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Molecular and Cellular Biology, July 2001, p. 4347-4368, Vol. 21, No. 13
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.13.4347-4368.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Transcriptional Profiling Shows that Gcn4p Is a Master Regulator of Gene Expression during Amino Acid Starvation in Yeast

Krishnamurthy Natarajan,1,dagger Michael R. Meyer,2 Belinda M. Jackson,1 David Slade,2 Christopher Roberts,2 Alan G. Hinnebusch,1,* and Matthew J. Marton2,*

Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, Maryland 20892,1 and Rosetta Inpharmatics, Kirkland, Washington 980342

Received 8 February 2001/Returned for modification 16 March 2001/Accepted 3 April 2001


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Starvation for amino acids induces Gcn4p, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. In an effort to identify all genes regulated by Gcn4p during amino acid starvation, we performed cDNA microarray analysis. Data from 21 pairs of hybridization experiments using two different strains derived from S288c revealed that more than 1,000 genes were induced, and a similar number were repressed, by a factor of 2 or more in response to histidine starvation imposed by 3-aminotriazole (3AT). Profiling of a gcn4Delta strain and a constitutively induced mutant showed that Gcn4p is required for the full induction by 3AT of at least 539 genes, termed Gcn4p targets. Genes in every amino acid biosynthetic pathway except cysteine and genes encoding amino acid precursors, vitamin biosynthetic enzymes, peroxisomal components, mitochondrial carrier proteins, and autophagy proteins were all identified as Gcn4p targets. Unexpectedly, genes involved in amino acid biosynthesis represent only a quarter of the Gcn4p target genes. Gcn4p also activates genes involved in glycogen homeostasis, and mutant analysis showed that Gcn4p suppresses glycogen levels in amino acid-starved cells. Numerous genes encoding protein kinases and transcription factors were identified as targets, suggesting that Gcn4p is a master regulator of gene expression. Interestingly, expression profiles for 3AT and the alkylating agent methyl methanesulfonate (MMS) overlapped extensively, and MMS induced GCN4 translation. Thus, the broad transcriptional response evoked by Gcn4p is produced by diverse stress conditions. Finally, profiling of a gcn4Delta mutant uncovered an alternative induction pathway operating at many Gcn4p target genes in histidine-starved cells.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

In response to environmental perturbations, Saccharomyces cerevisiae cells elicit rapid transcriptional reprogramming involving both activation and repression of gene expression. Transcriptional activator proteins function by binding to specific promoter elements, called upstream activating sequences (UASs) in yeast cells, and recruiting the transcriptional machinery. Thus, transcriptional stimulation requires the expression and function of an activator and the appropriate UAS element in the promoters of its target genes. A plethora of mechanisms are known to regulate the activity or expression of transcriptional activators in response to specific signals. For example, in cells grown on glucose, Gal80p inhibits the ability of Gal4p to activate transcription of genes encoding galactose-metabolizing enzymes, whereas Gal3p alleviates this inhibition on galactose medium (83). The transcriptional activators Pho4p, Swi5p, and Yap1p are regulated by the coupling of their nuclear localization to the levels of inorganic phosphate, cell cycle and mother-daughter status, or oxidative stress, respectively (reviewed in reference 52). Starvation for amino acids, purines, and glucose limitation induces the synthesis of Gcn4p, a bZIP transcriptional activator of amino acid biosynthetic genes in multiple pathways (35, 88, 108). This cross-pathway response is known as general amino acid control. Gcn2p, a translation initiation factor 2alpha (eIF2alpha ) kinase, mediates the derepression of GCN4 mRNA translation in nutrient-starved cells. The activity of Gcn2p is induced by high levels of uncharged tRNA, implying that uncharged tRNA is a critical upstream signal for derepression of GCN4 translation under starvation conditions (37).

Previous studies showed that transcription of at least 35 genes encoding amino acid biosynthetic enzymes is induced by Gcn4p (36). Given that starvation for diverse nutrients induces GCN4 translation, it seemed plausible that Gcn4p would have many other targets besides amino acid biosynthetic genes. Indeed, computational searches of the yeast genome revealed that the Gcn4p binding site, or UASGCRE, is present at the promoters of numerous genes not directly connected with amino acid biosynthesis (data not shown). Additionally, it was shown previously that several adenine biosynthetic genes (51, 72, 88), ATR1 (11), and LPD1 (109) are induced by Gcn4p in amino acid-starved cells. These observations led us to investigate whether the transcriptional activation function of Gcn4p greatly transcends the amino acid biosynthetic genes.

We used whole-genome expression profiling (62, 93) to identify the complete set of genes regulated by Gcn4p. Our results showed that more than 1,000 genes were induced in wild-type (WT) cells in response to starvation for histidine by treatment with 3-aminotriazole (3AT), a competitive inhibitor of His3p. To evaluate the contribution of Gcn4p to this massive regulatory response, we compared the expression profiles of isogenic WT and gcn4Delta strains treated with 3AT. We also compared the expression profiles under nonstarvation conditions of a WT strain and a GCN4c mutant that expresses high levels of Gcn4p constitutively (GCN4c/GCN4 experiment). These comparative profiling experiments indicated that at least 60% of the genes induced by 3AT are dependent on Gcn4p for high-level activation. As expected, these Gcn4p-dependent genes, called Gcn4p targets and numbering 539, encompass a larger number of amino acid biosynthetic genes than previously identified. They also include genes involved in cofactor biosyntheses, organelle biogenesis, mitochondrial transport, autophagy, and glycogen homeostasis. Furthermore, Gcn4p induced genes encoding 11 protein kinases and 26 transcription factors. Interestingly, treatment with the alkylating agent methyl methanesulfonate (MMS) induced GCN4 translation and a large proportion of Gcn4p target genes. Thus, it appears that Gcn4p is a master regulator of gene expression that evokes a broad range of transcriptional and signaling responses under conditions of nutrient limitation and other forms of cell stress.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Supplementary data. The complete data set for all of the experiments analyzed in Fig. 1 is available at http://www.rii.com/tech/pubs/mcb2001.htm. Copies of figures, including Fig. 1 in alternate colors, can be obtained at the above website.


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  		FIG. 1.   Hierarchical two-dimensional clustering analysis of the results from 10 microarray experiments involving 60 hybridizations. The color scale at the bottom represents the log10 ratio of hybridization signals obtained with the experimental samples to signals from control RNA samples, ranging from -1.0 (brightest green, 10-fold repressed) to 1.0 (brightest red, 10-fold induced). The log10 ratios for 2,528 genes with P values of <= 0.05 and at least twofold changes in expression were plotted along the x axis for the different experiments listed along the y axis. Details of experiments 1 to 3, 5, 7, and 10 are given in Table 2. Experiment 4 was conducted independently of experiments 1 and 2 with strain R491, and the data shown are the average expression ratios from one pair of hybridizations. The data from experiments 8 (WT ± 10 mM 3AT) (66) and 9 (WT with or without MMS) (48) were described previously. The data for experiment 6 were obtained by comparing the expression profiles of auxotrophic strain R491 grown with limiting amino acids and the same strain grown with abundant amino acids. The dendrogram on the left depicts the relatedness of expression profiles for the different experiments. Blocks R1, R3, and R4 shown below row 10 depict clusters of genes that were repressed by 3AT in WT but not in a gcn4Delta mutant and showed strong Gcn4p dependence in the GCN4/gcn4Delta experiment. Gene clusters R2, R5, R6, and R7 were repressed by 3AT in both WT and gcn4Delta strains and showed no Gcn4p dependence in the GCN4/gcn4Delta experiment. Clusters I1, I2, I4, and I6 depict genes that were induced in both WT and gcn4Delta strains and showed no Gcn4p dependence in the GCN4/gcn4Delta experiment. Clusters I3 and I5 represent canonical Gcn4p target genes that were induced by 3AT in WT but showed little or no induction in the gcn4Delta strain and displayed Gcn4p dependence in the GCN4/gcn4Delta and GCN4c/GCN4 experiments. Cluster I7 was repressed in the gcn4Delta strain and showed little or no induction in WT treated with 100 mM 3AT but was induced by 10 mM 3AT and displayed strong Gcn4p dependence in the GCN4/gcn4Delta experiment; hence, it was judged to contain Gcn4p target genes. A different version of this figure using alternative colors can be obtained at http://www.rii.com/tech/pubs/mcb2001.htm.

