From the Institut de Biochimie et Génétique Cellulaires, CNRS UMR 5095, 1 Rue Camille Saint-Saëns, F-33077 Bordeaux Cedex, France
Received for publication, August 30, 2000, and in revised form, October 12, 2000
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ABSTRACT |
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AMP and GMP are synthesized from IMP by specific
conserved pathways. In yeast, whereas IMP and AMP synthesis are
coregulated, we found that the GMP synthesis pathway is specifically
regulated. Transcription of the IMD genes, encoding the
yeast homologs of IMP dehydrogenase, was repressed by extracellular
guanine. Only this first step of GDP synthesis pathway is regulated,
since the latter steps, encoded by the GUA1 and
GUK1 genes, are guanine-insensitive. Use of mutants
affecting GDP metabolism revealed that guanine had to be transformed
into GDP to allow repression of the IMD genes.
IMD gene transcription was also strongly activated by
mycophenolic acid (MPA), a specific inhibitor of IMP dehydrogenase
activity. Serial deletions of the IMD2 gene promoter
revealed the presence of a negative cis-element, required
for guanine regulation. Point mutations in this guanine response
element strongly enhanced IMD2 expression, also making it
insensitive to guanine and MPA. From these data, we propose that the
guanine response element sequence mediates a repression process, which
is enhanced by guanine addition, through GDP or a GDP derivative, and
abolished in the presence of MPA.
Purine nucleotides are involved in many important cellular
processes, and therefore a balanced synthesis of AMP and GMP is required. Cells can synthesize purine nucleotides through the de
novo pathway, a 10-step pathway that produces IMP, which in turn
serves as the common precursor for AMP and GMP nucleotide biosynthesis
(Fig. 1). In yeast, the regulation of the
ADE genes involved in AMP biosynthesis has been
characterized (1, 2). Expression of the ADE genes is
repressed by extracellular adenine and activated by the transcription
factors Bas1p and Bas2p. However, in Saccharomyces
cerevisiae, GMP biosynthesis regulation has not received much
attention and is therefore poorly understood. IMP dehydrogenase
(IMPDH),1 catalyzing the
first step of de novo guanine nucleotide synthesis, has a
key role on growth of many cell types, including lymphocytes and
rapidly proliferating cells (see Ref. 3 for a recent review).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of purine
nucleotides de novo and salvage pathways.
Solid lines represent the plasma membrane. The following
abbreviations are used: Ade, adenine; ext,
extracellular medium; Gua, guanine; Hyp,
hypoxanthine; int, intracellular compartment;
PRPP, 5-phosphoribosyl-1-pyrophosphate. Gene names are
italicized and encode the following enzymatic activities:
AAH1, adenine deaminase; APT1, adenine
phosphoribosyltransferase; FCY2, purine cytosine permease;
GUA1, guanosine-5'-monophosphate synthetase;
GUK1, guanosine-5'-monophosphate kinase; HPT1,
hypoxanthine-guanine phosphoribosyltransferase; IMDs,
inosine-5'-monophosphate dehydrogenases; RNR, ribonucleotide
reductase; YNK1, nucleoside-5'-diphosphate kinase.
GMP synthesis and its regulation appear to play a crucial role in cell proliferation, since an increased level of IMPDH activity has been observed in rapidly proliferating cells (4), including human leukemic cell lines (6-9), solid tumor tissues (10), and B- and T-activated lymphocytes (5). Indeed, substances blocking IMPDH activity, such as mycophenolic acid (MPA), act as immunosuppressive drugs and are used to prevent allograft rejection (11-16; see Ref. 17 for recent reviews). Moreover, it was recently shown that a low level of IMPDH activity is necessary for p53-dependent growth suppression. Indeed, constitutive expression of IMPDH abolishes p53-dependent growth suppression (18), although the exact mechanism is not known.
