Cell-type-dependent repression of yeast a-specific genes requires Itc1p, a subunit of the Isw2p–Itc1p chromatin remodelling complex

Cristina Ruiz1, Victoria Escribano2, Eulalia Morgado2, María Molina1 and María J. Mazón2

1 Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain
2 Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, C/Arturo Duperier 4, 28029 Madrid, Spain

Correspondence
María J. Mazón
mjmazon{at}iib.uam.es


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In Saccharomyces cerevisiae MATa haploid cells, the a-specific genes are expressed, whereas in the MAT{alpha} haploid and MATa/{alpha} diploid cell types their transcription is repressed. It is shown in this report that Itc1p, a component of the ATP-dependent Isw2p–Itc1p chromatin remodelling complex, is required for the repression of a-specific genes. It has previously been reported that disruption of the ITC1 gene leads, in MAT{alpha} cells, to an aberrant cell morphology resembling the polarized mating projection of cells responding to pheromone. The activation of the pheromone signalling pathway in itc1 mutants of both mating types was examined and found to be constitutively active in MAT{alpha} itc1 but not in MATa itc1 cells. Furthermore, unlike the wild-type, MAT{alpha} itc1 and MATa/{alpha} itc1/itc1 cells secrete a-factor and express significant levels of other a-specific genes. The results indicate that the inappropriate a-factor production in a MAT{alpha} context, due to the derepression of the a-specific genes, produces an autocrine signalling loop that leads to the aberrant morphology displayed by MAT{alpha} itc1 cells. It is suggested that the Isw2p–Itc1p complex contributes to maintain the repressive chromatin structure described for the asg operator present in the promoters of a-specific genes.


Abbreviations: MAPK, mitogen-activated protein kinase; MAPKK(K), MAPK kinase (kinase)


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The budding yeast Saccharomyces cerevisiae has the capacity to respond to external signals by activating different mitogen-activated protein kinase (MAPK) cascades that will enable the cell to induce the appropriate response (for a review see Gustin et al., 1998). Among the external signals that yeast needs to respond to are mating pheromones, nutrient starvation and osmotic or cell wall stress. Peptide pheromones activate the mating signal transduction pathway which, among other physiological changes, induces a polarized cell growth toward the mating partner (Dohlman & Thorner, 2001). During the European Functional Analysis project (EUROFAN), we described that disruption of the YGL133w/ITC1 gene does not affect vegetative growth in different media but produces, only in the MAT{alpha} cell type, an aberrant cell morphology resembling the characteristic schmoo formed by cells exposed to mating factor and a decrease in the relative mating efficiency when assayed in liquid medium (Escribano & Mazón, 2000). No clues as to the biological function of the Itc1 protein were known when these phenotypes were reported. In a subsequent systematic screen for mutants displaying cell-wall-related phenotypes, also in the frame of EUROFAN, itc1 was identified among a group of mutants showing increased sensitivity to calcofluor white and constitutive activation of Slt2/Mpk1, the MAP kinase of the cell integrity signalling pathway (de Groot et al., 2001).

In an independent work, Itc1p was identified as the second subunit of an ATP-dependent chromatin-remodelling complex formed with Isw2p (Gelbart et al., 2001), shown to repress early meiotic genes during mitotic growth (Goldmark et al., 2000). The Isw2–Itc1p complex was subsequently shown to be involved in the repression of other genes related to the starvation response, such as INO1 (Sugiyama & Nikawa, 2001) and PHO3 (Kent et al., 2001). The ISW2 gene encodes a yeast homologue of the Drosophila ISWI chromatin-remodelling ATPase (Tsukiyama et al., 1999; Trachtulcova et al., 2000), which is essential for development as well as for cell viability (Deuring et al., 2000). Members of this class of remodelling factors have been found so far in yeasts, Drosophila, Xenopus and human (Goldmark et al., 2000), and play key roles in modulating transcription through the regulation of chromatin structure. Itc1p-related proteins have been identified in the pathogenic yeast Candida albicans and in higher organisms such as humans and mice.

To understand the mechanisms underlying the above-mentioned MAT{alpha}-specific phenotypes of the itc1 mutant, concerning cellular morphology and mating, we have analysed the mating pheromone response pathway in this mutant. In this pathway, the pheromone produced by cells of one mating type is recognized by cells of the opposite mating type, and this recognition triggers the activation of a signal transduction pathway which ultimately leads to induction of gene transcription, cell cycle arrest and changes in cellular morphology (Herskowitz, 1995). Each of the haploid cell types produces a different mating factor. Only MATa cells secrete a-factor, that binds to its specific receptor Ste3p, that is expressed only in MAT{alpha} cells. Conversely, MAT{alpha} cells secrete {alpha}-factor that binds to its specific receptor (Ste2p), expressed only in MATa cells. Activation of the receptor causes the dissociation of the G protein {alpha} subunit (G{alpha}) from the G{beta}{gamma} heterodimer which then activates the kinase cascade, formed by Ste11p (MAPKKK), Ste7p (MAPKK), and the MAPKs Fus3p or Kss1p. Linkage between the heterotrimeric G protein and the MAPK module involves the Ste20p kinase and Ste5p, a scaffold protein that associates with the members of the kinase cascade (Choi et al., 1994; Printen & Sprague, 1994). Activation of the MAPK module leads to phosphorylation of the transcription factor Ste12p, which, in association with Mcm1p, activates transcription of numerous genes (Kirkman-Correia et al., 1993) required for the pheromone response pathway itself and for cell fusion such as FUS1 (Hagen et al., 1991).

