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Molecular and Cellular Biology, July 2001, p. 4347-4368, Vol. 21, No. 13
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
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 gcn4 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 2 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 gcn4 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.
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
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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 gcn4 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
(eIF2) 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).
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
View larger version (57K):
[in a new window]
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
gcn4 mutant and showed strong Gcn4p dependence in the
GCN4/gcn4 experiment. Gene clusters R2, R5, R6, and
R7 were repressed by 3AT in both WT and gcn4 strains
and showed no Gcn4p dependence in the GCN4/gcn4
experiment. Clusters I1, I2, I4, and I6 depict genes that were induced
in both WT and gcn4 strains and showed no Gcn4p
dependence in the GCN4/gcn4 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 gcn4
strain and displayed Gcn4p dependence in the
GCN4/gcn4 and
GCN4c/GCN4 experiments.
Cluster I7 was repressed in the gcn4 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/gcn4 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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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
-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 (gcn1), and H2512
(gcn20) 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 -galactosidase activity as described above.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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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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strain
KNY124 when both were treated with 100 mM 3AT
(GCN4/gcn4 experiment). The results indicated
that 612 genes were expressed at levels 2-fold higher in
GCN4 than in gcn4 cells (Table 2,
GCN4/gcn4). The two-dimensional clustering analysis (Fig.
1) revealed that most genes showing higher expression in
GCN4 than in gcn4 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 gcn4 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/gcn4 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
gcn4 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 gcn4 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/gcn4 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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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 gcn4 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
(gcn4 ± 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 gcn4
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 gcn4 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/gcn4, and
gcn4 ± 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/gcn4 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 gcn4 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 gcn4
mutant. Hence, many genes displayed a strong, but incomplete, dependence on Gcn4p for induction by 100 mM 3AT.
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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 gcn4 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 gcn4 ± 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/gcn4 experiments but experienced little
or no repression in the gcn4 ± 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/gcn4 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 gcn4 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/gcn4 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 gcn4 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 gcn4 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/gcn4
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).
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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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.
strain (Fig. 5, columns B and C), lacking the eIF2 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
gcn1 and gcn20 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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and gcn2 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/gcn4 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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(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 -ketoglutarate and ammonia by the isozymes encoded by
GDH1 and GDH3 or from -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).
-ketoglutarate as the precursor.
Citrate is converted to -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, -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, -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
gcn4 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 -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 -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 gcn4 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 gcn4 cells (Fig.
6B, Gly).
(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) at272 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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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
-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 that Gcn4p induces transcription of 10 members of this family: ARG11/ORT1, YHM1, OAC1, YMC1, YMC2, CRC1, YER053C, YOR222W, YPR021C, and YPR128C (Fig. 7B, MCF; also data not shown). Transcription of two others, CTP1 and YFR045W, was induced by 3AT, but their Gcn4p dependence is unclear. The fact that a mutation in ARG11/ORT1 produced a leaky arginine auxotrophy (13) established the importance of this MCF protein in arginine biosynthesis. OAC1 encodes the mitochondrial transporter for oxaloacetate and sulfate (81). Given that oxaloacetate is the immediate precursor of aspartate, export of oxaloacetate (a TCA cycle intermediate) from mitochondria by Oac1p may be important for efficient aspartate synthesis by Aat2p in the cytosol. If enzymes of the Met pathway responsible for sulfate assimilation are mitochondrial, then transport of sulfate into this organelle by Oac1p could also facilitate Met biosynthesis.
Autophagy genes.
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, 76).
Our analysis revealed that APG1, APG13, and
APG14 are Gcn4p targets; however, APG14 also was
induced by 3AT in the gcn4 strain (Fig. 7C, 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.
cells also produced autophagic vesicles
(Fig. 8), suggesting that Gcn4p is not critically required for the
response. We found that apg1, apg13, and
apg14 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.
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Amino acid transporters. Yeast cells transport external amino acids through general and specific amino acid permeases. It might be expected that expression of these genes would be induced by Gcn4p to increase amino acid uptake under starvation conditions. Indeed, the genes encoding the general amino acid permease (GAP1), a basic amino acid permease (CAN1), leucine permease (BAP2), and the broad substrate permease (AGP1) were strongly induced by 3AT in a Gcn4p-dependent manner (Fig. 7C, Tsp). Additionally, MUP3, encoding a low-affinity Met permease, and LYP1 (data not shown), encoding a lysine permease, were identified as Gcn4p targets. Expression of several amino acid-specific permeases was repressed or remained unchanged in response to 3AT. Presumably, Gcn4p-mediated activation of the GAP1 and AGP1 general permeases would stimulate increased transport of most amino acids under starvation conditions. VHT1, a member of the allantoate permease family involved in biotin uptake, and YGL186C (data not shown), a member of the purine/cytosine permease family, also showed Gcn4p-dependent induction. Thus, uptake of vitamins, purines, and pyrimidines may all be induced in response to amino acid limitation.
