RESUMO
In eukaryotes combinatorial activation of transcription is an important component of gene regulation. In the budding yeast Saccharomyces cerevisiae, Adr1-Cat8 and Adr1-Oaf1/Pip2 are pairs of activators that act together to regulate two diverse sets of genes. Transcription activation of both sets is regulated positively by the yeast AMP-activated protein kinase homolog, Snf1, in response to low glucose or the presence of a non-fermentable carbon source and negatively by two redundant 14-3-3 isoforms, Bmh1 and Bmh2. Bmh regulates the function of these pairs at a post-promoter binding step by direct binding to Adr1. However, how Bmh regulates transcription after activator binding remains unknown. In the present study we analyzed Bmh-mediated regulation of two sets of genes activated independently by these pairs of activators. We report that Bmh inhibits mRNA synthesis when the second activator is absent. Using gene fusions we show that Bmh binding to the Adr1 regulatory domain inhibits an Adr1 activation domain but not a heterologous activation domain or artificially recruited Mediator, consistent with Bmh acting at a step in transcription downstream of activator binding. Bmh inhibits the assembly and the function of a preinitiation complex (PIC). Gene expression studies suggest that Bmh regulates Adr1 activity through the coactivators Mediator and Swi/Snf. Mediator recruitment appeared to occur normally, but PIC formation and function were defective, suggesting that Bmh inhibits a step between Mediator recruitment and PIC activation.
Assuntos
Proteínas 14-3-3/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas 14-3-3/metabolismo , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Cinética , Mutação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Evolutionarily conserved 14-3-3 proteins have important functions as dimers in numerous cellular signaling processes, including regulation of transcription. Yeast 14-3-3 proteins, known as Bmh, inhibit a post-DNA binding step in transcription activation by Adr1, a glucose-regulated transcription factor, by binding to its regulatory domain, residues 226 to 240. The domain was originally defined by regulatory mutations, ADR1(c) alleles that alter activator-dependent gene expression. Here, we report that ADR1(c) alleles and other mutations in the regulatory domain impair Bmh binding and abolish Bmh-dependent regulation both directly and indirectly. The indirect effect is caused by mutations that inhibit phosphorylation of Ser230 and thus inhibit Bmh binding, which requires phosphorylated Ser230. However, several mutations inhibit Bmh binding without inhibiting phosphorylation and thus define residues that provide important interaction sites between Adr1 and Bmh. Our proposed model of the Adr1 regulatory domain bound to Bmh suggests that residues Ser238 and Tyr239 could provide cross-dimer contacts to stabilize the complex and that this might explain the failure of a dimerization-deficient Bmh mutant to bind Adr1 and to inhibit its activity. A bioinformatics analysis of Bmh-interacting proteins suggests that residues outside the canonical 14-3-3 motif might be a general property of Bmh target proteins and might help explain the ability of 14-3-3 to distinguish target and nontarget proteins. Bmh binding to the Adr1 regulatory domain, and its failure to bind when mutations are present, explains at a molecular level the transcriptional phenotype of ADR1(c) mutants.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sítios de Ligação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dados de Sequência Molecular , Mutação , Fosforilação , Ligação Proteica , Multimerização Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
AMP-activated protein kinase, the "energy sensor of the cell," responds to low cellular energy stores by regulating enzymes and transcription factors that allow the cell to adapt to limiting nutritional conditions. Snf1, the yeast ortholog of AMP-activated protein kinase, has an essential role in respiratory metabolism in Saccharomyces cerevisiae that includes activating the transcription factor Adr1. How Snf1 regulates Adr1 activity is poorly understood. We used an analog-sensitive allele, SNF1(as)(I132G), that is inhibited by 2-naphthylmethyl pyrazolopyrimidine 1 to study the role of Snf1 in transcriptional regulation of glucose-repressible genes. When Snf1(as) was inhibited at the time of glucose depletion, there was a promoter-specific response with some Snf1-dependent genes being activated by low levels of inhibitor, whereas all Snf1-dependent genes were inhibited at high levels. Transcript accumulation was more sensitive to Snf1(as) inhibition than Adr1 or RNA polymerase (pol) II binding or acetylation of promoter nucleosomes. When Snf1(as) was inhibited after gene activation, Adr1 and RNA pol II remained at promoters, and RNA pol II remained in the ORF with associated nascent transcripts. However, cytoplasmic mRNAs were lost at a rapid rate compared with their decay following chemical or genetic inactivation of RNA pol II. In conclusion, Snf1 appears to affect multiple steps in gene regulation, including transcription factor binding, RNA polymerase II activity, and cytoplasmic mRNA stability.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Glucose/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Proteínas de Ligação a DNA/genética , Glucose/genética , Nucleossomos/genética , Nucleossomos/metabolismo , Regiões Promotoras Genéticas/fisiologia , Proteínas Serina-Treonina Quinases/genética , RNA Polimerase II/genética , Estabilidade de RNA/fisiologia , RNA Fúngico/biossíntese , RNA Fúngico/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Gênica/fisiologiaRESUMO
