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EUKARYOTIC CELL, Apr. 2010, p. 514–531 Vol. 9, No. 41535-9778/10/$12.00 doi:10.1128/EC.00251-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

The TEA Transcription Factor Tec1 Confers Promoter-Specific GeneRegulation by Ste12-Dependent and -Independent Mechanisms�†

Barbara Heise,‡ Julia van der Felden,‡ Sandra Kern, Mario Malcher,Stefan Bruckner, and Hans-Ulrich Mosch*

Department of Genetics, Philipps-Universitat Marburg, D-35043 Marburg, Germany

Received 28 August 2009/Accepted 20 January 2010

In Saccharomyces cerevisiae, the TEA transcription factor Tec1 is known to regulate target genes together with asecond transcription factor, Ste12. Tec1-Ste12 complexes can activate transcription through Tec1 binding sites(TCSs), which can be further combined with Ste12 binding sites (PREs) for cooperative DNA binding. However,previous studies have hinted that Tec1 might regulate transcription also without Ste12. Here, we show that in vivo,physiological amounts of Tec1 are sufficient to stimulate TCS-mediated gene expression and transcription of theFLO11 gene in the absence of Ste12. In vitro, Tec1 is able to bind TCS elements with high affinity and specificitywithout Ste12. Furthermore, Tec1 contains a C-terminal transcriptional activation domain that confers Ste12-independent activation of TCS-regulated gene expression. On a genome-wide scale, we identified 302 Tec1 targetgenes that constitute two distinct classes. A first class of 254 genes is regulated by Tec1 in a Ste12-dependent mannerand is enriched for genes that are bound by Tec1 and Ste12 in vivo. In contrast, a second class of 48 genes can beregulated by Tec1 independently of Ste12 and is enriched for genes that are bound by the stress transcription factorsYap6, Nrg1, Cin5, Skn7, Hsf1, and Msn4. Finally, we find that combinatorial control by Tec1-Ste12 complexesstabilizes Tec1 against degradation. Our study suggests that Tec1 is able to regulate TCS-mediated gene expressionby Ste12-dependent and Ste12-independent mechanisms that enable promoter-specific transcriptional control.

In Saccharomyces cerevisiae and related yeast species, the tran-scription factors Tec1 and Ste12 are a paradigm for studying themechanisms of combinatorial and promoter-specific target genecontrol (5, 30, 37, 53). These DNA binding proteins can interactwith each other and control various developmental programs,including vegetative adhesion, filament formation, and phero-mone-induced sexual mating (13, 15–17, 35, 37, 41, 42, 46). Tec1belongs to the family of the TEA transcription factors, whichcontrol several transcriptional programs governing cellular differ-entiation in many eukaryotes. Common to this family is the TEADNA binding domain (DBD) composed of a three-helix bundle,which binds to conserved TEA consensus sequence (TCS) ele-ments (2, 37). S. cerevisiae Ste12 is the founding member of theSte12-like transcription factors that are found preferentially infungi, where they control cellular development and pathogenesis(15, 33, 34, 50). Ste12 binds to pheromone response elements(PREs), and two or more of these elements are necessary toconfer pheromone-responsive transcriptional control by the Fus3/Kss1 mitogen-activated protein kinase (MAPK) pathway (13, 14,23, 52).

For combinatorial target gene control, Tec1 and Ste12 forma complex through the C-terminal part of Tec1 (10). For targetgene binding, initial in vitro studies have shown that Tec1-Ste12 complex formation enables cooperative binding to com-bined filamentation and invasion response elements (FREs),

which consist of a TCS and a PRE (37). More recent studiesshow that the promoter regions of many Tec1 target genes donot contain FREs but contain one or several TCSs (10). Inaddition, Tec1 and Ste12 are often present at the same pro-moters in vivo (5, 6, 53). It has thus been suggested that Tec1-Ste12 complexes regulate transcription predominantly via TCSelements, with Tec1 providing the DNA binding domain andSte12 the transcriptional activation domain (AD) (10). Tec1-Ste12 complexes can be further regulated by the protein Dig1,whose inhibitory function on Ste12 is relieved during mating byphosphorylation through the pheromone-stimulated MAPKsFus3 and Kss1 (4, 10, 12, 45, 49). In addition, Tec1 is directlyphosphorylated by the MAPK Fus3 in response to pheromone,which triggers ubiquitin-mediated degradation of the tran-scription factor (3, 8, 9). Finally, Ste12 directly controls TEC1gene expression via PREs, and TEC1 transcript levels are pher-omone inducible (43). As a consequence, TEC1 transcript andTec1 protein levels are drastically reduced in the absence ofSte12 (30).

In the study presented here, we have explored the possibilitythat Tec1 confers Ste12-independent transcriptional regula-tion, because we have previously shown that overexpression ofTEC1 in ste12� mutant strains leads to activation of TCS-driven reporter genes (30). We now demonstrate that physio-logical levels of Tec1 can bind to TCS elements in vitro andregulate TCS-driven genes in vivo in the absence of Ste12. Ona genome-wide scale we show that Tec1 is able to control asubset of its target genes without Ste12 through a C-terminaltranscriptional activation domain. We also show that complexformation between Tec1 and Ste12 provides Tec1 stability con-trol. Our study suggests that Tec1 is able to regulate TCS-mediated transcription by Ste12-dependent as well as Ste12-independent mechanisms to confer promoter-specific control.

* Corresponding author. Mailing address: Philipps-Universitat Mar-burg, Department of Genetics, Karl-von-Frisch-Strasse 8, D-35043Marburg, Germany. Phone: (49) 6421 282 3014. Fax: (49) 6421 2823032. E-mail: [email protected].

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

‡ These authors contributed equally to the study.� Published ahead of print on 29 January 2010.

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MATERIALS AND METHODSYeast strains and growth conditions. All yeast strains used in this study are

listed in Table 1. Yeast strains carrying the dig1�::kanMX4 deletion allele wereobtained by transformation with a corresponding deletion cassette and verifiedby Southern blot analysis. Different TEC1 versions and TEC1-STE12 hybrid

constructs were introduced into the yeast genome by targeting of the appropriateplasmid to the leu2::hisG locus. TCS and PRE reporter gene plasmids weretargeted to the ura3-52 locus. Yeast culture medium was prepared as describedpreviously (21). Adhesive growth tests were as described earlier (46). For cyclo-heximide-induced translational shutoff experiments, yeast cultures grown to an

