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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.

Dengue fever and dengue hemorrhagic fever are mosquito-borne viral diseases. Dendritic cell–specific ICAM-3 grabbing nonintegrin (DC-SIGN1, encoded by CD209), an attachment receptor of dengue virus, is essential for productive infection of dendritic cells1,2. Here, we report strong association between a promoter variant of CD209, DCSIGN1-336, and risk of dengue fever compared with dengue hemorrhagic fever or population controls. The G allele of the variant DCSIGN1-336 was associated with strong protection against dengue fever in three independent cohorts from Thailand, with a carrier frequency of 4.7% in individuals with dengue fever compared with 22.4% in individuals with dengue hemorrhagic fever (odds ratio for risk of dengue hemorrhagic fever versus dengue fever: 5.84, P = 1.4 × 10−7) and 19.5% in controls (odds ratio for protection: 4.90, P = 2 × 10−6). This variant affects an Sp1-like binding site and transcriptional activity in vitro. These results indicate that CD209 has a crucial role in dengue pathogenesis, which discriminates between severe dengue fever and dengue hemorrhagic fever. This may have consequences for therapeutic and preventive strategies. Supplementary information The online version of this article (doi:10.1038/ng1550) contains supplementary material, which is available to authorized users.

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; Although the primary determinant of cell tropism is the interaction of viral envelope or capsid proteins with cellular receptors, other viral elements can strongly modulate viral replication. While the HIV-1 promoter is polymorphic for a variety of transcription factor binding sites, the impact of these polymorphisms on viral replication in vivo is not known. To address this issue, we engineered isogenic SIVmac239 chimeras harboring the core promoter/enhancer from HIV-1 clades B, C, and E. Here it is shown that the clade C and E core promoters/enhancers bear a noncanonical activator protein-1 (AP-1) binding site, absent from the corresponding clade B region. Relative ex vivo replication of chimeras was strongly dependent on the tissue culture system used. Notably, in thymic histocultures, replication of the clade C chimera was favored by IL-7 enrichment, which suggests that the clade C polymorphism in the AP-1 and NF-kappaB binding sites is involved. Simultaneous infection of rhesus macaques with the 3 chimeras revealed a strong predominance of the clade C chimera during primary infection. Thereafter, the B chimera dominated in all tissues. These data show that the clade C promoter is particularly adapted to sustain viral replication in primary viremia and that clade-specific promoter polymorphisms constitute a major determinant for viral replication.

International audience; We have reported recently that the chemokine interleukin 8 (IL-8)/CXCL8 was overexpressed in invasive estrogen receptor (ERalpha)-negative breast cancer cells compared with ERalpha-positive breast cancer cells. We now demonstrate that histone deacetylases (HDACs) play an essential role in the regulation of IL-8 gene expression in ERalpha-positive MCF-7 breast cancer cells. Treatment of MCF-7 cells with the HDAC inhibitor trichostatin A (TSA) led to a strong up-regulation of IL-8 protein and RNA levels in MCF-7 cells. The up-regulation of IL-8 in MCF-7 cells was time- and concentration-dependent. Moreover, run-on and transfection experiments demonstrated that IL-8 induction by HDAC inhibitors was transcriptional and involved mainly the nuclear factor-kappaB (NF-kappaB) site of the IL-8 promoter. These observations are corroborated by an up-regulation of NF-kappaB activity in MCF-7 cells in the presence of TSA. In addition, blocking NF-kappaB pathway by adenoviral delivery of a dominant-negative IkappaBorIkappaB kinase complex 2 (IKK2) mutant abolished IL-8 gene induction by histone deacetylase inhibitors. HDAC inhibitors triggered IKK phosphorylation and up-regulated p65 nuclear translocation, although they decreased the protein levels of IkappaBalpha, which accounts for NF-kappaB activation. TSA increased binding of acetylated histone 3 to the IL-8 gene promoter. In summary, our results demonstrate that NF-kappaB pathway repression by HDAC is responsible for the low expression of IL-8 in ERalpha-positive breast cancer cells.

