[Show abstract][Hide abstract] ABSTRACT: The transcription factor Msn2 mediates a significant proportion of the environmental stress response, in which a common cohort of genes changes expression in a stereotypic fashion upon exposure to any of a wide variety of stresses. We have applied genome-wide chromatin immunoprecipitation and nucleosome profiling to determine where Msn2 binds under stressful conditions and how that binding affects, and is affected by, nucleosome positioning. We concurrently determined the effect of Msn2 activity on gene expression following stress and demonstrated that Msn2 stimulates both activation and repression. We found that some genes responded to both intermittent and continuous Msn2 nuclear occupancy while others responded only to continuous occupancy. Finally, these studies document a dynamic interplay between nucleosomes and Msn2 such that nucleosomes can restrict access of Msn2 to its canonical binding sites while Msn2 can promote reposition, expulsion and recruitment of nucleosomes to alter gene expression. This interplay may allow the cell to discriminate between different types of stress signaling.
Nucleic Acids Research 03/2014; · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Conditions of chronic stress are associated with genetic instability in many organisms, but the roles of stress responses in mutagenesis have so far been elucidated only in bacteria. Here, we present data demonstrating that the environmental stress response (ESR) in yeast functions in mutagenesis induced by proteotoxic stress. We show that the drug canavanine causes proteotoxic stress, activates the ESR, and induces mutagenesis at several loci in an ESR-dependent manner. Canavanine-induced mutagenesis also involves translesion DNA polymerases Rev1 and Polζ and non-homologous end joining factor Ku. Furthermore, under conditions of chronic sub-lethal canavanine stress, deletions of Rev1, Polζ, and Ku-encoding genes exhibit genetic interactions with ESR mutants indicative of ESR regulating these mutagenic DNA repair processes. Analyses of mutagenesis induced by several different stresses showed that the ESR specifically modulates mutagenesis induced by proteotoxic stress. Together, these results document the first known example of an involvement of a eukaryotic stress response pathway in mutagenesis and have important implications for mechanisms of evolution, carcinogenesis, and emergence of drug-resistant pathogens and chemotherapy-resistant tumors.
[Show abstract][Hide abstract] ABSTRACT: Nucleotide degradation is a universal metabolic capability. Here we combine metabolomics, genetics and biochemistry to characterize the yeast pathway. Nutrient starvation, via PKA, AMPK/SNF1, and TOR, triggers autophagic breakdown of ribosomes into nucleotides. A protein not previously associated with nucleotide degradation, Phm8, converts nucleotide monophosphates into nucleosides. Downstream steps, which involve the purine nucleoside phosphorylase, Pnp1, and pyrimidine nucleoside hydrolase, Urh1, funnel ribose into the nonoxidative pentose phosphate pathway. During carbon starvation, the ribose-derived carbon accumulates as sedoheptulose-7-phosphate, whose consumption by transaldolase is impaired due to depletion of transaldolase's other substrate, glyceraldehyde-3-phosphate. Oxidative stress increases glyceraldehyde-3-phosphate, resulting in rapid consumption of sedoheptulose-7-phosphate to make NADPH for antioxidant defense. Ablation of Phm8 or double deletion of Pnp1 and Urh1 prevent effective nucleotide salvage, resulting in metabolite depletion and impaired survival of starving yeast. Thus, ribose salvage provides means of surviving nutrient starvation and oxidative stress.
