EUKARYOTIC CELL, Aug. 2009, p. 1268–1277 Vol. 8, No. 81535-9778/09/$08.00�0 doi:10.1128/EC.00116-09Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Cooperative Regulation of ADE3 Transcription by Gcn4p and Bas1pin Saccharomyces cerevisiae�†

Yoo Jin Joo,1 Jung-Ae Kim,1 Joung Hee Baek,1 Ki Moon Seong,1 Kyung-Duk Han,1 Jae Mahn Song,1Jin Young Choi,2 and Joon Kim1*

Laboratory of Biochemistry, School of Life Sciences & Biotechnology, Korea University, Seoul 136-701, South Korea,1 andDepartment of Computer Science, Korea University, Seoul 136-701, South Korea2

Received 17 April 2009/Accepted 5 June 2009

The one-carbon response regulon is essential for the biosynthesis of nucleic acids as well as several aminoacids. The ADE3 gene is known to encode a crucial one-carbon regulon enzyme, tetrahydrofolate synthase,which is involved in the biosynthesis of purine and the amino acids methionine and glycine. Therefore, themechanism through which ADE3 transcription is regulated appears to be critical for the cross-talk amongthese metabolic pathways. Even so, the direct involvement of ADE3 transcription through gene-specifictranscription factors has not been shown clearly. In this study, the promoter structure of the ADE3 gene wasinvestigated in detail, and a genuine Gcn4p responsive element (GCRE) was confirmed among three putativeGCRE elements in vivo and in vitro. Through gene deletion studies of Gcn4p and Bas1p, it was established thatboth factors are involved in the transcriptional regulation of the ADE3 gene. Direct binding to this GCRE andthe occupancy of the ADE3 promoter by these factors were also confirmed. Taking these results together, weconcluded that Gcn4p is responsible for the basal and inducible expression of the ADE3 gene, while Bas1p isrequired for its basal expression.

One-carbon metabolism, using folate as a coenzyme, is im-portant for methyl group biosynthesis in yeast (3, 27). Thisfolate-dependent metabolic pathway is required for the bio-genesis of various amino acids and purines, and genes encod-ing the enzymes participating in this metabolism are termedthe one-carbon regulon. Purine biosynthesis, one of the majormetabolic pathways influenced by one-carbon metabolism, isexecuted by many ADE genes (4). It has been known that theexpression of the one-carbon regulon and ADE genes wereregulated mostly in a cooperative manner at the transcriptionallevel (18, 24, 33). Gcn4p and Bas1p are well-known transcrip-tion factors mediating this regulation (26, 39).

The well-studied mechanism through which yeast adapts toamino acid starvation is known as general amino acid control,a series of processes controlled by the yeast transcription factorGcn4p (for general control of non-derepressible) (10). Gcn4pcontains a bZIP DNA binding motif that is highly conservedamong eukaryotic DNA binding proteins (19, 31); the mam-malian transcription activator protein AP-1, the Candida albi-cans protein CaGcn4, Aspergillus niger protein CPCA, and theSaccharomyces cerevisiae protein Gcn4p all regulate transcrip-tion by binding to the same DNA consensus sequence [ATGA(C/G)TCAT] (2, 11, 14, 15, 41, 42). This element, called theAP-1 site, is located in the promoter region of various Gcn4ptarget genes in Saccharomyces cerevisiae. Gcn4p functions as apositive transcriptional regulator of many metabolic genes dur-ing UV stress and amino acid, glucose, or purine depletion (8,

12, 23, 25). Under these conditions, Gcn4p induction is regu-lated in a translational manner through the activity of theprotein kinase Gcn2p. In amino acid-starved cells, unchargedtRNA binds to and activates this kinase, which then phosphor-ylates the alpha subunit of eIF2 (for eukaryotic initiation factor2), leading to elevated Gcn4p translation (10). In yeast, theexpression of genes encoding amino acid biosynthetic enzymesis upregulated by Gcn4p in response to amino acid starvation.This derepression is induced by 3-aminotriazole (3-AT), acompetitive inhibitor of His3p. Gcn4p also activates the tran-scription of genes responsible for purine biosynthesis (43). Forexample, previous studies demonstrated that Gcn4p activatesADE4 (25), and microarray results using GCN4 wild-type anddeletion strains showed that Gcn4p stimulates the transcrip-tion of purine and pyrimidine biosynthetic genes following3-AT treatment (26). This result implies that Gcn4p controlsthe expression of genes related not only to amino acid biosyn-thesis but also to central metabolism.

Bas1p is a myb-related transcription factor that binds toTGACTC, a specific DNA sequence similar to that of GCRE(13, 29, 40). Bas1p is required for the activation of adenylatebiosynthetic genes under conditions of adenine depletion andin an SAGA- and SWI/SNF-dependent manner (18, 33). Inaddition, Bas1p activates not only adenylate biosynthesis butalso the one-carbon metabolic regulon under glycine-repleteconditions (39).

