INTRODUCTION

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In the budding yeast, Saccharomyces cerevisiae, the transcription factor SBF (SCB binding factor) induces cell-cycle-dependent expression of a large group of genes at the G₁/S-phase transition or Start (reviewed in reference 8). SBF is a heterodimeric complex composed of two proteins, Swi4 and Swi6, which bind the repeated upstream regulatory sequence CACGAAA (SCB, for Swi4,6-dependent cell cycle box [8, 32]). Biochemical studies show that Swi4 is the component of SBF that specifically binds the SCB sequence through an N-terminal helix-turn-helix DNA-binding domain (55). In contrast, Swi6 has no DNA binding activity but is present in SBF because of its interaction with Swi4 via the carboxy-terminal regions of the two proteins (3, 48). Swi6 is also a component of the transcription complex MBF (MCB-binding factor) that is composed of Swi6 and the DNA binding protein Mbp1 and binds the promoter element ACGCGT (MCB, for MluI cell cycle box [8]).

DNA microarray experiments provide an overview of cell-cycle-regulated genes in budding yeast; more than 200 genes show peak expression in late G₁ phase (G₁ genes [12, 52]). Most of the G₁ genes have at least one SCB or MCB element in their upstream sequences, implicating SBF and MBF in the induction of many G₁ genes. The G₁ group of genes includes the Cdc28-associated G₁ cyclins CLN1 and CLN2 and the Pho85-associated G₁ cyclins PCL1 and PCL2. These four cyclins activate their cognate cyclin-dependent kinase (Cdk) in late G₁ and are required for G₁-to-S phase progression (reviewed in reference 2). SBF is essential for the expression of the G₁-specific HO gene and is required for maximal G₁-specific induction of CLN1, CLN2, PCL1, PCL2, and various cell wall genes (8, 28, 46). Recently, about 200 additional putative targets of SBF and MBF were identified using yeast intergenic arrays coupled with chromatin immunoprecipitation (ChIP) (29).

SBF-mediated gene expression is controlled at multiple levels, including binding of SBF to SCBs, changes in the subcellular localization of Swi6, and the activation of SBF at Start. In vivo footprinting and ChIP experiments show that SBF is bound to the SCBs upstream of three different SBF-dependent genes throughout late M and G₁ phases (13, 23, 33). SBF binding to the SCBs of the HO promoter depends on chromatin remodeling events (13, 34), suggesting that chromatin remodeling may be a feature of SBF binding to the upstream regulatory regions of a variety of genes. The binding of SCBs by SBF is not coincident with SBF-mediated transcription; rather, a second event must occur for SBF activation, and the Cln3-Cdc28 Cdk plays an important role in this process (19, 54). In fact, DNA microarray experiments show that expression of most G₁ genes is induced by overexpression of CLN3 (52). However, the mechanism of Cln3-dependent activation of SBF remains unclear, and direct interaction of Cln3 with SBF has not been reported.

Strains lacking SBF arrest in G₁, and many G₁ genes have at least one copy of the SCB element in their promoters; therefore, a key role of SBF is to promote G₁-specific transcription. However, there are fewer than 300 genes whose transcription peaks at Start but more than 1,155 genes whose promoters contain matches to the SCB consensus sequence (http://cgsigma.cshl.org/jian/). A comparison of the number of SCB sites upstream of G₁ genes with the frequency of SCB sites upstream of a control group of non-cell-cycle-regulated genes, reveals that SCBs are found more frequently upstream of non-cell-cycle-regulated genes than MCB sites or sites for a G₂-specific transcription factor (MCM/SFF sites [52]). This analysis suggests that SCB elements and SBF may regulate the transcription of many genes other than those induced at Start.

One pathway that may regulate SBF outside of Start is the protein kinase C (PKC1) pathway (reviewed in reference 25). PKC1 encodes an essential serine-threonine-specific protein kinase that is the yeast homolog of members of the mammalian PKC family of genes (37). Pkc1 activates a mitogen-activated protein kinase (MAPK) cascade that consists of (i) the MEKK (MAPK kinase kinase) Bck1, (ii) the redundant MEKs (MAPK kinases) Mkk1 and Mkk2, and (iii) the MAPK Slt2/Mpk1. pkc1 mutants have thin cell walls and an osmoremedial sensitivity to a variety of cell wall stresses such as heat shock. Strains carrying a deletion of BCK1, SLT2, or both MKK1 and MKK2 are sensitive to high temperature but are viable at 25°C (35). Since PKC1 mutants are inviable, PKC1 must have other roles besides activation of the Slt2-MAPK pathway. Consistent with these genetic results, recent studies show that PKC1 is required for both the depolarization and the repolarization of the actin cytoskeleton upon cell wall stress (18). However, components of the PKC1-MAPK pathway module are required only for repolarization; both slt2 and bck1 mutants have a depolarized actin cytoskeleton, with delocalization of actin cortical spots, abnormal accumulation of secretory vesicles, and defects in polarized cell growth (14, 18, 43). These studies suggest that the PKC1-MAPK pathway is required for cell polarization and for maintenance of cell wall integrity, while an as-yet-uncharacterized PKC1-dependent pathway is required for depolarization of the actin cytoskeleton. Slt2 kinase is activated by heat shock (31) and hypo-osmotic shock (17) during periods of polarized growth, such as bud formation and mating projection formation (59), and in response to actin perturbation (24).

