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Eukaryotic Cell, November 2005, p. 1882-1891, Vol. 4, No. 11
1535-9778/05/$08.00+0     doi:10.1128/EC.4.11.1882-1891.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Repressors Nrg1 and Nrg2 Regulate a Set of Stress-Responsive Genes in Saccharomyces cerevisiae§

Valmik K. Vyas,1,{dagger} Cristin D. Berkey,2 Takenori Miyao,2,{ddagger} and Marian Carlson1,2*

Integrated Program in Cellular, Molecular and Biophysical Studies,1 Department of Genetics and Development, Columbia University, New York, New York2

Received 27 July 2005/ Accepted 7 September 2005


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The yeast Saccharomyces cerevisiae responds to environmental stress by rapidly altering the expression of large sets of genes. We report evidence that the transcriptional repressors Nrg1 and Nrg2 (Nrg1/Nrg2), which were previously implicated in glucose repression, regulate a set of stress-responsive genes. Genome-wide expression analysis identified 150 genes that were upregulated in nrg1{Delta} nrg2{Delta} double mutant cells, relative to wild-type cells, during growth in glucose. We found that many of these genes are regulated by glucose repression. Stress response elements (STREs) and STRE-like elements are overrepresented in the promoters of these genes, and a search of available expression data sets showed that many are regulated in response to a variety of environmental stress signals. In accord with these findings, mutation of NRG1 and NRG2 enhanced the resistance of cells to salt and oxidative stress and decreased tolerance to freezing. We present evidence that Nrg1/Nrg2 not only contribute to repression of target genes in the absence of stress but also limit induction in response to salt stress. We suggest that Nrg1/Nrg2 fine-tune the regulation of a set of stress-responsive genes.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
To survive in natural environments, microorganisms must be able to adapt to changes in environmental conditions, including changes in nutrient availability, temperature, pH, osmolarity, and the presence of oxidative agents. One of the mechanisms by which microorganisms adapt is by changing their genomic expression programs (24, 40). The budding yeast Saccharomyces cerevisiae responds to environmental stress by rapidly altering the expression of sets of genes (5, 12). Each expression program is specific to the particular stress with respect to the genes affected, the magnitudes of the changes in RNA levels, and the temporal patterns of expression (12). However, a large set of genes responds similarly to many kinds of environmental change by what have been called the environmental stress response (ESR) (12) and the common environmental response (5), which involve ~900 and ~500 genes, respectively, that are induced or repressed. Many of the induced genes function in protection against stress or generation of energy, and many of the repressed genes are involved in protein synthesis and growth. A hallmark of stress responses, with the exception of adaptation to nutrient changes, is that most alterations in gene expression are rapid but transient (5, 12, 31). After the cell adapts to the changed environment, its gene expression resets to a program close to that observed in the absence of stress.

Stress-responsive genes are regulated by multiple mechanisms in S. cerevisiae. Some mechanisms are specific to particular stress conditions; for example, the transcriptional activator Yap1 is particularly important for the adaptive response to oxidative stress (18), whereas Hot1 is specific for osmotic induction (36). In contrast, the activators Msn2 and Msn4 (Msn2/Msn4) are broadly involved in responses to nutrient starvation, heat, acidic pH, DNA damage, osmotic stress, and oxidative stress (3, 5, 11, 12, 25, 26, 41). Msn2/Msn4 bind the stress response element (STRE; C4T) (26, 41), which is present in the promoters of many stress-induced genes (29).

In this work, we report evidence that two repressors, Nrg1 and Nrg2 (Nrg1/Nrg2), regulate a set of stress-responsive genes. Nrg1/Nrg2 have highly similar C2H2 zinc fingers and function as transcriptional repressors (33, 47). Previous evidence implicated Nrg1/Nrg2 in glucose repression; Nrg1/Nrg2 repress the glucose-repressed STA, FLO11, SUC2, DOG2, and GAL genes (21, 33, 47, 49) and negatively regulate haploid invasive growth and initiation of biofilm formation (21, 46), which are cellular responses to glucose limitation (8, 37). In addition, both repressors interact with Snf1 protein kinase, a component of a major glucose signaling pathway (47). Nrg1/Nrg2 have also been implicated in responses to other environmental conditions. The alkaline pH response regulator Rim101 represses NRG1, and Nrg1 represses two alkaline pH-induced genes, ZPS1 and the Na+ pump gene ENA1; correspondingly, mutation of NRG1 confers ion tolerance and suppresses the rim101{Delta} defect in growth at pH 9 (22). Nrg1 acts with Rim101 to repress the sporulation-specific genes DIT1 and DIT2 (39). Nrg1/Nrg2 also negatively regulate diploid pseudohyphal growth (21), which occurs in response to nitrogen limitation; this function may be related to that of Candida albicans Nrg1 (CaNrg1), which represses filamentous growth and expression of hypha-specific genes (4, 19, 30).