The yeast ADE3 gene product has been identified as thecytoplasmic trifunctional enzyme C1-tetrahydrofolate (C1-THF) synthase (35). C1-THF synthase plays a critical role inpurine biosynthesis, as it is responsible for synthesizing 10-formyl-THF, the essential C-2 and C-8 purine base donor (6).ADE3 gene regulation is predominantly pretranslational;steady-state levels of the mRNA encoding C1-THF synthaseare two- to threefold higher in derepressed cells than in normal

* Corresponding author. Mailing address: School of Life Sciences &Biotechnology, Anam-dong, Seongbuk-Ku, Korea University, Seoul136-701, Korea. Phone: 82-2-3290-3442. Fax: 82-2-927-9028. E-mail:joonkim@korea.ac.kr.

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

� Published ahead of print on 12 June 2009.

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cells (1). However, there is some controversy about the tran-scriptional regulation of ADE3. Although adenine depletiondoes alter the expression of most ADE genes through theactivities of Gcn4p, Bas1p, and Bas2p, it does not alter thetranscription of the ADE3 gene (4). In another study with 106yeast transcription factors, it was found that the ADE3 pro-moter did not bind Bas1p using chromatin immunoprecipita-tion (ChIP)-chip analysis (21). However, Mieczkowski and co-workers recently revealed that the ADE3 gene is a target sitefor Bas1p by using another set of ChIP-chip analyses (24). Italso has been reported that ADE3 transcription is activated ina Bas1p-dependent manner under glycine-replete conditions(39). To understand the regulation mechanism of these closelyrelated metabolic pathways, it is very important to understandhow yeast regulates ADE3 transcription.

In this study, we examined cis and trans elements of theADE3 gene. First, it was revealed that the ADE3 promoterregion has complex and overlapping sets of potential regula-tory motifs, including a TATA box and three putative Gcn4presponsive elements (GCREs). We next demonstrated thatone of these along with Gcn4p itself both are required for thesteady-state expression of the ADE3 gene. It was found thatthe basal transcription levels of the BAS1 and GCN4 deletionstrains were significantly decreased. Moreover, we discoveredthat these proteins bound to the same GCRE in vitro. Finally,the promoter occupancy of the ADE3 gene by these two factorswas confirmed. Taken together, we present a simple regulatorymodel of ADE3 transcription that proposes that Gcn4p is re-sponsible for the basal and inducible transcription of the geneduring starvation, while Bas1p is responsible for the basaltranscription only.

MATERIALS AND METHODS

Plasmids and primers. Primers used in this study are summarized in Table S1in the supplemental material. The vector pJS80 has a 6.0-kb fragment containingan ADE3(1-333)-lacZ fusion gene with an intact ADE3 promoter (36). Themulticopy plasmid p410 was constructed from pJS80 and YEp24, which containsthe URA3 marker gene in addition to a replicating origin (the 2� sequence). TheADE3(1-333)-lacZ fusion gene in pJS80 was excised with BamHI and SnaBI andinserted into the BamHI and PvuII sites in YEp24, generating p410-WT (�516to ��1). The plasmids p410-d1 (�516 to ��400, �236 to ��1), p410-d2(�516 to ��382, �207��1), p410-d3 (�516 to ��434, �117 to ��1), andp410-d4 (�516 to ��373, �41 to ��1) contain internal deletions of the ADE3promoter constructed by the digestion of ADE3(1-333)-lacZ using a Bal31 and aBstEII site. The digested DNA was treated with Klenow enzyme to generateblunt ends, followed by ligation. The junctions of the deleted regions wereverified by DNA sequencing. A low-copy YA410 plasmid containing the wild-type ADE3 promoter was constructed using the p410-WT and pRS316 vectors(NEB). The ADE3(1-333)-lacZ fusion reporter was fragmented using TthIII andNcoI and inserted into the NcoI and PvuII sites of the pRS316 vector, whichcontains URA3 within a CEN/ARS cassette. Plasmids p414-WT, p414-d1, p414-d2, and p414-d4 were constructed by partial digestion with EcoRI to remove the2� sequences from p410-WT, p410-d1, p410-d2, p410-d3, and p410-d4 (this stepwas required in order for the plasmid to integrate in the URA3 locus of thechromosome without replicating). Plasmids p414-m1 (mutant GCRE1), p414-m2(mutant GCRE2), p414-m3 (mutant GCRE3), p414-m4 (mutant GCRE1 andGCRE3), p414-m5 (mutant GCRE1, GCRE2), p414-m6 (mutant GCRE2 andGCRE3), and p414-m7 (mutant GCRE1, GCRE2, and GCRE3) contain theindicated site-directed mutations of the putative GCREs in the ADE3 promoterand were constructed in two steps. First, site-directed mutations in the ADE3promoter were introduced using PCR. Oligonucleotides carrying specific muta-tions in the ADE3 promoter sequences were as follows: S-128f and S-128r for themutation of GCRE1, S-129f and S-129r for the mutation of GCRE2, and S-130fand S-130r for the mutation of GCRE3. Point mutations were confirmed byDNA sequencing. Wild-type and mutant promoter-reporter genes integrated at

the ura3 locus in the GT48 chromosome lack a replication origin, and single-copyintegration was confirmed by the Southern blotting of the genomic sequence.