Molecular Systems Biology 05/2013; 9:665. · 14.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nucleotide degradation is a universal metabolic capability. Here we combine metabolomics, genetics and biochemistry to characterize the yeast pathway. Nutrient starvation, via PKA, AMPK/SNF1, and TOR, triggers autophagic breakdown of ribosomes into nucleotides. A protein not previously associated with nucleotide degradation, Phm8, converts nucleotide monophosphates into nucleosides. Downstream steps, which involve the purine nucleoside phosphorylase, Pnp1, and pyrimidine nucleoside hydrolase, Urh1, funnel ribose into the nonoxidative pentose phosphate pathway. During carbon starvation, the ribose-derived carbon accumulates as sedoheptulose-7-phosphate, whose consumption by transaldolase is impaired due to depletion of transaldolase's other substrate, glyceraldehyde-3-phosphate. Oxidative stress increases glyceraldehyde-3-phos-phate, resulting in rapid consumption of sedoheptulose-7-phosphate to make NADPH for antioxidant defense. Ablation of Phm8 or double deletion of Pnp1 and Urh1 prevent effective nucleotide salvage, resulting in metabolite depletion and impaired survival of starving yeast. Thus, ribose salvage provides means of surviving nutrient starvation and oxidative stress. Molecular Systems Biology 9: 665; published online 14 May 2013; doi:10.1038/msb.2013.21
[Show abstract][Hide abstract] ABSTRACT: All cells perceive and respond to environmental stresses through elaborate stress-sensing networks. Yeast cells sense stress through diverse signaling pathways that converge on the transcription factors Msn2 and Msn4, which respond by initiating rapid, idiosyncratic cycles into and out of the nucleus. To understand the role of Msn2/4 nuclear localization dynamics, we combined time lapse studies of Msn2-GFP localization in living cells with computational modeling of stress-sensing signaling networks. We find that several signaling pathways, including Ras/Protein kinase A, AMP activated kinase, the HOG map kinase pathway, and protein phosphatase 1 (PP1), regulate activation of Msn2 in distinct ways in response to different stresses. Moreover, we find that bursts of nuclear localization elicit a more robust transcriptional response than does sustained nuclear localization. Using stochastic modeling, we reproduce in silico the responses of Msn2 to different stresses and demonstrate that bursts of localization arise from noise in the signaling pathways amplified by the small number of Msn2 molecules in the cell. This noise imparts diverse behaviors to genetically identical cells, allowing cell populations to "hedge their bets" in responding to an uncertain future, and to balance growth and survival in an unpredictable environment.
Molecular biology of the cell 04/2013; · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A number of transcriptional control elements are activated when Saccharomyces cerevisiae cells are submitted to various stress conditions, including high hydrostatic pressure (HHP). Exposure of Saccharomyces cerevisiae cells to HHP results in global transcriptional reprogramming, similar to that observed under other industrial stresses, such as temperature, ethanol and oxidative stresses. Moreover, treatment with a mild hydrostatic pressure renders yeast cells multi-stress tolerant. In order to identify transcriptional factors involved in coordinating response to high hydrostatic pressure, we performed a time series microarray expression analysis on a wild S. cerevisiae strain exposed to 50 MPa for 30 min followed by recovery at atmospheric pressure (0.1 MPa) for 5, 10 and 15 min. We identified transcription factors and corresponding DNA and RNA motifs targeted in response to hydrostatic pressure. Moreover, we observed that different motif elements are present in the promoters of induced or repressed genes during HHP treatment. Overall, as we have already published, mild HHP treatment to wild yeast cells provides multiple protection mechanisms, and this study suggests that the TFs and motifs identified as responding to HHP may be informative for a wide range of other biotechnological and industrial applications, such as fermentation, that may utilize HHP treatment.
Current pharmaceutical biotechnology 10/2012; · 3.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Availability of key nutrients, such as sugars, amino acids, and nitrogen compounds, dictates the developmental programs and the growth rates of yeast cells. A number of overlapping signaling networks-those centered on Ras/protein kinase A, AMP-activated kinase, and target of rapamycin complex I, for instance-inform cells on nutrient availability and influence the cells' transcriptional, translational, posttranslational, and metabolic profiles as well as their developmental decisions. Here I review our current understanding of the structures of the networks responsible for assessing the quantity and quality of carbon and nitrogen sources. I review how these signaling pathways impinge on transcriptional, metabolic, and developmental programs to optimize survival of cells under different environmental conditions. I highlight the profound knowledge we have gained on the structure of these signaling networks but also emphasize the limits of our current understanding of the dynamics of these signaling networks. Moreover, the conservation of these pathways has allowed us to extrapolate our finding with yeast to address issues of lifespan, cancer metabolism, and growth control in more complex organisms.
[Show abstract][Hide abstract] ABSTRACT: High hydrostatic pressure (HHP) is a stress that exerts broad effects on microorganisms with characteristics similar to those of common environmental stresses. In this study, we aimed to identify genetic mechanisms that can enhance alcoholic fermentation of wild Saccharomyces cerevisiae isolated from Brazilian spirit fermentation vats. Accordingly, we performed a time course microarray analysis on a S. cerevisiae strain submitted to mild sublethal pressure treatment of 50 MPa for 30 min at room temperature, followed by incubation for 5, 10 and 15 min without pressure treatment. The obtained transcriptional profiles demonstrate the importance of post-pressurisation period on the activation of several genes related to cell recovery and stress tolerance. Based on these results, we over-expressed genes strongly induced by HHP in the same wild yeast strain and identified genes, particularly SYM1, whose over-expression results in enhanced ethanol production and stress tolerance upon fermentation. The present study validates the use of HHP as a biotechnological tool for the fermentative industries.
