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International audience; Transcriptional memory of transient signals can be imprinted on living systems and influence their reactivity to repeated stimulations. Although they are classically ascribed to structural chromatin rearrangements in eukaryotes, such behaviors can also rely on dynamic memory circuits with sustained self-amplification loops. However, these phenomena are either of finite duration, or conversely associated to sustained phenotypic changes. A mechanism is proposed, in which only the responsiveness of the target gene is durably reset at a higher level after primary stimulation, using the celebrated but still puzzling vitellogenesis memory effect. The basic ingredients of this system are: 1), a positive autoregulation of the estrogen receptor α gene; 2), a strongly cooperative action of the estradiol receptor on vitellogenin expression; and 3), a variant isoform of the estradiol receptor with two autonomous transcription-activating modules, one of which is signal-independent and the other, signal-dependent. Realistic quantification supports the possibility of a multistationary situation in which ligand-independent activity is unable by itself to prime the amplification loop, but can click the system over a memory threshold after a primary stimulation. This ratchet transcriptional mechanism can have developmental and ecotoxicological importance and explain lifelong imprinting of past exposures without apparent phenotypic changes before restimulation and without need for persistent chromatin modifications.

International audience; An essential step towards understanding life would be to identify the very basic mechanisms responsible for the discerning behaviour of living biochemical systems, absent from randomly reacting chemical soups. One intuitively feels that this question goes beyond the particular nature of the biological molecules and should relate to general physical principles. The pre-eminent physicist Ludwig Boltzmann early envisioned life as a struggle for entropy, in concordance with the subsequent principle of self-organization out of equilibrium. Re-examination of elementary steady state biochemical systems from a statistical perspective supports this view and shows that sigmoidal responses arising from microstates elimination, are sufficient to explain innermost characteristics of life, including its capacity to convert random molecular interactions into accurate biological reactions. A primary operating strategy to achieve this goal is the introduction of time-irreversible transitions in molecular state conversion cycles by injection of free energy, which confers decisional capacity to single macromolecules. Selected examples from various fields of molecular biology such as enzymology and gene expression, are provided to show that these non-equilibrium steady state mechanisms remain important in contemporary biochemical systems. But in addition, information archiving allowed the emergence of the time-reversible counterparts of these mechanisms, mediated by evolutionary pre-organized macromolecular complexes capable of generating discernment in a non-dissipative manner.

International audience; The main purpose of this article is to study potential biological effects of low-power millimeter waves (MMWs) on endoplasmic reticulum (ER), an organelle sensitive to a wide variety of environmental insults and involved in a number of pathologies. We considered exposure frequencies around 60 GHz in the context of their near-future applications in wireless communication systems. Radiations within this frequency range are strongly absorbed by oxygen molecules, and biological species have never been exposed to such radiations in natural environmental conditions. A set of five discrete frequencies has been selected; three of them coincide with oxygen spectral lines (59.16, 60.43, and 61.15 GHz) and two frequencies correspond to the spectral line overlap regions (59.87 and 60.83 GHz). Moreover, we used a microwave spectroscopy approach to select eight frequencies corresponding to the spectral lines of various molecular groups within 59-61 GHz frequency range. The human glial cell line, U-251 MG, was exposed or sham-exposed for 24 h with a peak incident power density of 0.14 mW/cm(2). The average specific absorption rate (SAR) within the cell monolayer ranges from 2.64 +/- 0.08 to 3.3 +/- 0.1 W/kg depending on the location of the exposed well. We analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) the level of expression of two endogenous ER-stress biomarkers, namely, the chaperones BiP/GRP78 and ORP150/GRP170. It was found that exposure to low-power MMW does not significantly modify the mRNA levels of these stress-sensitive genes suggesting that ER homeostasis is not altered by low-power MMW at the considered frequencies.

