolytique très difficile a identifier, car elle est résistante à la plupart des inhibiteurs de protéases, mais elle est très sensible au fractionnement biochimique. En plus, les HCF-1pro-repeats sont un site de protéolyse complexe qui ne sert pas seulement au clivage des protéines HCF- chez les vertébrés mais aussi à recruter l'enzyme responsable de la O- GlcNAcylation nommée OGT. La protéine OGT et la O-GlcNAcylatio d'HCF-1 sont toutes les deux importantes pour le clivage protéolytique des HCF1pro-repeats. Curieusement, la protéine OGT humaine produite dans des cellules d'insectes est capable de cliver les HCF-1pro repeats in vitro et cela suggère que OGT soit (i) induit le clivage autocatalytique cl'HCF-1, soit (ii) est elle-même l'activité protéolytique qui clive HCF4, soit (iii) est associée à une activité protéolytique conservée dans les cellules d'insectes qui a été co-purifiée avec OGT. En conclusion, OGT joue un rôle important dans la maturation protéolytique d'HCF-1 et peut-être aussi un rôle plus large dans les fonctions biologiques de la protéine HCF-1.

Molecular basis of messenger RNA recognition by the specific bacterial repressing clamp RsmA/CsrA.

Proteins of the RsmA/CsrA family are global translational regulators in many bacterial species. We have determined the solution structure of a complex formed between the RsmE protein, a member of this family from Pseudomonas fluorescens, and a target RNA encompassing the ribosome-binding site of the hcnA gene. The RsmE homodimer with its two RNA-binding sites makes optimal contact with an 5'-A/UCANGGANGU/A-3' sequence in the mRNA. When tightly gripped by RsmE, the ANGGAN core folds into a loop, favoring the formation of a 3-base-pair stem by flanking nucleotides. We validated these findings by in vivo and in vitro mutational analyses. The structure of the complex explains well how, by sequestering the Shine-Dalgarno sequence, the RsmA/CsrA proteins repress translation.

Characterization of a genomic signature of pregnancy identified in the breast.

The objective of this study was to comprehensively compare the genomic profiles in the breast of parous and nulliparous postmenopausal women to identify genes that permanently change their expression following pregnancy. The study was designed as a two-phase approach. In the discovery phase, we compared breast genomic profiles of 37 parous with 18 nulliparous postmenopausal women. In the validation phase, confirmation of the genomic patterns observed in the discovery phase was sought in an independent set of 30 parous and 22 nulliparous postmenopausal women. RNA was hybridized to Affymetrix HG_U133 Plus 2.0 oligonucleotide arrays containing probes to 54,675 transcripts, scanned and the images analyzed using Affymetrix GCOS software. Surrogate variable analysis, logistic regression, and significance analysis of microarrays were used to identify statistically significant differences in expression of genes. The false discovery rate (FDR) approach was used to control for multiple comparisons. We found that 208 genes (305 probe sets) were differentially expressed between parous and nulliparous women in both discovery and validation phases of the study at an FDR of 10% and with at least a 1.25-fold change. These genes are involved in regulation of transcription, centrosome organization, RNA splicing, cell-cycle control, adhesion, and differentiation. The results provide initial evidence that full-term pregnancy induces long-term genomic changes in the breast. The genomic signature of pregnancy could be used as an intermediate marker to assess potential chemopreventive interventions with hormones mimicking the effects of pregnancy for prevention of breast cancer.

Mechanism of HFR1 function in adaptive growth responses in "Arabidopsis thaliana"

