Cooperative binding of estrogen receptor to imperfect estrogen-responsive DNA elements correlates with their synergistic hormone-dependent enhancer activity.

The Xenopus vitellogenin (vit) gene B1 estrogen-inducible enhancer is formed by two closely adjacent 13 bp imperfect palindromic estrogen-responsive elements (EREs), i.e. ERE-2 and ERE-1, having one and two base substitutions respectively, when compared to the perfect palindromic consensus ERE (GGTCANNNTGACC). Gene transfer experiments indicate that these degenerated elements, on their own, have a low or no regulatory capacity at all, but in vivo act together synergistically to confer high receptor- and hormone-dependent transcription activation to the heterologous HSV thymidine kinase promoter. Thus, the DNA region upstream of the vitB1 gene comprising these two imperfect EREs separated by 7 bp, was called the vitB1 estrogen-responsive unit (vitB1 ERU). Using in vitro protein-DNA interaction techniques, we demonstrate that estrogen receptor dimers bind cooperatively to the imperfect EREs of the vitB1 ERU. Binding of a first receptor dimer to the more conserved ERE-2 increases approximately 4- to 8-fold the binding affinity of the receptor to the adjacent less conserved ERE-1. Thus, we suggest that the observed synergistic estrogen-dependent transcription activation conferred by the pair of hormone-responsive DNA elements of the vit B1 ERU is the result of cooperative binding of two estrogen receptor dimers to these two adjacent imperfect EREs.

The redox potential interferes with the expression of laminin binding molecules in Bacteroides fragilis

The Bacteroides fragilis ATCC strain was grown in a synthetic media with contrasting redox potential (Eh) levels [reduced (-60 mV) or oxidised (+100mV)] and their adhesion capacity to extracellular matrix components was evaluated. The strain was capable of adhering to laminin, fibronectin, fibronectin + heparan sulphate and heparan sulphate. A stronger adherence to laminin after growing the strain under oxidising conditions was verified. Electron microscopy using ruthenium red showed a heterogeneous population under this condition. Dot-blotting analyses confirmed stronger laminin recognition by outer membrane proteins of cells cultured at a higher Eh. Using a laminin affinity column, several putative laminin binding proteins obtained from the cultures kept under oxidising (60 kDa, 36 kDa, 25 kDa and 15 kDa) and reducing (60 kDa) conditions could be detected. Our results show that the expression of B. fragilis surface components that recognise laminin are influenced by Eh variations.

The RNA-binding protein HuR regulates DNA methylation through stabilization of DNMT3b mRNA

The molecular basis underlying the aberrant DNA-methylation patterns in human cancer is largely unknown. Altered DNA methyltransferase (DNMT) activity is believed to contribute, as DNMT expression levels increase during tumorigenesis. Here, we present evidence that the expression of DNMT3b is post-transcriptionally regulated by HuR, an RNA-binding protein that stabilizes and/or modulates the translation of target mRNAs. The presence of a putative HuR-recognition motif in the DNMT3b 3'UTR prompted studies to investigate if this transcript associated with HuR. The interaction between HuR and DNMT3b mRNA was studied by immunoprecipitation of endogenous HuR ribonucleoprotein complexes followed by RT-qPCR detection of DNMT3b mRNA, and by in vitro pulldown of biotinylated DNMT3b RNAs followed by western blotting detection of HuR. These studies revealed that binding of HuR stabilized the DNMT3b mRNA and increased DNMT3b expression. Unexpectedly, cisplatin treatment triggered the dissociation of the [HuR-DNMT3b mRNA] complex, in turn promoting DNMT3b mRNA decay, decreasing DNMT3b abundance, and lowering the methylation of repeated sequences and global DNA methylation. In summary, our data identify DNMT3b mRNA as a novel HuR target, present evidence that HuR affects DNMT3b expression levels post-transcriptionally, and reveal the functional consequences of the HuR-regulated DNMT3b upon DNA methylation patterns.