International audience; The LEAFY (LFY) protein is a key regulator of flower development in angiosperms. Its gradually increased expression governs the sharp floral transition, and LFY subsequently controls the patterning of flower meristems by inducing the expression of floral homeotic genes. Despite a wealth of genetic data, how LFY functions at the molecular level is poorly understood. Here, we report crystal structures for the DNA-binding domain of Arabidopsis thaliana LFY bound to two target promoter elements. LFY adopts a novel seven-helix fold that binds DNA as a cooperative dimer, forming base-specific contacts in both the major and minor grooves. Cooperativity is mediated by two basic residues and plausibly accounts for LFY's effectiveness in triggering sharp developmental transitions. Our structure reveals an unexpected similarity between LFY and helix-turn-helix proteins, including homeodomain proteins known to regulate morphogenesis in higher eukaryotes. The appearance of flowering plants has been linked to the molecular evolution of LFY. Our study provides a unique framework to elucidate the molecular mechanisms underlying floral development and the evolutionary history of flowering plants.

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.

Permanent silencing of E2F-dependent genes is a hallmark of the irreversible cell cycle exit that characterizes terminally differentiated and senescent cells. The determinant of this silencing during senescence has been proposed to be the binding of the retinoblastoma protein Rb and the consequent methylation of H3K9. During ex vivo skeletal muscle differentiation, while most cells terminally differentiate and form myotubes, a subset of myoblasts remains quiescent and can be reinduced by growth factor stimulation to enter the cell cycle. Thus, differentiating cells are composed of two different populations: one in which E2F-dependent genes are permanently repressed and the other not. We observed that, in a manner reminiscent to senescent cells, permanent silencing of the E2F-dependent cdc6, dhfr, and p107 promoters in myotubes was associated with a specific increase in H3K9 trimethylation. To investigate the role of Rb in this process, we developed a reliable method to detect Rb recruitment by chromatinimmunoprecipitation. Surprisingly, we observed that Rb was recruited to these promoters more efficiently in quiescent cells than in myotubes. Thus, our data indicate that during muscle differentiation, permanent silencing and H3K9 trimethylation of some E2F-dependent genes are not directly specified by Rb binding, in contrast to what is proposed for senescence. (Mol Cancer Res 2008;6(3):418–25)

Although chromosome X open reading frame 6 (CXorf6) has been shown to be a causative gene for hypospadias, its molecular function remains unknown. To clarify this, we first examined CXorf6 protein structure, identifying homology to mastermind-like 2 (MAML2) protein, which functions as a co-activator in canonical Notch signaling. Transactivation analysis for wild-type CXorf6 protein by luciferase assays showed that CXorf6 significantly transactivated the promoter of a noncanonical Notch target gene hairy/enhancer of split 3 (Hes3) without demonstrable DNA-binding capacity. Transactivation analysis was also performed for the previously described three apparently pathologic nonsense mutations, indicating that E124X and Q197X proteins had no transactivation function, whereas R653X protein retained a nearly normal transactivation function. Subcellular localization analysis revealed that wild-type and R653X proteins co-localized with MAML2 protein in nuclear bodies, whereas E124X and Q197X proteins were incapable of localizing to nuclear bodies. Thus, further studies were performed for R653X, revealing the occurrence of nonsense mediated mRNA decay in vivo. Next, transient knockdown of CXorf6 was performed using small interfering RNA, showing reduced testosterone production in mouse Leydig tumor cells. Furthermore, steroidogenic factor 1 (SF1) protein bound to a specific sequence in the upstream of the CXorf6 coding region and exerted a transactivation activity. These results suggest that CXorf6 transactivates the Hes3 promoter, augments testosterone production, and contains the SF1 target sequence, thereby providing the first clue to clarify the biological role of CXorf6. We designate CXorf6 as MAMLD1 (mastermind-like domain-containing 1) based on its characteristic structure.

Epigénome er paléogénomie; International audience; Nucleosome positioning plays an essential role in cellular processes by modulating accessibility of DNA to proteins. Here, using only sequence-dependent DNA flexibility and intrinsic curvature, we predict the nucleosome occupancy along the genomes of Saccharomyces cerevisiae and Drosophila melanogaster and demonstrate the predictive power and universality of our model through its correlation with experimentally determined nucleosome occupancy data. In yeast promoter regions, the computed average nucleosome occupancy closely superimposes with experimental data, exhibiting a

Hormonal Carcinogenesis V : Proceedings of the Fifth International Symposium, Li JJ, Li SA, Mohla S, Rochefort H, Maudelonde T (Ed.) (2008) 129-138

