Transcriptional plasticity through differential assembly of a multiprotein activation complex

Cell adaptation to the environment often involves induction of complex gene expression programs under the control of specific transcriptional activators. For instance, in response to cadmium, budding yeast induces transcription of the sulfur amino acid biosynthetic genes through the basic-leucine zipper activator Met4, and also launches a program of substitution of abundant glycolytic enzymes by isozymes with a lower content in sulfur. We demonstrate here that transcriptional induction of PDC6, which encodes a pyruvate decarboxylase isoform with low sulfur content, is directly controlled by Met4 and its DNA-binding cofactors the basic-helix–loop–helix protein Cbf1 and the two homologous zinc finger proteins Met31 and Met32. Study of Cbf1 and Met31/32 association with PDC6 allowed us to find a new mechanism of recruitment of Met4, which allows PDC6 being differentially regulated compared to sulfur amino acid biosynthetic genes. Our findings provide a new example of mechanism allowing transcriptional plasticity within a regulatory network thanks to a definite toolbox comprising a unique master activator and several dedicated DNA-binding cofactors. We also show evidence suggesting that integration of PDC6 to the Met4 regulon may have occurred recently in the evolution of the Saccharomyces cerevisiae lineage

CO 2 Migration Monitoring Methodology in the Shallow Subsurface: Lessons Learned From the CO 2 FIELDLAB Project

International audienceA CO 2 migration field laboratory for testing of monitoring methods has been established in the glaciofluvial-glaciomarine Holocene deposits of the Svelvik ridge, near Oslo. A shallow CO 2 injection experiment was conducted in September 2011 in which approximately 1700 kg of CO 2 was injected at 18 m depth below surface. The objectives of this experiment were to (i) detect and, where possible, quantify migrated CO 2 concentrations, (ii) evaluate the sensitivity of the monitoring tools and (iii) study the impact of the vadose zone on measurements. This paper describes the injection, discusses the joint interpretation of the results and suggests some recommendations for further work

Mécanismes d activation transcriptionnelle du régulon Met4 chez Saccharomyces cerevisiae

Met4 est l activateur transcriptionnel des gènes de la voie de biosynthèse des acides aminés soufrés, les gènes MET. Il est actif lorsque le milieu est carencé en acides aminés soufrés ou encore lorsqu il contient du cadmium (Cd2+), ceci afin d assurer la synthèse de la cystéine nécessaire à la formation du glutathion, un tripeptide permettant la détoxification du Cd2+. Nous avons montré que Met4 recrute au moins trois coactivateurs en réponse à ces deux conditions: le Médiateur, SAGA et les TAF. Certaines composantes de ces trois complexes ne semblent être nécessaires que dans une des conditions d induction, suggérant un mécanisme d activation par Met4 différentiel en fonction du signal. D autre part, dans le cadre du phénomène d économie en soufre déclenché en réponse au Cd2+, nous nous sommes intéressé aux mécanismes assurant le remplacement de la forme principale de la pyruvate décarboxylase Pdc1 par une isoforme, Pdc6, de teneur plus faible en acides aminés soufrés, et avons montré (1) que l induction de PDC6 dépend directement de Met4 et de ses cofacteurs mais repose sur un site de liaison à l ADN atypique, (2) que le recrutement de la machinerie transcriptionnelle au niveau de PDC6 et l accumulation des transcrits sont retardés par rapport aux gènes MET et (3) que l activation de PDC6 nécessite des concentrations de Cd2+ plus importantes. Nous avons montré une implication du complexe corépresseur Ssn6/Tup1 dans la répression de PDC1 et ceci vraisemblablement par altération de la structure chromatinienne via l histone déacétylase Rpd3. Enfin, nous avons aussi observé ce remplacement de Pdc1 par Pdc6 en carence en acides aminés soufrés.Met4 is the transcriptional activator which controls the MET gene network responsible for the biosynthesis of sulfur-containing amino acids. It is actif under conditions of limitation in sulfur and also upon exposure to cadmium (Cd2+) and, therefore, leading to synthesis of the cystein required to formation of glutathione, a thiol tripeptide necessary to complex and detoxify Cd2+. We showed that Met4 recruits at least three coactivators in response to these two conditions: the Mediator, SAGA and TAF. However, some of the subunits of these three complexes seem to be required in only one of the two induction conditions, suggesting a differential activation mechanism by Met4 according to the signal. Within the framework of the global sulfur sparing response in answer to Cd2+, we focused on the molecular mechanisms that govern the switch in expression between Pdc1 and Pdc6, two isoforms of pyruvate decarboxylase with differents contents of sulfur-containing amino acids. We showed that (1) transcriptional activation of PDC6 depends directly on Met4 and its cofactors but involves on atypical DNA binding site, (2) recruitment of the transcriptional machinery at PDC6 promoter and accumulation of PDC6 transcripts are delayed compared to MET genes and (3) PDC6 activation requires high concentrations of Cd2+. In addition, we showed involvement of the corepressor complexe Ssn6/Tup1 in the PDC1 repression, this probably through changes in chromatin structure via the histone deacetylase Rpd3. Finally, this transcriptional switch was also observed under condition of limitation in sulfur.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Association of the Mediator complex with enhancers of active genes