Common variants of the liver fatty acid binding protein gene influence the risk of type 2 diabetes and insulin resistance in Spanish population

SUMMARY The main objective was to evaluate the association between SNPs and haplotypes of the FABP1-4 genes and type 2 diabetes, as well as its interaction with fat intake, in one general Spanish population. The association was replicated in a second population in which HOMA index was also evaluated. METHODS 1217 unrelated individuals were selected from a population-based study [Hortega study: 605 women; mean age 54 y; 7.8% with type 2 diabetes]. The replication population included 805 subjects from Segovia, a neighboring region of Spain (446 females; mean age 52 y; 10.3% with type 2 diabetes). DM2 mellitus was defined in a similar way in both studies. Fifteen SNPs previously associated with metabolic traits or with potential influence in the gene expression within the FABP1-4 genes were genotyped with SNPlex and tested. Age, sex and BMI were used as covariates in the logistic regression model. RESULTS One polymorphism (rs2197076) and two haplotypes of the FABP-1 showed a strong association with the risk of DM2 in the original population. This association was further confirmed in the second population as well as in the pooled sample. None of the other analyzed variants in FABP2, FABP3 and FABP4 genes were associated. There was not a formal interaction between rs2197076 and fat intake. A significant association between the rs2197076 and the haplotypes of the FABP1 and HOMA-IR was also present in the replication population. CONCLUSIONS The study supports the role of common variants of the FABP-1 gene in the development of type 2 diabetes in Caucasians.

Characterization of a cAMP-regulated enhancer-binding protein.

The transcription regulation of many hormone genes is modulated by intracellular second messengers such as cAMP. The cAMP response element binding protein, CREB, binds to the 8 base pair CRE enhancer, TGACGTCA, that is found in the 5'-flank of certain genes including those for somatostatin and the alpha-subunit of human chorionic gonadotropin. The recent characterization of CREB and CREB-related cDNA clones, combined with Southwesterns and Northern blot analyses, reveals a family of transcription factors that dimerize via a leucine zipper motif and bind to the CRE through positively charged basic regions. The CREB cDNA encoding a 327 residue protein is transcriptionally activated via phosphorylation by protein kinases, including the cAMP-dependent protein kinase-A.

Molecular analysis of an odorant-binding protein gene in two sympatric species of Lutzomyia longipalpis s.l.

Lutzomyia longipalpis s.l. is the main vector of American visceral leishmaniasis (AVL) and occurs as a species complex. DNA samples from two Brazilian sympatric species that differ in pheromone and courtship song production were used to analyse molecular polymorphisms in an odorant-binding protein ( obp29 ) gene. OBPs are proteins related to olfaction and are involved in activities fundamental to survival, such as foraging, mating and choice of oviposition site. In this study, the marker obp29 was found to be highly polymorphic in Lu. longipalpis s.l. , with no fixed differences observed between the two species. A pairwise fixation index test indicated a moderate level of genetic differentiation between the samples analysed.

The N-terminal DNA-binding 'zinc finger' of the oestrogen and glucocorticoid receptors determines target gene specificity.

Steroid hormone receptors activate specific gene transcription by binding as hormone-receptor complexes to short DNA enhancer-like elements termed hormone response elements (HREs). We have shown previously that a highly conserved 66 amino acid region of the oestrogen (ER) and glucocorticoid (GR) receptors, which corresponds to part of the receptor DNA binding domain (region C) is responsible for determining the specificity of target gene activation. This region contains two sub-regions (CI and CII) analogous to the 'zinc-fingers' of the transcription factor TFIIIA. We show here that CI and CII appear to be separate domains both involved in DNA binding. Furthermore, using chimaeric ERs in which either the first (N-terminal) (CI) or second (CII) 'zinc finger' region has been exchanged with that of the GR, indicates that it is the first 'zinc finger' which largely determines target gene specificity. We suggest that receptor recognition of the HRE is analogous to that of the helix-turn-helix DNA binding motif in that the receptor binds to DNA as a dimer with the first 'zinc finger' lying in the major groove recognizing one half of the palindromic HRE, and that protein-DNA interaction is stabilized through non-specific DNA binding and dimer interactions contributed by the second 'zinc finger'.