AbstractPlants are sessile organisms, which have evolved an astonishing ability to sense changes in their environment. Depending on the surrounding conditions, such as changes in light and temperature, plants modulate the activity of important transcriptional regulators. The shade avoidance syndrome (SAS) is one important mechanism for shade-intolerant plants to adapt their growth in high vegetative density. In shaded conditions plants sense a diminished red/far-red ratio via the phytochrome system and respond with morphological changes such as elongation growth of stems and petioles. The Phytochrome Interacting Factors 4 and 5 (PIF4 and PIF5) are positive regulators of the SAS and required for a full response (Lorrain et al, 2008). They regulate the SAS by inducing the expression of shade avoidance marker genes such as PIL1, ATHB2, XTR7 and HFR1 (Hornitschek et al, 2009; Lorrain et al, 2008).I investigated the molecular mechanism underlying the regulation of the SAS by HFR1 (long Hypocotyl in FR light). Although HFR1 is a PIF-related bHLH transcription factor, we discovered that HFR1 is a non-DNA binding protein. Moreover, we revealed that HFR1 inhibits an exaggerated SAS by forming non-DNA binding heterodimers with PIF4 and PIF5 (Hornitschek et al, 2009). This negative feedback loop is an important mechanism to limit elongation growth also in elevated temperatures. HFR1 accumulation and activity are highly temperature-dependent and the increased activity of HFR1 at warmer temperatures also provides an important restraint on PIF4-driven elongation growth (Foreman et al, 2011).Finally we performed a genome-wide analysis to determine how PIF4 and PIF5 regulate growth in response to shade. We identified potential PIF5- target genes, which represent many well-known shade-responsive genes. Our analysis of gene expression also revealed a role of PIF4 and PIF5 in simulated sun possibly via the regulation of auxin sensitivity.RésuméLes plantes sont des organismes sessiles ayant développé une capacité surprenante à détecter des changements dans leur environnement. En fonction des conditions extérieures, telles que les variations de lumière ou de température, elles adaptent l'activité d'importants régulateurs transcriptionnels. Le syndrome d'évitement de l'ombre (SAS), est un mécanisme important pour les plantes intolérantes à l'ombre leur permettant d'adapter leur croissance lorsqu'elles se développent dans des conditions de végétations très denses. Dans ces conditions, les plantes détectent une réduction de la quantité relative de lumière rouge par rapport à la lumière rouge-lointain (rapport R/FR). Ce changement, perçu via le système des phytochromes, induit des modifications morphologiques telle qu'une élongation des tiges et des pétioles. Les protéines PIF4 et PIF5 (Phytochrome Interacting Factors) sont des régulateurs positifs du SAS et sont nécessaires pour une réponse complète (Lorrain et al, 2008). Ces facteurs de transcription régulent le SAS en induisant l'expression de gènes marqueurs de cette réponse tels que PIL1, ATHB2, XTR7 et HFR1 (Hornitschek et al, 2009; Lorrain et al, 2008).J'ai étudié les mécanismes moléculaires sous-jacents à la régulation du SAS par HFR1 (long Hypocotyl in FR light). HFR1 est un facteur de transcription type bHLH de la famille des PIF, quoique nous ayons découvert que HFR1 est une protéine ne se liant pas à Γ ADN. Nous avons montré que HFR1 inhibe un SAS exagéré en formant des heterodimères avec PIF4 et PIF5 (Hornitschek et al, 2009). Nous avons également montré que cette boucle de régulation négative est également un mécanisme important pour limiter la croissance de l'élongation dans des conditions de fortes températures. De plus l'accumulation et l'activité de HFR1 augmentent avec la température ce qui permet d'inhiber plus fortement l'effet activateur de PIF4 sur la croissance.Enfin, nous avons effectué une analyse génomique à large échelle afin de déterminer comment PIF4 et PIF5 régulent la croissance en réponse à l'ombre. Nous avons identifié les gènes cibles potentiels de PIF5, correspondant en partie à des gènes connus dans la réponse de l'évitement de l'ombre. Notre analyse de l'expression des gènes a également révélé un rôle important de PIF4 et PIF5 dans des conditions de croissance en plein soleil, probablement via la régulation de la sensibilité à l'auxine.

No evidence for mutations of CTCFL/BORIS in Silver-Russell syndrome patients with IGF2/H19 imprinting control region 1 hypomethylation.