TABLE 1. Yeast strains used in this study

Strain Relevant genotype Reference

RH2500 MATa tec1�::HIS3 ura3-52 leu2::hisG trp1::hisG 30RH2501 MATa tec1�::HIS3 ste12�::TRP1 ura3-52 leu2::hisG 30RH2765 RH2500 with TCS-CYC1-lacZ::URA3 30RH2767 RH2500 with CYC1(�UAS)-lacZ::URA3 30RH2768 RH2500 with TCS-PRE-CYC1-lacZ::URA3 30RH2774 RH2501 with TCS-CYC1-lacZ::URA3 30RH2777 RH2501 with TCS-PRE-CYC1-lacZ::URA3 30RH2662 MATa flo11�::kanR ura3-52 7EGY48-p1840 MAT� ura3 his3 trp1 leu2 lexAop-lacZ-URA3 22YHUM1631 RH2765 with PURA3-TEC1::LEU2 This studyYHUM1634 RH2765 with PURA3-TEC11–280::LEU2 This studyYHUM1635 RH2765 with PURA3-TEC11–280-TEC1377–486::LEU2 This studyYHUM1640 RH2774 with PURA3-TEC11–280::LEU2 This studyYHUM1641 RH2774 with PURA3-TEC11–280-TEC1377–486::LEU2 This studyYHUM1642 RH2774 with PURA3-TEC11–280-STE121–688::LEU2 This studyYHUM1643 RH2774 with PURA3-TEC11–280-STE121–250::LEU2 This studyYHUM1644 RH2774 with PURA3-TEC11–280-STE121–400::LEU2 This studyYHUM1645 RH2774 with PURA3-TEC11–280-STE12380–688::LEU2 This studyYHUM1646 RH2774 with PURA3-TEC11–280-STE121–688-TEC1377–486::LEU2 This studyYHUM1647 RH2774 with PURA3-TEC11–280-STE121–250-TEC1377–486::LEU2 This studyYHUM1648 RH2774 with PURA3-TEC11–280-STE121–400-TEC1377–486::LEU2 This studyYHUM1649 RH2774 with PURA3-TEC11–280-STE12380–688-TEC1377–486::LEU2 This studyYHUM1676 RH2501 with PURA3-TEC1::LEU2 This studyYHUM1694 RH2500 with PTEC1-TEC1::LEU2 This studyYHUM1696 RH2500 with tcs(ACATTCcT)-CYC1-lacZ::URA3 This studyYHUM1697 RH2500 with tcs(ACATTCaT)-CYC1-lacZ::URA3 This studyYHUM1698 RH2500 with tcs(AtATTCTT)-CYC1-lacZ::URA3 This studyYHUM1699 RH2500 with tcs(ACATgCTT)-CYC1-lacZ::URA3 This studyYHUM1700 MATa tec1�::HIS3 ura3-52 trp1::hisG This studyYHUM1701 MATa tec1�::HIS3 ste12�::TRP1 ura3-52 This studyYHUM1712 RH2774 with PTEC1-TEC1::LEU2 This studyYHUM1713 RH2774 with PURA3-TEC1T273 M::LEU2 This studyYHUM1718 YHUM1676 with TCS-CYC1-lacZ::URA3 This studyYHUM1720 YHUM1649 with dig1�::kanMX4 This studyYHUM1721 MATa tec1�::HIS3 PTEC1-TEC1::LEU2 TCS-CYC1-lacZ::URA3 This studyYHUM1723 MATa tec1�::HIS3 PURA3-TEC1T273 M::LEU2 TCS-CYC1-lacZ::URA3 This studyYHUM1724 MATa tec1�::HIS3 TCS-CYC1-lacZ::URA3 This studyYHUM1727 MATa tec1�::HIS3 PURA3-TEC1::LEU2 TCS-CYC1-lacZ::URA3 This studyYHUM1731 RH2500 with tcs(ACtTTCTT)-CYC1-lacZ::URA3 This studyYHUM1732 RH2500 with tcs(ACAgTCTT)-CYC1-lacZ::URA3 This studyYHUM1733 RH2768 with PURA3-TEC11–280-TEC1377–486::LEU2 This studyYHUM1734 RH2777 with PURA3-TEC11–280-TEC1377–486::LEU2 This studyYHUM1735 RH2777 with PURA3-TEC11–280::LEU2 This studyYHUM1736 RH2768 with PURA3-TEC11–280::LEU2 This studyYHUM1737 RH2777 with PURA3-TEC11–280-STE121–688-TEC1377–486::LEU2 This studyYHUM1738 RH2777 with PURA3-TEC11–280-STE12380–688-TEC1377–486::LEU2 This studyYHUM1739 RH2777 with PURA3-TEC1::LEU2 This studyYHUM1740 RH2768 with PURA3-TEC1::LEU2 This studyYHUM1743 YHUM1727 with dig1�::kanMX4 This studyYHUM1744 YHUM1718 with dig1�::kanMX4 This studyYHUM1745 YHUM1644 with dig1�::kanMX4 This studyYHUM1746 YHUM1645 with dig1�::kanMX4 This studyYHUM1747 YHUM1648 with dig1�::kanMX4 This studyYHUM1749 RH2768 with PTEC1-TEC1::LEU2 This studyYHUM1750 RH2777 with PTEC1-TEC1::LEU2 This studyYHUM1751 RH2777 with PURA3-TEC11–280-STE121–250-TEC1377–486::LEU2 This studyYHUM1752 RH2777 with PURA3-TEC11–280-STE121–400-TEC1377–486::LEU2 This studyYHUM1846 RH2777 with PURA3-TEC11–280-STE121–688::LEU2 This studyYHUM1847 RH2777 with PURA3-TEC11–280-STE121–250::LEU2 This studyYHUM1848 RH2777 with PURA3-TEC11–280-STE121–400::LEU2 This studyYHUM1849 RH2777 with PURA3-TEC11–280-STE12380–688::LEU2 This study

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optical density at 600 nm (OD600) of 1 were treated with 200 �g/ml cyclohexi-mide and samples were taken at the indicated time points.

Plasmids. All plasmids used in this study are listed in Table 2. PlasmidsBHUM1622 to BHUM1627, carrying point-mutated TCS elements upstream ofthe CYC1-lacZ reporter gene, were obtained by substituting a 430-bp XhoIfragment containing the CYC1 UAS of plasmid pLI4 for synthetic linkers withthe indicated mutated TCS element and XhoI-cohesive ends. Linkers wereprepared by annealing of the corresponding primers (see Table S3 in the sup-plemental material), and a single insertion was confirmed by sequencing. Plas-mids BHUM1384, BHUM1388, BHUM1389, BHUM1394, and BHUM1401were obtained by site-directed mutagenesis using the QuikChange site-directedmutagenesis kit (Stratagene, La Jolla, CA) and BHUM388 as the template.Similarly, plasmids BHUM1480 to BHUM1484 were obtained by site-directedmutagenesis using pME2289 as the template. Plasmid BHUM1616 was con-structed by introduction of a 3.1-kb PstI/BamHI fragment from pME2068, car-

rying PTEC1-TEC1, into YIplac128. For BHUM1258, BHUM1259, BHUM1347,BHUM1350, BHUM1357 to BHUM1365, BHUM1464, and BHUM1465, theURA3 promoter region was amplified from the yeast genome by PCR withprimers BHURA3-1 and BHURA3-2, yielding a 430-bp product with a newlycreated upstream SphI site and a new SalI site behind the ATG start codon. Thefragment was inserted into YIplac128 to obtain plasmid YIplac128-PURA3. A2.1-kb SalI/BamHI fragment carrying TEC1 or TEC1T273M was released frompME2068 or pME2102 and placed downstream of the URA3 promoter to createBHUM1258 and BHUM1259. Plasmids with truncated TEC1 versions andTEC1-STE12 fusions were constructed by the following strategy. (i) First, a BglIIrestriction site was inserted into the TEC1 open reading frame (ORF) behindcodon 280 by whole-vector PCR with primers JFTEC1-280-1 and -2 using the2.1-kb SalI/BamHI TEC1 ORF from pME2068 inserted into pBluescript II KS�as a template. The PCR product served as a whole-vector PCR template tointroduce an additional XhoI site behind codon 377 (primers JFTEC1-377-1

TABLE 2. Plasmids used in this study

Plasmid Genotypea Reference

BHUM29 PGAL1-TEC1 in pRS316 30BHUM30 TEC1 in pRS202 30BHUM212 pLG669-Z with TCS-PRE-CYC1-lacZ 37BHUM213 pLG669-Z with TCS-pre-CYC1-lacZ 37BHUM214 pLG669-Z with tcs-PRE-CYC1-lacZ 37BHUM388 MBP-TEC1-FLAG in pMAL-c2 37BHUM389 MBP-STE12-FLAG in pMAL-c2 37BHUM752 TEC1281-486 in pEG202 This workBHUM1258 PURA3-TEC1 in YIplac128 This workBHUM1259 PURA3-TEC1T273M in YIplac128 This workBHUM1347 PURA3-TEC1Bgl280Xho486 in YIplac128 This workBHUM1350 PURA3-TEC1Bgl280Xho377 in YIplac128 This workBHUM1357 PURA3-TEC1

1–280-STE121–688::LEU2 in YIplac128 This work

BHUM1358 PURA3-TEC11–280-STE121–250::LEU2 in YIplac128 This workBHUM1359 PURA3-TEC11–280-STE121–400::LEU2 in YIplac128 This workBHUM1360 PURA3-TEC11–280-STE12380–688::LEU2 in YIplac128 This workBHUM1362 PURA3-TEC11–280-STE121–688-TEC1377–486::LEU2 in YIplac128 This workBHUM1363 PURA3-TEC11–280-STE12

1–250-TEC1377–486::LEU2 in YIplac128 This work

BHUM1364 PURA3-TEC11–280-STE121–400-TEC1377–486::LEU2 in YIplac128 This workBHUM1365 PURA3-TEC11–280-STE12380–688-TEC1377–486::LEU2 in YIplac128 This workBHUM1384 MBP-TEC1R164A-FLAG in pMAL-c2 This workBHUM1388 MBP-TEC1L144A-FLAG in pMAL-c2 This workBHUM1389 MBP-TEC1L146A-FLAG in pMAL-c2 This workBHUM1394 MBP-TEC1

