The Saccharomyces cerevisiae acetyltransferase Gcn5 exerts antagonistic pleiotropic effects on chronological ageing.

Compared to replicative lifespan, epigenetic regulation of chronological lifespan (CLS) is less well understood in yeast. Here, by screening all the viable mutants of histone acetyltransferase (HAT) and histone deacetylase (HDAC), we demonstrate that Gcn5, functioning in the HAT module of the SAGA/SLIK complex, exhibits an epistatic relationship with the HDAC Hda1 to control the expression of starvation-induced stress response and respiratory cell growth. Surprisingly, the gcn5Δ mutants lose their colony-forming potential early in the stationary phase but display a longer maximum CLS than their WT counterparts, suggesting the contradictory roles of Gcn5 in lifespan regulation. Integrative analyses of the transcriptome, metabolome and ChIP assays reveal that Gcn5 is necessary for the activation of two regulons upon glucose starvation: the Msn2/4-/Gis1-dependent stress response and the Cat8-/Adr1-mediated metabolic reprogramming, to enable pro-longevity characteristics, including redox homeostasis, stress resistance and maximal storage of carbohydrates. The activation of Cat8-/Adr1-dependent regulon also promotes the pyruvate dehydrogenase (PDH) bypass, leading to acetyl-CoA synthesis, global and targeted H3K9 acetylation. Global H3K9 acetylation levels mediated by Gcn5 and Hda1 during the transition into stationary phase are positively correlated with senescent cell populations accumulated in the aged cell cultures. These data suggest that Gcn5 lies in the centre of a feed-forward loop between histone acetylation and starvation-induced gene expression, enabling stress resistance and homeostasis but also promoting chronological ageing concomitantly.

The metabolic growth limitations of petite cells lacking the mitochondrial genome.

Eukaryotic cells can survive the loss of their mitochondrial genome, but consequently suffer from severe growth defects. 'Petite yeasts', characterized by mitochondrial genome loss, are instrumental for studying mitochondrial function and physiology. However, the molecular cause of their reduced growth rate remains an open question. Here we show that petite cells suffer from an insufficient capacity to synthesize glutamate, glutamine, leucine and arginine, negatively impacting their growth. Using a combination of molecular genetics and omics approaches, we demonstrate the evolution of fast growth overcomes these amino acid deficiencies, by alleviating a perturbation in mitochondrial iron metabolism and by restoring a defect in the mitochondrial tricarboxylic acid cycle, caused by aconitase inhibition. Our results hence explain the slow growth of mitochondrial genome-deficient cells with a partial auxotrophy in four amino acids that results from distorted iron metabolism and an inhibited tricarboxylic acid cycle.

Starvation signals in yeast are integrated to coordinate metabolic reprogramming and stress response to ensure longevity.

Studies on replicative and chronological aging in Saccharomyces cerevisiae have greatly advanced our understanding of how longevity is regulated in all eukaryotes. Chronological lifespan (CLS) of yeast is defined as the age-dependent viability of non-dividing cell populations. A number of nutrient sensing and signal transduction pathways (mainly TOR and PKA) have been shown to regulate CLS, yet it is poorly understood how the starvation signals transduced via these pathways lead to CLS extension. Using reporters whose expressions are induced by glucose starvation, we have screened the majority of the 'signaling' mutants in the yeast genome and identified many genes that are necessary for stress response. Subsequent analyses of the 'signaling' mutants not only revealed novel regulators of CLS, such as the GSK-3 ortholog Mck1, but also demonstrated that starvation signals transmitted by SNF1/AMPK, PKC1 and those negatively regulated by TOR/PKA, including Rim15, Yak1 and Mck1 kinases, are integrated to enable metabolic reprogramming and the acquisition of stress resistance. Coordinated metabolic reprogramming ensures the accumulation of storage carbohydrates for quiescent cells to maintain viability. We provide new evidence that Yak1, Rim15 and Mck1 kinases cooperate to activate H2O2-scanvenging activities, thus limiting the levels of ROS in cells entering quiescence. These findings support the recent advances in higher organisms that the flexibility of metabolic reprogramming and the balance between energetics and stress resistance are the unifying principles of lifespan extension. Future work to reveal how the metabolic switch and stress response is coordinated will help delineate the molecular mechanisms of aging in yeast and shed novel insight into aging/anti-aging principles in higher organisms.