Strains and media. All strains were derived from S288c and are listed in Table 1. Strain R176 was transformed with p238 (74) containing the constitutive GCN4 allele or vector YCp50 to construct strains R4760 and R6257, respectively. For all microarray experiments, cells were cultured in the synthetic complete (SC) medium whose composition is as follows: 1.6 g of yeast nitrogen base without ammonium sulfate and amino acids, 5 g of ammonium sulfate, 11 g of succinic acid, 6.9 g of sodium hydroxide, 1.4 g of a mixture of amino acids (called "C" powder; described below), and 20 g of dextrose per liter. The pH of the medium was adjusted to 5.8 with sodium hydroxide. C powder contains 1 g each of adenine, histidine, methionine, uracil, and arginine; 2.5 g of phenylalanine; 3 g each of lysine and tyrosine; 4 g each of tryptophan, leucine, and isoleucine; 5 g each of glutamic acid and aspartic acid; 7.5 g of valine; 10 g of threonine; and 20 g of serine. Complete (YPD) and minimal (SD) media were described previously (96).

                              
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  		TABLE 1.   Strains used in this study

Growth conditions. Treatment of cells with 100 mM 3AT for 1 h was conducted as described previously (66). For the GCN4c/GCN4 experiment, strains R4760 and R6257 were grown in SC medium lacking uracil to a density of 107 cells/ml, harvested, and lysed as described previously for 3AT treatment (66). To impose a mild amino acid starvation, strain R176, auxotrophic for leucine and histidine, was grown overnight in SC medium to an optical density at 600 nm (OD600) of 0.5 and used to inoculate SC medium containing 0.65 g of C powder (0.5× amino acids) or 1.4 g of C powder (1× amino acids) per liter. Cells were grown to a density of 107 cells/ml, harvested, and lysed as described previously (66).

Generation and analysis of microarray data. Poly(A)+ RNA preparation, cDNA labeling, cDNA microarray production, hybridization, washing, scanning, and image analysis were conducted as described previously (43, 66). Each individual microarray data set was generated by a fluor-reversed set of hybridizations as described previously (66), herein referred to as pairs of hybridizations. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer was performed as described previously (42). Use of an error model to determine P values for the data was described previously (43). The MMS data from the work of Jelinsky and Samson (48) were imported into the Rosetta Resolver system, and data were analyzed using the methods described in reference 44 for handling single-channel data.

GCN4-lacZ induction by MMS. Strain H187 was grown in duplicate in YPD medium to an OD600 of 0.9, MMS (Aldrich) was added to one culture to 0.07% (vol/vol), and both cultures were incubated with shaking at 30°C for 1 h. The MMS treatment reduced cell viability to 31% of that of untreated cultures, as judged by colony formation on YPD medium. Cells were collected by centrifugation and washed with sterile water, and the beta -galactosidase activity was assayed in cell extracts as described previously (71). Strain H1895, bearing GCN4-lacZ integrated in the chromosome, was transformed to uracil prototrophy with p585 (GCN2 CEN4 ARS1), p2201 (gcn2-m2 CEN6 ARSH4), p614 (gcn2-psk CEN4 ARS1), or a vector (URA3 2µm). As expected, p585 complemented the 3AT-sensitive phenotype of H1895 while the other plasmids did not. Strains F113 (WT), H2079 (gcn1Delta ), and H2512 (gcn20Delta ) were transformed with p180 (GCN4-lacZ CEN4 ARS1). The transformants of H1895, F113, H2079, and H2512 were cultured in SC-Ura medium with or without MMS (0.07%) and assayed for beta -galactosidase activity as described above.


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Gcn4p stimulates transcription of a large fraction of the yeast genome. We used cDNA microarray technology to compare the genome-wide expression profiles of a WT strain (KNY164) grown in SC medium and the same strain grown in SC medium containing 100 mM 3AT (WT ± 3AT experiment). Gcn4p is expressed at low levels on SC medium and rapidly induced in response to histidine starvation imposed by 3AT (1, 36). The results showed that, of the 3,940 genes which were measured with a statistical significance (P value) of 0.05 or less, 949 genes (24% of the total) were induced by 3AT by a factor of 2 or more (Table 2, set C, rows 1 to 4). Interestingly, almost an equal fraction (28%) of genes were repressed by 3AT in the same experiment (Table 2, set C, rows 5 to 7). Multiple experiments were carried out under identical growth conditions using a second, nonisogenic GCN4 strain (R491). Whereas 322 genes were highly induced (>= 4-fold) by 3AT in KNY164, a somewhat smaller number of genes were induced to this extent in the second strain (Table 2, sets A and B versus C). This may be attributable to the greater sensitivity of the ink-jet platform used for set C compared with that of cDNA spotted arrays (see reference 42 for a comparison of the two technologies). Nevertheless, large fractions of the genome were induced or repressed by 3AT in both GCN4 strains. A hierarchical two-dimensional clustering analysis of genes showing >= 2-fold changes in gene expression and with a P value of <= 0.05 was conducted to determine whether the same group of genes was induced or repressed by 3AT in all experiments described above. As shown in Fig. 1, the vast majority of genes that were found to be induced (shown in red) or repressed (shown in green) by 3AT using data set C behaved similarly using data sets A, B, and D (compare rows 1 to 4).

                              
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  		TABLE 2.   Summary of expression profiles in different experiments

An additional experiment was carried out using a lower concentration of 3AT (10 mM) to impose less severe histidine starvation on GCN4 strain R491. Additionally, we determined expression profiles of strain R491, auxotrophic for leucine and histidine, in medium containing limiting amounts of the amino acids (0.5×) versus medium replete with amino acids (1×). The two-dimensional clustering analysis in Fig. 1 shows that the majority of genes that were induced or repressed by the mild amino acid limitation imposed in these two experiments (rows 6 and 8) also were induced or repressed, respectively, in the multiple experiments using 100 mM 3AT (rows 1 to 4). As might be expected, the magnitude of induction or repression for most genes was diminished in the last two experiments compared to the 100 mM 3AT experiments (Fig. 1).

To evaluate whether the majority of genes induced by 3AT were dependent on Gcn4p for this response, we compared the expression profiles of GCN4 strain KNY164 and isogenic gcn4Delta strain KNY124 when both were treated with 100 mM 3AT (GCN4/gcn4Delta experiment). The results indicated that 612 genes were expressed at levels >= 2-fold higher in GCN4 than in gcn4Delta cells (Table 2, GCN4/gcn4Delta ). The two-dimensional clustering analysis (Fig. 1) revealed that most genes showing higher expression in GCN4 than in gcn4Delta cells treated with 3AT (red bars) also were induced by 3AT in the GCN4 cells (compare row 5 with rows 1 to 4). The same correlation holds when comparing the genes with lower expression in GCN4 than in gcn4Delta cells (green bars) and those that were repressed by 3AT in GCN4 cells (Fig. 1).

To compare the data obtained in the WT ± 3AT (set C) and GCN4/gcn4Delta experiments in greater detail, the log-ratio scatter plot shown in Fig. 2A was constructed for the 2,372 genes for which statistically significant data (P value of <= 0.05) were obtained in both experiments. Overall, the expression ratios in the two experiments were highly correlated, with a correlation coefficient of 0.81. Closer inspection of the plot revealed that 64% of the 635 genes induced >= 2-fold by 3AT in GCN4 cells also showed >= 2-fold-higher expression in GCN4 than in gcn4Delta cells. The 408 genes with this behavior are enclosed in the box in the upper right quadrant of the plot. Additionally, 64% of the 781 genes repressed by a factor of >= 2 by 3AT in GCN4 cells also showed reduced expression in GCN4 cells compared with that in gcn4Delta cells (Fig. 2A, black stars enclosed in the box in the lower left quadrant). A small fraction of genes that were induced (54 genes) or repressed (47 genes) in the WT ± 3AT experiment were negatively correlated in the GCN4/gcn4Delta experiment (gray stars in Fig. 2A). We conclude that a large fraction of genes induced by 100 mM 3AT are dependent on Gcn4p for maximal expression under these starvation conditions. Furthermore, Gcn4p contributes to the repression of most genes whose expression is reduced by severe histidine limitation.