The ability of cells to respond to changes in their environment is mediated by transcription factors that remodel chromatin and reprogram expression of specific subsets of genes. In Saccharomyces cerevisiae, changes in carbon source lead to gene induction by Adr1 and Cat8 that are known to require the upstream function of the Snf1 protein kinase, the central regulator of carbon metabolism, to exert their activating effect. How Snf1 facilitates transcription activation by Adr1 and Cat8 is not known. Here we show that under derepressing conditions, deletion of SNF1 abolishes the increase of histone H3 acetylation at the promoter of the glucose-repressed ADY2 gene, and as a consequence profoundly affects the chromatin structural alterations accompanying transcriptional activation. Adr1 and Cat8 are not required to regulate the acetylation switch and show only a partial influence on chromatin remodelling at this promoter, though their double deletion completely abolishes mRNA accumulation. Finally, we show that under derepressing conditions the recruitment of the histone acetyltransferase Gcn5 is abolished by SNF1 deletion, possibly explaining the lack of increased histone H3 acetylation and nucleosome remodelling. The results highlight a mechanism by which signalling to chromatin provides an essential permissive signal that is required for activation by glucose-responsive transcription factors.
Assuntos
Montagem e Desmontagem da Cromatina/genética , Proteínas de Membrana Transportadoras , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Ativação Transcricional , Acetilação/efeitos dos fármacos , Carbono/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/genéticaRESUMO
The zinc-finger transcription factor, Mxr1 activates methanol utilization and peroxisome biogenesis genes in the methylotrophic yeast, Pichia pastoris. Expression of Mxr1-dependent genes is regulated in response to various carbon sources by an unknown mechanism. We show here that this mechanism involves the highly conserved 14-3-3 proteins. 14-3-3 proteins participate in many biological processes in different eukaryotes. We have characterized a putative 14-3-3 binding region at Mxr1 residues 212-225 and mapped the major activation domain of Mxr1 to residues 246-280, and showed that phenylalanine residues in this region are critical for its function. Furthermore, we report that a unique and previously uncharacterized 14-3-3 family protein in P. pastoris complements Saccharomyces cerevisiae 14-3-3 functions and interacts with Mxr1 through its 14-3-3 binding region via phosphorylation of Ser215 in a carbon source-dependent manner. Indeed, our in vivo results suggest a carbon source-dependent regulation of expression of Mxr1-activated genes by 14-3-3 in P. pastoris. Interestingly, we observed 14-3-3-independent binding of Mxr1 to the promoters, suggesting a post-DNA binding function of 14-3-3 in regulating transcription. We provide the first molecular explanation of carbon source-mediated regulation of Mxr1 activity, whose mechanism involves a post-DNA binding role of 14-3-3.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Metanol/metabolismo , Pichia/enzimologia , Pichia/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Teste de Complementação Genética , Dados de Sequência Molecular , Pichia/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Homologia de Sequência de AminoácidosRESUMO
Transcriptional regulation of Snf1-dependent genes occurs in part by histone-acetylation-dependent binding of the transcription factor Adr1. Analysis of previously published microarray data indicated unscheduled transcription of a large number of Snf1- and Adr1-dependent genes when either the histone H3 or H4 tail was deleted. Quantitative real-time PCR confirmed that the tails were important to preserve stringent transcriptional repression of Snf1-dependent genes when glucose was present. The absence of the tails allowed Adr1 and RNA Polymerase II to bind promoters in normally inhibitory conditions. The promoters escaped glucose repression to a limited extent and the weak constitutive ADH2 transcription induced by deletion of the histone tails was transcription factor- and Snf1-independent. These effects were apparently due to a permissive chromatin structure that allowed transcription in the absence of repression mediated by the histone tails. Deleting REG1, and thus activating Snf1 in the H3 tail mutant enhanced transcription in repressing conditions, indicating that Snf1 and the H3 tail influence transcription independently. Deleting REG1 in the histone H4 tail mutant appeared to be lethal, even in the absence of Snf1, suggesting that Reg1 and the H4 tail have redundant functions that are important for cell viability.