A constitutively active mutant of the alpha 1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins G9 alpha and G11 alpha than the wild-type receptor.

Rat 1 fibroblasts transfected to express either the wild-type hamster alpha 1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288-294 to encode the equivalent region of the human beta 2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAM alpha 1B-adrenergic receptor was greater than for the wild-type receptor, The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAM alpha 1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA, receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the alpha subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins G9 and G11 in cells expressing either the wild-type or the CAM alpha 1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAM alpha 1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of G9 alpha/G11 alpha degradation between cells expressing the wild-type or the CAMalpha 1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAM alpha 1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAM alpha 1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to G9 and G11.

Chromatin domain boundaries delimited by a histone-binding protein in yeast.

When located next to chromosomal elements such as telomeres, genes can be subjected to epigenetic silencing. In yeast, this is mediated by the propagation of the SIR proteins from telomeres toward more centromeric regions. Particular transcription factors can protect downstream genes from silencing when tethered between the gene and the telomere, and they may thus act as chromatin domain boundaries. Here we have studied one such transcription factor, CTF-1, that binds directly histone H3. A deletion mutagenesis localized the barrier activity to the CTF-1 histone-binding domain. A saturating point mutagenesis of this domain identified several amino acid substitutions that similarly inhibited the boundary and histone binding activities. Chromatin immunoprecipitation experiments indicated that the barrier protein efficiently prevents the spreading of SIR proteins, and that it separates domains of hypoacetylated and hyperacetylated histones. Together, these results suggest a mechanism by which proteins such as CTF-1 may interact directly with histone H3 to prevent the propagation of a silent chromatin structure, thereby defining boundaries of permissive and silent chromatin domains.

Complex organization of CTF/NF-I, C/EBP, and HNF3 binding sites within the promoter of the liver-specific vitellogenin gene.

Vitellogenin genes are expressed specifically in the liver of female oviparous vertebrates under the strict control of estrogen. To explain this tissue-specific expression, we performed a detailed analysis of the Xenopus laevis vitellogenin gene B1 promoter by DNase I footprinting and gel mobility-shift assays. We characterized five binding sites for the ubiquitous factor CTF/NF-I. Two of these sites are close to the TATA-box, whereas the others are located on both sides of the estrogen responsive unit formed by two imperfect estrogen response elements. Moreover two liver-enriched factors, C/EBP and HNF3, were found to interact with multiple closely spaced proximal promoter elements in the first 100 base pairs upstream of the TATA-box. To confirm the physiological significance of this in vitro analysis, in vivo DNase I footprinting experiments were carried out using the ligation-mediated polymerase chain reaction technique. The various cis-elements characterized in vitro as binding sites for known transcription factors and more particularly for liver-enriched transcription factors are efficiently recognized in vivo as well, suggesting that they play an important role in the control of the liver-specific vitellogenin gene B1 expression.

Global GacA-steered control of cyanide and exoprotease production in Pseudomonas fluorescens involves specific ribosome binding sites.