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  		FIG. 2.   Log10 ratio scatter plots comparing expression profiles from different experiments. The log10 ratios of expression for all genes with P values of <= 0.05 in two experiments being compared were plotted against one another. Black stars depict genes whose expression profiles were correlated, while gray stars depict genes with negatively correlated expression profiles. A trend line was generated for the genes depicted by black stars. Genes induced (log10 ratio >=  +0.30) or repressed (log10 ratio <=  -0.30) twofold or more are enclosed in a box in the upper right or the lower left quadrants, respectively. (A) Comparison of data set C (WT ± 100 mM 3AT) with the GCN4/gcn4Delta data set. (B) Comparison of data set B (WT ± 100 mM 3AT) with the GCN4c/GCN4 data set. (C) Comparison of data set B (WT ± 100 mM 3AT) with the 0.5×/1× amino acid data set.

We used an independent means of identifying Gcn4p-inducible genes by comparing the expression profiles in WT and GCN4c mutant cells. This mutant expresses induced levels of WT Gcn4p under nonstarvation conditions (74). The cluster analysis in Fig. 1 shows that the profile of gene induction associated with constitutive expression of Gcn4p (row 7) most closely resembles that elicited by moderate amino acid starvation (rows 6 and 8). The log-ratio scatter plot in Fig. 2B for the 346 genes represented in both the GCN4c/GCN4 and WT ± 3AT (set B) experiments shows that 68% of the genes induced >= 2-fold by 3AT in WT cells also showed >= 2-fold-higher expression in GCN4c than in GCN4 cells (black stars enclosed in the box in the upper right quadrant in Fig. 2B). This correlation provides additional evidence that the majority of genes induced by 3AT are under Gcn4p control.

Interestingly, the scatter plot in Fig. 2B showed a poor correlation between genes that were repressed by 100 mM 3AT in WT cells (stars below zero in the y axis) and those repressed by constitutive expression of Gcn4p under nonstarvation conditions (stars to the left of zero in the x axis). Expression of only 12% of the genes was reduced in both experiments (Fig. 2B, black stars enclosed in the box in the lower left quadrant). Moreover, the genes that were most highly repressed by 3AT in WT cells (stars with the most negative y coordinates) showed little or no repression in the GCN4c/GCN4 strain. Thus, high-level expression of Gcn4p under nonstarvation conditions in the GCN4c mutant was insufficient to evoke the extensive repression of genes that occurred in WT cells under severe histidine starvation conditions. Similarly, the scatter plot in Fig. 2C shows that relatively few genes were repressed by moderate Leu-His starvation. Hence, the widespread repression of genes observed in the WT ± 3AT experiments seems to require a combination of high-level Gcn4p and severe amino acid limitation.

Finally, we examined a gcn4Delta mutant in the presence or absence of 100 mM 3AT to determine which genes can be induced or repressed by severe histidine starvation in the absence of Gcn4p (gcn4Delta  ± 3AT experiment). The cluster analysis in Fig. 1 shows that many genes that were induced by 3AT in GCN4 cells also were induced in the gcn4Delta mutant (rows 1 to 4 versus row 10). In addition, many such genes were dependent on Gcn4p for maximal induction by 3AT in the WT, showing higher expression in GCN4 than in gcn4Delta cells (compare rows 5 and 10). The overlap between these different gene sets is depicted graphically in Fig. 3A for 613 genes that produced significant data (P <=  0.05) in the WT ± 100 mM 3AT (data set C), GCN4/gcn4Delta , and gcn4Delta  ± 100 mM 3AT experiments and had an induction ratio of 2 or more in one of these experiments. There were 229 genes induced in both the WT ± 100 mM 3AT and the GCN4/gcn4Delta experiments, indicating a dependence on Gcn4p for maximal induction (Fig. 3A, sectors A and B). Interestingly, 78 of these genes also were induced by 3AT in the gcn4Delta mutant (sector A in Fig. 3A), including canonical Gcn4p target genes encoding amino acid biosynthetic enzymes, such as HIS5 and ARG4. As shown in Fig. 3B, the magnitude of 3AT induction of this latter class of genes was reduced in the gcn4Delta mutant. Hence, many genes displayed a strong, but incomplete, dependence on Gcn4p for induction by 100 mM 3AT.


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  		FIG. 3.   A fraction of Gcn4p target genes is induced by 3AT in gcn4Delta cells. (A) Venn diagram depicting the overlap among 613 genes for which statistically significant data were obtained (P <=  0.05) and that showed an induction ratio of >= 2 in the WT ± 3AT (set C), GCN4/gcn4Delta , or gcn4Delta  ± 3AT experiments. There are 229 Gcn4p target genes (sectors A and B) found at the intersection between 462 genes induced by 3AT in the WT (sectors A, B, C, and D) and 311 genes showing Gcn4p dependence for induction in the GCN4/gcn4Delta experiment (sectors F, B, and A). There were 280 genes induced by 3AT in the gcn4Delta  ± 3AT experiment (sectors A, C, and E). The 151 genes in sector B are Gcn4p targets that were not induced by 3AT in the gcn4Delta strain and thus are completely dependent on Gcn4p for induction. Sector A contains the subset of Gcn4p target genes that were also induced by 3AT in the gcn4Delta mutant. These 78 genes are subject to dual regulation and require Gcn4p only for maximal induction by 3AT. Sector C is comprised of the 133 genes induced by 3AT in both gcn4Delta and WT cells which showed little or no dependence on Gcn4p in the GCN4/gcn4Delta experiment (induction ratio of <2-fold). These genes are induced by 3AT independently of Gcn4p. Thus, ~50% of the genes induced by 3AT in the WT are designated Gcn4p targets because of their dependence on Gcn4p for maximal induction ([A + B]/[A + B + C + D]), and 34% of these target genes (A/[A + B]) can be induced by 3AT even in the absence of Gcn4p. The genes in sector F showed greater expression in WT cells than in gcn4Delta cells in the presence of 3AT but were not induced by 3AT in the WT. This class includes additional Gcn4p targets that depend on Gcn4p primarily to prevent repression in histidine-starved cells (see text). The genes in sector E were induced only in gcn4Delta cells and thus belong to the class of genes induced by 3AT independently of Gcn4p. The results obtained for the 100 genes in sector D marked with an asterisk are paradoxical in that any genes induced by 3AT in the WT should be either dependent on (sector A or B) or independent of (sector C) Gcn4p for their induction. Close examination of this group revealed that 28 genes were induced only in the single WT ± 3AT experiment used to construct this diagram (data set C) but not in other experiments of the same type (data set A, B, or D); hence, these 28 genes probably are not 3AT inducible. Fifty of the remaining genes in sector D showed induction ratios between 1.5 and 2.0 in the GCN4/gcn4Delta experiment and most likely belong to sector A or B, comprising the Gcn4p targets. Fifteen of the remaining genes had induction ratios between 1.5 and 2.0 in the gcn4Delta  ± 3AT experiment and probably belong to sector C or E, comprising the Gcn4p-independent 3AT-inducible genes. Among the 133 genes assigned to sector C, 53 had induction ratios between 1.5- and 2-fold in the GCN4/gcn4Delta experiment, suggesting that they belong to sector A: Gcn4p targets that are also inducible by a Gcn4p-independent mechanism. (B) Color display plot of the expression ratios of selected Gcn4p target genes that were induced >= 1.9-fold by 3AT in the gcn4Delta strain. The log10 ratios of expression are depicted using the color code described for Fig. 1, with the brightest red depicting log10 ratios of >= 1.0 and black signifying no significant change in expression (P > 0.05). Data were taken from the different experiments listed across the top (as defined in Fig. 1 and Table 2) for the genes listed on the right.

The data in Fig. 1 also uncovered a sizable group of genes that were highly induced by 3AT in the gcn4Delta mutant and showed little or no dependence on Gcn4p for maximal induction in the WT (Fig. 1, clusters I1, I2, I4, and I6 in row 10). The lack of Gcn4p dependence for these genes can be seen from the GCN4/gcn4 results shown in Fig. 1 (dull red, black, or green bars in row 5). Genes in this category fall into sector C of the Venn diagram shown in Fig. 3A, representing roughly half of the genes that are induced by 3AT in gcn4Delta cells. Either Gcn4p plays no role in their induction, or they can be induced equally well by a Gcn4p-independent mechanism in cells lacking Gcn4p. A number of genes in this group are known to be induced by hydrogen peroxide, including CTA1, CTT1, GTT2, HSP26, HSP42, and HSP78, consistent with the fact that 3AT inhibits catalase activity (30). Other highly induced Gcn4p-independent genes include BTN2, GAL7, IME2, INO1, NRG1, RPN4, and SSA4.