To identify the set of genes that is regulated by Nrg1/Nrg2, we have carried out genome-wide expression analysis of nrg1{Delta} and nrg2{Delta} single and double mutants. We also used genomic expression analysis to further explore the role of Nrg1/Nrg2 in regulation of carbon source-responsive genes. We found that many of the genes that are repressed by Nrg1/Nrg2 in unstressed, glucose-grown cells contain STRE and STRE-like elements in their promoters and are regulated in response to a variety of stress conditions. We present evidence that mutation of NRG1 and NRG2 alters the stress resistance of cells and that Nrg1/Nrg2 limit the induction of Nrg-repressed genes in response to salt stress.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Strains and genetic methods. S. cerevisiae strains used in this study are listed in Table 1. The gal83{Delta} allele has been described previously (46). msn2{Delta}::kanMX4 and msn4{Delta}::kanMX4 alleles were recovered from genomic DNA of mutant strains (Open Biosystems, Huntsville, AL) and were introduced into MCY5314 and MCY5326 by transformation. The natMX4 and hphMX4 deletion alleles were made by transformation of strains containing kanMX4 or kanMX6 alleles with DNA containing the natMX4 or hphMX4 cassettes, respectively (14). Rich medium was yeast extract-peptone (YEP) containing 2% glucose (YPD) or the indicated carbon source (38). Cells were grown at 30°C unless otherwise specified.


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  		TABLE 1. Strains used in this studya

 
Preparation of RNA. Cells (50 ml) were grown to an optical density at 600 nm of 1 in YPD and collected by filtration, except that for shifts to low glucose, cells were collected by centrifugation, washed, resuspended in YEP plus 0.05% glucose for the indicated time, and collected by centrifugation. Cells were resuspended in 0.7 ml of TES buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 0.5% sodium dodecyl sulfate) and frozen in liquid nitrogen. An equal volume of acid phenol was added to each sample. Samples were incubated at 65°C for 1.5 h with vortexing 10 times for 30 s at 3-min intervals and then for 30 s every 10 min. Samples were extracted four times with equal volumes of phenol and then extracted twice with chloroform. RNA was precipitated with ethanol and resuspended in water.

Gene expression analysis. Total RNA (30 µg) was used as a template for cDNA synthesis with the T7-(dT)24 oligonucleotide, as described by Affymetrix (Santa Clara, CA). Biotin-labeled cRNA was transcribed using an Enzo BioArray high-yield transcript-labeling kit (Affymetrix), fragmented, and hybridized to Affymetrix yeast genome S98 arrays using the manufacturer's protocols. Absolute and comparative analyses of expression data were performed with Affymetrix Microarray Suite 5.0, following the manufacturer's guidelines and using the default settings. For each strain and condition, samples were prepared in duplicate. All pairwise comparisons between mutant and wild-type (WT) samples were analyzed, yielding four signal log ratios (SLRs; base 2) for each mutant-to-WT comparison, which were exported into Microsoft Excel and then averaged. All comparisons were between strains with matched auxotrophies, except MCY4710 (nrg1{Delta}), which is His–. Genes that showed an average SLR of 1 (twofold difference) and that also displayed significant changes in at least two of these comparisons (as measured by the SLR P value determined by Affymetrix Microarray Suite 5.0) were kept for further analysis. Data from probe sets not conforming to standard open reading frame nomenclature guidelines (6) were not included in subsequent analysis. Filtered datasets were exported into Cluster and Treeview (10), where they were K-means clustered and visualized. Original data are available in Tables S1, S2, and S3 in the supplemental material.

Northern blot analysis. RNAs (40 µg) were separated by electrophoresis on a 1.2% agarose-MOPS (morpholinepropanesulfonic acid) gel containing formaldehyde. RNAs were transferred to Hybond N+ membrane (Amersham Biosciences). Probes contained ~250 to 1,500 bp of coding sequence and were 32P labeled using Ready-To-Go DNA-labeling beads (Amersham Biosciences). Levels of RNA relative to those of loading controls were quantified with a Molecular Dynamics PhosphorImager (Amersham Biosciences) and ImageJ software (1). The GSY1 probe did not cross-hybridize with GSY2.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Whole-genome expression analysis of nrg1{Delta} and nrg2{Delta} single and double mutants. To assess the roles of Nrg1/Nrg2 in genomic expression, we prepared RNAs from wild-type, nrg1{Delta}, nrg2{Delta}, and nrg1{Delta} nrg2{Delta} cells ({Sigma}1278b genetic background) grown to mid-log phase in rich medium containing 2% glucose. The growth rates of the wild-type and double mutant strains were identical in three independent experiments (data not shown). RNAs were analyzed by hybridization to a whole-genome DNA oligonucleotide microarray, and measurements of RNA levels were organized by clustering (10) to group genes according to their similarity in terms of expression pattern. Expression of 150 genes was increased twofold or more in the nrg1{Delta} nrg2{Delta} double mutant relative to that in the wild type, and RNA levels of 41 genes were increased at least threefold (Fig. 1; see Table S1 in the supplemental material). Surprisingly, most of these genes (119) also showed increased expression (>1.5-fold) in each single mutant, indicating that both Nrg1 and Nrg2 are required for full repression; only 11 genes showed <1.5-fold-increased expression in both single mutants, indicating that either Nrg1 or Nrg2 is sufficient for repression. Thus, Nrg1 and Nrg2 repress highly overlapping sets of genes, consistent with the strong similarity of their DNA-binding domains (84% identity). It is possible that Nrg1/Nrg2 regulate some of these genes by indirect mechanisms.