G163A-FLAG in pMAL-c2 This work

BHUM1401 MBP-TEC1S187A

-FLAG in pMAL-c2 This workBHUM1464 PURA3-TEC11–280::LEU2 in YIplac128 This workBHUM1465 PURA3-TEC11–280-TEC1377–486::LEU2 in YIplac128 This workBHUM1480 PTEC1-TEC1L146A in YCplac111 This workBHUM1482 PTEC1-TEC1G163A in YCplac111 This workBHUM1483 PTEC1-TEC1S187A in YCplac111 This workBHUM1484 PTEC1-TEC1R164A in YCplac111 This workBHUM1616 PTEC1-TEC1 in YIplac128 This workBHUM1622 pLI4 with tcs(ACATTCcT)-CYC1-lacZ This workBHUM1623 pLI4 with tcs(ACATTCaT)-CYC1-lacZ This workBHUM1624 pLI4 with tcs(ACATgCTT)-CYC1-lacZ This workBHUM1625 pLI4 with tcs(AtATTCTT)-CYC1-lacZ This workBHUM1626 pLI4 with tcs(ACtTTCTT)-CYC1-lacZ This workBHUM1627 pLI4 with tcs(ACAgTCTT)-CYC1-lacZ This workBHUM1687 TEC1350–486 in pEG202 This workpEG202 HIS3-based two-hybrid vector with lexA-DBD 22pJG4-5 TRP1-based two-hybrid vector with B42-AD 22pLI4 CYC1-lacZ in URA3-based integrative vector 30pME2051 pLI4 with TCS-CYC1-lacZ 30pME2055 pLI4 with TCS-TCS-CYC1-lacZ 30pME2068 PTEC1-TEC1 in YCplac33 30pME2102 PTEC1-TEC1T273M in YCplac33 30pME2289 PTEC1-TEC1 in YIplac111 30YIplac128 LEU2-based integrative vector 19YCplac111 LEU2-based CEN vector 19YEplac195 URA3-based 2�m vector 19

a TCS-PRE corresponds to the sequence of FRE(Ty1) (37).

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and -2) or behind codon 486 (primers JFTEC1-486-1 and -2). MutagenizedTEC1 ORFs were released by SalI/BamHI digestion and inserted intoYIplac128-PURA3. To replace a natural XhoI site in the TEC1 terminator regionby BamHI, PURA3-TEC1Bgl280Xho486 and PURA3-TEC1Bgl280Xho377 were amplifiedfrom these plasmids with primers BHURA3-1 and JFTEC1-1918-BamHI, re-sulting in 2.4-kb SphI/BamHI cassettes that were subcloned into YIplac128 toobtain BHUM1347 and BHUM1350. (ii) BHUM1464 and BHUM1465 wereobtained by replacing the BglII/XhoI TEC1 fragments in BHUM1347 andBHUM1350 by a synthetic linker which was prepared by annealing of primersBglII/XhoI linker 1 and BglII/XhoI linker 2. (iii) STE12 ORF fragments wereamplified from the yeast genome by PCR with primers generating a new BglIIsite at the 5� end and a XhoI site at the 3� end of each product. To obtain theTEC1-STE12 fusion constructs BHUM1357 to BHUM1365, STE12 fragmentswere inserted into BglII/XhoI sites of plasmids BHUM1347 and BHUM1350.

Protein analysis. (i) Preparation of yeast cell extracts. Preparation of total cellyeast extracts was performed as previously described (32, 51). Briefly, yeastcultures grown to an OD600 of 1 were concentrated to 1 ml, mixed with 150 �llysis buffer (1.85 M NaOH, 7.5% [vol/vol] �-mercaptoethanol), and incubated onice for 10 min. Samples were mixed with 150 �l trichloroacetic acid (55%) andcentrifuged for 10 min at 13,000 rpm. One hundred microliters of urea buffer(5% SDS, 8 M urea, 92.6 mM Na2HPO4, 107.4 mM NaH2PO4, 1.5% dithiothre-itol, 0.1 mM EDTA [pH 6.8], bromophenol blue) and 2 �l of 2 M Tris-Cl wereadded to the pellet and shaken for 20 min at 37°C. Samples were centrifuged for5 min at 13,000 rpm, and supernatants were further analyzed.

(ii) Generation of polyclonal anti-Tec1 antibodies. GST-Tec11–280 was purifiedby cultivation of Escherichia coli BL21(DE3) cells (Novagen, NJ) containingplasmid pME2676 (GST-TEC11-280 in pETM30) (8) in LB medium supple-mented with kanamycin. Target protein expression was induced with 0.1 mMIPTG (isopropyl-�-D-thiogalactopyranoside) for 4 h at 20°C, and cells wereresuspended in collection buffer (50 mM Tris-HCl [pH 7.5], 500 mM NaCl, 5 mMEDTA, 0.5 mM phenylmethylsulfonyl fluoride) and disrupted in a fluidizer.GST-Tec1M1-E280 was purified by column chromatography on a glutathione-Sepharose column, and the glutathione S-transferase (GST) tag was proteolyti-cally removed by TEV protease digestion and Ni-nitrilotriacetic acid (NTA)purification. Purified Tec1M1-E280 was used for standard immunization proce-dure by the Pineda Antibody Service (Berlin, Germany) to obtain polyclonalrabbit anti-Tec11–280 antibodies.

(iii) Immunoblot analysis. Equal amounts of proteins were separated by 12%SDS-PAGE and transferred to nitrocellulose membranes. Tec1 and Tec1-Ste12fusion proteins or Tub1 was detected using enhanced chemiluminescence (ECL)technology after incubation of membranes with polyclonal rabbit anti-Tec11–280

or monoclonal mouse anti-Tub1 antibodies (Calbiochem, Darmstadt, Germany).As secondary antibodies, horseradish peroxidase-coupled goat anti-rabbit (SantaCruz, CA) or goat anti-mouse (Dianova, Hamburg, Germany) antibodies wereused, respectively. Signals were quantified using a scanner and the ImageQuantTL software (GE Healthcare, Freiburg, Germany).

Electrophoretic mobility shift assays. Maltose binding protein (MBP), MBP-Tec1 and MBP-Ste12 proteins were prepared from E. coli using plasmids pMAL-c2, BHUM388, and BHUM389 essentially as described earlier (37). E. coliprotein extracts containing different Tec1 variants were obtained using appro-priate plasmids. 32P-labeled DNA was prepared by PCR amplification of 97-bp(TCS), 113-bp (TCS-TCS), or 111-bp (TCS-PRE, TCS-pre, and tcs-PRE) DNAfragments using the primer pair BHCYC1-2/BHCYC1-3 and plasmidBHUM212, BHUM213, BHUM214, pME2051, or pME2055. Five picomoles ofthe resulting DNA was incubated with 100 �Ci of [�-32P]ATP and T4 polynu-cleotide kinase, followed by purification using a QIAquick nucleotide removal kit(Qiagen, Hilden, Germany). Cy5-labeled 46-bp (TCS), 50-bp (PRE-tcs* andPRE-PRE-PRE) and 51-bp (TCS-PRE, TCS-pre, and tcs-PRE) DNA fragmentswere obtained by annealing synthetic 5� Cy5-labeled (Metabion, Martinsried,Germany) and unlabeled complementary oligonucleotides. To obtain unlabeledcompetitor DNA, 31-bp synthetic oligonucleotides were annealed.

For protein-DNA complex formation, the indicated amounts of labeled DNAfragments and purified proteins or E. coli protein extract were incubated for 30min at room temperature with 0.2 �g poly(dI-dC) � poly(dI-dC), Complete pro-tease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), 20 �g bovineserum albumin (BSA), 2 mM dithiothreitol, 50 mM NaCl, 10 mM Tris-Cl (pH7.5), and 0.1 mM EDTA. When used together, MBP-Tec1 and MBP-Ste12proteins were premixed and incubated on ice for 30 min before addition to DNA.Nonlabeled competitor DNA was added in a 100, 500, or 1,000 molar excessbefore addition of the proteins. After incubation, samples were mixed withloading buffer and separated on 6% native polyacrylamide gels for 1.5 h at 110to 150 V. Gels with Cy5-labeled DNA were directly scanned on a Typhoon Triovariable-mode manager (GE Healthcare, Freiburg, Germany). Gels with 32P-

labeled DNA were fixed in 10% acetic acid solution for 15 min, dried, andsubjected to autoradiography. Signals were quantified using the ImageQuant TLsoftware and analyzed using the Prism5 software (GraphPad, La Jolla, CA).

