Organizational constraints on Ste12 cis-elements for a pheromone response in Saccharomyces cerevisiae Ting-Cheng Su1,2, Elena Tamarkina1 and Ivan Sadowski1 Department of Biochemistry and Molecular Biology, Molecular Epigenetics, LSI, University of British Columbia, Vancouver, Canada Graduate Program in Genetics, University of British Columbia, Vancouver, Canada Keywords gene regulation; pheromone response; PRE, Ste12; yeast Correspondence I Sadowski, Department of Biochemistry and Molecular Biology, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, V6T 1Z3, Canada Fax: +1 604 822 9311 Tel: +1 604 822 4524 E-mail: sadowski@interchange.ubc.ca (Received April 2010, revised 30 May 2010, accepted June 2010) doi:10.1111/j.1742-4658.2010.07728.x Ste12 of Saccharomyces cerevisiae binds to pheromone-response cis-elements (PREs) to regulate several classes of genes Genes induced by pheromones require multimerization of Ste12 for binding of at least two PREs on responsive promoters We have systematically examined nucleotides of the consensus PRE for binding of wild-type Ste12 to DNA in vitro, as well as the organizational requirements of PREs to produce a pheromone response in vivo Ste12 binds as a monomer to a single PRE in vitro, and two PREs upstream of a minimal core promoter cause induction that is proportional to their relative affinity for Ste12 in vitro Although consensus PREs are arranged in a variety of configurations in the promoters of responsive genes, we find that there are severe constraints with respect to how they can be positioned in an artificial promoter to cause induction Two closely-spaced PREs can induce transcription in a directly-repeated or tail-to-tail orientation, although PREs separated by at least 40 nucleotides are capable of inducing transcription when oriented in a head-to-head or tail-to-tail configuration We characterize several examples of promoters that bear multiple consensus PREs or a single PRE in combination with a PRE-like sequence that match these requirements A significant number of responsive genes appear to possess only a single PRE, or PREs in configurations that would not be expected to enable induction, and we suggest that, for many pheromone-responsive genes, Ste12 must activate transcription by binding to cryptic or sub-optimal sites on DNA, or may require interaction with additional uncharacterized DNA bound factors Introduction Ste12 protein of the budding yeast Saccharomyces cerevisiae has attracted considerable interest as a model eukaryotic transcription factor because, much like metazoan factors with a similar function, it regulates multiple distinct classes of genes in response to combinations of signal transduction pathways In haploid yeast, Ste12 activates genes required for mating between MATa and MATa cells to form diploids, in response to peptide pheromones produced by the opposite mating type [1] Ste12 also activates genes necessary for filamentous growth in response to nutrient limitation in a process known as invasive or pseudohyphal growth In both cases, Ste12 activity is regulated by two inhibitor proteins, Dig1 and Dig2 [2], whose functions are considered to be antagonized by a prototypical mitogen-activated protein kinase (MAPK) signaling cascade [3–5] Genes induced by pheromones include those that encode many of the Abbreviations EMSA, electrophoretic mobility shift assay; FRE, filamentous response element; MAPK, mitogen-activated protein kinase; PRE, pheromone response element; RCS, relative competition strength; TCS, Tec1 binding site FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS 3235 Organization of PREs for a pheromone response T.-C Su et al mating pheromone response pathway components, proteins that cause G1 cell cycle arrest along with the morphological alterations necessary for mating, and gene products that eventually contribute to downregulation of the pheromone response, allowing reentry into the cell cycle following mating [6,7] Nutrient limitation induces filamentous growth through up-regulation of genes that alter cell cycle progression, budding pattern, formation of an elongated cellular morphology, increased agar invasiveness and enhanced cellular adhesion [8,9] The regulation of this response involves Ste12 in combination with a host of additional DNA bound factors, including Tec1, Phd1, Flo8 and Sok2 [10], through signals transmitted by the pheromone response MAPK, RascAMP-protein kinase A and Snf1 ⁄ AMP-activated protein kinase pathways [11,12] The capacity of Ste12 to activate these multiple distinct classes of genes in response to pheromone and nutrient signals is considered to involve the binding to DNA at pheromone-response elements (PREs), with the consensus 5¢-ATGAAACA-3¢ [13] in combination with additional factors bound to adjacent sites [7,14– 16] For example, the function of Ste12 with respect to the activation of genes involved in filamentous growth requires interaction with another transcription factor, Tec1 [17] Some filamentous response genes have a PRE adjacent to a Tec1 binding site (TCS) element, and this combination of cis-elements is designated a filamentous response element (FRE) [15] Ste12 and Tec1 bind cooperatively to FREs from the TEC1, FLO11 and TY1 promoters in vitro [15] Several different classes of genes can also be distinguished amongst the pheromone-responsive genes MATa and MATaspecific pheromone-inducible genes, including those encoding the peptide-mating pheromones and their receptors, appear to be regulated by Ste12 bound to DNA in combination with Mcm1 and a1 protein, respectively [14,16] By contrast, pheromone-responsive genes common to both MATa and MATa haploids are considered to require multimerization of Ste12 for binding to multiple adjacent PREs Additionally, genes that become activated later during the pheromone response, such as KAR3 and PRM2 involved in karyogamy, may be regulated by Ste12 in combination with Kar4, whose expression is itself induced by a pheromone [18] Despite having served as an important model for eukaryotic signal-responsive transcription factors for several decades, there is presently little mechanistic or structural information available regarding how Ste12 forms multimers and interacts with additional factors for the regulation of these different classes of genes 3236 Global localization of Ste12 indicates that there are more than 800 target genes in untreated cells [7,19,20], presumably representing those involved in both pheromone and filamentous responses It is generally accepted that Ste12 activates genes for the filamentous response when bound cooperatively to DNA at PREs closely positioned to a binding site for Tec1 [15,21] However, an examination of the arrangement of Ste12 and Tec1 binding sites in promoters of this class reveals a variety of spacing and orientations between PREs and TCS elements, and the FRE-like orientation as characterized from the TY1 and TEC1 promoters is quite rare An implication of this observation is that cooperative interaction between Ste12 and Tec1 must be accommodated by a variety of orientations between their sites Similarly, haploidspecific pheromone-responsive genes, common to both MATa and MATa haploid cells, are presumed to be solely activated by Ste12 multimers bound to adjacent PREs [2] Global expression analysis indicates