Organizational constraints on Ste12 cis-elements for a
pheromone response in Saccharomyces cerevisiae
Ting-Cheng Su
1,2
, Elena Tamarkina
1
and Ivan Sadowski
1
1 Department of Biochemistry and Molecular Biology, Molecular Epigenetics, LSI, University of British Columbia, Vancouver, Canada
2 Graduate Program in Genetics, University of British Columbia, Vancouver, Canada
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 regu-
lates multiple distinct classes of genes in response to
combinations of signal transduction pathways. In hap-
loid 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 nutri-
ent 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
Keywords
gene regulation; pheromone response; PRE,

sess 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.
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
mating pheromone response pathway components,
proteins that cause G
1
cell cycle arrest along with the
morphological alterations necessary for mating, and
gene products that eventually contribute to down-
regulation of the pheromone response, allowing re-
entry 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 elon-
gated 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, Ras-
cAMP-protein kinase A and Snf1⁄ AMP-activated pro-
tein kinase pathways [11,12].
The capacity of Ste12 to activate these multiple dis-
tinct classes of genes in response to pheromone and

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.
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 gen-
erally accepted that Ste12 activates genes for the fila-
mentous 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 obser-
vation is that cooperative interaction between Ste12
and Tec1 must be accommodated by a variety of ori-
entations between their sites. Similarly, haploid-
specific 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 min 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

ments 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 strin-
gent 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 orienta-
tions are necessary to cause induction in vivo.Wefind
that there is a direct linear relationship between the
response to pheromone and the combined strength of
the two PREs positioned in an optimal orientation.
Many natural pheromone-responsive promoters do not
possess PREs in optimal orientations [7] and, for these
genes, we propose that Ste12 must activate transcrip-
tion when bound to cryptic or sub-optimal sites, or in
cooperation with additional uncharacterized transcrip-
tion factors.
Results
Recombinant wild-type Ste12 binds as a
monomer to a single PRE in vitro
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 com-
plexes in vitro with an oligonucleotide (S26D) contain-
ing two directly-repeated PREs from the FUS1

73 amino acids is able to form multiple complexes on
Fig. 1. Organization of a selection of strongly inducible pheromone-
responsive 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 min 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.
T C. Su et al. Organization of PREs for a pheromone response
FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS 3237
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 1 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 terni-
ary complexes with Tec1 in vitro, is excluded from
forming multimerized complexes with two closely-
spaced PREs in vitro, which indicates that the binding
of wild-type Ste12 to multiple PREs in vivo may
require additional factors or post-translational modifi-
cations. We are currently investigating the significance
of this feature with respect to the pheromone response,
and we discuss the implications of these observations

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
AB
Fig. 3. 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 reac-
tion. (B) Reactions were performed with in vitro translated Ste12
1–476 (lanes 1, 3 and 4), 1–350 (lanes 2, 3, 4, 5, 7 and 8) or 1–215
(lanes 6–8). The Ste12 derivatives were synthesized separately
in vitro and then mixed prior to EMSA (lanes 3 and 7) or were
co-translated (lanes 4 and 8).
AC
B
Fig. 2. Recombinant Ste12 produced in insect cells binds to a sin-
gle 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 6 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 com-
plex on an FRE in vitro. EMSA reactions using a labeled FRE probe

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 oligonucleo-
tide 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 disrup-
tion. We found that none of the PREs inserted individ-
ually 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 phero-

comparable to the full FUS1 promoter (Fig. 5A, lines
1 and 5).
Because the inducibility of reporters bearing
two directly-repeated PREs appeared to be approxi-
mately 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 phero-
mone 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 compro-
mises 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 phero-
mone (lines 3–5). The latter mutation also completely
prevents binding of Ste12 in vitro (not shown) and, in
effect, the reporters indicated in lines 4 and 5 of
Fig. 5B possess only a single functional PRE. We also
examined the effect that mutations in both directly-
repeated 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,
comprising mutations that have a relatively minor
effect on binding Ste12 in vitro, cause an approxi-

III ATGAAACg 0.26
a
PREs represented in the FUS1 promoter (Fig. 4B).
b
RCS for each
oligo was calculated from the concentration of unlabeled competi-
tor 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.
A
B
C
Fig. 5. The pheromone response conferred by two directly-
repeated PREs in vivo is proportional to their relative affinity for
Ste12 in vitro. (A) Strains bearing single-copy integrations of a mini-
mal 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 min (blue bars) prior to harvesting the cells and assaying b-galac-
tosidase 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 5 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.

