RESUMO
Mediator is a large coregulator complex conserved from yeast to humans and involved in many human diseases, including cancers. Together with general transcription factors, it stimulates preinitiation complex (PIC) formation and activates RNA polymerase II (Pol II) transcription. In this study, we analyzed how Mediator acts in PIC assembly using in vivo, in vitro, and in silico approaches. We revealed an essential function of the Mediator middle module exerted through its Med10 subunit, implicating a key interaction between Mediator and TFIIB. We showed that this Mediator-TFIIB link has a global role on PIC assembly genome-wide. Moreover, the amplitude of Mediator's effect on PIC formation is gene-dependent and is related to the promoter architecture in terms of TATA elements, nucleosome occupancy, and dynamics. This study thus provides mechanistic insights into the coordinated function of Mediator and TFIIB in PIC assembly in different chromatin contexts.
Assuntos
Complexo Mediador/metabolismo , Regiões Promotoras Genéticas/fisiologia , Saccharomyces cerevisiae/fisiologia , Fator de Transcrição TFIIB/metabolismo , Cromatina/metabolismo , Complexo Mediador/genética , Mutação , Ligação Proteica/genética , Multimerização Proteica/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Mediator 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 activator-independent, 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 activator-independent 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.
Assuntos
Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica , Cisteína Sintase/genética , Proteínas de Ligação a DNA/metabolismo , Mutação , Regiões Promotoras Genéticas , Fatores de Transcrição de Fator Regulador X , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Ligação a TATA-Box/metabolismo , Fator de Transcrição TFIIB/metabolismo , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Autophagy is a key lysosomal degradative mechanism allowing a prosurvival response to stresses, especially nutrient starvation. Here we investigate the mechanism of autophagy induction in response to sulfur starvation in Saccharomyces cerevisiae. We found that sulfur deprivation leads to rapid and widespread transcriptional induction of autophagy-related (ATG) genes in ways not seen under nitrogen starvation. This distinctive response depends mainly on the transcription activator of sulfur metabolism Met4. Consistently, Met4 is essential for autophagy under sulfur starvation. Depletion of either cysteine, methionine or SAM induces autophagy flux. However, only SAM depletion can trigger strong transcriptional induction of ATG genes and a fully functional autophagic response. Furthermore, combined inactivation of Met4 and Atg1 causes a dramatic decrease in cell survival under sulfur starvation, highlighting the interplay between sulfur metabolism and autophagy to maintain cell viability. Thus, we describe a pathway of sulfur starvation-induced autophagy depending on Met4 and involving SAM as signaling sulfur metabolite.
Assuntos
Autofagia , S-Adenosilmetionina , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Transdução de Sinais , Enxofre , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Autofagia/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Enxofre/metabolismo , S-Adenosilmetionina/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Metionina/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Proteínas Quinases , Fatores de Transcrição de Zíper de Leucina BásicaRESUMO
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.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Regulação Fúngica da Expressão Gênica , Piruvato Descarboxilase/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transativadores/metabolismo , Ativação Transcricional , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Sítios de Ligação , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismoRESUMO
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.
Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Fatores de Transcrição de Zíper de Leucina Básica/química , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Histonas/metabolismo , Modelos Genéticos , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , RNA Polimerase II/metabolismo , RNA Mensageiro/análise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/genéticaRESUMO
Mediator 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.
Assuntos
Complexo Mediador/metabolismo , RNA Polimerase II/metabolismo , Iniciação da Transcrição Genética , Quinases Ciclina-Dependentes/genética , Complexo Mediador/genética , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Mot1 stably associates with the TATA-binding protein (TBP), and it can dissociate TBP from DNA in an ATP-dependent manner. Mot1 acts as a negative regulator of TBP function in vitro, but genome-wide transcriptional profiling suggests that Mot1 positively affects about 10% of yeast genes and negatively affects about 5%. Unexpectedly, Mot1 associates with active RNA polymerase (Pol) II and III promoters, and it is rapidly recruited in response to activator proteins. At Pol II promoters, Mot1 association requires TBP and is strongly correlated with the level of TBP occupancy. However, the Mot1/TBP occupancy ratio at both Mot1-stimulated and Mot1-inhibited promoters is high relative to that at typical promoters, strongly suggesting that Mot1 directly affects transcriptional activity in a positive or negative manner, depending on the gene. The effect of Mot1 at the HIS3 promoter region depends on the functional quality and DNA sequence of the TATA element. Unlike TBP, Mot1 association is largely independent of the Srb4 component of Pol II holoenzyme, and it also can occur downstream of the promoter region. Mot1 removes TBP, but not TBP complexes or preinitiation complexes, from inappropriate genomic locations. Mot1 inhibits the association of NC2 with promoters, suggesting that the TBP-Mot1 and TBP-NC2 complexes compete for promoter occupancy in vivo. We speculate that Mot1 does not form transcriptionally active TBP complexes but rather regulates transcription in vivo by modulating the activity of free TBP and/or by affecting promoter DNA structure.
