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International audience; The transcription factor CCAAT/enhancer binding protein-alpha (C/EBP alpha) is involved in the control of cell differentiation and proliferation, and has been suggested to act as a tumor suppressor in several cancers. By using microarray analysis, we have previously shown that hypoxia and estrogen down-regulate C/EBP alpha mRNA in T-47D breast cancer cells. Here, we have examined the mechanism by which the down-regulation by hypoxia takes place. Using the specific RNA polymerase II inhibitor 5,6-dichlorobenzimidazole-1-beta-D-ribofuranoside, the mRNA stability was analyzed under normoxia or hypoxia by quantitative reverse transcription-PCR. Hypoxia reduced the half-life of C/EBP alpha mRNA by approximately 30%. C/EBP alpha gene promoter studies indicated that hypoxia also repressed the transcription of the gene and identified a hypoxia-responsive element (-522; -527 bp), which binds to hypoxia-inducible factor (HIF)-1 alpha, as essential for down-regulation of C/EBP alpha transcription in hypoxia. Immunocytochemical analysis showed that C/EBP alpha was localized in the nucleus at 21% O(2), but was mostly cytoplasmic under 1% O(2). Knockdown of HIF-1 alpha by RNAi restored C/EBP alpha to normal levels under hypoxic conditions. Immunohistochemical studies of 10 tumor samples did not show any colocalization of C/EBP alpha and glucose transporter 1 (used as a marker for hypoxia). Taken together, these results show that hypoxia down-regulates C/EBP alpha expression in breast cancer cells by several mechanisms, including transcriptional and posttranscriptional effects. The down-regulation of C/EBP alpha in hypoxia is mediated by HIF-1.

International audience; Transcriptional activators, several different coactivators, and general transcription factors are necessary to access specific loci in the dense chromatin structure to allow precise initiation of RNA polymerase II (Pol II) transcription. Histone acetyltransferase (HAT) complexes were implicated in loosening the chromatin around promoters and thus in gene activation. Here we demonstrate that the 2 MDa GCN5 HAT-containing metazoan TFTC/STAGA complexes contain a histone H2A and H2B deubiquitinase activity. We have identified three additional subunits of TFTC/STAGA (ATXN7L3, USP22, and ENY2) that form the deubiquitination module. Importantly, we found that this module is an enhancer of position effect variegation in Drosophila. Furthermore, we demonstrate that ATXN7L3, USP22, and ENY2 are required as cofactors for the full transcriptional activity by nuclear receptors. Thus, the deubiquitinase activity of the TFTC/STAGA HAT complex is necessary to counteract heterochromatin silencing and acts as a positive cofactor for activation by nuclear receptors in vivo.

International audience; BubR1 is a key protein mediating spindle checkpoint activation. Loss of this checkpoint control results in chromosomal instability and aneuploidy. The transcriptional regulation of the cell cycle regulated human BUB1B gene, which encodes BubR1, was investigated in this report. A minimal BUB1B gene promoter containing 464 bp upstream from the translation initiation codon was sufficient for cell cycle regulated promoter activity. A pivotal role for transcription factor hStaf/ZNF143 in the expression of the BUB1B gene was demonstrated through gel retardation assays, transient expression of mutant BUB1B promoter-reporter gene constructs and chromatin immunoprecipitation assay. Two phylogenetically conserved hStaf/ZNF143-binding sites (SBS) were identified which are indispensable for BUB1B promoter activity. In addition, we found that the domain covering the transcription start sites contains conserved boxes homologous to initiator (Inr), cell cycle dependent (CDE) and cell cycle genes homology regions (CHR) elements. Mutations within the CDE and CHR elements led to diminished cell cycle regulation of BUB1B transcription. These results demonstrate that BUB1B gene transcription is positively regulated by hStaf/ZNF143, a ubiquitously expressed factor, and that the CDE-CHR tandem element was essential for G2/M-specific transcription of the BUB1B gene.

We studied Tcirg1 gene expression on RANKL-induced osteoclastic differentiation of the mouse model RAW264.7 cells. We identified a mechanism involving PARP-1 inhibition release and JunD/Fra-2 binding, which is responsible for Tcirg1 gene upregulation. Introduction: The Tcirg1 gene encodes the a3 isoform of the V-ATPase a subunit, which plays a critical role in the resorption activity of the osteoclast. Using serial deletion constructs of the Tcirg1 gene promoter, we performed a transcriptional study to identify factor(s) involved in the regulation of the RANKL-induced gene expression. Materials and Methods: The promoter activity of serial-deletion fragments of the Tcirg1 gene promoter was monitored throughout the RAW264.7 cells differentiation process. We next performed sequence analysis, EMSA, UV cross-linking, qPCR, and gel supershift experiments to identify the factor(s) interacting with the promoter. Results: A deletion of the −1297−1244 region led to the disappearance of the RANKL-induced promoter activity. EMSA experiments showed the binding of two factors that undergo differential binding on RANKL treatment. Supershift experiments led us to identify the dimer JunD/Fra-2 as the binding activity associated with the −1297/−1268 Tcirg1 gene promoter sequence in response to RANKL. Moreover, we observed poly(ADP-ribose) polymerase-1 (PARP-1) binding to an adjacent site (−1270/−1256), and this interaction was disrupted after RANKL treatment. Conclusions: We provide data that identify junD proto-oncogene (JunD) and Fos-related antigen 2 (Fra-2) as the activator protein-1 (AP-1) factors responsible for the RANKL-induced upregulation of the mouse Tcirg1 gene expression. Moreover, we identified another binding site for PARP-1 that might account for the repression of Tcirg1 gene expression in pre-osteoclastic cells.

P-cadherin expression is restricted to the basal layer of stratified epithelia including that of the mammary gland. Although evidence for an important role of P-cadherin in mammary morphogenesis and tumorigenesis is increasing, the mechanisms that regulate its expression are poorly understood. We show that in basal mammary epithelial cells, beta-catenin is associated with the P-cadherin promoter and activates its expression independently of LEF/TCF in a cell-type specific manner. Down-regulation of endogenous beta-catenin levels by RNA interference technique inhibited P-cadherin promoter activity. In vivo, in skin and mammary gland of mutant mice, activation of beta-catenin signalling correlates with up-regulation of P-cadherin expression. These data suggest that beta-catenin-dependent modulation of P-cadherin expression can contribute to the establishment of the basal phenotype.

The emergence of mutations in the androgen receptor (AR) gene is a recurrent event during progression of prostate cancer (PCa) on androgen ablation therapy. In this study, we show that nonsense mutations that lead to carboxyl-terminal end truncated ARs are found at high frequency in metastatic PCas. Transcriptional activities of the Q640X mutant AR in the androgen-sensitive LNCaP cell line differ to those of the wild-type AR. Indeed, this mutant AR exhibits strong and ligand-independent transcriptional activities from an artificial promoter construct containing two repeats of androgen-responsive elements, but is inactive on the human PSA gene promoter. Nevertheless, the expression of the Q640X mutant AR in LNCaP cells is accompanied by an increase in the level of PSA protein, and by an increase in the expression of the endogenous AR gene. This enhanced expression of the endogenous AR gene is not limited to the sole transfected cells, but is observed in non-transfected neighboring cells. Additionally, in co-cultures of transfected and non-transfected LNCaP cells, the Q640X mutant AR leads to an unpredicted nuclear localization of the endogenous AR protein in the two cellular populations and this, in the absence of androgen. These data indicate that cells expressing the Q640X mutant AR acquire the property to emit a signal that activates the AR in neighboring cells by a paracrine mechanism and in a ligand-independent manner. Our data strongly support the notion of cooperation among tumor cells in PCa and could be of relevance for the understanding of progression on androgen ablation therapy.