that more than 200 genes become induced within 30 of treatment with mating pheromone [6,7] Examination of the promoters of a group of the most strongly induced pheromone-responsive genes does not reveal a simple correlation between either the number or arrangement of predicted consensus pheromone response elements (PREs) and the relative level of inducibility (Fig 1), and there are also a significant number of pheromoneinduced genes that appear to completely lack PREs (not shown in Fig 1) [6,7] It might be concluded that there are few restrictions on the arrangement of multiple PREs to enable cooperative interaction for DNA binding of Ste12 for activation of pheromone response Most analyses of Ste12- and pheromone-responsive transcription have been performed in the context of the FUS1 promoter, which contains four PREs within a 100 nucleotide upstream sequence (Fig 1), and whose expression is strongly induced in both MATa and MATa haploid cells in response to a- and a-factor, respectively [13,22] Within the FUS1 promoter, a single PRE was found to confer some responsiveness to pheromone, although a minimum of two were shown to be necessary for a significant response Deletion of all four PREs eliminated the response to pheromone, and the response could be restored by insertion of oligonucleotides bearing the PRE consensus [13] The contribution of spatial and orientation differences between multiple PREs to produce pheromone response was not examined in this previous study and, in any case, the experiments were performed using high copy reporter genes, making it difficult to compare requirements for the expression of chromosomal genes FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS T.-C Su et al Organization of PREs for a pheromone response response to pheromone and the combined strength of the two PREs positioned in an optimal orientation Many natural pheromone-responsive promoters not possess PREs in optimal orientations [7] and, for these genes, we propose that Ste12 must activate transcription when bound to cryptic or sub-optimal sites, or in cooperation with additional uncharacterized transcription factors Results Recombinant wild-type Ste12 binds as a monomer to a single PRE in vitro Fig Organization of a selection of strongly inducible pheromoneresponsive promoters Schematic representation of the organization of consensus PREs within nine of the 35 most strongly induced pheromone response genes (excluding pseudogenes and genes without obvious PREs), as identified by global expression analysis (30 of a-factor treatment) [6,7] Numbers between any two PREs indicate the spacing in nucleotides, whereas the number furthest to the right indicates the distance to the translation start site The promoters are arranged in the relative order of inducibility (top to bottom) STE12 is within the top 100 pheromone-inducible genes, and was included here because we have examined this promoter in some detail Given the apparently relaxed organizational requirements for PREs on pheromone-responsive genes, we expected that it should be relatively trivial to produce artificial pheromone-responsive promoters Instead, in the present study, we find that there are rather stringent constraints on how two consensus PREs can be positioned within a minimal artificial promoter to enable a response to pheromone Wild-type Ste12 binds to a single PRE as a monomer in vitro, and a minimum of two PREs positioned in specific orientations are necessary to cause induction in vivo We find that there is a direct linear relationship between the Several previous studies have examined the binding of recombinant maltose-binding domain-Ste12 fusions or Ste12 DNA-binding domain fragments to an FRE [15], or the FUS1 promoter in vitro [23] We have expressed 6-His-Ste12 in insect cells using baculovirus, and found that the protein is capable of forming complexes in vitro with an oligonucleotide (S26D) containing two directly-repeated PREs from the FUS1 promoter, previously shown to be capable of conferring pheromone-responsiveness in vivo (Fig 2A, lane 2) Antibodies recognizing various Ste12 regions inhibit the formation of the complex (Fig 2A, lanes 8–10) but not control antibodies (Fig 2A, lane 11) Additionally, competition with unlabeled wild-type S26D oligo inhibits complex formation (Fig 2A, lane 3) but not competition with an oligonucleotide bearing a ciselement for an unrelated transcription factor (Fig 2A, lane 6), demonstrating that recombinant wild-type Ste12 protein produced in insect cells forms a sequence-specific interaction with a PRE-containing oligonucleotide in vitro The complex that we observed in an electrophoretic mobility shift assay (EMSA) likely represents the binding of Ste12 to a single PRE on the oligo because competition with an unlabeled competitor bearing a mutation of only one of the PREs does not prevent its formation (Fig 2A, lane 4), although a competitor bearing mutations of both PREs does not compete for binding of Ste12 (Fig 2A, lane 5) Furthermore, oligonucleotide probes containing only a single PRE produce a complex with identical mobility to that produced by oligos with two PREs (not shown; Figs and 4) Recombinant fulllength Ste12 appears to have an autoinhibitory effect because the addition of greater concentrations of protein causes the loss of DNA binding activity altogether (not shown), rather than producing multiple complexes This effect appears to require the C-terminus because a truncated derivative lacking the C-terminal 73 amino acids is able to form multiple complexes on FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS 3237 Organization of PREs for a pheromone response A T.-C Su et al C A B B Fig Recombinant Ste12 produced in insect cells binds to a single PRE in vitro (A) EMSA reactions were performed with extracts of Sf21 insect cells producing recombinant Ste12 protein (lanes 2–11) or uninfected cells (lane 1) using an oligonucleotide probe containing two directly-repeated PREs (sites II and III from the FUS1 promoter, S26D) Unlabeled oligonucleotide competitor oligos were added at ten-fold molar excess (lanes 3–5), as indicated in (B) The binding reaction in lane contained a ten-fold molar excess of an RBEIII oligonucleotide [37] Antibodies against Ste12 (lanes 8–10) or preimmune serum (lane 11) were added to the binding reactions (C) Full-length recombinant Ste12 and Tec1 form a complex on an FRE in vitro EMSA reactions using a labeled FRE probe (CN140 ⁄ 141) derived from the TY1 LTR were performed with Ste12 (lane 1), Tec1-flag (lane 2) or both Ste12 and Tec-1 flag (lanes and 4) Anti-flag sera were added to the binding reaction in lane this same probe (not shown) By contrast, recombinant Ste12 and Tec1, both produced in insect cells, are capable of binding individually to an FRE-containing oligonucleotide in vitro (Fig 2C, lanes and 2), and form a higher-order complex when added together in binding reactions (Fig 2C, lane 3) This indicates that recombinant Ste12, although capable of forming terniary complexes with Tec1 in vitro, is excluded from forming multimerized complexes with two closelyspaced PREs in vitro, which indicates that the binding of wild-type Ste12 to multiple PREs in vivo may require additional factors or post-translational modifications We are currently investigating the