Filamentation genes are regulated through TCS elements.
Based on the current model that the cooperative binding of Ste12 and Tec1 to FREs regulates the transcription of filamentation genes (29
), promoters of filamentation genes should contain FREs (a PRE adjacent to a TCS). However, we noticed that many filamentation genes do not have a sequence that resembles an FRE in their promoter region. To further address this, we extracted 1,000-bp upstream sequences of Ste12-regulated mating and filamentation genes that have been identified by genome-wide transcription analyses (dig1 dig2
versus wild type) (21
) and determined the existence of PRE (TGAAACR) and TCS (CATTCY) elements in these promoters. Because both motifs are rather short and can appear randomly at high frequency (PRE, 0.34 sites per 1,000 bp; TCS, 1.04 sites per 1,000 bp), we compared the promoter regions of these genes with the promoters of their orthologs from three other closely related Saccharomyces
). The information on PRE and TCS positions in these promoters is compiled in three figures that are available in the supplemental material. Genuine transcription factor binding motifs are likely to be conserved among all four species, while a random occurrence is generally not conserved (23
). PREs and TCS that are conserved among Ste12-regulated genes in all four species are compiled in Table . All Ste12-regulated genes have either PREs, TCS, or both elements in their promoters. Not surprisingly, all the genes that have only PREs are involved in mating (Table ; see Fig. S1 in the supplemental material). Only a few mating genes have both PREs and TCS in their promoters, including FUS1
, and PRM1
(Table ; see Fig. S3 in the supplemental material). In contrast, most genes that contain only TCS are involved in filamentation (Table ; see Fig. S2 in the supplemental material). A few filamentation genes also have PREs in their promoters (Table ; see Fig. S3 in the supplemental material). Therefore, it appears that the major difference between mating and filamentation genes lies in the presence of PREs versus TCS in their promoters.
Comparative promoter analysis for conserved Ste12 and Tec1 binding sites in four closely related Saccharomyces speciesa
We searched further for potential FREs among the genes that have both a PRE and a TCS in their promoter. In addition to two previously reported genes, TEC1
), we found only two additional genes, YDR249C
, that contain a potential FRE (Table ; see Fig. S3 in the supplemental material). S. cerevisiae FLO11
contains a single nucleotide mismatch in a potential PRE right upstream of a TCS, and the combination was previously suggested to be a potential FRE. However, the potential PRE is not in the promoters of FLO11
orthologs in the other three species (see Fig. S2 in the supplemental material) and, thus, might not be functional. Because the FLO11
promoter has been shown to be much larger than 1,000 bp (38
), we extended our sequence comparison of FLO11
promoters to 3,000 bp. No FREs but only two conserved TCS and a separate PRE were found. Therefore, FLO11
is more likely regulated through TCS than through an FRE, although the upstream PRE may play a role in FLO11
regulation, too. In total, all filamentation genes have TCS in their promoters, but only four of them (Ty1
, and SVS1
) contain an FRE. Our promoter analysis suggests that TCS and not FREs are the prevailing cis
elements upstream of filamentation genes.
Our promoter analysis is in agreement with transcription patterns of these genes. Fus3 plays a positive role in the transcriptional activation of Ste12, whereas it specifically phosphorylates Tec1 and triggers ubiquitin-mediated Tec1 degradation during the pheromone response (1
). Therefore, transcription of TCS-driven genes in a fus3
mutant is expected to be higher than that in wild type, while PRE-driven transcription in fus3
mutants should not be higher than that in wild type during the pheromone response. Ratios of gene expression in fus3
mutants treated with 50 nM α-factor versus wild type treated with 50 nM α-factor have been determined by Hughes et al. (21
) and are listed in Table . As expected, all genes with only PREs have a ratio of ≤1.0, and genes with only TCS unanimously have a ratio of >1.0 (Table ). The ratios also correlate well with the functional assignments for genes with both PREs and TCS (Table , right column).
Tec1 is in a complex with Ste12 and Dig1, which is distinct from the complex of Ste12 with Dig1 and Dig2.