In many systems, the major targets of MAPK cascades are transcription factors, and activation of the cascade leads to altered gene expression (reviewed in reference 56). Indeed, recent DNA microarray work showed transcriptional modulation of 90 genes in a strain expressing an activated allele of PKC1 (27, 47). Currently, only two transcription factors have been identified as targets of Slt2: the MADS-box transcription factor Rlm1 (20, 58) and SBF (39). A genome-wide survey for genes whose expression was altered after expression of a constitutively active MKK1^S386P allele for 4 h identified 25 affected genes (30). Twenty-four of the MKK1^S386P-induced genes were partially dependent on RLM1. This pattern suggests that, after 4 h of Slt2 activation, Rlm1 may mediate the majority of the Slt2 effects. However, the phenotypes of rlm1 strains are much less severe than those seen in PKC1-MAPK pathway mutants (57), suggesting that Rlm1 is not the only important transcriptional target of Slt2. In contrast, swi4 and swi6 mutant strains share phenotypes similar to those of strains mutated for PKC1 pathway genes. Some swi4 strains show a temperature-sensitive growth defect that is suppressed by sorbitol (28, 39), and both swi4 and swi6 strains are sensitive to cell wall stressors (28). Further, like PKC1-MAPK pathway mutants, swi4 mutants also exhibit defects in both bud emergence and projection formation (22, 39). These phenotypic similarities suggested that SBF might be a major transcriptional target of Slt2. Indeed, genetic studies, coimmunoprecipitation experiments, and kinase assays have established SBF as a target of Slt2 kinase (39).

Although the role of Slt2 in SBF regulation is not known, several observations suggest that Slt2 may activate SBF toward a subset of target genes. First, genetic evidence suggests a role for the PKC1-MAPK pathway at Start (41, 43), and this role may be discharged through SBF. Second, the Pkc1 pathway and Slt2 are required for maximal heat shock-dependent induction of only a subset of SBF-dependent G₁ genes, including PCL1 and PCL2 (39), and cell wall genes (28). Finally, overexpression of the G₁ cyclins PCL1 and PCL2, but not CLN1 or CLN2, suppresses the cell lysis defects of a slt2 strain (39).

In this study, we test the hypothesis that Slt2 may act to modulate SBF to induce transcription of only a subset of SBF-dependent genes. We began by exploring the regulation of the SBF- and Slt2-dependent gene, PCL1. Our genetic approaches, coupled with DNA microarray analysis and ChIP experiments, establish a new Slt2-dependent mode of Swi4 regulation and suggest roles for Swi4 beyond cell cycle control.

	MATERIALS AND METHODS

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Plasmid constructs The PCL1 promoter-lacZ reporter plasmid, prPCL1_751-146 (pBA537), was previously described as pSS-HCS26 (46). To construct a PCL2 promoter-lacZ reporter construct, the PCL2 promoter from 982 to 82 bp from the start site was amplified by using primers with the following sequences: 5'-AACGCGTCGACCGTAATTCTATCGATGGACC-3' and 5'-AACGCGTCGACGGAGAATTATAAAGTG-3'. The PCR product was digested with SalI and cloned into SalI-digested pSSBglII to create plasmid prPCL2_982-82 (pBA1306).

Strains and medium. Yeast strains used in this study are listed in Table 1. Standard methods and media were used for yeast growth and transformation. Most of the yeast strains used in this study are isogenic derivatives of BY263 (an S288C derivative [45]). Disruption and epitope tagging of SLT2 was achieved by homologous recombination at its chromosomal locus using a PCR-based method (38). The slt2 strain was verified by PCR and phenotypic assays. The GFP(S65T)-SLT2 allele was verified by PCR and the functionality of the Slt2 fusion protein was confirmed by Western blotting, kinase assays, and complementation of slt2 phenotypes.

-Galactosidase assays. To assay the activity of CYC::lacZ reporter genes in log-phase cultures, yeast transformants were grown in 5 ml of SD-URA medium to an optical density at 600 nm (OD₆₀₀) of 0.6 at 30°C. For heat shock experiments, cultures were grown to an OD₆₀₀ of 0.6 at 30°C and then transferred to test tubes that had been preincubated at 39°C, and cultures were incubated at 39°C for 30 min. To arrest cells with -factor, cells were grown in minimal medium supplemented with 1 M sorbitol. Once the cultures reached an OD₆₀₀ of 0.3, -factor (Procyon Biopharma) was added to the cultures to a final concentration of 5 µM for 2 h before harvesting. The cells were harvested by centrifugation at 3,000 × g for 5 min, the cell pellets were quickly frozen in liquid nitrogen, and the pellets were stored at 80°C. Liquid -galactosidase assays were performed on the frozen pellets as described previously (44). Data are presented as the mean values from triplicate experiments.