To assess the molecular mechanisms governing guanylic nucleotide synthesis, we have proceeded to study the regulation of GTP synthesis in Saccharomyces cerevisiae. In this yeast, GMP synthetase, GMP kinase, and NDP kinase, required for catalysis of the latter steps of guanylic nucleotide biosynthesis, are encoded by the GUA1, GUK1, and YNK1 genes, respectively (Fig. 1) (19-21). IMPDH, catalyzing the first committed step in guanylic nucleotide biosynthesis, has been isolated from a variety of eukaryotic and prokaryotic sources and exhibits a high level of amino acid conservation (22-27). Molecular mass, kinetic parameters, and reaction mechanism are also similar, demonstrating a high degree of conservation among species (28-30). By using these features and the complete sequencing of the S. cerevisiae genome, we have identified four yeast homologs for IMPDHs, YAR073w, YHR216w, YLR432w, and YML056c, that we named IMD1, IMD2, IMD3, and IMD4 genes, respectively. These genes encode proteins with more than 80% amino acid identity to each other and that share about 60% amino acid residues with human IMPDHs type I and type II.
In this paper we have studied the regulation of the yeast GMP synthesis
genes, and we found that GMP synthesis is differently regulated from
IMP and AMP synthesis. We show that the IMD genes transcript
levels are impaired by guanine but not by adenine. IMDs
transcriptional expression is strongly activated by MPA. Mutational
analysis of the sequences upstream of IMD2 ATG allowed us to
identify a cis-element (TATAAATA), named guanine response element (GRE), affecting guanine regulation; several
trans-mutations affecting regulation of IMDs are
also presented. We propose that IMD2 regulation by MPA and
guanine takes place through the cis-acting GRE, in response
to intracellular concentrations of GDP or a derivative of GDP.
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EXPERIMENTAL PROCEDURES |
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Yeast Media-- SD is 2% glucose, 0.17% nitrogen base, and 0.5% ammonium sulfate. Adenine or guanine were optionally added to a final concentration of 0.15 and 0.13 mM, respectively. Hydroxyurea (HU) was used at a concentration of 240 mM. SD casa is SD supplemented with 0.2% casamino acids (Difco).
Yeast Strains--
Two sets of isogenic strains were used as
follows: Y350 (MATa; ura3-52; leu2-3, 112;
lys2201), 216 (MATa; ura3-52; leu2-3, 112; lys2
201; hpt1-2), 220 (MATa; ura3-52; leu2-3, 112;
lys2
201, guk1-3), Y349 (MAT
;
ura3-52; leu2-3, 112; lys2
201; his3
200), 119 (MAT
; ura3-52;
leu2-3, 112; lys2
201; his3
200; fcy2-1); and D451-3 (MATa; leu2-3, 112;
ura3-52), D451-3
ynk1 (MATa;
leu2-3, 112; ynk1::URA3).
Plasmids--
The lacZ fusions were constructed in
the vectors described by Myers and coworkers (31). Except for P777, all
of them are URA3 vectors. The IMD1-lacZ fusion
(P677) was constructed as follows: a 389-bp
KpnI-BglII restriction fragment, starting 357 bp
upstream from the ATG initiation codon, was cloned in YEp353 linearized with BamHI and KpnI. For IMD2-lacZ
(P354), a 2321-bp BamHI-HindIII fragment from
P260 (a plasmid carrying the IMD2 gene in YEp13), starting
1186 bp upstream from the ATG initiation codon, was cloned in YEp356
linearized with BamHI and HindIII. A
IMD2-lacZ fusion (P777) was also constructed in a plasmid
carrying the LEU2 selection gene, by cloning the
BamHI-HindIII fragment of P354 in the YEp366 vector, described by Myers and coworkers (31). The IMD3-lacZ fusion was constructed as follows: a 588-bp
EcoRI-EcoRV restriction fragment, starting 555 bp
upstream from the ATG initiation codon, was cloned in YEp356R
linearized with SmaI and EcoRI. For
IMD4-lacZ, a 2573-bp HindIII-EcoRI
restriction fragment, starting 668 bp upstream from the ATG initiation
codon, was cloned in YEp356R linearized with HindIII and
EcoRI. The GUA1-lacZ fusion was constructed by
inserting the BamHI-BglII restriction fragment
from pFL44/T1-BE (19) in YEp358R linearized with BamHI.
Construction of GUK1-lacZ was done as follows: a 1657-bp
EcoRI fragment starting 1486 bp upstream from the ATG
initiation codon was cloned in YEp356R linearized with
EcoRI. lacZ fusion plasmids used for
characterization of the IMD2 gene promoter were constructed
as follows. All the desired IMD2 fragments were obtained by
polymerase chain reaction (PCR) using the appropriate promoter-coding
strand synthetic oligonucleotide and the noncoding strand IMP22
synthetic oligonucleotide. The resulting PCR fragments were digested
with BamHI-HindIII and cloned at the
BamHI-HindIII sites of the YEp356 vector (31).