Chronological Lifespan in Yeast Is Dependent on the Accumulation of Storage Carbohydrates Mediated by Yak1, Mck1 and Rim15 Kinases.

Upon starvation for glucose or any other macronutrient, yeast cells exit from the mitotic cell cycle and acquire a set of characteristics that are specific to quiescent cells to ensure longevity. Little is known about the molecular determinants that orchestrate quiescence entry and lifespan extension. Using starvation-specific gene reporters, we screened a subset of the yeast deletion library representing the genes encoding 'signaling' proteins. Apart from the previously characterised Rim15, Mck1 and Yak1 kinases, the SNF1/AMPK complex, the cell wall integrity pathway and a number of cell cycle regulators were shown to be necessary for proper quiescence establishment and for extension of chronological lifespan (CLS), suggesting that entry into quiescence requires the integration of starvation signals transmitted via multiple signaling pathways. The CLS of these signaling mutants, and those of the single, double and triple mutants of RIM15, YAK1 and MCK1 correlates well with the amount of storage carbohydrates but poorly with transition-phase cell cycle status. Combined removal of the glycogen and trehalose biosynthetic genes, especially GSY2 and TPS1, nearly abolishes the accumulation of storage carbohydrates and severely reduces CLS. Concurrent overexpression of GSY2 and TSL1 or supplementation of trehalose to the growth medium ameliorates the severe CLS defects displayed by the signaling mutants (rim15Δyak1Δ or rim15Δmck1Δ). Furthermore, we reveal that the levels of intracellular reactive oxygen species are cooperatively controlled by Yak1, Rim15 and Mck1, and the three kinases mediate the TOR1-regulated accumulation of storage carbohydrates and CLS extension. Our data support the hypothesis that metabolic reprogramming to accumulate energy stores and the activation of anti-oxidant defence systems are coordinated by Yak1, Rim15 and Mck1 kinases to ensure quiescence entry and lifespan extension in yeast.

Lst4, the yeast Fnip1/2 orthologue, is a DENN-family protein.

The folliculin/Fnip complex has been demonstrated to play a crucial role in the mechanisms underlying Birt-Hogg-Dubé (BHD) syndrome, a rare inherited cancer syndrome. Lst4 has been previously proposed to be the Fnip1/2 orthologue in yeast and therefore a member of the DENN family. In order to confirm this, we solved the crystal structure of the N-terminal region of Lst4 from Kluyveromyces lactis and show it contains a longin domain, the first domain of the full DENN module. Furthermore, we demonstrate that Lst4 through its DENN domain interacts with Lst7, the yeast folliculin orthologue. Like its human counterpart, the Lst7/Lst4 complex relocates to the vacuolar membrane in response to nutrient starvation, most notably in carbon starvation. Finally, we express and purify the recombinant Lst7/Lst4 complex and show that it exists as a 1 : 1 heterodimer in solution. This work confirms the membership of Lst4 and the Fnip proteins in the DENN family, and provides a basis for using the Lst7/Lst4 complex to understand the molecular function of folliculin and its role in the pathogenesis of BHD syndrome.

Arabidopsis Yak1 protein (AtYak1) is a dual specificity protein kinase.

Yak1 is a member of dual-specificity Tyr phosphorylation-regulated kinases (DYRKs) that are evolutionarily conserved. The downstream targets of Yak1 and their functions are largely unknown. Here, a homologous protein AtYAK1 was identified in Arabidopsis thaliana and the phosphoprotein profiles of the wild type and an atyak1 mutant were compared on two-dimensional gel following Pro-Q Diamond phosphoprotein gel staining. Annexin1, Annexin2 and RBD were phosphorylated at serine/threonine residues by the AtYak1 kinase. Annexin1, Annexin2 and Annexin4 were also phosphorylated at tyrosine residues. Our study demonstrated that AtYak1 is a dual specificity protein kinase in Arabidopsis that may regulate the phosphorylation status of the annexin family proteins.

The Yeast GSK-3 Homologue Mck1 Is a Key Controller of Quiescence Entry and Chronological Lifespan.