In the present work we show that the MAT{alpha} itc1 mutant inappropriately produces a-factor leading to the constitutive activation of the mating pheromone response pathway. Also, we confirm the activation of the cell integrity pathway and find it to be cell type-independent. Finally, we show that the a-specific genes are derepressed in the MAT{alpha} itc1 and MATa/{alpha} itc1/itc1 mutant cells, thus providing first evidence that Itc1p participates in the cell type-dependent repression of a-specific genes in wild-type cells.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Yeast strains and growth media.
The strains used in this study are haploid derivatives of strain FY1679 (MATa/MAT{alpha} ura3-52/ura3-52 his3{Delta}200/HIS3 leu2{Delta}1/LEU2 trp1{Delta}63/TRP1) carrying an itc1 : : kanMX4 gene disruption in heterozygosis (Escribano & Mazón, 2000). ITC1 gene disruption, sporulation and tetrad analysis were carried out as described by Escribano & Mazón (2000). Wild-type and itc1/itc1 homozygous mutant autodiploids were generated from MAT{alpha} ITC1 and MAT{alpha} itc1 strains by transformation with a centromeric YCp50 plasmid carrying the HO gene (Russell et al., 1986) and counterselection of Ura+ transformants in fluoroorotic acid-containing plates. Strains carrying the isw2 : : kanMX4 gene disruption and their isogenic wild-type, were obtained from the EUROSCARF collection (European Saccharomyces cerevisiae Archive for Functional Analysis; http://www.uni-frankfurt.de/fb15/mikro/euroscarf) and are derivatives of BY4741 (MATa his3{Delta}1 leu2{Delta}0 met15{Delta}0 ura3{Delta}0) and BY4742 (MAT{alpha} his3{Delta}1 leu2{Delta}0 met15{Delta}0 ura3{Delta}0). Strains FYDK (MATa his3-{Delta}200 ura3-52 trp1-63 leu2-1 slt2 : : URA3) (de Nobel et al., 2000), JTY2519 (MATa ade2-101 trp1-63 leu2-1 ura3-52 his3-{Delta}200 lys2-801 fus3 : : LEU2) (Bardwell et al., 1998) and JTY2520 (MATa ade2-101 trp1-63 leu2-1 ura3-52 his3-{Delta}200 lys2-801 kss1 : : HIS3) (Ma et al., 1995) were used as controls in the immunoblotting experiments. Yeast was grown on 1 % yeast extract, 2 % peptone and 2 % glucose (YPD) or, when required, on glucose minimal medium (SD) containing 6·7 g yeast nitrogen base without amino acids (YNB) l-1 and 2 % glucose. SD medium was supplemented with the appropriate requirements (added at 0·1 mg ml-1) except for those used for selection. Solid media contained 2 % agar.

Molecular biology techniques.
MATa and MAT{alpha} strains carrying the wild-type or the itc1 : : kanMX4 alleles of ITC1 were disrupted for STE20 or STE3 genes by the one-step gene disruption technique (Rothstein, 1991), by transformation with the corresponding cassette, ste20 : : URA3 (Martín et al., 1997) or ste3 : : URA3 (kindly donated by G. F. Sprague, Institute of Molecular Biology, University of Oregon, USA). In each case the correct replacement of the gene by the URA3 cassette at the target locus was tested by analytical PCR using genomic DNA of the transformants. FUS1lacZ reporter plasmid was constructed from pDH17 (Hagen et al., 1991) by cloning a SalI–PstI fragment containing the FUS1lacZ fusion into pRS424 (Christianson et al., 1992). Yeast transformation was carried out by the lithium acetate method (Ito et al., 1983).

{beta}-Galactosidase assays.
{beta}-Galactosidase induction experiments were performed as described by Hagen et al. (1991). FUS1lacZ plasmid-bearing strains were grown to saturation in selective medium, diluted in YPD medium to OD660 0·2 and allowed to grow in this medium for one doubling (usually 3 h). The cells were then centrifuged and resuspended in YPD or pheromone-containing YPD and further incubated at 30 °C with agitation for 2·5 h. The a-factor was the culture filtrate of the MATa ITC1 strain grown in YPD medium for 48 h. The {alpha}-factor was purchased from Sigma and was added to the YPD medium at a final concentration of 1 µg ml-1. {beta}-Galactosidase activity was measured in whole-cell extracts prepared with glass beads as described by Leber et al. (2001).

Immunoblot analysis.
Whole-cell extracts were prepared from mid-exponential phase cells and equal protein amounts were fractionated by SDS-PAGE and transferred to nitrocellulose membranes as previously described (Martín et al., 2000). Detection of dually phosphorylated Slt2p, Kss1p and Fus3p was performed with anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibodies from New England Biolabs. The blots were stripped and reprobed with specific antibodies against each MAPK to verify the identity of the phosphoprotein bands and as a protein loading control. Slt2p was detected using anti-GST–Slt2 antibodies (Martín et al., 1993). Fus3p and Kss1p were detected with specific antibodies purchased from Santa Cruz Biotechnology.