The respiratory syncytial virus (RSV) phosphoprotein (P) is a major polymerase co-factor that interacts with both the large polymerase fragment (L) and the nucleoprotein (N). The N-binding domain of RSV P has been investigated by co-expression of RSV P and N proteins in Escherichia coli. Pull-down assays performed with a series of truncated forms of P fused to glutathione S-transferase (GST) revealed that the region comprising the last nine C-terminal amino acid residues of P (233-DNDLSLEDF-241) is sufficient for efficient binding to N. Site-directed mutagenesis shows that the last four residues of this peptide are crucial for binding and must be present at the end of a flexible C-terminal tail. The presence of the P oligomerization domain (residues 100–160) was an important stabilizing factor for the interaction. The tetrameric full-length P fused to GST was able to pull down both helical and ring structures, whereas a monomeric C-terminal fragment of P (residues 161–241) fused to GST pulled down exclusively RNA–N rings. Electron-microscopy analysis of the purified rings showed the presence of two types of complex: undecamers (11N) and decamers (10N). Mass-spectrometry analysis of the RNA extracted from rings after RNase A treatment showed two peaks of 22 900 and 24 820 Da, corresponding to a mean RNA length of 67 and 73 bases, respectively. These results suggest strongly that each N subunit contacts 6 nt, with an extra three or four bases further protected from nuclease digestion by the ring structure at both the 5′ and 3′ ends.

The number of mRNAs and proteins that can be produced from a single gene is known to be increased by the number of start sites and by multiple splicing of products. A few genes have been found to generate extraordinarily large numbers of splicing isoforms. In the human, the largest number, nearly 2000 mRNA isoforms, has been reported for the neurexin 3α gene. However, the biological significance of alternative splicing often remains unclear because many alternative transcripts contain early translational stops and are thought to be rapidly degraded. We demonstrate here that human basonuclin 2 (bn2; approved gene symbol BNC2) transcripts are initiated from six promoters, are alternatively spliced at multiple positions, and are polyadenylated at four sites. Characterization of nearly 100 bn2 mRNA isoforms suggests that each promoter, splice site, and poly(A) addition site is used independently. The bn2 gene has therefore the potential to generate up to 90,000 mRNA isoforms encoding more than 2000 different proteins. Because alternative exons affect the position of the first methionine codon, the length of the coding region, and the position of the translational stop, the encoded proteins range in size from 43 to 1211 amino acids and some bear no sequence similarity to others. PCR analysis and transient expression in HeLa cells show that the major bn2 mRNA isoforms are stable and are translated into equally stable proteins, even when the mRNA bears an early translational stop.

International audience; PromAn is a modular web-based tool dedicated to promoter analysis that integrates distinct complementary databases, methods and programs. PromAn provides automatic analysis of a genomic region with minimal prior knowledge of the genomic sequence. Prediction programs and experimental databases are combined to locate the transcription start site (TSS) and the promoter region within a large genomic input sequence. Transcription factor binding sites (TFBSs) can be predicted using several public databases and user-defined motifs. Also, a phylogenetic footprinting strategy, combining multiple alignment of large genomic sequences and assignment of various scores reflecting the evolutionary selection pressure, allows for evaluation and ranking of TFBS predictions. PromAn results can be displayed in an interactive graphical user interface, PromAnGUI. It integrates all of this information to highlight active promoter regions, to identify among the huge number of TFBS predictions those which are the most likely to be potentially functional and to facilitate user refined analysis. Such an integrative approach is essential in the face of a growing number of tools dedicated to promoter analysis in order to propose hypotheses to direct further experimental validations. PromAn is publicly available at http://bips.u-strasbg.fr/PromAn.

International audience; 12-Lipoxygenase utilizes arachidonic acid to synthesize 12(S)-hydroperoxyeicosatetraenoic acid, which is converted to the end product 12(S)-hydroxyeicosatetraenoic acid, an eicosanoid that promotes tumorigenesis and metastasis. Increased expression of 12-lipoxygenase has been documented in a number of carcinomas. When overexpressed in human prostate or breast cancer, 12-lipoxygenase promotes tumor angiogenesis and growth in vivo. The present study was undertaken to delineate the mechanisms by which 12-lipoxygenase enhances angiogenesis. Herein we report that nordihydroguaiaretic acid, a pan inhibitor of lipoxygenases and baicalein, a selective inhibitor of 12-lipoxygenase, reduced VEGF expression in human prostate cancer PC-3 cells. Overexpression of 12-lipoxygenase in PC-3 cells resulted in a 3-fold increase in VEGF protein level when compared with vector control cells. An increase in PI 3-kinase activity was found in 12-LOX-transfected PC-3 cells and inhibition of PI 3-kinase by LY294002 significantly reduced VEGF expression. Northern blot and real time PCR analyses revealed an elevated VEGF transcript level in PC-3 cells transfected with a 12-lipoxygenase expression construct. Using a VEGF promoter luciferase construct (-1176/+54), we found a 10-fold increase in VEGF promoter activity in 12-lipoxygenase-transfected PC-3 cells. The region located between -88 and -66 of the VEGF promoter was identified as 12-lipoxygenase responsive using VEGF promoter-based luciferase assays. Further analysis with mutant constructs indicated Sp1 as a transcription factor required for 12-lipoxygenase stimulation of VEGF. Neutralization of VEGF by a function-blocking antibody significantly decreased the ability of 12-lipoxygenase-transfected PC-3 cells to stimulate endothelial cell migration, suggesting VEGF as an important effector for 12-lipoxygenase-mediated stimulation of tumor angiogenesis.

International audience; Tumor necrosis factor-alpha (TNF-alpha) has been shown to induce the expression of adhesion molecules in airway resident cells and contribute to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and NF-kappaB in TNF-alpha-induced expression of vascular cell adhesion molecule (VCAM)-1 were investigated in human tracheal smooth muscle cells (HTSMCs). TNF-alpha-enhanced expression of VCAM-1 protein and mRNA as well as phosphorylation of p42/p44 MAPK, p38, and JNK were significantly attenuated by inhibitors of MEK1/2 (U0126), p38 (SB202190), and JNK (SP600125). Transfection with dominant negative mutants of MEK1/2, ERK1, ERK2, p38, and JNK attenuated TNF-alpha-induced VCAM-1 expression. Furthermore, TNF-alpha-induced VCAM-1 expression was significantly blocked by a selective NF-kappaB inhibitor helenalin. TNF-alpha-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha was blocked by helenalin, but not by U0126, SB202190, or SP600125. VCAM-1 promoter activity was enhanced by TNF-alpha in HTSMCs transfected with VCAM-1-Luc, which was inhibited by helenalin, U0126, SB202190, and SP600125. Most surprisingly, VCAM-1 expression was also significantly blocked by a selective inhibitor of p300, curcumin. NF-kappaB transcription factor and p300 were associated with the VCAM-1 promoter, which was dynamically linked to histone H3 acetylation stimulated by TNF-alpha, as determined by chromatin immunoprecipitation assay. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells (PMNs) to monolayer of HTSMCs, which was blocked by helenalin, U0126, SB202190, or SP600125. These results suggest that in HTSMCs, activation of MAPK pathways, NF-kappaB, and p300 is essential for TNF-alpha-induced VCAM-1 expression.