Assuntos
DNA Helicases/metabolismo , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Proteína de Ligação a TATA-Box/metabolismo , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases , DNA Helicases/genética , Genes Fúngicos , Hidroliases/genética , Hidroliases/metabolismo , Fosfoproteínas/genética , Ligação Proteica , Subunidades Proteicas , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Polimerase III/genética , RNA Polimerase III/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , TATA Box/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
Saccharomyces cerevisiae GimC (mammalian Prefoldin) is a hexameric (Gim1-6) cytoplasmic complex involved in the folding pathway of actin/tubulin. In contrast to a shared role in GimC complex, we show that absence of individual Gim proteins results in distinct stress responses. No concomitant alteration in F-actin integrity was observed. Transcription of stress responsive genes is altered in gim2Δ, gim3Δ and gim6Δ mutants: TRX2 gene is induced in these mutants but with a profile diverging from type cells, whereas CTT1 and HSP26 fail to be induced. Remaining gimΔ mutants display stress transcript abundance comparable to wild type cells. No alteration in the nuclear localization of the transcriptional activators for TRX2 (Yap1) and CTT1/HSP26 (Msn2) was observed in gim2Δ. In accordance with TRX2 induction, RNA polymerase II occupancy at TRX2 discriminates the wild type from gim2Δ and gim6Δ. In contrast, RNA polymerase II occupancy at CTT1 is similar in wild type and gim2Δ, but higher in gim6Δ. The absence of active RNA polymerase II at CTT1 in gim2Δ, but not in wild type and gim1Δ, explains the respective CTT1 transcript outputs. Altogether our results put forward the need of Gim2, Gim3 and Gim6 in oxidative and osmotic stress activated transcription; others Gim proteins are dispensable. Consequently, the participation of Gim proteins in activated-transcription is independent from the GimC complex.
Assuntos
Pressão Osmótica/fisiologia , Estresse Oxidativo/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/genética , Actinas/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Chaperonas Moleculares/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Chromatin immunoprecipitation (ChIP) is one of the most powerful methods to identify and characterize the association of proteins with specific genomic regions in the context of intact cells. In this method, cells are first treated with formaldehyde to crosslink protein-protein and protein-DNA complexes in situ. Next, the crosslinked chromatin is sheared by sonication to generate small chromatin fragments, and the fragments associated with the protein of interest are immunoprecipitated using antibodies to the protein. Finally, protein-DNA crosslinks are reversed and the DNA is examined for the presence of particular sequences by quantitative polymerase chain reaction (PCR). Enrichment of specific sequences in the precipitate indicates that the sequences are associated with the protein of interest in vivo. The ChIP method described here is intended for studying protein-DNA association in the budding yeast Saccharomyces cerevisiae, but it can be easily implemented in other cell types, including fly, mammalian, and plant cells.
Assuntos
Cromatina/química , Proteínas de Ligação a DNA/análise , DNA/análise , Testes de Precipitina/métodos , Animais , Cromatina/metabolismo , Reagentes de Ligações Cruzadas/química , DNA/isolamento & purificação , Proteínas de Ligação a DNA/imunologia , Proteínas de Ligação a DNA/metabolismo , Formaldeído/química , Microesferas , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína Estafilocócica ARESUMO
The 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.
Assuntos
Elementos Facilitadores Genéticos , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Substâncias Macromoleculares , Complexos Multiproteicos , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , RNA Fúngico/genética , RNA Fúngico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Ativação TranscricionalRESUMO
In yeast, TFIID strongly associates with nearly all ribosomal protein (RP) promoters, but a TAF-independent form of TBP preferentially associates with other active promoters. RP promoters are regulated in response to growth stimuli, in most cases by a Rap1-containing activator. This Rap1-dependent activator is necessary and sufficient for TFIID recruitment, whereas other activators do not efficiently recruit TFIID. TAFs are recruited to RP promoters even when TBP and other general transcription factors are not associated, suggesting that TFIID recruitment involves a direct activator-TAF interaction. Most RP promoters lack canonical TATA elements, and they are preferentially activated by the Rap1-containing activator. These results demonstrate activator-specific recruitment of TFIID in vivo, and they suggest that TFIID recruitment is important for coordinate expression of RP genes.
Assuntos
DNA Fúngico/metabolismo , Proteínas Ribossômicas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição TFII/metabolismo , Cromatina/genética , DNA Fúngico/genética , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Modelos Genéticos , Testes de Precipitina , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Ribossômicas/biossíntese , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , TATA Box , Transativadores/fisiologia , Fator de Transcrição TFIID , Proteínas rap1 de Ligação ao GTP/fisiologiaRESUMO
The ubiquitin system has been recently implicated in various aspects of transcriptional regulation, including proteasome-dependent degradation of transcriptional activators. In yeast, the activator Met4 is inhibited by the SCF(Met30) ubiquitin ligase, which recognizes and oligo-ubiquitylates Met4. Here, we demonstrate that in minimal media, Met4 is ubiquitylated and rapidly degraded in response to methionine excess, whereas in rich media, Met4 is oligo-ubiquitylated but remains stable. In the latter growth condition, oligo-ubiquitylated Met4 is not recruited to MET gene promoters, but is recruited to the SAM genes, which are required for production of S-adenosylmethionine, an unstable metabolite that is not present in rich medium. Thus, ubiquitylation not only regulates Met4 by distinct degradation-dependent and -independent mechanisms, but also controls differential recruitment of a single transcription factor to distinct promoters, thereby diversifying transcriptional activator specificity.