The plasmid pRSFRgcn4 was constructed to create the �gcn4 strain (YJK101)from the wild-type GCN4 gene (JS143-7D). The upstream 382-bp sequence andthe downstream 284-bp sequence of the GCN4 open reading frame were ac-quired by PCR. These two fragments possessed BglII sites on one end and eitherHindIII or XbaI sequence on the other. All PCR products were digested ac-cordingly and inserted into the pRS305 vector (NEB). The plasmid pRS305contains an LEU2 marker lacking its own replicating origin to allow integrationinto a specific host chromosome locus.

Yeast strains and media. The yeast strains used are listed in Table 1. TheYJK101 strain was derived from the JS143-7D strain (34) by replacing thechromosomal GCN4 gene with pRSFRgcn4. The retention of the gcn4::LEU2allele was verified on synthetic complete (SC) medium lacking leucine. Mutantswere scored as exhibiting no growth on medium containing 20 mM 3-AT andconfirmed by PCR. KY803 and GT48 strains were used for the reporter assay.Wild-type and mutant promoter-reporter fusion genes were integrated into thehost chromosomal DNA of these strains. Single-copy integration was confirmedin parallel with a marker test and genomic Southern blotting. The YJK309 strainwas generated from BY4741�bas1. The LEU2 deletion cassette introduced intothe GCN4 open reading frame for site-specific deletion was constructed by PCR.For ChIP, the YJK102 and YJK111 strains were generated from JS143-7D byPCR-based gene tagging. The C-terminal tri-hemagglutinin (HA) tagging cas-sette was obtained by PCR using pFA6a-HA-TRP as a template (22). Theamplified tagging cassette was transformed, and strain generation was confirmedby genomic DNA PCR and Western blotting with an anti-HA antibody. Alltransformations performed in this work were executed by means of a generaltransformation method using lithium acetate.

All strains were cultured in YPD or SC medium. YPD contained 2% Bacto-pepton (BD), 1% yeast extract (BD), and 2% glucose. SC medium contained 2%glucose and 0.67% yeast nitrogen base without amino acid (BD) and was sup-plemented with appropriate amino acids as previously described (6).

For the induction of galactose, cultured cells in SC medium were transferredinto SRG medium (0.67% yeast nitrogen base without amino acid [BD], 2%galactose, 2% raffinose) after reaching early log phase. After 10 h of mediumtransfer, cells were harvested for RNA and protein analysis.

Assay of �-galactosidase activity. Strains to be assayed were grown to late logphase in SC medium supplemented with appropriate amino acids at 30°C, fol-lowed by a 1:10 dilution with fresh SC medium and growth at 30°C to an opticaldensity at 600 nm of 1 to �2. A growth culture of 1 ml was harvested, and theactivities of the harbored reporter constructs were measured using chloroform.The composition of the solutions as well as the data calculations were performedas previously described (4). Values provided were acquired from triplicate assaysof three independent colonies.

Electrophoretic mobility shift assay (EMSA). The GST, GST-Gcn4, and GST-Bas1 fusion proteins were overexpressed in Escherichia coli BL21(DE3, RIL),which contains a pGEX vector carrying each gene downstream of a T7 promoter.Fifty milliliters of cultured cells was harvested after 3 h of isopropyl-�-D-thioga-lactopyranoside (IPTG) induction and resuspended in 1 ml of phosphate-buff-ered saline (PBS) containing protease inhibitors (1 mM phenylmethylsulfonylfluoride [PMSF], 1 �g/ml pepstatin A, 5 �g/ml leupeptin, and 1 �g/ml aprotinin).

TABLE 1. S. cerevisiae strains used in this study

Strain Relevant genotype Parent Source orreference

GT48 MATa ade3-130 ser1-171ura3-52

35

KY803 MATa trp1-� ura3-52 leu2-P1gcn4-�1

12

JS143-7D MATa leu2-3,112 trp1-�1ura3-52

36

YJK101 JS143-7D, except gcn4::LEU2 JS143-7D This studyYJK102 JS143-7D, except

gcn4::GCN4-HA3

JS143-7D This study

YJK111 JS143-7D, exceptbas1::BAS1-HA3

JS143-7D This study

BY4741 MATa his3-�1 leu2-�0 met15-�,ura3-�0

Clontech

BY4741�gcn4 BY4741, except gcn4::KANMX BY4741 ClontechBY4741�bas1 BY4741, except bas1::KANMX BY4741 ClontechYJK309 BY4741�bas1, except