Assuntos
Histonas/genética , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transcrição Gênica , Proteínas de Ligação a DNA/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Deleção de Sequência , Fatores de Transcrição/metabolismoRESUMO
Eukaryotes utilize fatty acids by beta-oxidation, which occurs in the mitochondria and peroxisomes in higher organisms and in the peroxisomes in yeast. The AMP-activated protein kinase regulates this process in mammalian cells, and its homolog Snf1, together with the transcription factors Adr1, Oaf1, and Pip2, regulates peroxisome proliferation and beta-oxidation in yeast. A constitutive allele of Adr1 (Adr1(c)) lacking the glucose- and Snf1-regulated phosphorylation substrate Ser-230 was found to be Snf1-independent for regulation of peroxisomal genes. In addition, it could compensate for and even suppress the requirement for Oaf1 or Pip2 for gene induction. Peroxisomal genes were found to be regulated by oleate in the presence of glucose, as long as Adr1(c) was expressed, suggesting that the Oaf1/Pip2 heterodimer is Snf1-independent. Consistent with this observation, Oaf1 binding to promoters in the presence of oleate was not reduced in a snf1Delta strain. Exploring the mechanism by which Adr1(c) permits Snf1-independent peroxisomal gene induction, we found that strength of promoter binding did not correlate with transcription, suggesting that stable binding is not a prerequisite for enhanced transcription. Instead, enhanced transcriptional activation and suppression of Oaf1, Pip2, and Snf1 by Adr1(c) may be related to the ability of Adr1(c) to suppress the requirement for and enhance the recruitment of transcriptional coactivators in a promoter- and growth medium-dependent manner.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Peroxissomos/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sítios de Ligação , Imunoprecipitação da Cromatina , DNA Fúngico/genética , DNA Fúngico/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Mutagênese Sítio-Dirigida , Mutação/genética , Ácido Oleico/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Serina/química , Serina/genética , Serina/metabolismo , Ativação TranscricionalRESUMO
The AMP-activated protein kinase in yeast, Snf1, coordinates expression and activity of numerous intracellular signaling and developmental pathways, including those regulating cellular differentiation, response to stress, meiosis, autophagy, and the diauxic transition. Snf1 phosphorylates metabolic enzymes and transcription factors to change cellular physiology and metabolism. Adr1 and Cat8, transcription factors that activate gene expression after the diauxic transition, are regulated by Snf1; Cat8 through direct phosphorylation and Adr1 by dephosphorylation in a Snf1-dependent manner. Adr1 and Cat8 coordinately regulate numerous genes encoding enzymes of gluconeogenesis, the glyoxylate cycle, ß-oxidation of fatty acids, and the utilization of alternative fermentable sugars and nonfermentable substrates. To determine the roles of Adr1, Cat8, and Snf1 in metabolism, two-dimensional gas chromatography coupled to time-of-flight mass spectrometry and liquid chromatography coupled to tandem mass spectrometry were used to identify metabolites whose levels change after the diauxic transition in wild-type-, ADR1-, CAT8-, and SNF1-deficient yeast. A discovery-based approach to data analysis utilized chemometric algorithms to identify, quantify, and compare 63 unique metabolites between wild type, adr1∆, cat8∆, adr1∆cat8∆, and snf1∆ strains. The primary metabolites found to differ were those of gluconeogenesis, the glyoxylate and tricarboxylic acid cycles, and amino acid metabolism. In general, good agreement was observed between the levels of metabolites derived from these pathways and the levels of transcripts from the same strains, suggesting that transcriptional control plays a major role in regulating the levels of metabolites after the diauxic transition.