�-Galactosidase assays. Yeast strains carrying TCS-CYC1-lacZ or FRE-CYC1-lacZ reporters were grown to exponential phase in appropriate media, andextracts were prepared and assayed for �-galactosidase activity as previouslydescribed (8). �-Galactosidase activity was normalized to the total protein ineach extract with the following formula: (optical density at 420 nm � 0.304)/(0.0045 � protein concentration � extract volume � time). Assays were per-formed in triplicate on at least three transformants, and the mean values werecalculated. Standard deviations did not exceed 20%.

Yeast one-hybrid assay. Yeast strain EGY48-p1840 (20) carrying plasmidpEG202, pEG-TEC1281–486 (BHUM752), or pEG-TEC1350–486 (BHUM1687)and plasmid pJG4-5 (22) was cultivated to exponential growth in liquid SC-Trp-His medium supplemented with 2% galactose before �-galactosidase was mea-sured as described above.

Microarray analysis. Transcriptional profiling of yeast strains YHUM1642,YHUM1676, YHUM1694, YHUM1700, and YHUM1701 was performed usingYeast Genome 2.0 expression arrays (Affymetrix, High Wycombe, United King-dom) following standard protocols. Yeast strains were grown in synthetic com-plete medium lacking leucine and histidine with 2% glucose. For each strain twoindependent colonies were used. Overnight cultures were diluted into 30 ml offresh medium to an OD600 of 0.25 and grown to an OD600 of 1 at 30°C beforecells were harvested by centrifugation and rapidly frozen in liquid nitrogen. TotalRNA was prepared using an RNeasy mini kit (Qiagen, Hilden, Germany) fol-lowing Yeast Protocol 1c for mechanical disruption. RNA yield and purity weredetermined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Boblin-gen, Germany). All subsequent steps were conducted according to the AffymetrixGene Chip Expression Technical Manual (Affymetrix, High Wycombe, UnitedKingdom). Briefly, one cycle of cDNA synthesis was performed with 8 �g of totalRNA. In vitro transcription labeling was carried out for 16 h. The fragmentedsamples were hybridized for 16 h on Affymetrix Yeast Genome 2.0 expressionarrays, washed, and stained using the GeneChip Hybridization, Wash, and StainKit (P/N 900720) and an Affymetrix Fluidics 450 station. Arrays were scanned onan Affymetrix GeneArrayScanner 3000 7G, and nonscaled RNA signal intensity(CEL) files were generated using the Affymetrix GeneChip Command Consolesoftware. The resulting CEL files were loaded into the Affymetrix ExpressionConsole software, and CHP files were created using Quantile normalization andProbe Logarithmic Intensity Error Estimation (PLIER). Differentially expressedgenes were defined as having an average signal intensity of more than 50 and afold change of at least 1.5 (calculated as the fold change of the average expres-sion in the duplicate measurements).

Quantitative real-time PCR. Quantitative real-time PCR was performed byreverse transcription of 8 �g of total RNA into cDNA using the GeneChipExpression 3�-Amplification One Cycle cDNA synthesis kit (Affymetrix, HighWycombe, United Kingdom). Quantitative real-time PCR experiments wereperformed in duplicate in 96-well plates using the MyiQ single-color real-timePCR detection system (Bio-Rad, Munich, Germany). Independent PCRs wereperformed using the same cDNA for both the gene of interest and CDC28 as areference. Gene-specific primers are shown in Table S3 in the supplementalmaterial. The reaction mix (25-�l final volume) consisted of 12.5 �l of 2� iQSYBR green supermix (Bio-Rad, Munich, Germany), 1 �l of each primer (0.4�M final concentration), 0.5 �l fluorescein isothiocyanate (FITC) (0.02 �M finalconcentration), 9 �l H2O, and 1 �l of a 1/10 dilution of the cDNA preparation.A control without template was incorporated in each assay. The thermocyclingprogram consisted of one hold at 95°C for 3 min, followed by 45 cycles of 15 s at95°C, 30 s at 60°C, and 30 s at 72°C. After completion of these cycles, meltingcurve data were collected to determine PCR specificity, contamination, and theabsence of primer dimers. The detection of the threshold cycle by was doneautomatically by the cycler software. Quantification of gene expression wascarried out with the Gene Expression Analysis for iQ real-time PCR detectionsystem software (Bio-Rad, Munich, Germany).

Microarray data accession number. Array data are available from theArrayExpress database (http://www.ebi.ac.uk) under accession numberE-MEXP-2207.

RESULTS

In vivo, physiological amounts of Tec1 are sufficient to acti-vate TCS-mediated gene expression and FLO11-mediated ad-hesion in the absence of Ste12. We have previously shown thatTec1 is able to activate expression of TCS-driven reporters and



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natural target genes in ste12� mutant strains (30), suggestingthat Tec1 can regulate transcription independently of Ste12.These former experiments were done by expression of TEC1from high-copy plasmids, because efficient TEC1 transcriptiondepends on Ste12. To more accurately measure the influenceof Ste12 on the transcriptional activity of Tec1 in vivo, wecreated yeast strains that contain physiological amounts ofTec1 even when Ste12 is absent. These strains carry a singlegenomic copy of a TEC1 gene under the control of the Ste12-independent URA3 promoter to uncouple TEC1 transcriptionfrom Ste12 control. Using highly specific Tec1 antibodies di-rected against the TEA domain, we found that PURA3-TEC1expressing STE12 strains contain Tec1 protein levels that veryclosely match the Tec1 amounts detected in normal TEC1STE12 strains (Fig. 1A). In strains lacking STE12, Tec1 proteinlevels dropped more than 14-fold when TEC1 was driven bythe TEC1 promoter but only 2-fold in the case of PURA3-TEC1.

We next used the PURA3-TEC1-expressing yeast strains toquantify Tec1-dependent activation of TCS- and FRE-driventranscriptional reporters, and we related these activities to theTec1 protein levels present in the different strains. These quan-titative measurements revealed that the presence or absence ofSte12 had no obvious effect on the Tec1-dependent expressionof the TCS reporter (Fig. 1A). For the FRE reporter, we found

that Tec1 was able to stimulate expression even in the absenceof Ste12 (Fig. 1A, compare tec1� and PURA3-TEC1) and thatTec1-mediated expression was similar to that found for theTCS reporter. However, Tec1-mediated expression of the FREreporter, but not that of the TCS reporter, was further stimu-lated by the presence of Ste12, which reflects the cooperativebinding of Tec1 and Ste12 to the combined FRE element.These data suggest that there may be no fundamental differ-ence between the functions of Tec1 at the TCS versus the FREreporter.

We also measured adhesive growth and FLO11 expression inthe different TEC1 and PURA3-TEC1 expressing yeast strains inorder to relate these processes to the different Tec1 protein levels.As expected, TEC1 strains grow adhesively in the presence, butnot in the absence, of STE12 (Fig. 1B). In contrast, PURA3-TEC1confers adhesive growth even in a ste12� strain, although withlower efficiency than in a STE12 strain or compared to a ste12�strain carrying TEC1 on a high-copy plasmid (Fig. 1B). In agree-ment with these data, we found that Tec1 was able to significantlyactivate FLO11 transcription in the absence of Ste12 (Fig. 1C)and that in general FLO11 transcript levels correlated with Tec1protein levels.

Together, these data demonstrate that Tec1 is able to activateTCS-mediated gene expression in the absence of Ste12 and they

FIG. 1. Activation of TCS- and FRE-driven reporter genes and FLO11-dependent adhesion by Tec1 in the presence and absence of Ste12.(A) Reporter gene expression was determined by measuring �-galactosidase activities in STE12 or ste12� strains expressing no TEC1 (tec1�) orsingle genomic copies of TEC1 driven from the endogenous promoter (TEC1) or the Ste12-independent URA3 promoter (PURA3-TEC1) andcarrying integrated TCS- or FRE-driven CYC1-lacZ genes. Tec1 protein levels were determined by quantitative immunoblot analysis using specificantibodies against Tec1 and Tub1 as an internal control and are indicated as percentages of Tec1 protein present in a control TEC1 STE12 strain.Relative reporter gene activation corrected to Tec1 protein levels is shown in italic. (B) Adhesive growth of yeast strains of the indicated genotypeswas determined after growth on solid SC-Ura medium. Plates were photographed before (total growth) and after (adhesive growth) removal ofnonadhesive cells by a wash assay. For high-copy TEC1 expression (hc TEC1) plasmid BHUM30 was used; all other strains carry control plasmidYEplac195. (C) FLO11 expression in strains of the indicated genotypes was determined by quantitative real-time PCR. Expression is presentedas the percentage of the level measured in a TEC1 STE12 control strain, which was set to 100. Error bars indicate standard deviations.