gcn4::LEU2BY4741 This study

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The crude soluble extract was obtained by sonication. Each fusion protein taggedby glutathione S-transferase (GST) was purified with glutathione-Sepharose 4B(Amersham Biosciences) by following general methods of batch purification.Probes were prepared by annealing with two complementary single-strandedDNAs (see Table S1 in the supplemental material). These double-stranded DNAprobes were end labeled with 32P by T4 polynucleotide kinase, after which 15-�lbinding reaction mixtures were prepared that contained 20,000 cpm of theradiolabeled DNA probe each. Probes from the different constructs were incu-bated with different amounts of eluted recombinant Gcn4p in EB buffer [20 mMTris-HCl (pH 8.0), 60 mM KCl, 0.1 mM EDTA, 20% glycerol, 0.25 mg/ml bovineserum albumin, and 0.05 �g/�l poly(dI/dC)]. Binding was allowed to proceed for30 min at 30°C, followed by the analysis of the binding mixtures by electrophore-sis on 5% Tris-borate-EDTA (TBE)–polyacrylamide gels (100 mA for 1 h in0.5� TBE buffer). Autoradiography signals were detected with a BAS-2500image reader (Fujifilm).

Total RNA preparation and Northern blotting. Total yeast RNA was isolatedfrom early-log-phase cells by the hot phenol extraction method (30). A total of5 to 10 �g RNA was separated on a 1% formaldehyde–agarose gel with RNArunning buffer (1� morpholinepropanesulfonic acid), transferred to a nylonmembrane (Gene Screen Plus; NEN) using 20� SSC (1� SSC is 0.15 M NaClplus 0.015 M sodium citrate) buffer, and then UV fixed (UVC; 1,500 J) by a UVcross-linker. The probes were labeled with digoxigenin for ADE3, TRP3, HIS4,and ACT1, and detection was performed using the procedures recommended bythe manufacturer (Boehringer Mannheim).

Western blotting. To prepare the protein lysate, PBS-washed cell pellets wereresuspended in FA lysis buffer composed of 50 mM HEPES-HCl (pH 7.5), 150mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 25 mM�-glycerophosphate, 25 mM NaF, 1 mM PMSF, 1 �g/ml pepstatin A, 5 �g/mlleupeptin, and 1 �g/ml aprotinin. Cells were broken with glass beads using a beadbeater (MP), and lysate samples were generated by centrifugation at 10,000 � gfor 20 min at 4°C. The protein concentrations from different samples weredetermined. Equal amounts of each lysate were separated by sodium dodecylsulfate (SDS)–10% polyacrylamide gel electrophoresis, followed by the electro-transfer of proteins to a 0.45-�m nitrocellulose membrane (Bio-Rad). Immuno-blotting was performed with anti-c-myc (Santa Cruz Biotechnology), anti-HA(Santa Cruz Biotechnology), and anti-Pgk1p (Molecular Probes).

ChIP. Chromatin DNA preparation, immunoprecipitation, and semiquantita-tive PCR were performed as previously described, with several modifications(20). Cell cultures (200 ml; grown to an absorbance at 600 nm of 1.0) weretreated with 1% formaldehyde and gently swirled for 20 min at room tempera-ture. Glycine was added to a final concentration of 350 mM, and cells wereswirled for 5 min, followed by being harvested and washed twice with FA lysisbuffer. The cell pellet then was resuspended with an equal amount of FA lysisbuffer. After combining the FA buffer with the acid-washed glass beads, the cellswere broken by being vortexed with a bead beater, and the lysate was preparedby centrifugation. The input chromatin DNA fraction was obtained by ultracen-trifugation, followed by immunoprecipitation with an HA antibody (2 �g) for 2 hat 4°C. Protein A agarose beads were added to recover the immunocomplex,followed by incubation for two additional hours. Coprecipitated chromatinDNAs were analyzed by quantitative PCR using primer pairs corresponding tospecific regions of the ADE3 promoter (JK0660/JK0661), HIS3 promoter(JK0487/JK0488), and POL1 coding sequences (CDS; JK0450/JK0451). PCRwas performed with 1/80 of the immunoprecipitated DNA and 1/10,000 of thetotal input chromatin DNA as a template. The PCR products were analyzed ona 1% agarose gel.

RESULTS

The GCRE1 element in the ADE3 promoter is a functional,cis-acting element that promotes basal-level transcription. Toidentify the cis element of the ADE3 gene, we analyzed thesequence of the promoter. We found that the promoter se-quence contains putative DNA binding sites for transcriptionfactors such as TBP, Gcn4p, and Bas1p (Fig. 1A). We furthercompared the sequence of three putative GCREs to the con-sensus GCRE sequence (Fig. 1B). Among the three GCREs,GCRE1 most closely resembles the consensus sequence. Usinginternal deletions, we next generated a series of ADE3 pro-moter constructs upstream of the lacZ expression reporters(p410, p410-WT, p410-d1, p410-d2, p410-d3, and p410-d4)

(Fig. 1C, left). These reporter gene constructs were cloned intomulticopy plasmids and transformed into the GT48 strain,followed by the performance of the �-galactosidase assay (Fig.1C, right). The promoter activities of all constructs containingGCRE1 (p410-WT and p410-d1) were much higher than thoselacking GCRE1 (p410-d2, p410-d3, and p410-d4). We alsofound that the TATA box is important for adequate ADE3transcription, which is supported by the fact that the promoteractivity of the TATA-less promoter construct (p410-d4) wasdiminished to the same level as that of the vector control(p410). From these data, it was suggested that there are twofunctional cis elements in the ADE3 promoter region, GCRE1and the TATA box.