Activation of genes encoding transcription factors. We observed induction and repression of numerous transcription factors when cells were cultured in 3AT medium. Of the 159 known or predicted DNA-binding transcription factors in the MIPS functional catalog (69), 26 were identified as Gcn4p targets. These include regulators of amino acid and purine biosynthesis (ARG80, LEU3, LYS14, MET4, MET28, BAS1, and GLN3), TCA cycle intermediates (RTG3), and peroxisome biogenesis (PIP2), most of which are discussed above. Additional Gcn4p targets included transcriptional activators involved in utilization of poor nitrogen sources (GAT1 and UGA3), catabolism of maltose (MAL13), heat shock response (HSF1), copper homeostasis (CUP9), meiosis (RIM101), and Ty transcription (TEA1). For most of the latter, known target genes were not induced in a Gcn4p-dependent manner. Perhaps the influence of Gcn4p would be observed only under the physiological conditions that normally stimulate these activators and their target genes, e.g., by heat shocking amino acid-starved cells.
Regulation of protein kinases and phosphatases by Gcn4p. Eleven genes in the MIPS protein kinase group were found to be Gcn4p targets, including APG1 (discussed above), DBF20, STE11, and NPR1. In nutrient-poor medium, Npr1p is active and promotes Gap1p stabilization and degradation of the tryptophan permease Tat2p (95). Perhaps induction of Npr1p by Gcn4p can augment the induction of GAP1 transcription by Gcn4p. Consistently, TAT2 expression was repressed by 3AT. Two of the three genes encoding PKA catalytic subunits, TPK1/SRA3 and TPK2, were also identified as Gcn4p targets. It was shown recently that a tpk1 mutant has reduced expression of the Gcn4p target genes BAT1 and ILV5 in YPD medium (87). Additionally, several other genes that are dependent on TPK1 or TPK2 for high-level expression (87) were identified as Gcn4p targets. Thus, Gcn4p may increase PKA expression, which in turn can promote increased expression of a subset of the genes induced in a Gcn4p-dependent manner.
Several regulatory subunits of protein phosphatases were identified as Gcn4p targets, including GIP1, PTP1, and SAP4. The essential type 1 phosphatase Glc7p has multiple roles, including stimulation of glycogen accumulation, and it was implicated previously as a negative regulator of general amino acid control (105). As Gip1p interacts with Glc7p (85), the induction of GIP1 transcription by Gcn4p could influence the negative effect of Glc7p on the general control response.Role for Gcn4p in glycogen metabolism. Forty-five genes encoding enzymes or regulatory proteins involved in energy generation (69) were identified as Gcn4p targets. These included GLG1, GSY1, GSY2, and GLC3, involved in the biosynthesis of the polysaccharide glycogen. Glycogen accumulates under various nutrient starvation conditions or with decreasing growth rates (25, 60), leading to the notion that it serves as a storage carbohydrate and is used as an energy source when cells resume rapid growth. Interestingly, GPH1 and GDB1/YPR184W (encoding glycogen phosphorylase and glycogen debranching enzyme, respectively), involved in the breakdown of glycogen, also were induced by Gcn4p in 3AT medium. Thus, Gcn4p seems to induce the enzymatic capacity to maintain glycogen homeostasis.
We used a semiquantitative assay based on iodine staining of intact cells to monitor glycogen levels (9) under amino acid starvation conditions. WT or gcn4 strains were grown to
saturation in minimal (SD) medium and resuspended at the original cell
density in fresh SD medium or in SD medium containing SM to impose
branched-chain amino acid starvation. After 2 or 4 h of
incubation, in which less than one cell doubling occurred, aliquots of
cells were removed and assayed for glycogen by iodine staining. As
expected, the stationary-phase cells prior to resuspension in fresh
medium stained intense brown, indicating high glycogen content (Fig.
9, 0 h). Following incubation of the
GCN4 cells in fresh SD medium, the glycogen levels decreased
in the presence or absence of SM; however, glycogen levels were
consistently higher in the gcn4 strain treated with SM
(Fig. 9, 4 h). These results suggest that Gcn4p prevents accumulation of glycogen under amino acid starvation conditions. Presumably, inducing the catabolic enzymes encoded by GPH1
and GDB1/YPR184W overrides the simultaneous induction of
glycogen anabolic enzymes by Gcn4p. We speculate that the glucose
released by glycogen breakdown during amino acid starvation would
stimulate amino acid precursor biosynthesis via glycolysis and early
steps of the TCA cycle. In contrast to our results obtained with amino acid-starved cells, it was reported previously that glucose deprivation leads to glycogen accumulation dependent on Gcn2p (108).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Gcn4p is a master regulator of gene expression.