The plasmids containing 442, 355, 311, 287, 263, 234, 197, 159, or 108 bp of the IMD2 promoter and 1135 bp of its coding sequence
were named P853, P855, P892, P895, P857, P1527, P1528, P860 and P890,
respectively, and were obtained with the oligonucleotide IMP2-442,
IMP2-355, IMP2-311, IMP2-287, IMP2-263, IMP2-234, IMP2-197, IMP2-159,
or IMP2-108, respectively. The plasmids containing triple CCC mutations in the 311 to
287 region of the IMD2 promoter, named
P909, P963, P911, P913, P915, P917, P919, P921, and P923, were obtained
with the oligonucleotide IMP2-C309, IMP2-308, IMP2-C300, IMP2-C297, IMP2-C294, IMP2-C291, or IMP2-C288, respectively. The plasmids containing a single C mutation in the
308 to
301 region of the IMD2 promoter, named P1369, P1370, P1372, P1326, P1328,
P1330, P1332, and P1334, were obtained with the oligonucleotide
IMP2-C1, IMP2-C2, IMP2-C3, IMP2-C4, IMP2-C5, IMP2-C6, IMP2-C7, or
IMP2-C8, respectively. The oligonucleotides used to PCR amplify the
IMD2 fragments from the S288C genomic DNA template were as
follows: IMD2 promoter, coding strand, IMP2-442,
5'-CGGGATCCCGTGTCTGTTCTCTACA-3'; IMP2-355,
5'-CGGGATCCCGAAGAAAGCGGAAAAATAA-3'; IMP2-311,
5'-CGGGATCCAAGTATAAATAGTGAAGAC-3'; IMP2-287,
5'-CGGGATCCCATTTGATATTTGGTA-3'; IMP2-263,
5'-CGGGATCCCGGCTGGAAGTTTTTTGC-3'; IMP2-232,
5'-CGGGATCCGGAGAATCTTGGTGGCTT-3'; IMP2-197,
5'-CGGGATCCTTATACATTTTACCTAGT-3'; IMP2-159,
5'-CGGGATCCCGTATTCTATTCTATTCCT-3'; IMP2-108,
5'-CGGGATCCCAATTTCAATAATACTT-3'; IMP2-C309,
5'-CGGGATCCGTATAAATAGTGAAGACT-3'; IMP2-308,
5'-CGGGATCCCTATAAATAGTGAAGACT-3'; IMP2-C306,
5'-CGGGATCCAACCCTAAATAGTGAAGACTTTTTC-3'; IMP2-C303, 5'-CGGGATCCAAGTACCCATAGTGAAGACTTTTTCC-3'; IMP2-C300,
5'-CGGGATCCAAGTATAACCCGTGAAGACTTTTTCCAT-3'; IMP2-C297,
5'-CGGGATCCAAGTATAAATACCCAAGACTTTTTCCATTTG-3'; IMP2-C294, 5'-CGGGATCCAAGTATAAATAGTGCCCACTTTTTCCATTTGATA-3'; IMP2-C291,
5'-CGGGATCCAAGTATAAATAGTGAAGCCCTTTTCCATTTGATATTTG-3'; IMP2-C288,5'-CGGGATCCAAGTATAAATAGTGAAGACTCCCTCCATTTGATATTTGGT-3'; IMP2-C1, 5'-CGGGATCCAAGCATAAATAGTGAAGACT-3'; IMP2-C2,
5'-CGGGATCCAAGTCTAAATAGTGAAGACT-3'; IMP2-C3,
5'-CGGGATCCAAGTACAAATAGTGAAGACT-3'; IMP2-C4,
5'-CGGGATCCAAGTATCAATAGTGAAGACT-3'; IMP2-C5,
5'-CGGGATCCAAGTATACATAGTGAAGACT-3'; IMP2-C6,
5'-CGGGATCCAAGTATAACTAGTGAAGACT-3'; IMP2-C7,
5'-CGGGATCCAAGTATAAACAGTGAAGACT-3'; IMP2-C8,
5'-CGGGATCCAAGTATAAATCGTGAAGACT-3'; and for the IMD2 open
reading frame, noncoding strand, IMP22, 5'-TCCCCCGGGCCCAAATACCGTA-3'.