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suggest that Tec1 can bind to TCS elements independently ofSte12.

In vitro, Tec1 binds to TCS elements with high affinity andspecificity independently of Ste12. We next investigated the invitro binding of Tec1 to TCS elements in the absence and pres-ence of Ste12. For this purpose, we performed quantitative gelretardation analysis using Tec1 and Ste12 proteins purified fromE. coli and different TCS-containing DNA fragments. Impor-tantly, we used Tec1 protein at nanomolar concentrations, whichcorresponds to the in vivo amounts of approximately 530 Tec1molecules per cell (18). Under these conditions, Tec1 efficientlybound to single TCSs without Ste12, and binding was not influ-enced by a neighboring PRE (Fig. 2A). However, Tec1 DNA

binding was much more efficient when a second TCS element waspresent on the target DNA, and it was drastically reduced whenthe TCS sequence was altered from the proposed consensus se-quence CATTCTT to CAaaCTT (Fig. 2A).

We further quantified the binding affinity of Tec1 to a singleTCS by performing titration gel shift analysis (Fig. 2B) and cal-culating the apparent dissociation constant (Kd) of the Tec1-TCScomplex. These experiments revealed that Tec1 binds to the con-sensus TCS with an approximate Kd of 56 nM (Fig. 2C and D).This value is comparable to that found for mammalian TEADfamily transcription factors, which bind to TCS elements with Kd

values of 16 to 45 nM (27).In higher eukaryotes, systematic analysis of target se-

FIG. 2. Tec1 DNA binding affinity in the absence of Ste12. (A) MBP-Tec1 protein at the indicated concentration was mixed with approximately0.5 nM 32P-labeled DNA fragments carrying TCS and/or PRE sequences or mutated versions (tcs and pre). Tec1-DNA complexes and free DNAwere visualized by autoradiography after separation by native gel electrophoresis. MBP alone was added as a negative control at 100 nM in laneswithout MBP-Tec1 (). (B) Titration analysis of Tec1 binding to single-TCS-containing DNA was performed by adding increasing amounts ofMPB-Tec1 at the indicated concentrations to 0.5 nM 32P-labeled TCS DNA fragments as described for panel A. (C) Tec1-TCS complex formationobtained for panel B was quantified by measuring bound and free TCS DNA and visualized by plotting the ratio (TCS bound/TCS total) versusthe Tec1 protein concentration. Error bars indicate standard deviations obtained from two independent measurements. (D) Scatchard plot analysisof the data obtained for panel C was used to calculate the apparent dissociation constant Kd of the Tec1-TCS complex.



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FIG. 3. Quantitative analysis of sequence requirement for Tec1-TCS binding in vitro and TCS-mediated reporter gene activation by Tec1 invivo. (A) Effects of single-nucleotide exchanges on Tec1 binding. Systematic competition gel shift analysis was performed with 50 nM Tec1 proteinand approximately 1 nM Cy5-labeled TCS DNA premixed with increasing amounts (100-, 500-, or 1,000-fold excess) of nonlabeled competitorDNA carrying the indicated TCS sequences. Nucleotides differing from the consensus TCS sequence ACATTCTT are shown in lowercase letters.The ratio of bound TCS to total TCS was determined by quantitative fluorescence imaging. Values multiplied by 100 are shown to indicate thepercentage of bound DNA. (B) Relative Tec1 binding affinities to different mutant TCS sequences. Values are shown as percentage of the control



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quences for TEAD family proteins revealed strong bindingto the consensus sequence ANATDCHN (2). Here, we de-termined the effects of all possible single-nucleotide ex-changes in an ACATTCTT target sequence on the Tec1binding affinity by performing a quantitative competitiontitration gel retardation analysis (Fig. 3A and B). This sys-tematic analysis revealed RMATTCYY as the consensussequence for strong and Ste12-independent Tec1 DNAbinding (Fig. 3B). We also found a good correlation be-tween in vitro DNA binding affinity and in vivo target generecognition when measuring Tec1-dependent activation ofCYC1-lacZ reporter genes driven by the consensus TCSACATTCTT or mutated versions (Fig. 3C).

We next asked whether (i) Ste12 affects binding of Tec1 tosingle TCSs and (ii) Tec1 influences the binding of Ste12 torepeated PREs. To address these issues, we performed gel retar-dation experiments using Tec1 together with Ste12. We foundthat Ste12 did not affect Tec1 binding to individual TCSs in vitro(Fig. 4A), suggesting that Tec1-Ste12 complex formation does not

increase the Tec1 DNA binding affinity. As previously described(37), we found that FREs are cooperatively bound by Tec1 andSte12 but not by Ste12 alone (Fig. 4A and B). Mutation of thePRE sequence in the FRE from TGAAACG to acttACG (pre)resulted in loss of cooperativity and led to a binding behavior ofTec1 as found for the individual TCS (Fig. 4B). However, muta-tion of the TCS in the FRE from CATTCT to CAaaCT (tcs) didnot completely disrupt cooperative binding of Tec1 and Ste12(Fig. 4B). This indicates that Ste12 can stimulate Tec1 binding atlow-affinity TCSs that are flanked by PREs. To further test thishypothesis, we looked for such configurations of Tec1 and Ste12binding sites in natural yeast promoters. We found that the TEC1promoter itself contains a TGAAACAGGAGATTCTT se-quence element at positions 93 to 77 relative to the transla-tional start site, which consists of a PRE and a low-affinity TCS(Fig. 4C, PRE-tcs*). Gel retardation analysis revealed significantcooperative binding of Tec1 and Ste12 to this element (Fig. 4C),indicating that low-affinity TCSs might be relevant in vivo if theyare flanked by Ste12 binding sites. Finally, we measured the effect

TCS affinity and were obtained by building the ratios between the values (1,000-fold excess of competitor) of control and mutant sequencesmultiplied by 100. The derived consensus sequence conferring strong Tec1 binding is shown at the bottom and was compared to the consensussequence for the human TEF-1 TEAD (2). (C) Reporter gene activation by different TCS sequences was determined by measuring �-galactosidaseactivities in yeast strains carrying integrated CYC1-lacZ genes driven by the indicated TCS sequences or no TCS element and expressing TEC1 froma low-copy plasmid. tec1� indicates expression of the control TCS reporter gene in the absence of Tec1. Error bars indicate standard deviations.

FIG. 4. Influence of Ste12 on Tec1 DNA binding and vice versa. (A to D) MBP-Tec1 and MBP-Ste12 proteins at the concentrations shown weremixed with approximately 1 nM Cy5-labeled DNA fragments containing the indicated combinations of TCS and/or PRE elements or mutated tcs,tcs*, or pre versions (for sequences, see the text). The percentage of bound DNA is shown at the bottom and was determined by quantitativefluorescence imaging as described for Fig. 2.



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of Tec1 on Ste12 binding to repeated PREs, which are found inpheromone-stimulated genes. Here, we analyzed a FUS1 pro-moter element that consists of three tandemly repeated PREs.We found that Ste12 binding was very efficient in both the ab-sence and the presence of Tec1 (Fig. 4D), indicating that Tec1-Ste12 complex formation does not affect Ste12 DNA binding torepeated PREs.

TCS-mediated transcriptional regulation by Tec1 dependson conserved residues in the TEA domain. In mammalian TEFproteins, structural and mutational analyses of the TEA domainhave revealed a number of residues that are crucial for DNAbinding (2). Because no comparable analysis has been performedfor Tec1, we analyzed a number of amino acid residues in thethree helices that form the TEA domain for their involvement inTCS binding in vitro and TCS recognition in vivo. We focused onresidues either that are conserved between Tec1 and mammalianTEA domains or that in mammalian TEA domains were associ-ated with DNA binding (Fig. 5A). This analysis revealed that two

single mutations introduced in helix H1, L144A and L146A, didnot affect DNA binding affinity in vitro, indicating that theseresidues are not crucial for TCS recognition (Fig. 5B). In vivo, theL146A mutation led to a 2-fold reduction in TCS reporter ex-pression (Fig. 5C), which might be caused by the slightly de-creased protein levels of this Tec1 variant when expressed in yeast(Fig. 5D). We also introduced two mutations in the highly con-served GRNEL sequence of helix H2 found in most TEA familytranscription factors. Both mutations, G163A and R164A, abol-ished TCS binding in vitro (Fig. 5B) and reporter gene activationin vivo (Fig. 5C), demonstrating the crucial role of these con-served residues in DNA binding. Finally, we found that the con-served residue S187 in helix H3, which has been suggested toconfer DNA recognition, is essential for DNA binding and TCS-mediated gene expression.