Upon starvation for glucose or any other core nutrient, yeast cells exit from the mitotic cell cycle and acquire a set of G0-specific characteristics to ensure long-term survival. It is not well understood whether or how cell cycle progression is coordinated with the acquisition of different G0-related features during the transition to stationary phase (SP). Here, we identify the yeast GSK-3 homologue Mck1 as a key regulator of G0 entry and reveal that Mck1 acts in parallel to Rim15 to activate starvation-induced gene expression, the acquisition of stress resistance, the accumulation of storage carbohydrates, the ability of early SP cells to exit from quiescence, and their chronological lifespan. FACS and microscopy imaging analyses indicate that Mck1 promotes mother-daughter cell separation and together with Rim15, modulates cell size. This indicates that the two kinases coordinate the transition-phase cell cycle, cell size and the acquisition of different G0-specific features. Epistasis experiments place MCK1, like RIM15, downstream of RAS2 in antagonising cell growth and activating stress resistance and glycogen accumulation. Remarkably, in the ras2∆ cells, deletion of MCK1 and RIM15 together, compared to removal of either of them alone, compromises respiratory growth and enhances heat tolerance and glycogen accumulation. Our data indicate that the nutrient sensor Ras2 may prevent the acquisition of G0-specific features via at least two pathways. One involves the negative regulation of the effectors of G0 entry such as Mck1 and Rim15, while the other likely to involve its functions in promoting respiratory growth, a phenotype also contributed by Mck1 and Rim15.

Synergistic effects of TOR and proteasome pathways on the yeast transcriptome and cell growth.

The proteasome has been implicated in gene transcription through a variety of mechanisms. How the proteasome regulates genome-wide transcription in relation to nutrient signalling pathways is largely unknown. Using chemical inhibitors to compromise the functions of the proteasome and/or TORC1, we reveal that the proteasome and TORC1 synergistically promote the expression of de novo purine and amino acid biosynthetic genes, and restrict the transcription of those associated with proteolysis, starvation and stress responses. Genetic analysis demonstrates that TORC1 negatively regulates both the Yak1 and Rim15 kinases to modulate starvation-specific gene expression mediated by the Msn2/4 and Gis1 transcription factors. Compromising proteasome function induces starvation-specific gene transcription in exponential-phase cells and abrogates the strict control of such expression by Yak1 and Rim15 in rapamycin-treated cells, confirming that the proteasome functions to ensure stringent control of the starvation response by the TOR pathway. Synergy between the two pathways is also exhibited on cell growth control. Rpn4-dependent upregulation of proteasomal genes and a catalytically competent 20S proteasome are essential for yeast cells to respond to reduced TORC1 activity. These data suggest that the proteasome and the TOR signalling pathway synergistically regulate a significant portion of the genome to coordinate cell growth and starvation response.

The genetic control of growth rate: a systems biology study in yeast.

BACKGROUND: Control of growth rate is mediated by tight regulation mechanisms in all free-living organisms since long-term survival depends on adaptation to diverse environmental conditions. The yeast, Saccharomyces cerevisiae, when growing under nutrient-limited conditions, controls its growth rate via both nutrient-specific and nutrient-independent gene sets. At slow growth rates, at least, it has been found that the expression of the genes that exert significant control over growth rate (high flux control or HFC genes) is not necessarily regulated by growth rate itself. It has not been determined whether the set of HFC genes is the same at all growth rates or whether it is the same in conditions of nutrient limitation or excess. RESULTS: HFC genes were identified in competition experiments in which a population of hemizygous diploid yeast deletants were grown at, or close to, the maximum specific growth rate in either nutrient-limiting or nutrient-sufficient conditions. A hemizygous mutant is one in which one of any pair of homologous genes is deleted in a diploid, These HFC genes divided into two classes: a haploinsufficient (HI) set, where the hemizygous mutants grow slower than the wild type, and a haploproficient (HP) set, which comprises hemizygotes that grow faster than the wild type. The HI set was found to be enriched for genes involved in the processes of gene expression, while the HP set was enriched for genes concerned with the cell cycle and genome integrity. CONCLUSION: A subset of growth-regulated genes have HFC characteristics when grown in conditions where there are few, or no, external constraints on the rate of growth that cells may attain. This subset is enriched for genes that participate in the processes of gene expression, itself (i.e. transcription and translation). The fact that haploproficiency is exhibited by mutants grown at the previously determined maximum rate implies that the control of growth rate in this simple eukaryote represents a trade-off between the selective advantages of rapid growth and the need to maintain the integrity of the genome.