Pathogenic apicomplexan parasites like Toxoplasma and Plasmodium (malaria) have complex life cycles consisting of multiple stages. The ability to differentiate from one stage to another requires dramatic transcriptional changes, yet there is a paucity of transcription factors in these protozoa. In contrast, we show here that Toxoplasma possesses extensive chromatin remodeling machinery that modulates gene expression relevant to differentiation. We find that, as in other eukaryotes, histone acetylation and arginine methylation are marks of gene activation in Toxoplasma. We have identified mediators of these histone modifications, as well as a histone deacetylase (HDAC), and correlate their presence at target promoters in a stage-specific manner. We purified the first HDAC complex from apicomplexans, which contains novel components in addition to others previously reported in eukaryotes. A Toxoplasma orthologue of the arginine methyltransferase CARM1 appears to work in concert with the acetylase TgGCN5, which exhibits an unusual bias for H3 [K18] in vitro. Inhibition of TgCARM1 induces differentiation, showing that the parasite life cycle can be manipulated by interfering with epigenetic machinery. This may lead to new approaches for therapy against protozoal diseases and highlights Toxoplasma as an informative model to study the evolution of epigenetics in eukaryotic cells.

The cell cycle and the circadian clock are endogenous pacemakers, which coexist in most eukaryotic cells and share a number of conceptual features. In the zebrafish, light directly regulates the timing of both clocks, although the signaling and transcriptional pathways that convey photic information to essential nuclear regulators have yet to be deciphered. We have previously established the Z3 cell line, which recapitulates the features of zebrafish circadian clock and represents an ideal system to study light-dependent signaling and gene regulation. We conducted a search for light-responsive transcription factors and found that AP-1 DNA binding is highly induced. Light induces the expression of zWee1 , a cell cycle gene essential for G 2 /M transition, and zCry1a , a clock gene of the feedback regulatory loop. We have found consensus AP-1 sites in the regulatory regions of both zWee1 and zCry1a genes, and we show that light inducibility of both genes is abrogated by inhibition of AP-1 function. Light also elicits chromatin remodeling by stimulating hyperacetylation at Lys-14 of histone H3 at both zWee1 and zCry1a promoters, as assessed by chromatin immunoprecipitation assays by using anti-Fos antibody. These findings provide strong evidence that circadian and cell cycle clocks share unique light-responsive pathways in zebrafish.

International audience; The differentiation potential of stem cells is determined by the ability of these cells to establish and maintain developmentally regulated gene expression programs that are specific to different lineages. Although transcriptionally potentiated epigenetic states of genes have been described for haematopoietic progenitors, the developmental stage at which the formation of lineage-specific gene expression domains is initiated remains unclear. In this study, we show that an intergenic cis-acting element in the mouse lambda5-VpreB1 locus is marked by histone H3 acetylation and histone H3 lysine 4 methylation at a discrete site in embryonic stem (ES) cells. The epigenetic modifications spread from this site toward the VpreB1 and lambda5 genes at later stages of B-cell development, and a large, active chromatin domain is established in pre-B cells when the genes are fully expressed. In early B-cell progenitors, the binding of haematopoietic factor PU.1 coincides with the expansion of the marked region, and the region becomes a center for the recruitment of general transcription factors and RNA polymerase II. In pre-B cells, E2A also binds to the locus, and general transcription factors are distributed across the active domain, including the gene promoters and the intergenic region. These results suggest that localized epigenetic marking is important for establishing the transcriptional competence of the lambda5 and VpreB1 genes as early as the pluripotent ES cell stage.

International audience; The loss of E-cadherin expression and the translocation of beta-catenin to the nucleus are frequently associated with the metastatic conversion of epithelial cells. In the nucleus, beta-catenin binds to the TCF/LEF-1 (T-cell factor/ lymphoid enhancer factor) transcription factor family resulting in the activation of several genes, some of them having important implications in tumour progression. In our study, we investigated the potential regulation of monocyte chemotactic protein-1 (MCP-1/CCL2) expression by the beta-catenin/TCF pathway. This CC-chemokine has been implicated in tumour progression events such as angiogenesis or tumour associated macrophage (TAM) infiltration. We thus demonstrated that MCP-1 expression correlates with the reorganization of the E-cadherin/beta-catenin complexes. Indeed, MCP-1 was expressed by invasive breast cancer cells (MDA-MB-231, BT549 and Hs578T), which do not express E-cadherin but was not produced by noninvasive breast cancer cell lines (MCF7 and T47D) expressing high level of E-cadherin. In addition, the MCP-1 promoter was activated in BT549 breast cancer cells transfected with beta-catenin and TCF-4 cDNAs. The MCP-1 mRNA level was similarly upregulated. Moreover, we showed that MCP-1 mRNA was downregulated after transfection with a siRNA against beta-catenin in both BT549 and Hs578T cells. Our results therefore identify MCP-1 as a target of the beta-catenin/TCF/LEF pathway in breast tumour cells, a regulation which could play a key role in breast tumour progression.

International audience; Increased expression of vascular endothelial growth factor (VEGF) contributes to the growth of many tumors by increasing angiogenesis. Although hypoxia is a potent inducer of VEGF, we previously showed that epidermal growth factor receptor amplification and loss of PTEN, both of which can increase phosphatidylinositol-3-kinase (PI3K) activity, increase VEGF expression. Using both adenoviral vectors and a cell line permanently expressing constitutively active myristoylated Akt (myrAkt), we show that activation of Akt, which is downstream of PI3K, increases VEGF expression in vitro and increases angiogenesis in a Matrigel plug assay. Transient transfection experiments using reporter constructs containing the VEGF promoter showed that up-regulation of VEGF by Akt is mediated through Sp1 binding sites located in the proximal promoter. Small interfering RNA directed against Sp1 prevented the induction of VEGF mRNA in response to myrAkt but not to hypoxia. Expression of myrAkt is associated with increased phosphorylation of Sp1 and its increased binding to a probe corresponding to the -88/-66 promoter region. In conclusion, our results indicate that Sp1 is required for transactivation of the VEGF by Akt. Others have proposed that the PI3K/Akt pathway can increase VEGF expression via the hypoxia-inducible factor 1 (HIF-1); however, our results suggest an alternative mechanism can also operate.

International audience; We have previously cloned and characterized three estrogen receptors (ER) in the zebrafish (zfERalpha, zfERbeta1 and zfERbeta2). We have also shown that they are functional in vitro and exhibit a distinct expression pattern, although partially overlapping, in the brain of zebrafish. In this paper, we have shown that the hepatic expression of these zfER genes responds differently to estradiol (E2). In fact, a 48-h direct exposure of zebrafish to E2 resulted in a strong stimulation of zfERalpha gene expression while zfERbeta1 gene expression was markedly reduced and zfERbeta2 remained virtually unchanged. To establish the potential implication of each zfER in the E2 upregulation of the zfERalpha gene, the promoter region of this gene was isolated and characterized. Transfection experiments with promoter-luciferase reporter constructs together with different zfER expression vectors were carried out in different cell contexts. The data showed that in vivo E2 upregulation of the zfERalpha gene requires ERalpha itself and a conserved transcription unit sequence including at least an imperfect estrogen-responsive element (ERE) and an AP-1/ERE half site at the proximal transcription initiation site. Interestingly, although in the presence of E2 zfERalpha was the most potent at inducing the expression of its own gene, the effect of E2 mediated by zfERbeta2 represented 50% of the zfERalpha activity. In contrast, zfERbeta1 was unable to upregulate the zfERalpha gene whereas this receptor form was able to tightly bind E2 and activate a reporter plasmid containing a consensus ERE. Altogether, these results indicated that the two ERbeta forms recently characterized in teleost fish could have partially distinct and not redundant functions.

