EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer.

The Notch1 gene has an important role in mammalian cell-fate decision and tumorigenesis. Upstream control mechanisms for transcription of this gene are still poorly understood. In a chemical genetics screen for small molecule activators of Notch signalling, we identified epidermal growth factor receptor (EGFR) as a key negative regulator of Notch1 gene expression in primary human keratinocytes, intact epidermis and skin squamous cell carcinomas (SCCs). The underlying mechanism for negative control of the Notch1 gene in human cells, as well as in a mouse model of EGFR-dependent skin carcinogenesis, involves transcriptional suppression of p53 by the EGFR effector c-Jun. Suppression of Notch signalling in cancer cells counteracts the differentiation-inducing effects of EGFR inhibitors while, at the same time, synergizing with these compounds in induction of apoptosis. Thus, our data reveal a key role of EGFR signalling in the negative regulation of Notch1 gene transcription, of potential relevance for combinatory approaches for cancer therapy.

Segregation in a mycorrhizal fungus alters rice growth and symbiosis-specific gene transcription

Arbuscular mycorrhizal fungi (AMF) form symbioses with the majority of plants, improving plant nutrition and diversity. Evidence exists suggesting that AMF contain populations of genetically different nucleotypes coexisting in a common cytoplasm. This potentially has two important consequences for their genetics. First, by random distribution of nuclei at spore formation, new offspring of an AMF could receive different complements of nucleotypes compared to the parent or siblings-we consider this as segregation. Second, genetic exchange between AMF would allow the mixing of nuclei, altering nucleotype diversity in new spores. Because segregation was assumed not to occur and genetic exchange has only recently been demonstrated, no attempts have been made to test whether this affects the symbiosis with plants. Here, we show that segregation occurs in the AMF Glomus intraradices and can enhance the growth of rice up to five times, even though neither parental nor crossed AMF lines induced a positive growth response. This process also resulted in an alteration of symbiosis-specific gene transcription in rice. Our results demonstrate that manipulation of AMF genetics has important consequences for the symbiotic effects on plants and could be used to enhance the growth of globally important crops.

SOCS-1 protein prevents Janus Kinase/STAT-dependent inhibition of beta cell insulin gene transcription and secretion in response to interferon-gamma.

In the pathogenesis of type I diabetes mellitus, activated leukocytes infiltrate pancreatic islets and induce beta cell dysfunction and destruction. Interferon (IFN)-gamma, tumor necrosis factor-alpha and interleukin (IL)-1 beta play important, although not completely defined, roles in these mechanisms. Here, using the highly differentiated beta Tc-Tet insulin-secreting cell line, we showed that IFN-gamma dose- and time-dependently suppressed insulin synthesis and glucose-stimulated secretion. As described previously IFN-gamma, in combination with IL-1 beta, also induces inducible NO synthase expression and apoptosis (Dupraz, P., Cottet, S., Hamburger, F., Dolci, W., Felley-Bosco, E., and Thorens, B. (2000) J. Biol. Chem. 275, 37672--37678). To assess the role of the Janus kinase/signal transducer and activator of transcription (STAT) pathway in IFN-gamma intracellular signaling, we stably overexpressed SOCS-1 (suppressor of cytokine signaling-1) in the beta cell line. We demonstrated that SOCS-1 suppressed cytokine-induced STAT-1 phosphorylation and increased cellular accumulation. This was accompanied by a suppression of the effect of IFN-gamma on: (i) reduction in insulin promoter-luciferase reporter gene transcription, (ii) decrease in insulin mRNA and peptide content, and (iii) suppression of glucose-stimulated insulin secretion. Furthermore, SOCS-1 also suppressed the cellular effects that require the combined presence of IL-1 beta and IFN-gamma: induction of nitric oxide production and apoptosis. Together our data demonstrate that IFN-gamma is responsible for the cytokine-induced defect in insulin gene expression and secretion and that this effect can be completely blocked by constitutive inhibition of the Janus kinase/STAT pathway.

Involvement of nuclear factor-kappaB in macrophage migration inhibitory factor gene transcription up-regulation induced by interleukin- 1 beta in ectopic endometrial cells.