International audience; BAG6/Scythe/Bat3 is a cochaperone of the heat shock protein HSP70 and is involved in various developmental processes, cellular stress and viability. BAG6 interferes with the protein-refolding activity of HSP70 but its precise involvement in proteotoxic stresses remains unknown. We show that BAG6 is required for the accumulation of HSP70 upon heat shock and that conversely, once accumulated, HSP70 leads to the massive and CHIP-independent degradation of BAG6 through the ubiquitin-proteasome system. These reciprocal influences between BAG6 and HSP70 upon heat shock suggest that BAG6 is a central regulator of the cellular content of HSP70. The HSP70-driven degradation of BAG6, following the BAG6-dependent accumulation of HSP70, could allow the protein-refolding activity of HSP70 and limit the extent of its induction.

International audience; Clusterin is a usually secreted glycoprotein with chaperone properties. Recently, it has been suggested that clusterin isoforms reside in the nuclear and cytosolic compartments of human cell types, where they can influence various cellular programs including DNA repair, transcription and apoptosis. Several mechanisms have been proposed to explain this atypical location, including alternative transcription initiation and alternative splicing. However, none of these have been unequivocally established as occurring in live cells. Here we provide direct experimental evidence that in live intact cells, under certain stress conditions, clusterin can evade the secretion pathway and reach the cytosol. This was demonstrated using several complementary approaches. Flow cytometry and selective permeabilization of U251 cell membranes with digitonin allowed detection of cytosolic clusterin in stressed U251 cells. In addition, a stringent enzymatic assay reliant upon the exclusively cytosolic deubiquitinase enzymes confirmed that clusterin synthesized with its hydrophobic secretion signal sequence can reach the cytosol of U251 cells. The retrotranslocation of clusterin is likely to occur through a mechanism similar to the endoplasmic reticulum (ER)-associated protein degradation pathway and involves passage through the Golgi apparatus. We also report that the ER-associated ubiquitin ligase Hrd1/synoviolin can interact with, and ubiquitinate clusterin. The possible biological functions of these novel behaviours of clusterin are discussed.

International audience; Protein transduction domains (PTDs) are promising tools for transducing presynthesized polypeptides across the plasma membrane. However, the development and optimization of PTDs are hampered by many technical problems and artifacts resulting notably from the tight binding of PTDs to the cell surface and the difficulty in discriminating, through imagery analyses, truly cytosolic from cytoplasmic vesicular compartments. To circumvent these problems, we have developed an unambiguous enzymatic assay of the cytosolic uptake of PTD-driven proteins, based on the processing by ubiquitin-specific C-terminal proteases (DUBs). This method, coupled with fluorometry and fluorescence microscopy, shows that the TAT PTD derived from human immunodeficiency virus type 1 is rapidly taken up by cells but fails to reach their cytosol, except when dendritic cells, which are known to take up circulating antigens for cross-presentation, are used. In addition to its usefulness in assessing cytosolic uptake, DUB processing of PTD-linked proteins can ensure the intracellular release of cargo proteins, which might prove helpful for MHC-I-based vaccination or intracellular delivery of biologically active polypeptides.

The heat shock factor 1 (HSF1) is the central actor of the response to hyperthermia in eukaryotic cells. In mammals, male germ cells are an exception among all cellular populations for their HSF1 activation occurs at low temperature. This feature was believed to be specific of homeotherms whose testicular compartment is located outside the main body cavity, where temperature is lower. In the present study, we show that in the poikilotherm rainbow trout, the maximal heat shock response of male germ cells, that are located in the same body compartment than the other organs, occurs also at a significantly lower temperature (22 degrees C) than for somatic cells (28 degrees C), regardless of culture conditions before heat shock. In addition, the acquisition of the HSE-binding activity of HSF1 upon heat shock is not associated with the classical hsp70 mRNA accumulation. Taken together, these results strongly suggest the existence of a particular mode of heat shock response that could be specific of male germ cells but not restricted to homeotherms.