Cell cycle synchronization, heat shock, and ChIP. For heat shock experiments, 50-ml cultures of cells were grown to an OD₆₀₀ of 0.6. One culture was held at 30°C, while two other cultures were shifted to 39°C for either 30 or 60 min. Samples of cells were cross-linked with 1% formaldehyde for 15 min with shaking at either 30°C or 39°C. For cell cycle or -factor treatment experiments, 500-ml cultures were grown at 30°C to an OD₆₀₀ of 0.3, and -factor (Procyon Bio-Pharma) was added directly to the medium to a final concentration of 5 µM. Cultures were incubated with -factor for 2 h until at least 95% of the cells were arrested in G₁ phase as determined by microscopy. Cells were pelleted and then resuspended in fresh yeast extract-peptone-dextrose (YPD) medium. Samples (50 ml) were taken before the arrest, before the release, and every 10 min after release and then cross-linked with 1% formaldehyde for 15 min at 30°C. In both cases, cross-linking was quenched by the addition of glycine to 125 mM. Cells were pelleted at 3,000 × g for 5 min and washed twice with ice-cold TBS (150 mM NaCl, 20 mM Tris-HCl; pH 7.6), and whole-cell extracts were prepared for use in ChIPs basically as described previously (53). Cell lysis was performed in 400 µl of lysis buffer (50 mM HEPES-KOH [pH 7.5], 140 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, and one EDTA-Free Protease Inhibitor Pellet [Boehringer Mannheim] for every 50 ml) and 400 µl of glass beads. The suspension was sonicated four times for 10 s each time (resulting in an average fragment size of 0.5 kb) and clarified by centrifugation for 15 min at 14,000 × g. Protein concentration for each sample was determined by Bradford assay (Bio-Rad). Immunoprecipitations were performed with 1 mg of extract at 4°C overnight with rotation on a nutator and either 15 µl of protein A-Sepharose (pA) alone or 15 µl of pA plus 10 µl of affinity-purified Swi4 polyclonal antibody or 15 µl of pA plus 5 µl of affinity-purified Swi6 polyclonal antibody. Precipitates were washed twice for 10 min each time in 1 ml of lysis buffer and twice for 10 min each time in 1 ml of TBS. Finally, the samples were processed for DNA purification as described previously (53).

PCR analysis of immunoprecipitated DNA. PCRs were carried out in 25-µl volumes. Serial dilutions of the immunoprecipitated material and the input material for the whole-cell extracts (WCEs) were performed to ensure the PCRs were performed in the linear range. Typically, PCRs were performed with 1/2,000 of the immunoprecipitated material and 1/20,000 of the input material for WCEs. PCR was performed using Platinum Taq polymerase (Gibco-BRL) with 25 pmol of each primer and 0.5 µCi of [-³²P]dATP. For multiplex PCR with the PCL2, PCL1, PHO5, and CLN1 promoter primer pairs, 4.0 mM MgCl₂ was used; for PCR with only one primer pair, 1.5 mM MgCl₂ was used. The promoters were amplified using a cycling program of an initial 2 min of denaturation at 95°C; followed by 25 cycles of 30 s at 95°C, 30 s at 53°C, and 60 s at 70°C; and a final extension step of 5 min at 70°C. The gene-specific primers were designed as 20-mers with a roughly 50% GC content. The PCR primer sequences used for the amplification of promoter regions in PHO5, PCL1, PCL2, CLN1, RLM1, YLR110c, CWP1, SRL1/YOR248c, and GIC1 are available upon request. The PCR products were separated on a 5% polyacrylamide gel, dried, and exposed to Kodak Biomax-MR film.

Fluorescence microscopy. Localization of Slt2-GFP(S65T) and DNA DAPI (4',6'-diamidino-2-phenylindole) staining of live yeast cells was performed using cultures of BY263 and BY1343 grown to early log phase in YPD. Cultures were then treated with 5 µM -factor for 2 h and washed with a buffer containing 0.1 µg of DAPI/ml. Cells were observed at a magnification of ×100 using Nomarski optics or fluorescence through a fluorescein isothiocyanate (FITC) filter to observe GFP(S65T). Photographs were taken with a Micromax 1300y high-speed digital camera (Princeton Instruments, Trenton, N.J.) mounted on a Leica DM-LB microscope. Images from the camera were analyzed with Metaview software (Universal Imaging, Media, Pa.).