Although GCRE1 is the core region for ADE3 transcrip-tional regulation, as shown in Fig. 1C, 5�-to-3� serial deletionpromoter constructs could not exclude the possibility thatGCRE2 and GCRE3 participate in ADE3 transcription.Therefore, we generated seven promoter constructs containingone or more GCRE mutants [from ATGA(C/G)TCAT to ATGAATTCAT] capable of abolishing the direct binding ofGcn4p (see Fig. 5) (15). We next integrated these into the ura3locus of the GT48 strain and measured the promoter activitiesby �-galactosidase assay (Fig. 2). All of the constructs possess-ing the GCRE1 mutation (p414-m1, p414-m4, p414-m5, andp414-m7) showed significantly lower levels of activity (20 to�30%) than that of the wild type (100%). However, mutationsin GCRE2 and GCRE3 did not change promoter activity at all.A comparison of the activities of the WT, m2, and m3 con-structs clearly revealed that GCRE2 and GCRE3 are not func-tional for the basal-level transcription. In addition, the resultsfrom constructs containing multiple mutations (p414-m4,p414-m5, p414-m6, and p414-m7) clearly showed that therewas no cooperativity among GCREs. From these results, weconcluded that only GCRE1 is the functional element for basaltranscription, not GCRE2 and GCRE3.

ADE3 transcription is upregulated in a Gcn4p-dependentmanner. After finding the functional cis element in the ADE3promoter region, trans elements were examined. Since thefunctional GCRE was present, we monitored ADE3 mRNAlevels under stress conditions such as amino acid starvationand 3-amino-1,2,4-triazole (3-AT) treatment using Northernblotting. As shown in Fig. 3A, the transcription levels of the�gcn4 strain were much lower than those of wild-type GCN4under the tested conditions. Moreover, mRNA induction un-der derepression conditions was diminished in the �gcn4strain. We evaluated the expression of endogenous Gcn4p andfound that the expression levels corresponded exactly to theincreases generated under derepression conditions (data notshown). We therefore speculated that ADE3 transcription isdirectly correlated with the expression of Gcn4p. To furtherverify Gcn4p-dependent transcription, a reporter assay wasperformed. We transformed the low-copy YA410 plasmid con-taining ADE3(1-333)-lacZ into the isogenic GCN4 (JS143-7D)and �gcn4 (YJK101) strains. The ADE3 promoter activity ofthe low-copy plasmid was about threefold higher in the straincontaining wild-type GCN4 (Fig. 3B). To confirm that thisphenomenon is true in the multicopy state, we transformed themulticopy wild-type construct (p410-WT) into the KY803strain (gcn genotype) and GT48 strain (wild type). The ADE3promoter activity in GT48 was about 13-fold higher than that

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in KY803 (Fig. 3C). The results show that the level of increase(threefold) in the low-copy-number state was less robust thanthat in the multicopy state. Northern blotting was performed todetermine the ADE3 mRNA level following exogenous Gcn4p-myc7 overexpression using a galactose-inducible promoter.The transcript levels of overexpressed ADE3 and TRP3, whichis known to have a single Gcn4p response element (9, 23), weremuch higher than that of the vector control (Fig. 3D). Fromthis result, we concluded that Gcn4p is responsible for thetranscription of the ADE3 gene, some portion of basal tran-scription, and transcription under derepression conditionscaused by amino acid starvation.

Both Gcn4p and Bas1p are needed for ADE3 gene expres-sion. As shown in Fig. 3A, there was a slight increase in ADE3transcription in the �gcn4 strains under amino acid starvation.Moreover, the basal ADE3 transcript level in �gcn4 was abouthalf of that in the wild type. Specifically, the loss of the GCRE1region diminished promoter activity by about 20% comparedto that of the constructs containing GCRE1 (p410-d1 andp410-d2) (Fig. 1C). Accordingly, we suggested that anotherregulatory factor acts through GCRE1. To examine this, wesearched for other factors containing the GCRE1 sequencethat activate transcription. Among the many transcription fac-tors in yeast, the well-known protein Bas1p was presented as a

strong candidate, as it is bound to a sequence similar to that ofGCRE, 5�-TGACTC-3�, and also is known to regulate theADE genes and the one-carbon regulon. However, unlikeother ADE genes, it was reported that the ADE3 gene was notactivated by Bas1p under adenine limitation (4). Why is theADE3 gene regulated differently from other ADE genes? Al-though the mutation of ADE3 leads to a requirement for pu-rines, this is not directly required for the de novo pathway ofpurine biosynthesis. ADE3 encodes an enzyme participating inthe one-carbon metabolism. Therefore, there was a possibilitythat ADE3 gene transcription is regulated by Bas1p only underbasal conditions, similarly to other one-carbon metabolic andhistidine biosynthetic genes, and not under the derepressionconditions caused by adenine limitation, unlike other ADEgenes. To investigate this further, we examined the transcriptlevels of the wild-type, �gcn4, and �bas1 strains (Fig. 4). Sur-prisingly, the basal transcription levels of both deletion strainswere lower than that of the wild type. The level of basal ADE3mRNA in �gcn4 and �bas1 was 30 and 40% of the level ofwild-type mRNA, respectively. Although the basal level wasremarkably decreased in both deletion strains, the derepres-sion patterns were quite different under amino acid starvation.Even though all of the expression levels were decreased, thepattern of gene expression in �bas1 was not too different from

