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
). 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 , set C, rows 1 to 4). Interestingly, almost an equal fraction (28%) of genes were repressed by 3AT in the same experiment (Table , 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 , 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. , 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).
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. 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. ).
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 gcn4Δ strain KNY124 when both were treated with 100 mM 3AT (GCN4/gcn4Δ experiment). The results indicated that 612 genes were expressed at levels ≥2-fold higher in GCN4 than in gcn4Δ cells (Table , GCN4/gcn4Δ). The two-dimensional clustering analysis (Fig. ) revealed that most genes showing higher expression in GCN4 than in gcn4Δ 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 gcn4Δ cells (green bars) and those that were repressed by 3AT in GCN4 cells (Fig. ).
To compare the data obtained in the WT ± 3AT (set C) and GCN4/gcn4Δ experiments in greater detail, the log-ratio scatter plot shown in Fig. A 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 gcn4Δ 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 gcn4Δ cells (Fig. A, 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/gcn4Δ experiment (gray stars in Fig. A). 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.
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 (more ...)
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. 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. B 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. B). This correlation provides additional evidence that the majority of genes induced by 3AT are under Gcn4p control.
Interestingly, the scatter plot in Fig. B 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. B, 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. C 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 gcn4Δ 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 (gcn4Δ ± 3AT experiment). The cluster analysis in Fig. shows that many genes that were induced by 3AT in GCN4 cells also were induced in the gcn4Δ 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 gcn4Δ cells (compare rows 5 and 10). The overlap between these different gene sets is depicted graphically in Fig. A for 613 genes that produced significant data (P ≤ 0.05) in the WT ± 100 mM 3AT (data set C), GCN4/gcn4Δ, and gcn4Δ ± 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/gcn4Δ experiments, indicating a dependence on Gcn4p for maximal induction (Fig. A, sectors A and B). Interestingly, 78 of these genes also were induced by 3AT in the gcn4Δ mutant (sector A in Fig. A), including canonical Gcn4p target genes encoding amino acid biosynthetic enzymes, such as HIS5 and ARG4. As shown in Fig. B, the magnitude of 3AT induction of this latter class of genes was reduced in the gcn4Δ mutant. Hence, many genes displayed a strong, but incomplete, dependence on Gcn4p for induction by 100 mM 3AT.
FIG. 3 A fraction of Gcn4p target genes is induced by 3AT in gcn4Δ 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 (more ...)
The data in Fig. also uncovered a sizable group of genes that were highly induced by 3AT in the gcn4
Δ mutant and showed little or no dependence on Gcn4p for maximal induction in the WT (Fig. , clusters I1, I2, I4, and I6 in row 10). The lack of Gcn4p dependence for these genes can be seen from the GCN4
results shown in Fig. (dull red, black, or green bars in row 5). Genes in this category fall into sector C of the Venn diagram shown in Fig. A, representing roughly half of the genes that are induced by 3AT in gcn4
Δ 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
consistent with the fact that 3AT inhibits catalase activity (30
). Other highly induced Gcn4p-independent genes include BTN2
The results of the gcn4Δ ± 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/gcn4Δ experiments but experienced little or no repression in the gcn4Δ ± 3AT experiment (Fig. , 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. , clusters R2, R5, R6, and R7 in row 10) and displayed minimal Gcn4p dependence for this response in the GCN4/gcn4Δ 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 gcn4Δ 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/gcn4Δ 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 gcn4Δ mutant (sector C in Fig. A). 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 gcn4Δ strain (Fig. , 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. C). 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. , cluster I7).
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 (more ...)
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
, 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
), 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
). Gcn4p can also bind to naturally occurring variants of this sequence (TGATTCA, TGACTCT, TGACTGA, and TGACTAT) found in the ILV2
), 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
) 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/gcn4
Δ 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. , 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. , 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. , 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).
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/gcn4Δ experiment. (more ...) 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. , 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. , 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 gcn2
Δ strain (Fig. , columns B and C), lacking the eIF2α 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. , 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
). 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 gcn1
Δ and gcn20
Δ strains lacking positive effectors required for activation of Gcn2p in amino acid-starved cells (26
). 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.
FIG. 5 MMS induces GCN4-lacZ expression dependent on translational activators of GCN4. The β-galactosidase activity expressed from a GCN4-lacZ fusion was assayed in extracts from untreated (black bars) or MMS-treated (striped bars) cultures of prototrophic (more ...)