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  		FIG. 1. Cluster analysis of 150 genes with increased expression in the nrg1{Delta} nrg2{Delta} double mutant relative to that in the wild type. Clusters are indicated to the left. (Lanes 1 to 3) Expression of genes in glucose-grown nrg1{Delta}, nrg2{Delta}, and nrg1{Delta} nrg2{Delta} strains (MCY4710, MCY4706, and MCY4714) is compared to that of the wild type (WT, MCY4577). Genes with twofold-or-greater increases in expression in the double mutant relative to that of the wild type are shown; black bars mark 41 genes with expression increased by more than threefold. (Lanes 4 to 7) Glucose-grown WT cells (MCY4565) were shifted to 0.05% glucose for 15, 30, 45, and 60 min; expression is compared to that at time point zero. (Lanes 8 and 9) Expression of genes in glucose-grown reg1{Delta} (MCY4569) and reg1{Delta} gal83{Delta} (MCY4571) cells is compared to that in WT (MCY4565). (Lane 10) Expression in reg1{Delta} cells is compared to that in reg1{Delta} gal83{Delta} cells. Scale indicates increase (red) or decrease (green) (n-fold) relative to that of the control (black). See Table S1 in the supplemental material for data.

 
In addition, expression of 265 genes was decreased at least twofold in the nrg1{Delta} nrg2{Delta} mutant, and 187 genes showed decreased expression in each single mutant (see Table S2 in the supplemental material). These findings suggest that Nrg1/Nrg2 play positive regulatory roles either indirectly, by Nrg1/Nrg2-mediated repression of genes encoding repressors, or directly. The possibility that Nrg1 directly activates some promoters, as do other repressors (32, 34), was raised by evidence that, in mutant cells lacking the Ssn6-Tup1 corepressor, DNA-bound LexA-Nrg1 activated transcription of a reporter (2). However, previous studies have characterized Nrg1/Nrg2 as repressors (21, 22, 33, 39, 47, 49), and we have therefore focused on the 150 genes that were upregulated in the mutant.

Northern blot analysis of representative genes yielded results parallel to those of the microarray analysis (Fig. 2 and data not shown). For GSY1, PTR2, PGU1, PHO84, RPI1, and INH1, RNA levels were similarly increased in the single and double mutants relative to the loading control level, confirming that both Nrg1 and Nrg2 are necessary for full repression. For SGA1, ENA, and RSB1, RNA levels in the double mutant were substantially higher than that in either single mutant, confirming that either Nrg1 or Nrg2 alone confers repression. Finally, RNA levels of CTS1, a representative downregulated gene, were decreased in the mutants.



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  		FIG. 2. Northern blot analysis. RNA was prepared from glucose-grown cultures of WT, nrg1{Delta}, nrg2{Delta}, and nrg1{Delta} nrg2{Delta} strains (MCY4577, MCY4710, MCY4706, and MCY4714) and subjected to Northern blot analysis. Blots were hybridized with probes specific for each of the indicated RNAs and for the loading control, the small nucleolar RNA U3 encoded by SNR17A and SNR17B.

 
Functions of Nrg-repressed genes. Previous studies implicated Nrg1 and/or Nrg2 in glucose repression, haploid invasive and diploid pseudohyphal growth, adaptation to alkaline pH, and ion tolerance (21, 22, 33, 47, 49). In accord with these findings, the Nrg1/Nrg2-repressed (Nrg-repressed) genes identified in this study are involved in mitochondrial function, carbon utilization and signaling, nitrogen utilization, cell wall and cell surface function, transcriptional control, mating, transport of nutrients and ions, and other cellular processes (Table 2). The largest category comprises genes involved in mitochondrial function, many of which are regulated in response to the carbon source. IMP2, NCE102, PUT4, XBP1, and YOR161C are important for invasive or pseudohyphal growth (28, 43). Mutation of the ENA locus, PHO84, or SHC1 impairs growth at alkaline pHs (13, 16, 17). Many of the Nrg-repressed genes are important for growth on media containing NaCl or sorbitol, including not only the ENA genes but also AGA2, ADK2, ALD4, CUP9, ELO1, GSP2, LSP1, NCE102, PGU1, YMR003W, YNL274C, YMR194C-A, and ZMS1 (13). We note that several targets of Nrg1/Nrg2 are important for multiple processes that cross simple classification lines; for example, NCE102, which was identified by its role in nonclassic export (7), encodes a protein that purifies with mitochondria (42) and is important for invasive growth, growth on glycerol, and resistance to NaCl (13).