We also determined the effects of selected mutations in theTec1 TEA domain on yeast adhesive growth in order to ana-lyze the correlation between in vitro DNA binding, in vivo TCS

FIG. 5. Characterization of the Tec1 TEA domain. (A) Sequence alignment of Tec1 and human TEF-1 TEA domains. The diagram indicatesthe positions of helices and intermediate loops. Numbering refers to Tec1 residues, identical amino acids are marked by lines, triangles indicateTec1 residues analyzed by point mutagenesis, and asterisks refer to TEF-1 residues involved in DNA binding (2). (B) The effects of single aminoacid exchanges on Tec1 DNA binding were measured by quantitative gel retardation analysis using identical amounts of bacterially expressed Tec1proteins and Cy5-labeled TCS DNA fragments. Percentages of bound DNA and relative DNA binding activities compared to unaltered Tec1 areshown at the bottom. (C) TCS reporter gene activation by different TEC1 variants was determined by measuring �-galactosidase activities in yeaststrain YHUM1363 carrying an integrated TCS-CYC1-lacZ gene and expressing no TEC1 (tec1�) or TEC1 variants with the indicated mutationsfrom low-copy plasmids. Error bars indicate standard deviations. (D) Protein levels of different Tec1 variants in the strains described for panel Cwere determined by quantitative immunoblot analysis using specific antibodies against Tec1 and Cdc28 (as an internal control) and are indicatedas percentages of Tec1 protein present in the control TEC1 strain. (E) Activation of adhesive growth by different TEC1 variants with the indicatedmutations in the yeast strains described for panel C was determined using a wash assay after growth on solid SC-Leu medium for 5 days at 30°C.



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reporter gene activation, and regulation of a Tec1-dependentdevelopmental process. We found that G163A, R164A, andS187A, which had severe defects in DNA binding, were alsodefective in adhesive growth. In contrast, the L146A mutant,which did not exhibit obvious defects in binding to TCS, wascompetent in adhesive growth (Fig. 5E).

The C-terminal domain of Tec1 confers Ste12-independenttranscriptional activation and can be replaced by a Ste12activation domain. We have previously shown that deletions inthe C-terminal part of Tec1 cause a loss of TCS-mediated geneactivation (30). Therefore, we further analyzed the function ofthis part of Tec1 with respect to reporter gene activation. Forthis purpose, we expressed TEC11–280 from the URA3 pro-moter, a construct that encodes the TEA DNA binding domain(DBD) and lacks the C-terminal Ste12 interaction domain(Fig. 6A and B). We found that the relative transcriptionalactivity of Tec11–280 was drastically reduced in the presenceand absence of Ste12 (Fig. 6C). We then fused the C-terminalTEC1377–486 domain back to TEC11–280 and found a signifi-cant, yet not a full, reconstitution of the transcriptional activityin both the presence and the absence of Ste12 (Fig. 6C).Together, these data indicate that the C-terminal part of Tec1carries a Ste12-independent transcriptional activation domain.To independently corroborate this hypothesis, we performed ayeast one-hybrid analysis using the lexA-based yeast two-hybridsystem (22). Here, we found that fusion of the lexA DNAbinding domain to the C-terminal Tec1281–486 or Tec1350–486

portions led to a significant stimulation of a lexAop-driven lacZreporter gene compared to lexA alone (Fig. 6D). Also, Tec1281–486

led to a better activation than Tec1350–486, a finding that corre-lates with the results obtained with the TCS reporter.

We next asked whether fusion of Ste12 or different Ste12 do-mains that were previously identified as a DNA binding domainand two independent transcriptional activation domains, ADIand ADII (29), would confer transcriptional activity to the Tec1TEA domain. For this purpose, we constructed a series ofTEC1TEA-STE12 hybrid genes (Fig. 6A and B) and measured theactivity of the encoded synthetic proteins with respect to TCS-and FRE-mediated reporter gene activation and adhesion. Allgenes were expressed as single genomic copies driven by theURA3 promoter in tec1� ste12� mutant strains.

With respect to TCS-mediated reporter gene activation, wefound that addition of the DBD carrying Ste121–250 portion toTec11–280 did not render an efficient transcriptional activity(Fig. 6E, construct 4). In contrast, fusion of the Ste12 ADI orADII to Tec11–280 resulted in hybrid proteins that activated theTCS-driven reporter gene as efficiently as the full-length Tec1protein in the absence of Ste12 (Fig. 6E, constructs 6 and 8).An even better effect was obtained by fusion of the full-lengthSte12 protein to Tec11–280, which stimulated TCS-driven re-porter expression more than 6-fold (Fig. 6E, construct 10). Wealso fused the Tec1377–486 domain to the different Tec11–280-Ste12 hybrid proteins, and in all cases we found an increase inthe relative TCS-mediated transcriptional activities (Fig. 6E,constructs 5, 7, 9, and 11). This finding further supports theview that the C-terminal part of Tec1 carries an AD. We alsodetermined the relative transcriptional activities of differentTec1-Ste12 hybrid proteins at the FRE reporter. As found forthe TCS reporter, we measured a strong stimulation of activ-ities by inserting either Ste12 ADI, Ste12 ADII, or full-length

Ste12 into Tec11–280-Tec1377–486. Interestingly, insertion of theSte12 DBD alone into Tec11–280-Tec1377–486 conferred a 9.1-fold stimulation of the relative transcriptional activity at theFRE (compare Fig. 6C, construct 3, with Fig. 6E, construct 5),indicating cooperative DNA binding of this construct at FREsites.

We then determined activation of FLO11-mediated adhe-sive growth by the different Tec1-Ste12 hybrid proteins. We didnot observe a full correlation between stimulation of adhesionand activation of the TCS and FRE reporter genes. Specifi-cally, fusions between the Tec1 TEA domain and Ste12 ADIIwere unable to activate adhesive growth (Fig. 6F, constructs 6and 7), although their transcriptional activities at isolated TCSand FRE sites were significantly higher than that of the naturalTec1-Ste12 complex. This unexpected finding indicates thatthe activity of this hybrid protein not only requires a TCS butmight be additionally influenced by neighboring sequence el-ements for, e.g., further DNA binding proteins that appear todiffer between the TCS reporter and the natural FLO11 gene.In the case of fusions between the Tec1 TEA domain andSte12 ADI or full-length Ste12, reporter gene activation wasmirrored by a strong stimulation of adhesion (Fig. 6F, con-structs 8, 9, 10, and 11). These data point to a specific functionof Ste12 ADI in FLO11-mediated adhesion.

In vivo, Tec1 has been found to form a complex with Dig1 viaassociation with Ste12. Therefore, we measured how Dig1 af-fects the Ste12-dependent and Ste12-independent activity ofTec1 and that of different Tec1-Ste12 hybrid proteins. Inagreement with previous studies, we found that TCS-mediatedactivity of the natural Tec1-Ste12 complex and adhesivegrowth are under negative control of Dig1 (see Fig. S1A and Bin the supplemental material). However, the Ste12-indepen-dent stimulation of the TCS reporter and of adhesion by Tec1was not influenced by Dig1. We also found a negative effect ofDig1 on the transcriptional activities of hybrids between theTec1 TEA domain and Ste12 ADII (see Fig. S1A, constructs 6and 7, in the supplemental material) but not for the Tec1-Ste12ADI hybrids (Fig. S1A, constructs 8 and 9), indicating thatDig1 inhibits Ste12 activity via ADII.

In summary, the data in this section show that Tec1 is ableto activate TCS-mediated transcription in the absence of Ste12depending on its C-terminal domain, which can be replaced byan activation domain of Ste12.