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
, RAD26, RAD55
. Thus, transcriptional stimulation of these genes by Gcn4p might be important for efficient repair of MMS-induced DNA damage. However, we found that gcn4
Δ and gcn2
Δ 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.
(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 α-ketoglutarate and ammonia by the isozymes encoded by GDH1
or from α-ketoglutarate and glutamine by glutamate synthase (Glt1p) (64
). In our experiments, GLT1
was weakly induced by Gcn4p, whereas GDH1
were repressed, and GDH3
showed little response to 3AT (Fig. A, Glu and Gln).
Gln3p is a transcriptional activator of GLN1
), and many other genes involved in utilization of poor nitrogen sources (64
). Interestingly, GLN3
was found to be a Gcn4p target (Fig. A), 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. A, 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
because Ure2p would be inactive (64
Glutamate biosynthesis requires α-ketoglutarate as the precursor. Citrate is converted to α-ketoglutarate by the sequential action of the tricarboxylic acid (TCA) cycle enzymes aconitase and NAD-dependent isocitrate dehydrogenase, encoded by ACO1
, respectively. Expression of ACO1
was moderately induced by 3AT, but independently of Gcn4p, and IDH1
was not judged to be a Gcn4p target (Fig. C, α-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
can functionally substitute for the IDH1
), and IDP1
was found to be a Gcn4p target. Similarly, ACO2/YJL200C,
encoding an isozyme of aconitase, is a Gcn4p target (Fig. C, α-KGA). Two of the three genes encoding citrate synthase, CIT2
(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 gcn4
Δ strains, its expression was induced ~2-fold in the GCN4c/GCN4
experiment (Fig. C, Citrate). IDP1
, ACO2, CIT2,
elements in their 5′ noncoding sequences, consistent with direct activation by Gcn4p. We conclude that Gcn4p-mediated induction of IDP1
may play an important role in stimulating α-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. A, 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,
) are Gcn4p targets and that most were strongly induced by 3AT (Fig. A, Arg).
Lysine is synthesized from α-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. A, Lys). LYS14,
encoding a pathway-specific activator (24
), was found to be a Gcn4p target (Fig. A, 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
) or functional (LYS4
, -9, -20,
) variants of UASGCRE
occur in the promoters of all LYS
genes except LYS5
(iii) Aromatic family: Trp, Phe, and Tyr.
The aromatic amino acids tryptophan, tyrosine, and phenylalanine are synthesized from chorismate (51
). As shown previously (21
), 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
, 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. A and B, Aro). As noted previously (94
was not induced by Gcn4p. Although expression of TRP1
could not be ascertained (see legend to Fig. ), there is previous evidence that its expression is not induced by Gcn4p (5
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. B, 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
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
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 gcn4
Δ strain (Fig. B, 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
. Our data show that SER1
is a Gcn4p target, in agreement with previous results (68
), as are SER3
, whereas SER2
was repressed by 3AT (Fig. B, Ser). As SER2
contains two consensus UASGCRE
s 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
belongs to the category of Gcn4p-dependent genes that are not highly induced by 3AT but show reduced expression in starved gcn4
Δ cells (Fig. B, 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,
), 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,
(encoding the cytoplasmic SHMT) were all induced by 3AT, and of these genes, ACO2
, ICL1, YFL030W,
were judged to be Gcn4p targets (Fig. B, Ser/Gly). The presence of a Gcn4p site(s) upstream of GCV1, GCV2, GCV3
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
). Surprisingly, CYS3
were either repressed or unaffected by 3AT (Fig. B, 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. B, Asp). Both genes contain consensus Gcn4p sites in their promoters. Asparagine is synthesized from aspartate by asparagine synthetase, encoded by ASN1
. In accordance with previous findings (15
), both genes were found to be Gcn4p targets (Fig. B, Asn) and to contain Gcn4p sites. Threonine is synthesized from aspartate by the products of HOM3
. In keeping with published findings (36
), HOM3, HOM2
were found to be Gcn4p targets (Fig. B, Thr), and they contain several UASGCRE
s in their promoters. AAT2
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. B).