-Galactosidase Assays--
For
-galactosidase
assays, cells were grown overnight in SD casa and then diluted in the
same medium supplemented or not with adenine, guanine, HU, or MPA.
After 6 h at 30 °C (or 12 h for HU assays), the
-galactosidase assays were performed on these exponentially growing
cells. Cells were made permeable by the method of Kippert (32) with
N-lauroyl sarcosine. Units of
-galactosidase activity
were calculated by the Equation 1,
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(Eq. 1) |
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(Eq. 2) |
Northern Blots-- Yeast strains Y350 and 220 were grown in SD casa, supplemented or not with 0.13 mM guanine or 0.03 µg/ml mycophenolic acid, to an A600 = 0.5. After centrifugation, glass beads were added to the cell pellet, and total RNA was isolated using either the CLONsep Total RNA Isolation Kit (CLONTECH) (for experiments in Fig. 2 and 7) or the TRI Reagent RNA/DNA/PROTEIN Isolation Reagent (EUROMEDEX) (for experiments in Fig. 8). RNA blots were prepared as described previously (2) and probed with a labeled PCR fragment, specific for the IMD2 gene. After autoradiography, bound probes were removed from the membranes and then probed again with the ACT1 gene, as described previously (2). The oligonucleotides used to PCR-amplify the IMD2 probe from the S288C genomic DNA template are as follows: IMP2coli, 5'-TTTTTGCATGCCGCCATTAGAGACTAC-3', and IMP22, 5'-TCCCCCGGGCCCAAATACCGTA-3'. Radiolabeling of the probes was carried out using the random-priming procedure (Kit Prime-a-Gene labeling system, Promega).
IMD2 mRNA 5'-Mapping--
The transcription start for
IMD2 was determined as follows: 10 µg of total RNAs were
treated with DNase and RNasin for 1 h at 37 °C, followed by a
phenol/chloroform extraction and RNA precipitation. The 5' extremity of
the IMD2 transcript was then mapped with the Roche
Molecular Biochemicals 5'/3'-rapid amplification of cDNA ends kit
using the following primers: SP1-5'-CCCAAGCTTAGGCTAACTTCAGAGG-3', SP2-5'-CCCAAGCTTGCAAAATCGACTAAACCT-3', and
SP3-5'-CCCAAGCCCCCATTGCTTTTGCTACT-3'.
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RESULTS |
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Expression of IMD Genes Is Repressed by Guanine but Not by
Adenine--
To investigate whether GDP biosynthesis genes are
coregulated with the AMP pathway genes, i.e. repressed by
external addition of adenine, fusions between the GDP biosynthesis
genes and the lacZ reporter gene were constructed. The
-galactosidase activity of these fusions was measured in the
presence or absence of adenine. As shown in Fig.
2, expression of all the tested
lacZ fusions was not affected by adenine addition.
Expression of the IMD1-lacZ fusion was hardly detectable. In
fact, IMD1 is thought to be a silent gene, since it carries
a frameshift in its coding sequence, and its transcript was not
detectable (33).
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Expression of the lacZ fusion genes was also tested in the
presence of external guanine. Guanine clearly impaired expression of
the IMD2, -3, and -4 lacZ fusions,
whereas it did not affect expression of GUA1-lacZ and
GUK1-lacZ fusions (Fig. 2A). Thus, only the first
step of GDP synthesis appeared to be regulated by guanine. Expression
of IMD2 was also monitored in the presence or absence of
guanine by Northern blot analysis. Results presented in Fig.
2B and quantification of the blots (data not shown) revealed that the IMD2 transcript was 3-fold less abundant in the
presence of guanine. In this experiment, cross-hybridization with
IMD3 and IMD4 transcripts cannot be totally
excluded, since the IMD2 probe used is 85 and 87% identical
to IMD3 and IMD4, and coding sequences of these
three genes have approximately the same size. However, if this happens,
it further suggests that IMDs are coregulated, in agreement
with -galactosidase results. Because IMD2 is the most
repressed gene, we focused on the regulation of this member of the
IMPDH family.