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  		TABLE 2. Functional categorization of Nrg-repressed genes

 
Nrg1/Nrg2 regulate carbon source-responsive genes. Previous work showed that Nrg1/Nrg2 contribute to repression of various glucose-repressed genes (21, 33, 47, 49). To determine whether the Nrg-repressed genes are in general regulated by glucose repression, we carried out genomic expression analyses of wild-type cells that were grown in high (2%) glucose and then shifted to low (0.05%) glucose; aliquots were taken for analysis of RNA at 15-min intervals for 1 h after the shift. Many of the 150 Nrg-repressed genes were also glucose repressed: the expression of 60 genes increased more than twofold at all time points after the shift, and 94 genes showed increased expression at two or more time points (Fig. 1). However, some Nrg1/Nrg2 target genes were downregulated in response to glucose limitation (Fig. 1, cluster III); this subset includes six genes involved in mating (see Table 2).

To further explore the connections between Nrg-repressed genes and regulation in response to carbon source, we examined gene expression in reg1{Delta} mutant cells, which exhibit a general release of glucose repression. Reg1 is a regulatory subunit of protein phosphatase 1. One of its functions is targeting protein phosphatase 1 to Snf1 to negatively regulate its catalytic activity; hence, in a reg1{Delta} mutant, the Snf1 protein kinase signaling pathway is aberrantly activated in glucose-grown cells (23, 27, 44). Microarray analysis revealed that 90 of the 150 Nrg-repressed genes were upregulated twofold or more in glucose-grown reg1{Delta} cells relative to wild type (Fig. 1). Together, these findings support a broad role for Nrg1/Nrg2 in regulation of carbon source-responsive genes.

Relationship of Nrg1/Nrg2 and Snf1-Gal83 protein kinase. Genetic studies implicated Snf1 protein kinase containing the ß-subunit isoform Gal83, called Snf1-Gal83, in antagonizing Nrg1/Nrg2 repressor function with respect to STA2 and FLO11 expression and invasive growth (46). To assess the role of Snf1-Gal83 in the control of other Nrg-repressed genes, we examined gene expression in gal83{Delta} mutant cells after a shift to low glucose. Most of the Nrg-repressed genes that were upregulated in the wild type were similarly upregulated in gal83{Delta} cells (data not shown); however, previous work has shown that upregulation is usually complex, involving both relief of repression and activation. We then asked whether the increased gene expression in glucose-grown reg1{Delta} cells relative to that in wild-type cells depends on Snf1-Gal83. Of the 90 genes that were upregulated in the reg1{Delta} mutant, expression of 89 was less elevated in the reg1{Delta} gal83{Delta} mutant (Fig. 1), suggesting that Snf1-Gal83 and Nrg1/Nrg2 regulate overlapping sets of genes.

Many Nrg1/Nrg2-repressed genes contain STREs and STRE-like sequences. To analyze sequence elements in the promoters of the 150 Nrg-repressed genes, we used regulatory sequence analysis tools to retrieve sequences 600 bp upstream of the ATG. Motifs were found using the oligo-analysis program with the default settings (45). The STRE sequence, C4T, was identified most frequently and is present 131 times in 73 genes, and C3TC was also frequent (Fig. 3; see Table S4 in the supplemental material). CaNrg1 binds C4T in vitro, and many C. albicans genes that are regulated by CaNrg1 carry a related sequence with the consensus (A/C)(A/C/G)C3T (30). This consensus sequence is present 156 times in the promoters of 93 Nrg-repressed genes (Fig. 3), and a recent study showed that Nrg1 binds in vitro to an oligonucleotide containing AAC3T (39). We note that the Nrg1 footprint at the STA1 promoter (33) includes both TC4T and ATC3T. The closely related sequence GGaC3T was identified as a consensus binding site for Nrg1 by genome-wide location analysis (15) and is present 25 times in 23 of the 150 Nrg-repressed genes. Together, these data suggest that Nrg1/Nrg2 bind STRE-like sequences.