The methionine biosynthetic genes have been well characterized in yeast (for a review, see reference 98
). Previous studies have shown that Gcn4p regulates MET16
), encoding two enzymes in the pathway, and MET4
), encoding a transcriptional activator of the MET
genes. There were also previous indications that Gcn4p induces MET3
, and MET6
, although only in methionine-starved cells (36
). Our findings confirmed that MET16, MET17, MET3,
are Gcn4p targets (Fig. C, 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,
encoding other Met pathway enzymes, and MET28,
encoding a transcriptional activator of the pathway, are Gcn4p targets, as are SUL1
, encoding high-affinity sulfate transporters. The transcriptional activators MET31
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. C, 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
expression, it may indirectly activate MET
genes by stimulating these pathway-specific activators. Additionally, Gcn4p can activate MET16
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
, encoding AdoMet synthetase, were repressed two- to fivefold by 3AT (Fig. C, 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
. Our data show that all of these genes, excluding ILV5,
are Gcn4p targets (Leu, Ile, and Val in Fig. C). These results confirm previous findings (36
), except for ILV3
, 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. ). 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. C). 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
. As LEU3
is induced by Gcn4p (109a) (Fig. C), the activation of LEU4
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
, 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
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. C, Ala).
Purine-pyrimidine biosynthetic enzymes.
The purine biosynthetic genes ADE1, ADE2, ADE3, ADE4, ADE8
were induced by 3AT, and Gcn4p contributed to this response at ADE1, ADE3, ADE8
. Previously, it was reported that ADE4
transcription was induced in a mutant strain containing high constitutive levels of Gcn4p (72
) and that ADE1
, -5, -7,
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
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.
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
]) and it also binds to TGACTC elements (14
). 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
), and SHM2
), 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
, 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
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
, encoding the latter enzyme, and YEL029C
, whose product has ~38% identity to human pyridoxine kinase, were both identified as Gcn4p targets (Fig. A, Pdx). It was shown recently that fungal proteins highly related to yeast Snz1p (and perhaps Sno1p) are involved in pyridoxine (vitamin B6
) biosynthesis (22
), although an enzymatic activity has not been ascribed to them. 3AT treatment led to 20- to 50-fold induction of the SNZ1
pair, which was completely Gcn4p dependent (Fig. A, 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
, only SNZ1
transcription was induced by Gcn4p (Fig. A, Pdx), and consistently, only the SNZ1
promoter has consensus Gcn4p sites.
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 (more ...)
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
, the two other predicted genes in this pathway (56
), were all identified as Gcn4p targets in our experiments (Fig. A, Ntm). BIO3
whose products are involved in biotin synthesis, also were found to be Gcn4p targets, although BIO2
was not (Fig. A, 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. A, 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. A, 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
genes. Our results showed that transcription of RIB1
is Gcn4p dependent (Fig. A, Rib). Although THI6
, encoding an enzyme in thiamine biosynthesis, was induced by 3AT, it was only weakly Gcn4p dependent (Fig. A, Thi).
In response to starvation for nitrogen, carbon, sulfate, phosphate, or amino acids, cytosolic proteins are targeted to the vacuole for bulk degradation in membrane-bound autophagosomes, in a process known as autophagy (55
). Our analysis revealed that APG1
are Gcn4p targets; however, APG14
also was induced by 3AT in the gcn4
Δ strain (Fig. C, Apg). Thus, APG14
shows Gcn4p-dependent and -independent induction in response to histidine starvation. APG16
was induced by 3AT independently of Gcn4p. A consensus Gcn4p site is present upstream of APG13
, a degenerate site (TGACTT) occurs at APG1,
and no Gcn4p site is evident at APG14.
Consistent with transcriptional induction of APG genes by Gcn4p, 3AT treatment induced dense bodies exhibiting Brownian motion inside the vacuoles, known as autophagic vesicles (Fig. , GCN4, SD versus 3AT). However, gcn4Δ cells also produced autophagic vesicles (Fig. ), suggesting that Gcn4p is not critically required for the response. We found that apg1Δ, apg13Δ, and apg14Δ strains were not impaired for growth in the presence of 3AT or sulfometuron methyl (SM), an inhibitor of branched-chain amino acid biosynthesis (data not shown), indicating that autophagy is not required for growth of GCN4 cells under amino acid starvation conditions where cell division is still occurring. Presumably, the salvage of amino acids from proteins by autophagy would lessen the impact of more severe amino acid starvation conditions than were imposed here. LAP4, encoding a vacuolar aminopeptidase, and AAP1, encoding alanine/arginine aminopeptidase, were found to be Gcn4p targets. The induction of these vacuolar proteases may accelerate the degradation of proteins transported to the vacuole by the autophagy pathway.
FIG. 8 Autophagy is induced by amino acid starvation. Strains KNY201 (GCN4) and KNY202 (gcn4Δ) were cultured in YPD medium to an OD600 of 1.0, harvested, washed with minimal (SD) medium, resuspended in SD medium or in SD medium containing 40 mM 3AT (3AT), (more ...)