The plastid genome of dicotyledonous plants is transcribed by three different RNA polymerases; an eubacterial-type enzyme, PEP; and two phage-type enzymes, RPOTp and RPOTmp. RPOTp plays an important role in chloroplast transcription, biogenesis, and mesophyll cell proliferation. RPOTmp fulfills a specific function in the transcription of the rrn operon in proplasts/amyloplasts during seed imbibition/germination and a more general function in chloroplasts during later developmental stages. In chloroplasts, RPOTmp is tightly associated with thylakoid membranes indicating that functional switching of RPOTmp is connected to thylakoid association. By using the yeast two-hybrid system, we have identified two proteins that interact with RPOTmp. The two proteins are very similar, both characterized by three N-terminal transmembrane domains and a C-terminal RING domain. We show that at least one of these proteins is an intrinsic thylakoid membrane protein that fixes RPOTmp on the stromal side of the thylakoid membrane, probably via the RING domain. A model is presented in which light by triggering the synthesis of the RING protein determines membrane association and functional switching of RPOTmp.

Class-switch recombination (CSR) enables IgM-producing B cells to switch to the production of IgG, IgE, and IgA. The process requires germ-line (GL) transcription that initiates from promoters upstream of switch (S) sequences and is regulated by the 3′ regulatory region (3′RR) located downstream of the Ig heavy chain (IgH) locus. How the 3′RR effect its long-range activation is presently unclear. We generated a mouse line in which Iγ3 GL promoter was replaced by Iγ1. We found that GL transcription could initiate from the inserted Iγ1 promoter and was induced by increased concentrations of IL-4 and that the transcripts were normally spliced. However, when compared with GL transcripts derived from the endogenous Iγ1 promoter in the same stimulation conditions, those from the inserted Iγ1 promoter were less abundant. CSR to Cγ3 was abrogated bothin vivoandin vitro. The results strongly suggest that the endogenous Iγ1 promoter insulates the inserted Iγ1 from the long-range activating effect of the 3′RR. The implications of our findings are discussed in light of the prominent models of long-distance activation in complex loci.

International audience; Carcinoma progression is associated with the loss of epithelial features, and the acquisition of mesenchymal characteristics and invasive properties by tumour cells. The loss of cell-cell contacts may be the first step of the epithelium mesenchyme transition (EMT) and involves the functional inactivation of the cell-cell adhesion molecule E-cadherin. Repression of E-cadherin expression by the transcription factor Snail is a central event during the loss of epithelial phenotype. Akt kinase activation is frequent in human carcinomas, and Akt regulates various cellular mechanisms including EMT. Here, we show that Snail activation and consequent repression of E-cadherin may depend on AKT-mediated nuclear factor-kappaB (NF-kappaB) activation, and that NF-kappaB induces Snail expression. Expression of the NF-kappaB subunit p65 is sufficient for EMT induction, validating this signalling module during EMT. NF-kappaB pathway activation is associated with tumour progression and metastasis of several human tumour types; E-cadherin acts as a metastasis suppressor protein. Thus, this signalling and transcriptional network linking AKT, NF-kappaB, Snail and E-cadherin during EMT is a potential target for antimetastatic therapeutics.

International audience; The mitochondrial transcription factor A (Tfam) is essential for transcription initiation and replication of mitochondrial DNA. It was previously reported that transcription factors Sp1, NRF-1, NRF-2 were critical for maintaining the normal transcription levels of the mammalian TFAM gene. In this work, investigation of the transcriptional regulation of the human TFAM gene revealed the presence of two cross-species conserved binding sites for the transcription factor hStaf/ZNF143. By using promoter binding assays, transient expression of mutant TFAM reporter gene constructs and chromatin immunoprecipitation experiments, we provided insight into the involvement of hStaf/ZNF143 in promoter activity. Furthermore, we reported the identification of two other functionally important elements. Altogether, our data led to the conclusion that the promoter of the human TFAM gene harbors a complex organization with at least six transcriptional regulatory elements.