Halo assays.
The spot halo assay indirectly measures the amount of pheromone that has been secreted by the cell to the culture medium. This assay is based on the growth arrest caused by the pheromone secreted by cells of one mating type on cells of the opposite mating type. To improve the sensitivity of the assay, sst2 mutants, defective in the adaptation to mating signal, are used as tester strains. a-Factor was prepared from ITC1 or itc1 strains of both mating types, grown in 10 ml SD dropout medium, by rinsing the culture flasks with methanol and concentrating to dryness, as described by Nijbroek & Michaelis (1998). The dried sample was resuspended in 5 µl methanol and serial dilutions of ten- to twofold increments were prepared in YPD containing 0·25 mg BSA ml-1. {alpha}-Factor was prepared by concentrating to dryness 1 ml of the culture filtrate of the ITC1 or itc1 strains of both mating types. After resuspension in YPD, twofold dilutions were prepared in YPD. Aliquots (2 µl) of each dilution were spotted onto a lawn of supersensitive MAT{alpha} sst2 or MATa sst2 : : URA3 tester strains, respectively, and the plates were incubated for 3 days at 23 °C to determine a- or {alpha}-factors. About 1 ng synthetic factors (purchased from Sigma) was used as a positive control on each plate. Halo plates were YPD plates overlaid with 0·3 OD660 units of the tester strain resuspended in YPD-top-agar (3 ml, 0·7 % agar).

RT-PCR semi-quantitative analysis.
Exponentially growing haploid, wild-type or itc1, and diploid, wild-type or itc1/itc1 cells were collected and RNA was obtained with TRIzol (Gibco). Samples were tested by electrophoresis on 1·2 % agarose/2·2 M formaldehyde gels, and by PCR to confirm that there was no contaminating DNA. Reverse transcription was performed using Promega Reverse Transcription System with 500 ng total RNA to yield 20 µl cDNA. PCRs were then performed to determine the linear range of amplification for each gene that would allow a semi-quantitative assessment of expression levels. The optimal parameters determined for each PCR were 95 °C, 15 s; 60 °C, 1 min, and 18 cycles for ACT1, 20 for STE12, 22 for STE2 and GPA1, and 24 for ASG7 and BAR1. The primers used (Table 1) were designed to yield small amplicons (ACT1, 77 bp; ASG7, 75 bp; BAR1, 64 bp; GPA1, 77 bp; STE2, 106 bp and STE12, 82 bp) to improve the efficiency and reproducibility of the PCR. Ten microlitres of each DNA sample were separated on a 2 % agarose gel, stained with ethidium bromide and photographed.


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Table 1. Oligonucleotides used in the RT-PCR assays

 

   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Deletion of ITC1 results in MAT{alpha}-specific constitutive activation of the pheromone signalling pathway
In {alpha} mating type cells, disruption of ITC1 has been shown to produce an aberrant morphology strongly resembling that of cells challenged by mating pheromone (Escribano & Mazón, 2000). To elucidate the mechanisms underlying the observed defect, the state of activation of the pheromone signalling pathway was studied by determining the transcriptional activation of the pheromone-responsive gene FUS1, one of the targets of the pathway, required for cell fusion during the mating process. For this purpose, a FUS1–lacZ fusion was transformed into wild-type and itc1 mutant cells, either MATa or MAT{alpha}, and {beta}-galactosidase activity was measured in pheromone treated and untreated cells. As shown in Fig. 1(a), while the MATa itc1 mutant showed activation of the reporter gene only when stimulated with {alpha} pheromone, the MAT{alpha} itc1 cells showed high levels of FUS1 expression even in the absence of a-factor. This result indicates a constitutive activation of the pheromone signalling pathway in the MAT{alpha} itc1 strain.



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Fig. 1. The pheromone signalling pathway is constitutively activated in a MAT{alpha} itc1 mutant. (a) FUS1 expression is induced in MAT{alpha} itc1 mutant cells in the absence of added pheromone. FUS1 expression was determined for wild-type, itc1, ste20 and itc1 ste20 strains, either MATa or MAT{alpha}, in the presence (dark grey bars) or absence (light grey bars) of added pheromone. The indicated strains were transformed with the FUS1lacZ construct and {beta}-galactosidase activity was measured in whole-cell extracts of the transformants as descrbed by Leber et al. (2001). All assays were carried out in duplicate. Data shown are the mean of the {beta}-galactosidase specific activities determined in at least three independent experiments. Variability between experiments was {els]15 %. (b) Kss1p and Fus3p MAPKs are phosphorylated in growing MAT{alpha} itc1 mutant cells. Protein extracts were prepared from mid-exponential phase cultures of the indicated strains, fractionated by SDS-PAGE and transferred to nitrocellulose membranes as described in Methods. Detection of dually phosphorylated Slt2p, Kss1p and Fus3p was performed with anti-phospho-p44/42 MAPK (Thr202/Tyr204) antibodies. The blots were stripped and reprobed with specific antibodies against each MAPK to verify the identity of the phosphoprotein bands and as a loading control.