BACKGROUND: Silver-Russell syndrome (SRS) is a genetically and clinically heterogeneous disease. Although no protein coding gene defects have been reported in SRS patients, approximately 50% of SRS patients carry epimutations (hypomethylation) at the IGF2/H19 imprinting control region 1 (ICR1). Proper methylation at ICR1 is crucial for the imprinted expression of IGF2, a fetal growth factor. CTCFL, a testis-specific protein, has recently been proposed to play a role in the establishment of DNA methylation at the murine equivalent of ICR1. A screen was undertaken to assess whether CTCFL is mutated in SRS patients with hypomethylation, to explore a link between the observed epimutations and a genetic cause of the disease. METHODOLOGY/PRINCIPAL FINDINGS: DNA was obtained from 36 SRS patients with hypomethylation at ICR1. All CTCFL coding exons were sequenced and analyzed for duplications/deletions using both multiplex ligation-dependent probe amplification, with a custom CTCFL probe set, and genomic qPCR. Novel SNP alleles were analyzed for potential differential splicing in vitro utilizing a splicing assay. Neither mutations of CTCFL nor duplications/deletions were observed. Five novel SNPs were identified and have been submitted to dbSNP. In silico splice prediction suggested one novel SNP, IVS2-66A>C, activated a cryptic splice site, resulting in aberrant splicing and premature termination. In vitro splicing assays did not confirm predicted aberrant splicing. CONCLUSIONS/SIGNIFICANCE: As no mutations were detected at CTCFL in the patients examined, we conclude that genetic alterations of CTCFL are not responsible for the SRS hypomethylation. We suggest that analysis of other genes involved in the establishment of DNA methylation at imprinted genes, such as DNMT3A and DNMT3L, may provide insight into the genetic cause of hypomethylation in SRS patients.

The role of NFI-C liver regeneration and its potential function as a general regulator of regenerative processes

Collectively, research aimed to understand the regeneration of certain tissues has unveiled the existence of common key regulators. Knockout studies of the murine Nuclear Factor I-C (NFI-C) transcription factor revealed a misregulation of growth factor signaling, in particular that of transforming growth factor ß-1 (TGF-ßl), which led to alterations of skin wound healing and the growth of its appendages, suggesting it may be a general regulator of regenerative processes. We sought to investigate this further by determining whether NFI-C played a role in liver regeneration. Liver regeneration following two-thirds removal of the liver by partial hepatectomy (PH) is a well-established regenerative model whereby changes elicited in hepatocytes following injury lead to a rapid, phased proliferation. However, mechanisms controlling the action of liver proliferative factors such as transforming growth factor-ßl (TGF-ß1) and plasminogen activator inhibitor-1 (PAI-1) remain largely unknown. We show that the absence of NFI-C impaired hepatocyte proliferation due to an overexpression of PAI-1 and the subsequent suppression of urokinase plasminogen (uPA) activity and hepatocyte growth factor (HGF) signaling, a potent hepatocyte mitogen. This indicated that NFI-C first acts to promote hepatocyte proliferation at the onset of liver regeneration in wildtype mice. The subsequent transient down regulation of NFI-C, as can be explained by a self- regulatory feedback loop with TGF-ßl, may limit the number of hepatocytes entering the first wave of cell division and/or prevent late initiations of mitosis. Overall, we conclude that NFI-C acts as a regulator of the phased hepatocyte proliferation during liver regeneration. Taken together with NFI-C's actions in other in vivo models of (re)generation, it is plausible that NFI-C may be a general regulator of regenerative processes. - L'ensemble des recherches visant à comprendre la régénération de certains tissus a permis de mettre en évidence l'existence de régulateurs-clés communs. L'étude des souris, dépourvues du gène codant pour le facteur de transcription NFI-C (Nuclear Factor I-C), a montré des dérèglements dans la signalisation de certains facteurs croissance, en particulier du TGF-ßl (transforming growth factor-ßl), ce qui conduit à des altérations de la cicatrisation de la peau et de la croissance des poils et des dents chez ces souris, suggérant que NFI-C pourrait être un régulateur général du processus de régénération. Nous avons cherché à approfondir cette question en déterminant si NFI-C joue un rôle dans la régénération du foie. La régénération du foie, induite par une hépatectomie partielle correspondant à l'ablation des deux-tiers du foie, constitue un modèle de régénération bien établi dans lequel la lésion induite conduit à la prolifération rapide des hépatocytes de façon synchronisée. Cependant, les mécanismes contrôlant l'action de facteurs de prolifération du foie, comme le facteur de croissance TGF-ßl et l'inhibiteur de l'activateur du plasminogène PAI-1 (plasminogen activator inhibitor-1), restent encore très méconnus. Nous avons pu montrer que l'absence de NFI-C affecte la prolifération des hépatocytes, occasionnée par la surexpression de PAI-1 et par la subséquente suppression de l'activité de la protéine uPA (urokinase plasminogen) et de la signalisation du facteur de croissance des hépatocytes HGF (hepatocyte growth factor), un mitogène puissant des hépatocytes. Cela indique que NFI-C agit en premier lieu pour promouvoir la prolifération des hépatocytes au début de la régénération du foie chez les souris de type sauvage. La subséquente baisse transitoire de NFI-C, pouvant s'expliquer par une boucle rétroactive d'autorégulation avec le facteur TGF-ßl, pourrait limiter le nombre d'hépatocytes qui entrent dans la première vague de division cellulaire et/ou inhiber l'initiation de la mitose tardive. L'ensemble de ces résultats nous a permis de conclure que NFI-C agit comme un régulateur de la prolifération des hépatocytes synchrones au cours de la régénération du foie.