On a genome-wide scale, Ste12-dependent and Ste12-inde-pendent Tec1-regulated genes constitute two distinct classes.Our strains expressing TEC1 from the Ste12-independentURA3 promoter enabled us to analyze and compare genome-wide Tec1-regulated gene expression in the presence and ab-sence of Ste12, an issue which was not addressed in previousstudies (6, 38). For this purpose, we compared transcriptionalprofiles of TEC1 STE12 and tec1� STE12 strains as well as ofPURA3-TEC1 ste12� and tec1� ste12� strains grown under nu-trient-rich conditions using high-density oligonucleotide arrays(Affymetrix GeneChips). We found 289 genes that are regu-lated at least 1.5-fold by Tec1 in the presence of Ste12 and 48genes that are regulated by Tec1 in the absence of Ste12, with35 genes overlapping between the two groups (Fig. 7A and B;see Table S1 in the supplemental material). Thus, the total of302 Tec1 target genes can be divided into two distinct classes,with 254 genes (84%) being Tec1 regulated in a strictly Ste12-



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FIG. 6. Activities of different Tec1 and Ste12 proteins. (A) Diagrams showing Tec1 with TEA and Ste12-binding domains and Ste12 with the DNAbinding domain (DBD) and activation domains (ADI and ADII) (44). (B) Diagrams showing different TEC1-STE12 hybrid constructs driven by theURA3 promoter. Encoded Tec1 and Ste12 amino acid residues and functional domains are indicated. (C) Relative activation of TCS and FRE reportergenes by Tec1 (lanes 1), Tec11–280 (lanes 2), and Tec11–280-Tec1377–486 (lanes 3) was determined in STE12 or ste12� strains expressing single genomiccopies of corresponding TEC1 variants and TCS- or FRE-driven CYC1-lacZ genes as described for Fig. 5. (D) Activation of a lexAop-lacZ reporter geneby lexA (pEG202), lexA-TEC1281–486 (BHUM752), or lexA-TEC1350–486 (BHUM1687). Yeast strain EGY48-p1840 (22) was transformed with theseplasmids and pJG4-5, followed by quantitative measurement of �-galactosidase activities using SC-Trp-His medium supplemented with 2% galactose.Error bars indicate standard deviations. (E) Relative activation of TCS and FRE reporter genes by different Tec1-Ste12 hybrid proteins was determinedin ste12� tec1� strains expressing single genomic copies of corresponding TEC1-STE12 variants and TCS- or FRE-driven CYC1-lacZ reporter genes.(F) Adhesive growth of the yeast strains described for panels C and E was determined after 5 days of incubation on solid yeast extract-peptone-dextrose(YEPD) medium.

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dependent manner (Fig. 7C, class I) and 48 genes (16%) thatare Ste12-independently regulated by Tec1 (Fig. 7C, class II).

To uncover possible differences in the promoters of class Iand class II genes, we performed a concurrent enrichmentanalysis using the ChIPCodis web server (1), which is based onavailable chip-on-chip (25) and TFBS data (36) for 203 yeasttranscription factors. We found that class I was specificallyenriched for a total of 38 genes that are bound by Tec1, Ste12,and Dig1 under nutrient-rich conditions (Fig. 7D) (25). Wethen analyzed these class I genes for the presence of Tec1binding sites and found high-affinity TCS elements matchingthe RMATTCYY consensus sequence in 50% of the promot-ers (Fig. 7D; see Table S2 in the supplemental material). Thisindicates that these class I genes are regulated by Tec1-Ste12complexes via TCS binding. In contrast, class II was specificallyenriched for 32 genes that were previously identified to bebound by the transcription factors Yap6, Nrg1, Cin5, Skn7,Hsf1, and Msn4 under hyperoxic stress conditions (Fig. 7D)(25). Notably, most of these class II genes were previously notfound to bind Tec1 under nutrient-rich conditions (25). Nev-ertheless, we detected high-affinity RMATTCYY TCS ele-

ments in the promoter regions of 59% of these genes (Fig. 7D).Thus, efficient in vivo binding of Tec1 to these class II genesmight depend on nonstandard conditions or additional tran-scription factors.

Because we found that the Tec11–280-Ste121–688 hybrid pro-tein, which represents the Tec1 TEA domain fused to thefull-length Ste12 (Tec1TEA-Ste12), is a very potent activator ofTCS-driven reporters, we wondered how this protein wouldregulate the different classes of Tec1 target genes. Transcrip-tional profiling revealed that 199 (78%) of class I genes arenormally regulated by Tec1TEA-Ste12, whereas only 55 genes(22%) are deregulated (Fig. 7C and D). This indicates that alarge proportion of class I genes are regulated by Tec1-Ste12complexes without the need of the Tec1 C-terminal domain. Asignificantly higher proportion (72%) of class II genes enrichedfor Yap6, Nrg1, Cin5, Skn7, Hsf1, or Msn4 binding were de-regulated by Tec11–280-Ste121–688 (Fig. 7D), indicating thatnormal regulation of these genes specifically depends on theTec1 C-terminal activation domain.

In summary, our genome-wide analysis reveals that Tec1target genes constitute two distinct classes that are defined by

FIG. 6—Continued.



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their Ste12-dependent and Ste12-independent regulatory pat-terns and their promoter characteristics.

Ste12 controls Tec1 protein stability independently of Dig1and the Cdc4 phosphodegron (CPD) at Thr273 but involvesthe Tec1 C-terminal part. We noticed that Tec1 protein lev-els are lower in the absence of Ste12, even when TEC1 isexpressed from the Ste12-independent URA3 promoter(Fig. 1A). Because Ste12 does not affect TEC1 transcriptlevels in PURA3-TEC1-carrying strains, we tested whetherSte12 might influence Tec1 protein stability. Therefore, wedetermined in vivo decay rates of Tec1 in the presence andabsence of Ste12 by performing cycloheximide-inducedtranslational shutoff experiments. We followed the decrease ofendogenous Tec1 protein levels in the PURA3-TEC1-expressingSTE12 and ste12� strains after addition of cycloheximide by

quantitative immunoblot analysis (Fig. 8A). These experimentsrevealed that the rate of decay of Tec1 increases more than5-fold when STE12 is deleted (Fig. 8B), indicating that Ste12positively controls Tec1 protein stability. To corroborate thisfinding, we performed a promoter shutoff experiment using theTEC1 gene under the control of the glucose-repressible GAL1promoter. Again, we found that the Tec1 half-life decreasesroughly 5-fold in strains lacking STE12 (see Fig. S2 in thesupplemental material), supporting the view that Tec1-Ste12complex formation significantly enhances Tec1 protein stabil-ity. We also explored the possibility that the Ste12-dependentTec1 stability control is conferred by Dig1, which associateswith Tec1-Ste12 complexes (10). However, Dig1 had no effecton the Tec1 half-life in either the presence or absence of Ste12(Fig. 8C).

FIG. 7. Classification of Tec1-regulated genes by transcriptional profiling. (A) Venn diagram indicating the number of genes differentiallyexpressed in yeast strains YHUM1694 (TEC1 STE12) and YHUM1700 (tec1� STE12) (blue) or YHUM1676 (PURA3-TEC1 ste12�) andYHUM1701 (tec1� ste12�) (red). Numbers are given for genes whose expression differs at least 1.5-fold between the yeast strains compared andfor genes shared between the different groups. (B) Display of a subset of Tec1 target genes differentially expressed in strains of the indicatedgenotypes (combinations A to C). Numbers indicate the fold changes in expression levels when comparing the different strain pairs. (C) Classi-fication of all 302 Tec1-regulated genes identified in this study into two major classes. Numbers in parentheses indicate the number of genes presentin the different classes. (D) Concurrent enrichment analysis of 203 transcription factors bound by 302 Tec1-regulated genes was performed usingthe ChIPCodis web server (1) based on available yeast chip-on-chip (25) and TFBS (36) data. Statistically overrepresented transcription factors(P � 0.02) bound by the indicated class I and class II genes are shown. Transcription factor binding is indicated in green. The presence ofhigh-affinity RMATTCYY TCS sequences in the promoter regions of the different genes is indicated by red boxes. Normal regulation by theTec1TEA-Ste12 fusion protein is shown by blue boxes.



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Previous studies have shown that Tec1 stability is decreasedin response to pheromone by Fus3-mediated phosphorylationof a Cdc4 phosphodegron (CPD) motif centered at positionThr273 of Tec1 and that mutation of Thr273 or absence ofFus3 increases Tec1 stability (3, 8, 9). Therefore, we testedwhether Ste12-dependent Tec1 stability control might also bemediated by Thr273. For this purpose, we constructed STE12and ste12� strains carrying a single genomic copy of theTEC1T273M allele under the control of the URA3 promoter asthe single source for functional Tec1 protein. We found thatSte12 affected the decay rate of Tec1T273M comparably to thatof regular Tec1 (Fig. 8D), indicating that the CPD motif atT273 is not involved in regulation of Tec1 stability by Ste12.