 
Activation of the pheromone signalling pathway was also checked by testing the dual phosphorylation of the MAPKs Fus3p and Kss1p, two downstream elements of the pheromone-dependent kinase cascade initiated by the MAPKKK Ste11p and the MAPKK Ste7p. There has been some controversy in the literature as to whether or not Fus3p and Kss1p have specialized functions in mating and invasive growth, respectively, and it has been proposed that Kss1p participates in mating only in the absence of Fus3p (Madhani et al., 1997). However, it has been recently shown that both MAPKs are activated by mating pheromones in wild-type cells, and that this activation is required for normal pheromone-induced gene expression (Sabbagh et al., 2001). To analyse Fus3p and Kss1p activation, we used an antibody that specifically recognizes the dually phosphorylated form of MAPKs bearing a TEY motif in the activation domain (Rodríguez-Pachón et al., 2002). The results (Fig. 1b) show an increase in the phosphorylated and therefore activated form of both Kss1p and Fus3p in MAT{alpha} itc1 cells not treated with pheromone, as compared with its corresponding wild-type, whereas in MATa cells this increase is not observed. In these experiments, the identity of the phosphoprotein bands was verified by immunoblotting with specific anti-Kss1p and anti-Fus3p antibodies. An increase in both the amount and activation of Kss1p can be observed in the fus3 mutant strain (Fig. 1b), indicating a negative role for Fus3p in the regulation of Kss1p protein levels and/or phosphorylation. In fact, it has been recently reported that active Fus3p limits the magnitude and duration of Kss1p phosphorylation (Sabbagh et al., 2001). Remarkably, in the MAT{alpha} itc1 mutant both MAPKs co-exist in their activated form. The constitutive activation of the Fus3p and Kss1p MAPKs, and of the FUS1lacZ reporter gene, suggests that the aberrant morphology detected specifically in MAT{alpha} itc1 cells may be caused by signalling through the pheromone pathway.

itc1 mutants show mating type-independent activation of the cell integrity pathway
ITC1 has been identified among a group of genes whose deletion results in defects in cell wall integrity (de Groot et al., 2001). The systematic screening of 620 MATa mutants for different cell wall-related phenotypes revealed the itc1 mutant to be sensitive to the presence of calcofluor white or caffeine in the growth medium, and to show constitutive activation of Slt2p/Mpk1p, the MAPK of the cell wall integrity signalling pathway. Therefore, in the same set of experiments designed to detect MAPK phosphorylation (Fig. 1b) we also analysed the activation state of Slt2p in itc1 strains of both mating types. To confirm the identity of the Slt2 phosphoprotein band we used specific anti-Slt2p antibodies. As shown in Fig. 1, Slt2p was found to be activated in both itc1 strains, thus indicating that the previously reported activation of this MAPK (de Groot et al., 2001) is not mating type-specific. These results suggest that the aberrant morphology and the constitutive activation of the pheromone pathway, detected only in the MAT{alpha} itc1 mutant cells, are not related to the cell wall phenotype.

MAT{alpha} itc1 mutant cells require an intact pheromone signalling pathway to activate the expression of FUS1lacZ
To determine which components of the pheromone signalling pathway were required for the activation of the FUS1 reporter, we introduced an ste20 : : URA3 mutation into the wild-type and itc1 mutant strains. The STE20 gene product is a protein kinase linking the activation of the pheromone receptor-coupled G protein to the pheromone-dependent MAPK cascade, and its deletion is known to block the pheromone response in a wild-type background (Leberer et al., 1992). The ste20 disruptants were transformed with the FUS1lacZ fusion and {beta}-galactosidase activity was measured in crude cell extracts. The levels of {beta}-galactosidase activity in the presence of pheromone were found to be dependent on Ste20p in wild-type and itc1 mutant strains of both mating types (Fig. 1a). The constitutive activation of the reporter gene in the MAT{alpha} itc1 strain was also abolished in the absence of Ste20p, indicating that an intact pheromone response pathway is required for the observed mating type-specific constitutive signalling. Accordingly, in the double mutant MAT{alpha} itc1 ste20, the phosphorylation of the Fus3p and Kss1p MAPKs was eliminated (Fig. 2a) and the morphological defect was rescued (Fig. 2b), thus confirming that the aberrant morphology shown by the MAT{alpha} itc1 mutant is due to the activation of the pheromone pathway. In contrast, the Slt2p MAPK remained activated after disruption of the pathway, although its degree of phosphorylation in the itc1 ste20 double mutant was lower than in the corresponding itc1 single mutant (Fig. 2a).



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Fig. 2. An intact pheromone response pathway is required for the mating type-specific constitutive signalling and aberrant cell morphology in the MAT{alpha} itc1 mutant. (a) STE20 and STE3 are required for constitutive signalling in MAT{alpha} itc1 cells. Cell extracts, Western blots and detection of Slt2p, Kss1p and Fus3p dually phosphorylated forms were performed as described in Fig. 1(b). Slt2p detection with anti-GST–Slt2 antibodies was used as a protein loading control. (b) STE20 and STE3 gene disruption rescues the abnormal morphology of MAT{alpha} itc1 cells. Growing cells of the itc1 single mutant, and itc1 ste20 or itc1 ste3 double mutant strains were photographed with a Nikon Eclipse TE2000-U microscope and the percentage of aberrant cells was determined by counting four independent fields (at least 200 cells were counted for each strain). Aberrant cells were 48·5 % (MAT{alpha} itc1), 2·2 % (MAT{alpha} itc1 ste20) and 1·9 % (MAT{alpha} itc1 ste3) of the total. The small percentage of abnormal cells remaining in the double mutants showed a less pronounced morphological phenotype.

 
Except for the pheromones and their receptors, the components of the mating signal transduction pathway are common to both mating types. We reasoned that signalling in MAT{alpha} itc1 cells through this pathway should depend on the pheromone receptor, Ste3p. To test this hypothesis, we checked the activation of the MAPKs in MAT{alpha} wild-type and itc1 cells carrying an STE3 gene disruption. It was found that the activation of Fus3p and Kss1p was completely abolished (Fig. 2a) and that normal morphology was recovered (Fig. 2b) in MAT{alpha} itc1 ste3 cells. Thus, the constitutive activation of the two MAPKs and also the aberrant morphology require the presence of the pheromone receptor.