significance of this feature with respect to the pheromone response, and we discuss the implications of these observations below To determine the stoichiometry of Ste12 bound to a single PRE in vitro, we expressed a series of C-terminal truncations for use in the analysis of hetero-complex formation Wild-type Ste12 protein produced in insect cells (Fig 3A, lane 1) or truncated versions of Ste12 containing residues 1–476 (Fig 3A, lane 2), 1–350 3238 Fig Ste12 binds to a PRE as a monomer (A) EMSA reactions were performed with a labeled oligo containing a single PRE (IS1430 ⁄ 1431) and full-length Ste12 (lane 1), Ste12 1–476 (lane 2), Ste12 1–350 (lane 3) and Ste12 1–215 (lane 4) Full-length Ste12 was mixed with 1–476 (lane 5), 1–350 (lane 6) or 1–215 (lane 7) prior to adding the labeled oligo and performing the binding reaction (B) Reactions were performed with in vitro translated Ste12 1–476 (lanes 1, and 4), 1–350 (lanes 2, 3, 4, 5, and 8) or 1–215 (lanes 6–8) The Ste12 derivatives were synthesized separately in vitro and then mixed prior to EMSA (lanes and 7) or were co-translated (lanes and 8) (Fig 3A, lane 3) or 1–215 (Fig 3A, lane 4), produced by in vitro transcription and translation, each were capable of forming complexes with a single PRE-containing oligo in EMSA We then mixed the full-length protein together with the truncated forms in vitro prior to adding the labeled oligonucleotide probe and performing EMSA In these experiments, none of the truncated species caused the production of an intermediate complex in combination with wild-type Ste12 (Fig 3A, lanes 5–7), which would be expected if there were multiple protein molecules bound to a single PRE Because it is possible that co-translation of Ste12 may be necessary for hetero-complex formation, as is the case with proteins such as GCN4 and GAL4 [24,25], we also performed this experiment using cotranslation of the truncated Ste12 derivatives (Fig 3B) We found that when the 1–476 and 1–350 or 1–350 and 1–215 derivatives are produced by co-translation (Fig 3B, lanes and 8, respectively), we also not observe intermediate-sized complexes that would indicate formation of hetero-multimers From these results, we argue that Ste12 protein likely binds to a single PRE as a monomer Sequence requirement of the PRE for binding Ste12 in vitro The sequence requirements for binding of Ste12 to DNA have largely been inferred from a comparative FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS T.-C Su et al A B C Fig Nucleotides required for binding of full-length Ste12 to the consensus PRE in vitro (A) EMSA reactions were performed with recombinant wild-type Ste12 and a labeled oligonucleotide bearing a single consensus PRE (RS010 ⁄ 011) Binding reactions contained no competitor (lane 1), or a 0.625- (lanes and 7), 1.25- (lanes and 8), 2.5- (lanes and 9), 5- (lanes and 10) or 10- (lanes and 11) fold molar excess of unlabeled consensus oligo (lanes 2–6) or the indicated mutant oligos (lanes 7–11) Mutant oligos (lines 1–7) contained a single nucleotide substitution from the consensus PRE (Table S1) (B) The sequence of the FUS1 promoter indicating the position of four PREs (designated sites I, II, III and IV, 5¢–3¢) EMSA reactions were performed as in (A) but using a labeled oligonucleotide bearing PRE IV (IS1428 ⁄ 1429), and with the unlabeled competitors as indicated (C) The RCS was calculated for each mutant oligo (Table 1) The effect that mutation of each nucleotide of the consensus PRE has on the binding of Ste12 in vitro is indicated proportional to the font size for each residue analysis of pheromone-responsive promoters and genomic localization of Ste12 protein in vivo [7,10,19] To characterize residues of the PRE that are necessary for affinity of Ste12 in vitro, we performed a systematic analysis using competitions with mutant oligonucleotides in EMSA (Fig 4A) Within the eight nucleotide consensus (ATGAAACA), we found that mutation of Organization of PREs for a pheromone response each of the residues impairs the ability to compete for binding to the wild-type oligo (Fig 4A, PRE mutants) In particular, mutations of residues A5 and A6 of the central AAA trinucleotide to G significantly impair competition (Fig 4A, lines and 6), as does substitution of G3 with a pyrimidine (C or T) (Fig 4A, line 2; Table 1) We also compared the relative affinities of the four PREs within the FUS1 promoter (Fig 4B, designated I, II, III and IV, 5¢–3¢, top) Amongst these, site II is identical to the eight nucleotide consensus, sites III and IV have substitutions of the outer 3¢ and 5¢ nucleotides, respectively, and site I has a substitution of A5 within the AAA trinucleotide Using competition experiments, we were able to rank the relative strengths of PREs within the FUS1 promoter as sites II, IV, III and I (Fig 4B, strongest to weakest; Table 1) Because higher concentrations of recombinant Ste12 produce an autoinhibitory effect, we were unable to determine affinity constants using EMSA with this reagent However, for each mutant oligonucleotide, we calculated a relative competition strength (RCS) value, which represents the ratio of competitor oligonucleotide required to compete for 50% binding of total Ste12 relative to the consensus oligonucleotide within the same experiment (Fig S1 and Table 1) From the RCS values, we predict the relative contribution of each nucleotide within the consensus PRE for binding of wild-type Ste12 in vitro, as shown in Fig 4C Relative affinity of Ste12 for PREs in vitro correlates directly with the pheromone response in vivo To determine by how much the relative affinity of Ste12 for PREs in vitro contributes to the pheromone response in vivo, we inserted oligonucleotides bearing the consensus or mutant PREs into a reporter with a minimal GAL1 core promoter upstream of LacZ, which were integrated in single copy at a lys2 disruption We found that none of the PREs inserted individually upstream of the GAL1 core element were capable of inducing a response to pheromone, even with the strongest of the PREs from the FUS1 promoter (not shown) By contrast, reporters with an insertion of two identical directly-repeated PREs, in either orientation relative to the transcriptional start site (not shown), and arranged in the same context as FUS1 PREs II and III (Fig 4B), all produced a response to pheromone and, interestingly, the level of inducibility correlated with the RCS values for the PREs as determined in vitro (Fig 5A) Accordingly, a duplicated PRE with a substitution of residue A5 of the central AAA FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS 3239 Organization of PREs for a pheromone response T.