DNA microarray analysis. Yeast strains were grown in rich medium at 25°C to an A₆₀₀ of 0.3 to 0.6. For heat shock experiments, cells were grown to an A₆₀₀ of 0.3 at 25°C and then shifted to 39°C for 45 min. Cells were harvested by centrifugation and then quickly frozen in liquid nitrogen. Poly(A)⁺ mRNA was isolated as described previously (42). Alternatively, total RNA was isolated by hot phenol extraction as described previously (4) with minor modifications. Total RNA was precipitated from the final aqueous layer and run over a Qiagen RNeasy column to remove smaller RNAs. The final eluate was used as the source of mRNA for fluorescently labeled cDNA. In order to generate labeled cDNA, ca. 2 to 3 µg of mRNA or 50 µg of total RNA was incubated with Superscript II reverse transcriptase (Gibco-BRL) and Cy3- or Cy5-dCTP (Mandel) at 42°C for 2 h as described elsewhere (http://www.oci.utoronto.ca/microarrays).

Batch affinity chromatography. GST and GST-Slt2 were purified from Escherichia coli harboring appropriate expression plasmids as previously described (45). GST and GST-Slt2 were bound to glutathione-Sepharose 4B beads (Pharmacia) at concentrations of 1 and 3 µg/µl of beads, respectively. For affinity chromatography with in vitro-transcribed and translated Swi4 and Swi6, 20 µl of GST or GST-Slt2 beads was incubated with 18 µl of lysis buffer (100 mM Tris-HCl [pH 8.0], 1 mM EDTA, 10 mM MgCl₂, 100 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 0.5% Triton X-100; two EDTA-Free Protease Inhibitor cocktail tablets [Boehringer Mannheim] per 50 ml) and 2 µl of either in vitro-translated Swi4 or Swi6 (5) for 2.5 h at 4°C. The beads were harvested by centrifugation at 2,000 rpm in an Eppendorf centrifuge for 2 min, and the unbound supernatant was collected. The beads were washed three times each in 100 µl of lysis buffer. After the final wash, the beads were resuspended in 20 µl of 1× SDS gel sample buffer and boiled. The bound and unbound supernatant fractions were separated by SDS-6% polyacrylamide gel electrophoresis (PAGE). The gels were fixed, treated with Amplify (Amersham), dried, and exposed to X-ray film.

Northern blot analysis. Yeast strains were grown at 30°C in YPD medium to an OD₆₀₀ of 0.4. For -factor treatment, cells were treated with 5 µM -factor (Procyon BioPharma) for 2 h. Cells were pelleted at 3,000 × g in a 4°C centrifuge for 3 min, and RNA was isolated and Northern blotting was done as described previously (45). The probes used for the Northern blot analysis were a 600-bp EcoRI-HindIII fragment of the ACT1 gene (44), PCR products containing the coding sequence of PCL1 (45), YLR110c, SRL1, and GIC1. Sequences of PCR primers are available upon request. Probes were labeled using random-primed synthesis with Klenow DNA polymerase in the presence of [-³²P]dATP. For RNA quantitation, Northern blots were exposed on a Molecular Dynamics screen and scanned with a Molecular Dynamics PhosphorImager and analyzed with ImageQuant software (version 3.3).

	RESULTS

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Heat shock induction of PCL1 requires Slt2 and Swi4 but not Swi6. Upon heat shock, Slt2 is required for a two- to threefold increase in transcript levels for numerous cell-cycle-regulated genes, including the G₁ cyclins PCL1 and PCL2 (28, 39). To characterize the molecular mechanism of the Slt2-dependent transcriptional response, we turned to an in vivo assay to analyze the roles of Swi6 and Swi4 in the Slt2-dependent transcription of PCL1. A PCL1 reporter gene was constructed, and sequences spanning from 751 to 146 relative to the PCL1 ATG were placed upstream of a CYC::lacZ reporter gene on a yeast vector to create the reporter plasmid prPCL1_751-146 (Fig. 1). To assay the contribution of SBF and Slt2 to the upstream activating sequence (UAS) activity of the prPCL1_751-146 reporter, we transformed the wild type and swi4, swi6, mbp1, and slt2 mutant strains with the prPCL1_751-146 plasmid and the control plasmid CYC1::lacZ and then measured -galactosidase activity. None of the transformants containing the control reporter gene produced -galactosidase activity (data not shown), while a wild-type strain containing the prPCL1_751-146 reporter gene had significant -galactosidase activity at 30°C (Fig. 1). Upon heat shock, the -galactosidase activity was induced twofold (Fig. 1). Thus, the behavior of the PCL1 reporter gene in response to heat shock mirrors that of the endogenous PCL1 gene (39). As previously shown, expression of the prPCL1_751-146 reporter gene was completely dependent on Swi4 (Fig. 1 [46]), under normal growth conditions; heat shock-dependent induction of the PCL1 reporter was also largely dependent on Swi4 (Fig. 1). Surprisingly, we discovered that the upstream activation sequence (UAS) activity from the promoter of prPCL1_751-146 was independent of Swi6 (Fig. 1). Even though there is one consensus MCB element in the PCL1 promoter, deletion of MPB1 did not affect prPCL1_751-146 reporter activity. Furthermore, heat shock-dependent induction of the PCL1 reporter gene was completely eliminated in the slt2 strain (Fig. 1), a finding consistent with previous Northern blot experiments (39). The Swi4-Slt2-dependent but Swi6-independent regulation of the reporter gene was also seen using an upstream segment of the PCL1 promoter lacking all consensus SCB and MCB elements but containing degenerate matches to the SCB sequence (751 to 363; data not shown). In addition to the reporter gene assays, Northern blot analysis was also performed to assay PCL1 transcript levels in log phase cultures of wild-type, swi4, and swi6 strains (see Fig. 6). These blots confirmed that expression of PCL1 was dependent on Swi4 but independent of Swi6.