Again, as observed upon STE20 disruption, the Slt2p MAPK remained partially activated in the absence of the receptor (Fig. 2a). All together the data on Slt2p suggest that its activation in the itc1 mutants is mainly due to the activation of the cell integrity pathway by a still unknown cell wall alteration. Consistent with this interpretation is the fact that addition of 0·8 M sorbitol, known to provide osmotic stabilization to a weakened cell wall, remedied the Slt2p activation in itc1 mutants of both mating types (Fig. 3a), while the cell type-specific Kss1p and Fus3p constitutive activation and the aberrant morphology remained unaltered (Fig. 3b).



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Fig. 3. Mating type-independent activation of the cell integrity pathway in itc1 mutants. (a) Slt2p activation is rescued by the addition of sorbitol to itc1 mutants of both cell types. The indicated strains were grown in YPD medium in the absence or presence of 0·8 M sorbitol. Cell extracts, Western blots and detection of Slt2p, Kss1p and Fus3p dually phosphorylated forms were performed as described in Fig. 1(b). Slt2p detection with anti-GST–Slt2 antibodies was used as a protein loading control. (b) Abnormal morphology of MAT{alpha} itc1 cells remains unaltered in the presence of 0·8 M sorbitol. Cells of the MAT{alpha} itc1 mutant strain were grown in YPD or in YPD+0·8 M sorbitol and photographed with an Olympus BH-2 microscope.

 
MAT{alpha} itc1 mutant cells produce and secrete a-factor
Since the presence of the Ste3p receptor is required for the signalling through the pheromone response pathway observed in the absence of added pheromone in the MAT{alpha} itc1 mutant, we tested this mutant strain for production of a-factor. To determine the presence of the pheromones in the culture media, we measured their biological activity by performing halo assays with pheromones isolated from wild-type and mutant cultures. The results (Fig. 4a) clearly showed that MAT{alpha} itc1 cells secrete a-factor into the medium, although to a lesser extent than either wild-type or mutant MATa cells. Therefore, the constitutive activation of the pheromone pathway in the MAT{alpha} itc1 mutant may be explained by autocrine stimulation through Ste3p.



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Fig. 4. a-Specific genes are derepressed in MAT{alpha} itc1 mutant cells. (a) Spot halo assay showing relative levels of a- and {alpha}-factor production by wild-type and itc1 strains. a-Factor produced by the indicated strains was prepared as described in Methods. Serial dilutions (from left to right: 102, 103, 2x103, 4x103 and 8x103) of the concentrated factor, 2 µl each, were spotted onto a lawn of the supersensitive MAT{alpha} sst2 strain. About 1 ng synthetic a-factor was used as the positive control. {alpha}-Factor was prepared by concentrating to dryness 1 ml of the culture filtrate of the indicated strains. After resuspension, the concentrated {alpha}-factor and twofold dilutions in YPD, 5 µl each, were spotted onto a lawn of MATa sst2 : : URA3 supersensitive cells. Synthetic {alpha}-factor (1 ng) was used as the positive control. Halo plates were prepared as described in Methods. The plates shown are representative of at least three separate experiments performed with two different preparations of the factors. (b) RT-PCR semi-quantitative analysis of three a-specific genes. Exponentially growing wild-type and itc1 cells of both mating types were collected and RNA was isolated. Reverse transcription and PCR reactions with the specific primers listed in Table 1 were performed for ASG7, BAR1 and STE2 genes. Shown is the electrophoresis in a 2 % agarose gel of 10 µl of each PCR reaction. RT-PCR of the actin gene was performed as an internal control. RNA extraction was performed twice and the results shown are representative of four independent RT-PCR experiments.

 
The results also showed (Fig. 4a) that the quantity of {alpha}-pheromone produced and/or secreted by the MAT{alpha} itc1 mutant was considerably lower than that produced by the corresponding wild-type. This fact might explain the lower mating ability shown by these cells, since it has been reported that defects in {alpha}-factor production correlate quantitatively with mating efficiency (Caplan & Kurjan, 1991).

a-Specific genes are derepressed in the MAT{alpha} itc1 mutant
The results obtained with the halo assay led us to conclude that not only MFA1 and MFA2, the genes encoding the a-factor, but also STE6, the ABC transporter required for a-factor secretion, must be expressed in the MAT{alpha} mutant. We then tested the possibility that other a-specific genes could also be expressed by performing semi-quantitative RT-PCR analysis, using as template RNA isolated from wild-type and itc1 strains of both mating types. Among the a-specific genes, we analysed STE2, the gene encoding the pheromone {alpha}-factor receptor (Blumer et al., 1988; Jenness et al., 1983); BAR1, encoding the protease that degrades {alpha}-factor (barrier protease) (Mackay et al., 1988); and ASG7, which has been recently shown to be involved in the regulation of the zygotic transition to vegetative growth (Roth et al., 2000). As shown in Fig. 4(b), STE2, BAR1 and ASG7 were found to be expressed in MATa wild-type cells, as expected, and to a similar extent in MATa itc1 mutant cells. Remarkably, significant expression levels of these genes were also found in MAT{alpha} itc1 cells, thus pointing to an extensive alteration of the transcriptional repression of a-specific genes in this mutant.