response, we compared the responses of a GAL1 mini-
mal 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 orienta-
tion 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 orien-
tation completely prevented the response to phero-
mone (Fig. 7A, line 3). By contrast, two consensus
PREs from the STE12 promoter positioned in a tail-
to-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 con-
straints for closely-positioned PREs that must limit
Fig. 6. 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. 5 was calculated as log(RCS
PRE1
· RCS
PRE2
) and
plotted against the respective pheromone responsiveness for each
reporter (b-galactosidase activity (· 10
)3
).
A

sufficient length of intervening DNA is required to
bend or twist into a conformation enabling an inter-
action 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 pro-
moters 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-frag-
ment 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 1 and 2) and, furthermore, that a sub-
fragment 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 1 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 5 and
6). These observations indicate that, although PREs
may be scattered throughout the promoters of phero-
mone-responsive genes, in some cases, the majority of
pheromone response may involve only two properly
spaced and oriented binding sites for Ste12.

untreated cells (basal expression, left) or
cells treated with a-factor for 60 min
(pheromone induction).
Organization of PREs for a pheromone response T C. Su et al.
3242 FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS
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 head-
to-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 pro-
moter 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
produce a pheromone response by Ste12 multimers ori-
ented in a head-to-head conformation approximately
40 nucleotides away from a consensus PRE, and we
had demonstrated this effect with the artificial promo-
ters. Taken together, these results indicate that, for
some pheromone-responsive genes, Ste12 must activate
transcription from sub-optimal binding sites, in combi-
nation with a single consensus PRE whose arrange-
ment falls within specific organizational constrains.
A

T C. Su et al. Organization of PREs for a pheromone response
FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS 3243
We note, however, that we have only examined sub-
fragments 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 trans-
mitted through MAP kinase cascades. However,
despite almost 20 years of intensive research, there
remain many unanswered questions regarding the func-
tion of Ste12, including the molecular mechanisms that
control its activity by upstream MAPKs, how it causes
transcriptional activation, and the nature of its interac-
tion with PREs on DNA. To begin addressing the lat-
ter issue, we have performed a systematic analysis of
Ste12 binding to the PRE in vitro, and studied the rela-
tionship between binding affinity and spatial orienta-
tion 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 mul-
timerization in vivo to bind DNA and activate the
haploid-specific pheromone response because a mini-
mum of two PREs are required.
Surprisingly, based on analysis of artificial promot-
ers containing two PREs, there appear to be serious
constraints with respect to how these can be positioned

vening DNA is able to bend or twist into a conforma-
tion 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 phero-
mone 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 con-
sider 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 com-

the PRE by mutagenesis, and have compared the rela-
tive affinity of natural sites within the FUS1 promoter
for binding of wild-type Ste12 in vitro. Using an artifi-
cial reporter bearing two PREs arranged in a directly-
repeated 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 sug-
gests that, in pheromone-treated cells, using a concen-
tration 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 induc-
tion, 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

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 orienta-
tion. We find such examples within the FIG1 and
PRM4 promoters (Fig. 1).
There also genes that are strongly induced by phero-
mone 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 addi-
tional DNA binding proteins, such as Mcm1 [16,31]
and Kar4 [18], and perhaps with previously unrecog-
nized additional factors. Consistent with this possibil-
ity, it was recently shown that there is a strong
correlation between the association of Ste12 on phero-
mone-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 func-
tion 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 stimu-
latory factor [32,33].

gene integrants at the lys2 disruption were identified by rep-
lica plating, and single copy integration was verified by
analysis of chromosomal DNA using PCR [34]. The phero-
mone responsiveness of strains bearing the reporter genes
was assayed in cultures grown in yeast extract peptone dex-
trose until A
600
of 0.6 was reached. Pheromone was added
at a concentration of 2 lgÆ mL
)1
. The cells were collected
and b-galactosidase activity was assayed as described previ-
ously [36]; the results represent an average of three indepen-
dent experiments.
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-ter-
minal 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), respec-
tively. 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, 1 mm dithiothreitol, 5% glycerol,
2.5 mm MgCl
2
,1mm Na
3
VO

ture. EMSA reactions contained 1 lL of labeled oligonu-
cleotide probe (2 pmol), 2 l g of poly(dI-dC) (Sigma,
St Louis, MO, USA), 2.5 m m MgCl
2
, 1% glycerol, 20 mm
Tris (pH 8.0), 40 mm NaCl and 1 l L 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 min pre-incubation on ice with unla-
beled competitor oligos or specific antibodies. Binding reac-
tions were performed at room temperature for 30 min and
the reactions were resolved on nondenaturing polyacryl-
amide gels containing 0.5 · TBE (89 mm Tris, 89 mm Boric
acid, 2 mm EDTA, pH 8.0) buffer and 1% glycerol at
200 V for 3 h. Signals produced in the EMSA reactions
were quantitated using imagequant software (GE Health-
care, Milwaukee, WI, USA).
Acknowledgements
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).
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should be addressed to the authors.
Organization of PREs for a pheromone response T C. Su et al.
3248 FEBS Journal 277 (2010) 3235–3248 ª 2010 The Authors Journal compilation ª 2010 FEBS