Assuntos
Metabolômica/métodos , Mutação , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/métodos , Regulação Fúngica da Expressão Gênica , Gluconeogênese , Metaboloma , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Glucose represses transcription of a network of co-regulated genes in Saccharomyces cerevisiae, ensuring that it is utilized before poorer carbon sources are metabolized. Adr1 is a glucose-regulated transcription factor whose promoter binding and activity require Snf1, the yeast homologue of the AMP-activated protein kinase in higher eukaryotes. In this study we found that a temperature-sensitive allele of MED14, a Mediator middle subunit that tethers the tail to the body, allowed a low level of Adr1-independent ADH2 expression that can be enhanced by Adr1 in a dose-dependent manner. A low level of TATA-independent ADH2 expression was observed in the med14-truncated strain and transcription of ADH2 and other Adr1-dependent genes occurred in the absence of Snf1 and chromatin remodeling coactivators. Loss of ADH2 promoter nucleosomes had occurred in the med14 strain in repressing conditions and did not require ADR1. A global analysis of transcription revealed that loss of Med14 function was associated with both up- and down- regulation of several groups of co-regulated genes, with ADR1-dependent genes being the most highly represented in the upregulated class. Expression of most genes was not significantly affected by the loss of Med14 function.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Complexo Mediador/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Álcool Desidrogenase/metabolismo , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Complexo Mediador/genética , Nucleossomos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaRESUMO
The transcription factors Adr1 and Cat8 act in concert to regulate the expression of numerous yeast genes after the diauxic shift. Their activities are regulated by Snf1, the yeast homolog of the AMP-activated protein kinase of higher eukaryotes. Cat8 is regulated directly by Snf1, but how Snf1 regulates Adr1 is unknown. Mutations in Adr1 that alleviate glucose repression are clustered between amino acids 227 and 239. This region contains a consensus sequence for protein kinase A, RRAS(230)F, and Ser230 is phosphorylated in vitro by both protein kinase A and Ca(++) calmodulin-dependent protein kinase. Using an antiphosphopeptide antibody, we found that the level of Adr1 phosphorylated on Ser230 was highest in glucose-grown cells and decreased in a Snf1-dependent manner when glucose was depleted. A nonphosphorylatable Ser230Ala mutant was no longer Snf1 dependent for activation of Adr1-dependent genes and could suppress Cat8 dependence at genes coregulated by Adr1 and Cat8. Contrary to expectation, neither protein kinase A (PKA) nor Ca(++) calmodulin-dependent protein kinase appeared to have an important role in Ser230 phosphorylation in vivo, and a screen of 102 viable kinase deletion strains failed to identify a candidate kinase. We conclude that either Ser230 is phosphorylated by multiple protein kinases or its kinase is encoded by an essential gene. Using the Ser230Ala mutant, we explain a long-standing observation of synergy between Adr1 constitutive mutants and Snf1 activation and conclude that dephosphorylation of Ser230 via a Snf1-dependent pathway appears to be a major component of Adr1 regulation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Serina/metabolismo , Fatores de Transcrição/metabolismo , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Genótipo , Glucose/metabolismo , Glucose/farmacologia , Mutação , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas Serina-Treonina Quinases/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Serina/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genéticaRESUMO
Activated transcription in eukaryotes requires the aid of numerous co-factors to overcome the physical barriers chromatin poses to activation, bridge the gap between activators and polymerase, and ensure appropriate regulation. S. cerevisiae has long been a model organism for studying the role of co-activators in the steps leading up to gene activation. Detailed studies on the recruitment of these co-activators have been carried out for more than a dozen promoters. Taking a step back to survey these results, however, suggests that there are few generalizations that could be used to guide future studies of uncharacterized promoters.