-C Su et al Table RCS of mutant PREs for binding of wild-type Ste12 to a PRE consensus (ATGAAACA) in vitro FUS1 PREa Sequence RCSb II IV ATGAAACA tTGAAACA AaGAAACA ATaAAACA ATcAAACA ATtAAACA ATGcAACA ATGgAACA ATGAgACA ATGAAgCA ATGAAAgA ATGAAACg 1.00 0.27c 0.14 0.81 0.03c 0.01c 0.69 0.20c 0.02c 0.05c 0.30 0.26 A I III PREs represented in the FUS1 promoter (Fig 4B) b RCS for each oligo was calculated from the concentration of unlabeled competitor oligonucleotide required to compete 50% of total Ste12 protein bound to the consensus PRE, relative to competition in the same experiment with a wild-type PRE (Fig S1) c Concentrations of oligo required for 50% competition was calculated by extrapolation B a trinucleotide to G, which seriously inhibits binding of Ste12 in vitro, produces a small but detectable level of inducibility (Fig 5A, line 4), whereas the duplicated consensus PRE causes a level of pheromone response comparable to the full FUS1 promoter (Fig 5A, lines and 5) Because the inducibility of reporters bearing two directly-repeated PREs appeared to be approximately proportional to the relative affinity for Ste12 in vitro, we were interested in determining the extent that mutations of one PRE would have in combination with a strong consensus element To address this, we introduced mutations of the central AAA trinucleotide into the 3¢ PRE of the artificial reporter constructs Mutation of the central A5 residue of the trinucleotide, causes an approximately three-fold reduction in pheromone inducibility in combination with a consensus PRE (compare Fig 5B, line 1, with Fig 5A, line 1) Mutation of two of the central A residues compromises the response by approximately ten-fold (Fig 5B, line 2), and a PRE bearing substitution of all three A residues completely prevents the response to pheromone (lines 3–5) The latter mutation also completely prevents binding of Ste12 in vitro (not shown) and, in effect, the reporters indicated in lines and of Fig 5B possess only a single functional PRE We also examined the effect that mutations in both directlyrepeated PREs have on pheromone response, and observed that inducibility was reduced significantly when both elements have mutations that limit binding of Ste12 in vitro For example, directly-repeated PREs with substitutions of residues A1 and A8, respectively, 3240 C Fig The pheromone response conferred by two directlyrepeated PREs in vivo is proportional to their relative affinity for Ste12 in vitro (A) Strains bearing single-copy integrations of a minimal GAL1-LacZ reporter bearing two copies of the indicated PRE (lines 1–4) were left untreated (red bars) or treated with a-factor for 60 (blue bars) prior to harvesting the cells and assaying b-galactosidase activity The shading of the boxes containing the PRE sequence indicates the relative competition strength for Ste12 in vitro, with the stronger PREs being shaded darker and the weaker PREs shaded lighter Line shows results from a strain bearing the full FUS1-LacZ promoter (B) Reporter genes bearing a consensus PRE and PREs containing substitutions of the central AAA trinucleotide were assayed as in (A) (C) Combinations of consensus PREs and PREs bearing the indicated mutations were assayed in the same context as described above comprising mutations that have a relatively minor effect on binding Ste12 in vitro, cause an approximately four-fold defect in inducibility relative to two consensus PREs (Fig 5C, line 5) Combinations of PREs that have more serious defects in binding Ste12 produce proportionally less response (Fig 5C, lines and 7), although even two quite weak directly-repeated PREs retain a detectable level of inducibility (Fig 5C, line 8) These results demonstrate that a significant FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS T.-C Su et al response to pheromone can be conferred by a single strong consensus PRE in combination with much weaker adjacent PREs, with a level of inducibility proportional to the relative strength of the second PRE Additionally, duplicated PREs with substitutions that inhibit Ste12 binding are capable of inducing a response to pheromone, but at significantly lower levels Interestingly, when we examined the effect of the combined RCS of two directly-repeated PREs on the response to pheromone, we observed a direct and simple linear relationship between the product of the RCS values and pheromone responsiveness (Fig 6) This analysis indicates that, in the context of the minimal GAL1 promoter, the limiting factor for transcriptional activation in pheromone-treated cells appears to be binding of Ste12 multimers to DNA Organizational constraints on multiple PREs for a pheromone response When examining the promoters of some of the most strongly induced pheromone response genes (Fig 1), we noted that PREs are arranged in a variety of configurations Most promoters have PREs in a directlyrepeated orientation, although there are many instances of PREs arranged in a tail-to-tail configuration (PRM6, FUS1, AGA1 and STE12) Also, there is considerable variability in spacing between multiple PREs (Fig 1) To examine the significance that these differences in configuration have for pheromone Fig The combined relative strength of two directly-repeated PREs produces a proportionally linear response to pheromone A combined relative PRE strength for each of the reporter genes described in Fig was calculated as log(RCSPRE1 · RCSPRE2) and plotted against the respective pheromone responsiveness for each reporter (b-galactosidase activity (· 10)3) Organization of PREs for a pheromone response response, we compared the responses of a GAL1 minimal promoter bearing two consensus PREs positioned at different orientations with respect to each other (Fig 7) In the FUS1 promoter, two PREs (sites II and III) are positioned in a directly-repeated orientation separated by three nucleotides (Fig 7A, line 2) (i.e the same context as the experiments described above) We found that inverting one of the PREs such that they are positioned in a head-to-head orientation completely prevented the response to pheromone (Fig 7A, line 3) By contrast, two consensus PREs from the STE12 promoter positioned in a tailto-tail configuration, separated by a single nucleotide, caused considerably greater induction compared to the directly-repeated PREs from FUS1 (Fig 7A, line 1) This indicates that there are severe organizational constraints for closely-positioned PREs that must limit A B Fig Organizational constraints on closely-spaced PREs for pheromone response in vivo (A) Pheromone responsiveness of minimal promoters containing PREs II and III from the STE12 promoter in a tail-to-tail orientation (line 1), directly-repeated consensus PREs from the FUS1 promoter (PRE II, line 2) or with the second consensus PRE inverted into a head-to-head orientation (line 3) (B) The consensus PREs from the FUS1 promoter were moved apart to produce an intervening spacing of ten (lines 7–9), 20 (lines 4–6) or 40 (lines 1–3) nucleotides, with the orientation of the PREs as indicated FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS 3241 Organization of PREs for a pheromone response T.