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  		FIG. 3. Cluster analysis of Nrg-repressed genes under various growth and stress conditions. Clusters are indicated to the left. Data for the 150 Nrg-repressed genes from Fig. 1 were combined with data from DeRisi et al. (9), Gasch et al. (12), and Causton et al. (5). Top rows show expression of NRG1 and NRG2. Triangles indicate a time course; in each case the zero time point is the reference. Expression during steady-state growth in ethanol (Eth), galactose (Gal), raffinose (Raf), glucose (Glu), mannose (Man), and sucrose (Suc) was compared to the pooled data for all carbon sources. Nitrogen depletion was achieved by a shift to limiting ammonium sulfate. Diamide, peroxide (H2O2), NaCl, and sorbitol were added to final concentrations of 1.5 mM, 0.4 mM, 1 M, and 1.5 M, respectively. Heat shock refers to a shift from 25°C to 37°C, and cold shock, vice versa. Heat transitions were shifts from various temperatures (17°C to 37°C) to 37°C for 20 min. Alkali and acid indicate media at pH 7.9 and at pH 4.0, respectively. The rightmost columns show expression in the msn2{Delta} msn4{Delta} mutant exposed to acid. Scale indicates change (n-fold) relative to the unstressed condition or time point zero. Bars mark genes containing C4T, C3TC, or (A/G)(A/G/C)C3T, and the shade of blue corresponds to the number of elements, as indicated in the scale.

 
The presence of STRE sites raised the possibility that the upregulation of these genes reflects a stress response triggered by reduced fitness of the nrg1{Delta} nrg2{Delta} mutant cells. Three lines of evidence suggest that this is not the case. First, the mutant cells exhibited a wild-type growth rate under the conditions employed. Second, only 25 of the 150 upregulated genes and 28 of the 265 downregulated genes were classified as part of the ESR (12). Third, some of the genes that were upregulated in the mutant are in fact downregulated in wild-type cells in response to stress (Fig. 3).

Regulation of Nrg1/Nrg2-repressed genes in response to stress. The presence of STRE sites and the regulation of many Nrg1/Nrg2 target genes in response to glucose limitation, which can be viewed as carbon stress, suggested that these genes may be broadly regulated in response to stress signals. To address this possibility, we combined our data with data sets derived from microarray analyses of gene expression under different environmental conditions (5, 9, 12) and clustered the Nrg-repressed genes with respect to their expression patterns (Fig. 3; also see Table S3 in the supplemental material).

Figure 3 further supports the view that the Nrg-repressed genes are regulated by carbon availability. Many of these genes were induced during the diauxic shift, when cells shift from fermentative to respiratory growth in response to the depletion of glucose, and during entry into stationary phase (clusters I and II). These genes were also more highly expressed during growth in less-preferred carbon sources (ethanol, galactose, and raffinose) than during growth in more-preferred carbon sources (glucose, mannose, and sucrose). In contrast, genes that were downregulated in response to glucose limitation (cluster III) were also correspondingly repressed under other conditions of poor carbon availability.

Many of the 150 Nrg-repressed genes were also regulated in response to multiple stresses, including nitrogen depletion, oxidative stress, various transitions to heat, cold shock, salt and osmotic stress, and both alkaline and acidic pHs. The genes in cluster I, which were strongly upregulated in response to glucose limitation, were also induced in response to all of these different stress conditions, except cold shock. The genes in cluster II, which were also upregulated in response to glucose limitation, were less strongly affected by other stresses, except for an initial marked downregulation in response to salt and osmotic stress. The genes in cluster III were generally downregulated in response to glucose limitation and other environmental changes. Thus, the Nrg-repressed genes are regulated by stresses unrelated to carbon availability and exhibit three broadly different patterns of response to environmental change.

Evidence indicates that many of the 150 Nrg-repressed genes are also targets of Msn2/Msn4. The induction of 78 of these genes by acidic pH depends on Msn2/Msn4 (Fig. 3; columns at right); of the 91 genes that were upregulated at both the 10-min and 20-min time points in wild-type cells, 29 were less upregulated in the msn2{Delta} msn4{Delta} mutant, and 49 were downregulated, suggesting that Msn2/Msn4 function to overcome transcriptional repression (5). In addition, 106 of the Nrg-repressed genes were induced to some extent by overexpression of Msn2 and/or Msn4 (12).

Regulation of NRG1/NRG2 expression by stress. Our genomic expression analysis showed a 4.3-fold increase in NRG1 RNA levels and a slight elevation in NRG2 levels during a 1-h shift from high to low glucose (Fig. 3, top rows). These results are in accord with previous evidence that NRG1 RNA levels, and Nrg1 protein levels, are elevated in response to glucose limitation or growth in nonpreferred carbon sources, while NRG2 RNA levels remain nearly constant (2, 9, 12).

Expression of NRG1 is also regulated in response to other stresses, and NRG1 RNA levels showed a pattern broadly similar to those of the genes in cluster I, with particularly strongly increased expression upon the addition of salt or sorbitol (5, 12) (Fig. 3, top row). The downregulation of NRG1 in response to alkaline pH represents a difference from the pattern in cluster I but is in accord with another report (22). We confirmed the regulation of NRG1 RNA in response to salt stress by Northern blot analysis; NRG1 RNA levels were elevated relative to that of the loading control, with similar results for the {Sigma}1278b, W303, and S288C genetic backgrounds (Fig. 4 and data not shown). In addition, NRG1 RNA was induced by salt stress and glucose limitation in the msn2{Delta} msn4{Delta} mutant (Fig. 4 and data not shown).