International audience; Growing evidences show that functionally relevant polymorphisms in various promoters alter both transcriptional activity and affinities of existing protein-DNA interactions, and thus influence disease progression in humans. We previously reported the -94G>T CFTR promoter variant in a female CF patient in whom any known disease-causing mutation has been detected. To investigate whether the -94G>T could be a regulatory variant, we have proceeded to in silico analyses and functional studies including EMSA and reporter gene assays. Our data indicate that the promoter variant decreases basal CFTR transcriptional activity in different epithelial cells and alters binding affinities of both Sp1 and USF nuclear proteins to the CFTR promoter. The present report provides evidence for the first functional polymorphism that negatively affects the CFTR transcriptional activity and demonstrates a cooperative role of Sp1 and USF transcription factors in transactivation of the CFTR gene promoter.

International audience; Ribosome biogenesis requires equimolar amounts of four rRNAs and all 79 ribosomal proteins (RP). Coordinated regulation of rRNA and RP synthesis by eukaryotic RNA polymerases (Pol) I, III, and II is a key requirement for growth control. Using a novel global genetic approach, we showed that the absence of Hmo1 becomes lethal when combined with mutations of components of either the RNA Pol II or Pol I transcription machineries, of specific RP, or of the TOR pathway. Hmo1 directly interacts with both the region transcribed by Pol I and a subset of RP gene promoters. Down-regulation of Hmo1 expression affects RP gene expression. Upon TORC1 inhibition, Hmo1 dissociates from ribosomal DNA (rDNA) and some RP gene promoters simultaneously. Finally, in the absence of Hmo1, TOR-dependent repression of RP genes is alleviated. Therefore, we show here that Saccharomyces cerevisiae Hmo1 is directly involved in coordinating rDNA transcription by Pol I and RP gene expression by Pol II under the control of the TOR pathway.

International audience; The nuclear factor-kappaB (NF-kappaB) transcription factor family has a crucial role in rapid responses to stress and pathogens. We show that the NF-kappaB subunit RelB is functionally associated with the aryl hydrocarbon receptor (AhR) and mediates transcription of chemokines such as IL-8 via activation of AhR and protein kinase A. RelB physically interacts with AhR and binds to an unrecognized RelB/AhR responsive element of the IL-8 promoter linking two signaling pathways to activate gene transcription. We found a time-dependent recruitment of AhR to the RelB/AhR responsive element site of IL-8 mediated by the AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) and via activation of protein kinase A. Furthermore, NF-kappaB-binding sites that are preferentially recognized by RelB/p52 are a target for RelB/AhR complexes without addition of any stimuli, implicating the endogenous function of the AhR. RelB/AhR complexes are also found to bind on xenobiotic responsive element, and RelB drastically increases the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced xenobiotic responsive element reporter activity. The interaction of RelB with AhR signaling, and AhR with NF-kappaB RelB signaling pathways represent a new mechanism of cross talk between the two transcription factors.

International audience; We analyzed the effects of Rho GTPases on XSnail2 expression during neural crest (NC) ontogeny in Xenopus laevis embryos. The ectopic expression of both dominant-negative (N-) and constitutively active (V-) Rho GTPase mutants after RNA or DNA microinjection disrupted the endogenous expression of XSnail2, XFoxD3, and XSnail1. V14RhoA and N17Rac1 were inhibitory, whereas N19RhoA and V12Rac1 increased NC marker gene expression. In reporter assays using a XSnail2 promoter-green fluorescent protein (GFP) construct (alpha700BA-GFP), the ectopic expression of V14RhoA, N17Rac1, or the Rac1 inhibitor NSC 23766 decreased reporter expression in NC-neural plate, whereas N19RhoA or the RhoA inhibitor Y27632 and V12Rac1 enhanced it. Similarly, transgenic embryos expressing Rho GTPase mutants and GFP under control of the alpha700BA promoter displayed variations similar to those observed for ectopic RNA and DNA expression. These results show that Rho GTPases can regulate the expression of XSnail2 during NC ontogeny.

International audience; Transcriptional activation of silent genes can require the erasure of epigenetic marks such as DNA methylation at CpGs (cytosine-guanine dinucleotide). Active demethylation events have been observed, and associated processes are repeatedly suspected to involve DNA glycosylases such as mCpG binding domain protein 4, thymine DNA glycosylase (TDG), Demeter, and repressor of silencing 1. A complete characterization of the molecular mechanisms occurring in metazoan is nonetheless awaited. Here, we report that activation of the endogenous vitronectin gene in P19 cells by the nuclear receptor chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is observed in parallel with the recruitment of TDG and p68 RNA helicase, two components of a putative demethylation complex. Interestingly, when activated, the vitronectin gene was loaded with DNA methyltransferases 3a and 3b (Dnmt3a/b), and a strand-biased decrease in CpG methylation was detected. Dnmt3a was further found to associate with COUP-TFI and TDG in vivo, and cotransfection experiments demonstrated that Dnmt3a/b can enhance COUP-TFI-mediated activation of a methylated reporter gene. These results suggest that Dnmt3a/b could cooperate with the orphan receptor COUP-TFI to regulate transcription of the vitronectin gene.