We used
whole-genome expression profiling to investigate the cellular response
to amino acid starvation, focusing on the role of the transcriptional
activator Gcn4p. Combining data from multiple experiments, we found
that histidine starvation by treatment with 100 mM 3AT led to a global
alteration in gene expression, with 23% of the genome surveyed (1,400 out of 6,038 genes) being induced and 23% of the genes being repressed
by a factor of 2 or more. By comparing the expression profiles among
isogenic GCN4, GCN4c, and
gcn4 strains, we determined that 3AT induction of about 60% of the 897 genes that yielded significant data in multiple experiments was dependent on Gcn4p for maximal induction. These 539 genes were designated Gcn4p targets.
|
cells
accumulated higher levels of glycogen than did WT cells under amino
acid starvation conditions, suggesting that Gcn4p induction produces a
net breakdown of glycogen.
A large number of genes encoding regulatory proteins were identified as
Gcn4p targets, including genes encoding transcription factors (26 genes), protein kinases (11 genes), and protein phosphatase regulatory
subunits (4 genes) (Fig. 10). Thus, induction of Gcn4p may elicit a
cascade of regulatory events leading to gene activation or repression
and the mobilization of various signal transduction pathways. It is
possible that these secondary regulatory responses require an
additional environmental stimulus, in which case Gcn4p would serve to
intensify (or dampen) the response when there is also amino acid limitation.
Genes whose transcription is regulated directly by Gcn4p should possess
one or more Gcn4p binding sites, UASGCREs. We
found that 235 of the 539 Gcn4p target genes contain a predicted Gcn4p binding site between positions 20 and 600 relative to the start codon. The remaining Gcn4p targets may contain Gcn4p sites in the
coding region or 3' untranslated region, or they may be regulated indirectly through induction of other transcription factors by Gcn4p.
The latter explanation is consistent with the fact that a much higher
proportion of the canonical Gcn4p targets encoding amino acid
biosynthetic enzymes contain a Gcn4p binding site (~84%), compared
to the occurrence of the UASGCRE among Gcn4p
target genes at large (~44%).
Genes that were induced by Gcn4p were much more likely to contain a
binding site in the 5'-proximal 300 nucleotides than were those whose
maximal repression is Gcn4p dependent. Hence, Gcn4p probably does not
bind to the promoters of genes repressed by 3AT. Rather, we predict
that such genes contain promoter elements that function ineffectively
in amino acid-starved cells. The high level of Gcn4p may squelch a
transcription factor and exacerbate reduced promoter activity at these
genes in starved cells.
It is remarkable that Gcn4p function is required, directly or
indirectly, for activation of at least 539 genes, since microarray studies of the transcriptional response to the activators Gal4p, Yap1p,
Pdr1p/Pdr3p, Mac1p, and Ace1p (10, 18, 19, 29, 63, 89)
yielded only a handful of target genes. Ndt80p was implicated in
activation of about 200 genes, and mutant analysis showed that most of
these genes were only partially dependent on Ndt80p (10);
moreover, Zap1p was implicated in the activation of 111 genes
(63), still far below the number of Gcn4p target genes
identified here.
Induction of Gcn4p target genes by other treatments.
While
this work was in progress, Jia et al. determined the expression profile
of a subset of the yeast genome (1,529 genes) in cells treated with SM,
an inhibitor of branched-chain amino acid biosynthesis
(49). Treatment of yeast cells with SM, like treatment
with 3AT, induces GCN4 translation (36). In
accordance with our findings, SM induced multiple amino acid and
vitamin biosynthetic genes (49). In contrast to 3AT,
however, SM repressed genes encoding enzymes of methionine and purine
biosynthesis and sulfur assimilation pathways. Additionally, SM altered
the expression of fewer genes in a gcn4 mutant
(49) than did 3AT treatment of the gcn4
strain reported here. Additional work is required to understand the
nature of the Gcn4p-independent response to 3AT treatment.
-irradiation, which causes DNA damage, did not induce the Gcn4p target genes (47). Moreover, we found that induction of
Gcn4p by MMS did not require the DNA damage checkpoint proteins Dun1p, Mec1p, and Rad53p. Thus, protein methylation rather than DNA damage may
be the stimulus that triggers increased GCN4 translation in MMS-treated cells. On the other hand, UV irradiation stimulates the
transcriptional activation function of Gcn4p, and possibly GCN4 translation, and gcn4 mutants show a modest
increase in UV sensitivity (23). Hence, certain forms of
DNA damage seem to activate Gcn4p at different levels, and MMS may
stimulate both the activation function of Gcn4p and its synthesis.