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  		FIG. 4. Regulation of stress response genes by Nrg1/Nrg2. Strains were (A and B) MCY5326, MCY5378, MCY5338, and MCY5385 ({Sigma}1278b) and (C and D) MCY5444 and MCY5451 (W303). Cultures were grown to mid-log phase in YPD. (Panel A) Cells were collected by filtration and transferred to YPD containing 1 M NaCl. (Panels B to D) YPD containing 5 M NaCl was added to a final concentration of (panel B) 75 mM, (panel C) 0.5 M, or (panel D) 0.3 M NaCl. Samples were collected at the indicated times. RNAs were prepared, separated by denaturing agarose gel electrophoresis, and subjected to Northern blot analysis. Note that the 15-min sample for the nrg1{Delta} nrg2{Delta} mutant in panel C is underloaded, as judged by the loading control, the ACT1 (actin gene) RNA.

 
NRG2 RNA levels showed only modest changes during exposure to stress conditions (5, 12) (Fig. 3). Northern blot analysis confirmed a very modest response to salt stress (data not shown).

Nrg1/Nrg2 limit induction of target genes in response to salt stress. To assess the effects of Nrg1/Nrg2 on gene expression during salt stress, we first examined the regulation of three Nrg-repressed, stress-induced genes, CTT1, CYC7, and RSB1. Nrg1 binds directly to the CYC7 and RSB1 promoters (2, 15), and osmotic induction of CTT1 and CYC7 depends on Msn2/Msn4 (35). We carried out Northern blot analysis of RNAs prepared from cells ({Sigma}1278b background) exposed to 1 M NaCl (Fig. 4A). Basal expression and induction of all three RNAs were increased in the nrg1{Delta} nrg2{Delta} mutant relative to that in the wild type, and upregulation of NRG1 showed a similar temporal pattern. Induction was reduced in the msn2{Delta} msn4{Delta} mutant and was partially restored in the msn2{Delta} msn4{Delta} nrg1{Delta} nrg2{Delta} quadruple mutant.

In {Sigma}1278b strains, the response to 1 M NaCl occurred much more slowly than in other characterized strains. Exposure of {Sigma}1278b strains to 75 mM NaCl elicited a more rapid response (Fig. 4B [note that time points are different from those in Fig. 4A]). In addition to CYC7, we examined SGA1 and ENA ({Sigma}1278b has only one ENA gene [48]), which were independently identified as Nrg1 targets by genome-wide location analysis (15). All three showed not only increased basal expression but also increased upregulation in the nrg1{Delta} nrg2{Delta} mutant relative to that in the wild type.

We next examined the regulation of several Nrg-repressed, stress-induced genes in W303 cells, which respond rapidly to salt stress. Induction of XBP1, SGA1, and CTT1 in response to 0.5 M NaCl was increased in the nrg1{Delta} nrg2{Delta} mutant relative to that in the wild type (Fig. 4C). In this experiment, the temporal pattern of transient induction was evident and was unperturbed in the mutant. To further assess the role of Nrg1/Nrg2 in tempering the amplitude of induction, we exposed W303 cells to a mild stress, 0.3 M NaCl. The induction response was very brief, and the presence of Nrg1/Nrg2 reduced induction, particularly in the case of SGA1 (Fig. 4D). Thus, these studies support a role for Nrg1/Nrg2 in limiting the amplitude of induction during the salt stress response.

With respect to the Nrg1/Nrg2 target genes that are repressed during exposure to stress (Fig. 3, cluster III), the simple model is that Nrg1/Nrg2 contribute to the repression response. We examined the regulation of one of these genes, TIP1, in the nrg1{Delta} nrg2{Delta} mutant during exposure to salt. Although the overall level of expression was higher in the mutant than in the wild type, the absence of Nrg1/Nrg2 did not prevent the transient repression response (Fig. 4C) (confirmed by quantitative analysis of the phosphorimaging data).

Altered resistance of nrg1{Delta} nrg2{Delta} mutant cells to salt and to oxidative and cold stresses. To assess the physiological importance of Nrg1/Nrg2 in stress responses, we tested nrg1{Delta} nrg2{Delta} mutant cells for the ability to tolerate several different stress conditions. Previously, the nrg1{Delta} mutant was reported to be salt tolerant (22). Both single and double mutants of the S288C genetic background grew better than the wild type on medium containing 1 M NaCl (Fig. 5A). Similarly, the nrg1{Delta} nrg2{Delta} mutant of the {Sigma}1278b background showed increased salt tolerance relative to the wild type (Fig. 5B), although the latter cells were sensitive to 150 mM NaCl ({Sigma}1278b cells have defects at the ENA locus [48]). These findings are consistent with evidence that Nrg1/Nrg2 repress a number of genes, including ENA genes, that are induced by salt stress and contribute to salt resistance.



