Nianshu Zhang

A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations

A large proportion of the 6,000 genes present in the genome of Saccharomyces cerevisiae, and of those sequenced in other organisms, encode proteins of unknown function. Many of these genes are “silent,” that is, they show no overt phenotype, in terms of growth rate or other fluxes, when they are deleted from the genome. We demonstrate how the intracellular concentrations of metabolites can reveal phenotypes for proteins active in metabolic regulation. Quantification of the change of several metabolite concentrations relative to the concentration change of one selected metabolite can reveal the site of action, in the metabolic network, of a silent gene. In the same way, comprehensive analyses of metabolite concentrations in mutants, providing “metabolic snapshots,” can reveal functions when snapshots from strains deleted for unstudied genes are compared to those deleted for known genes. This approach to functional analysis, using comparative metabolomics, we call FANCY—an abbreviation for functional analysis by co-responses in yeast.

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.

Hybridization array technology coupled with chemostat culture: Tools to interrogate gene expression in Saccharomyces cerevisiae

Hybridization array technology is increasingly being used for the analysis of gene expression in the yeast Saccharomyces cerevisiae. It is a powerful technique in which the relative abundance of all the mRNA molecules transcribed under a particular condition may be simultaneously measured. However, most studies performed using this technique are carried out in batch culture where the growth rate and environment are continuously changing. Often, the experimental condition being studied also impacts on the growth rate of the cells. Changes in growth rate affect the pattern of gene expression. Consequently, the analysis and interpretation of experimental results obtained in this way are inherently problematic due to the difficulty in discriminating between effects due to the experimental condition per se and concomitant growth rate-related effects. Here, we present a method that addresses this problem by exploiting chemostat culture, in which the cells can be grown at a fixed growth rate, in combination with hybridization array technology. We use two experimental examples to illustrate the advantages of using this approach and then describe a specific application of this approach to investigate the effect of carbon and nitrogen limitation at the transcriptome level.

Glycosylation deficiency phenotypes resulting from depletion of GDP-mannose pyrophosphorylase in two yeast species.

The genes encoding GDP‐mannose pyrophosphorylase from Saccharomyces cerevisiae (SRB1/PSA1) and Candida albicans (CaSRB1) were expressed under the control of the tightly regulated promoters of MET3 and CaMET3 respectively. Northern analysis showed that the addition of methionine effectively blocks the transcription of pMET3‐SRB1/PSA1 and pCaMET3CaSRB1 expression cassettes, which had been integrated into the genomes of appropriate mutants. Methionine‐mediated repression of CaSRB1 caused loss of viability in C. albicans, demonstrating that, as in S. cerevisiae, the gene is essential for growth. Depletion of GDP‐mannose pyrophosphorylase had a highly pleiotropic effect in the two yeasts. The major phenotypes observed were lysis, failure of cell separation and/or cytokinesis, impaired bud growth and buds site selection, clumping and flocculation, as well as increased sensitivity to a wide range of antifungal drugs and cell wall inhibitors, and impaired hyphal switching ability. These phenotypes resulted from defects in glycosylation, as demonstrated by reduced affinity for Alcian blue and sensitivity to hygromycin B. Our results provide new information about the roles of protein glycosylation in yeast and, in particular, the steps that require GDP‐mannose in the fungal pathogen C. albicans.

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.

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. Copyright

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.

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. Copyright

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

BackgroundControl 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.ResultsHFC 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.ConclusionA 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.

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.