OBJECTIVE: To investigate the involvement of the nuclear factor (NF)-kappaB in the interleukin (IL)-1 beta-mediated macrophage migration inhibitory factor (MIF) gene activation. DESIGN: Prospective study. SETTING: Human reproduction research laboratory. PATIENT(S): Nine women with endometriotic lesions. INTERVENTION(S): Endometriotic lesions were obtained during laparoscopic surgery. MAIN OUTCOME MEASURE(S): The MIF protein secretion was analyzed by ELISA, MIF mRNA expression by quantitative real-time polymerase chain reaction (PCR), NF-kappaB translocation into the nucleus by electrophoresis mobility shift assay, I kappaB phosphorylation and degradation by Western blot, and human MIF promoter activity by transient cell transfection. RESULT(S): This study showed a significant dose-dependent increase of MIF protein secretion and mRNA expression, the NF-kappaB translocation into the nucleus, I kappaB phosphorylation, I kappaB degradation, and human MIF promoter activity in endometriotic stromal cells in response to IL-1 beta. Curcumin (NF-kappaB inhibitor) significantly inhibited all these IL-1 beta-mediated effects. Analysis of the activity of deletion constructs of the human MIF promoter and a computer search localized two putative regulatory elements corresponding to NF-kappaB binding sites at positions -2538/-2528 bp and -1389/-1380 bp. CONCLUSION(S): This study suggests the involvement of the nuclear transcription factor NF-kappaB in MIF gene activation in ectopic endometrial cells in response to IL-1 beta and identifies a possible pathway of endometriosis-associated inflammation and ectopic cell growth.

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.

Genetic variability in Arbuscular Mycorrhizal Fungi : effect on gene transcription of "Oryza Sativa"

AbstractArbuscular Mycorrhizal Fungi (AMF) form obligate symbioses with the majority of land plants. These fungi influence the diversity and productivity of plants. AMF are unusual organisms, harbouring genetically different nuclei in a common cytoplasm (known as heterokaryosis). Genetic variability has been shown between AMF individuals coming from the same population. Recent findings showed that genetic exchange between genetically different AMF individuals was possible. Additionnaly, segregation was shown to occur at spore formation in AMF. These two processes were shown to increase genetic variability between AMF individuals.Because of the difficulty to study these organisms, almost nothing is known about the effect of intra-specific genetic variability in AMF on the plant transcriptome. The aim of this thesis was to bring insights into the effect of intra-specific genetic variability in AMF on plant gene transcription. We demonstrated that genetic exchange could influence expression of some symbiosis specific plant genes and the timing of the colonization of the fungi in plant roots. We also showed that segregation could have a large impact on plant gene transcription. Taken together, these results demonstrated that AMF intra-specific variability could profoundly affect the life of plants by altering various molecular pathways. Moreover, results obtained on rice open a field of research on AMF genetics in impromvment of growth in agricultural plants and should be taken into account for future experiments.RésuméLes champignons endomycorhiziens arbusculaires (CEA) forment une symbiose obligatoire avec la majorité des plantes sur terre. Ces champignons peuvent influencer la diversité et la productivité des plantes avec lesquelles ils forment la symbiose. Les CEA sont des organismes particuliers de part le fait qu'ils possèdent des noyaux génétiquement différents (appelés hétérocaryosis) dans un cytoplasme commun. Il a été montré qu'il existait de la variabilité génétique intra-specific chez les CEA. De plus, des études recentes ont montré que l'échange génétique chez les CEA était possible entre des individus génétiquement différents tout comme la ségrégation qui a aussi été démontrée au moment de la formation des nouvelles spores chez les CEA. Ces deux processus ont été montrés comme pouvant créer aussi de la variabilité génétique intra-specific.Du fait de la difficulté de travailler avec les CEA et à cause de la nouveauté de ces recherches, très peu de choses sont connues sur l'effet de l'échange génétique et de la ségrégation chez les CEA sur les plantes, et particulièrement au niveau moléculaire. Le but de cette thèse a été d'apporter la lumière sur les effets de la viariabilité génétique intra-specific chez les CEA, sur la transcription des gènes chez la plante. Nous avons pu montrer que l'échange génétique pouvait avoir des effets sur l'expression de gènes spécifiques à cette symbiose mais aussi pouvait influencer le timing de colonisation des racines de plantes par les CEA. Nous avons aussi montré que la ségrégation pouvait grandement influencer le transcriptome complet de la plante, et pas seulement les voies métaboliques spécifiques à la symbiose comme cela avait été montré auparavant.L'ensemble de ces résultats démontre l'importance de la variation intra-specific chez les CEA sur les plantes et leur implication sur leur cycle de vie en changeant l'expression de voies métaboliques. De plus, ces résultats obtenus sur le riz ouvrent un champ de recherches sur les plantes destinées à l'agriculture et devraient être pris en compte pour des expériences futures.