International audienceThe multiprotein Mediator complex has been shown to interact with gene-specific regulatory proteins and RNA polymerase II in vitro. Here, we use chromatin immunoprecipitation to analyze the recruitment of Mediator to GAL genes of yeast in vivo. We find that Mediator associates exclusively with transcriptionally active and not inactive GAL genes. This association maps to the upstream activating sequence, rather than the core promoter, and is independent of RNA polymerase II, general transcription factors, and core promoter sequences. These findings support the idea of Mediator as a primary conduit of regulatory information from enhancers to promoters in eukaryotic cells

Independent Recruitment of Mediator and SAGA by the Activator Met4

Mediator is a key RNA polymerase II (Pol II) cofactor in the regulation of eukaryotic gene expression. It is believed to function as a coactivator linking gene-specific activators to the basal Pol II initiation machinery. In support of this model, we provide evidence that Mediator serves in vivo as a coactivator for the yeast activator Met4, which controls the gene network responsible for the biosynthesis of sulfur-containing amino acids and S-adenosylmethionine. In addition, we show that SAGA (Spt-Ada-Gcn5-acetyltransferase) is also recruited to Met4 target promoters, where it participates in the recruitment of Pol II by a mechanism involving histone acetylation. Interestingly, we find that SAGA is not required for Mediator recruitment by Met4 and vice versa. Our results provide a novel example of functional interplay between Mediator and coactivators involved in histone modification

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Mediator, known as a coactivator, can act in transcription initiation in an activator-independent manner in vivo

International audienceMediator is an evolutionarily conserved complex best known for its role as a coactivator responsible for transducing regulatory signals from DNA-bound activators to the basal RNA polymerase II (Pol II) machinery that initiates transcription from promoters of protein-encoding genes. By exploiting our in vivo activator-independent transcription assay in Saccharomyces cerevisiae, in combination with new temperature sensitive (ts) mutants of Med14 N-terminal half exhibiting widespread transcriptional defects, and existing ts mutants of Kin28 and Med17, we show that, in the absence of activator: (i) Mediator can associate with a promoter as a form devoid of the Cyclin-dependent kinase 8 (CDK8) module, and this association remains regulated by Kin28; (ii) Mediator can stimulate the assembly of the entire Pol II initiation machinery. Although the literature emphasizes the role of the interaction between activators and Mediator, together our results support the view that Mediator is able to act through a dual mechanism in vivo, activator-dependent but also activator-independent, therefore not always as a coactivator

Mediator is an intrinsic component of the basal RNA polymerase II machinery in vivo

International audienceMediator is a prominent multisubunit coactivator that functions as a bridge between gene-specific activators and the basal RNA polymerase (Pol) II initiation machinery. Here, we study the poorly documented role of Mediator in basal, or activatorindependent, transcription in vivo. We show that Mediator is still present at the promoter when the Pol II machinery is recruited in the absence of an activator, in this case through a direct fusion between a basal transcription factor and a heterologous DNA binding protein bound to the promoter. Moreover, transcription resulting from activatorindependent recruitment of the Pol II machinery is impaired by inactivation of the essential Mediator subunit Med17 due to the loss of Pol II from the promoter. Our results strongly support that Mediator is an integral component of the minimal machinery essential in vivo for stable Pol II association with the promoter

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