Applied Microbiology and Biotechnology 08/2012; · 3.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Allostery and covalent modification are major means of fast-acting metabolic regulation. Their relative roles in responding to environmental changes remain, however, unclear. Here we examine this issue, using as a case study the rapid decrease in pyruvate kinase flux in yeast upon glucose removal. The main pyruvate kinase isozyme (Cdc19) is phosphorylated in response to environmental cues. It also exhibits positively cooperative (ultrasensitive) allosteric activation by fructose-1,6-bisphosphate (FBP). Glucose removal causes accumulation of Cdc19's substrate, phosphoenolpyruvate. This response is retained in strains with altered protein-kinase-A or AMP-activated-protein-kinase activity or with CDC19 carrying mutated phosphorylation sites. In contrast, yeast engineered with a CDC19 point mutation that ablates FBP-based regulation fail to accumulate phosphoenolpyruvate. They also fail to grow on ethanol and slowly resume growth upon glucose upshift. Thus, while yeast pyruvate kinase is covalently modified in response to glucose availability, its activity is controlled almost exclusively by ultrasensitive allostery.
[Show abstract][Hide abstract] ABSTRACT: Combinatorial sensor arrays, such as the olfactory system, can detect a large number of analytes using a relatively small number of receptors. However, the complex pattern of receptor responses to even a single analyte, coupled with the non-linearity of responses to mixtures of analytes, makes quantitative prediction of compound concentrations in a mixture a challenging task. Here we develop a physical model that explicitly takes receptor-ligand interactions into account, and apply it to infer concentrations of highly related sugar nucleotides from the output of four engineered G-protein-coupled receptors. We also derive design principles that enable accurate mixture discrimination with cross-specific sensor arrays. The optimal sensor parameters exhibit relatively weak dependence on component concentrations, making a single designed array useful for analyzing a sizable range of mixtures. The maximum number of mixture components that can be successfully discriminated is twice the number of sensors in the array. Finally, antagonistic receptor responses, well-known to play an important role in natural olfactory systems, prove to be essential for the accurate prediction of component concentrations.
[Show abstract][Hide abstract] ABSTRACT: Most promoters in yeast contain a nucleosome-depleted region (NDR), but the mechanisms by which NDRs are established and maintained in vivo are currently unclear. We have examined how genome-wide nucleosome placement is altered in the absence of two distinct types of nucleosome remodeling activity. In mutants of both SNF2, which encodes the ATPase component of the Swi/Snf remodeling complex, and ASF1, which encodes a histone chaperone, distinct sets of gene promoters carry excess nucleosomes in their NDRs relative to wild-type. In snf2 mutants, excess promoter nucleosomes correlate with reduced gene expression. In both mutants, the excess nucleosomes occupy DNA sequences that are energetically less favorable for nucleosome formation, indicating that intrinsic histone-DNA interactions are not sufficient for nucleosome positioning in vivo, and that Snf2 and Asf1 promote thermodynamic equilibration of nucleosomal arrays. Cells lacking SNF2 or ASF1 still accomplish the changes in promoter nucleosome structure associated with large-scale transcriptional reprogramming. However, chromatin reorganization in the mutants is reduced in extent compared to wild-type cells, even though transcriptional changes proceed normally. In summary, active remodeling is required for distributing nucleosomes to energetically favorable positions in vivo and for reorganizing chromatin in response to changes in transcriptional activity.
Molecular biology of the cell 06/2011; 22(12):2106-18. · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We conducted a phenotypic, transcriptional, metabolic, and genetic analysis of quiescence in yeast induced by starvation of prototrophic cells for one of three essential nutrients (glucose, nitrogen, or phosphate) and compared those results with those obtained with cells growing slowly due to nutrient limitation. These studies address two related questions: (1) Is quiescence a state distinct from any attained during mitotic growth, and (2) does the nature of quiescence differ depending on the means by which it is induced? We found that either limitation or starvation for any of the three nutrients elicits all of the physiological properties associated with quiescence, such as enhanced cell wall integrity and resistance to heat shock and oxidative stress. Moreover, the starvations result in a common transcriptional program, which is in large part a direct extrapolation of the changes that occur during slow growth. In contrast, the metabolic changes that occur upon starvation and the genetic requirements for surviving starvation differ significantly depending on the nutrient for which the cell is starved. The genes needed by cells to survive starvation do not overlap the genes that are induced upon starvation. We conclude that cells do not access a unique and discrete G(0) state, but rather are programmed, when nutrients are scarce, to prepare for a range of possible future stressors. Moreover, these survival strategies are not unique to quiescence, but are engaged by the cell in proportion to nutrient scarcity.