Differential control of Notch1 gene transcription by Klf4 and Sp3 transcription factors in normal versus cancer-derived keratinocytes.

In specific cell types like keratinocytes, Notch signaling plays an important pro-differentiation and tumor suppressing function, with down-modulation of the Notch1 gene being associated with cancer development. Besides being controlled by p53, little else is known on regulation of Notch1 gene expression in this context. We report here that transcription of this gene is driven by a TATA-less "sharp peak" promoter and that the minimal functional region of this promoter, which extends from the -342 bp position to the initiation codon, is differentially active in normal versus cancer cells. This GC rich region lacks p53 binding sites, but binds Klf4 and Sp3. This finding is likely to be of biological significance, as Klf4 and, to a lesser extent, Sp3 are up-regulated in a number of cancer cells where Notch1 expression is down-modulated, and Klf4 over-expression in normal cells is sufficient to down-modulate Notch1 gene transcription. The combined knock-down of Klf4 and Sp3 was necessary for the reverse effect of increasing Notch1 transcription, consistent with the two factors exerting an overlapping repressor function through their binding to the Notch1 promoter.

Histone deacetylase inhibitors repress macrophage migration inhibitory factor (MIF) expression by targeting MIF gene transcription through a local chromatin deacetylation.

The cytokine macrophage migration inhibitory factor plays a central role in inflammation, cell proliferation and tumorigenesis. Moreover, macrophage migration inhibitory factor levels correlate with tumor aggressiveness and metastatic potential. Histone deacetylase inhibitors are potent antitumor agents recently introduced in the clinic. Therefore, we hypothesized that macrophage migration inhibitory factor would represent a target of histone deacetylase inhibitors. Confirming our hypothesis, we report that histone deacetylase inhibitors of various chemical classes strongly inhibited macrophage migration inhibitory factor expression in a broad range of cell lines, in primary cells and in vivo. Nuclear run on, transient transfection with macrophage migration inhibitory factor promoter reporter constructs and transduction with macrophage migration inhibitory factor expressing adenovirus demonstrated that trichostatin A (a prototypical histone deacetylase inhibitor) inhibited endogenous, but not episomal, MIF gene transcription. Interestingly, trichostatin A induced a local and specific deacetylation of macrophage migration inhibitory factor promoter-associated H3 and H4 histones which did not affect chromatin accessibility but was associated with an impaired recruitment of RNA polymerase II and Sp1 and CREB transcription factors required for basal MIF gene transcription. Altogether, this study describes a new molecular mechanism by which histone deacetylase inhibitors inhibit MIF gene expression, and suggests that macrophage migration inhibitory factor inhibition by histone deacetylase inhibitors may contribute to the antitumorigenic effects of histone deacetylase inhibitors.

MALT1 auto-proteolysis is essential for NF-κB-dependent gene transcription in activated lymphocytes.

Mucosa-associated lymphoid tissue 1 (MALT1) controls antigen receptor-mediated signalling to nuclear factor κB (NF-κB) through both its adaptor and protease function. Upon antigen stimulation, MALT1 forms a complex with BCL10 and CARMA1, which is essential for initial IκBα phosphorylation and NF-κB nuclear translocation. Parallel induction of MALT1 protease activity serves to inactivate negative regulators of NF-κB signalling, such as A20 and RELB. Here we demonstrate a key role for auto-proteolytic MALT1 cleavage in B- and T-cell receptor signalling. MALT1 cleavage occurred after Arginine 149, between the N-terminal death domain and the first immunoglobulin-like region, and did not affect its proteolytic activity. Jurkat T cells expressing an un-cleavable MALT1-R149A mutant showed unaltered initial IκBα phosphorylation and normal nuclear accumulation of NF-κB subunits. Nevertheless, MALT1 cleavage was required for optimal activation of NF-κB reporter genes and expression of the NF-κB targets IL-2 and CSF2. Transcriptome analysis confirmed that MALT1 cleavage after R149 was required to induce NF-κB transcriptional activity in Jurkat T cells. Collectively, these data demonstrate that auto-proteolytic MALT1 cleavage controls antigen receptor-induced expression of NF-κB target genes downstream of nuclear NF-κB accumulation.