The results of the gcn4Delta  ± 3AT experiment also confirmed that most of the genes that were repressed by 3AT treatment of WT cells were dependent on Gcn4p for maximal repression. Such genes showed a repression ratio of >= 2 in both the WT ± 3AT and the GCN4/gcn4Delta experiments but experienced little or no repression in the gcn4Delta  ± 3AT experiment (Fig. 1, clusters R1, R3, and R4 in rows 1 to 5 and 10). However, a sizable group of genes were also strongly repressed in the gcn4 mutant (Fig. 1, clusters R2, R5, R6, and R7 in row 10) and displayed minimal Gcn4p dependence for this response in the GCN4/gcn4Delta experiment (black or dull red bars, row 5). Included in this category are ALD6, ADH1, ACS2, RPE1, SAM2, and ALG7. It should be noted that treatment of a gcn4Delta mutant with 100 mM 3AT severely impedes growth because high-level induction of histidine biosynthetic enzymes cannot occur in the absence of Gcn4p. Hence, this represents a more extreme starvation condition than that when WT cells were treated with 100 mM 3AT.

Summarizing the results described thus far, expression of 539 genes was induced >= 2-fold in at least one of the WT ± 3AT experiments and also displayed significant Gcn4p dependence for this response, showing an induction ratio of >= 2.0 in the GCN4/gcn4Delta or GCN4c/GCN4 experiments. Henceforth, we refer to this large group of genes as the Gcn4p targets. As noted above, many genes induced by 3AT in the WT were induced equally well, or more strongly, in the gcn4Delta mutant (sector C in Fig. 3A). It is conceivable that some of these genes are Gcn4p targets that can be induced to high levels by histidine starvation through an alternative mechanism in cells lacking Gcn4p. Hence, the number of Gcn4p target genes may have been underestimated by demanding dependence on Gcn4p for induction by 3AT.

Interestingly, a small subset of 29 Gcn4p target genes were not strongly induced in WT cells by 100 mM 3AT but required Gcn4p to maintain high-level expression in starved cells; hence, these genes were repressed by 100 mM 3AT in the gcn4Delta strain (Fig. 1, cluster I7, rows 1 to 4 versus row 10). Among the genes exhibiting this behavior are ILV1, ILV2, LEU1, and BAT1 (see below in Fig. 6C). One way to interpret this behavior is to propose that the promoters of these genes contain regulatory elements that mediate reduced transcription in response to severe histidine starvation and that Gcn4p counteracts this repression. Consistent with this explanation, these genes were induced most effectively under the less extreme starvation conditions of 10 mM 3AT and also by the GCN4c allele in nonstarved cells (Fig. 1, cluster I7).

The microarray results revealed that transcription of GCN4 was not induced by 3AT treatment for 1 h. However a twofold increase in GCN4 mRNA was previously observed at 2 h of 3AT induction (1). Our microarray data also showed that expression of the Gcn4p translational activators, GCN1, GCN3, and GCN20, was not substantially altered, whereas GCN2 expression was increased twofold by 3AT treatment, consistent with an earlier observation (92). Thus, stimulation of GCN4 mRNA translation, via the upstream open reading frames (ORFs) and activation of Gcn2p by uncharged tRNA (reviewed in references 38 and 39), seems to be the predominant mechanism for inducing Gcn4p during the first hour of histidine starvation.

Relationship between Gcn4p-dependent gene expression and occurrence of Gcn4p binding sites in the promoter. If the Gcn4p-induced genes identified above are regulated directly by Gcn4p, they should contain one or more copies of the UASGCRE in their promoters. Previous in vitro studies showed that Gcn4p binds to the TGA(C/G)TCA sequence, with the critical central C · G base pair flanked by TGA half-sites (34, 77). Gcn4p can also bind to naturally occurring variants of this sequence (TGATTCA, TGACTCT, TGACTGA, and TGACTAT) found in the ILV2 and HIS4 promoters (2, 34), and ATGACTCT was found to be a functional UASGCRE in the HIS3 promoter in vivo (34). A computer algorithm used to scan the promoters of several amino acid biosynthetic genes, including known genes under Gcn4p control, predicted a consensus Gcn4p site, RRRWGASTCA (with R = purine, W = T or A, and S = G or C), that closely matched the previous findings (41). Hence, we used a motif search program called CoSMoS (28) to calculate what fraction of the genes with the greatest dependence on Gcn4p for induction by 3AT contained a copy of the sequence TGASTCW or one of the known functional variants in the 5' noncoding DNA. Of the 210 genes showing an induction ratio of >= 4-fold in the GCN4/gcn4Delta experiment, 52% contained one or more copies of the UASGCRE located between 20 and 600 nucleotides upstream from the translation start site. It is possible that Gcn4p-dependent genes which lack a Gcn4p binding site in this interval would contain a functional UASGCRE in the coding region or 3' noncoding sequences. Alternatively, these genes may be induced indirectly by Gcn4p, as numerous transcriptional activators are among the Gcn4p targets (see below).

In a complementary approach, we scanned the complete genome sequence for those genes containing UASGCRE in the 5' noncoding DNA. We found that 7 genes harbored three copies, 78 contained two copies, and 822 had a single UASGCRE in the -600 to -20 interval. Of the genes containing two or more copies of UASGCRE (for which we also obtained sufficient data to analyze their expression and Gcn4p dependence), 64% showed Gcn4p-dependent induction by 3AT in the GCN4/gcn4 experiment, whereas only a single gene in this group showed Gcn4p-dependent repression by 3AT (Fig. 4, column G). For the genes containing only a single UASGCRE located between -20 and -300, ~50% showed Gcn4p-dependent induction while only 6% displayed Gcn4p-dependent repression (Fig. 4, columns A to C). For the remaining genes containing only a single UASGCRE upstream of -300, there was a nearly equal probability of ~22 to 25% that the gene was induced or repressed by 3AT in a Gcn4p-dependent manner (Fig. 4, columns D to F). Thus, genes containing a UASGCRE within 300 nucleotides upstream of the gene are much more likely to be induced than to be repressed by Gcn4p in 3AT-treated cells. We interpret this strong bias to indicate that induced genes that fit these criteria (numbering 149) are activated by direct binding of Gcn4p to the UASGCRE in the promoter. Furthermore, the role of Gcn4p in gene repression is probably indirect (see below).


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  		FIG. 4.   Correlation between Gcn4p-dependent induction by 3AT and the presence of Gcn4p binding sites in the 5' noncoding DNA. The location of the Gcn4p binding site TGASTCW was determined for all genes identified in the GCN4/gcn4Delta experiment. Genes that harbor two or more binding sites between -20 and -600 were assigned to a single group (column G), and those with a single site were divided into separate groups (columns A to F) depending on the location of the site. The gray bars represent the total numbers of genes in each category; the striped bars depict the numbers of genes with induction ratios of >= 2-fold, and the black bars represent the numbers with repression ratios of >= 2-fold, in the GCN4/gcn4Delta experiment.

Ribosomal proteins (RP) are strongly repressed when Gcn4p is highly induced in amino acid-starved cells. About 1,000 genes were repressed by a factor of 2 or more in 3AT-treated cells, and our analysis showed that 66% of these genes showed an unmistakable dependence on Gcn4p for strong repression by 100 mM 3AT. The 90 RPL and RPS genes encoding the RP formed the largest group of genes with a common function that was repressed by 3AT in a Gcn4p-dependent manner. This group also included numerous genes encoding general translation initiation factors. This behavior can be rationalized as a mechanism for coordinating a decrease in ribosome production and protein synthesis with the induction of amino acid biosynthetic capacity under conditions of amino acid limitation. In this sense, it is analogous to the stringent response of Escherichia coli (8). Repression of the RP genes occurs in response to various conditions of starvation or stress (7, 10, 19, 48). What seems unique here is that Gcn4p contributes to the magnitude of the repression under amino acid starvation conditions. Most of the RPL and RPS genes lack the UASGCRE in the promoter; thus, Gcn4p probably contributes indirectly to their repression in histidine-starved cells.