Proliferation and differentiation of progenitor stem cells are mainly controlled by diffusible and adhesion molecules. Stem cell factor (SCF), an essential regulator of spermatogenesis produced by Sertoli cells, utilize both modes of cell to cell communication. Indeed, SCF exists in soluble (SCFs) and membrane-bound (SCFm) forms, which are required for a complete spermatogenesis, and are generated by alternative splicing of optional exon 6, encoding sites of proteolysis. We show that in the mouse testis, the alternative splicing of SCF is developmentally regulated. SCFs predominates in fetal and neonatal gonads and is then replaced by SCFm in the prepubertal and adult gonads. By sequencing SCF exon 6, we show that the flanking intronic sequences perfectly follow the gt-at rule, suggesting that the basal splicing machinery might not be responsible by itself for exon 6 skipping. Moreover, freshly isolated Sertoli cells mainly express SCFm, but a switch to SCFs occurs after 48 h of culture. We found that this change can be prevented by acidification of the culture medium at pH 6.3 or by addition of lactate. The sustained synthesis of SCFm at low pH was no longer observed in the presence of cycloheximide, suggesting that SCF exon 6 skipping requires de novo protein synthesis. Accordingly, UV cross-linking experiments show that nuclear Sertoli cell protein(s) bind in a sequence-specific manner to exon 6. Together, our data allow the proposal of an integrated mechanism in which the synthesis of lactate by Sertoli cells is used in the same time as an energetic substrate for germ cells and as a promoter of their survival/proliferation through the production of SCFm.

A systematic scanning of nucleic acid databases for DNA elements made of combinations of RGGTCA nuclear receptor half sites, has revealed that identical 19 nucleotide-long motifs composed of two inverted RGGTCA sites with a spacing of 7 nucleotides (IR7), are present upstream of the regions coding for the human TR2 and of the sea urchin SpSHR2 orphan receptors. We have developed an experimental strategy based on PCR, to check if this IR7 could correspond to an unusually long cis-element, conserved along evolution and regulating the TR2 genes. We found that indeed IR7 is present in the 5' untranslated region of TR2 genes from all species tested, including Xenopus, rainbow trout, zebrafish and mouse. The exact conservation throughout the animal kingdom of such a long, non repetitive and non coding genomic region, highly suggests that it should ensure important biological functions. In addition, this work has allowed the identification of a new, non coding, upstream exon in the mouse TR2 gene present in testicular TR2 mRNAs.

International audience; The quasi-equilibrium approximation is acceptable when molecular interactions are fast enough compared to circuit dynamics, but is no longer allowed when cellular activities are governed by rare events. A typical example is the lactose operon (lac), one of the most famous paradigms of transcription regulation, for which several theories still coexist to describe its behaviors. The lac system is generally analyzed by using equilibrium constants, contradicting single-event hypotheses long suggested by Novick and Weiner (1957). Enzyme induction as an all-or-none phenomenon. Proc. Natl. Acad. Sci. USA 43, 553-566) and recently refined in the study of (Choi et al., 2008. A stochastic single-molecule event triggers phenotype switching of a bacterial cell. Science 322, 442-446). In the present report, a lac repressor (LacI)-mediated DNA immunoprecipitation experiment reveals that the natural LacI-lac DNA complex built in vivo is extremely tight and long-lived compared to the time scale of lac expression dynamics, which could functionally disconnect the abortive expression bursts and forbid using the standard modes of lac bistability. As alternatives, purely kinetic mechanisms are examined for their capacity to restrict induction through: (i) widely scattered derepression related to the arrival time variance of a predominantly backward asymmetric random walk and (ii) an induction threshold arising in a single window of derepression without recourse to nonlinear multimeric binding and Hill functions. Considering the complete disengagement of the lac repressor from the lac promoter as the probabilistic consequence of a transient stepwise mechanism, is sufficient to explain the sigmoidal lac responses as functions of time and of inducer concentration. This sigmoidal shape can be misleadingly interpreted as a phenomenon of equilibrium cooperativity classically used to explain bistability, but which has been reported to be weak in this system.