View larger version (34K): [in this window] [in a new window]   FIG. 1.   Slt2/Swi4-dependent and Swi6-independent activation of a PCL1-lacZ reporter gene. Schematic diagram of the PCL1 upstream regulatory region used to create a CYC::lacZ reporter construct. Locations of consensus SCB and MCB motifs are indicated. Wild-type (Wt, BY263), swi4 (BY108), swi6 (BY107), mbp1 (BY551), and slt2 (BY1342) strains were transformed with the prPCL1751-146 reporter plasmid and grown at 30°C to log phase (open bars) or grown at 30°C to log phase and then heat shocked for 30 min at 39°C (filled bars). Cell lysates were made and -galactosidase activity (Miller units) was determined. Activity depicted is the mean of three experiments; the error bars show the standard deviation for the three experiments.

Localization of Swi4 and Swi6 to the promoters of PCL1 and PCL2 during G₁ phase, pheromone-treatment, and heat shock. PCL1 gene expression peaks in late G₁ (52) and the promoter of PCL1 contains multiple SCB elements (46). Although Swi6 is only required for the proper cell cycle-dependent expression of a number of SBF target genes, we were surprised to find that the heat shock-dependent induction of PCL1 was independent of Swi6 (see above). Studies to date show that both Swi4 and Swi6 are required for SCB-driven gene expression; in the absence of Swi6, Swi4 is unable to bind SCBs both in vitro (5) and in vivo (23, 33).

View larger version (49K): [in this window] [in a new window]   FIG. 2.   Swi4 and Swi6 localization to the promoters of PCL1 and PCL2 during the cell cycle and after -factor and heat shock treatment. (A) Cell cycle ChIP using affinity-purified Swi4 antibodies (left panel) and Swi6 antibodies (right panel). A wild-type strain (BY263) was grown to mid- log phase and then blocked in G1 with -factor. Cells were inoculated into fresh medium lacking -factor, and samples were taken every 10 min. Samples taken of the unsynchronized log-phase culture (log), the G1 arrested cells (-factor), and the 10-min time points were cross-linked with formaldehyde. WCEs were made, and ChIP was done using pA alone or pA plus affinity-purified Swi4 or Swi6 antibodies as indicated. In the top panels multiplex PCR was performed to amplify the promoter regions of PCL2, PCL1, PHO5, and CLN1 in each of the ChIP samples, in an immunoprecipitation with extract which was not cross-linked (NX), and in WCEs. The graphs depict the results of phosphorimager analysis of each PCR product. The results are expressed as a percentage of the product in the WCE. The experiment shown is representative of three independent experiments with Swi4 antiserum and two with Swi6 antiserum. (B) Heat shock ChIPs. Cultures of a wild-type strain (BY263) grown at either 30°C (white bars) or 30°C, followed by heat shock at 39°C for 30 min (gray bars) or 60 min (black bars) were cross-linked with formaldehyde. Lysates were made, and ChIP was performed using pA or with affinity-purified Swi4 antibodies (-Swi4) or affinity-purified Swi6 antibodies (-Swi6). PCR was performed to amplify the promoters of PCL1, PCL2, and PHO5 in the ChIP samples and in WCE. The bar graphs depict the results of phosphorimager analysis of each PCR product. The results are expressed as a percentage of the product in WCE.

-Factor-dependent induction of PCL2 expression is dependent on Slt2 and SBF. Since we saw increased localization of SBF to PCL promoters during pheromone treatment, we next investigated the role of Slt2 in this process. First, we asked whether SBF and Slt2 are required for the pheromone-dependent induction of PCL2 expression. We used both a reporter gene construct containing sequences 5' to the PCL2 transcriptional start (Fig. 3A) and Northern blot analysis (Fig. 3B) to assay PCL2 expression. The sequences upstream of PCL2 contain a consensus binding site or PRE (pheromone response element) for Ste12 (reviewed in reference 40). As expected, the -factor-dependent induction of both the PCL2 reporter gene and the endogenous PCL2 gene were fully dependent on Ste12 (Fig. 3A and B). However, induction of PCL2 during -factor treatment was also dependent on Swi4 and Swi6 and partially dependent on Slt2 (Fig. 3; also see Discussion). Our results suggest that there may be an activation event specific to SBF on the promoter of PCL2 during the pheromone response.