Free in PubMed Central : http://www.pubmedcentral.nih.gov/picrender.fcgi?tool=pmcentrez&artid=1951289&blobtype=pdf; The heterodimer Ku70/80 Ku is the DNA-binding component of the DNA-PK complex required for the nonhomologous end-joining pathway. It participates in numerous nuclear processes, including telomere and chromatin structure maintenance, replication, and transcription. Ku interacts with retroviral preintegration complexes and is thought to interfere with the retroviral replication cycle, in particular the formation of 2-long terminal repeat (LTR) viral DNA circles, viral DNA integration, and transcription. We describe here the effect of Ku80 on both provirus integration and the resulting transgene expression in cells transduced with retroviral vectors. We found that transgene expression was systematically higher in Ku80-deficient xrs6 cells than in Ku80-expressing CHO cells. This higher expression was observed irrespective of the presence of the viral LTR and was also not related to the nature of the promoter. Real-time PCR monitoring of the early viral replicative steps demonstrated that the absence of Ku80 does not affect the efficiency of transduction. We analyzed the transgene distributions localization in nucleus by applying a three-dimensional reconstruction model to two-dimensional fluorescence in situ hybridization images. This indicated that the presence of Ku80 resulted in a bias toward the transgenes being located at the periphery of the nucleus associated with their being repressed; in the absence of this factor the transgenes tend to be randomly distributed and actively expressed. Therefore, although not strictly required for retroviral integration, Ku may be involved in targeting retroviral elements to chromatin domains prone to gene silencing.

acs encodes a high-affinity enzyme that permits survival during carbon starvation. As befits a survival gene, its transcription is subject to complex regulation. Previously, we reported that cAMP receptor protein (CRP) activates acs transcription by binding tandem DNA sites located upstream of the major acsP2 promoter and that the nucleoid protein IHF (integration host factor) binds three specific sites located just upstream. In vivo, the sequence that includes these IHF sites exerts a positive effect on CRP-dependent transcription, while a construct containing only the most proximal site exhibits reduced transcription compared with the full-length promoter or with a construct lacking all three IHF sites. Here, we defined the minimal system required for this IHF-dependent inhibition, showing it requires the promoter-distal CRP site and an amino acid residue located within activation region 2 (AR2), a surface determinant of CRP that interacts with RNA polymerase (RNAP). Surprisingly, for a Class III promoter, disruption of AR2 caused significant changes in the activity and structure of both the full-length promoter and the construct with the single proximal IHF site. We propose that AR2, together with IHF, mediates formation of a multi-protein complex, in which RNAP is stabilized in an open complex that remains poised on the promoter ready to respond rapidly to environmental changes.

Jérôme N. Feige, Laurent Gelman (Both authors contributed equally to the reported work); International audience; The ability of pollutants to affect human health is a major concern, justified by the wide demonstration that reproductive functions are altered by endocrine disrupting chemicals. The definition of endocrine disruption is today extended to broader endocrine regulations, and includes activation of metabolic sensors, such as the peroxisome proliferator-activated receptors (PPARs). Toxicology approaches have demonstrated that phthalate plasticizers can directly influence PPAR activity. What is now missing is a detailed molecular understanding of the fundamental basis of endocrine disrupting chemical interference with PPAR signaling. We thus performed structural and functional analyses that demonstrate how monoethyl-hexyl-phthalate (MEHP) directly activates PPARgamma and promotes adipogenesis, albeit to a lower extent than the full agonist rosiglitazone. Importantly, we demonstrate that MEHP induces a selective activation of different PPARgamma target genes. Chromatin immunoprecipitation and fluorescence microscopy in living cells reveal that this selective activity correlates with the recruitment of a specific subset of PPARgamma coregulators that includes Med1 and PGC-1alpha, but not p300 and SRC-1. These results highlight some key mechanisms in metabolic disruption but are also instrumental in the context of selective PPAR modulation, a promising field for new therapeutic development based on PPAR modulation.