Assuntos
Saccharomyces cerevisiae/fisiologia , Fatores de Transcrição/metabolismo , Transcrição Gênica , DNA Fúngico/metabolismo , Modelos Biológicos , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
The effect of sampling time in the context of growth conditions on a dynamic metabolic system was investigated in order to assess to what extent a single sampling time may be sufficient for general application, as well as to determine if useful kinetic information could be obtained. A wild type yeast strain (W) was compared to a snf1Delta mutant yeast strain (S) grown in high-glucose medium (R) and in low-glucose medium containing ethanol (DR). Under these growth conditions, different metabolic pathways for utilizing the different carbon sources are expected to be active. Thus, changes in metabolite levels relating to the carbon source in the growth medium were anticipated. Furthermore, the Snf1 protein kinase complex is required to adapt cellular metabolism from fermentative R conditions to oxidative DR conditions. So, differences in intracellular metabolite levels between the W and S yeast strains were also anticipated. Cell extracts were collected at four time points (0.5, 2, 4, 6 h) after shifting half of the cells from R to DR conditions, resulting in 16 sample classes (WR, WDR, SR, SDR) x (0.5, 2, 4, 6 h). The experimental design provided time course data, so temporal dependencies could be monitored in addition to carbon source and strain dependencies. Comprehensive two-dimensional (2D) gas chromatography coupled to time-of-flight mass spectrometry (GC x GC-TOFMS) was used with discovery-based data mining algorithms ( Anal. Chem. 2006, 78, 5068-5075 (ref 1); J. Chromatogr., A 2008, 1186, 401-411 (ref 2)) to locate regions within the 2D chromatograms (i.e., metabolites) that provided chemical selectivity between the 16 sample classes. These regions were mathematically resolved using parallel factor analysis to positively identify the metabolites and to acquire quantitative results. With these tools, 51 unique metabolites were identified and quantified. Various time course patterns emerged from these data, and principal component analysis (PCA) was utilized as a comparison tool to determine the sources of variance between these 51 metabolites. The effect of sampling time was investigated with separate PCA analyses using various subsets of the data. PCA utilizing all of the time course data, averaged time course data, and each individual time point data set independently were performed to discern the differences. For the yeast strains examined in the current study, data collection at either 4 or 6 h provided information comparable to averaged time course data, albeit with a few metabolites missing using a single sampling time point.
Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/metabolismo , Glucose/metabolismo , Espectrometria de Massas , Mutação/genética , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Fatores de TempoRESUMO
In Saccharomyces cerevisiae, glucose depletion causes a profound alteration in metabolism, mediated in part by global transcriptional changes. Many of the transcription factors that regulate these changes act combinatorially. We have analyzed combinatorial regulation by Adr1 and Cat8, two transcription factors that act during glucose depletion, by combining genome-wide expression and genome-wide binding data. We identified 32 genes that are directly activated by Adr1, 28 genes that are directly activated by Cat8, and 14 genes that are directly regulated by both. Our analysis also uncovered promoters that Adr1 binds but does not regulate and promoters that are indirectly regulated by Cat8, stressing the advantage of combining global expression and global localization analysis to find directly regulated targets. At most of the coregulated promoters, the in vivo binding of one factor is independent of the other, but Adr1 is required for optimal Cat8 binding at two promoters with a poor match to the Cat8 binding consensus. In addition, Cat8 is required for Adr1 binding at promoters where Adr1 is not required for transcription. These data provide a comprehensive analysis of the direct, indirect, and combinatorial requirements for these two global transcription factors.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Regulação Fúngica da Expressão Gênica , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Genes Fúngicos , Genoma Fúngico , Glucose/fisiologia , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
A yeast metabolome exhibiting oscillatory behavior was analyzed using comprehensive two-dimensional gas chromatography-time-of-flight-mass spectrometry (GC x GC-TOF-MS) and in-house developed data analysis software methodology, referred to as a signal ratio method (S(ratio) method). In this study, 44 identified unique metabolites were found to exhibit cycling, with a depth-of-modulation amplitude greater than three. After the initial locations are found using the S(ratio) software, and identified preliminarily using ChromaTOF software, the refined mass spectra and peak volumes were subsequently obtained using parallel factor analysis (PARAFAC). The peak volumes provided by PARAFAC deconvolution provide a measurement of the cycling depth-of-modulation amplitude that is more accurate than the initial S(ratio) information (which serves as a rapid screening procedure to find the cycling metabolites while excluding peaks that do not cycle). The S(ratio) reported is a rapid method to determine the depth-of-modulation while not constraining the search to specific cycling frequencies. The phase delay of the cycling metabolites ranged widely in relation to the oxygen consumption cycling pattern.