International audience; Chaperone synthesis in response to proteotoxic stress is dependent on a family of transcription factors named heat shock factors (HSFs). The two main factors in this family, HSF1 and HSF2, are co-expressed in numerous tissues where they can interact and form heterotrimers in response to proteasome inhibition. HSF1 and HSF2 exhibit two alternative splicing isoforms, called α and β, which contribute to additional complexity in HSF transcriptional regulation, but remain poorly examined in the literature. In this work, we studied the transcriptional activity of HSF1 and HSF2 splicing isoforms transfected into immortalized Mouse Embryonic Fibroblasts (iMEFs) deleted for both Hsf1 and Hsf2, under normal conditions and after proteasome inhibition. We found that HSF1α is significantly more active than the β isoform after exposure to the proteasome inhibitor MG132. Furthermore, we clearly established that, while HSF2 had no transcriptional activity by itself, short β isoform of HSF2 exerts a negative role on HSF1β-dependent transactivation. To further assess the impact of HSF2β inhibition on HSF1 activity, we developed a mathematical modelling approach which revealed that the balance between each HSF isoform in the cell regulated the strength of the transcriptional response. Moreover, we found that cellular stress such as proteasome inhibition could regulate the splicing of Hsf2 mRNA. All together, our results suggest that relative amounts of each HSF1 and HSF2 isoforms quantitatively determine the cellular level of the proteotoxic stress response.

Clusterin cDNA has been isolated as a copy of a mRNA overexpressed in a wide variety of biological disorders, including tissue regression, brain injuries and oncogenic cell transformation. While the molecular cloning of the rat and the human clusterin genes has revealed a high degree of conservation of the genomic organization between mammals, the avian locus described here illustrates several divergent features. The avian gene has the particularity to be transcribed from at least two different promoters, both of which are active in transient expression assays using the quail QT6 transformed cell line. The detection of the two clusterin mRNA species by reverse-transcription-mediated PCR reveals a coordinated initiation of transcription from both promoters in all organs tested. In possible relation to the bipartite organization of the avian regulatory region, the putative cis-elements described in the unique mammalian promoters appear divided among the two avian promoters. In addition, the sequence comparison of avian and mammalian regulatory sequences has allowed the identification of a conserved putative cis-element which appears to be the target for specific DNA-binding factors.

International audience; A single heat shock factor (HSF), mediating the heat shock response, exists from yeast to Drosophila, whereas several related HSFs have been found in mammals. This raises the question of the specific or redundant functions of the different members of the HSF family and in particular of HSF1 and HSF2, which are both ubiquitously expressed. Using immortalized mouse embryonic fibroblasts (iMEFs) derived from wild-type, Hsf1(-/-), Hsf2(-/-) or double-mutant mice, we observed the distinctive behaviors of these mutants with respect to proteasome inhibition. This proteotoxic stress reduces to the same extent the viability of Hsf1(-/-)- and Hsf2(-/-)-deficient cells, but through different underlying mechanisms. Contrary to Hsf2(-/-) cells, Hsf1(-/-) cells are unable to induce pro-survival heat shock protein expression. Conversely, proteasome activity is lower in Hsf2(-/-) cells and the expression of some proteasome subunits, such as Psmb5 and gankyrin, is decreased. As gankyrin is an oncoprotein involved in p53 degradation, we analyzed the status of p53 in HSF-deficient iMEFs and observed that it was strongly stabilized in Hsf2(-/-) cells. This study points a new role for HSF2 in the regulation of protein degradation and suggests that pan-HSF inhibitors could be valuable tools to reduce chemoresistance to proteasome inhibition observed in cancer therapy.

International audience; Certain transcription factors have recently been shown to interact with DNA in living cells, through very short binding cycles, contrasting with the data previously obtained in vitro, and with the view of a stepwise building of transcription initiation complexes. These short cycles are triggered by active dissociation mechanisms, suggesting that they ensure important biological functions. Various interpretations of these observations have been proposed, including a mechanism allowing the cell to switch off gene expression after removal of the inducer, or increasing the availability of free transcription factors. The interpretation examined here is that the brevity of the transcription factor turnovers favors the determinism of gene expression. For the genes with open chromatin and subject to this mode of interaction, the differential dynamics between promoter occupancy and the following processes mediating protein accumulation, can be essential for the dosage of gene expression. Biological activities and quantitative conditions allowing to increase the frequency of DNA-protein binding cycles are proposed. The unexpected dynamics of certain DNA-protein interactions can provide a concrete example of the notion of apparent gradation of single-site occupancy, which is a general solution allowing to extend the mass action determinism to low copy number molecules.