Since Ste12 can regulate filamentation genes without binding to PREs, there must be an alternative way for Ste12 to associate with the promoters. One likely model for Ste12 to control TCS-driven transcription is by direct association of Ste12 with Tec1. Ste12 is suspected to interact with Tec1 because Tec1 and Ste12 can bind cooperatively to FREs both in vitro and in vivo (5
), although it has not been shown whether the two proteins can interact in the absence of their binding to adjacent cis
elements on DNA. The only report of Ste12 association with Tec1 independent of cis
elements was from a systematic identification of protein complexes by mass spectrometry in cells that overexpressed TEC1-FLAG
from the GAL1
). To further investigate whether Tec1 interacts with Ste12 and Ste12-associated proteins Dig1 and Dig2, we tagged these proteins at their C termini with either HA3
on chromosomes. The tagged proteins were expressed from their own promoters and were functional (data not shown). Fractionation experiments with epitope-tagged Ste12 showed that over 90% of Ste12 was in the pellet fraction with DNA after an ultracentrifugation when 150 mM NaCl was used for protein extraction, whereas Ste12 was effectively extracted when 300 mM NaCl was used (data not shown). Therefore, 300 mM NaCl was used in all of our in vivo immunoprecipitation experiments in this study. Immunoprecipitation of Tec1-HA with an anti-HA antibody was able to bring down similar amounts of Dig1-myc and Ste12-myc (Fig. ), despite the fact that Dig1 was more abundant than Ste12 in the whole-cell lysate. The interaction was specific, as immunoprecipitation of the cell extracts of untagged strains did not pull down any myc-tagged proteins. Reciprocal immunoprecipitation of Dig1 or Ste12 also detected Tec1 (data not shown). In contrast to Dig1 and Ste12, immunoprecipitation of Tec1-HA did not bring down a substantial amount of Dig2 (Fig. , lane 3). Repeating the Tec1 immunoprecipitation showed that Tec1 consistently pulled down Dig1 and Ste12, but the Dig2 signal in the Tec1 immunoprecipitation (IP) was sometimes weak and sometimes undetectable. As Dig1 is more abundant than Dig2 in whole-cell lysate, one may argue that the lack of detectable Dig2 in the Tec1 IP could be caused by the difference in protein abundance. But this is unlikely because immunoprecipitation of Dig2-HA specifically brought down Ste12-myc, as previously reported (43
), as well as Dig1-myc (Fig. ), but the amount of Tec1 in the Dig2 IP was barely detectable, despite the fact that similar levels of Ste12 and Tec1 were in the cell lysate (Fig. ). Therefore, Tec1 and Dig2 are likely in two different complexes with Ste12 and Dig1. The Ste12/Dig1/Dig2 complex is the known Ste12 complex for the mating program (34
), while the newly identified Tec1/Ste12/Dig1 complex is likely for the filamentation program.
FIG. 1. Tec1 forms a complex with Ste12 and Dig1 but not with Dig2. (A) Immunoprecipitation of Tec1-HA. Protein lysates were subjected to immunoprecipitation with an anti-HA antibody, and the precipitation products were resolved by SDS-polyacrylamide gel electrophoresis (more ...) TCS-lacZ expression and LexA-Tec1 transcriptional activity are inhibited by Dig1 but not Dig2.
Dig1 and Dig2 are two functionally redundant inhibitors of Ste12, and PRE-lacZ
expression or Ste12 activity is high in dig1 dig2
double mutants but not in either dig1
single mutants (9
is also highly expressed in a dig1 dig2
double mutant (4
), but its expression in dig1
single mutants has not been reported. If only Dig1, but not Dig2, is present in the complex with Tec1 and Ste12, TCS-driven expression is expected to be high in a dig1
strain. To investigate whether Dig1 and Dig2 play different roles in the regulation of TCS-driven transcription, we assayed TCS-lacZ
) expression in dig1
, and dig1 dig2
mutants. For comparison, we also assayed the expression of PRE(FUS1)-lacZ
) and FRE(Ty1)-lacZ
) reporters in the dig1
, and dig1 dig2
mutants. Expression of TCS-lacZ
, but not PRE(FUS1)-lacZ
, was significantly elevated in dig1
mutants (Fig. ). In contrast, TCS-lacZ
expression was not increased in dig2
mutants. Deletion of both DIG1
increased the expression of all three reporters, as expected for TCS-lacZ
and as previously reported for FRE(Ty1)-lacZ
). Therefore, a dig1
single mutant is able to release the inhibition on FRE- or TCS-driven expression. This is consistent with a genome-wide transcription analysis which shows that Dig1 is the primary negative regulator for the expression of filamentation genes (6
FIG. 2. Differential regulation of Ste12 and Tec1 transcriptional activities by Dig1 and Dig2. Relative β-galactosidase activities of PRE(FUS1)-lacZ, FRE(Ty1)-lacZ, and TCS-lacZ (pHL710) in wild-type (10560-4A), dig1 (HLY3315), dig2 (HLY3316), and dig1 (more ...)