As noted above, high-level Gcn4p expression in the GCN4c mutant did not elicit strong gene repression under nonstarvation conditions, suggesting that amino acid limitation is additionally required. To explain this dual requirement for repression, we propose that promoters of RP genes are down-regulated under amino acid starvation conditions by a mechanism that involves the activator Rap1p (71) and signal transduction by protein kinase A (PKA) (54). Induction of Gcn4p in amino acid-starved cells would intensify the response by sequestering one or more transcription factors required at RP promoters (squelching) (84). Squelching alone by overexpressing Gcn4p in nonstarvation conditions (GCN4c mutant) would be insufficient for strong repression. Moreover, when strain R491 was grown with limiting amounts of the required amino acids, either the induction of Gcn4p was not extensive enough or the starvation was not severe enough to elicit strong repression of RP genes.

Overlap between the induction profiles of MMS and 3AT. It was shown recently that MMS treatment induced the transcription of about 40 genes involved in amino acid metabolism among a total of 1,324 genes induced >= 2-fold (48). We compared the expression profiles during 3AT and MMS treatment and found that, of the 409 genes induced by 3AT, 309 (90%) were also induced by MMS, whereas only 28% of the MMS-induced genes were induced by 3AT (Fig. 1, row 9 versus rows 1 to 4). Based on this comparison, it seemed likely that Gcn4p was responsible for activating a substantial fraction (~28%) of the MMS-induced genes. As the steady-state level of GCN4 mRNA was unchanged by MMS treatment (48), we predicted that MMS would induce GCN4 at the translational level.

In agreement with this prediction, treatment of WT strain H187 on nutrient-rich medium with 0.07% (vol/vol) MMS induced a GCN4-lacZ reporter 7.4-fold (Fig. 5, GCN2, column A). Somewhat lower induction ratios were observed for two other WT strains (GCN2, columns B and F). Because strain H187 is prototrophic for all amino acids, we conclude that MMS does not induce GCN4-lacZ expression indirectly by interfering with amino acid uptake in an auxotroph. Importantly, GCN4-lacZ induction by MMS was completely impaired in the gcn2Delta strain (Fig. 5, columns B and C), lacking the eIF2alpha kinase Gcn2p required for translational induction of GCN4. Uncharged tRNA activates Gcn2p in amino acid-starved cells by binding to a domain related to histidyl-tRNA synthetase (HisRS) located adjacent to the kinase domain (107). As shown in Fig. 5, the MMS induction of GCN4-lacZ was defective in a gcn2-m2 mutant with point mutations in the HisRS-like domain that impair tRNA binding and kinase activity (107, 110). MMS induction of GCN4-lacZ also was absent in the gcn2-psk mutant, bearing a two-codon substitution in a degenerate kinase domain located N-terminal to the authentic kinase domain (106), and in gcn1Delta and gcn20Delta strains lacking positive effectors required for activation of Gcn2p in amino acid-starved cells (26, 65, 103). These results indicate that the same regulatory elements are required for induction of GCN4 translation in response to MMS or 3AT treatment. The fact that MMS induction of GCN4-lacZ required the tRNA-binding activity of Gcn2p could indicate that MMS interferes with aminoacylation of one or more tRNAs and thereby generates the same activating ligand as does amino acid starvation. Alternatively, binding of uncharged tRNA may be an unconditional prerequisite for Gcn2p activation, and methylation of Gcn2p (or an unknown negative regulator of Gcn2p) by MMS could lower the threshold of uncharged tRNA required to activate the kinase.


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  		FIG. 5.   MMS induces GCN4-lacZ expression dependent on translational activators of GCN4. The beta -galactosidase activity expressed from a GCN4-lacZ fusion was assayed in extracts from untreated (black bars) or MMS-treated (striped bars) cultures of prototrophic strain H187 grown in YPD medium (column A). The same fusion was assayed in extracts from gcn2Delta strains harboring plasmids bearing GCN2 (column B), no allele (column C), the gcn2-m2 allele (column D), or the gcn2-psk allele (column E) cultured in SC medium with or without MMS. Finally, GCN4-lacZ was assayed in isogenic WT (column F), gcn1Delta (column G), or gcn20Delta (column H) strains grown in SC medium with or without MMS. Error bars depict the standard errors of the means of activities measured from at least three independent cultures or transformants.

The checkpoint proteins Mec1p, Rad53p, and Dun1p are required for a response to DNA damage (reviewed in reference 104). We found that null mutations in these genes did not impair MMS induction of GCN4-lacZ expression (data not shown). Thus, it appears that Gcn2p is activated in MMS-treated cells independently of the major signal transduction system for responding to DNA damage. Interestingly, certain genes induced by Gcn4p are involved in the repair of DNA damage, including RAD5, RAD14, RAD26, RAD55, ECM32, NTG1, and APN1. Thus, transcriptional stimulation of these genes by Gcn4p might be important for efficient repair of MMS-induced DNA damage. However, we found that gcn4Delta and gcn2Delta mutants were not more sensitive to the toxic effects of MMS than were isogenic WT strains (data not shown). Hence, the induction of Gcn4p target genes is not a critical aspect of the cellular response to DNA damage by MMS under laboratory growth conditions.

Amino acid biosynthetic genes induced by Gcn4p. We used the MIPS functional categories (69) in an effort to assign the 539 Gcn4p target genes to different pathways. There are 119 genes in the MIPS amino acid biosynthesis category, and 78 of them were induced by 3AT in a WT strain. In accordance with previous work (36), 73 were identified here as Gcn4p target genes. All amino acid biosynthetic pathways contain multiple genes induced by Gcn4p in our studies, except for the Cys pathway, which has none. And even for the Cys pathway, genes involved in the biosynthesis of the precursors serine and homocysteine were induced by Gcn4p. As discussed below, there were other instances where genes involved in producing precursors for amino acid biosynthetic pathways were induced by Gcn4p. Taken together, all of these biosynthetic genes account for 77 of the 539 Gcn4p target genes that we identified.

(i) Histidine pathway. The microarray data showed that six of the seven histidine biosynthetic genes were Gcn4p targets (Fig. 6A, His), as expected from previous work (36, 57). HIS2 showed the weakest induction by 3AT and the least dependence on Gcn4p (Fig. 6A), consistent with the absence of a consensus Gcn4p site in the -20 to -600 region. HIS6 was judged not to be a Gcn4p target, based primarily on data from the WT ± 3AT (set C) and GCN4/gcn4Delta experiments (Fig. 6A), despite the presence of a Gcn4p binding site. The magnitude of HIS gene induction by 3AT was not significantly greater than that of many other amino acid biosynthetic pathways (e.g., Arg, Lys, and Met [Fig. 6A and B]), consistent with the absence of histidine-specific transcriptional repression in yeast. Histidine, tryptophan, and adenine biosyntheses require phosphoribosyl pyrophosphate (PRPP) (51), and numerous TRP and ADE genes also are Gcn4p target genes (Fig. 6B and data not shown). However, four of the five PRS genes encoding PRPP synthetase (33) were repressed more than twofold by 3AT, whereas PRS2 expression was not substantially affected (data not shown).


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  		FIG. 6.   Color display plots of the expression ratios of genes involved in amino acid biosynthesis. Genes that are known or predicted to participate in the biosynthesis of amino acids, or amino acid precursors, are indicated on the right, and the log10 ratios of expression for the experiments listed along the top are displayed using the color code described for Fig. 1. (Gray indicates that no data were obtained for that gene.) Genes that were not judged to be Gcn4p targets are shown in parentheses, and those for which insufficient data exist to assess Gcn4p dependence are enclosed in brackets. (Expression of TRP1 and LEU2 could not be assessed because most of the strains that we analyzed have trp1 and leu2 mutations. Although strains R6257 and R4760 used in the GCN4c/GCN4 experiment carry TRP1, statistically significant data were not obtained for TRP1 in that experiment.) Asterisks indicate Gcn4p target genes lacking a recognizable Gcn4p binding site (TGASTCW, TGACTGA, or TGATTCA) in the -20 to -600 region of the promoter. (A) Results for genes in the His, Glu, Gln, Pro, Arg, Lys, or aromatic (Aro) amino acid biosynthetic pathways. (B) Results for genes in the aromatic, Ser, Gly, Cys, Asp, Asn, and Thr biosynthetic pathways. (C) Results for genes in the Met, Leu, Ile, Val, Ala, alpha -ketoglutarate (alpha -KGA), and citrate biosynthetic pathways.