-C Su et al binding and activation by Ste12 We then examined how the spacing between two directly-repeated consensus PREs affects the observed response, and found that they could not be moved apart without seriously compromising induction (Fig 7B) Separation of PREs by even one nucleotide completely prevented induction, as did separation by three, five, seven (not shown), 10 or 20 nucleotides (Fig 7B, lines 4–9) Curiously, however, two PREs spaced 40 nucleotides apart in either a head-to-head or tail-to-tail orientation produced a significant level of pheromone response (Fig 7B, lines and 3, respectively) Taken together, these results indicate that there must be structural constraints on Ste12 that allow binding to closely-spaced PREs in several different configurations Additionally, the fact that head-to-head and tail-to-tail PREs separated by 40 nucleotides allow induction implies that a sufficient length of intervening DNA is required to bend or twist into a conformation enabling an interaction between Ste12 proteins bound to these PREs We discuss the possible implications of these results further below PREs from the STE12 promoter demonstrate organizational constraints To examine whether the organizational constraints that we observe on artificially produced arrangements of PREs are representative of pheromone-responsive promoters in vivo, we examined the contribution of PREs within the STE12 promoter, which contains four PREs: three in the forward orientation and one in the reverse orientation (Fig 1, bottom) We found that a sub-fragment bearing only the three 5¢ elements (sites I, II and III) caused an elevated level of basal expression, which is dependent upon STE12 (Fig 8, basal expression, compare lines and 2) and, furthermore, that a subfragment bearing only the inverted PREs II and III could account for almost all pheromone inducibility of the STE12 promoter (Fig 8, pheromone induction, line 1, compare lines and 4) Similarly, mutation of site I had only a small negative effect on the response (Fig 8, line 3), whereas mutation of either sites II or III completely prevented induction (Fig 8, lines and 6) These observations indicate that, although PREs may be scattered throughout the promoters of pheromone-responsive genes, in some cases, the majority of pheromone response may involve only two properly spaced and oriented binding sites for Ste12 Pheromone response of promoters with a single consensus PRE Considering the results reported above, we questioned how it is possible that a number of genes amongst those that are strongly induced by pheromone have only a single consensus PRE (Fig 1) [7] CIK1, for example, is one of the most strongly induced genes in pheromone-treated cells, and apparently has only a single consensus PRE We examined the CIK1 promoter to determine whether there were potential weaker binding sites for Ste12 falling within the constraints that we observed on the artificial promoters described above Accordingly, we noted that the CIK1 PRE is positioned only three nucleotides downstream of a PRE-like sequence with substitution at residues T1 and A6 of the consensus (Figs 4C and 9A, top) A portion of the CIK1 promoter bearing these elements inserted upstream of a minimal promoter was found to be strongly induced by pheromone, although deletion Fig Orientation and spacing of PREs contributing to response of the STE12 promoter The sequence of the STE12 promoter region containing the three most distal PREs (designated I, II, and III, 5¢–3¢) is indicated An oligonucleotide representing this sequence, or bearing mutations or deletions as indicated, was inserted upstream of the minimal GAL1 core promoter-LacZ reporter gene The expression of the reporter was measured in untreated cells (basal expression, left) or cells treated with a-factor for 60 (pheromone induction) 3242 FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS T.-C Su et al Organization of PREs for a pheromone response of the PRE-like sequence completely prevented the response (Fig 9A), indicating that this element does contribute to induction by Ste12 multimers in vivo Similarly, on the PRM3 promoter, we observed the PRE-like sequence 5¢-ATAAAACA-3¢ 36 nucleotides upstream of the consensus PRE, positioned in a headto-head orientation (Fig 9B) In vitro, we found that an oligonucleotide bearing this sequence competes for binding to Ste12 only slightly less efficiently than does a consensus PRE (Table 1) A region including these elements inserted upstream of the GAL1 core promoter was responsive to pheromone (Fig 9B, line 1), although the response was reduced considerably when the PRE-like sequence was deleted (Fig 9B, line 2) These results indicate that this PRE-like sequence can A B C A D B Fig A single consensus PRE can confer pheromone responsiveness in conjunction with PRE-like sequences (A) Sequence of the CIK1 promoter region, indicting the consensus PRE and a PRE-like sequence An oligonucleotide representing this sequence, or bearing a deletion of the PRE-sequence, was inserted upstream of the minimal GAL1 core promoter-LacZ reporter, and expression was measured in untreated and pheromone-treated cells (B) Sequence of the PRM3 promoter indicating the location of a consensus PRE and PRE-like sequence The pheromone responsiveness of the minimal promoter bearing oligonucleotides representing the wild-type or mutant promoter sequences was measured in untreated and pheromone-treated cells Fig 10 Structural constraints on Ste12 for binding closely-positioned PREs Schematic representation of a possible mechanism for the recognition of closely-spaced PREs in different conformations by Ste12 multimers Interaction with directly-repeated PREs, positioned three nucleotides apart (A) or in a tail-to-tail orientation (B) may involve an interaction with C-terminal sequences separated from the N-terminal DNA binding domain by a flexible linker region Some closely-spaced configurations appear to be excluded from binding Ste12 multimers, as in a closely-spaced head-to-head orientation (C) Head-to-head and tail-to-tail orientations may be accommodated providing that the sites are separated sufficiently to allow bending or twisting of the intervening DNA to enable binding of Ste12 multimers (D) produce a pheromone response by Ste12 multimers oriented in a head-to-head conformation approximately 40 nucleotides away from a consensus PRE, and we had demonstrated this effect with the artificial promoters Taken together, these results indicate that, for some pheromone-responsive genes, Ste12 must activate transcription from sub-optimal binding sites, in combination with a single consensus PRE whose arrangement falls within specific organizational constrains FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS 3243 Organization of PREs for a pheromone response T.-C Su et al We note, however, that we have only examined subfragments for both of these promoters, and there are likely to be additional factors that contribute to response In this vein, it is important to note that both were shown to be Kar4-dependent [18] Discussion The pheromone response pathway of Saccharomyces has provided an important model for understanding how genes are regulated in response to signals transmitted through MAP kinase cascades However, despite almost 20 years of intensive research, there remain many unanswered questions regarding the function of Ste12, including the molecular mechanisms that control its activity by upstream MAPKs, how it causes transcriptional activation, and the nature of its interaction with PREs on DNA To begin addressing the latter issue, we have performed a systematic analysis of Ste12 binding to the PRE in vitro, and studied the relationship between binding affinity and spatial orientation between two PREs for pheromone responsiveness in vivo Ste12 likely binds to a single PRE in vitro as a monomer, and therefore the protein must require multimerization in vivo to bind DNA and activate the haploid-specific pheromone response because a minimum of two PREs are required Surprisingly, based on analysis of artificial promoters containing two PREs, there appear to be