A sizable number of Gcn4p target genes were induced by the drug
rapamycin (31). Rapamycin inhibits the TOR1 and TOR2
proteins, which negatively regulate Gln3p function. Thus, rapamycin
induces Gln3p target genes involved in the utilization of poor nitrogen sources (7, 31). Of the 228 genes induced twofold or more by rapamycin (31), we found that 71 (31%) are Gcn4p
targets. These include certain arginine biosynthetic genes, the
putative pyridoxine biosynthetic gene SNO1, and the general
amino acid permease GAP1. However, none of the lysine
biosynthetic genes, all prominent Gcn4p targets, were induced by
rapamycin, indicating that only a fraction of Gcn4p targets are induced
by rapamycin. A recent study indicates that Gcn4p is required for full
induction of a subset of the genes induced by rapamycin in cells
growing on rich medium, including DAL5, DAL1, and
HIS3. Induction of DAL5 and DAL1 by
rapamycin is also dependent on Gln3p. Interestingly, rapamycin induced
GCN4 expression on rich medium to only ~1/10 the level
achieved by 3AT on minimal medium (101). The small amount
of Gcn4p induced by rapamycin may be inadequate for induction of most
Gcn4p target genes, but it may contribute substantially to activation
of promoters containing the UASGCRE that are
bound simultaneously by Gln3p.
Brown and colleagues described a set of about 868 genes that were
regulated in a stereotypical manner in response to diverse stress
conditions, designated the environmental stress response (ESR)
(27). About 586 genes were repressed and 282 genes were induced in the ESR. Ninety-four of the genes induced in the ESR were
identified here as Gcn4p targets, including ARO9,
ARO10, HSP12, HSP78, CTT1,
GTT1, GSY2, APG1, NPR1, and
PKA1. It is possible that Gcn4p contributes to the induction
of these 94 genes under stress conditions besides amino acid starvation
that induce the ESR. This would be akin to the contribution of Gcn4p to
induction of Gcn4p targets by rapamycin discussed above.
Gcn4p-independent response to amino acid starvation.
A sizable
number of the genes induced by 3AT in the WT were not dependent on
Gcn4p for this induction, showing expression ratios close to unity in
the GCN4/gcn4 comparison and high-level induction by 3AT in a gcn4 mutant (Fig. 3A, sectors C and
E). Thus, these genes are induced in histidine-starved cells by a transcriptional mechanism independent of Gcn4p. Interestingly, a number
of Gcn4p targets also were induced by 3AT in the gcn4 mutant. These genes are dependent on Gcn4p for maximal induction by
3AT, showing lower induction ratios in the gcn4 mutant
than in WT cells. Several canonical Gcn4p targets involved in amino acid biosynthesis belong to this group of genes that respond to an
alternative induction pathway in histidine-starved cells, including HIS5, GLN3, ARG4, MET28,
and BIO3 (Fig. 3B). ARO9, ARO10,
YOR391C, and YNR069C are other Gcn4p target genes
with particularly high 3AT induction ratios in the gcn4
mutant. It is possible that the transcriptional machinery itself is
altered to permit increased transcription from certain promoters under
starvation conditions independently of Gcn4p. For example, an altered
form of the SAGA complex containing histone acetyltransferase Gcn5p
accumulates in histidine-starved cells (4). Identification
of the factor(s) mediating this Gcn4p-independent transcriptional
response would shed new light on the regulatory networks that govern
transcriptional control in amino acid-starved cells.
| |
ACKNOWLEDGMENTS |
|---|
We thank Steve Elledge, Orna Cohen-Fix, Ted Weinert, and Yoshinori Ohsumi for yeast strains. We thank Allan Jones and Chris Armour for coordination of the microarray experiments, including the GCN4 constitutive experiment; Tom Dever, Ronda Rolfes, Orna Cohen-Fix, Minerva Garcia, and members of the Hinnebusch laboratory for helpful discussions; and Tom Dever and Klaus Nielsen for critical reading of the manuscript. We also thank Zhixiong Xue for communicating results prior to publication. We thank the Center for Information Technology at NIH for assistance with Excel programming, and we acknowledge the Saccharomyces Genome Database and Yeast Protein Database for access to gene annotations.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address for Alan G. Hinnebusch: Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, Bldg. A, Rm. B1A13, Bethesda, MD 20892. Phone: (301) 496-4480. Fax: (301) 496-6828. E-mail: ahinnebusch{at}nih.gov. Mailing address for Matthew J. Marton: Rosetta Inpharmatics, Kirkland, WA 98034. Phone: (425) 636-6563. Fax: (425) 636-6501. E-mail: mmarton{at}rii.com.
Present address: School of Life Sciences, Jawaharlal Nehru
University, New Delhi 110067, India.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.13.4347-4368.2001
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