(ii) Glutamate family: Glu/Gln, Arg, Pro, and Lys. Glutamate and glutamine are the key amino group donors in the biosynthesis of amino acids, nucleotides, and other nitrogen-containing compounds (51). Glutamine is produced from glutamate and ammonia in a reaction catalyzed by Gln1p, an enzyme shown previously to be induced by Gcn4p (70). Glutamate can be synthesized from alpha -ketoglutarate and ammonia by the isozymes encoded by GDH1 and GDH3 or from alpha -ketoglutarate and glutamine by glutamate synthase (Glt1p) (64). In our experiments, GLT1 was weakly induced by Gcn4p, whereas GDH1 and GLN1 were repressed, and GDH3 showed little response to 3AT (Fig. 6A, Glu and Gln).

Gln3p is a transcriptional activator of GLN1 (70), GLT1 (102), and many other genes involved in utilization of poor nitrogen sources (64). Interestingly, GLN3 was found to be a Gcn4p target (Fig. 6A), and it contains a consensus UASGCRE in its promoter. Perhaps GLN1 was not induced by 3AT because Gln3p was impaired by its negative regulator Ure2p, owing to the presence of ammonia as a nitrogen source (64). In fact, URE2 was induced by Gcn4p in response to 3AT treatment (Fig. 6A, Glu and Gln). In this event, the modest induction of GLT1 that we observed likely involved Gln3p-independent activation by Gcn4p. Consistently, GLT1 contains a UASGCRE in its promoter. Presumably, induction of GLN3 by Gcn4p in cells starved for glutamine would induce GLN1 and GLT1 because Ure2p would be inactive (64).

Glutamate biosynthesis requires alpha -ketoglutarate as the precursor. Citrate is converted to alpha -ketoglutarate by the sequential action of the tricarboxylic acid (TCA) cycle enzymes aconitase and NAD-dependent isocitrate dehydrogenase, encoded by ACO1 and IDH1/IDH2, respectively. Expression of ACO1 was moderately induced by 3AT, but independently of Gcn4p, and IDH1 was not judged to be a Gcn4p target (Fig. 6C, alpha -KGA). Although IDH2 was induced about twofold by 3AT in the WT, its Gcn4p dependence could not be ascertained. However, the NADP-dependent isocitrate dehydrogenases encoded by IDP1 and IDP2 can functionally substitute for the IDH1 and IDH2 products (32), and IDP1 was found to be a Gcn4p target. Similarly, ACO2/YJL200C, encoding an isozyme of aconitase, is a Gcn4p target (Fig. 6C, alpha -KGA). Two of the three genes encoding citrate synthase, CIT2 and CIT3 (encoding the peroxisomal and mitochondrial isozymes, respectively), were judged to be Gcn4p targets. Although CIT2 was strongly induced by 3AT in both the WT and gcn4Delta strains, its expression was induced ~2-fold in the GCN4c/GCN4 experiment (Fig. 6C, Citrate). IDP1, ACO2, CIT2, and CIT3 contain UASGCRE elements in their 5' noncoding sequences, consistent with direct activation by Gcn4p. We conclude that Gcn4p-mediated induction of IDP1, ACO2, CIT2, and CIT3 may play an important role in stimulating alpha -ketoglutarate synthesis for glutamate production during amino acid starvation on glucose medium.

Arginine and proline are synthesized directly from glutamate (51). Of the three genes involved in proline biosynthesis, only PRO2 was judged to be a Gcn4p target. Surprisingly, PRO1 was repressed by 3AT (Fig. 6A, Pro), consistent with a previous report that PRO1 is not induced by histidine starvation (59). As expected (36), we found that nine of the arginine biosynthetic genes (ARG1, -2, -3, -4, -5, 6, -7, and -8; CPA1; and CPA2) are Gcn4p targets and that most were strongly induced by 3AT (Fig. 6A, Arg).

Lysine is synthesized from alpha -ketoglutarate by a pathway of eight enzymes (51). In agreement with previous findings (36), all eight LYS genes were found to be Gcn4p targets (Fig. 6A, Lys). LYS14, encoding a pathway-specific activator (24), was found to be a Gcn4p target (Fig. 6A, Lys), suggesting that derepression of LYS genes by Gcn4p is mediated partly by induction of Lys14p (at least under lysine starvation conditions). It is probable that Gcn4p also activates LYS genes directly (86) and that consensus (LYS1, -12, and -2) or functional (LYS4, -9, -20, and -21) variants of UASGCRE occur in the promoters of all LYS genes except LYS5 and LYS9.

(iii) Aromatic family: Trp, Phe, and Tyr. The aromatic amino acids tryptophan, tyrosine, and phenylalanine are synthesized from chorismate (51). As shown previously (21, 36, 50), all four genes encoding enzymes required for chorismate synthesis (ARO1, ARO2, ARO3, and ARO4) and three of the four genes encoding enzymes that convert chorismate to tryptophan (TRP2, TRP3, TRP4, and TRP5) were found to be Gcn4p targets. ARO2 belongs to the class of Gcn4p targets that were not strongly induced by 3AT but required Gcn4p to prevent its repression in severely starved cells (Fig. 6A and B, Aro). As noted previously (94), ARO7 was not induced by Gcn4p. Although expression of TRP1 could not be ascertained (see legend to Fig. 6), there is previous evidence that its expression is not induced by Gcn4p (5).

TYR1 and PHA2, whose products carry out the second steps in tyrosine and phenylalanine biosynthesis, respectively, were not judged to be Gcn4p targets, although TYR1 was induced by 3AT independently of Gcn4p (Fig. 6B, Aro). ARO8, encoding aromatic aminotransferase I, which functions in the last step of Tyr and Phe biosynthesis (100), was strongly regulated by Gcn4p, in accordance with previous findings (46).

ARO9 encodes aromatic aminotransferase II, which functions in the first step of tryptophan degradation (46). The ARO10 product may catalyze the second step (decarboxylation) of this pathway, and both ARO9 and ARO10 were induced by tryptophan on medium containing a poor nitrogen source (45). Both genes displayed significant dependence on Gcn4p for their induction by 3AT and also were highly induced in the gcn4Delta strain (Fig. 6B, Aro, and data not shown).

(iv) Serine family: Ser, Gly, and Cys. In addition to their roles in protein synthesis, serine and glycine serve as precursors for the one-carbon units carried by tetrahydrofolate (THF) derivatives. The latter participate as coenzymes in single-carbon transfer reactions in purine and pyrimidine biosynthesis, amino acid metabolism, and methyl group biogenesis. During growth on fermentable carbon sources, the majority of serine is derived from 3-phosphoglycerate, a glycolytic intermediate, by sequential action of SER3/SER33, SER1, and SER2. Our data show that SER1 is a Gcn4p target, in agreement with previous results (68), as are SER3 and -33, whereas SER2 was repressed by 3AT (Fig. 6B, Ser). As SER2 contains two consensus UASGCREs in its promoter, it is possible that Gcn4p can induce this gene in serine-deprived cells but is prevented from doing so in histidine-starved cells by a serine-specific transcriptional repression. Glycine can be produced from serine by serine hydroxymethyltransferase (SHMT); however, the major synthetic route is catalyzed by threonine aldolase encoded by GLY1 (61). GLY1 belongs to the category of Gcn4p-dependent genes that are not highly induced by 3AT but show reduced expression in starved gcn4Delta cells (Fig. 6B, Gly).

An alternative pathway for serine and glycine biosynthesis operates during growth on nonfermentable carbon sources, proceeding from citrate to glycine via glyoxylate in reactions catalyzed by aconitase (encoded by ACO1/2), isocitrate lyase (encoded by ICL1), and alanine-glyoxylate aminotransferase (probably encoded by YFL030W). A portion of the glycine is decarboxylated by glycine decarboxylase (encoded by GCV1, GCV2, GCV3, and LPD1), forming 5,10-methylene THF, which serves as the one-carbon donor for production of serine from glycine by SHMT (51). As indicated above, SHMT can also utilize serine to generate glycine and 5,10-methylene-THF. ACO2, ICL1, YFL030W, GCV1, GCV2, LPD1, and SHM2 (encoding the cytoplasmic SHMT) were all induced by 3AT, and of these genes, ACO2, ICL1, YFL030W, and LPD1 were judged to be Gcn4p targets (Fig. 6B, Ser/Gly). The presence of a Gcn4p site(s) upstream of GCV1, GCV2, GCV3, SHM2, ACO2, ICL1, and LPD1 is consistent with direct activation of these genes by Gcn4p.