Finally, we tested whether the C-terminal part of Tec1 mightaffect protein stability, because we noticed that steady-statelevels of the Tec11–280 variant are significantly increased (Fig.6C). Indeed, we found that the half-life of Tec11–280 lackingthe C-terminal part is 3-fold higher than that of full-lengthTec1 and, more importantly, completely uncoupled from Ste12control (Fig. 8E). Moreover, fusing the Tec1377–486 part backto Tec11–280 not only lowered the half-life 3-fold to match thevalue observed for regular Tec1 but also reconstituted stabilitycontrol by Ste12 (Fig. 8E).

In summary these data indicate that Ste12-mediated Tec1stability control involves the C-terminal part of Tec1 but notDig1 or the CPD at Thr273.

DISCUSSION

The mechanisms by which the S. cerevisiae transcription fac-tors Tec1 and Ste12 regulate their target genes have become avaluable paradigm for combinatorial and program-specifictranscriptional control. Previous studies have shown that thesemechanisms include cooperative binding of Tec1 and Ste12 tocombined TCS-PRE target sequences (37), Tec1-Ste12 com-plex formation and interaction with the inhibitor protein Dig1(4, 10, 12, 45, 49), control of Tec1 protein stability by theMAPK Fus3 (3, 8, 9), and regulation of TEC1 transcription bySte12 (30, 43). In addition, both regulators have been found tocolocalize at many target gene promoters in vivo, suggestingthat Tec1-Ste12 complex formation confers program-specifictarget gene control (6, 53). Our study provides (i) new insightsinto the mechanisms by which Tec1-Ste12 complex formationcontributes to TCS-mediated target gene control and (ii) evi-dence that Tec1 is capable of controlling target gene expres-sion by previously unknown Ste12-independent mechanisms.

FIG. 8. Dependence of Tec1 protein stability on Ste12 and Dig1. (A) The rate of decay of Tec1 in yeast strains YHUM1727 (STE12PURA3-TEC1) and YHUM1718 (ste12� PURA3-TEC1) was determined after addition of cycloheximide to exponentially growing cultures bymeasuring Tec1 protein levels at the indicated time points using quantitative immunoblot analysis. (B) The Ste12-dependent half-life of Tec1 wascalculated by plotting the relative Tec1 protein levels obtained in panel A versus time, with starting amounts set to 100%. (C) Dig1-dependentdecay of Tec1 in yeast strains of the indicated genotype and expressing PURA3-driven TEC1 was measured as described for panel A. (D) Ste12-dependent decay of Tec1T273M was determined in strains YHUM1723 (STE12 PURA3-TEC1T273M) and YHUM1713 (ste12� PURA3-TEC1T273M).(E) The Ste12-dependent half-lives of Tec11–280 and Tec11–280-Tec1377–486 proteins were determined using the strains described for Fig. 6.



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With respect to regulation by Tec1-Ste12 complexes, wehave identified a large class of Tec1 target genes that areregulated by Ste12-dependent mechanisms. Our bioinformaticanalysis reveals that many of these genes contain high-affinityTCS elements, often without a flanking PRE, and that manyclass I genes are bound by Tec1, Ste12, and Dig1 in vivo (25,53). We further find that Tec1 efficiently binds to TCSs in vitroand that this activity is not affected by Ste12. Also, our Tec1deletion and Tec1-Ste12 hybrid analyses have revealed thatTCS reporters and many TCS-carrying class I genes are nor-mally regulated by chimeras between the Tec1 TEA domainand Ste12. Together, these findings suggest that TCS-contain-ing class I genes can be regulated by Tec1-Ste12 complexes byat least two different mechanisms (Fig. 9). We conclude that atTCS promoters without PREs, Tec1-Ste12 complexes can bindvia the Tec1 TEA domain and activate transcription throughthe different ADs of Ste12. This conclusion is in agreementwith the study performed by Chou et al. (10). Our data do notrule out, however, that additional factors present at TCS pro-moters might affect recruitment or transcriptional activity ofTec1-Ste12 complexes. At promoters carrying TCSs flanked bya PRE, we find that Tec1-Ste12 complexes bind cooperatively,which is in line with initial studies (37). However, our study hasrevealed that there might be no fundamental difference inTec1 functions at TCS- versus TCS-PRE-containing promot-ers. Finally, we show that class I genes can be regulated byDig1. Our data indicate that Dig1 regulation is mediated bySte12 ADII (Fig. 9), a finding that is supported by the fact thatSte12 associates with Dig1 through the ADII domain (44).

A completely novel outcome of our study is that Tec1-Ste12complex formation confers Tec1 stability. So far, only a fewexamples of stability control by interacting transcription factorshave been found in S. cerevisiae, e.g., in the case of the matingtype transcription factors MATa1 and MAT�2 (26). Here, wefind that Ste12-mediated Tec1 stability control is independentof T273 phosphorylation and Fus3 (3, 8, 9) but instead involvesthe C-terminal part of Tec1. Given the fact that Tec1-Ste12complex formation depends on the C-terminal portion of Tec1(10), our results indicate that Ste12 might mask a destabilizingsignal in this part of Tec1 (Fig. 9). What is the role of thismechanism? An interesting possibility is that this might be aregulatory device that ensures appropriate discrimination be-tween class I and class II Tec1 target genes. For instance, sucha mechanism might prevent erroneous activation of class Igenes in the absence of Ste12 and/or ensure efficient expres-

sion in its presence. It might also stabilize class I gene expres-sion over a broader range of Tec1 concentrations, e.g., whenTec1 protein levels fluctuate due to varying Fus3 activity withina population (11). Thus, future studies of Ste12-mediated Tec1stability control could reveal novel insights into the significanceof such mechanisms in population development and dynamics.

A central and novel finding of our investigation is the dis-covery of a second class of Tec1 target genes that can beregulated by Ste12-independent mechanisms. How does Tec1regulate these genes? Our biochemical and genetic analyseshave shown that Tec1 efficiently binds TCSs without Ste12 andthat class II gene activation depends on the C-terminal domainof Tec1. Our bioinformatic analysis has revealed that a largenumber of class II gene promoters contain high-affinity TCSs.Yet, we find that only a few class II promoters have previouslybeen identified to be bound by Tec1 under nutrient-rich con-ditions (25). Therefore, efficient Tec1 recruitment to class IIpromoters might depend on nonstandard conditions and onthe interaction with further regulators. So far, no genome-wideTec1 binding analysis has been performed under a regimen ofdifferent stress conditions. However, we found that class II isenriched for genes that are bound by the transcriptional reg-ulators Yap6, Nrg1, Cin5, Skn7, Hsf1, and Msn4 under hyper-oxic stress (25). This opens the possibility that Tec1 might berecruited to class II promoters under certain stress conditionsand interact with these stress-related DNA binding proteins.Based on previously identified functions of these regulators,such conditions might include oxidative and salt stress (e.g.,Yap6, Cin5, and Skn7) (40, 48), heat shock (e.g., Hsf1 andMsn4) (24, 39) or glucose starvation (e.g., Nrg1) (31). Whetherand how Tec1 might interact with any of these regulatorsremains to be investigated. Alternatively, Tec1 might regulateclass II genes also indirectly via transcription factors that areunder direct Tec1 control. With respect to this possibility, wenoticed that class II includes RPI1, a gene which encodes aputative transcriptional regulator involved in the heat shockresponse and entry into stationary phase (28, 47). Interestingly,the promoter of RPI1 contains high-affinity TCS elements andis bound by Tec1, but not Ste12, under nutrient-rich conditions(25). This opens the possibility that class II genes might beregulated by Tec1 via Rpi1.

In summary, our study reveals that the mechanisms by whichTec1 is able to control target gene expression in S. cerevisiaeare much more complex than previously anticipated. Becausewe find that TCS-mediated transcriptional control by Tec1

FIG. 9. Mechanisms for promoter-specific gene regulation by Tec1 and Ste12. Deg, degradation; Txn, transcription; AD, activation domain;DBD, DNA binding domain; CPD, Cdc4 phosphodegron; TEA, TEA DNA binding domain. For further details, see the text.



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depends on conserved residues in the TEA domain, our find-ings also contribute to a better understanding of the functionsof TEA transcription factors in eukaryotes in general.

ACKNOWLEDGMENTS

We thank Hiten Madhani and Gerald R. Fink for generous gifts ofplasmids and strains. We are grateful to Diana Kruhl for technicalassistance and to Gunther Dohlemann for help in performing microar-ray analysis and quantitative real-time PCR.

This work was supported by grants from the Deutsche Forschungs-gemeinschaft (DFG).

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