The promoter of the gene encoding 3',5'-cyclic adenosine monophosphate (cAMP) response element binding protein contains cAMP response elements: evidence for positive autoregulation of gene transcription.

The transcriptional transactivational activities of the phosphoprotein cAMP-response element-binding protein (CREB) are activated by the cAMP-dependent protein kinase A signaling pathway. Dimers of CREB bind to the palindromic DNA element 5'-TGACGTCA-3' (or similar motifs) called cAMP-responsive enhancers (CREs) found in the control regions of many genes, and activate transcription in response to phosphorylation of CREB by protein kinase A. Earlier we reported on the cyclical expression of the CREB gene in the Sertoli cells of the rat testis that occurred concomitant with the FSH-induced rise in cellular cAMP levels and suggested that transcription of the CREB gene may be autoregulated by cAMP-dependent transcriptional proteins. We now report the structure of the 5'-flanking sequence of the human CREB gene containing promoter activity. The promoter has a high content of guanosines and cytosines and lacks canonical TATA and CCAAT boxes typically found in the promoters of genes in eukaryotes. Notably, the promoter contains three CREs and transcriptional activities of a promoter-luciferase reporter plasmid transfected to placental JEG-3 cells are increased 3- to 5-fold over basal activities in response to either cAMP or 12-O-tetradecanoyl phorbol-14-acetate, and give 6- to 7-fold responses when both agents are added. The CREs bind recombinant CREB and endogenous CREB or CREB-like proteins contained in placental JEG-3 cells and also confer cAMP-inducible transcriptional activation to a heterologous minimal promoter. Our studies suggest that the expression of the CREB gene is positively autoregulated in trans.

Genetic exchange in an arbuscular mycorrhizal fungus results in increased rice growth and altered mycorrhiza-specific gene transcription.

Arbuscular mycorrhizal fungi (AMF) are obligate symbionts with most terrestrial plants. They improve plant nutrition, particularly phosphate acquisition, and thus are able to improve plant growth. In exchange, the fungi obtain photosynthetically fixed carbon. AMF are coenocytic, meaning that many nuclei coexist in a common cytoplasm. Genetic exchange recently has been demonstrated in the AMF Glomus intraradices, allowing nuclei of different Glomus intraradices strains to mix. Such genetic exchange was shown previously to have negative effects on plant growth and to alter fungal colonization. However, no attempt was made to detect whether genetic exchange in AMF can alter plant gene expression and if this effect was time dependent. Here, we show that genetic exchange in AMF also can be beneficial for rice growth, and that symbiosis-specific gene transcription is altered by genetic exchange. Moreover, our results show that genetic exchange can change the dynamics of the colonization of the fungus in the plant. Our results demonstrate that the simple manipulation of the genetics of AMF can have important consequences for their symbiotic effects on plants such as rice, which is considered the most important crop in the world. Exploiting natural AMF genetic variation by generating novel AMF genotypes through genetic exchange is a potentially useful tool in the development of AMF inocula that are more beneficial for crop growth.

Phosphorylation by casein kinase 2 facilitates rRNA gene transcription by promoting dissociation of TIF-IA from elongating RNA polymerase I.

The protein kinase casein kinase 2 (CK2) phosphorylates different components of the RNA polymerase I (Pol I) transcription machinery and exerts a positive effect on rRNA gene (rDNA) transcription. Here we show that CK2 phosphorylates the transcription initiation factor TIF-IA at serines 170 and 172 (Ser170/172), and this phosphorylation triggers the release of TIF-IA from Pol I after transcription initiation. Inhibition of Ser170/172 phosphorylation or covalent tethering of TIF-IA to the RPA43 subunit of Pol I inhibits rDNA transcription, leading to perturbation of nucleolar structure and cell cycle arrest. Fluorescence recovery after photobleaching and chromatin immunoprecipitation experiments demonstrate that dissociation of TIF-IA from Pol I is a prerequisite for proper transcription elongation. In support of phosphorylation of TIF-IA switching from the initiation into the elongation phase, dephosphorylation of Ser170/172 by FCP1 facilitates the reassociation of TIF-IA with Pol I, allowing a new round of rDNA transcription. The results reveal a mechanism by which the functional interplay between CK2 and FCP1 sustains multiple rounds of Pol I transcription.

Critical role for Ets, AP-1 and GATA-like transcription factors in regulating mouse Toll-like receptor 4 (Tlr4) gene expression.