In Arabidopsis, the basic leucine zipper transcription factor ABI5 activates several late embryogenesis-abundant genes, including AtEm1 and AtEm6. However, the expression of many other seed maturation genes is independent of ABI5. We investigated the possibility that ABI5 homologs also participate in the regulation of gene expression during seed maturation. We identified 13 ABI5-related genes in the Arabidopsis genomic sequence. RNA gel blot analysis showed that seven of these genes are active during seed maturation and that they display distinct expression kinetics. We isolated and characterized two mutant alleles of one of these genes, AtbZIP12/EEL. Unlike abi5, the eel mutations did not inhibit the expression of any of the maturation marker genes that we monitored. On the contrary, the accumulation of the AtEm1 and AtEm6 mRNAs was enhanced in eel mutant seeds compared with wild-type seeds. Gel mobility shift assays, combined with analysis of the genetic interactions among the eel and abi5 mutations, indicated that ABI5 and EEL compete for the same binding sites within the AtEm1 promoter. This study illustrates how two homologous transcription factors can play antagonistic roles to fine-tune gene expression.

cDNA expression vectors of Potato leafroll virus (PLRV) were used to analyse specific mutations in the proteinase and replicase domains of the proteins encoded by ORF1 and ORF2. Agrobacterium-mediated DNA transfer was used to introduce a PLRV RNA expression unit, controlled by the 35S promoter of Cauliflower mosaic virus, into potato leaf cells. Expression of unmodified PLRV cDNA led to the replication of viral genomic and subgenomic RNAs and accumulation of the viral capsid protein, whereas alteration of amino acids GDD513–515 of the replicase to VHD abolished PLRV replication. Mutations in the presumed H-D-S catalytic triad of the viral proteinase abolished the formation of viral genomic and subgenomic RNAs as well as synthesis of the viral capsid protein. Co-agroinoculation of the GDD mutant along with any of the proteinase mutants restored virus replication in leaf discs, showing that these mutants are able to complement each other. Moreover, mutation of the postulated serine residue of the catalytic triad of the proteinase altered the pattern of proteins synthesized in vitro in comparison to wild-type, further supporting the relevance of the H-D-S motif.

International audience; Formaldehyde cross-linking was used in a kinetic analysis of RNA polymerase-lacUV5 promoter interactions in open complexes (RP(o)). RP(o) quenched from 37 degrees C to 14 degrees C isomerised to a closed, competitor resistant, complex (RP(LT)). We observed that contacts of the beta' and sigma subunits with the positions -3, -5 of the non-template DNA strand disappeared very quickly during the first 30 seconds after the temperature downshift. The re-annealing of the DNA downstream of the transcription start site takes place in the same time scale. However re-annealing of the upstream part of the transcription bubble was slower and completed within five minutes. The results support a two-step model of promoter melting and suggest that conformational changes in the RNA polymerase occur concurrently with the melting around the transcription start site.

Recently it has been shown that dominant mutations in the human hepatocyte nuclear factor 1alpha (HNF1alpha) gene, encoding for a homeoprotein that is expressed in liver, kidney, pancreas and intestine, result in maturity onset diabetes of the young type 3 (MODY3). HNF1alpha-null mice are diabetic, but at the same time suffer from a renal Fanconi syndrome characterized by urinary glucose loss. Here we show that MODY3 patients are also characterized by a reduced tubular reabsorption of glucose. The renal murine defect is due to reduced expression of the low affinity/high capacity glucose cotransporter (SGLT2). Our results show that HNF1alpha directly controls SGLT2 gene expression. Together these data indicate that HNF1alpha plays a key role in glucose homeostasis in mammals.

International audience; The FruR regulator of Escherichia coli controls the initiation of transcription of several operons encoding a variety of proteins involved in carbon and energy metabolism. The sequence determinants of the FruR-binding site were analysed by using 6x His-tagged FruR and a series of double-stranded randomized oligonucleotides. FruR consensus binding sites were selected and characterized by several consecutive rounds of the polymerase chain reaction-assisted binding-site selection method (BSS) using nitrocellulose-immobilized DNA-binding protein. FruR was demonstrated to require, for binding, an 8 bp left half-site motif and a 3 bp conserved right half-site with the following sequence: 5'-GNNGAATC/GNT-3'. In this sequence, the left half-site AATC/ consensus tetranucleotide is a typical motif of the DNA-binding site of the regulators of the GalR-Lacl family. On the other hand, the high degree of degeneracy found in the right half-site of this palindrome-like structure indicated that FruR, which is a tetramer in solution, interacts asymmetrically with the two half-sites of its operator. However, potentially FruR-target sites showing a high degree of symmetry were detected in 13 genes/operons. Among these, we have focused our interest on the pfkA gene, encoding phosphofructo-kinase-1, which is negatively regulated by FruR.

International audience; The unique LAMMER (or Clk) protein kinase of Drosophila is encoded at the Doa locus. To better understand the pleiotropic effects of Doa mutations, we describe the structure and expression of the multiple RNA and protein products of the locus, as well as their evolutionary conservation among Drosophila. The gene produces at least six different protein isoforms, primarily through alternative promoter usage, generating kinases with virtually identical catalytic domains but variable N-terminal noncatalytic domains. The single known alternative splicing event generates a kinase with the insertion of six additional amino-acids in the catalytic domain. Two independent predicted genes nested within Doa introns actually encode additional alternative N-termini of the locus. An alternative polyadenylation site utilized exclusively during early embryogenesis generates a transcript with a short half-life, apparently to ensure a "burst" of kinase expression at the onset of development. Ecdysone induction of Doa transcripts affects all isoforms during pupariation. Finally, extensive conservation of amino-acid sequences of both the catalytic and N-terminal noncatalytic exons observed in alignments between D. melanogaster exons and the genome sequences of 11 other Drosophila species suggest that the multiple isoforms serve important and nonredundant functions.

MAP kinase phosphatase 1 (MKP1) is a negative regulator for the mitogen-activated protein kinase (MAPK)—mediated signal transduction, a key pathway that leads to the regulated expression of circadian clock genes. Here the authors analyzed mkp1 expression by in situ hybridization and found that mkp1 is a light-inducible and clock-controlled gene expressed in the central pacemaker neurons of the hypothalamic SCN. Interestingly, mkp1 presents a marked similarity to the clock core gene per1 in terms of the gene expression profiles as well as the gene promoter organization. Both mkp1 and per1 are subject to bimodal regulation in the SCN: the external light-dependent acute up-regulation and the functional clock-dependent circadian oscillation. Consistent with this, the authors show that mkp1 gene has a per1-like promoter that contains 2 functionally distinct elements: cAMP-responsive element (CRE) and E-box. CRE sites present in the mkp1 promoter constitute the functional binding sites for the CRE binding protein (CREB), which serves as an important regulator that mediates the light-induced signaling cascades in the SCN neurons. Furthermore, the authors show that the E-box present in the mkp1 promoter is necessary and sufficient for transcriptional control exerted by circadian clock core regulators that include a positive complex CLOCK/BMAL1 and a negative factor CRY1. The authors' studies on mkp1 have identified for the first time a gene encoding a phosphatase that functions in light-dependent and time-of-day-dependent manners in the mammalian central clock structure SCN.