serious constraints with respect to how these can be positioned relative to one another to enable pheromone response of an artificial promoter Two directly-repeated PREs cause activation only when located within three nucleotides of each other By contrast, PREs inverted in a tail-to-tail conformation separated by a single nucleotide produce a very strong response Additionally, PREs oriented in head-to-head or tail-to-tail configurations are only able to cause a pheromone response when separated by approximately 40 nucleotides Taken together, these observations indicate that Ste12 must have structural features that can accommodate multimerization for binding of closely-spaced sites oriented in several different conformations (Fig 10), such that binding to closely-positioned PREs in either a directly-repeated (Fig 10A) or tail-to-tail conformation (Fig 10B) may form multimers through interaction between surfaces on the Ste12 protein that are separated from the DNA-binding domain by a flexible linker in order to accommodate different orientations Because PREs oriented in a head-to-head manner not produce a response, the flexibility of Ste12 may not be able to accommodate this particular orientation, or perhaps the N-terminal DNA binding domain 3244 is sterically precluded from such an interaction (Fig 10C) PREs oriented in either a head-to-head or tail-to-tail conformation are capable of inducing a pheromone response if positioned 40 nucleotides apart (i.e approximately four helical turns of DNA), suggesting that Ste12 is capable of forming multimers that can bind these configurations, provided that the intervening DNA is able to bend or twist into a conformation that can accommodate the interaction (Fig 10D) An additional possibility is that Ste12 multimerization in vivo, enabling accommodation of various PRE arrangements, may require additional nuclear factors Accordingly, Ste12 was shown to associate on pheromone response promoters in vivo with both inhibitor proteins Dig1 and Dig2 [2], and so it is possible these proteins facilitate the binding of Ste12 to PREs arranged in various configurations However, we consider this to be unlikely considering that the activation of Ste12-dependent genes appears to be constitutive in dig1 dig2 null strain backgrounds [3–5], presumably including genes requiring a variety of PRE orientations for a pheromone response Curiously, recombinant wild-type Ste12 produced in insect cells is incapable of forming multimers on oligos containing two PREs in vitro, despite the fact that the same arrangement of PREs confers a strong response to pheromone in vivo Furthermore, full-length Ste12 appears to have an autoinhibitory function because high concentrations of protein completely prevent binding to DNA Because the deletion of the C-terminus prevents these effects (not shown), we suggest that multimerization of Ste12 in vivo must be regulated through a mechanism involving the C-terminus Ste12 produced in insect cells becomes phosphorylated on most of the same residues that we have observed in yeast [26,27], and we find that mild treatment with phosphatase in vitro produces slower migrating complexes with oligos containing two PREs (T.-C Su and I Sadowski, unpublished results), suggesting that phosphorylation may regulate the ability to bind multiple adjacent PREs By contrast, recombinant wild-type Ste12 does produce terniary complexes with Tec1 on an FRE-containing oligo in vitro (Fig 2C) These results suggest that activation of haploid-specific pheromone-responsive genes, but not Ste12 ⁄ Tec1-responsive genes, may require additional regulation in vivo involving dephosphorylation The results obtained in the present study also raise the important question of why two PREs are required for pheromone response if wild-type Ste12 is able to bind to a single PRE in vitro This indicates that either the activation domain of Ste12 is incapable of activating transcription when bound to a single site, or that binding to a single site FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS T.-C Su et al in vivo is limited by additional factors Consistent with the latter possibility, it was shown that Ste12 does not interact with filamentous response promoters (containing a single PRE) in the absence of Tec1 [2], indicating that Ste12 is prevented from binding a single PRE in vivo on its own This effect is likely mediated by the inhibitor proteins Dig1 and ⁄ or Dig2 [3,28] and, consistent with this, we found that binding of wild-type Ste12 to a single PRE in vitro is inhibited by the addition of recombinant Dig1 and Dig2 (T.-C Su and I Sadowski, unpublished results) We have systematically examined nucleotides within the PRE by mutagenesis, and have compared the relative affinity of natural sites within the FUS1 promoter for binding of wild-type Ste12 in vitro Using an artificial reporter bearing two PREs arranged in a directlyrepeated orientation, we find that there is a significant and simple linear relationship between the combined relative strength of the two PREs in vitro and the level of pheromone responsiveness in vivo (Fig 6) This suggests that, in pheromone-treated cells, using a concentration of pheromone where presumably Ste12 is free of inhibition by the regulatory proteins Dig1 and Dig2 [2], the association of Ste12 protein with cis-elements on DNA is probably the limiting interaction for induction, at least in the context of our artificial promoters However, we envisage that many, if not most, natural promoters controlled by Ste12 will also be subject to the additional effects of nucleosome positioning, which likely would significantly alter the effects produced by combinations of PREs with different affinities for Ste12 protein, as previously shown for transcriptional activation by Pho4 [29,30] Upon cursory examination of the most strongly induced pheromone-responsive promoters in vivo, it could not be predicted that there should be such severe constraints on the organization of PREs for induction by pheromone (Fig 1) Most of these promoters appear to have PREs arranged without any particular defined conformation, some promoters appear to only have a single PRE, and other pheromone-responsive promoters have none (not shown in Fig 1) On the basis of the results obtained in the present study, we expect that many pheromone-responsive genes must rely on nonconsensus weaker binding sites for Ste12, which are positioned adjacent to consensus PREs in a conformation that can accommodate the binding of Ste12 multimers We have detailed such instances on sub-fragments of the CIK1 and PRM3 promoters (Fig 9) Both of these genes are also regulated by Kar4 [18], and it will be interesting to determine how these factors interact within the context of their full promoters to promote induction during pheromone Organization of PREs for a pheromone response response On several promoters, including FUS1 and STE12, we find that only two PREs oriented in an optimal configuration can account for the majority of pheromone response, and this suggests that many genes strongly induced by pheromone may only require two properly oriented PREs Many pheromone-responsive promoters bear consensus PREs positioned some distance apart, and the results obtained in the present study indicate that two consensus elements oriented in a