Guanine Must Be Metabolized to GDP to Repress IMD2 Expression-- Since IMD2 was down-regulated in the presence of extracellular guanine, we investigated the signaling pathway controlling guanine-responsive genes. A IMD2-lacZ fusion was studied in several yeast genetic backgrounds in which guanine utilization is impaired (Fig. 1). The importance of guanine uptake was first analyzed by assaying guanine repression in a strain mutated at the fcy2 locus, encoding purine-cytosine permease. Data presented in Table I show that the fcy2 mutant failed to repress the IMD2 gene expression in response to guanine (compare Y349 to 119), indicating that guanine uptake is strictly required for repression. Once inside the cell, guanine can be metabolized into GMP by the hypoxanthine-guanine phosphoribosyltransferase enzyme, encoded by the HPT1 gene (Fig. 1) (34). Analysis of the IMD2-lacZ fusion expression in a strain mutated at the HPT1 locus (35) showed that the hypoxanthine phosphoribosyltransferase enzyme is necessary to transduce the repression signal (Table I, compare Y350 to 216). GMP can then be phosphorylated to GDP. This step is catalyzed by GMP kinase, encoded by the GUK1 essential gene. Expression of the IMD2-lacZ fusion was assayed in the guk1-3 mutant strain strongly impaired for the GMP kinase activity (36). Results presented in Table I clearly show that regulation by guanine of the IMD2-lacZ gene is completely abolished in this strain (compare Y350 to 220). Thus, guanine must be transformed into GDP to repress IMD genes expression.
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Further metabolism of GDP involves its reduction to dGDP by
ribonucleotide reductase or its phosphorylation to GTP by nucleotide diphosphate kinase. The importance of deoxyribonucleotides in this
transduction pathway can be assessed with HU, a drug that specifically
inhibits ribonucleotide reductase activity (37, 38). In the absence of
HU, guanine addition led to a 5-fold reduction of IMD2-lacZ
expression (data not shown). In the presence of 240 mM HU,
a dose that resulted in 96% of growth inhibition (see "Experimental
Procedures"), guanine provoked a comparable (6-fold) reduction in
IMD2-lacZ fusion expression (data not shown). Thus, we found
that HU did not affect repression by guanine, even at doses that
strongly inhibit cell growth. This result clearly shows that dGDP does
not participate in the transduction pathway of guanine repression.
Expression of the IMD2-lacZ fusion was assayed in a strain
deleted for the nucleotide diphosphate kinase-encoding gene,
YNK1, and in an isogenic wild-type strain (21). In the absence of guanine, activity of IMD2-lacZ in the
YNK1 and ynk1 strains was 241 and 234 -galactosidase units, respectively. Guanine addition
decreased IMD2-lacZ expression to 18 and 30
-galactosidase units, in the YNK1 and
ynk1 strains, respectively. Thus, IMD2 repression
by guanine was not severely affected by ynk1 disruption, meaning that the nucleotide diphosphate kinase encoded by the YNK1 gene is not necessary for the transduction of the
guanine repression signal. Altogether, these results indicate that the signal molecule responsible for IMD2 repression by guanine
could be either GDP itself or a yet unidentified derivative of GDP.
Expression of the IMD2 Gene Is Activated by the Immunosuppressive
Drug MPA--
We then examined the effect of MPA, a potent and
specific inhibitor of eukaryotic IMPDHs and an immunosuppressive drug
(see Introduction) on the expression of the IMD2 gene. We
reasoned that since GDP or a derivative of GDP represses
IMD2 gene expression, MPA inhibition of XMP synthesis should
starve the cells for guanine nucleotides, which should result in
enhanced expression of IMD2. A range of MPA concentrations
was tested for both growth inhibition and expression of the
IMD2-lacZ fusion. Results presented in Fig. 3 show that MPA inhibited growth in a
dose-dependent manner. In parallel, MPA activated the IMD2-lacZ fusion gene
expression, and this response was maximal between 0.03 and 0.3 µg/ml
MPA. Addition of guanine completely reversed both MPA toxicity (data not shown) and its effect on IMD2-lacZ expression (Fig. 3),
thus confirming the high specificity of this drug. A concentration of
0.03 µg/ml MPA fully activated IMD2-lacZ fusion and only
slightly affected growth; therefore, this drug concentration was chosen for further experiments.