Megakaryocytic (MK) lineage is an attractive target for cell/gene therapy approaches, aiming at correcting platelet protein deficiencies. However, MK cells are short-lived cells, and their permanent modification requires modification of hematopoietic stem cells with an integrative vector such as a lentiviral vector. Glycoprotein (Gp) IIb promoter, the most studied among the MK regulatory sequences, is also active in stem cells. To strictly limit transgene expression to the MK lineage after transduction of human CD34+ hematopoietic cells with a lentiviral vector, we looked for a promoter activated later during MK differentiation. Human cord blood, bone marrow, and peripheral-blood mobilized CD34+ cells were transduced with a human immunodeficiency virus-derived self-inactivating lentiviral vector encoding the green fluorescent protein (GFP) under the transcriptional control of GpIbα, GpIIb, or EF1α gene regulatory sequences. Both GpIbα and GpIIb promoters restricted GFP expression (analyzed by flow cytometry and immunoelectron microscopy) in MK cells among the maturing progeny of transduced cells. However, only the GpIbα promoter was strictly MK-specific, whereas GpIIb promoter was leaky in immature progenitor cells not yet engaged in MK cell lineage differentiation. We thus demonstrate the pertinence of using a 328-base-pair fragment of the human GpIbα gene regulatory sequence, in the context of a lentiviral vector, to tightly restrict transgene expression to the MK lineage after transduction of human CD34+ hematopoietic cells. Disclosure of potential conflicts of interest is found at the end of this article.

Delta-lactoferrin is a cytoplasmic lactoferrin isoform that can locate to the nucleus, provoking antiproliferative effects and cell cycle arrest in S phase. Using macroarrays, the expression of genes involved in the G(1)/S transition was examined. Among these, Skp1 showed 2-3-fold increased expression at both the mRNA and protein levels. Skp1 (S-phase kinase-associated protein) belongs to the Skp1/Cullin-1/F-box ubiquitin ligase complex responsible for the ubiquitination of cellular regulators leading to their proteolysis. Skp1 overexpression was also found after delta-lactoferrin transient transfection in other cell lines (HeLa, MDA-MB-231, HEK 293) at comparable levels. Analysis of the Skp1 promoter detected two sequences that were 90% identical to those previously known to interact with lactoferrin, the secretory isoform of delta-lactoferrin (GGCACTGTAC-S1(Skp1), located at - 1067 bp, and TAGAAGTCAA-S2(Skp1), at - 646 bp). Both gel shift and chromatin immunoprecipitation assays demonstrated that delta-lactoferrin interacts in vitro and in vivo specifically with these sequences. Reporter gene analysis confirmed that delta-lactoferrin recognizes both sequences within the Skp1 promoter, with a higher activity on S1(Skp1). Deletion of both sequences totally abolished delta-lactoferrin transcriptional activity, identifying them as delta-lactoferrin-responsive elements. Delta-lactoferrin enters the nucleus via a short bipartite RRSDTSLTWNSVKGKK(417-432) nuclear localization signal sequence, which was demonstrated to be functional using mutants. Our results show that delta-lactoferrin binds to the Skp1 promoter at two different sites, and that these interactions lead to its transcriptional activation. By increasing Skp1 gene expression, delta-lactoferrin may regulate cell cycle progression via control of the proteasomal degradation of S-phase actors.

International audience; Recognition of the -10 promoter consensus element by region 2 of the bacterial RNA polymerase sigma subunit is a key step in transcription initiation. sigma also functions as an elongation factor, inducing transcription pausing by interacting with transcribed DNA non-template strand sequences that are similar to the -10 element sequence. Here, we show that the region 1.2 of Escherichia coli sigma70, whose function was heretofore unknown, is strictly required for efficient recognition of the non-template strand of -10-like pause-inducing DNA sequence by sigma region 2, and for sigma-dependent promoter-proximal pausing. Recognition of the fork-junction promoter DNA by RNA polymerase holoenzyme also requires sigma region 1.2 and thus resembles the pause-inducing sequence recognition. Our results, together with available structural data, support a model where sigma region 1.2 acts as a core RNA polymerase-dependent allosteric switch that modulates non-template DNA strand recognition by sigma region 2 during transcription initiation and elongation.

International audience; In vertebrates, the actin-binding proteins tropomyosins are encoded by four distinct genes that are expressed in a complex pattern during development and muscle differentiation. In this study, we have characterized the transcriptional machinery of the alpha-tropomyosin (alpha-Tm) gene in muscle cells. Promoter analysis revealed that a 284-bp proximal promoter region of the Xenopus laevis alpha-Tm gene is sufficient for maximal activity in the three muscle cell types. The transcriptional activity of this promoter in the three muscle cell types depends on both distinct and common cis-regulatory sequences. We have identified a 30-bp conserved sequence unique to all vertebrate alpha-Tm genes that contains an MCAT site that is critical for expression of the gene in all muscle cell types. This site can bind transcription enhancer factor-1 (TEF-1) present in muscle cells both in vitro and in vivo. In serum-deprived differentiated smooth muscle cells, TEF-1 was redistributed to the nucleus, and this correlated with increased activity of the alpha-Tm promoter. Overexpression of TEF-1 mRNA in Xenopus embryonic cells led to activation of both the endogenous alpha-Tm gene and the exogenous 284-bp promoter. Finally, we show that, in transgenic embryos and juveniles, an intact MCAT sequence is required for correct temporal and spatial expression of the 284-bp gene promoter. This study represents the first analysis of the transcriptional regulation of the alpha-Tm gene in vivo and highlights a common TEF-1-dependent regulatory mechanism necessary for expression of the gene in the three muscle lineages.

The Cyclin E1 gene ( CCNE1 ) is an ideal model to explore the mechanisms that control the transcription of cell cycle-regulated genes whose expression rises transiently before entry into S phase. E2F-dependent regulation of the CCNE1 promoter was shown to correlate with changes in the level of H3-K9 acetylation/methylation of nucleosomal histones positioned at the transcriptional start site region. Here we show that, upon growth stimulation, the same region is subject to variations of H3-R17 and H3-R26 methylation that correlate with the recruitment of coactivator-associated arginine methyltransferase 1 (CARM1) onto the CCNE1 and DHFR promoters. Accordingly, CARM1-deficient cells lack these modifications and present lowered levels and altered kinetics of CCNE1 and DHFR mRNA expression. Consistently, reporter gene assays demonstrate that CARM1 functions as a transcriptional coactivator for their E2F1/DP1-stimulated expression. CARM1 recruitment at the CCNE1 gene requires activator E2Fs and ACTR, a member of the p160 coactivator family that is frequently overexpressed in human breast cancer. Finally, we show that grade-3 breast tumors present coelevated mRNA levels of ACTR and CARM1 , along with their transcriptional target CCNE1 . All together, our results indicate that CARM1 is an important regulator of the CCNE1 gene.

The cocaine and amphetamine regulated transcript (CART), an anorexigenic peptide responding to leptin, is expressed in various areas of the hypothalamus. The role of CART in humans and its potential contribution to abnormalities in feeding control are mostly unknown. Since CART plays an important role in the hypothalamic regulation of energy balance by reducing food intake and increasing lipid substrate utilization, it might affect cholesterol metabolism as Neuropeptide Y or pro-opiomelanocortin do. In the present work, we studied the potential effects of three SNPs of the CART promoter in a WHO–MONICA general population from North of France ( n =840), untreated for hypercholesterolemia, hypertension, or diabetes mellitus since any treatment is likely to interfere with lipoprotein/lipid variables. Our results show associations between these SNPs and plasma LDL-cholesterol level and the LDL/HDL ratio, a marker of atherogenicity. A haplotypic study suggests that these effects are mainly attributable to the functional SNP −3608C>T. Subjects bearing the −3608 C allele present a plasma lipid profile protective against atherogenesis: decrease of plasma LDL-cholesterol level ( p =0.001) and of the LDL/HDL ratio ( p =0.0003). This result offers new evidences for a potential implication of the CART gene in lipid metabolism and in atherogenesis.