International audience; Scythe (BAT3; HLA-B associated transcript 3, Bag 6) is a protein that has been implicated in apoptosis because it can modulate the Drosophila melanogaster apoptotic regulator, Reaper. Mice lacking Scythe show pronounced defects in organogenesis and in the regulation of apoptosis and proliferation during mammalian development. However, the biochemical pathways important for Scythe function are unknown. We report here multiple levels of interaction between Scythe and the apoptogenic mitochondrial intermembrane protein AIF (apoptosis-inducing factor). Scythe physically interacts with AIF and regulates its stability. AIF stability is markedly reduced in Scythe(-/-) cells, which are more resistant to endoplasmic reticulum stress induced by thapsigargin. Reintroduction of Scythe or overexpression of AIF in Scythe(-/-) cells restores their sensitivity to apoptosis. Together, these data implicate Scythe as a regulator of AIF.

International audience; Clusterin is a secreted protein chaperone up-regulated in several pathologies, including cancer and neurodegenerative diseases. The present study shows that accumulation of aberrant proteins, caused by the proteasome inhibitor MG132 or the incorporation of the amino acid analogue AZC (L-azetidine-2-carboxylic acid), increased both clusterin protein and mRNA levels in the human glial cell line U-251 MG. Consistently, MG132 treatment was capable of stimulating a 1.3 kb clusterin gene promoter. Promoter deletion and mutation studies revealed a critical MG132-responsive region between -218 and -106 bp, which contains a particular heat-shock element, named CLE for 'clusterin element'. Gel mobility-shift assays demonstrated that MG132 and AZC treatments induced the formation of a protein complex that bound to CLE. As shown by supershift and chromatin-immunoprecipitation experiments, CLE is bound by HSF1 (heat-shock factor 1) and HSF2 upon proteasome inhibition. Furthermore, co-immunoprecipitation assays indicated that these two transcription factors interact. Gel-filtration analyses revealed that the HSF1-HSF2 heterocomplexes bound to CLE after proteasome inhibition have the same apparent mass as HSF1 homotrimers after heat shock, suggesting that HSF1 and HSF2 could heterotrimerize. Therefore these studies indicate that the clusterin is a good candidate to be part of a cellular defence mechanism against neurodegenerative diseases associated with misfolded protein accumulation or decrease in proteasome activity

International audience; HSF2 is an enigmatic member of the heat shock factor family, identified in the homeotherm classes of birds and mammals. We report the characterization of HSF2 from an evolutionary ancient vertebrate, the fish rainbow trout (rtHSF2). rtHSF2 appears closely related to its mammalian counterparts at structural and functional levels. The conservation of the distinctive features of HSF2 from fish to human suggests that it should ensure important biological functions, not redundant with those of HSF1. Proteasome inhibition, reported as a potent stimulator of HSF2, leads to the stabilization and to a striking nuclear trafficking of rtHSF2-GFP fusion protein. Upon treatment with the proteasome inhibitor MG132, rtHSF2-GFP accumulates into PML nuclear bodies (NBs) independently of its sumoylation and, if expressed at moderate level, moves to nucleoli. The translocation of rtHSF2-GFP from NBs to nucleoli is greatly favored by overexpression of the heat shock protein Hsp70. The mammalian counterpart mouse HSF2 (mHSF2) also exhibited changes in intracellular distribution upon MG132 treatment. mHSF2 partitioned between a juxtanuclear area that we characterized as an aggresome and the nucleoli. These relocalizations are likely to reflect common structural changes of mouse and trout HSF2 upon activation.

International audience; Clusterin is a puzzling protein upregulated in many diseased tissues, presented as either a survival or a death protein. The role of clusterin might depend on the final maturation and localization of the protein, which can be secreted or reside inside cells, either after in situ synthesis or uptake of extracellular clusterin. We studied the biological effects of intracellular clusterin and observed that clusterin forms containing the alpha-chain region strongly accumulated in an ubiquitinated form in juxtanuclear aggregates meeting the main criterions of aggresomes and leading to profound alterations of the mitochondrial network. The viability of cells transfected by intracellular forms of clusterin was improved by overexpression of Bcl-2, and caspase inhibition was capable of rescuing cells expressing clusterin, which presented an altered mitochondrial permeability. We propose that, although it might be an inherently pro-survival and anti-apoptotic protein expressed by cells under stress in an attempt to protect themselves, clusterin can become highly cytotoxic when accumulated in the intracellular compartment. This activity might reconcile the opposite purported influences of clusterin on cell survival and explain how clusterin can be causally involved in neurodegeneration.