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Identification of a GRE in the IMD2 Promoter--
As a first step
toward identification of promoter cis-elements responsible
for IMD2 regulation, we mapped the IMD2
transcription start. 5'-Rapid amplification of cDNA ends mapping
was done on RNA extracted from cells grown in the presence of either
guanine or MPA. In both cases transcription started at the C nucleotide 107 bp upstream the ATG. The IMD2-lacZ plasmid used in the
previous experiments contained 853 bp of 5'-flanking sequence. To map
the 5'-boundary of the regulatory region of the IMD2 gene,
sequential deletions of the 5'-flanking sequence of
IMD2-lacZ were constructed, from 853 to 108 bp upstream from
the IMD2 ATG. Expression and regulation by guanine of these
constructs were assayed in a wild-type strain (Y350).
IMD2-lacZ fusion genes containing 442, 355, and 311 bp
upstream of the start codon (Fig. 4,
plasmids P853, P855, and P892) were repressed by
guanine, as the control IMD2-lacZ plasmid (Fig. 4,
P354). In contrast, an IMD2-lacZ fusion gene containing 287 bp of the 5'-flanking sequence (Fig. 4, P895)
was insensitive to guanine and was expressed at a much higher level than the control fusion. A similar derepression profile was found for
IMD2-lacZ fusion genes containing 263, 234, 197, 159, or 108 bp of the 5'-flanking sequence (Fig. 4, P857, P1527, P1528,
P860, and P890), although they were less expressed than
the 287 fusion gene. These results suggested the presence of a
cis-negative GRE whose 5'-boundary would be between
311
and
287 bp. Positive regulatory cis-elements might also be
present between
287 and
108 bp.
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To investigate more precisely the importance of the region between
311 and
287 bp in the IMD2 promoter, this region was submitted to site-directed mutagenesis. Triplet CCC mutations were
created in the 24 nucleotides spanning the interval
311 to
287.
Constructs carrying the clustered mutations were assayed for their
effects on
-galactosidase expression under both guanine-repressing and -derepressing conditions. The results shown in Fig.
5 indicate that the CCC substitutions in
clusters GTA, TAA, and ATA (plasmids P911, P913, and P915) abolished
guanine regulation and enhanced expression of these fusions, as
compared with the parental construct. On the other hand, all the other
clustered substitutions had no effect, neither on guanine regulation
nor on the expression levels of the IMD2-lacZ gene fusion.
Comparison of data obtained with plasmids P909 and P963 show that the G
to C mutation of the GTA cluster does not affect guanine regulation of
the IMD2 gene. In summary, results from this cluster
mutations experiment demonstrate that changing the octanucleotide
TATAAATA into CCTAAATA, TACCCATA, or TATAACCC completely prevents
guanine regulation of the IMD2 gene. To identify more
precisely the critical nucleotides contained therein, each nucleotide
of the TATAAATA box was mutated to a C. The lacZ fusion
constructs, each containing a C substitution in the octanucleotide and
311 bp of the 5'-flanking sequence of the IMD2 gene, were
tested in guanine-repressing and -derepressing conditions. As shown in
Fig. 6, each single mutation introduced led to a guanine-insensitive derepression of the IMD2-lacZ
expression, thus revealing that all eight nucleotides of the TATAAATA
sequence are required for transcriptional regulation of the
IMD2 gene. The TATAAATA box was named GRE. To ensure that
this regulation occurs at the transcriptional level, we measured the
expression of a wild-type GRE and a mutant GRE IMD2-lacZ
fusion in a wild-type strain grown in the presence or absence of
external guanine by Northern blot analysis. We first confirmed that
regulation by guanine was maintained for the
311 IMD2-lacZ
fusion (Fig. 7). In contrast, an
IMD2-lacZ fusion gene containing a mutant sequence in the
TATAAATA element was expressed at a much higher level, in a
guanine-insensitive manner (Fig. 7). These results confirm that guanine
regulation of IMD2 takes place at the transcriptional level,
and only in the presence of a functional GRE.
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MPA and Guanine Signals for IMD2 Regulation Are Transduced through the Same Pathway-- IMD genes are activated by MPA and repressed by guanine. To find out whether these regulations are transduced through the same pathway, IMD2 regulation by MPA was analyzed in hpt1 and guk1 mutant strains, deficient for transduction of the guanine repression signal. Results presented in Table II and Fig. 8 confirmed that in a wild-type strain, MPA addition enhanced IMD2 expression levels. This effect was reversed by guanine addition, which decreased IMD2 expression level (Fig. 8 and Table II, line 1). In hpt1 or guk1 mutants, guanine addition was unable to repress IMD2 expression or to reverse MPA activation effect (Fig. 8 and Table II, line 1). We conclude that MPA reversion and guanine repression act through the same pathway. In this hypothesis, a mutation of the GRE box should also disable MPA regulation of the IMD2 gene. This was tested by monitoring the effect of MPA on the regulation of an IMD2-lacZ fusion carrying a mutant GRE. Results show that this fusion was expressed at a constant high level, regardless of guanine or MPA presence (Table II, line 2), meaning that IMD2 regulation by guanine or MPA is possible only in presence of the wild-type GRE box. This same fusion, in a hpt1 or guk1 mutant, behaved as in the wild-type strain, making IMD2 insensitive to guanine or MPA addition (Table II, lines 4 and 6).