The transcription/replication activity of ribonucleoproteins derived from influenza A primary isolates of human (A/Paris/908/97) or avian origin (A/Mallard/Marquenterre/MZ237/83, A/Hong Kong/156/97) was compared upon reconstitution in mammalian or avian cells, using viral-like reporter RNAs synthesized under the control of the human and chicken RNA polymerase I promoters, respectively. In avian cells, transcription/replication activities were in the same range with all ribonucleoproteins tested. In human cells, ribonucleoproteins derived from A/Mallard/Marquenterre/MZ237/83 showed reduced transcription/replication activity and reduced NP binding to the PB1-PB2-PA complex (P) or to the isolated PB2 subunit, as compared to the ribonucleoproteins derived from A/Paris/908/97. Both defects were restored when PB2 residue Glu-627 was changed to a Lys. Ribonucleoproteins derived from the human A/Hong Kong/156/97 H5N1 isolate showed efficient NP-P interaction in human cells, and high levels of activity which were determined mostly by the PB2 and PA proteins. Our data suggest that PB2 might play a pivotal role in molecular interactions involving both the viral nucleoprotein and cellular proteins.

International audience; Neural stem cells are known to give rise to distinct subtypes of neurons and glial cells over time by changing their competency. However, precise characterization of neural stem cells at various developmental stages remains to be performed. For such analysis, a tool to manipulate neural stem cells at different time points is necessary. Here, we generated transgenic mice that express Cre-ER(T2) in the ventricular zone of the developing nervous system under the control of the nestin promoter and enhancer (Nes-CreER(T2)). In mice expressing Cre-ER(T2) at appropriate levels, Cre recombinase activity was mostly inactive but efficiently activated by tamoxifen within 1 day. When such mice were crossed with the ROSA-26 or Z/EG reporter mice, neural stem cells were permanently labeled after administration of tamoxifen. Thus, Nes-CreER(T2) mice offer a powerful tool to manipulate neural stem cells genetically at desired time points.

Plasma membrane transport of single amino-acid methionine in yeast is shown to be mediated by at least seven different permeases whose activities are transcriptionaly and post-transcriptionaly regulated by different ubiquitin-dependent mechanisms. Upon high extracellular methionine exposure, three methionine-permease genes are repressed while four others are induced. SCF(Met30), SCF(Grr1) and Rsp5 ubiquitin ligases are the key actors of the ubiquitin-dependent remodeling of methionine transport. In addition to regulating the activity of Met4, the SCF(Met30) ubiquitin ligase is shown to convey an intracellular signal to a membrane initiated signaling pathway by controlling the nuclear concentration of the Stp1 transcription factor. By coupling intra- and extracellular metabolite sensing, SCF(Met30) thus allows yeast cells to accurately adjust the intermediary sulfur metabolism to the growth conditions. The multiple ubiquitin-dependent mechanisms that function in methionine transport regulation further exemplify the pervasive role of ubiquitin in the adaptation of single-cell organisms to environmental modifications.

International audience; The Ikaros transcription factor is both a key regulator of lymphocyte differentiation and a tumor suppressor in T lymphocytes. Mice carrying a hypomorphic mutation (Ik(L/L)) in the Ikaros gene all develop thymic lymphomas. Ik(L/L) tumors always exhibit strong activation of the Notch pathway, which is required for tumor cell proliferation in vitro. Notch activation occurs early in tumorigenesis and may precede transformation, as ectopic expression of the Notch targets Hes-1 and Deltex-1 is detected in thymocytes from young Ik(L/L) mice with no overt signs of transformation. Notch activation is further amplified by secondary mutations that lead to C-terminal truncations of Notch 1. Strikingly, restoration of Ikaros activity in tumor cells leads to a rapid and specific downregulation of Notch target gene expression and proliferation arrest. Furthermore, Ikaros binds to the Notch-responsive element in the Hes-1 promoter and represses Notch-dependent transcription from this promoter. Thus, Ikaros-mediated repression of Notch target gene expression may play a critical role in defining the tumor suppressor function of this factor.

BackgroundThe persistence of latent HIV-1 reservoirs is the principal barrier preventing the eradication of HIV-1 infection in patients by current antiretroviral therapy. It is thus crucial to understand the molecular mechanisms involved in the establishment, maintenance and reactivation of HIV-1 latency. Since chromatin remodeling has been implicated in the transcriptional reactivation of the HIV-1 promoter, we assessed the role of the histone deacetylase inhibitor sodium butyrate (NaB) on two HIV-1 latently infected cell lines (U1 and ACH-2) gene expression.ResultsAnalysis of microarrays data led us to select two candidate genes:NCoA3(Nuclear Receptor Coactivator 3), a nuclear receptor coactivator andIRF8(Interferon Regulatory Factor 8), an interferon regulatory factor.NCoA3gene expression is upregulated following NaB treatment of latently infected cells whereasIRF8gene expression is strongly downregulated in the promonocytic cell line following NaB treatment. Their differential expressions were confirmed at the transcriptional and translational levels. Moreover,NCoA3gene expression was also upregulated after treatment of U1 and ACH-2 cells with phorbol myristyl acetate (PMA) but not trichostatin A (TSA) and after treatment with NaB of two others HIV-1 latently infected cell lines (OM10.1 and J1.1).IRF8gene is only expressed in U1 cells and was also downregulated after treatment with PMA or TSA. Functional analyses confirmed that NCoA3 synergizes with Tat to enhance HIV-1 promoter transcription and that IRF8 represses the IRF1-mediated activation through the HIV-1 promoter Interferon-stimulated response element (ISRE).ConclusionThese results led us to postulate that NCoA3 could be involved in the transcriptional reactivation of the HIV-1 promoter from latency and that IRF8 may contribute to the maintenance of the latent state in the promonocytic cell line. Implication of these factors in the maintenance or reactivation of the viral latency may provide potential new targets to control HIV-1 replication in latent viral reservoirs.

Antigen receptor gene assembly is governed by transcriptional promoters and enhancers that communicate over large distances and modulate chromatin accessibility to V(D)J recombinase. The precise role of these cis-acting elements in opening chromatin at recombinase targets and the mechanisms underlying their crosstalk remain unclear. We show that the TCRbeta enhancer (Ebeta) directs long-range chromatin opening over both DbetaJbeta clusters. Strikingly, chromatin associated with the Dbeta1 gene segment is refractory to Ebeta-mediated opening. Accessibility at Dbeta1 is accompanied by the formation of a stable holocomplex between a Dbeta-proximal promoter and Ebeta. These findings indicate a stepwise process for Dbeta --> Jbeta recombination that relies on distinct aspects of Ebeta activity: an intrinsic function that directs general chromatin opening and a cooperative function that facilitates the assembly of a promoter/enhancer holocomplex, unmasks the Dbeta1 gene segment, and triggers TCRbeta gene assembly.