To determine whether the high TCS-lacZ
expression in the dig1
strain reflects the inhibitory effect of Dig1 on Tec1 transcriptional activity, we constructed a fusion of Tec1 to the DNA-binding domain of bacterial LexA, and the lexA-TEC1
expression was under the control of the ADH1
promoter so that the lexA-TEC1
expression was not influenced by Ste12 activity. Tec1 transcriptional activity was assayed in strains carrying a lacZ
reporter under the regulation of lexA
operators. Similar to the TCS reporter, LexA-Tec1 activity was up by ~45-fold in the dig1
mutant (Fig. ). In contrast to LexA-Tec1, LexA-Ste12 activity was high only in the dig1 dig2
double mutant and not in the dig1
single mutant, as previously reported (34
). Therefore, Dig1 is the major inhibitor of Tec1 activity. Since PRE-lacZ
expression or LexA-Ste12 activity was similar in dig1
single mutants, the observed difference in TCS-lacZ
expression or LexA-Tec1 activity between dig1
strains is unlikely due to the difference in Dig1 and Dig2 protein abundance but could be explained by the protein composition in the Tec1/Ste12/Dig1 and Ste12/Dig1/Dig2 complexes.
Tec1 binds to the same region of Ste12 as Dig2.
Since the two Ste12 complexes differ in Tec1 and Dig2, a possible mechanism that could give two distinct Ste12 complexes is one whereby Tec1 and Dig2 bind to the same region on Ste12 in a mutually exclusive way. Dig2 is known to bind to the N-terminal DNA binding region of Ste12 (34
). We found that Tec1 also associated with the N-terminal region of Ste12 by immunoprecipitation (Fig. ). A myc-tagged N-terminal fragment of Ste12(1-215)
in an ste12
mutant was used for IP to avoid the potential interaction of the Ste12 N terminus with full-length Ste12. Because Ste12(1-215)
is not sufficient for the expression of Ste12-regulated genes, HA-tagged Tec1 was expressed from the ADH1
promoter. The N-terminal DNA binding region (residues 1 to 215) of Ste12 is sufficient for interaction with Tec1; other regions of Ste12 were not required for the interaction with Tec1, as immunoprecipitation of Ste12 with deletions between residues 253 to 355, 387 to 512, and 512 to 669 could still pull down Tec1 (data not shown).
Dig1 interaction with Tec1 requires Ste12.
Because Dig1 can bind Ste12 and inhibit Ste12 activity (9
) and because Ste12 is a component of the Tec1 immunocomplex, it is possible that the interaction of Dig1 with Tec1 is not direct but is mediated through Ste12. To test this possibility, we examined whether Dig1 and Tec1 still interact in the absence of Ste12. Because TEC1
expression is Ste12 dependent, we placed TEC1-HA
under the control of the ADH1
promoter. Dig1-myc was detected in the Tec1-HA immunoprecipitation in wild type but not in an ste12
mutant (Fig. ). Therefore, Ste12 is required for Tec1 interaction with Dig1.
FIG. 3. Ste12 interaction with Tec1 is essential for the transcriptional activation of Tec1. (A) The Dig1 and Tec1 interaction is Ste12 dependent. Yeast strains HLY3335 (Tec1-HA Dig1-myc) and HLY3343 (Tec1-HA Dig1-myc ste12) with TEC1 under its endogenous promoter (more ...) Tec1 transcriptional activity is dependent on its association with Ste12.