Assuntos
Fatores Biológicos/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Saccharomyces cerevisiae/metabolismo , Fatores Biológicos/química , Análise de Componente Principal , Saccharomyces cerevisiae/crescimento & desenvolvimentoRESUMO
The transcription factor Adr1 directly activates the expression of genes encoding enzymes in numerous pathways that are upregulated after the exhaustion of glucose in the yeast Saccharomyces cerevisiae. ADH2, encoding the alcohol dehydrogenase isozyme required for ethanol oxidation, is a highly glucose-repressed, Adr1-dependent gene. Using a genetic screen we isolated >100 mutants in 12 complementation groups that exhibit ADR1-dependent constitutive ADH2 expression on glucose. Temperature-sensitive alleles are present among the new constitutive mutants, indicating that essential genes play a role in ADH2 repression. Among the genes we cloned is MOT1, encoding a repressor that inhibits TBP binding to the promoter, thus linking glucose repression with TBP access to chromatin. Two genes encoding proteins involved in vacuolar function, FAB1 and VPS35, and CDC10, encoding a nonessential septin, were also uncovered in the search, suggesting that vacuolar function and the cytoskeleton have previously unknown roles in regulating gene expression. Constitutive activation of ADH2 expression by Adr1 is SNF1-dependent in a strain with a defective MOT1 gene, whereas deletion of SNF1 did not affect constitutive ADH2 expression in the mutants affecting vacuolar or septin function. Thus, the mutant search revealed previously unknown Snf1-dependent and -independent pathways of ADH2 expression.
Assuntos
Álcool Desidrogenase/genética , Regulação Fúngica da Expressão Gênica , Proteínas Serina-Treonina Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Álcool Desidrogenase/química , Alelos , Cromatina/metabolismo , Deleção de Genes , Teste de Complementação Genética , Glucose/metabolismo , Modelos Genéticos , Mutação , Plasmídeos/metabolismo , Regulação para CimaRESUMO
In the yeast Saccharomyces cerevisiae, the switch from respiratory metabolism to fermentation causes rapid decay of transcripts encoding proteins uniquely required for aerobic metabolism. Snf1, the yeast ortholog of AMP-activated protein kinase, has been implicated in this process because inhibiting Snf1 mimics the addition of glucose. In this study, we show that the SNF1-dependent ADH2 promoter, or just the major transcription factor binding site, is sufficient to confer glucose-induced mRNA decay upon heterologous transcripts. SNF1-independent expression from the ADH2 promoter prevented glucose-induced mRNA decay without altering the start site of transcription. SNF1-dependent transcripts are enriched for the binding motif of the RNA binding protein Vts1, an important mediator of mRNA decay and mRNA repression whose expression is correlated with decreased abundance of SNF1-dependent transcripts during the yeast metabolic cycle. However, deletion of VTS1 did not slow the rate of glucose-induced mRNA decay. ADH2 mRNA rapidly dissociated from polysomes after glucose repletion, and sequences bound by RNA binding proteins were enriched in the transcripts from repressed cells. Inhibiting the protein kinase A pathway did not affect glucose-induced decay of ADH2 mRNA. Our results suggest that Snf1 may influence mRNA stability by altering the recruitment activity of the transcription factor Adr1.