Hypoxia and the Net ternary complex factor (TCF) regulate similar processes (angiogenesis, wound healing, and cellular migration) and genes (PAI-1, c-fos, erg-1, NOS-2, HO-1, and vascular endothelial growth factor genes), suggesting that they are involved in related pathways. We show here that hypoxia regulates Net differently from the other TCFs and that Net plays a role in the hypoxic response in vivo in mice and in cells. Hypoxia induces Net depletion from target promoters, nuclear export, ubiquitylation, and proteasomal degradation. Key mediators of the hypoxic response, the prolyl-4-hydroxylases containing domain proteins (PHDs), regulate Net. PHD downregulation in normoxia leads to Net degradation, and PHD overexpression delays Net downregulation by hypoxia. Net inhibition by RNA interference or mutation leads to altered regulation by hypoxia of the Net targets PAI-1, c-fos, and egr-1. We propose that hypoxia stimulates transcription of target promoters through removal of the repressor function of Net. Interestingly, the hematocrit response to a chemical inducer of hypoxia-like responses (cobalt chloride) is strongly altered in Net mutant mice. Our results show that the Net TCF is part of the biological response to hypoxia, adding a new component to an important pathological and physiological process.

International audience; Biallelic inactivating mutations of the transcription factor 1 gene (TCF1), encoding hepatocyte nuclear factor 1alpha (HNF1alpha) were identified in 50% of hepatocellular adenomas (HCA) phenotypically characterized by a striking steatosis. To understand the molecular basis of this aberrant lipid storage, we performed a microarray transcriptome analysis validated by quantitative reverse transcription-PCR, Western blotting, and lipid profiling. In mutated HCA, we showed a repression of gluconeogenesis coordinated with an activation of glycolysis, citrate shuttle, and fatty acid synthesis predicting elevated rates of lipogenesis. Moreover, the strong down-regulation of liver fatty acid-binding protein suggests that impaired fatty acid trafficking may also contribute to the fatty phenotype. In addition, transcriptional profile analysis of the observed deregulated genes in non-HNF1alpha-mutated HCA as well as in non-tumor livers allowed us to define a specific signature of the HNF1alpha-mutated HCA. In these tumors, lipid composition was dramatically modified according to the transcriptional deregulations identified in the fatty acid synthetic pathway. Surprisingly, lipogenesis activation did not operate through sterol regulatory element-binding protein-1 (SREBP-1) and carbohydrate-response element-binding protein (ChREBP) that were repressed. We conclude that steatosis in HNF1alpha-mutated HCA results mainly from an aberrant promotion of lipogenesis that is linked to HNF1alpha inactivation and that is independent of both SREBP-1 and ChREBP activation. Finally, our findings have potential clinical implications since lipogenesis can be efficiently inhibited by targeted therapies.

International audience; In lung adenocarcinoma, expression of Regulated upon Activation, Normal T cell Expressed and presumably Secreted (RANTES) is a predictor of survival while that of interleukin (IL)-8 is associated with a poor prognosis. In several models, tumorigenesis is abolished by RANTES, while it is facilitated by IL-8. We studied the regulation of RANTES and IL-8 expression in A549 lung adenocarcinoma cells. The effects of tumor necrosis factor (TNF)-alpha and regulators of protein kinases C (PKC)alpha/beta were tested because these have been shown to modulate cancer development and progression. TNF-alpha stimulated expression of both chemokines, while the PKCalpha/beta activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced only expression of IL-8 and inhibited TNF-alpha-induced RANTES expression. The PKCalpha/beta inhibitor G?76 increased TNF-alpha-induced RANTES production and prevented its down-regulation by TPA. In contrast, it decreased TNF-alpha or TPA-induced IL-8 release. The differential regulation of RANTES and IL-8 expression was further analyzed. Site-directed mutagenesis indicated that regulation of RANTES promoter activity required two nuclear factor (NF)-kappaB response elements but not its activator protein (AP)-1 binding sites. An AP-1 and a NF-kappaB recognition sites were necessary for full induction of IL-8 promoter activity by TNF-alpha and TPA. Moreover, electrophoretic mobility shift assays demonstrated that NF-kappaB response elements from the RANTES promoter were of lower affinity than that from the IL-8 promoter. Immunoblotting experiments showed that TPA was more potent than TNF-alpha to induce in a PKCalpha/beta dependent manner the p44/p42 mitogen-activated protein kinases (MAPK) signaling cascade which controls AP-1 activity. Conversely, TPA inhibited TNF-alpha-induced NF-kappaB signaling and was a weak activator of this pathway. Thus, TPA did not sufficiently activate NF-kappaB to increase transcription through the low affinity NF-kappaB binding sites on RANTES promoter and its inhibitory effect on TNF-alpha-induced NF-kappaB signaling resulted in a reduced transcription rate. On IL-8 promoter, increased transcription through the high affinity NF-kappaB binding site occurred even with poorly activated NF-kappaB and the functional AP-1 response element compensated any loss of transcription rate. These data provide a mechanistic insight into the differential regulation of IL-8 and RANTES expression by PKCalpha/beta in lung adenocarcinoma cells.