View larger version (74K): [in this window] [in a new window]   FIG. 3.   Slt2 and SBF activity during -factor treatment. (A) PCL2 reporter gene assay. Above the graph is a schematic diagram of the PCL2 promoter region used to create the PCL2-lacZ reporter construct prPCL2-989-82. The locations of the consensus SCB, MCB, and PRE elements are indicated. The graph shows -factor-induced expression of -galactosidase from the prPCL2989-82 reporter plasmid. Wild-type (wt BY263), swi4 (BY1321), swi6 (BY891), slt2 (BY1342), and ste12 (BY332) strains were transformed with prPCL2989-82. The transformants were grown to mid-log phase (open bars) or grown to mid-log phase and treated with 5 µM -factor for 2 h (filled bars). Cell lysates were made, and the -galactosidase activity (Miller units) of each strain was determined. Activity depicted is the mean of three experiments; the error bars show the standard deviations for the three experiments. (B) PCL2 Northern blots. The indicated strains were grown to log phase or grown to log phase and treated with 5 µM -factor for 2 h. Total RNA was isolated and probed with PCL2, followed by the loading control ACT1. Indicated across the bottom is the fold induction of PCL2 expression after -factor treatment for each strain. Gel shown is representative of three independent experiments. (C) Subcellular localization of Slt2 after -factor treatment. BY1343 cells (SLT2::GFP) were grown in rich medium to early log phase and then treated with 5 µM -factor for 2 h. Cells were then washed once with buffer containing DAPI and once with water before examination by fluorescence microscopy at a magnification of ×1,000. Cells were exposed to fluorescent light through an FITC filter for 400 ms in order to visualize Slt2-GFP. Wild-type (BY263) cells treated identically showed no fluorescence. Photographs of the same fields viewed with Nomarski optics (DIC) and stained with DAPI to visualize cell nuclei are shown.

Localization of SBF to the promoters of PCL1 and PCL2 upon heat shock and pheromone treatment is partially dependent on Slt2. Since we saw increased binding of SBF to the promoters of PCL1 and PCL2 in two conditions that activate Slt2, we next tested the localization of SBF to PCL promoters in a slt2 strain. We used Swi4 and Swi6 polyclonal antibodies to immunoprecipitate chromatin from formaldehyde cross-linked wild-type and slt2 cells that were either in log phase, heat shocked at 39°C for 60 min, or treated with -factor for 2 h. Since both heat shock and -factor treatment cause cell lysis in slt2 mutant cells, both the wild-type and slt2 cells were grown in medium containing sorbitol. The addition of sorbitol did not affect -factor induction of PCL2 promoter activity or heat shock induction of PCL1 promoter activity (data not shown). In cells grown at 30°C, Swi4 and Swi6 associated with the PCL1 and PCL2 promoters at similar levels in both wild-type and slt2 cells (Fig. 4, log columns). Upon heat shock of wild-type cells, the localization of Swi4 and Swi6 to both the PCL1 and PCL2 promoters increased (Fig. 2B and 4). However, in slt2 cells, the enhanced localization of Swi4 and Swi6 to the promoters of PCL1 and PCL2 was reproducibly reduced (Fig. 4B). As described above, -factor treatment of wild-type cells also resulted in increased localization of Swi4 and Swi6 to the PCL1 and PCL2 promoters (Fig. 3 and 4). As for heat shock, the localization of SBF to the PCL1 and PCL2 promoters after pheromone treatment was reproducibly reduced but not eliminated in slt2 cells (Fig. 4). We conclude that Slt2 contributes to the increased localization of SBF to the promoters of PCL1 and PCL2 upon heat shock and -factor treatment.

View larger version (35K): [in this window] [in a new window]   FIG. 4.   Slt2 requirement for SBF localization to the promoters of PCL1 and PCL2. (A) PCR amplification of ChIPs from wild-type and slt2 strains. Wild-type (BY263) and slt2 (BY1342) cultures grown at 30°C (log), heat shocked for 1 h at 39°C (HS), or treated with -factor for 2 h () were cross-linked with formaldehyde. Lysates were made, and ChIPs were performed using pA alone, Swi4 antibodies (Swi4-Ip), or Swi6 antibodies (Swi6-Ip). Multiplex PCR was used to amplify the promoter regions of PCL2, PCL1, PHO5, and CLN1 from each of the immunoprecipitated chromatin reactions and the WCE. (B) PhosphorImager analysis of each PCR product was performed, and the localization of Swi4 and Swi6 to the promoters of PCL1, PCL2, PHO5, and CLN1 is depicted as percent WCE immunoprecipitated (%WCE Iped). Open bars, wild-type ChIPs; shaded bars, slt2 ChIPs. The data shown are representative of two separate experiments.