Contributions of Saccharomyces cerevisiae to understanding mammalian gene function and therapy.

Due to its genetic tractability and ease of manipulation, the yeast Saccharomyces cerevisiae has been extensively used as a model organism to understand how eukaryotic cells grow, divide, and respond to environmental changes. In this chapter, we reasoned that functional annotation of novel genes revealed by sequencing should adopt an integrative approach including both bioinformatics and experimental analysis to reveal functional conservation and divergence of complexes and pathways. The techniques and resources generated for systems biology studies in yeast have found a wide range of applications. Here we focused on using these technologies in revealing functions of genes from mammals, in identifying targets of novel and known drugs and in screening drugs targeting specific proteins and/or protein-protein interactions.

The role of proteosome-mediated proteolysis in modulating potentially harmful transcription factor activity in Saccharomyces cerevisiae.

MOTIVATION: The appropriate modulation of the stress response to variable environmental conditions is necessary to maintain sustained viability in Saccharomyces cerevisiae. Particularly, controlling the abundance of proteins that may have detrimental effects on cell growth is crucial for rapid recovery from stress-induced quiescence. RESULTS: Prompted by qualitative modeling of the nutrient starvation response in yeast, we investigated in vivo the effect of proteolysis after nutrient starvation showing that, for the Gis1 transcription factor at least, proteasome-mediated control is crucial for a rapid return to growth. Additional bioinformatics analyses show that potentially toxic transcriptional regulators have a significantly lower protein half-life, a higher fraction of unstructured regions and more potential PEST motifs than the non-detrimental ones. Furthermore, inhibiting proteasome activity tends to increase the expression of genes induced during the Environmental Stress Response more than those in the rest of the genome. Our combined results suggest that proteasome-mediated proteolysis of potentially toxic transcription factors tightly modulates the stress response in yeast. CONTACT: jasmin.fisher@microsoft.com

JmjN interacts with JmjC to ensure selective proteolysis of Gis1 by the proteasome.

A group of JmjC domain-containing proteins also harbour JmjN domains. Although the JmjC domain is known to possess histone demethylase activity, the function of the JmjN domain remains largely undetermined. Previously, we have demonstrated that the yeast Gis1 transcription factor, bearing both JmjN and JmjC domains at its N terminus, is subject to proteasome-mediated selective proteolysis to downregulate its transcription activation ability. Here, we reveal that the JmjN and JmjC domains interact with each other through two ß-sheets, one in each domain. Removal of either or both ß-strands or the entire JmjN domain leads to complete degradation of Gis1, mediated partially by the proteasome. Mutating the core residues essential for histone demethylase activity demonstrated for other JmjC-containing proteins or deleting both Jumonji domains enhances the transcription activity of Gis1, but has no impact on its selective proteolysis by the proteasome. Together, these data suggest that JmjN and JmjC interact physically to form a structural unit that ensures the stability and appropriate transcription activity of Gis1.

The transcription activity of Gis1 is negatively modulated by proteasome-mediated limited proteolysis.

The transcriptional response to environmental changes has to be prompt but appropriate. Previously, it has been shown that the Gis1 transcription factor is responsible for regulating the expression of postdiauxic shift genes in response to nutrient starvation, and this transcription regulation is dependent upon the Rim15 kinase. Here we demonstrate that the activity of Gis1 is negatively modulated by proteasome-mediated limited proteolysis. Limited degradation of Gis1 by the proteasome leads to the production of smaller variants, which have weaker transcription activities than the full-length protein. The coiled-coil domain, absent from the smaller variants, is part of the second transcription activation domain in Gis1 and is essential for both the limited proteolysis of Gis1 and its full activity. Endogenous Gis1 and its variants, regardless of their transcription capabilities, activate transcription in a Rim15-dependent manner. However, when the full-length Gis1 accumulates in cells due to overexpression or inhibition of the proteasome function, transcription activation by Gis1 is no longer solely controlled by Rim15. Together, these data strongly indicate that the function of the limited degradation is to ensure that Gis1-dependent transcription is strictly regulated by the Rim15 kinase. Furthermore, we have revealed that the kinase activity of Rim15 is essential for this regulation.