The conserved two-component regulatory system GacS/GacA determines the expression of extracellular products and virulence factors in a variety of Gram-negative bacteria. In the biocontrol strain CHA0 of Pseudomonas fluorescens, the response regulator GacA is essential for the synthesis of extracellular protease (AprA) and secondary metabolites including hydrogen cyanide. GacA was found to exert its control on the hydrogen cyanide biosynthetic genes (hcnABC) and on the aprA gene indirectly via a posttranscriptional mechanism. Expression of a translational hcnA'-'lacZ fusion was GacA-dependent whereas a transcriptional hcnA-lacZ fusion was not. A distinct recognition site overlapping with the ribosome binding site appears to be primordial for GacA-steered regulation. GacA-dependence could be conferred to the Escherichia coli lacZ mRNA by a 3-bp substitution in the ribosome binding site. The gene coding for the global translational repressor RsmA of P. fluorescens was cloned. RsmA overexpression mimicked partial loss of GacA function and involved the same recognition site, suggesting that RsmA is a downstream regulatory element of the GacA control cascade. Mutational inactivation of the chromosomal rsmA gene partially suppressed a gacS defect. Thus, a central, GacA-dependent switch from primary to secondary metabolism may operate at the level of translation.

Identification of potential small molecule binding pockets on Rho family GTPases

Rho GTPases are conformational switches that control a wide variety of signaling pathways critical for eukaryotic cell development and proliferation. They represent attractive targets for drug design as their aberrant function and deregulated activity is associated with many human diseases including cancer. Extensive high-resolution structures (.100) and recent mutagenesis studies have laid the foundation for the design of new structure-based chemotherapeutic strategies. Although the inhibition of Rho signaling with drug-like compounds is an active area of current research, very little attention has been devoted to directly inhibiting Rho by targeting potential allosteric non-nucleotide binding sites. By avoiding the nucleotide binding site, compounds may minimize the potential for undesirable off-target interactions with other ubiquitous GTP and ATP binding proteins. Here we describe the application of molecular dynamics simulations, principal component analysis, sequence conservation analysis, and ensemble small-molecule fragment mapping to provide an extensive mapping of potential small-molecule binding pockets on Rho family members. Characterized sites include novel pockets in the vicinity of the conformationaly responsive switch regions as well as distal sites that appear to be related to the conformations of the nucleotide binding region. Furthermore the use of accelerated molecular dynamics simulation, an advanced sampling method that extends the accessible time-scale of conventional simulations, is found to enhance the characterization of novel binding sites when conformational changes are important for the protein mechanism.

The loss of GLUT2 expression in the pancreatic beta-cells of diabetic db/db mice is associated with an impaired DNA-binding activity of islet-specific trans-acting factors.

GLUT2 expression is reduced in the pancreatic beta-cells of several diabetic animals. The transcriptional control of the gene in beta-cells involves at least two islet-specific DNA-binding proteins, GTIIa and PDX-1, which also transactivates the insulin, somatostatin and glucokinase genes. In this report, we assessed the DNA-binding activities of GTIIa and PDX-1 to their respective cis-elements of the GLUT2 promoter using nuclear extracts prepared from pancreatic islets of 12 week old db/db diabetic mice. We show that the decreased GLUT2 mRNA expression correlates with a decrease of the GTIIa DNA-binding activity, whereas the PDX-1 binding activity is increased. In these diabetic animals, insulin mRNA expression remains normal. The adjunction of dexamethasone to isolated pancreatic islets, a treatment previously shown to decrease PDX-1 expression in the insulin-secreting HIT-T15 cells, has no effect on the GTIIa and PDX-1 DNA-binding activities. These data suggest that the decreased activity of GTIIa, in contrast to PDX-1, may be a major initial step in the development of the beta-cell dysfunction in this model of diabetes.

Tumor necrosis factor and transforming growth factor β regulate clock genes by controlling the expression of the cold inducible RNA-binding protein (CIRBP).

The circadian clock drives the rhythmic expression of a broad array of genes that orchestrate metabolism, sleep wake behavior, and the immune response. Clock genes are transcriptional regulators engaged in the generation of circadian rhythms. The cold inducible RNA-binding protein (CIRBP) guarantees high amplitude expression of clock. The cytokines TNF and TGFβ impair the expression of clock genes, namely the period genes and the proline- and acidic amino acid-rich basic leucine zipper (PAR-bZip) clock-controlled genes. Here, we show that TNF and TGFβ impair the expression of Cirbp in fibroblasts and neuronal cells. IL-1β, IL-6, IFNα, and IFNγ do not exert such effects. Depletion of Cirbp is found to increase the susceptibility of cells to the TNF-mediated inhibition of high amplitude expression of clock genes and modulates the TNF-induced cytokine response. Our findings reveal a new mechanism of cytokine-regulated expression of clock genes.