head-to-head or tail-to-tail orientation at least 40 nucleotides apart can confer a significant response Such configurations are observed on many natural pheromone-responsive promoters, including FUS3 and PRM6 (Fig 1) Furthermore, PRE I of the FUS1 promoter is oriented in a tail-to-tail conformation with respect to the three more proximal sites (II, III, and IV) and, consequently, this may allow activation by Ste12 multimers from any combination of these proximal three sites Several other promoters, with either a single consensus PRE or with two PREs in orientations that should occlude a pheromone response based on our data, have potential weaker Ste12 binding sites positioned in a tail-to-tail orientation We find such examples within the FIG1 and PRM4 promoters (Fig 1) There also genes that are strongly induced by pheromone but appear to lack a consensus PRE, including many of the PRMs, ASG7, FIG2, FIG3, ECM18 and MCH2 (not shown) In these cases, Ste12 must activate from multiple nonconsensus binding sites or through cooperative interaction on weaker elements with additional DNA binding proteins, such as Mcm1 [16,31] and Kar4 [18], and perhaps with previously unrecognized additional factors Consistent with this possibility, it was recently shown that there is a strong correlation between the association of Ste12 on pheromone-responsive promoters with potential binding sites for Flo8, suggesting that pheromone response for many genes may involve an association between these factors [7] Accordingly, it is interesting that the function of Ste12 with respect to activating transcription in response to the pheromone-response MAPK pathway is remarkably similar to TFII-I, which is a protein in mammalian cells that performs this function in response to MAPK signaling downstream of RAS through cooperative interactions on upstream elements with a number of factors, including serum response factor, PHOX1, nuclear factor-jB and upstream stimulatory factor [32,33] The results reported in the present study demonstrate that many aspects of Ste12 regulation at the molecular level are still not well understood This protein appears to bind as a monomer to a single PRE in vitro, FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS 3245 Organization of PREs for a pheromone response T.-C Su et al although at least two properly configured PREs are necessary for a pheromone response in vivo It will be important to elucidate the structural features of Ste12 that impose these restrictions, as well as the mechanisms controlling the interaction of this factor with multiple PREs in vivo to mediate pheromone response Materials and methods Oligoucleotides, plasmids and yeast strains Sequences of oligonucleotides for construction of minimal promoter reporters are detailed in Table S2 Oligonucleotides for construction of reporter genes were annealed and cloned into the XhoI ⁄ XbaI sites of pIS341, which is a lys2 disintegrator vector [34], bearing the GAL1 core promoter region upstream of LacZ and the ADH1 terminator All experiments were performed in a W303-1A strain background (MATa ade2 leu2 trp1 ura3 can1) Reporter gene plasmids were linearized by digestion with NruI prior to transformation into yeast using the LiAc technique [35] URA+ transformants were allowed to grow nonselectively on yeast extract peptone dextrose for days to allow rearrangement of the disintegrator, prior to streaking for single colonies on 5-fluoroorotic acid Strains bearing reporter gene integrants at the lys2 disruption were identified by replica plating, and single copy integration was verified by analysis of chromosomal DNA using PCR [34] The pheromone responsiveness of strains bearing the reporter genes was assayed in cultures grown in yeast extract peptone dextrose until A600 of 0.6 was reached Pheromone was added at a concentration of lgỈmL)1 The cells were collected and b-galactosidase activity was assayed as described previously [36]; the results represent an average of three independent experiments TNT T7 Quick Coupled Transcription ⁄ Translation System (Promega, Madison, WI, USA) Briefly, plasmid pSC4, which contains a full-length genomic clone of STE12, was used as template for amplification with oligonucleotide oIS1144, in combination with oVT2, oET30 and oIS1146 (Table S3), to produce fragments with a 5¢ T7 RNA polymerase promoter and encoding Ste12 (1–215), Ste12 (1–350) and Ste12 (1–479), respectively The Ste12 derivatives were produced individually or by co-translation in 50 lL reactions containing lL of T7 RNA polymerase and 40 lL of rabbit reticulocyte lysate The reactions were carried out at 30 °C for 90 and then assayed immediately for DNA binding activity Oligonucleotides used for EMSA are detailed in Table S1, and were annealed and labeled using Klenow (New England Biolabs, Beverly, MA, USA) with [32P]adATP and [32P]adTTP, as described previously [37] The 5¢ overhangs of unlabeled competitor oligonucleotides were filled in using Klenow and an unlabeled dNTP mixture EMSA reactions contained lL of labeled oligonucleotide probe (2 pmol), lg of poly(dI-dC) (Sigma, St Louis, MO, USA), 2.5 mm MgCl2, 1% glycerol, 20 mm Tris (pH 8.0), 40 mm NaCl and lL of Sf21 extract or in vitro translation reaction in a total volume of 20 lL Labeled oligonucleotide probes were added to the binding reactions after a 30 pre-incubation on ice with unlabeled competitor oligos or specific antibodies Binding reactions were performed at room temperature for 30 and the reactions were resolved on nondenaturing polyacrylamide gels containing 0.5 · TBE (89 mm Tris, 89 mm Boric acid, mm EDTA, pH 8.0) buffer and 1% glycerol at 200 V for h Signals produced in the EMSA reactions were quantitated using imagequant software (GE Healthcare, Milwaukee, WI, USA) Acknowledgements Recombinant proteins and EMSA Full-length Ste12 protein was expressed as an N-terminal 6-His fusion in insect cells using baculovirus in the Sf21 insect cell line [26] Tec1 was expressed with a 6-His-N-terminal and C-terminal flag epitope tag using the Bac-to-Bac system (Invitrogen, Carlsbad, CA, USA) Antibodies A3, B3 and F3 were raised against Escherichia coli TrpE fused to Ste12 residues (265–688), (314–688) and (1–215), respectively Sf21 cells infected with Ste12 and Tec1 virus were collected and washed in ice-cold lysis buffer (20 mm Tris, pH 8.0, 40 mm NaCl, mm dithiothreitol, 5% glycerol, 2.5 mm MgCl2, mm Na3VO4, mm EGTA, 50 mm NaF and 20 mm b-glycerol phosphate) The cells were lysed by forcing through a 27-gauge needle ten times, and then sonicated for 10 s A clarified supernatant was obtained by centrifugation at 10 000 g for 10 Ste12 proteins were produced by in vitro transcription and translation using the 3246 We thank LeAnn Howe, Mike Kobor, Viven Measday, Sheetal Raithatha and Kris Barretto for their helpful comments This research was supported by funds from the Canadian Cancer Society Research Institute (grant 018436) References Fields S, Chaleff DT & Sprague GF Jr (1988) Yeast STE7, STE11, and STE12 genes are required for expression of cell-type-specific genes Mol Cell Biol 8, 551–556 Chou S, Lane S & Liu H (2006) Regulation of mating and filamentation genes by two distinct Ste12 complexes in Saccharomyces cerevisiae Mol Cell Biol 26, 4794– 4805 Tedford K, Kim S, Sa D, Stevens K & Tyers M (1997) Regulation of the mating pheromone and invasive FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS T.