It is thought that cysteine is synthesized in yeast exclusively from serine and homocysteine, an intermediate of methionine biosynthesis, by the products of CYS3 and CYS4 (78). Surprisingly, CYS3 and CYS4 were either repressed or unaffected by 3AT (Fig. 6B, Cys). Nevertheless, Gcn4p might stimulate cysteine biosynthesis by induction of the serine and methionine biosynthetic pathways, leading to increased production of the precursors of cysteine.

(v) Aspartate family: Asp, Asn, Thr, and Met. Aspartate is synthesized by transamination of oxaloacetate via glutamate, in a reaction catalyzed by aspartate aminotransferase, encoded by AAT1 (mitochondrial) and AAT2 (cytoplasmic and peroxisomal). The regulation of AAT1 by Gcn4p was equivocal, whereas AAT2 was judged to be a Gcn4p target (Fig. 6B, Asp). Both genes contain consensus Gcn4p sites in their promoters. Asparagine is synthesized from aspartate by asparagine synthetase, encoded by ASN1 and ASN2. In accordance with previous findings (15), both genes were found to be Gcn4p targets (Fig. 6B, Asn) and to contain Gcn4p sites. Threonine is synthesized from aspartate by the products of HOM3, HOM2, HOM6, THR1, and THR4. In keeping with published findings (36), HOM3, HOM2, THR1, and THR4 were found to be Gcn4p targets (Fig. 6B, Thr), and they contain several UASGCREs in their promoters. AAT2, ASN1, ASN2, and THR1 are examples of genes that were not induced by 100 mM 3AT but were dependent on Gcn4p to prevent repression under these starvation conditions (Fig. 6B).

The methionine biosynthetic genes have been well characterized in yeast (for a review, see reference 98). Previous studies have shown that Gcn4p regulates MET16 and MET17 (75), encoding two enzymes in the pathway, and MET4 (73), encoding a transcriptional activator of the MET genes. There were also previous indications that Gcn4p induces MET3, MET14, and MET6, although only in methionine-starved cells (36). Our findings confirmed that MET16, MET17, MET3, and MET14 are Gcn4p targets (Fig. 6C, Met). MET6 belongs to the class of genes dependent on Gcn4p to prevent its repression in 100 mM 3AT. Additionally, we found that MET10, MET1, MET13, MET22, and MET2, encoding other Met pathway enzymes, and MET28, encoding a transcriptional activator of the pathway, are Gcn4p targets, as are SUL1 and SUL2, encoding high-affinity sulfate transporters. The transcriptional activators MET31 and MET32 do not appear to be regulated by Gcn4p; while MET4 was strongly induced by 3AT, it showed little dependence on Gcn4p in this response (Fig. 6C, Met). Although Gcn4p was thought to have a limited role in MET gene expression under methionine-limiting conditions (98), data from our studies and others (75) indicate strong Gcn4p-dependent induction of MET genes in cells starved for histidine or tryptophan.

Because Gcn4p induces Met4 and Met28 expression, it may indirectly activate MET genes by stimulating these pathway-specific activators. Additionally, Gcn4p can activate MET16 and MET17/25 independently of Met4p, suggesting direct activation of these genes by Gcn4p (75). The cellular level of S-adenosylmethionine (AdoMet) is the regulatory signal for methionine abundance, and at high levels of AdoMet, the SCFMet30 complex targets Met4p for degradation and thereby represses MET gene transcription (91). Interestingly, SAM1 and SAM2, encoding AdoMet synthetase, were repressed two- to fivefold by 3AT (Fig. 6C, Met). It is possible, therefore, that repression of AdoMet synthetase in 3AT medium decreases the AdoMet pool and activates MET gene transcription by reducing SCFMet30-mediated degradation of Met4p.

(vi) Pyruvate family: Ile, Val, Leu, and Ala. Isoleucine, valine, and leucine are synthesized from threonine and pyruvate by the sequential action of ILV1, ILV2, ILV6, ILV5, ILV3, BAT1, BAT2, LEU4, LEU1, and LEU2. Our data show that all of these genes, excluding ILV5, are Gcn4p targets (Leu, Ile, and Val in Fig. 6C). These results confirm previous findings (36), except for ILV3 and ILV6, which had not been analyzed in this regard. LEU2 expression was not induced by 3AT, and its Gcn4p dependence could not be established in our strains (see the legend to Fig. 6). However, there is previous evidence that LEU2 is not under general control (40). The expression pattern of ILV5 resembles that of those genes that require Gcn4p only to prevent their repression by 100 mM 3AT. In fact, all of the genes in these pathways exhibit strong Gcn4p dependence but relatively low 3AT induction ratios (Fig. 6C). Hence, they may have promoter elements in common that mediate reduced expression in response to severe amino acid starvation.

Leu3p is a transcriptional activator of all three LEU genes and probably also ILV2 and ILV5. As LEU3 is induced by Gcn4p (109a) (Fig. 6C), the activation of LEU4, ILV2, and ILV5 by Gcn4p could be indirect. LEU4 transcription is also activated directly by Gcn4p (36). Additionally, all of the genes involved in Leu, Ile, or Val biosynthesis, except for BAT2, ILV2, and LEU2, contain a consensus Gcn4p binding site, suggesting a direct role for Gcn4p in their induction. Since the biosynthesis of alanine and threonine, precursors of this pathway, seems to be induced by Gcn4p, this may provide an additional stimulatory effect of Gcn4p on the biosynthesis of Ile, Val, and Leu.

Biosynthesis of alanine is thought to occur by transamination of pyruvate (51). YLR089c and YDR111c have sequence similarity to bacterial alanine aminotransferases and likely encode isozymes of the corresponding yeast enzyme. Whereas YLR089c was judged to be a Gcn4p target gene, data for YDR111c were below the P value threshold, and its dependence on Gcn4p could not be ascertained (Fig. 6C, Ala).

(vii) Aminoacyl-tRNA synthetases. It was shown previously that KRS1/GCD5, ILS1, and MES1 genes, encoding aminoacyl-tRNA synthetases for Lys, Ile, and Met, respectively, were induced by Gcn4p in amino acid-starved cells (36, 58). We found that KRS1 and the genes encoding AspRS (DPS1), ArgRS (YDR341C), and a protein related to ThrRS (YGL219C) are all Gcn4p targets. Presumably, the induced levels of these enzymes lead to higher levels of the corresponding aminoacylated tRNAs under conditions of histidine limitation. By contrast, transcription of the genes encoding GlnRS (GLN4), PheRS (FRS1 and FRS2), and SerRS (SES1) was repressed by 3AT. Thus, regulation of these enzymes is akin to that of other components of the translational machinery. Expression of MES1, ILS1, DED81 (encoding AsnRS), TYS1 (encoding TyrRS), and YHR020W (encoding a protein related to ProRS) was not substantially altered by 3AT, although DED81 probably depends on Gcn4p to prevent its repression in 100 mM 3AT. Thus, the different genes encoding aminoacyl-tRNA synthetases exhibit diverse responses to 3AT and a varying dependence on Gcn4p, which is not currently understood.

Purine-pyrimidine biosynthetic enzymes. The purine biosynthetic genes ADE1, ADE2, ADE3, ADE4, ADE8, ADE12, and ADE17 were induced by 3AT, and Gcn4p contributed to this response at ADE1, ADE3, ADE8, ADE12, and ADE17. Previously, it was reported that ADE4 transcription was induced in a mutant strain containing high constitutive levels of Gcn4p (72) and that ADE1, -2, -4, -5, -7, and -8 were moderately induced by Gcn4p upon 3AT treatment (88). As the purine ring of ATP is partially consumed in histidine biosynthesis, an increase in adenine nucleotide biosynthesis could be viewed as a strategy to support increased histidine biosynthesis. On the other hand, the pathway to AMP consumes PRPP, glycine, aspartate, glutamine, and THF derivatives, and its induction could be viewed as counterproductive under amino acid starvation conditions. It was shown previously that adenine limitation in medium replete with amino acids induces GCN4 mRNA translation and that mutations in GCN4 or its translational activator GCN1 or GCN2 impair cell growth under adenine starvation conditions (88). Thus, the contribution of Gcn4p to ADE gene expression in adenine-starved cells, demonstrable for ADE8 in particular, seems to be required for adequate adenine nucleotide biosynthesis under adenine starvation conditions and may have little to do with amino acid biosynthesis.