FIG. 1. Deletion analysis of the ADE3 promoter. (A) The 5�-untranslated region of the ADE3 gene promoter is indicated. (B) Comparison ofthe three putative GCRE sequences to the sequence of the consensus Gcn4p binding site. Sequences matched to the consensus sequence areenlarged and shown in boldface. (C) Reporter assay with five internal deletion promoter constructs containing various putative binding sites. Thedirection of an arrowhead indicates the orientation of each element. Multicopy plasmids containing mutant constructs (left) were transformed intothe GT48 strain. The promoter activities of every construct were examined and calculated as a relative percentage of wild-type promoter(p410-WT) activity. All values indicated for the �-galactosidase assays were averages from three independent experiments with three independentcolonies.

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that of the wild type, indicating that the basal transcriptionlevel of this strain was affected. However, for �gcn4, there wasa dramatic change upon amino acid starvation (Fig. 4B, lane5), as evidenced by the affected induction level. Under adeninedepletion, factor-dependent transcription and derepressionunder starvation were not detected. Thus, we conclude thatGcn4p is required for both basal and inducible ADE3 tran-scription, and that Bas1p is required only for the basal-levelexpression of ADE3.

Gcn4 and Bas1 bind directly to GCRE1 in vitro. We nextinvestigated the direct binding of Gcn4p and Bas1p to theGCRE1 of ADE3. Accordingly, EMSA was performed with32P-end-labeled probes harboring ADE3 promoter regions thatcontained the wild-type sequence and the mutated GCRE1sequences GST-Gcn4 and GST-Bas1 (Fig. 5A). Both proteinswere incapable of binding to the mutant probe (A3Gm) despitestrong interaction with the wild-type promoter (A3GW). Theextent of DNA-protein complex formation was dependentupon the concentration of GST-Gcn4 and GST-Bas1 in thebinding mixture. To examine whether both proteins bind si-multaneously to the GCRE1 element, EMSA was performedwith a fixed concentration of GST-Gcn4 and an increasedconcentration of GST-Bas1 (Fig. 5B, left) or vice versa (Fig.5B, right). Interestingly, we found that increasing the amountof one recombinant protein competed with the binding of theother. These results suggest that Gcn4 and Bas1 bind directlyto the same GCRE1 as trans-activators; however, both proteinscould not bind to the element at the same time.

Gcn4p and Bas1p are associated with the ADE3 promoterregion in vivo. To confirm promoter occupancy, strains con-taining Gcn4-HA3 (YJK101) and Bas1-HA3 (YJK111) weregenerated from the JS143-7D strain and subjected to ChIPanalysis. Yeast strains were verified by PCR, and normal func-tionalities of these tagging proteins were confirmed by theNorthern blotting of transcriptional target genes of each fac-

tor. ChIP assays for Gcn4p and Bas1p binding were performedafter 2 h of 3-AT treatment. Since previous studies have shownthe association of Gcn4p with the HIS3 promoter (16, 17), weused primers specific for the HIS3 promoter region as a posi-tive control and a specific primer set for the POL1 CDS as anegative control, similarly to a previous study (28). Comparedto the input and coimmunoprecipitated signals, it was clearthat Gcn4p and Bas1p weakly bind to the ADE3 promoterunder basal conditions. However, only Gcn4p increased bind-ing to this region under amino acid starvation (Fig. 6). Theseresults confirm that Gcn4p and Bas1p associate with the ADE3promoter region in vivo, activating the basal transcription ofADE3. In addition, Gcn4p, but not Bas1p, binds increasinglyfor the bulk of ADE3 gene induction during amino acid star-vation.