Genes & development 02/2011; 25(4):336-49. · 12.64 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic cell proliferation is controlled by growth factors and essential nutrients, in the absence of which cells may enter into a quiescent (G(0)) state. In yeast, nitrogen and/or carbon limitation causes downregulation of the conserved TORC1 and PKA signaling pathways and, consequently, activation of the PAS kinase Rim15, which orchestrates G(0) program initiation and ensures proper life span by controlling distal readouts, including the expression of specific genes. Here, we report that Rim15 coordinates transcription with posttranscriptional mRNA protection by phosphorylating the paralogous Igo1 and Igo2 proteins. This event, which stimulates Igo proteins to associate with the mRNA decapping activator Dhh1, shelters newly expressed mRNAs from degradation via the 5'-3' mRNA decay pathway, thereby enabling their proper translation during initiation of the G(0) program. These results delineate a likely conserved mechanism by which nutrient limitation leads to stabilization of specific mRNAs that are critical for cell differentiation and life span.
[Show abstract][Hide abstract] ABSTRACT: The origin recognition complex (ORC) marks chromosomal sites as replication origins and is essential for replication initiation. In yeast, ORC also binds to DNA elements called silencers, where its primary function is to recruit silent information regulator (SIR) proteins to establish transcriptional silencing. Indeed, silencers function poorly as chromosomal origins. Several genetic, molecular, and biochemical studies of HMR-E have led to a model proposing that when ORC becomes limiting in the cell (such as in the orc2-1 mutant) only sites that bind ORC tightly (such as HMR-E) remain fully occupied by ORC, while lower affinity sites, including many origins, lose ORC occupancy. Since HMR-E possessed a unique non-replication function, we reasoned that other tight sites might reveal novel functions for ORC on chromosomes. Therefore, we comprehensively determined ORC "affinity" genome-wide by performing an ORC ChIP-on-chip in ORC2 and orc2-1 strains. Here we describe a novel group of orc2-1-resistant ORC-interacting chromosomal sites (ORF-ORC sites) that did not function as replication origins or silencers. Instead, ORF-ORC sites were comprised of protein-coding regions of highly transcribed metabolic genes. In contrast to the ORC-silencer paradigm, transcriptional activation promoted ORC association with these genes. Remarkably, ORF-ORC genes were enriched in proximity to origins of replication and, in several instances, were transcriptionally regulated by these origins. Taken together, these results suggest a surprising connection among ORC, replication origins, and cellular metabolism.
[Show abstract][Hide abstract] ABSTRACT: Genes required for ribosome biogenesis in yeast, referred to collectively as the Ribi regulon, are tightly regulated in coordination with nutrient availability and cellular growth rate. The promoters of a significant fraction of Ribi genes contain one or more copies of the RNA polymerases A and C (PAC) and/or ribosomal RNA-processing element (RRPE) motifs. Prompted by recent studies showing that the yeast protein Dot6 and its homolog Tod6 can bind to a PAC motif sequence in vitro and are required for efficient Ribi gene repression in response to heat shock, we have examined the role of Dot6 and Tod6 in nutrient control of Ribi gene expression in vivo. Our results indicate that PAC sites function as Dot6/Tod6-dependent repressor elements in vivo. Moreover, Dot6 and Tod6 mediate different nutrient signals, with Tod6 responsible for efficient repression of Ribi genes after inhibition of the nitrogen-sensitive TORC1 pathway and Dot6 responsible for repression after inhibition of the carbon-sensitive protein kinase A signaling pathway. Consistently, Dot6 and Tod6 are required for efficient repression of Ribi gene repression immediately after nutrient deprivation and for successful adaptation to nutrient limitation. Thus, these results establish Dot6/Tod6 as a direct link between nutrient availability, Ribi gene regulation, and growth control.
Proceedings of the National Academy of Sciences 11/2009; 106(47):19928-33. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several well-studied promoters in yeast lose nucleosomes upon transcriptional activation and gain them upon repression, an observation that has prompted the model that transcriptional activation and repression requires nucleosome remodeling of regulated promoters. We have examined global nucleosome positioning before and after glucose-induced transcriptional reprogramming, a condition under which more than half of all yeast genes significantly change expression. The majority of induced and repressed genes exhibit no change in promoter nucleosome arrangement, although promoters that do undergo nucleosome remodeling tend to contain a TATA box. Rather, we found multiple examples where the pre-existing accessibility of putative transcription factor binding sites before glucose addition determined whether the corresponding gene would change expression in response to glucose addition. These results suggest that selection of appropriate transcription factor binding sites may be dictated to a large extent by nucleosome prepositioning but that regulation of expression through these sites is dictated not by nucleosome repositioning but by changes in transcription factor activity.