Despite the economic and sanitary problems caused by harmful biofilms, biofilms are nonetheless used empirically in industrial environmental and bioremediation processes and may be of potential use in medical settings for interfering with pathogen development. Escherichia coli is one of the bacteria with which biofilm formation has been studied in great detail, and it is especially appreciated for biotechnology applications because of its genetic amenability. Here we describe the development of two new genetic tools enabling the constitutive and inducible expression of any gene or operon of interest at its native locus. In addition to providing valuable tools for complementation and overexpression experiments, these two compact genetic cassettes were used to modulate the biofilm formation capacities of E. coli by taking control of two biofilm-promoting factors, autotransported antigen 43 adhesin and the bscABZC cellulose operon. The modulation of the biofilm formation capacities of E. coli or those of other bacteria capable of being genetically manipulated may be of use both for reducing and for improving the impact of biofilms in a number of industrial and medical applications.

International audience; In lung adenocarcinoma, expression of Regulated upon Activation, Normal T cell Expressed and presumably Secreted (RANTES) is a predictor of survival while that of interleukin (IL)-8 is associated with a poor prognosis. In several models, tumorigenesis is abolished by RANTES, while it is facilitated by IL-8. We studied the regulation of RANTES and IL-8 expression in A549 lung adenocarcinoma cells. The effects of tumor necrosis factor (TNF)-alpha and regulators of protein kinases C (PKC)alpha/beta were tested because these have been shown to modulate cancer development and progression. TNF-alpha stimulated expression of both chemokines, while the PKCalpha/beta activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced only expression of IL-8 and inhibited TNF-alpha-induced RANTES expression. The PKCalpha/beta inhibitor G?76 increased TNF-alpha-induced RANTES production and prevented its down-regulation by TPA. In contrast, it decreased TNF-alpha or TPA-induced IL-8 release. The differential regulation of RANTES and IL-8 expression was further analyzed. Site-directed mutagenesis indicated that regulation of RANTES promoter activity required two nuclear factor (NF)-kappaB response elements but not its activator protein (AP)-1 binding sites. An AP-1 and a NF-kappaB recognition sites were necessary for full induction of IL-8 promoter activity by TNF-alpha and TPA. Moreover, electrophoretic mobility shift assays demonstrated that NF-kappaB response elements from the RANTES promoter were of lower affinity than that from the IL-8 promoter. Immunoblotting experiments showed that TPA was more potent than TNF-alpha to induce in a PKCalpha/beta dependent manner the p44/p42 mitogen-activated protein kinases (MAPK) signaling cascade which controls AP-1 activity. Conversely, TPA inhibited TNF-alpha-induced NF-kappaB signaling and was a weak activator of this pathway. Thus, TPA did not sufficiently activate NF-kappaB to increase transcription through the low affinity NF-kappaB binding sites on RANTES promoter and its inhibitory effect on TNF-alpha-induced NF-kappaB signaling resulted in a reduced transcription rate. On IL-8 promoter, increased transcription through the high affinity NF-kappaB binding site occurred even with poorly activated NF-kappaB and the functional AP-1 response element compensated any loss of transcription rate. These data provide a mechanistic insight into the differential regulation of IL-8 and RANTES expression by PKCalpha/beta in lung adenocarcinoma cells.

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.