DNA microarray analysis of swi4 and slt2 mutants. Thus far, our experiments have identified one gene, PCL1, whose expression depends on SLT2 and SWI4. To determine whether PCL1 is representative of a larger group of genes, we used DNA microarrays to assay for genes regulated by both SLT2 and SWI4. We analyzed the genome-wide changes in transcription by using log-phase cultures of slt2, swi4, and bck1 strains grown at 25°C. We expected that the subtle effects of Slt2 on gene expression in log-phase cells meant that this analysis would provide an incomplete view of Slt2-Swi4-regulated genes. However, both Slt2 and Swi4 clearly have roles in the normal mitotic cell cycle, and we expected that strongly regulated genes may be uncovered (see Materials and Methods). We also assayed slt2 and swi4 cells at 39°C since transient heat shock causes a G₁ delay and activation of Slt2, thus biasing ourselves to a population of cells in which both Swi4 and Slt2 are essential.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   DNA microarray analysis. Experiments are listed along the horizontal axis, and genes are listed along the vertical axis. Three independent DNA microarray datasets were generated (slt2-25°C, swi4-25°C, and bck1-25°C) and compared to two related data sets from previously published data (swi4 and swi6 [27]). Genes whose expression levels were reduced >1.8-fold in swi4 and/or slt2 cells are shown. Three control experiments were performed with wild-type RNA (wild-type by wild-type comparison) to validate the cutoff used in the experiments with the mutant strains (see Materials and Methods). Cell cycle expression data were obtained from the Stanford Cell Cycle Expression project and Spellman et al. (52). SCB and MCB elements were found using the SCPD and SGD databases available on the internet. Numbers below the colored squares indicate the intensity ratio between the mutant strain versus the wild-type strain grown under the same conditions and represent the average values from two independent experiments. A value of 1 indicates no change; only genes that were verified by Northern blot analysis are shown.

Swi6 independence of newly identified Slt2-Swi4-dependent genes. Our PCL1 experiments suggested that genes regulated by Slt2 and Swi4 may also be independent of Swi6. Indeed, our analysis of DNA microarray data revealed genes whose expression appears dependent on Swi4 but independent of Swi6 (Fig. 5 [27]). To further explore our microarray results, we performed both Northern blot analysis and Swi4 ChIP on the Swi4- and Slt2-dependent genes (YLR110c and GIC1) and one Swi4-dependent gene (SRL1) identified in our microarray experiments. Although PCL1 was not identified in our DNA microarray analysis, we included it in our analysis.

View larger version (38K): [in this window] [in a new window]   FIG. 6.   Northern blot analysis and Swi4 localization to the promoters of newly identified Swi4-Slt2-dependent genes. (A) Northern blot analysis of a subset of proposed Swi4- and/or Slt2-dependent genes. Wild-type (Wt, BY263), swi4 (BY108), swi6 (BY107), and slt2 (BY1342) cells were grown to mid-log phase at 30°C, and total RNA was isolated and probed with the indicated open reading frame (ORF). A probe for ACT1 was used as a loading control. Autoradiograms shown are representative of one of three experiments. PhosphorImager analysis was performed, and the signal of each ORF was standardized to the ACT1 loading control (volume ORF/volume ACT1). The average standardized signal of three experiments is indicated beneath each lane. The standard deviation for each triplicate was <10% of the average. (B) Swi4 localization to the promoters of Swi4-Slt2-dependent genes. Wild-type (Wt, BY263), swi6 (BY107), and swi4 (BY108) cultures grown at 30°C (log) were cross-linked with formaldehyde, WCEs were made, and ChIPs were performed using pA or Swi4 antibodies (Swi4-IP). Multiplex PCR was performed as indicated for the ChIP samples and on fivefold serial dilutions of the WCE to amplify the promoter regions of the genes listed to the left of the panel. RLM1 and PHO5 were included as negative controls. CWP1 was identified as a putative SBF target in a genome-wide ChIP experiment (29) but did not immunoprecipitate with Swi4 antibodies in our experiment.

Swi4 interacts directly with Slt2 in vitro. Activation of the PKC1 pathway by a number of stimuli results in Slt2-dependent phosphorylation of Swi6 (39). These results have led to the suggestion that Slt2 may regulate gene expression through phosphorylation of Swi6 (39). Several results suggest that phosphorylation of Swi6 by Slt2 may not be of regulatory significance. First, our promoter analysis of PCL1, along with DNA microarray and Northern blot analysis, suggests that expression of Swi4-Slt2-dependent genes may be partially or fully independent of Swi6. Second, our ChIP experiments showed Swi4 localization to the promoters of PCL1, YLR110c, and SRL1 genes in the absence of Swi6 (Fig. 6B). Finally, a phosphorylation site mutant of Swi6, Swi6-SA4 (50), which cannot be phosphorylated by Slt2 in vivo, does not display any obvious cell integrity defects (data not shown). As discussed previously, Swi6 independence is an unusual feature of Swi4-dependent genes, and no other SCB-containing reporter construct has displayed Swi6 independence. Together, these results suggest that Slt2 may not modulate the activity of Swi6. Instead, Slt2 may regulate Swi4, possibly through direct interaction of Slt2 with Swi4.