Not only was the interaction of Dig1 and Tec1 dependent on Ste12, but deleting STE12 in a dig1 mutant also blocked the elevated LexA-Tec1 transcriptional activity that was otherwise observed in a dig1 strain (Fig. , dig1 versus dig1 ste12). It is possible that Tec1-associated Ste12 is directly responsible for the induction of Tec1 transcriptional activity in the dig1 strain. In this case, the region of Tec1 that interacts with Ste12 should be required for Tec1 transcriptional activation. To test this possibility, we generated deletions of Tec1 from either the N or the C terminus. Deletions within the first 300 residues did not affect its association with Ste12, whereas a deletion of up to 400 residues was unable to bind Ste12 (Fig. ). A deletion from residue 401 to the C terminus was able to bind to Ste12, but deleting to residue 301 abolished Ste12 binding (Fig. ). These data suggest that the region between residues 301 to 400 of Tec1 is required for Tec1 to interact with Ste12. Although required, the region was not sufficient for binding with Ste12 (data not shown). The Tec1 deletions were then fused in frame to the DNA binding domain of LexA, and the LexA-Tec1 fusions were analyzed for Tec1 transcriptional activity in the dig1 strain. We found that C-terminal deletions of Tec1 abolished the elevated LexA-Tec1 transcriptional activity in the dig1 strain (Fig. ). In contrast, the region from the N terminus to residue 300 was not required for the high LexA-Tec1 activity in the dig1 mutant (Fig. , Tec1 regions 101 to 486, 201 to 486, and 301 to 486). These data suggest that the Ste12 binding region (residues 301 to 400) is required for LexA-Tec1 transcriptional activity. Therefore, Tec1 transcriptional activity is dependent on its association with Ste12, which is under the negative regulation of Dig1. Although the C terminus of Tec1 (from 401 to 486) was not essential for Ste12 binding, it was still required for lexA-Tec1 transcriptional induction in the dig1 strain (Fig. ). This suggests that, besides Ste12, there might be additional regulations on the C terminus of Tec1.
Stoichiometry of Ste12 interaction with Tec1, Dig1, and Dig2 in vitro.
To further characterize the interaction between Tec1 and Ste12, we generated Tec1-FLAG and Ste12 by in vitro transcription/translation and examined whether Tec1-FLAG could interact with Ste12 in vitro. As shown in Fig. , lane 4, IP with an anti-FLAG antibody brought down similar levels of 35S-labeled Tec1-FLAG and Ste12. Reciprocally, IP of 35S-labeled myc-Ste12 also brought down 35S-Tec1 at the molar ratio of about 1 Ste12 to 1.3 Tec1 (Fig. , lane 8).
FIG. 4. Formation of distinct Ste12 complexes in vitro. (A) Tec1 binds to Ste12 with an equal molar ratio. One microliter of in vitro translated and 35S-labeled Tec1-FLAG or Ste12 was loaded as input. A total of 15 μl of each was used for immunoprecipitation (more ...)
We also used in vitro translated proteins to confirm that Ste12 is required to mediate the interaction between Tec1 and Dig1. IP of Tec1-FLAG did not bring down Dig1 in the absence of Ste12 (Fig. B, lane 8); the weak Dig1 band in lane 8 was nonspecific, as a similar level of Dig1 was also seen in the IP with beads without Tec1-FLAG in lane 5. However, a significant amount of Dig1 was precipitated with Tec1-FLAG in the presence of Ste12 (Fig. , lane 10). Therefore, Tec1 does not interact with Dig1 directly; rather, Ste12 bridges Tec1 and Dig1 in the complex.