The glucocorticoid receptor (GR) is a transcription factor, subject to several types of posttranslational modifications including phosphorylation and ubiquitination. We showed that the GR is covalently modified by the small ubiquitin-related modifier-1 (SUMO-1) peptide in mammalian cells. We demonstrated that GR sumoylation is not dependent on the presence of the ligand and regulates the stability of the protein as well as its transcriptional activity. SUMO-1 overexpression induces dramatic GR degradation, abolished by proteasome inhibition. We also found that SUMO-1 stimulates the transactivation capacity of GRs to an extent largely exceeding those observed so far for other sumoylated transcription factors. Overexpression of SUMO-1 specifically enhances the ligand-induced transactivation of GR up to 8-fold. However, this hyperactivation occurs only in the context of a synergy between multiple molecules of GRs. It requires more than one receptor DNA-binding site in promoter and becomes more prominent as the number of sites increases. Interestingly, these observations may be related to the transcriptional properties of the synergy control region of GRs, which precisely contains two evolutionary conserved sumoylation sites. We propose a model in which SUMO-1 regulates the synergy control function of GR and serves as a unique signal for activation and destruction.

The developmental expression patterns of the nuclear orphan receptors COUP-TFs (chicken ovalbumin upstream promoter-transcription factors) have been correlated to neurogenesis in several animal species. Nevertheless, the role of COUP-TFs in neurogenesis remains unknown. We have studied the functional involvement of COUP-TFI in retinoic acid (RA)-induced neuronal differentiation of P19 embryonal carcinoma cells through two complementary approaches: 1) deregulated expression of COUP-TFI, and 2) inactivation of endogenous COUP-TFs by means of a dominant-negative COUP-TFI mutant. Low levels of wild-type (wt)COUP-TFI transgene expression did not inhibit neural cell fate and primarily enhanced neuron outgrowth from RA-treated P19 aggregates. In contrast, high COUP-TFI expression impeded the neuronal differentiation of P19 cells induced with RA, resulting in cell cultures lacking neurons. This morphological effect was correlated to an elevated level of E-cadherin mRNA. The dominant-negative COUP-TFI mutant induced cell packing after RA treatment and inhibited neurite extension and neuron outgrowth from aggregates. A RGD peptide interference assay indicated that endogenous COUP-TFs could favor migration of neurons through an integrin-dependent mechanism. Accordingly, vitronectin mRNA levels were shown to be up-regulated by COUP-TFI by RT-PCR analysis, and COUP-TFI stimulated the mouse vitronectin promoter activity in transient transfection assays. Taken together, these data indicate that COUP-TFI is not simply a global repressor of retinoid functions, but shows a high selectivity for regulating genes involved in cellular adhesion and migration processes that are particularly important for neuronal differentiation.

Clusterin expression is increased in tissues undergoing apoptosis, including neurodegenerative retina, but the causal relationships remain to be clarified. To test the hypothesis that overexpression of clusterin could induce apoptosis in neurons, transgenic mice were generated in which rat clusterin transgene was expressed in photoreceptor cells under the transcriptional control of the human interphotoreceptor retinoid-binding protein (IRBP) promoter. Photoreceptor cell death in the resulting transgenic mice was examined by histology and TUNEL techniques. The expression of the clusterin transgene was confirmed by in situ hybridization in the photoreceptor cells, and results in a complex pattern of clusterin protein distribution in the retina. A reduction in apoptotic staining in the transgenic retinas was observed from birth to postnatal day 15. These results suggest that clusterin is not causally involved in apoptotic mechanisms of photoreceptor cell death, but may relate to cytoprotective functions.