Background: The ACTH receptor (ACTH-R) is a member of the seven transmembrane domain receptor super-family. In non-functional adrenal adenomas and adrenocortical carcinomas, ACTH-R expression is low. However, no inhibitory factor for ACTH-R expression has been defined to date. DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene-1) is a general repressor of steroid production, inhibiting steroidogenic factor-1 (SF-1)-dependent expression of multiple steroidogenic enzymes. The aim of this study was to investigate whether ACTH-R gene transcription is affected by DAX-1 and whether this mechanism is involved in down-regulation of ACTH-R expression in adrenocortical tumors. Methods: We screened 22 adrenocortical tumors for ACTH-R and DAX-1 mRNA expression by Northern blot. For in vitro analyses we co-transfected mouse Y1 adrenocortical carcinoma cells with the luciferase reporter gene vector pGL3 containing full-length constructs of human (h) or mouse (m) ACTH-R promoter together with a DAX-1 expression plasmid. These experiments were also performed using ACTH-R promoter 5′-deletion constructs and constructs mutated at the SF-1-binding sites. Results: We found a negative correlation between DAX-1 and ACTH-R mRNA expression (R = −0.47, P < 0.02). Accordingly, in vitro expression of DAX-1 significantly reduced hACTH-R and mACTH-R promoter activity by 89 and 55% respectively. DAX-1 inhibition was also present in the shortest construct of a series of 5′-deletion constructs of the human promoter extending from −64 to +40 bp relative to the transcription start site. Mutation of the SF-1-binding sites within the hACTH-R promoter resulted in reduced or abolished DAX-1 inhibition, arguing for a mechanism that involves SF-1 for DAX-1 inhibition. Conclusions: These data support the concept that DAX-1 is a major repressor of ACTH-R gene expression in vitro and in vivo.

Integrin cytoplasmic domain-associated protein 1 (ICAP-1) has been shown to interact specifically with the β1 integrin cytoplasmic domain and to control cell spreading on fibronectin. Interestingly, ICAP-1 also is observed in the nucleus, by immunocytochemical staining, and after biochemical cell fractionation, suggesting that it has additional roles that have yet to be determined. We show that the nucleocytoplasmic shuttling capability of ICAP-1 is dependent on a functional nuclear localization signal. In addition, overexpression of β1 integrin strongly reduced this nuclear localization, suggesting that integrin activity could modulate ICAP-1 shuttling by sequestering it in the cytoplasm. Indeed, the nuclear localization of ICAP-1 is dependent on the stage of cell spreading on fibronectin, and we also show that ICAP-1 expression stimulates cellular proliferation in a fibronectin-dependent manner. This function is dependent on its nuclear localization. Moreover, ICAP-1 is able to activate the c-myc promoter in vitro. Together, these results demonstrate that ICAP-1 shuttles between the nucleus and cytoplasm in a β1 integrin-dependent manner. It could act as a messenger that relays information from sites of integrin-dependent cell adhesion to the nucleus for controlling gene expression and cell proliferation.

Evidence is accumulating about the role of individual AP-1 components in cell proliferation and transformation. Notably, Ras-mediated transformation is characterized by the upregulation of particular AP-1 members, such as c-Jun and Fra-1. The p14/p19ARF tumor suppressor gene is a key link between oncogenic Ras signaling and the p53 pathway. We explored the involvement of AP-1 dimers in the transcriptional regulation of the p14/p19ARF gene. We demonstrate that both the human and mouse ARF promoters are transcriptional targets of selective AP-1 dimers. The ARF promoter is regulated specifically by AP-1 heterodimers containing Fra-1. Overexpression of c-Jun approximately Fra-1 dimers in primary murine fibroblast cells led to the upregulation of the endogenous ARF protein and growth arrest. Conversely, inhibition of c-Jun or Fra-1 protein levels resulted in decreased ARF expression. In addition, we show that AP-1 dimers cooperate with oncogenic Ras in the transcriptional activation of the p14/p19ARF promoter. Thus, AP-1 heterodimers may contribute to the regulation of ARF expression upon oncogenic signaling.

Contains fulltext : 47464thebault.pdf (Publisher’s version ) (Closed access) TRPM8 (melastatine-related transient receptor potential member 8), a member of the transient receptor potential (TRP) superfamily of cation channels, has been shown to be a calcium-channel protein. TRPM8 mRNA has also been shown to be overexpressed in prostate cancer and is considered to play an important role in prostate physiology. This study was designed to determine the androgen-regulation mechanisms for TRPM8 mRNA expression and to identify the phenotype of TRPM8-expressing cells in the human prostate. Our findings show that trpm8 gene expression requires a functional androgen receptor. Furthermore, this article argues strongly in favour of the fact that the trpm8 gene is a primary androgen-responsive gene. Single-cell reverse transcriptase PCR and immunohistochemical experiments also showed that the trpm8 gene was mainly expressed in the apical secretory epithelial cells of the human prostate and trpm8 down-regulation occurred during the loss of the apical differentiated phenotype of the primary cultured human prostate epithelial cells. The androgen-regulated trpm8 expression mechanisms are important in understanding the progression of prostate cancer to androgen-independence. These findings may contribute to design a strategy to predict prostate cancer status from the TRPM8 mRNA level. Furthermore, as the TRPM8 channel is localized in human prostate cells, it will be interesting to understand its physiological function in the normal prostate and its potential role in prostate cancer development.

International audience; Peroxisome proliferator-activated receptor-gamma (PPARgamma) plays a role in adipocyte differentiation and insulin sensitization. We identified and characterized a new C/T substitution at position -689 (-689C>T) in the P2 promoter of PPARgamma in a putative GATA binding site. By electrophoretic mobility shift assay, both GATA2 and GATA3 proteins could bind weakly to the wild-type P2 -689 GATA binding site but not to the mutated site. Neither GATA2 nor GATA3 was able to regulate significantly the P2 promoter activity in a reporter-luciferase assay, whatever the allele at position -689 was, suggesting that the -689 putative GATA site was probably not a functional target for GATAs. However, the presence of the -689T allele rendered the P2 promoter less active at the basal state. We genotyped a population of 1155 men and women for the -689C>T polymorphism and looked for possible associations with anthropometric and lipid variables. The carriers of the -689T allele had elevated body weight and LDL-cholesterol concentrations compared with the homozygous for the common allele. Haplotype analyses including the -681C>G (P3 promoter), -689C>T (P2 promoter), and Pro12Ala (exon B) polymorphisms were performed. Carriers of the G-T-Ala haplotype (corresponding to the P3 -681C>G, P2 -689C>T and Pro12Ala polymorphisms in this order) had elevated LDL-cholesterol concentrations and body weight compared with C-C-Pro individuals. In conclusion, we identified a new polymorphism in the P2 promoter of PPARgamma. The P3 -681C>G, P2 -689C>T, and Pro12Ala polymorphisms and related haplotypes were associated with higher body weight and plasma LDL-cholesterol concentrations.

International audience; We have investigated the expression of c-fos in cervical carcinoma cells and in somatic cell hybrids derived therefrom. In malignant cells, c-fos was constitutively expressed even after serum starvation. Dissection of the c-fos promoter showed that expression was mainly controlled by the SRE motif, which was active in malignant cells, but repressed in their non-malignant counterparts. Constitutive SRE activity was not mediated by sustained mitogen-activated protein kinase activity but because of inefficient expression of the ternary complex factor Net, which was either very low or even barely discernible. Chromatin immunoprecipitation assays revealed that Net directly binds to the SRE nucleoprotein complex in non-tumorigenic cells, but not in malignant segregants. Small interfering RNA targeted against Net resulted in enhanced c-fos transcription, clearly illustrating its repressor function. Conversely, stable ectopic expression of Net in malignant cells negatively regulated endogenous c-fos, resulting in a disappearance of the c-Fos protein from the AP-1 transcription complex. These data indicate that loss of Net and constitutive c-fos expression appear to be a key event in the transformation of cervical cancer cells.