View larger version (37K): [in this window] [in a new window]   FIG. 7.   Binding of Slt2 to Swi4 and Swi6 in vitro. (A) Five micrograms of partially purified SBF derived from insect cell extracts was incubated with either GST or GST-Slt2 immobilized on glutathione beads. The unbound (U) and bound (B) fractions were separated by SDS-6% PAGE. The gels were blotted and incubated with Swi4 antiserum (lanes 1 to 4) or Swi6 antiserum (lanes 5 to 8) to identify Swi4 or Swi6 proteins. The migration positions of molecular mass markers are indicated to the left (in kilodaltons). (B) Two microliters of in vitro-translated Swi4 and Swi6 were incubated with either GST or GST-Slt2 immobilized on glutathione beads. The unbound (U) and bound (B) fractions were separately by SDS-6% PAGE. The migration positions of molecular mass markers are indicated to the left (in kilodaltons).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We have used genetic and biochemical approaches, along with DNA microarray analysis, to describe genes whose expression is dependent on both the cell cycle transcription factor Swi4 and the PKC1-activated MAPK Slt2. Genes that are sensitive to both Slt2 and Swi4 appear to be uniquely regulated, which may reflect the ability of Slt2 to modulate the activity of Swi4; in particular, regulation of Swi4 by Slt2 may allow Swi4-dependent transcription in the absence of Swi6 (summarized in Fig. 8 and discussed further below). Our study also implies roles for Swi4 beyond its prominent role in controlling cell-cycle-dependent transcription.

View larger version (27K): [in this window] [in a new window]   FIG. 8.   Model for Slt2 and Swi4 coordinate regulation of PCL1-like genes. Possible modes of regulation of Swi4 by Slt2 are diagrammed. Swi4 interacts with Swi6 to form SBF (in dashed box) to control expression of G1-specific transcripts (HO, CLN1, CLN2, PCL1, etc.) via binding to SCB elements. In our model, phosphorylation of Swi4 by Slt2, either on its own or complexed with Swi6, may relieve the autoinhibition of Swi4 DNA binding. Regulation of Swi4 by Slt2 may impart a unique Swi6-independent function to Swi4 that directs activation of a subset of Swi4-Slt2-dependent genes (PCL1, SRL1, etc.). Genes controlled by Swi4 and Slt2 include non-cell-cycle-regulated genes and define a novel role for Swi4 beyond its major role in controlling cell-cycle-dependent transcription.

Swi4 and Slt2 coregulate a subset of genes. In order to understand the transcriptional role of Swi4 and Slt2 in maintaining cell wall integrity, we performed DNA microarray analysis to identify genes that require both Slt2 and Swi4 for their expression. Through careful analysis of our microarray data and other published datasets, we identified genes whose expression was reduced in both SWI4 and SLT2 deletion strains (Fig. 5). Although Northern blot analysis and ChIP experiments generally support the validity of the microarray data, we are unlikely to have identified all Slt2-Swi4-dependent genes. First, we performed our microarrays using RNA probes from log-phase cells in an attempt to minimize cell cycle effects. Second, many Slt2-dependent transcriptional effects range from 1.2- to 2-fold changes (28, 30, 39), which is below the cutoff for our microarray analysis (1.8-fold). Indeed, our limits exclude the possibility of identifying known Slt2-Swi4-dependent genes, including PCL1 and several cell wall biosynthetic genes (28).

Role of the PKC1 pathway and SBF in pheromone response. In addition to regulating a subset of Swi4 targets during mitotic growth, our work suggests that Slt2 may also partner with Swi4 to regulate the pheromone-dependent induction of gene expression. DNA microarrays have been used to examine genome-wide patterns of gene expression in response to pheromone treatment (47). Interestingly, pheromone treatment induces both an early transcriptional response dependent on Ste12 and a later transcriptional response dependent on PKC1. The late-induced genes are not enriched for Ste12 binding sites (PREs) in their promoters, nor are they induced by overexpression of STE12. These results suggest that the PKC1-MAPK pathway likely activates other transcription factors necessary for the late pheromone-induced genes.

Slt2-dependent regulation of Swi4. As mentioned above, both our PCL1::lacZ reporter gene and Northern blot analyses show that PCL1 expression is dependent on Swi4 and partially dependent on Slt2 but independent of Swi6 (Fig. 1 and 6). Swi6 independence is not unique to the PCL1 gene. As discussed above, our microarray experiments identified other genes whose expression during log phase is dependent on Swi4 but independent of Swi6. Further, our Northern blot analysis confirmed that the log-phase levels of PCL1 and YLR110c mRNA are Swi6 independent and that the log phase levels of GIC1 and SRL1 mRNA are partially independent of Swi6. Surprisingly, Swi4 was also localized to the promoters of PCL1, SRL1, and YLR110c in the absence of Swi6. This result contrasts with a previous Swi4 ChIP study, which showed that Swi6 was required for Swi4 localization to the SCBs of the promoter of HO endonuclease (13), whose activity is fully dependent on Swi6 (9). In the same study, Swi6-independent localization of Swi4 to the promoter of CLN2 was detected; however, we find that expression of CLN2 is not affected by deletion of SLT2 (data not shown). Clearly, Swi4 regulation is complex, and we propose that, in the absence of Swi6, Slt2 may control the binding of Swi4 to only a subset of Swi4-dependent genes.