We have systematically examined nucleotides of the consen-sus PRE for binding of wild-type Ste12 to DNA in vitro, as well as the organizational requirements of PREs to produce a pheromon
Trang 1pheromone response in Saccharomyces cerevisiae
Ting-Cheng Su1,2, Elena Tamarkina1and Ivan Sadowski1
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,
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 1 April 2010, revised 30 May
2010, accepted 3 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 respon-sive promoters We have systematically examined nucleotides of the consen-sus 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 posi-tioned 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 pos-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.
Trang 2mating 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
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
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
fil-amentous response element (FRE) [15] Ste12 and
Tec1 bind cooperatively to FREs from the TEC1,
FLO11and TY1 promoters in vitro [15] Several
differ-ent classes of genes can also be distinguished amongst
the pheromone-responsive genes MATa and
MATa-specific 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
phero-mone 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
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 (PREs) and the relative level of inducibility (Fig 1), and there are also a significant number of pheromone-induced 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 bind-ing 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 com-pare requirements for the expression of chromosomal genes
Trang 3Given the apparently relaxed organizational
require-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 We find
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 promoter, previously shown to be capable of confer-ring pheromone-responsiveness in vivo (Fig 2A, lane 2) Antibodies recognizing various Ste12 regions inhi-bit the formation of the complex (Fig 2A, lanes 8–10) but not control antibodies (Fig 2A, lane 11) Addi-tionally, competition with unlabeled wild-type S26D oligo inhibits complex formation (Fig 2A, lane 3) but not competition with an oligonucleotide bearing a cis-element 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 contain-ing only a scontain-ingle PRE produce a complex with identi-cal mobility to that produced by oligos with two PREs (not shown; Figs 3 and 4) Recombinant full-length Ste12 appears to have an autoinhibitory effect because the addition of greater concentrations of pro-tein causes the loss of DNA binding activity altogether (not shown), rather than producing multiple com-plexes 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
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.
Trang 4this 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
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
(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-con-taining 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 per-forming EMSA In these experiments, none of the truncated species caused the production of an inter-mediate 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 co-translation 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 4 and 8, respectively), we also do not observe intermediate-sized complexes that would indi-cate 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
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).
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
(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 3 and 4) Anti-flag sera were added to the binding reaction in
lane 4.
Trang 5analysis of pheromone-responsive promoters and
geno-mic 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
oligonucleo-tides in EMSA (Fig 4A) Within the eight nucleotide
consensus (ATGAAACA), we found that mutation of
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 5 and 6), as does substitu-tion 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 substitu-tion of A5 within the AAA trinucleotide Using com-petition 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 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-mone and, interestingly, the level of inducibility corre-lated 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
A
B
C
Fig 4 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 2 and 7), 1.25- (lanes 3
and 8), 2.5- (lanes 4 and 9), 5- (lanes 5 and 10) or 10- (lanes 6 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
oligonucleo-tide bearing PRE IV (IS1428 ⁄ 1429), and with the unlabeled
compet-itors 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.
Trang 6trinucleotide 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
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-mately 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 6 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
Table 1 RCS of mutant PREs for binding of wild-type Ste12 to a
PRE consensus (ATGAAACA) in vitro.
a
PREs represented in the FUS1 promoter (Fig 4B).bRCS 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).cConcentrations 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.
Trang 7response 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
mini-mal GAL1 promoter, the limiting factor for
transcrip-tional 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
con-figurations Most promoters have PREs in a
directly-repeated orientation, although there are many
instances of PREs arranged in a tail-to-tail
configura-tion (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
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(RCSPRE1· RCS PRE2 ) and
plotted against the respective pheromone responsiveness for each
reporter (b-galactosidase activity (· 10)3).
A
B
Fig 7 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 pro-moter 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.
Trang 8binding and activation by Ste12 We then examined
how the spacing between two directly-repeated
consen-sus 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
induc-tion, 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 2 and 3, respectively) Taken together,
these results indicate that there must be structural
con-straints 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
sepa-rated by 40 nucleotides allow induction implies that 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¢ elesub-frag-ments (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
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 pro-moter to determine whether there were potential weaker binding sites for Ste12 falling within the con-straints 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 8 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 min (pheromone induction).
Trang 9of 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
B
Fig 9 A single consensus PRE can confer pheromone
responsive-ness 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
bear-ing 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
min-imal promoter bearing oligonucleotides representing the wild-type
or mutant promoter sequences was measured in untreated and
pheromone-treated cells.
A
B
C
D
Fig 10 Structural constraints on Ste12 for binding closely-posi-tioned PREs Schematic representation of a possible mechanism for the recognition of closely-spaced PREs in different conforma-tions 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 orien-tation (C) Head-to-head and tail-to-tail orienorien-tations may be accom-modated providing that the sites are separated sufficiently to allow bending or twisting of the intervening DNA to enable binding of Ste12 multimers (D).
Trang 10We 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
relative to one another to enable pheromone response
of an artificial promoter Two directly-repeated PREs
cause activation only when located within three
nucle-otides of each other By contrast, PREs inverted in a
tail-to-tail conformation separated by a single
nucleo-tide produce a very strong response Additionally,
PREs oriented in head-to-head or tail-to-tail
configura-tions 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
conforma-tion (Fig 10B) may form multimers through
interac-tion 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 do
not produce a response, the flexibility of Ste12 may
not be able to accommodate this particular
orienta-tion, or perhaps the N-terminal DNA binding domain
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), sug-gesting that Ste12 is capable of forming multimers that can bind these configurations, provided that the inter-vening DNA is able to bend or twist into a conforma-tion that can accommodate the interacconforma-tion (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 concon-sidering 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-plexes with oligos containing two PREs (T.-C Su and
I Sadowski, unpublished results), suggesting that phosphorylation may regulate the ability to bind multi-ple 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 pher-omone-responsive genes, but not Ste12⁄ Tec1-respon-sive 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