The ADE genes have one or more TGACTC elements in their promoters, consistent with a direct role for Gcn4p in activating these genes. However, Bas1p is an activator of multiple ADE genes (except ADE3 [17]) and it also binds to TGACTC elements (14, 99). We identified BAS1 as a Gcn4p target gene, and it contains TGACTG (a weak Gcn4p binding site) at -272 and a consensus Gcn4p site at -1038. Accordingly, the induction of Bas1p by Gcn4p in response to histidine or purine limitation may contribute to the activation of ADE genes under these starvation conditions. As Bas1p additionally activates HIS4 (3, 99), HIS7 (97), and SHM2 and MTD1 transcription (17), Gcn4p-dependent activation of one or more of these genes in 3AT medium could involve a contribution from the induced levels of Bas1p. Since ADE3 expression is independent of Bas1p (14), Gcn4p presumably activates this gene directly in histidine-starved cells.

The genes URA1 through -8, encoding the pyrimidine biosynthetic enzymes, were repressed by 3AT treatment, along with PRP1, encoding a transcriptional activator of the URA genes. This could be viewed as a means of limiting consumption of aspartate, glutamine, and PRPP, precursors of the pyrimidine pathway, under amino acid starvation conditions. Paradoxically, URA10 was highly induced by 3AT in a Gcn4p-dependent manner and contains a single Gcn4p binding site. URA10 contributes about 20% of the orotate phosphoribosyltransferase activity, with the remainder coming from URA5 (16).

Vitamin-cofactor biosynthetic pathways. An unanticipated finding of this study is that numerous vitamin biosynthetic genes are induced during amino acid starvation in a Gcn4p-dependent manner. These genes are required for biosynthesis of biotin, NAD, THF, riboflavin, pyridoxal phosphate, and coenzyme A. Because vitamins function as cofactors for various enzymes of intermediary metabolism, we propose that vitamin biosynthesis is induced by Gcn4p to support increased amino acid production.

Pyridoxal phosphate is synthesized from pyridoxine by the sequential action of pyridoxine kinase and pyridoxine (pyridoxamine) phosphate oxidase (67). PDX3, encoding the latter enzyme, and YEL029C, whose product has ~38% identity to human pyridoxine kinase, were both identified as Gcn4p targets (Fig. 7A, Pdx). It was shown recently that fungal proteins highly related to yeast Snz1p (and perhaps Sno1p) are involved in pyridoxine (vitamin B6) biosynthesis (22, 79), although an enzymatic activity has not been ascribed to them. 3AT treatment led to 20- to 50-fold induction of the SNZ1-SNO1 pair, which was completely Gcn4p dependent (Fig. 7A, Pdx). These two genes are divergently transcribed from a common promoter, and their transcription is induced in late stationary phase (80). Although two other highly related gene pairs occur in yeast, SNZ2-SNO2 and SNZ3-SNO3, only SNZ1-SNO1 transcription was induced by Gcn4p (Fig. 7A, Pdx), and consistently, only the SNZ1-SNO1 promoter has consensus Gcn4p sites.


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  		FIG. 7.   Color display plots of the expression ratios for genes involved in selected pathways that may contribute indirectly to amino acid biosynthesis or accumulation. The log10 ratios of expression for the genes indicated on the right in the experiments listed across the top are displayed using the color code defined for Fig. 1. (A) Results for genes known or suspected to be involved in biosynthesis of pyridoxal phosphate (Pdx), nicotinamide (Ntm), biotin (Bio), THF, riboflavin (Rib), and thiamine (Thi). (B) Results for genes involved in peroxisome (Pex) biogenesis or belonging to the MCF. (C) Results for genes involved in autophagy (Apg) or belonging to the transporter (Tsp) family.

Nicotinamide, derived from tryptophan, is a component of NAD and NADP, two coenzymes involved in dehydrogenase reactions. BNA1/HAD1 (encoding 3-hydroxyanthranilate 3,4-dioxygenase) and YBL098W and YFR047C, the two other predicted genes in this pathway (56), were all identified as Gcn4p targets in our experiments (Fig. 7A, Ntm). BIO3, BIO4, and BIO5, whose products are involved in biotin synthesis, also were found to be Gcn4p targets, although BIO2 was not (Fig. 7A, Bio), despite the presence of a Gcn4p site in its promoter. Biotin is the carrier of carboxyl groups in enzymatic carboxylation reactions. It was reported previously that ornithine transcarbomylase, encoded by ARG3, requires biotin (20). It is unclear which of the many ATP-dependent carboxylation reactions in amino acid biosynthetic pathways require biotin as a carrier.

Interestingly, MTD1 and ADE3, encoding the enzymes catalyzing formation of 5,10-methenyl-THF and 10-formyl-THF (a precursor of adenine biosynthesis) from 5,10-methylene-THF, both were induced by 3AT. ADE3 is clearly a Gcn4p target, whereas the Gcn4p dependence of MTD1 is less certain (Fig. 7A, THF). FOL2, an enzyme involved in folic acid synthesis, also was found to be a Gcn4p target, and it contains a consensus Gcn4p binding site in its promoter. Thus, several enzymes involved in formation of THF derivatives, besides SHMT and glycine decarboxylase, mentioned above, are regulated by Gcn4p. Gcn4p as mentioned above, may induce SHMZ and MTD1 indirectly via induction of Bas1p.

Pantothenate, a component of the acyl group carrier coenzyme A, is synthesized from pantoate. YDR531W, encoding pantothenate kinase, the first committed step in coenzyme A biosynthesis (6), is a Gcn4p target gene (Fig. 7A, CoA). Riboflavin (vitamin B2) is the precursor for flavin mononucleotide and flavin adenine dinucleotide, both of which function as coenzymes in various reactions of intermediary metabolism. Riboflavin is synthesized from GTP by the sequential action of the products of RIB1, RIB7, RIB2, RIB3, RIB4, and RIB5 genes. Our results showed that transcription of RIB1, -3, and -5 is Gcn4p dependent (Fig. 7A, Rib). Although THI6, encoding an enzyme in thiamine biosynthesis, was induced by 3AT, it was only weakly Gcn4p dependent (Fig. 7A, Thi).

Organellar involvement in amino acid biosynthesis. (i) Peroxisomal genes. Another unexpected finding was that Gcn4p activates peroxisomal genes. The yeast peroxisome is the sole site of beta -oxidation for catabolism of fatty acids. Recent evidence indicates that lysine biosynthetic enzymes Lys1p and Lys4p are localized in peroxisomes and that pex8 and pex15 mutants are leaky lysine auxotrophs (28). Thus, lysine biosynthesis occurs, at least partly, in peroxisomes and is dependent on the functions of Pex8p and Pex15p. We found that PEX1, PEX2, PEX5, PEX11, PEX14, PEX21, and PXA2 were induced in 3AT medium and that all but PEX1 and PEX2 were dependent on Gcn4p for this response (Fig. 7B, Pex). The products of these genes function in peroxisome biogenesis or are located in the peroxisomal membrane, and Pxa2p is required for fatty acid transport across the peroxisomal membrane. Interestingly, PIP2, encoding a transcriptional activator of oleate-induced genes (including PEX genes) and peroxisome proliferation (53, 90), was identified as a Gcn4p target. We speculate that Gcn4p induces peroxisome proliferation in amino acid-starved cells as a means of stimulating lysine biosynthesis.

(ii) Mitochondrial carrier proteins. Portions of certain amino acid biosynthetic pathways, including Arg, Lys, Ile, Val, and Leu, take place in the mitochondria; thus, precursors and intermediates in these pathways must be shuttled between the two compartments (51). The yeast genome encodes about 35 members of the mitochondrial carrier family (MCF) involved in small molecule transport between cytosol and mitochondria (82). We found t