a-Specific genes are derepressed in the homozygous itc1/itc1 mutant
Expression of a-specific genes is, in wild-type strains, restricted to MATa cells. The a-specific genes are strongly repressed in the other two cell types, MAT{alpha} haploid and MATa/{alpha} diploid. A 32 bp sequence present in every a-specific gene is responsible for this transcriptional regulation. This sequence, the asg operator, includes a Mcm1p binding site flanked by two recognition sequences for the Mat{alpha}2p repressor (Johnson, 1995). Mcm1p belongs to the MADS box family of transcription factors and is highly conserved across species. In MATa cells, Mcm1p binds to the asg operator and recruits the transcription factor Ste12p resulting in transcriptional activation of the a-specific genes. In MAT{alpha} and MATa/{alpha} cells, a Mat{alpha}2p dimer binds to DNA cooperatively with Mcm1p (Mak & Johnson, 1993; Smith & Johnson, 1992) and recruits the Ssn6p–Tup1p general transcription repressor complex (Keleher et al., 1992), thus bringing about full repression in the appropriate cell types, since Mat{alpha}2p is not expressed in MATa cells. Taking into account that the repressor complex and the DNA context are the same for MAT{alpha} and MATa/{alpha} cells, we considered the possibility that the repression of a-specific genes could be dependent on the presence of Itc1p not only in haploid but also in diploid cells. First, we tested the possible production of a-factor by performing halo assays, using pheromones obtained from wild-type or homozygous itc1/itc1 mutant diploid cells. It was found that the latter strain is able to produce and secrete a-factor (Fig. 5a) whereas no {alpha}-factor activity was detected (not shown). This finding implies that at least the MFA and STE6 genes must be expressed, pointing to the derepression of a-specific genes in the diploid mutant. This extreme was confirmed by semi-quantitative RT-PCR analysis. The results (Fig. 5b) showed that STE2, BAR1 and ASG7 are expressed in the homozygous itc1/itc1 mutant strain. All together these data show that Itc1p is involved in the repression of a-specific genes both in MAT{alpha} and MATa/{alpha} cells.



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Fig. 5. a-Specific genes are derepressed in MATa/{alpha} itc1/itc1 homozygous mutant cells. (a) Spot halo assay to determine a-factor production by wild-type and itc1/itc1 diploid strains. a-Factor was prepared from the indicated strains, serially diluted and assayed as described in Fig. 4(a). Synthetic a-factor (1 ng) was used as the positive control. The result shown is representative of two experiments performed with two different preparations of a-factor. (b) RT-PCR semi-quantitative analysis of three a-specific genes. Exponentially growing diploid cells, either wild-type or itc1/itc1, were collected and RNA obtained and processed for RT-PCR exactly as in Fig. 4(b). (c) RT-PCR semi-quantitative analysis of two haploid-specific genes. Exponentially growing haploid and diploid cells, either wild-type or itc1/itc1, were collected and RNA obtained and processed for RT-PCR as in Fig. 4(b) except that the primers used (listed in Table 1) were specific for GPA1 and STE12. (b, c) RT-PCR of the actin gene was performed as an internal control. RNA extraction was performed twice and the results shown are representative of four independent RT-PCR experiments.

 
Transcriptional regulation of haploid-specific genes is not affected in the homozygous itc1/itc1 mutant
The Ssn6p–Tup1p repressor complex is recruited to the asg operator through direct interaction of Tup1p with Mat{alpha}2p (Komachi et al., 1994). Thus, a defect in the formation and/or recruitment of this complex in the itc1 mutant could result in the observed derepression of a-specific genes. Mat{alpha}2p is also required, together with Mata1p, for repression of haploid-specific genes in MATa/{alpha} diploid cells. A heterodimer formed by these two regulatory proteins, co-expressed only in diploids, binds to the upstream sequence of the haploid-specific genes, the hsg operator, through their homeodomains (Goutte & Johnson, 1994) and then attracts the Ssn6p–Tup1p complex to the DNA through the interaction of Tup1p with Mat{alpha}2p. This complex is therefore involved in the repression of both a-specific and haploid-specific genes (Tzamarias & Struhl, 1994). We then checked the functionality of the repressor complex formed by Mat{alpha}2p–Mata1p–Tup1p–Ssn6p in the itc1/itc1 mutant by measuring the expression of GPA1, a haploid-specific gene (Miyajima et al., 1987) encoding the alpha subunit of the heterotrimeric G-protein of the pheromone response pathway, and of STE12, which is repressed 5- to 10-fold in diploid cells (Fields & Herskowitz, 1987). Semiquantitative RT-PCR analysis was performed in wild-type and mutant haploid and diploid strains. As shown in Fig. 5(c), the two genes are repressed in the diploid strains, whether wild-type or mutant, and expressed in the wild-type and mutant haploid strains of both mating types. These results indicate that, in the absence of Itc1p, the Ssn6p–Tup1p complex must be correctly formed and recruited to DNA by the Mat{alpha}2p–Mata1p regulatory dimer, and that Itc1p is not involved in the control of haploid-specific genes. The derepression of a-specific genes displayed by the itc1 mutant cannot thus be attributed to defects in the Ssn6p–Tup1p repressor complex.

Phenotype of isw2 mutant strains
As stated in the Introduction, it has been reported that Itc1p together with Isw2p constitute a chromatin remodelling complex and that Itc1p is essential for the in vivo function of this complex (Gelbart et al., 2001). Therefore, we investigated if the absence of the Isw2p subunit would reproduce the cell type-specific constitutive signalling through the mating pathway that we have shown for the itc1 mutant. For this purpose, using MATa and MAT{alpha} isw2 mutants, we first analysed the effect of the isw2 mutation on cellular morphology. The isw2 mutant strain exhibited a MAT{alpha}-specific morphological defect (Fig. 6a) that is not prevented by the presence of sorbitol in the medium (not shown). Moreover, the isw2 mutant showed MAT{alpha}-specific constitutive activation of the Kss1p and Fus3p MAPKs, and cell type-independent activation of the Slt2p MAPK (Fig. 6b). These results indicate that the absence of any one of the two subunits of the remodelling complex causes MAT{alpha}-specific constitutive activation of the mating pathway, and cell type-independent constitutive activation of the cell integrity pathway.