By using degenerate primers designed from glutamate decarboxylase (GAD) sequences of mammals, Xenopus and Drosophila, a 270-bp cDNA fragment was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) from cerebellum total RNA of rainbow trout. This partial cDNA shows 90% identity with mammalian GAD 65 and presents the Asn-Pro-His-Lys (NPHK) sequence corresponding to the pyridoxal-binding region of porcine DOPA decarboxylase or mammalian GAD. The distribution of GAD 65 mRNA-expressing neurons in the forebrain of the trout was studied by in situ hybridization using either digoxigenin- or 35S-labeled probes. The results demonstrate that gamma-amino butyric acid (GABA) neurons are widely distributed throughout the forebrain, with a high density in the periventricular regions. In this study, we report their precise distribution in the telencephalon and diencephalon. GAD mRNA-expressing cells were particularly abundant in the preoptic region and the mediobasal hypothalamus, two major neuroendocrine and estrogen-sensitive regions in fish. The presence of GAD mRNA-expressing neurons was observed in visually related structures such as the suprachiasmatic nucleus, the pretectal region, and the thalamus. Immunohistochemistry with antibodies directed against mouse GAD failed to demonstrate the presence of immunoreactive cell bodies, but showed a very high concentration of GAD-immunoreactive fibers in many brain regions, notably in the preoptic area, hypothalamus, and neurohypophyseal digitations of the pituitary, in particular in the proximal pars distalis. These results indicate that GABA neurons are ideally placed to modulate neuroendocrine activities at the hypothalamic and pituitary levels and to participate in the processing of sensorial information.

Existing conditional expression systems can be classified in two major categories that are based either on the induction or on the de-repression of transcription. The system described here combines both mechanisms, since a unique transcription factor can be shifted from a repression to a stimulation activity by simply changing its ligand. The resulting advantage of this system is the complete absence of basal expression before active induction. The principle of this method is based on the unexpected ability of the chimeric protein containing the DNA-binding domain of the yeast Gal4 transcription factor fused to the COOH half of the estradiol receptor (GalER), to act as a repressor when bound to the drug 4OHtamoxifen, in the context of a previously described optimized Gal4-responsive promoter. The efficacy of this system has been assessed in transient expression assays using the chloramphenicol acetyl transferase (CAT), and in situ, through the activity of a Gal4 responsive β-galactosidase gene.

Clusterin/apoJ is an intriguing gene frequently isolated by differential screening in laboratories from different areas of molecular biology, since it is overexpressed in numerous cases of degenerative diseases such as Alzheimer's disease and scrapie. While the dramatic increase of clusterin expression in injured tissues is well established, the molecular basis of the gene induction remains unclear. In this study, we have focused our attention on the only DNA region strictly conserved between clusterin gene proximal promoters from different vertebrate classes. We show that this 14-bp DNA element is specifically recognized by the HSF1 transcription factor and can mediate heat-shock-induced transcription in transient expression assays. Conversely, the avian clusterin proximal promoter, point-mutated at the level of this element, no longer transmits heat-shock activation. These findings provide a possible explanation for the high sensitivity of clusterin expression to environmental changes and allow the classification of clusterin as an extracellular version of heat-shock protein.

We have isolated the avian gene T64 corresponding to the mammalian clusterin, on the basis of high accumulation of its template mRNA in cells infected with oncogenic retroviruses. Since the clusterin was shown to have a protective effect against the immune system, its induction by oncogenic viruses is of major biological importance. The unique, short 5 kb-long T64 genomic locus is inactive in normal quail embryo fibroblasts in primary culture whereas it shows a high transcriptional activity after transformation by the Rous sarcoma virus. The 963 bp-long 5' flanking region is sufficient to drive the transcription of the chloramphenicol acetyltransferase reporter gene in a thermodependent manner when a thermosensitive version of pp60v-src is used. Deletion and point mutation analyses of the promoter show that the v-src response requires at least two separate elements: PUR and AP-1, located respectively at positions -167 to -152 and -25 to -19 relative to the single transcription initiation site. In addition, the binding of specific nuclear factors to these responsive elements correlates with the T64 promoter activation.