Assuntos
Álcool Desidrogenase/genética , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
The relative importance of gross chromosomal rearrangements to adaptive evolution has not been precisely defined. The Saccharomyces cerevisiae flor yeast strains offer significant advantages for the study of molecular evolution since they have recently evolved to a high degree of specialization in a very restrictive environment. Using DNA microarray technology, we have compared the genomes of two prominent variants of S. cerevisiae flor yeast strains. The strains differ from one another in the DNA copy number of 116 genomic regions that comprise 38% of the genome. In most cases, these regions are amplicons flanked by repeated sequences or other recombination hotspots previously described as regions where double-strand breaks occur. The presence of genes that confer specific characteristics to the flor yeast within the amplicons supports the role of chromosomal rearrangements as a major mechanism of adaptive evolution in S. cerevisiae. We propose that nonallelic interactions are enhanced by ethanol- and acetaldehyde-induced double-strand breaks in the chromosomal DNA, which are repaired by pathways that yield gross chromosomal rearrangements. This mechanism of chromosomal evolution could also account for the sexual isolation shown among the flor yeast.
Assuntos
Evolução Biológica , Cromossomos Fúngicos/genética , Amplificação de Genes , Rearranjo Gênico , Genoma Fúngico , Saccharomyces cerevisiae/genética , Alelos , Aneuploidia , Southern Blotting , Dosagem de Genes , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura AbertaRESUMO
Activated eukaryotic transcription requires the components of the Mediator complex, which can act as both a positive and negative regulator of transcription. This review of the yeast Saccharomyces cerevisiae Mediator complex describes the role of Mediator and its effects on transcriptional regulation. One focal point of the review is to summarize new information regarding the negative effect of Mediator on transcription and suggest a possible mechanism that encompasses the latest results.
Assuntos
Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
What has been will be again, what has been done will be done again; there is nothing new under the sun. -Ecclesiastes 1:9 (New International Version) Posttranscriptional regulation of gene expression has an important role in defining the phenotypic characteristics of an organism. Well-defined steps in mRNA metabolism that occur in the nucleus-capping, splicing, and polyadenylation-are mechanistically linked to the process of transcription. Recent evidence suggests another link between RNA polymerase II (Pol II) and a posttranscriptional process that occurs in the cytoplasm-mRNA decay. This conclusion appears to represent a conundrum. How could mRNA synthesis in the nucleus and mRNA decay in the cytoplasm be mechanistically linked? After a brief overview of mRNA processing, we will review the recent evidence for transcription-coupled mRNA decay and the possible involvement of Snf1, the Saccharomyces cerevisiae ortholog of AMP-activated protein kinase, in this process.
Assuntos
Estabilidade de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , Animais , Exorribonucleases/genética , Exorribonucleases/metabolismo , Humanos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/análise , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Stresses, such as glucose depletion, activate Snf1, the Saccharomyces cerevisiae ortholog of adenosine monophosphate-activated protein kinase (AMPK), enabling adaptive cellular responses. In addition to affecting transcription, Snf1 may also promote mRNA stability in a gene-specific manner. To understand Snf1-mediated signaling, we used quantitative mass spectrometry to identify proteins that were phosphorylated in a Snf1-dependent manner. We identified 210 Snf1-dependent phosphopeptides in 145 proteins. Thirteen of these proteins are involved in mRNA metabolism. Of these, we found that Ccr4 (the major cytoplasmic deadenylase), Dhh1 (an RNA helicase), and Xrn1 (an exoribonuclease) were required for the glucose-induced decay of Snf1-dependent mRNAs that were activated by glucose depletion. Unexpectedly, deletion of XRN1 reduced the accumulation of Snf1-dependent transcripts that were synthesized during glucose depletion. Deletion of SNF1 rescued the synthetic lethality of simultaneous deletion of XRN1 and REG1, which encodes a regulatory subunit of a phosphatase that inhibits Snf1. Mutation of three Snf1-dependent phosphorylation sites in Xrn1 reduced glucose-induced mRNA decay. Thus, Xrn1 is required for Snf1-dependent mRNA homeostasis in response to nutrient availability.