Molecular biology of the cell 07/2009; 20(15):3503-13. · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Modern computational methods are revealing putative transcription-factor (TF) binding sites at an extraordinary rate. However, the major challenge in studying transcriptional networks is to map these regulatory element predictions to the protein transcription factors that bind them. We have developed a microarray-based profiling of phage-display selection (MaPS) strategy that allows rapid and global survey of an organism's proteome for sequence-specific interactions with such putative DNA regulatory elements. Application to a variety of known yeast TF binding sites successfully identified the cognate TF from the background of a complex whole-proteome library. These factors contain DNA-binding domains from diverse families, including Myb, TEA, MADS box, and C2H2 zinc-finger. Using MaPS, we identified Dot6 as a trans-active partner of the long-predicted orphan yeast element Polymerase A & C (PAC). MaPS technology should enable rapid and proteome-scale study of bi-molecular interactions within transcriptional networks.
[Show abstract][Hide abstract] ABSTRACT: Maintaining balanced growth in a changing environment is a fundamental systems-level challenge for cellular physiology, particularly in microorganisms. While the complete set of regulatory and functional pathways supporting growth and cellular proliferation are not yet known, portions of them are well understood. In particular, cellular proliferation is governed by mechanisms that are highly conserved from unicellular to multicellular organisms, and the disruption of these processes in metazoans is a major factor in the development of cancer. In this paper, we develop statistical methodology to identify quantitative aspects of the regulatory mechanisms underlying cellular proliferation in Saccharomyces cerevisiae. We find that the expression levels of a small set of genes can be exploited to predict the instantaneous growth rate of any cellular culture with high accuracy. The predictions obtained in this fashion are robust to changing biological conditions, experimental methods, and technological platforms. The proposed model is also effective in predicting growth rates for the related yeast Saccharomyces bayanus and the highly diverged yeast Schizosaccharomyces pombe, suggesting that the underlying regulatory signature is conserved across a wide range of unicellular evolution. We investigate the biological significance of the gene expression signature that the predictions are based upon from multiple perspectives: by perturbing the regulatory network through the Ras/PKA pathway, observing strong upregulation of growth rate even in the absence of appropriate nutrients, and discovering putative transcription factor binding sites, observing enrichment in growth-correlated genes. More broadly, the proposed methodology enables biological insights about growth at an instantaneous time scale, inaccessible by direct experimental methods. Data and tools enabling others to apply our methods are available at http://function.princeton.edu/growthrate.
[Show abstract][Hide abstract] ABSTRACT: Addition of glucose to yeast cells increases their growth rate and results in a massive restructuring of their transcriptional output. We have used microarray analysis in conjunction with conditional mutations to obtain a systems view of the signaling network responsible for glucose-induced transcriptional changes. We found that several well-studied signaling pathways-such as Snf1 and Rgt-are responsible for specialized but limited responses to glucose. However, 90% of the glucose-induced changes can be recapitulated by the activation of protein kinase A (PKA) or by the induction of PKB (Sch9). Blocking signaling through Sch9 does not interfere with the glucose response, whereas blocking signaling through PKA does. We conclude that both Sch9 and PKA regulate a massive, nutrient-responsive transcriptional program promoting growth, but that they do so in response to different nutritional inputs. Moreover, activating PKA completely recapitulates the transcriptional growth program in the absence of any increase in growth or metabolism, demonstrating that activation of the growth program results solely from the cell's perception of its nutritional status.
Molecular Systems Biology 02/2009; 5:245. · 14.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: G-protein-coupled receptors (GPCR) are prime targets for therapies with small molecule-antagonists. Since yeast have GPCR triggered signaling pathways analogous to those present in mammalian cells, it is possible to express human receptors in yeast coupled to the pheromone responsive signaling cascade in variants that contain mammalian-yeast Galpha subunit chimeras. CXCR4 and CXCR4(N119S), a constitutively active mutant were expressed in yeast coupled to pheromone responsive reporter genes, HIS3, lacZ, or FUI, and tested for signaling activity. Compounds derived from T140, an inverse agonist for CXCR4, were screened for activity using yeast cells expressing CXCR4(N119S) and containing a FUS1-lacZ reporter gene. Levels of inhibition of beta-galactosidase activities triggered by constitutive activation of the pheromone response pathway that were obtained in the presence of the T140 derived compounds correlated with affinities measured in radioligand binding inhibition experiments. The yeast signaling system may provide an effective approach for screening chemokine receptor antagonists.
Methods in enzymology 02/2009; 460:399-412. · 1.90 Impact Factor