TLR4 (Toll-like receptor 4) is essential for sensing the endotoxin of Gram-negative bacteria. Mutations or deletion of the TLR4 gene in humans or mice have been associated with altered predisposition to or outcome of Gram-negative sepsis. In the present work, we studied the expression and regulation of the Tlr4 gene of mouse. In vivo, TLR4 levels were higher in macrophages compared with B, T or natural killer cells. High basal TLR4 promoter activity was observed in RAW 264.7, J774 and P388D1 macrophages transfected with a TLR4 promoter reporter vector. Analysis of truncated and mutated promoter constructs identified several positive [two Ets (E twenty-six) and one AP-1 (activator protein-1) sites] and negative (a GATA-like site and an octamer site) regulatory elements within 350 bp upstream of the transcriptional start site. The myeloid and B-cell-specific transcription factor PU.1 bound to the proximal Ets site. In contrast, none among PU.1, Ets-1, Ets-2 and Elk-1, but possibly one member of the ESE (epithelium-specific Ets) subfamily of Ets transcription factors, bound to the distal Ets site, which was indispensable for Tlr4 gene transcription. Endotoxin did not affect macrophage TLR4 promoter activity, but it decreased TLR4 steady-state mRNA levels by increasing the turnover of TLR4 transcripts. TLR4 expression was modestly altered by other pro- and anti-inflammatory stimuli, except for PMA plus ionomycin which strongly increased promoter activity and TLR4 mRNA levels. The mouse and human TLR4 genes were highly conserved. Yet, notable differences exist with respect to the elements implicated in gene regulation, which may account for species differences in terms of tissue expression and modulation by microbial and inflammatory stimuli.

Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line.

In pancreatic beta-cells, the high Km glucose transporter GLUT2 catalyzes the first step in glucose-induced insulin secretion by glucose uptake. Expression of the transporter has been reported to be modulated by glucose either at the protein or mRNA levels. In this study we used the differentiated insulinoma cell line INS-1 which expresses high levels of GLUT2 and show that the expression of GLUT2 is regulated by glucose at the transcriptional level. By run-on transcription assays we showed that glucose induced GLUT2 gene transcription 3-4-fold in INS-1 cells which was paralleled by a 1.7-2.3-fold increase in cytoplasmic GLUT2 mRNA levels. To determine whether glucose regulatory sequences were present in the promoter region of GLUT2, we cloned and characterized a 1.4-kilobase region of mouse genomic DNA located 5' of the translation initiation site. By RNase protection assays and primer extension, we determined that multiple transcription initiation sites were present at positions -55, -64, and -115 from the first coding ATG and which were identified in liver, intestine, kidney, and beta-cells mRNAs. Plasmids were constructed with the mouse promoter region linked to the reporter gene chloramphenicol acetyltransferase (CAT), and transiently and stably transfected in the INS-1 cells. Glucose induced a concentration-dependent increase in CAT activity which reached a maximum of 3.6-fold at 20 mM glucose. Similar CAT constructs made of the human GLUT2 promoter region and the CAT gene displayed the same glucose-dependent increase in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence identity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common sequences.

Myelin gene expression during demyelination and remyelination in aggregating brain cell cultures.

Remyelination can be studied in aggregating rat brain cell cultures after limited demyelination. Demyelination was induced using a monoclonal antibody against myelin/oligodendrocyte glycoprotein (MOG mAb), in the presence of complement. De- and remyelination were assessed by measuring myelin basic protein (MBP). Two days after removing the MOG mAb, MBP levels reached 50% of controls and after 7 days 93%. During this period, cell proliferation determined by [14C]thymidine incorporation was similar in remyelinating and control cultures. Hormones and growth factors were tested for possible stimulatory effect on remyelinating cultures. Bovine growth hormone (bGH), triiodothyronine (T3), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) did not improve remyelination. Only epidermal growth factor (EGF) increased the level of remyelination. PDGF increased the rate of cell proliferation in both control and remyelinating cultures. A significant proportion of oligodendrocytes entered the cell division cycle and were not available for remyelination. The results obtained with PDGF and FGF (inhibition) support the idea that a pool of progenitor cells was still present and able to proliferate and differentiate into myelinating oligodendrocytes. The levels of myelin protein mRNAs were investigated during de- and remyelination. During demyelination, myelin protein mRNA levels decreased to approximately 50% of control cultures and returned to normal during remyelination. These preliminary results indicate that normal levels of gene transcription are sufficient to meet the increased need for newly synthesized myelin proteins during remyelination.