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Fig. 6. Phenotype of isw2 strains. (a) MAT{alpha} isw2 mutant shows aberrant cell morphology. Wild-type and isw2 cells of both mating types were grown in YPD and photographed with a Nikon Eclipse TE2000-U microscope. (b) Kss1p and Fus3p MAPKs are phosphorylated in growing MAT{alpha} isw2 mutant cells while activation of the cell integrity pathway in the isw2 background is mating type-independent. Protein extracts, SDS-PAGE, transfer to nitrocellulose membranes and detection of dually phosphorylated Slt2p, Kss1p and Fus3p were performed as described in Fig. 1(a). The blots were stripped and reprobed with specific antibodies against GST–Slt2 as a loading control.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We present the first evidence that Itc1p, a component of the ATP-dependent chromatin remodelling complex Isw2p–Itc1p, plays a crucial role in the repression of a-specific genes. We had previously reported the aberrant morphology of a MAT{alpha} itc1 mutant but the mechanisms underlying this defect remained unknown.

Autocrine signalling causes constitutive activation of the pheromone signalling pathway in MAT{alpha} itc1 mutants
In this work we present evidence that MAT{alpha} itc1 cells have constitutively activated the mating pheromone signal transduction pathway. First, expression of FUS1, a pheromone responsive gene, was found to be induced in this mutant strain, in the absence of added pheromone. Second, Kss1p and Fus3p kinases, the MAPKs of the pheromone pathway, were found to be constitutively active. Both effects, the stimulation of FUS1 transcription and the activation of the MAPKs, occurred only in the MAT{alpha} itc1 mutant and not in the MATa background. Activation of Ste20p, the protein kinase that initiates the chain of events leading to changes in transcription during mating, is one of the primary effects of the addition of pheromone. The fact that STE20 was required for the constitutive activation of FUS1 expression in MAT{alpha} itc1 cells indicated that an intact pathway is necessary to display this phenotype. In addition, the requirement for the Ste3p a-factor receptor pointed to an extracellular signal as the reason for the pathway activation. We then showed that the MAT{alpha} itc1 mutant produces and secretes a-factor by measuring its ability to inhibit growth of a MAT{alpha} tester strain. Thus, the phenotype of the mutant may well be explained as the cell response to the continuous external stimulus by a-factor that leads to an autocrine signalling. The fact that disruption of STE20 or STE3 rescues the morphological defect further confirms that the reported phenotype requires signalling through the complete mating pheromone response pathway. We have also detected a-factor production in the homozygous itc1/itc1 mutant but, in this case, the absence of the Ste3p receptor precludes the autocrine signalling and thus the morphological phenotype.

Activation of the cell integrity pathway in itc1 mutants is independent of cell type
The constitutive activation of Slt2p, the MAPK of the cell integrity pathway, first reported in the MATa itc1 mutant strain (de Groot et al., 2001), is now confirmed and found to be independent of mating type. Slt2p is usually activated in response to environmental signals that alter cell wall stability. However, activation in the absence of external stimuli has been observed in mutants affected in cell wall functions, as a mechanism to compensate a weakened cell wall (de Nobel et al., 2000). Thus, the cell type-independent activation of the cell integrity pathway found in itc1 mutants may be triggered by structural cell wall defects that would determine the reported phenotype (de Groot et al., 2001), raising the interesting possibility that Itc1p participates in the regulation of a set of genes involved in cell wall synthesis and/or maintenance.

Recent work has revealed that components of the mating pathway are involved in the maintenance of cell integrity during vegetative growth. Ste20p, Ste11p and Ste7p were found to be required for survival of an och1 mutant, affected in the synthesis of cell wall mannan (Lee & Elion, 1999). Furthermore, mating type-independent activation of FUS1 expression was found in a series of mutants defective at various steps in mannose utilization and protein glycosylation (Cullen et al., 2000). However, the mating type specificity and the STE3 requirement for Fus3p and Kss1p activation displayed by the itc1 mutant strongly indicate that the FUS1 activation and morphological phenotype of MAT{alpha} itc1 cells is not due to cell wall alterations, but is channelled through the pheromone signalling pathway. This interpretation is consistent with our observation that Slt2p activation in itc1 mutants is remedied by osmotic stabilization, whereas that of Kss1p and Fus3p is not.

a-Specific genes are derepressed in MAT{alpha} itc1 and homozygous diploid MATa/{alpha} itc1/itc1 mutant cells
We also show in this work that not only a-factor and Ste6p, required for its transport, but other a-specific genes such as STE2, BAR1 and ASG7 are expressed in MAT{alpha} itc1 and MATa/{alpha} itc1/itc1 mutant cells. Expression of BAR1 in the context of an {alpha} cell type provides a feasible explanation for the low amount of {alpha}-factor detected in the halo assay, although a defective expression of the genes encoding this pheromone can not be discarded. The Bar1 protease present in the culture medium of the MAT{alpha} itc1 mutant may degrade {alpha}-pheromone and interfere with the establishment of an adequate {alpha}-factor gradient, thus explaining the reported mating defect in liquid medium. By the same token, the low amount of {alpha}-pheromone might explain why, even though the Ste2p receptor is inappropriately expressed in the MAT{alpha} itc1 mutant, no signalling through the mating pathway remained after disruption of the gene encoding the Ste3 receptor.