-C Su et al 10 11 12 13 14 15 16 17 growth responses in yeast by two MAP kinase substrates Curr Biol 7, 228–238 Bardwell L, Cook JG, Zhu-Shimoni JX, Voora D & Thorner J (1998) Differential regulation of transcription: repression by unactivated mitogen-activated protein kinase Kss1 requires the Dig1 and Dig2 proteins Proc Natl Acad Sci USA 95, 15400–15405 Cook JG, Bardwell L, Kron SJ & Thorner J (1996) Two novel targets of the MAP kinase Kss1 are negative regulators of invasive growth in the yeast Saccharomyces cerevisiae Genes Dev 10, 2831–2848 Roberts CJ, Nelson B, Marton MJ, Stoughton R, Meyer MR, Bennett HA, He YD, Dai H, Walker WL, Hughes TR et al (2000) Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles Science 287, 873–880 Zheng W, Zhao H, Mancera E, Steinmetz LM & Snyder M (2010) Genetic analysis of variation in transcription factor binding in yeast Nature 464, 1187–1191 Wittenberg C & La Valle R (2003) Cell-cycle-regulatory elements and the control of cell differentiation in the budding yeast Bioessays 25, 856–867 Gagiano M, Bauer FF & Pretorius IS (2002) The sensing of nutritional status and the relationship to filamentous growth in Saccharomyces cerevisiae FEMS Yeast Res 2, 433–470 Borneman AR, Leigh-Bell JA, Yu H, Bertone P, Gerstein M & Snyder M (2006) Target hub proteins serve as master regulators of development in yeast Genes Dev 20, 435–448 Zaman S, Lippman SI, Zhao X & Broach JR (2008) How Saccharomyces responds to nutrients Annu Rev Genet 42, 27–81 Orlova M, Ozcetin H, Barrett L & Kuchin S (2009) Roles of the Snf1-activating kinases during nitrogen limitation and pseudohyphal differentiation in Saccharomyces cerevisiae Eukaryot Cell 9, 208–214 Hagen DC, McCaffrey G & Sprague GF Jr (1991) Pheromone response elements are necessary and sufficient for basal and pheromone-induced transcription of the FUS1 gene of Saccharomyces cerevisiae Mol Cell Biol 11, 2952–2961 Sengupta P & Cochran BH (1991) MAT alpha can mediate gene activation by a-mating factor Genes Dev 5, 1924–1934 Madhani HD & Fink GR (1997) Combinatorial control required for the specificity of yeast MAPK signaling Science 275, 1314–1317 Yuan YO, Stroke IL & Fields S (1993) Coupling of cell identity to signal response in yeast: interaction between the alpha and STE12 proteins Genes Dev 7, 1584– 1597 Gavrias V, Andrianopoulos A, Gimeno CJ & Timberlake WE (1996) Saccharomyces cerevisiae TEC1 Organization of PREs for a pheromone response 18 19 20 21 22 23 24 25 26 27 28 29 30 31 is required for pseudohyphal growth Mol Microbiol 19, 1255–1263 Lahav R, Gammie A, Tavazoie S & Rose MD (2007) Role of transcription factor Kar4 in regulating downstream events in the Saccharomyces cerevisiae pheromone response pathway Mol Cell Biol 27, 818–829 Zeitlinger J, Simon I, Harbison CT, Hannett NM, Volkert TL, Fink GR & Young RA (2003) Programspecific distribution of a transcription factor dependent on partner transcription factor and MAPK signaling Cell 113, 395–404 Lefrancois P, Euskirchen GM, Auerbach RK, Rozowsky J, Gibson T, Yellman CM, Gerstein M & Snyder M (2009) Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing BMC Genomics 10, 37 Kohler T, Wesche S, Taheri N, Braus GH & Mosch HU (2002) Dual role of the Saccharomyces cerevisiae TEA ⁄ ATTS family transcription factor Tec1p in regulation of gene expression and cellular development Eukaryot Cell 1, 673–686 Fields S & Herskowitz I (1985) The yeast STE12 product is required for expression of two sets of cell-type specific genes Cell 42, 923–930 Yuan YL & Fields S (1991) Properties of the DNAbinding domain of the Saccharomyces cerevisiae STE12 protein Mol Cell Biol 11, 5910–5918 Carey M, Kakidani H, Leatherwood J, Mostashari F & Ptashne M (1989) An amino-terminal fragment of GAL4 binds DNA as a dimer J Mol Biol 209, 423–432 Hope IA & Struhl K (1987) GCN4, a eukaryotic transcriptional activator protein, binds as a dimer to target DNA EMBO J 6, 2781–2784 Nelson C, Goto S, Lund K, Hung W & Sadowski I (2003) Srb10 ⁄ Cdk8 regulates yeast filamentous growth by phosphorylating the transcription factor Ste12 Nature 421, 187–190 Hung W, Olson KA, Breitkreutz A & Sadowski I (1997) Characterization of the basal and pheromonestimulated phosphorylation states of Ste12p Eur J Biochem 245, 241–251 Olson KA, Nelson C, Tai G, Hung W, Yong C, Astell C & Sadowski I (2000) Two regulators of Ste12p inhibit pheromone-responsive transcription by separate mechanisms Mol Cell Biol 20, 4199–4209 Kim HD & O’Shea EK (2008) A quantitative model of transcription factor-activated gene expression Nat Struct Mol Biol 15, 1192–1198 Lam FH, Steger DJ & O’Shea EK (2008) Chromatin decouples promoter threshold from dynamic range Nature 453, 246–250 Hwang-Shum JJ, Hagen DC, Jarvis EE, Westby CA & Sprague GF Jr (1991) Relative contributions of MCM1 and STE12 to transcriptional activation of a- and FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS 3247 Organization of PREs for a pheromone response 32 33 34 35 36 37 T.-C Su et al alpha-specific genes from Saccharomyces cerevisiae Mol Gen Genet 227, 197–204 Chen J, Malcolm T, Estable MC, Roeder RG & Sadowski I (2005) TFII-I regulates induction of chromosomally integrated human immunodeficiency virus type long terminal repeat in cooperation with USF J Virol 79, 4396–4406 Kim DW, Cheriyath V, Roy AL & Cochran BH (1998) TFII-I enhances activation of the c-fos promoter through interactions with upstream elements Mol Cell Biol 18, 3310–3320 Sadowski I, Su TC & Parent J (2007) Disintegrator vectors for single-copy yeast chromosomal integration Yeast 24, 447–455 Gietz RD & Schiestl RH (2007) High-efficiency yeast transformation using the LiAc ⁄ SS carrier DNA ⁄ PEG method Nat Protoc 2, 31–34 Amberg DC, Burke DJ & Strathern JN (2005) Assay of b-galactosidase in yeast: assay of crude extracts In Methods in Yeast Genetics Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY Chen J, Malcolm T, Estable M, Roeder R & Sadowski I (2005) TFII-I regulates induction of chromosomally integrated human immunodeficiency virus type long 3248 terminal repeat in cooperation with USF J Virol 79, 4396–4406 Supporting information The following supplementary material is available: Fig S1 Graphical representation of competition experiments for Ste12 DNA binding in EMSA Table S1 Annealed double-stranded oligonucleotides for use as probes and competitors in EMSA reactions Table S2 Annealed oligonucleotides used for construction of reporter gene plasmids and yeast strains Table S3 Oligonucletides for production of templates for in vitro transcription and translation reactions This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS ... for binding of wild-type Ste12 to a PRE consensus (ATGAAACA) in vitro FUS1 PREa Sequence RCSb II IV ATGAAACA tTGAAACA AaGAAACA ATaAAACA ATcAAACA ATtAAACA ATGcAACA ATGgAACA ATGAgACA ATGAAgCA ATGAAAgA... This indicates that there are severe organizational constraints for closely-positioned PREs that must limit A B Fig Organizational constraints on closely-spaced PREs for pheromone response in vivo... PREs bearing the indicated mutations were assayed in the same context as described above comprising mutations that have a relatively minor effect on binding Ste12 in vitro, cause an approximately