Gcn4p and Bas1p are needed to fully induce ADE3 tran-scription. According to previous reports, the maximal expres-sion of several genes, including ADE2, ADE4, HIS4, and HIS7,appears to require the transcriptional activators Gcn4p andBas1p (7, 25, 37, 38). Our data also supported this for ADE3gene transcription. To prove this, we generated a GCN4 andBAS1 double deletion strain and then compared mRNA levelsof ADE3, TRP3, HIS4, and ACT1. As shown in Fig. 7A, thelevel of ADE3 transcript in the steady state of the doubledeletion strain was significantly lower than that of the wild typeor that of each of the single-deletion strains, as is the case forHIS4. Furthermore, the TRP3 mRNA level was not affected atall in the BAS1 deletion strain (Fig. 7B). As mentioned previ-ously, the TRP3 promoter has a single Gcn4p response ele-ment, while the HIS4 gene is regulated by two transcriptionfactors, Gcn4p and Bas1p. Therefore, we concluded thatGcn4p and Bas1p are responsible for basal-level transcriptionthrough the same response element, namely GCRE1, which islocated in the promoter region. Moreover, these two proteinsactivate transcription independently from each other under

FIG. 2. GCRE1 is a critical cis element in the ADE3 promoter. Seven constructs, each containing combinations of mutant GCRE elements,were generated. Point mutant elements are indicated by filled arrowheads. To check the activity of each mutant promoter, a general �-galactosidaseassay was performed. The reported values are averages calculated from the results of duplicate assays from at least three independent transfor-mants. The error bars represent standard deviations. (GCRE1 point mutant, ATGACTCCT3ATGAATTCCT; GCRE2 point mutant,ATGACTGAC3ATGAATTGAC; GCRE2 point mutant, TTGACTTGT3TTGAATTTGT.)

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FIG. 3. Gcn4p upregulates the expression of ADE3 mRNA under basal and inducible conditions. (A) Exponentially growing wild-type(JS143-7D) and �gcn4 (YJK101) strains were transferred into fresh SC-his medium alone (normal), SC-his medium lacking TRP (amino acidstarvation), and SC-his medium containing 80 mM 3-AT (3-AT). After 1 h of medium transfer, total RNA was precipitated and Northern blottingwas performed with probes specific for ADE3 and ACT1. mRNA band intensities were expressed numerically by a phosphorimager, and eachADE3/ACT1 mRNA ratio was calculated and normalized to levels for the WT normal sample. (B and C) Specific activities of ADE3(1-333)-lacZfusion proteins expressed from a single-copy plasmid (B) or multicopy plasmids (C) were measured by reporter assay. (B) The single-copy plasmidcarrying the ADE3(1-333)-lacZ fusion gene (YA410-WT) was transformed into GCN4 wild-type (JS143-7D) and �gcn4 (YJK101) strains.(C) p410-WT, a multicopy plasmid bearing the ADE3(1-333)-lacZ construct, was transformed into the KY803 (GCN-negative) and GT48(GCN-positive) strains. All indicated values for the �-galactosidase assays were averages from three independent experiments. Error bars representstandard deviations. (D) BY4741�gcn4 strain containing pGALGcn4-myc or pGAL-myc (32) was grown to early log phase and transferred to SRGmedium. After 12 h of galactose induction, cells were harvested for the preparation of protein and RNA sampling. Western blotting was performedwith antibodies specific against c-myc and Pgk1p. Northern blot analysis was performed with probe specific against ADE3, TRP3, and ACT1. IB,immunoblot.

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both normal and derepressed conditions induced by aminoacid starvation.

DISCUSSION

The folate-dependent pathway termed one-carbon metabo-lism is crucial for the biosynthesis of purines and several aminoacids, like glycine and methionine, and it encompasses themajority of methyl group biogenesis in yeast. Gcn4p, Bas1p,and Bas2p regulate most of the genes that compose the one-carbon pathway; for example, most of the purine biosyntheticgenes, such as ADE1, ADE2, ADE5,7, and ADE8 but excludingADE3, are activated in an adenine-dependent manner by

Bas1p (4, 5). Previous reports indicated that 3-AT treatmentinduced the expression of ADE1, ADE2, ADE3, ADE4, ADE8,ADE12, and ADE17, and a microarray study revealed that thisinduction depends upon Gcn4p in several cases (ADE1, ADE3,ADE8, ADE12, and ADE17) (26). Even with these extensivedata, the details of the transcriptional mechanism mediated byGcn4p and Bas1p on the ADE3 gene during purine biosynthe-sis have not yet been clearly investigated.

In this report, we focused on ADE3 transcription and re-vealed clear evidence of its dependence upon Gcn4p andBas1p. First, we investigated the promoter activities of thethree putative GCREs upstream of the ADE3 gene. It wasconfirmed that only GCRE1 is a functional cis-acting promoter

FIG. 4. Both Gcn4p and Bas1p are responsible for basal and inducible ADE3 gene expression. (A) Wild-type (WT; BY4741), �gcn4(BY4741�gcn4), and �bas1 (BY4741�bas1) strains were grown to log phase (optical density at 600 nm, 1.0 to 2.0) in SC medium. Cells werereseeded in new SC medium (no stress), SC-his medium (amino acid starvation), and SC-ade medium (adenine depletion), respectively. After 1 h,cells were collected, total RNA was precipitated, and Northern blotting was performed with ADE3 and ACT1 probes. (B) The level of ADE3mRNA relative to that of ACT1 mRNA was evaluated with a phosphorimager. The resulting values from triplicate experiments were quantified,and the ADE3/ACT1 ratio was normalized to the value for the wild type under no stress, which was set arbitrarily to 1. Error bars indicate standarddeviations.