It is unclear why the presence of a-factor elicits in the mutant some of the expected cellular responses, such as polarized cell growth and changes in gene transcription, while there is no growth arrest. It has been recently demonstrated that cell-cycle progression and morphological changes can occur concurrently in response to low levels of pheromones (Erdman & Snyder, 2001), pointing to a dose–response relationship for mating pheromone-induced cell cycle inhibition. The fact that the MAT{alpha} itc1 mutant does not secrete wild-type levels of a-pheromone might thus explain the observed lack of growth arrest.

A role for the Itc1p–Isw2p complex in maintaining a repressive chromatin structure on asg gene promoters
The finding that a-specific genes are derepressed in the absence of Itc1p points to the involvement of this protein in the transcriptional repression mechanisms of these genes in wild-type cells. How is the absence of Itc1p eliciting the derepression in the mutant cells? It has been recently reported that Itc1p exists in vivo exclusively in complex with Isw2p, and that both isw2 and itc1 mutants share identical phenotypes (Gelbart et al., 2001). We now show that isw2 mutant cells exhibit the same aberrant morphology and constitutive activation of the mating pheromone and cell integrity pathways observed in the itc1 mutant. Thus, the derepression of a-specific genes found in this mutant may be ascribed to the absence of the Isw2p–Itc1p complex. This complex negatively regulates the transcription of the early meiotic genes during vegetative growth of haploid cells by creating nuclease-inaccessible chromatin structure at the promoter of these genes through the alteration of nucleosome positions (Goldmark et al., 2000). It is tempting to speculate that the defect of the itc1 mutant in the repression of a-specific genes may be due to chromatin disorganization in the promoters of these genes as a consequence of the absence of the Isw2p–Itc1p complex. It is known from structural studies that chromatin around the asg operator is well organized in MAT{alpha} cells, with nucleosomes precisely and stably positioned flanking the operator (Shimizu et al., 1991). This organized chromatin, that is disrupted in MATa cells, is implicated in the mechanism of repression of a-specific genes. In contrast, no nucleosome positioning is needed for repression of haploid-specific genes in diploid cells (Huang et al., 1997). Therefore, the fact that absence of Itc1p affects the a-specific but not the haploid-specific gene expression, points to a role of the Itc1p–Isw2p complex in the maintenance of the chromatin structure around the asg operator in MAT{alpha} cells (Fig. 7).



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Fig. 7. Model describing the proposed function for the Isw2p–Itc1p complex in the repression of a-specific genes. Chromatin structure is quite different among different cell types in the region surrounding the asg operator. Nucleosomes are precisely positioned at the edges of the asg operator in MAT{alpha} cells in which a-specific genes are repressed, while in MATa cells this ordered structure is not observed, correlating with gene expression. In contrast to the highly structured chromatin adjacent to the asg operator, nucleosomes are not positioned in the promoters of haploid-specific genes. The complex formed by Itc1p and Isw2p could contribute, through its chromatin remodelling activity, to maintain the organized chromatin required for cell type-specific repression of a-specific genes.

 
Among the known targets of the Isw2p–Itc1p complex, early meiotic and INO1 genes share a common cis element, URS1, in their upstream sequences. The remodelling complex is recruited to the promoters of these genes by the transcriptional regulator Ume6p through its interaction with the Itc1p subunit (Goldmark et al., 2000). However, PHO3, also under the control of Isw2p–Itc1p (Kent et al., 2001), neither contains the URS1 sequence nor is regulated by Ume6p. Therefore, different sequence-specific transcription factors may attract the complex to different gene promoters. In the case of the a-specific genes, specific factors such as Mat{alpha}2p or Mcm1p, or even Ssn6p–Tup1p, could be necessary for the recruitment of Isw2p–Itc1p, playing an analogous role to the one assigned to Ume6p.

In summary, our analysis shows that in the MAT{alpha} itc1 mutant, at least two MAPK cascades, the mating pheromone and the cell wall integrity signalling pathways, are constitutively activated. The results presented here clearly indicate that the a-specific genes are derepressed in MAT{alpha} and MATa/{alpha} mutant cells. Our work points for the first time to the involvement of the Isw2p–Itc1p complex in the transcriptional regulation of a group of genes that are constitutively repressed in a cell type-dependent manner.


   ACKNOWLEDGEMENTS
 
We thank George F. Sprague for the gift of the FUS1lacZ fusion plasmid and the ste3 : : URA3 disruption cassette; Jeremy W. Thorner for the fus3 and kss1 mutant strains; José M. Rodríguez-Peña from the Centro de Genómica y Proteómica de la Universidad Complutense de Madrid, for help with the oligonucleotide and RT-PCR design; Santiago Vico for technical assistance with the construction of the ste3 mutant; Víctor J. Cid, Pilar Eraso, Juana M. Gancedo, Carlos Gancedo and Francisco Portillo for helpful suggestions and comments on the manuscript. Work was supported by funding from the Comunidad Autónoma de Madrid for strategic groups (CPGE1010/2000) to M. M. and from the Dirección General de Investigación (PB97-0054 and BMC2001-1517) to M. J. M. C. R. is a recipient of a predoctoral fellowship from the Comunidad Autónoma de Madrid.


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Received 31 July 2002; revised 22 October 2002; accepted 30 October 2002.