Gis1 is required for transcriptional reprogramming of carbon metabolism and the stress response during transition into stationary phase in yeast.

Transition from growth to the stationary phase in yeast is still poorly understood. Previously, we identified a group of yeast genes that are universally upregulated upon starvation for different macronutrients. Here, we demonstrate that the Gis1 transcription factor and the Rim15 kinase are responsible for the upregulation of many of these genes. In chemostat cultures, gis1 or rim15 mutant cells are outcompeted by their wild-type parents under conditions resembling the later stages of diauxie (glucose-limiting) and post-diauxie (ethanol as a carbon source). Whilst Gis1p and Rim15p have distinct functions in gene repression, the growth defects of gis1 or rim15 deletants can be accounted for by the overlapping functions of their protein products in promoting the expression of genes involved in glutamate biosynthesis, the glyoxylate cycle, the pentose phosphate pathway and the stress response. Further, we show that the sets of GIS1- and RIM15-dependent genes and the degree of their regulation change in response to the identity of the carbon source, suggesting the likely dynamics of gene regulation exerted by Rim15p and Gis1p during different phases of the transition into stationary phase. In particular, Rim15p is required for the expression of genes involved in gluconeogenesis/glycolysis and glycerol biosynthesis only when ethanol is used as the carbon source. In agreement with this, Rim15p is shown to act in parallel with Hog1p to defend cells against osmotic stress.

Growth control of the eukaryote cell: a systems biology study in yeast.

BACKGROUND: Cell growth underlies many key cellular and developmental processes, yet a limited number of studies have been carried out on cell-growth regulation. Comprehensive studies at the transcriptional, proteomic and metabolic levels under defined controlled conditions are currently lacking. RESULTS: Metabolic control analysis is being exploited in a systems biology study of the eukaryotic cell. Using chemostat culture, we have measured the impact of changes in flux (growth rate) on the transcriptome, proteome, endometabolome and exometabolome of the yeast Saccharomyces cerevisiae. Each functional genomic level shows clear growth-rate-associated trends and discriminates between carbon-sufficient and carbon-limited conditions. Genes consistently and significantly upregulated with increasing growth rate are frequently essential and encode evolutionarily conserved proteins of known function that participate in many protein-protein interactions. In contrast, more unknown, and fewer essential, genes are downregulated with increasing growth rate; their protein products rarely interact with one another. A large proportion of yeast genes under positive growth-rate control share orthologs with other eukaryotes, including humans. Significantly, transcription of genes encoding components of the TOR complex (a major controller of eukaryotic cell growth) is not subject to growth-rate regulation. Moreover, integrative studies reveal the extent and importance of post-transcriptional control, patterns of control of metabolic fluxes at the level of enzyme synthesis, and the relevance of specific enzymatic reactions in the control of metabolic fluxes during cell growth. CONCLUSION: This work constitutes a first comprehensive systems biology study on growth-rate control in the eukaryotic cell. The results have direct implications for advanced studies on cell growth, in vivo regulation of metabolic fluxes for comprehensive metabolic engineering, and for the design of genome-scale systems biology models of the eukaryotic cell.

The relative merits of the tetO2 and tetO7 promoter systems for the functional analysis of heterologous genes in yeast and a compilation of essential yeast genes with tetO2 promoter substitutions.

We have generated a collection of yeast strains, each of which has an essential yeast gene under the control of the tetracycline-responsive, tetO, promoter. Screens using first-generation promoter-swap strains uncovered the non-specific responsiveness of the tetO7 promoter to a known human transcription factor (hIRF-1). Non-specific regulation was not observed with the tetO2 promoter. Reporter assays have been used to demonstrate this phenomenon. Subsequent efforts to generate a collection of tetracycline-regulatable strains have focused on the tetO2 promoter. These strains are available to the yeast community and can be used for functional genomics studies.

Doxycycline, the drug used to control the tet-regulatable promoter system, has no effect on global gene expression in Saccharomyces cerevisiae.

The tet-regulatable promoter system is commonly used for genetic studies in many eukaryotic organisms. The promoter is regulated using doxycycline. There are no obvious phenotypic effects observed when doxycycline is added to the growth medium of yeast to control expression from the promoter. It is widely accepted that doxycycline is innocuous to yeast. Global genetic studies are now commonplace and the tetO-system is being used in transcriptome studies. Hence, we wanted to ensure that the absence of phenotypic effects, on addition of doxycycline to the growth medium, is mirrored in transcriptome data. We have demonstrated that doxycycline has no significant effect on global transcription levels and will continue to use the tetO-regulatable promoter system for genetic studies.