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element. When GCRE1 was mutated or deleted, the basalgene expression levels were reduced to 33% of that producedby the wild-type promoter (Fig. 2). Moreover, reduced expres-sion levels of ADE3 transcript were observed in the GCN4deletion strain compared to levels for the wild-type strain.These results confirm the role of Gcn4p as a positive regulatorof ADE3 gene expression. However, we also found several linesof evidence that another factor also is involved in ADE3 tran-scription.

The de novo syntheses of histidine and purines are intercon-nected due to the sharing of common substrates and interme-diates between these pathways. This connection is reflected inthe cross-pathway transcriptional regulation of certain HIS andADE genes by transcription factors such as Gcn4p and Bas1p/Bas2p (25, 29). Since the ADE3 gene product, C1-THF syn-thase, is involved in several important amino acid and purinebiosynthetic pathways, it is very possible that the ADE3 gene ishighly expressed in vivo, unlike other ADE genes. In fact, aprevious study showed that the promoter of ADE3 was muchmore active than those of ADE1 and ADE5,7 under normalconditions (4). Cells require a unique regulatory mechanism tomeet the cellular requirement for a high basal pool of C1-

synthase enzyme to overcome nutrient starvation. These fea-tures may explain why Bas1p-dependent transcription occursonly under basal conditions, as well as the unremarkable down-regulation of the ADE3 gene during adenine-limiting condi-tions. Moreover, we could detect more than two differentlengths of ADE3 transcripts (Fig. 4A and 7B). After checkingthe transcriptional start site by primer extension, we found thatmRNA of ADE3 was started at four sites, �27 bp, �30 bp, �38bp, and �42 bp, which are consistent with the results reportedby the National Center for Biological Information (NCBI). Wealso found that there are slightly different regulation patternsof these various types of transcripts. Therefore, it was sug-gested that the deletion of gcn4 or bas1 led to a change of thestart site of ADE3 transcription, which remains to be examineby further studies for the transcriptional regulation.

We hypothesized that both the Gcn4p and Bas1p transcrip-tion factors were responsible for the abundant transcript levelsunder basal conditions, as well as the robust transcriptionalactivation of ADE3 in response to amino acid starvation. Wetherefore evaluated Bas1p-dependent transcriptional regula-tion using BY4741 deletion variants. The Northern blot exper-iments in Fig. 4 and 7 clearly confirmed that Bas1p significantly

FIG. 5. Gcn4 and Bas1 directly bind to the GCRE1 sequence from the ADE3 promoter. (A) 32P-labeled DNA probes containing wild-type(A3GW; JK06101/JK06102) and mutated (A3Gm; JK06103/JK06104) GCRE1 (0.025 pmol) were mixed with the indicated amount of GST-Gcn4 orGST-Bas1. Following binding for 30 min at 30°C, the mixtures were analyzed on a 5% TBE–acrylamide gel, followed by being dried for detectionwith a BAS-2500 image reader. The mutation site of the A3Gm probe is identical to that of the GCRE1 mutant in Fig. 2. (B) EMSA was performedwith 32P-labeled A3GW probe, GST-Gcn4 (200 �g), and an increased concentration of GST-Bas1 (100, 200, 400, and 800 �g; left) or GST-Bas1(200 �g), as well as an increased concentration of GST-Gcn4 (100, 200, 400, and 800 �g; left). DNA-protein complex and unbound, free probeare indicated with an arrowhead.

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influenced basal ADE3 expression. Interestingly, Gcn4p andBas1p bind to the same DNA sequence. Our data suggest thatGcn4p and Bas1p are functional homologs in the case of ADE3transcription by acting through the same cis element. However,although it was predicted that derepressed Gcn4p stimulatesBAS1 expression under amino acid starvation (26), the expres-sion of Bas1p as well as the promoter occupancy of ADE3 byBas1p did not change after 3-AT treatment in our experiment(Fig. 6A). Therefore, we suggest that Gcn4p and Bas1p main-tain ADE3 gene expression at the basal level, while dere-pressed Gcn4p stimulates transcription that is capable of over-coming the stress condition by activating amino acidbiosynthetic genes such as ADE3 only through GCRE1.

Other reports showed that Bas2p is required for the regu-lated expression of ADE genes under adenine-limiting condi-tions in a Bas1-dependent manner (2). However, the expres-sion of Bas1p and Bas2p is not regulated by adenine (40, 44),indicating that adenine repression occurs by downregulatingactivator functions. Recently, a model was presented for theregulation of ADE genes (except ADE3) by Bas1p and Bas2pin an adenine-dependent manner (33). In the report, adenine-

dependent activation was achieved by protein-protein interac-tion between Bas1p and Bas2p. Furthermore, it also was re-vealed that transcription occurred in a SAGA- and SWI/SNF-dependent manner (18, 33). The absence of this activationmechanism on the ADE3 promoter may lead to the adenine-independent regulation of transcription.

ACKNOWLEDGMENTS

This work was supported in part by grants FRR05C2-390, KRF-2008-C00787, and KRF-2007-314-C00129.

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