ICER induced by hyperglycemia represses the expression of genes essential for insulin exocytosis.

The GTPases Rab3a and Rab27a and their effectors Granuphilin/Slp4 and Noc2 are essential regulators of neuroendocrine secretion. Chronic exposure of pancreatic beta-cells to supraphysiological glucose levels decreased selectively the expression of these proteins. This glucotoxic effect was mimicked by cAMP-raising agents and blocked by PKA inhibitors. We demonstrate that the transcriptional repressor ICER, which is induced in a PKA-dependent manner by chronic hyperglycemia and cAMP-raising agents, is responsible for the decline of the four genes. ICER overexpression diminished the level of Granuphilin, Noc2, Rab3a and Rab27a by binding to cAMP responsive elements located in the promoters of these genes and inhibited exocytosis of beta-cells in response to secretagogues. Moreover, the loss in the expression of the genes of the secretory machinery caused by glucose and cAMP-raising agents was prevented by an antisense construct that reduces ICER levels. We propose that induction of inappropriate ICER levels lead to defects in the secretory process of pancreatic beta-cells possibly contributing, in conjunction with other known deleterious effects of hyperglycemia, to defective insulin release in type 2 diabetes.

Antifungal drug resistance mechanisms in fungal pathogens from the perspective of transcriptional gene regulation.

Fungi are primitive eukaryotes and have adapted to a variety of niches during evolution. Some fungal species may interact with other life forms (plants, insects, mammals), but are considered as pathogens when they cause mild to severe diseases. Chemical control strategies have emerged with the development of several drugs with antifungal activity against pathogenic fungi. Antifungal agents have demonstrated their efficacy by improving patient health in medicine. However, fungi have counteracted antifungal agents in several cases by developing resistance mechanisms. These mechanisms rely on drug resistance genes including multidrug transporters and drug targets. Their regulation is crucial for the development of antifungal drug resistance and therefore transcriptional factors critical for their regulation are being characterized. Recent genome-wide studies have revealed complex regulatory circuits involving these genetic and transcriptional regulators. Here, we review the current understanding of the transcriptional regulation of drug resistance genes from several fungal pathogens including Candida and Aspergillus species.

IRF4 and BATF are critical for CD8(+) T-cell function following infection with LCMV.

CD8(+) T-cell functions are critical for preventing chronic viral infections by eliminating infected cells. For healthy immune responses, beneficial destruction of infected cells must be balanced against immunopathology resulting from collateral damage to tissues. These processes are regulated by factors controlling CD8(+) T-cell function, which are still incompletely understood. Here, we show that the interferon regulatory factor 4 (IRF4) and its cooperating binding partner B-cell-activating transcription factor (BATF) are necessary for sustained CD8(+) T-cell effector function. Although Irf4(-/-) CD8(+) T cells were initially capable of proliferation, IRF4 deficiency resulted in limited CD8(+) T-cell responses after infection with the lymphocytic choriomeningitis virus. Consequently, Irf4(-/-) mice established chronic infections, but were protected from fatal immunopathology. Absence of BATF also resulted in reduced CD8(+) T-cell function, limited immunopathology, and promotion of viral persistence. These data identify the transcription factors IRF4 and BATF as major regulators of antiviral cytotoxic T-cell immunity.