Amniotic fluid insulin-like growth factor binding-protein 3 concentration as early indicator of fetal growth restriction

Rapport de synthèse : Introduction : La croissance foetale infra-utérine dépend d'un grand nombre de facteurs maternels, placentaires et foetaux. Une inadéquation d'un ou plusieurs de ces facteurs peut induire un retard de croissance infra-utérin (RCIU) ou au contraire une macrosomie. Les principales causes de RCIU comprennent les infections maternelles, l'éclampsie, les cardiovasculopathies maternelles, la toxicomanie, les malformations foetales et les insuffisances placentaires. Les facteurs endocriniens constituent un petit pourcentage des causes de RCIU, mais méritent que l'on s'y intéresse de plus près. Les facteurs hormonaux les plus importants pour la croissance fatale sont l'insuline et les insuline-like growth factors (IGFs) et non l'hormone de croissance (GH) qui joue un rôle majeur dans la croissance postnatale. Notre attention s'est portée sur IGF-1 qui joue un rôle important dans la croissance intrautérine. Sa biodisponibilité dépend de plusieurs protéines plasmatiques, les IGF-binding proteins (IGFBP 1 à 9). IGFBP-3 est la principale de ces IGFBPs, autant d'un point de vue quantitatif que fonctionnel. Nous avons cherché à déterminer si les concentrations d'IGF-1 et d'IGFBP-3 dans le liquide amniotique au début du deuxième trimestre étaient prédictives de la croissance infra-utérine. Les gènes codant pour IGF-1 et IGFBP-3 contenant certaines séquences polymorphiques, nous avons également étudié leur influence sur la croissance foetale. L'analyse du liquide amniotique présente l'avantage de pouvoir être effectuée dès la 14ème semaine d'aménorrhée alors que la biométrie foetale échographique ne permet pas à ce stade de déceler des anomalies de la croissance infra-utérine. Méthode : Nous avons analysé des échantillons de liquide amniotique prélevés entre la 14ème et la 18ème semaine de grossesse chez 196 patientes. Les concentrations d'IGF-1 et d'IGFBP-3 ont été dosées par ELISA, les polymorphismes analysés par PCR. Ces résultats ont été ensuite analysés en fonction du poids de naissance des nouveaux-nés, répartis en trois groupes normal pour l'âge gestationnel (AGA), petit pour l'âge gestationnel (SGA) et grand pour l'âge gestationnel (LGA). Résultats : Les concentrations d'IGFBP3 dans le liquide amniotique sont significativement plus élevées (p = 0.030) dans le groupe SGA par rapport au groupe AGA, d'autant plus quand les taux sont ajustés en fonction de paramètres tels que l'âge gestationnel lors de l'amniocentèse (ANCOVA analysis : p = 0.009). La distribution du polymorphisme VNTR (variable number of tandem repeat) dans la région promotrice d'IGF-1 au sein du groupe SGA est significativement différente de celle du groupe AGA (p = 0.029). En effet, la fréquence de l'association allélique 19CA/20CA est diminuée dans le groupe SGA. Nous n'avons pas identifié de différence de distribution des séquences polymorphiques d'IGFBP-3 entre les différents groupes. Conclusion : Une concentration élevée d'IGFBP-3 dans le liquide amniotique au début du deuxième trimestre est associée à un risque plus élevé de retard de croissance alors que l'association allélique 19CA/20CA dans la région polymorphique IGF-1 VNTR est un facteur protecteur.