In contrast to Dig1, Dig2 was not detected in the Tec1-FLAG immunoprecipitation even in the presence of Ste12 (Fig. , lane 11). Interestingly, when both Ste12 and Dig1 were present, Tec1-FLAG could bring down a small, but detectable amount of Dig2 (Fig. , lane 12 as indicated by the arrow). Because 35S-Ste12 IP also produced a faint band at the same position as Dig2 (Fig. , lanes 7, 10, and 11), we repeated the Tec1-FLAG IP experiment with unlabeled Ste12 (Fig. ). IP of Tec1-FLAG in the presence of Ste12 could bring down Dig1 (lane 7) but not Dig2 (lane 5). In the presence of both Ste12 and Dig1, Tec1-FLAG could bring down a small amount of Dig2 (Fig. , lane 6). Therefore, a small amount of Dig2 is tethered to the Tec1/Ste12/Dig1 complex through its association with Dig1.
We also investigated the ability of Ste12 to interact with Dig1 and Dig2 by using a myc-tagged Ste12 for IP (3
). As shown by Bardwell et al., a small amount of Dig1 bound to myc-Ste12 (Fig. , lane 9). The molar ratio of this binding is about 1 Ste12 to 0.3 Dig1. The amount of Dig2 in the myc-Ste12 IP was also very small, at about 1 Ste12 to 0.2 Dig2 (Fig. , lane 10). Surprisingly, in the presence of both Dig1 and Dig2, the amount of Dig1 and Dig2 associated with myc-Ste12 reached a molar ratio of 1.4 Dig1 and 1.5 Dig2 to 1 Ste12 (Fig. , lane 11). Therefore, there is synergy between Dig1 and Dig2 in binding to myc-Ste12 in vitro. Identical results, both in terms of low levels of binding with Dig1 or Dig2 and the synergy between them, were observed with an Ste12-myc in which we fused myc13
to the C terminus of Ste12 (data not shown).
Ste12-associated Dig2 can be competed off by Tec1.
Because both Tec1 and Dig2 bind to the N-terminal region of Ste12 (Fig. ), we reasoned that they might bind to Ste12 in a competitive and, therefore, mutually exclusive manner to generate two distinct Ste12 complexes. To determine whether Tec1 can compete with Dig2 for binding to Ste12, an increasing amount of Tec1 was added to IP reaction mixtures that contained an equal amount of myc-Ste12 and 35S-labeled Dig1 and Dig2. Decreasing levels of Dig2 and Dig1 were found to be associated with myc-Ste12 in the presence of an increasing amount of Tec1 (Fig. ). This suggests that excess Tec1 could compete off Dig2 from Ste12. Interestingly, a significant amount of Dig1 also fell off with Dig2 from the Ste12 complex in the presence of a large excess of Tec1 (Fig. , lane 8 and 9), consistent with the observed synergistic interaction of Dig1 and Dig2 with Ste12. When we carried out the Tec1 competition experiment with myc-Ste12 and Dig1, the Ste12-bound Dig1 level did not change with an increasing amount of Tec1 (data not shown). When an increasing amount of Tec1 was added to myc-Ste12 and Dig2, Ste12-bound Dig2 decreased; however, the initial amount of Ste12-bound Dig2 was very small (data not shown).
FIG. 5. Tec1 competes with Dig2 for Ste12 binding both in vitro and in vivo. (A) Tec1 can compete off Dig2 from Ste12. Twenty microliters of myc-Ste12 and 10 μl of in vitro translated and 35S-labeled Dig1 and Dig2 were used in each immunoprecipitation (more ...)
We also examined whether Dig2 and Dig1 proteins could compete off Tec1 from Ste12. Interestingly, Tec1 could not be competed off from Ste12 with excess Dig2 and Dig1 proteins (Fig. ). The competition data suggest that Tec1 and Dig2 share an overlapping region of Ste12 for binding because Tec1 could replace Dig2 from Ste12, but their affinities are not identical. Tec1 may have a higher Ste12 binding affinity than that of Dig2, and, therefore, it cannot be replaced by Dig2 in binding to Ste12.
We further tested the ability of Tec1 to compete with Dig2 for Ste12 binding in vivo by an Ste12 IP in cells with and without overexpression of TEC1. When overexpressed from the GAL1 promoter, Tec1 significantly reduced the amount of Dig2 that was associated with Ste12 in yeast cells (Fig. ). Thus, our data show that Tec1 competes with Dig2 in binding with Ste12 both in vivo and in vitro.
The Tec1/Ste12/Dig1 complex binds to TCS of filamentation genes and PREs of mating genes.