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DISCUSSION |
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In this report, we show that the IMD genes, encoding
the S. cerevisiae homologs of IMPDH, are transcriptionally
repressed by guanine, which has to be metabolized into GDP or a
derivative of GDP to exert its regulatory role. Expression of
IMD2 is highly enhanced in the presence of MPA. A
cis-negative element, necessary to both guanine and MPA
regulation, was found in the IMD2 gene promoter. A simple
explanation could be that in response to guanylic nucleotides levels, a
repressor could bind to the IMD2 promoter and regulate its
transcription. A model for yeast regulation of the guanylic nucleotide
synthesis genes is presented in Fig.
9.
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Specific Regulation of the Guanylic Nucleotide Pathway by Its End Product-- We showed that expression of the GDP pathway genes is unaffected by adenine; they are also unaffected by bas1 or bas2 mutations,2 i.e. the signal and transcription factors involved in regulation of IMP and AMP synthesis pathways (1, 2). These results establish that AMP and GMP synthesis are differentially regulated in S. cerevisiae.
Mechanisms of purine biosynthesis regulation have been elucidated in two bacteria, Escherichia coli and Bacillus subtilis. In E. coli, all the genes for AMP and GMP synthesis are repressed by the PurR repressor in the presence of the corepressors hypoxanthine and/or guanine (39, 40). In B. subtilis, synthesis of AMP and GMP is repressed independently by adenine and guanine nucleotides, whereas adenine nucleotides regulate transcription initiation (via the PurR repressor) (41), and guanine nucleotides regulate transcription termination by an anti-termination mechanism (42). Therefore, in these bacteria, regulation of both AMP and GMP biosynthesis pathway genes respond to AMP and GMP precursors, either through a common transcription factor as in E. coli or through independent mechanisms as in B. subtilis. The situation is clearly different in yeast where AMP synthesis genes is poorly repressed by guanine (35) and where GMP synthesis genes is not affected by adenine (this study). This differential regulation could be a general feature for eukaryotic organisms since it has been shown that expression of human IMPDH is repressed in the presence of guanosine but not adenosine (43).
Negative Regulation of IMPDH Synthesis by Guanylic Nucleotide
Levels Is Conserved among Eukaryotes--
Extracellular guanine led to
a 3-7-fold decrease of IMD2 transcription, depending on the
experiment. The reasons for such variations of the repression factor,
which occurred both with Northern blot and -galactosidase
experiments, are unclear. In the fcy2, hpt1, or
guk1 mutant strains, guanine failed to repress the
IMD genes, indicating that levels of GDP or a GDP derivative negatively regulate IMD gene expression. Deletion of the
YNK1 gene, encoding NDP kinase, did not affect guanine
repression of IMD genes. Nevertheless, GTP cannot be totally
excluded from the guanine signaling pathway, since the ynk1
disrupted strain retained 10% of GDP to GTP conversion capacity, when
compared with wild-type cells (21). Inhibition of dNDPs biosynthesis by
a severely growth-inhibiting dose of hydroxyurea did not affect guanine
regulation of IMD2 gene. Therefore, whereas guanylic
ribonucleotide levels regulate IMDs expression, guanylic
deoxyribonucleotides apparently do not participate in the guanine
repression transduction pathway.
In the hpt1 or fcy2 mutant strains, IMD-lacZ fusions were expressed at wild-type levels in the absence of guanine, whereas in the guk1 strain, IMD-lacZ fusions were 2-fold more expressed. We interpret this result as follows: whereas the hpt1 or fcy2 mutations affect only preformed purine utilization, the guk1 mutation affects both salvage and de novo GDP synthesis. Consequently, GDP levels might be more limiting in a guk1 mutant than in a hpt1 or fcy2 mutant strains. Consistently, a pur5 mutant, which is allelic to GUK1 (36), was previously shown to affect IMPDH activity (see the Discussion in Ref. 34).