Neuronal apoptosis has been shown to require de novo RNA/protein synthesis. However, very few genes whose expression is necessary for inducing apoptosis have been identified so far. To systematically identify such genes, we have used genome-scale, long oligonucleotide microarrays and characterized the gene expression profile of cerebellar granule neurons in the early phase of apoptosis elicited by KCl deprivation. We identified 368 significantly differentially expressed genes, including most of the genes previously reported to be transcriptionally regulated in this paradigm. In addition, we identified several hundreds of genes whose transcriptional regulation has never been associated with neuronal apoptosis. We used automated Gene Ontology annotation, analysis of promoter sequences, and statistical tools to characterize these regulations. Although differentially expressed genes included some components of the apoptotic machinery, this functional category was not significantly over-represented among regulated genes. On the other hand, categories related to signal transduction were the most significantly over-represented group. This indicates that the apoptotic machinery is mainly constitutive, whereas molecular pathways that lead to the activation of apoptotic components are transcriptionally regulated. In particular, we show for the first time that signaling pathways known to be involved in the control of neuronal survival are regulated at the transcriptional level and not only by post-translational mechanisms. Moreover, our approach provides insights into novel transcription factors and novel mechanisms, such as the unfolded protein response and cell adhesion, that may contribute to the induction of neuronal apoptosis.

International audience; The efficient introduction of somatic mutations in a given gene at a given time and in specific cell types of the skin will greatly facilitate the studies of a number of genes expressed in the skin and the production of animal models for skin diseases. We describe here strategies and techniques to create spatiotemporally controlled somatic mutations of target genes in the skin using a conditional Cre/LoxP system. They are based on cell-specific expression of the chimeric Cre recombinase Cre-ERT2, whose activity is induced by antiestrogens such as Tamoxifen (Tam), and which is obtained by fusing the Cre recombinase with a mutated ligand binding domain of the human estrogen receptor ERalpha. As an example, we present ablation of the retinoid receptor RXRalpha in epidermal basal keratinocytes of adult mice.

International audience; NF-kappa B essential modulator/IKK-gamma (NEMO/IKK-gamma) plays a key role in the activation of the NF-kappa B pathway in response to proinflammatory stimuli. Previous studies suggested that the signal-dependent activation of the IKK complex involves the trimerization of NEMO. The minimal oligomerization domain of this protein consists of two coiled-coil subdomains named Coiled-coil 2 (CC2) and leucine zipper (LZ) (Agou, F., Traincard, F., Vinolo, E., Courtois, G., Yamaoka, S., Israel, A., and Veron, M. (2004) J. Biol. Chem. 279, 27861-27869). To search for drugs inhibiting NF-kappa B activation, we have rationally designed cell-permeable peptides corresponding to the CC2 and LZ subdomains that mimic the contact areas between NEMO subunits. The peptides were tagged with the Antennapedia/Penetratin motif and delivered to cells prior to stimulation with lipopolysaccharide. Peptide transduction was monitored by fluorescence-activated cell sorter, and their effect on lipopolysaccharide-induced NF-kappa B activation was quantified using an NF-kappa B-dependent beta-galactosidase assay in stably transfected pre-B 70Z/3 lymphocytes. We show that the peptides corresponding to the LZ and CC2 subdomains inhibit NF-kappa B activation with an IC(50) in the mum range. Control peptides, including mutated CC2 and LZ peptides and a heterologous coiled-coil peptide, had no inhibitory effect. The designed peptides are able to induce cell death in human retinoblastoma Y79 cells exhibiting constitutive NF-kappa B activity. Our results provide the "proof of concept" for a new and promising strategy for the inhibition of NF-kappa B pathway activation through targeting the oligomerization state of the NEMO protein.

Controlling gene expression during plant development is an efficient tool to explore gene function. In this paper, we describe a gene expression system driven by a heat-shock gene promoter (HSP18.2), to trigger the expression of an intron-containing inverted-repeat. RNA interference became a powerful way for gene functional analysis by reverse genetic approaches. However, constitutive gene silencing cannot be used with genes involved in fundamental processes such as embryo viability. Inducible promoters provide an alternative approach for temporal and spatial gene expression control and we described here a new system, complementary to those using chemical gene inducers. To evaluate the efficiency of this system, RNA corresponding to the phytoene desaturase gene of Arabidopsis thaliana was used as a reporter gene in transgenic plants and a comparative study was performed using either the CaMV35S constitutive promoter or the HSP18.2 inducible promoter.

Two upstream regions of the human urokinase (uPA) gene regulate its transcription: the minimal promoter (MP) and the enhancer element. The activity of the minimal promoter is essential for basal uPA transcription in prostate adenocarcinoma PC3 cells. Binding of a phosphorylated Sp1 transcription factor is, in turn, essential for the activity of the MP. Here we report that the Jun kinase (JNK) pathway is required for the basal activity of the MP and for the expression of the endogenous uPA gene in PC3 cells and for activated transcription in LNCaP cells. On the other hand, the p42/p44 mitogen-activated protein kinase (MAPK) pathway activates uPA gene expression through Sp1 phosphorylation in HeLa, LNCaP, and CCL39-derivative cells that do not typically express uPA in basal conditions. In HeLa cells the dominant-negative form of JNK interferes with the p42/p44 MAPK activation of the uPA-MP. The results suggest that the stress-activated protein kinase (SAPK)/JNK pathway plays an important role in the phosphorylation of Sp1, which, in turn, leads to basal or activated transcription from the uPA-MP element.

The expression of seed storage proteins is under tight developmental regulation and represents a powerful model system to study the regulation of gene expression during plant development. In this study, we show that three homologous B3 type transcription factors regulate the model storage protein gene, At2S3, via two distinct mechanisms: FUSCA3 (FUS3) and LEAFY COTYLEDON2 (LEC2) activate the At2S3 promoter in yeast suggesting that they regulate At2S3 by directly binding its promoter; ABSCISIC ACID INSENSITIVE3 (ABI3), however, appears to act more indirectly on At2S3, possibly as a cofactor in an activation complex. In accordance with this, FUS3 and LEC2 were found to act in a partially redundant manner and differently from ABI3 in planta: At2S3expression is reduced to variable and sometimes only moderate extent in fus3 and lec2 single mutants but is completely abolished in the lec2 fus3 double mutant. In addition, we found that FUS3and LEC2 expression patterns, together with an unsuspected regulation of FUS3 by LEC2, enable us to explain the intriguing expression pattern of At2S3 in lec2 or fus3 single mutants. Based on these results, we present a model of At2S3regulation and discuss its implications for other aspects of seed maturation.

International audience; Neuropilin-1 (NRP1) is a receptor for the vascular endothelial growth factor (VEGF) family of angiogenesis factors and for the semaphorin family of secreted neuronal guidance polypeptides. Very little is known, however, about how NRP1 gene expression is regulated. In this study, it was demonstrated that the tumor promoter, TPA (12-O-tetradecanoylphorbol-13-acetate) significantly up-regulated NRP1 mRNA levels by increasing its gene transcription rate in a manner dependent on de novo protein synthesis. To determine which elements regulate functional NRP1 expression, the promoter regions of human and mouse NRP1 genes were cloned and characterized. Promoter-reporter gene transfection experiments using deletion and point mutations demonstrated that two Sp1 elements are major contributors to both the constitutive and TPA-induced activity of the NRP1 promoter. Gel shift analysis showed a specific binding of the Sp1 transcription factor to those elements. Further mutational analysis revealed that an AP-1, and a CCAAT box also contributed to NRP1 constitutive and TPA-induced promoter activity. It was concluded that NRP1 expression is regulated by the cooperation of several regulatory elements including AP-1, Sp1, and a CCAAT box.