If filamentation genes are regulated by the Tec1/Ste12/Dig1 complex via the TCS and mating genes are regulated by the Ste12/Dig1/Dig2 complex via the PRE, we would expect to find Dig2 at the promoters of mating genes and Tec1 at those of filamentation genes. Ste12 and Dig1 should be present at the promoters of both groups. A study of the genome-wide location of Ste12, Dig1, and Tec1 has shown that Ste12 and Dig1 are present at both mating and filamentation genes, and Tec1 is present at filamentation genes as well as at some of the mating genes (47
). The localization of Dig2 in these genes is not known. Therefore, we compared the distribution of Tec1 and Dig2 at the promoters of mating and filamentation genes using ChIP analysis. Ste12 and Dig1 were included as controls. STE12-myc
, and DIG2-myc
strains were grown in YEPD (yeast extract, peptone, and dextrose) medium and harvested for ChIP analysis. The immunoprecipitated DNA was analyzed by PCR using primer pairs that are located about 100 bp upstream and downstream of either the TCS elements of filamentation genes or PRE elements of mating genes. Because we wanted to determine the differences between Ste12 complexes at PREs and at TCS, promoters containing both PRE and TCS sites were excluded from the analysis. In addition, only promoters that were efficiently bound by Ste12 and Tec1 in the whole-genome ChIP experiment were used (47
). Ste12, Tec1, and Dig1 were present in nearly equal amounts at the TCS of filamentation genes, whereas Dig2 was detected at a lower level (Fig. ). Since our in vitro binding experiments have shown that Tec1, Ste12, and Dig1 were present at a near equal ratio in the Tec1/Ste12 complex and that limited Dig2 was bound to the complex through the association with Dig1, the relative amounts of the four proteins detected at the filamentation genes are consistent with the localization of the Tec1/Ste12/Dig1 complex to the filamentation genes.
FIG. 6. Distribution and function of the Tec1/Ste12/Dig1 complex at TCS sites of filamentation genes and PREs of mating genes (A) Yeast strains HLY3320 (TEC1-myc), HLY3321 (STE12-myc), HLY3322 (DIG1-myc), HLY3323 (DIG2-myc), and 10560-4A (wild type) were grown (more ...)
To test whether the presence of Dig2 at the promoters of the filamentation gene promoters was due to its association with Dig1 in the Tec1/Ste12/Dig1 complex, instead of with Ste12, we compared Dig2-myc localization at the promoters of filamentation genes in a wild-type strain and a dig1 mutant by ChIP (Fig. ). The deletion of DIG1 greatly decreased the amount of Dig2 bound to the promoter of CHS7 (Fig. ) as well as CWP1 (data not shown), indicating that Dig2 associates with Dig1 to TCS. The observed decrease in Dig2 at the CHS7 promoter in the dig1 mutant was not due to a potential Dig1-dependent interaction of Dig2 with Ste12, as the same amount of Dig2 was detected at the PREs of FUS1 in wild-type and dig1 mutant cells (Fig. ).
PCR of selected mating genes from the above ChIPs shows that Tec1 was present, but at a much lower level than Ste12, Dig1, and Dig2, at the promoters of mating genes (Fig. ). Because Tec1 is only present in the Tec1/Ste12/Dig1 complex, the detection of the small amount of Tec1 at the mating genes suggested that there was some Tec1/Ste12/Dig1 complex at the promoters of mating genes via Ste12 binding to PREs. We suggest that both types of Ste12 complexes are present at the promoters of mating genes. The Ste12/Dig1/Dig2 complex is the major form, while the Tec1/Ste12/Dig1 complex is the minor form.
The biological significance for the presence of the Tec1/Ste12/Dig1 complex at the PREs of mating genes is not clear. But we did find that the PRE(FUS1)-lacZ expression level was slightly elevated in tec1 and was even higher in a tec1 dig1 double mutant (Fig. ). The synergistic effect between tec1 and dig1 was specific, as PRE(FUS1)-lacZ expression was not increased in a tec1 dig2 mutant, suggesting that Tec1 might function together with Dig1 in inhibiting Ste12 activity at the promoters of mating genes.