Global analysis of nutrient control of gene expression in Saccharomyces cerevisiae during growth and starvation.

Global gene expression in yeast was examined in five different nutrient-limited steady states and in their corresponding starvation-induced stationary phases. The use of chemostats, with their ability to generate defined and reproducible physiological conditions, permitted the exclusion of the confounding variables that frequently complicate transcriptome analyses. This approach allowed us to dissect out effects on gene expression that are specific to particular physiological states. Thus, we discovered that a large number of ORFs involved in protein synthesis were activated under ammonium limitation, whereas the expression of ORFs concerned with energy and metabolism was enhanced by carbon limitation. Elevated transcription of genes in high-affinity glucose uptake, the trichloroacetic acid cycle, and oxidative phosphorylation were observed in glucose-limiting, but not glucose-abundant, conditions. In contrast, genes involved in gluconeogenesis and, interestingly, genes subject to nitrogen catabolite repression increased their transcription when ethanol was the carbon source, even though ammonium was in excess. This result suggests that up-regulation of genes sensitive to nitrogen catabolite repression may contribute anapleurotic intermediates in ethanol-grown cells. The different starvation conditions produced two general types of transcription profiles, with carbon-starved cells transcribing far fewer genes than cells starved for any of the other macronutrients. Nonetheless, each starvation condition induced its own peculiar set of genes, and only 17 genes were induced >5-fold by all five starvations. In all cases, analysis of the upstream sequences of clusters of coregulated genes identified motifs that may be recognized by transcription factors specific for controlling gene expression in each of the physiological conditions examined.

An improved tetO promoter replacement system for regulating the expression of yeast genes.

Regulatable promoters are commonly used to control the expression of, especially, essential genes in a conditional manner. Integration of such promoters upstream of an ORF using one-step PCR-mediated homologous recombination should be particularly efficient. However, integration of the original KanMX4-tetO promoter cassette (Belli et al., 1998a) into the relatively short upstream regions of many yeast genes is often problematic, presumably due to the size (3.9 kb) of the replacement cassette. We have created a new, shorter, KanMX4-tetO cassette by removing the transactivator (tTA) sequence from the original cassette. The transactivator (tTA) has been integrated into the yeast genome to create a new strain for use with the new system, which has a greatly increased efficiency of promoter substitution. With it, we have been able to create strains that could not be made with the original cassette. To increase the throughput of promoter substitutions, we have developed a new assay for testing doxycycline sensitivity, based on liquid culture using microtitre trays. Altogether, the components of this new 'tool kit' greatly increase the efficiency of systematic promoter substitutions.

Using yeast to place human genes in functional categories.

The availability of the draft sequence of the human genome has created a pressing need to assign functions to each of the 35,000 or so genes that it defines. One useful approach for this purpose is to use model organisms for both bioinformatic and functional comparisons. We have developed a complementation system, based on the model eukaryote Saccharomyces cerevisiae, to clone human cDNAs that can functionally complement yeast essential genes. The system employs two regulatable promoters. One promoter, tetO (determining doxycycline-repressible expression), is used to control essential S. cerevisiae genes. The other, pMET3 (which is switched off in the presence of methionine), is employed to regulate the expression of mammalian cDNAs in yeast. We have demonstrated that this system is effective for both individual cDNA clones and for cDNA libraries, permitting the direct selection of functionally complementing clones. Three human cDNA libraries have been constructed and screened for clones that can complement specific essential yeast genes whose expression is switched off by the addition of doxycycline to the culture medium. The validity of each complementation was checked by showing that the yeast cells stop their growth in the presence of doxycycline and methionine, which represses the expression of the yeast and mammalian coding sequence, respectively. Using this system, we have screened 25 tetO replacement strains and succeeded in isolating human cDNAs complementing six essential yeast genes. In this way, we have uncovered a novel human ubiquitin-conjugating enzyme, have isolated a human cDNA clone that may function as a signal peptidase and have demonstrated that the functional segment of the human Psmd12 proteosome sub-unit contains a PINT domain.