This brief review discusses the recent characterization in the brain of a gene coding for a protein that may be involved in programmed cell death and/or brain plasticity. We will term it sulfated glycoprotein-2 (SGP-2), the name corresponding to the first cDNA characterized. Recent studies have demonstrated the overexpression of this sulfated glycoprotein in various CNS disorders, such as certain gliomas, Alzheimer's disease and epilepsy, as well as after experimental brain injury in animals where different cell types were undergoing tissue remodelling or cell death. In peripheral tissues, SGP-2 gene expression has been found to be strikingly increased following experimental manipulations in which cells of injured tissues were undergoing programmed cell death or apoptosis. The results reported thus far are intriguing and suggest the possible involvement of SGP-2 in apoptotic mechanisms as well as its interaction with components of the immune system possibly associated with cell death in neurodegenerative disorders. © 1992 Wiley-Liss, Inc.

Polypurine/polypyrimidine repetitive sequences occur with high frequency in eucaryotic genomes, particularly around transcription units. Since such sequences are known to adopt triple stranded-structures under appropriate conditions in vitro, it is of major interest to know if they occur in vivo, and thus if they can have some biological importance by inducing structural constraints in the genomic DNA. To this end, we have isolated a (TTCCC)48 sequence, present in the promoter of an avian gene, and tested its ability to form PU-PY-PY and PU-PU-PY triple helices in vitro, through the oligonucleotide gel shift technique and single strand-specific nuclease footprinting. We have then developed an oligonucleotide protection assay, which can be adapted to in vivo investigations. This strategy leads us to conclude that in vivo conditions allow preponderant formation of triplex of the PU-PU-PY class.

Synthetic human pancreatic growth hormone-releasing factor (hpGRF(1–40)-NH2) causes a 100% stimulation of cyclic AMP cell content in rat adenohypophysial cells in culture as early as 1 min after its addition while a maximal 33-fold increase is measured at 40 min. Somatostatin (10 nM) causes a 40%–60% inhibition of GRF-induced cyclic AMP accumulation while GH release is inhibited by 90%–95% at all time intervals. The inhibitory effect of somatostatin is exerted on the maximal effect of GRF while the ED50 values of GRF action on cyclic AMP cell content ( $ 1 nM) and GH release ( $ 0.1 nM) are not affected by the tetradecapeptide. Prostaglandin E2 causes a 2.5-fold increase in cyclic AMP levels within 1 min after its addition with a maximal 25-fold stimulation measured at 30 min. Although not completely additive, the stimulatory effects of PGE2 and GRF together on cyclic AMP cell content and GH release are more potent than when either substance is present alone. The inhibitory effects of somatostatin on PGE2 action are analogous to those observed in the presence of GRF. The present data suggest that the hypothalamic control of GH secretion by GRF and somatostatin results, at least to a large extent, from the balance between the stimulatory action of GRF and the inhibition of somatostatin on the adenylate cyclase system.

The stimulatory effect of maximal concentrations of synthetic human pancreatic growth hormone (GH)-releasing factor (GRF)(1–40)NH on cyclic AMP accumulation in rat anterior pituitary cells in culture is 4.5-fold increased following a 48-h preincubation with the potent glucocorticoid dexamethasone while the sensitivity of GRF action is increased by approximately 4-fold. Dexamethasone pretreatment, on the other hand, has no effect on basal cyclic AMP levels but approximately doubles both basal and GRF-induced GH release. The present data suggest that the potent stimulatory effect of glucocorticoids on GH secretion is exerted on the adenylate cyclase system at a step preceding cyclic AMP formation.

Normal apoptosis occurs continuously in the olfactory neuroepithelium of adult vertebrates, making it a useful model for studying neuronal apoptosis. Here we demonstrate that overexpression of the anti-apoptotic Bag-1 gene in olfactory neuronal cells confers a strong resistance to apoptosis. Conversely decreased levels of Bag-1 were found to precede a massive wave of olfactory neuronal apoptosis triggered by synaptic target ablation. We show that the decrease is brought about by ubiquitination and subsequent degradation of the Bag-1 protein. The ring finger protein Siah-2 is a likely candidate for the ubiquitination reaction since Siah-2 mRNA accumulated in lesioned olfactory neuroepithelium and overexpression of Siah-2 stimulated Bag-1 ubiquitination and degradation in transient expression assays. These results together identify destabilization of Bag-1 as a necessary step in olfactory neuronal apoptosis.