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Transcriptional response of steady-state yeast cultures to transient perturbations in carbon source.

ABSTRACT: To understand the dynamics of transcriptional response to changing environments, well defined, easily controlled, and short-term perturbation experiments were undertaken. We subjected steady-state cultures of Saccharomyces cerevisiae in chemostats growing on limiting galactose to two different size pulses of glucose, well known to be a preferred carbon source. Although these pulses were not large enough to change growth rates or cell size, approximately 25% of the genes changed their expression at least 2-fold. Using DNA microarrays to estimate mRNA abundance, we found a number of distinguishable patterns of transcriptional response among the many genes whose expression changed. Many of these genes were already known to be regulated by particular transcription factors; we estimated five potentially relevant transcription factor activities from the observed changes in gene expression (i.e., Mig1p, Gal4p, Cat8p, Rgt1p, Adr1p, and Rcs1p). With these estimates, for two regulatory circuits involving interaction among multiple regulators we could generate dynamical models that quantitatively account for the observed transcriptional responses to the transient perturbations.

SUBMITTER: Ronen M

PROVIDER: S-EPMC1326188 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

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Transcriptional response of steady-state yeast cultures to transient perturbations in carbon source.

Ronen Michal M Botstein David D

Proceedings of the National Academy of Sciences of the United States of America 20051228 2

To understand the dynamics of transcriptional response to changing environments, well defined, easily controlled, and short-term perturbation experiments were undertaken. We subjected steady-state cultures of Saccharomyces cerevisiae in chemostats growing on limiting galactose to two different size pulses of glucose, well known to be a preferred carbon source. Although these pulses were not large enough to change growth rates or cell size, approximately 25% of the genes changed their expression ...[more]

PMID: 16381818

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Project description:BackgroundControl effective flux (CEF) of a reaction is the weighted sum of all fluxes through that reaction, derived from elementary flux modes (EFM) of a metabolic network. Change in CEFs under different environmental conditions has earlier been proven to be correlated with the corresponding changes in the transcriptome. Here we use this to investigate the degree of transcriptional regulation of fluxes in the metabolism of Saccharomyces cerevisiae. We do this by quantifying correlations between changes in CEFs and changes in transcript levels for shifts in carbon source, i.e. between the fermentative carbon source glucose and nonfermentative carbon sources like ethanol, acetate, and lactate. The CEF analysis is based on a simple stoichiometric model that includes reactions of the central carbon metabolism and the amino acid metabolism.ResultsThe effect of the carbon shift on the metabolic fluxes was investigated for both batch and chemostat cultures. For growth on glucose in batch (respiro-fermentative) cultures, EFMs with no by-product formation were removed from the analysis of the CEFs, whereas those including any by-products (ethanol, glycerol, acetate, succinate) were omitted in the analysis of growth on glucose in chemostat (respiratory) cultures. This resulted in improved correlations between CEF changes and transcript levels. A regression correlation coefficient of 0.60 was obtained between CEF changes and gene expression changes in the central carbon metabolism for the analysis of 5 different perturbations. Out of 45 data points there were no more than 6 data points deviating from the correlation. Additionally, up- or down-regulation of at least 75% of the genes were in qualitative agreement with the CEF changes for all perturbations studied.ConclusionThe analysis indicates that changes in carbon source are associated with a high degree of hierarchical regulation of metabolic fluxes in the central carbon metabolism as the change in fluxes are correlating directly with the change in transcript levels of genes encoding their corresponding enzymes. For amino acid biosynthesis there was, however, not found to exist a similar correlation, and this may point to either post-transcriptional and/or metabolic regulation, or be due to the absence of a direct perturbation on the amino acid pathways in these experiments.

Project description:Saccharomyces cerevisiae and Schizosaccharomyces pombe are the most commonly studied yeast model systems, yet comparisons of global proteome remodeling between these yeast species are scarce. Here, we profile the proteomes of S. cerevisiae and S. pombe cultured with either glucose or pyruvate as the sole carbon source to define common and distinctive alterations in the protein landscape across species. In addition, we develop an updated streamlined-tandem mass tag (SL-TMT) strategy that substitutes chemical-based precipitation with more versatile bead-based protein aggregation method (SP3) prior to enzymatic digestion and TMT labeling. Our new workflow, SL-SP3-TMT, allow for near-complete proteome profiles in a single experiment for each species. The data reveal expected alterations in protein abundance and differences between species, highlighted complete canonical biochemical pathways, and provided insight into previously uncharacterized proteins. The techniques used herein, namely SL-SP3-TMT, can be applied to virtually any experiment aiming to study remodeling of the proteome using a high-throughput, comprehensive, yet streamlined mass spectrometry-based strategy. SIGNIFICANCE: Saccharomyces cerevisiae and Schizosaccharomyces pombe are single-celled eukaryotes that diverged from a common ancestor over a period of 100 million years, such that evolution has driven fundamental differences between the two species. Cellular metabolism and the regulation thereof are vital for living organisms. Here, we hypothesize that large scale proteomic alterations are prevalent upon the substitution of glucose with another carbon source, in this case pyruvate. To efficiently process our samples, we developed an updated streamlined-tandem mass tag (SL-TMT) strategy with more versatile bead-based protein aggregation. The data revealed expected alterations in protein abundance and illustrated differences between species. We highlighted complete canonical biochemical pathways and provided insight into previously uncharacterized proteins.

Project description:Cells must adjust their gene expression in order to compete in a constantly changing environment. Two alternative strategies could in principle ensure optimal coordination of gene expression with physiological requirement. First, the internal physiological state itself could feedback to regulated gene expression. Second, the expected physiological state could be inferred from the external environment, using evolutionary-tuned signaling pathways. Coordination of ribosomal biogenesis with the requirement for protein synthesis appears to be particularly important, since cells devote a large fraction of their biosynthetic capacity for ribosomal biogenesis. To define the relative importance of internal vs. external sensing to the regulation of ribosomal biogenesis gene expression, we subjected S. cerevisiae cells to conditions which decoupled the actual vs. environmentally-expected growth rate. Gene expression followed the environmental signal according to the expected, but not the actual, growth rate. Simultaneous monitoring of gene expression and growth rate in chemostat-grown cultures further confirmed that ribosome biogenesis genes responded rapidly to changes in the environments but were oblivious to longer-term changes in growth rate. Our results suggest that the capacity to anticipate and prepare for environmental changes presented a major selection force during yeast evolution. Keywords: Saccharomyces_Cerevisiae, Stress response, ADH1 deletion, time courses, chemostat Experiment 1: ADH1 deletion cells, which grow faster on glycerol rather then glucose medium, were grown in both glucose and glycerol medium. The cells expression on glycerol is compared to their expression glucose (5 microarrays). Experiment 2: Time course of ADH1 deletion cells upon growth on glycerol and on glucose (7 microarrays). Experiment 3: Response of steady state grown cells to environmental perturbations. Cells were grown in glucose or histidine limited chemostats and reached steady state. The cells were then subjected to perturbations such as DTT, heat shock, NaCl, Clotrimazole, H2O2, and addition of limiting factors (histidine and glucose, respectively). Overall we have examined 10 timecourses with 83 microarrays.

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Transcriptional response of steady-state yeast cultures to transient perturbations in carbon source.

Publications

Transcriptional response of steady-state yeast cultures to transient perturbations in carbon source.

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