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  		FIG. 5. Assays of nrg1{Delta} nrg2{Delta} mutants for salt, NaAsO2, H2O2, and cold tolerance. (A) WT and mutant strains (MCY4521, MCY4523, MCY4525, and MCY4549; S288C background) were grown overnight in YPD and diluted to an optical density at 600 nm of 0.3, and serial threefold dilutions were spotted on YPD and YPD plus 1 M NaCl. Plates were incubated for 5 days and photographed. (B) Strains MCY5326 and MCY5378 ({Sigma}1278b background) were grown and spotted as described for panel A on YPD and YPD plus 150 mM NaCl. Plates were photographed after 2 days. (C) Strains MCY5326 and MCY5378 were grown to mid-log phase in YPD, and equal amounts of cells were plated on YPD. Sterile disks soaked with 5 µl of H2O, 100 mM NaAsO2, or 1.1 M H2O2 were placed on the plates. Plates were incubated for 2 days and photographed. (D) Wild-type and mutant strains (MCY5314 and MCY5414; S288C background) were grown to mid-log phase, and multiple aliquots (5 µl) were spotted onto sterile Whatman filters. Filters were placed in sterile tubes and incubated at –20°C for the indicated times. Cells were then resuspended in 1 ml of YPD, incubated for 15 min at 30°C, and plated onto YPD. Viability is shown relative to that at the initial time point prior to freezing. Data in top and bottom panels are from two independent experiments using different tubes and different freezers.

 
Many Nrg-repressed genes are induced by oxidative stress, and we therefore tested nrg1{Delta} nrg2{Delta} cells for altered resistance to oxidative stress. Cells were spread on a plate and exposed to a disk containing NaAsO2 or H2O2. Both oxidative agents caused a larger zone of growth inhibition for wild-type cells than for nrg1{Delta} nrg2{Delta} cells ({Sigma}1278b background) (Fig. 5C).

The ability of yeast cells to tolerate low or freezing temperatures depends on the induction of trehalose synthesis (20). Although we did not identify genes for trehalose synthesis in this study, three of the Nrg-repressed genes, PGM1, GSY1, and GLC3, are important for the synthesis and metabolism of glycogen, another reserve carbohydrate. These three genes are among the many Nrg-repressed genes that are repressed in response to cold shock (12). We therefore tested nrg1{Delta} nrg2{Delta} cells for survival at freezing temperatures by use of the assay of Kandror et al. (20). The mutant cells showed reduced ability to survive exposure to –20°C. After 10 days, 26% of wild-type cells (S288C background) remained viable, compared to 2.1% of the mutant cells (Fig. 5D, top panel); similar results were obtained for cells of the {Sigma}1278b background, with 8.0% viability for the wild type (MCY5326) and 1.6% for the nrg1{Delta} nrg2{Delta} mutant (MCY5378). Survival seems to be dependent on the particular conditions for freezing; cells of the S288C background showed much-reduced viability in an independent experiment using different tubes and a different freezer, although again the nrg1{Delta} nrg2{Delta} cells showed viability that was much reduced relative to that of wild-type cells (Fig. 5D, bottom panel). This increased susceptibility to freezing temperatures is consistent with evidence that, prior to freezing, the nrg1{Delta} nrg2{Delta} mutant exhibits elevated expression of genes that are normally repressed in response to cold shock.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
By analysis of whole-genome expression, we have identified a set of 150 genes that were upregulated in the nrg1{Delta} nrg2{Delta} double mutant, relative to wild type, when cells were grown in rich medium containing glucose. These genes are involved in mitochondrial function, carbon utilization and signaling, nitrogen utilization, transport of nutrients and ions, cell wall and cell surface function, transcriptional control, mating, and other cellular processes. Of the 150 genes, 94 are glucose repressed and show increased expression when cells are limited for glucose, in accord with studies implicating Nrg1/Nrg2 in glucose repression of several specific genes (21, 33, 47, 49). Analysis of the upstream regions of the Nrg1/Nrg2 targets revealed that STRE and STRE-like elements are overrepresented, and a search of available expression data sets showed that many of these genes are regulated by a variety of stresses and by the stress-responsive transcriptional activators Msn2/Msn4. Thus, these studies implicate Nrg1/Nrg2 in regulation of a set of stress-responsive genes.