MPA addition resulted in a 7-fold increase of IMD2 transcription, suggesting that blocking XMP synthesis severely depletes guanylic nucleotide supplies and consequently increases IMDs expression. Guanine addition overcomes this effect, since it allows guanine nucleotides supply through the salvage pathway. Accordingly, in the fcy2, hpt1, or guk1 mutant strains, guanine can no longer bypass MPA effect on the IMD genes.
Therefore, a decrease in guanine nucleotide pools (by MPA or the guk1 mutation) causes an increase in the expression of IMD genes, whereas an increase in these pools (by guanine addition) causes a decrease in IMD genes expression.
A similar negative regulation by guanylic nucleotide levels was reported in mammalian cells. Glesne and coworkers (43) have shown that MPA induced a 4-fold increase in the IMPDH mRNA level, whereas guanosine addition decreased it to 20% of the control levels. Moreover, these authors suggested that changes in the levels of guanine ribonucleotide pools, and not deoxyguanine nucleotide pools, are the primary determinant regulating IMPDH gene expression (43). Therefore, regulation of guanylic nucleotide synthesis appears strongly conserved between yeast and mammals.
The GRE cis-Negative Element of IMD2-- Mapping of the regulatory cis-elements present in the promoter of the IMD2 gene allowed us to identify a GRE, localized 308 to 301 pb upstream of the ATG start codon. Single substitutions of each of the GRE nucleotides to a C prevented guanine regulation of the IMD2 gene and led to an increased transcription of IMD2. Surprisingly, the GRE sequence, TATAAATA, is very similar to the TATA box consensus sequence. Moreover, the TATA-binding protein was shown to bind to the TATAAATA sequence in vitro, although with reduced affinity as compared with binding to the TATATAA consensus sequence (44). Nevertheless, since the GRE in IMD2 is a negative element, it is not expected to behave as a regular TATA box. Therefore, even if TATA-binding protein binds in vivo to the GRE sequence, it should have a different function than in the basal transcription initiation complex. Interestingly, the GRE sequence is also found in the IMD3 and IMD4 genes, at about 300 and 200 bp upstream from their respective ATGs, suggesting that IMD3 and IMD4 might also be regulated through a GRE cis-element. Mutations in the GRE sequence lead to an increased expression of IMD2 gene, suggesting that a repressor transcription factor could bind to the GRE box and repress IMD2 expression. Mutations in the TUP1 gene, encoding a corepressor involved in many different repression processes (45-48), did not affect guanine regulation of the IMD genes (data not shown). Also, electrophoretic mobility shift assays attempting to show binding of a trans-acting factor to a wild-type GRE, but not to a mutant GRE, were unsuccessful. This could be due to the low concentration of transcription factors in the crude extract used as a protein source.
In mammals, increased expression of IMPDH is observed in rapidly
proliferating and deregulated cells, suggesting that in normal mammalian cells, IMPDH encoding genes are regulated by a repression mechanism. Moreover, it has recently been shown that in murine cells,
p53 needs to repress IMPDH to exert its normal tumor suppression role
(18). However, it is not yet known whether the p53 factor directly
binds to human IMPDH-encoding genes. Even though S. cerevisiae does not have any p53 homolog, the overall regulation
mechanisms could be conserved between yeast and mammals. Identification
of this factor is now necessary to unravel the mechanisms by which IMD2 transcription is controlled.
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Fink and Watanabe for sending yeast strains. We thank Dr. Fernandes for suggestions on the manuscript.
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FOOTNOTES |
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* This work was supported by a fellowship from Fundação para a Ciência e Tecnologia (to M. E.-H.), by ARC Research Grant 5259, Conseil Régional d'Aquitaine, and CNRS UMR Grant 5095.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 33 5 56 99 90 55; Fax: 33 5 56 99 90 59; E-mail: B. Daignan-Fornier@
ibgc.u-bordeaux2.fr.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M007926200
2 V. Denis and B. Daignan-Fornier, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: IMPDH, IMP dehydrogenase; HU, hydroxyurea; MPA, mycophenolic acid; GRE, guanine response element; PCR, polymerase chain reaction; bp, base pair.
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