We also note that Nrg1/Nrg2 may regulate a set of stress-responsive genes larger than that identified here. Our genomic expression analysis identified genes that, in the absence of Nrg1/Nrg2, exhibit increased expression during exponential growth in rich medium containing glucose (unstressed conditions). It seems likely that some, and perhaps many, Nrg1/Nrg2 target genes are activated primarily by stress-responsive activators and are simply not activated under such optimal growth conditions, despite release of Nrg-mediated repression. Thus, it is possible that Nrg1/Nrg2 repress additional stress-responsive genes. It is also possible that the effects of Nrg1/Nrg2 on some of the 150 genes identified in this study are indirect. Only 14 of the 150 genes were among the 128 targets of Nrg1 identified by genome-wide location analysis (P value < 0.001) carried out in cells exposed to hydrogen peroxide (SGA1, GSY1, RPI1, SHC1, PRY3, NDH1, FRE4, GAL7, IDH2, ELO1, ICY1, RSB1, YHR033W, and YGR050C) (15). However, there are at least two possible explanations for this minimal overlap: our study was carried out in unstressed cells, and the binding of Nrg1 to some promoters may not be easily detected by genome-wide location analysis. In accord with this view, the 128 identified targets did not include three genes shown to bind Nrg1 (CYC7, DIT1, and FLO11, which has an upstream region nearly identical to that of STA1; see references 2, 33, and 39).

The relationship of Nrg1/Nrg2 to Msn2/Msn4 is not clear. Many Nrg-repressed genes contain STRE and STRE-like elements. The footprint of Nrg1 in the STA promoter (33) and the binding of CaNrg1 to the STRE (30) suggest that the binding specificity of Nrg1/Nrg2 overlaps that of Msn2/Msn4, and it is possible that Nrg1/Nrg2 compete directly with Msn2/Msn4 for binding to some promoters. The individual roles of Nrg1 and Nrg2 are also unclear. For most of the 150 Nrg-repressed genes, RNA levels in glucose-grown nrg1{Delta} and nrg2{Delta} single mutants were similar to those of the double mutant, indicating that both Nrg1 and Nrg2 are required for full repression under unstressed conditions. Levels of the two proteins are similar in unstressed cells (in both wild types and single mutants; C. D. Berkey, unpublished data), and twofold-reduced levels of total Nrg1/Nrg2 protein may be insufficient for repression at most promoters. In response to stress, the level of NRG1 RNA increases more than that of NRG2 (2, 5, 12); this differential regulation suggests that Nrg1 is particularly important for adaptation to stress.

The phenotypes of mutants lacking Nrg1 and/or Nrg2 support the physiological importance of these repressors in the regulation of stress responses. Previous studies showed that the nrg1{Delta} mutation increases ion tolerance and suppresses the rim101{Delta} defect in alkaline pH tolerance (22) and that the nrg1{Delta} nrg2{Delta} mutant exhibits increased invasive and pseudohyphal growth in response to carbon and nitrogen limitation, respectively (21). Here we have documented increased resistance to salt and oxidative stress, consistent with the increased expression of stress-induced genes in the mutants. Conversely, we showed that the nrg1{Delta} nrg2{Delta} mutant is more sensitive to freezing temperatures, in accord with evidence that the mutant exhibits increased expression of genes that are repressed in response to cold shock.

We have examined the expression of selected Nrg-repressed, stress-induced genes in the nrg1{Delta} nrg2{Delta} mutant during salt stress. In the cases of CTT1, CYC7, RSB1, ENA, XBP1, and SGA1, the RNA was upregulated more strongly in the mutant than in the wild type, indicating that Nrg1/Nrg2 limit the amplitude of induction, presumably by counteracting the function of stress-responsive activators. Based on our initial identification of Nrg-repressed genes by virtue of their increased expression in glucose-grown nrg1{Delta} nrg2{Delta} cells, Nrg1/Nrg2 clearly function to keep some stress-induced genes securely off in the absence of stress. It is also possible that the repressive function of Nrg1/Nrg2 effectively raises the threshold for induction of the stress response.

Nrg1/Nrg2 alter the resistance of cells to various stresses in growth assays, and yet these repressors make only modest contributions to the regulation of the genes we have examined here. Although Nrg1/Nrg2 may play more substantial roles in controlling expression of other genes, or in responses to other stresses, another possibility is that subtle regulatory effects matter to the cell. Stress responses typically involve induction of large numbers of genes, and large energetic costs are associated with such a gene expression program. The contribution of Nrg1/Nrg2 to fine-tuning the regulation of stress-responsive genes may serve to minimize unnecessary energy expenditures and enhance competitiveness in natural environments.


   ACKNOWLEDGMENTS
 
We thank S. Kuchin, S.-P. Hong, and M.-D. Kim for useful discussions and G. Hovel-Miner for initial experiments.

This work was supported by NIH grant GM47259 to M.C.


   FOOTNOTES
 
* Corresponding author. Mailing address: 701 W. 168th Street, HSC922, New York, NY 10032. Phone: (212) 305-6314. Fax: (212) 305-1741. E-mail: mbc1{at}columbia.edu. Back

§ Supplemental material for this article may be found at http://ec.asm.org/. Back

{ddagger} Present address: S. Matsui & Associates, Tokyo 104-0061, Japan. Back


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Eukaryotic Cell, November 2005, p. 1882-1891, Vol. 4, No. 11
1535-9778/05/$08.00+0     doi:10.1128/EC.4.11.1882-1891.2005
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