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
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ABSTRACT |
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INTRODUCTION |
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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 Isw2Itc1p 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-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
cells. Conversely, MAT
cells secrete
-factor that binds to its specific receptor (Ste2p), expressed only in MATa cells. Activation of the receptor causes the dissociation of the G protein
subunit (G
) from the G
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 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
itc1 and MATa/
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.
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METHODS |
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Molecular biology techniques.
MATa and MAT 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 SalIPstI 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
).
-Galactosidase assays.
-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
-factor was purchased from Sigma and was added to the YPD medium at a final concentration of 1 µg ml-1.
-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-GSTSlt2 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.
-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
sst2 or MATa sst2 : : URA3 tester strains, respectively, and the plates were incubated for 3 days at 23 °C to determine a- or
-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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RESULTS |
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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
itc1 mutant cells, are not related to the cell wall phenotype.
MAT 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
-galactosidase activity was measured in crude cell extracts. The levels of
-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
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
itc1 ste20, the phosphorylation of the Fus3p and Kss1p MAPKs was eliminated (Fig. 2
a) and the morphological defect was rescued (Fig. 2b
), thus confirming that the aberrant morphology shown by the MAT
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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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. 3
a), while the cell type-specific Kss1p and Fus3p constitutive activation and the aberrant morphology remained unaltered (Fig. 3b
).
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a-Specific genes are derepressed in the MAT 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 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
-factor receptor (Blumer et al., 1988
; Jenness et al., 1983
); BAR1, encoding the protease that degrades
-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
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 haploid and MATa/
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
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
and MATa/
cells, a Mat
2p dimer binds to DNA cooperatively with Mcm1p (Mak & Johnson, 1993
; Smith & Johnson, 1992
) and recruits the Ssn6pTup1p general transcription repressor complex (Keleher et al., 1992
), thus bringing about full repression in the appropriate cell types, since Mat
2p is not expressed in MATa cells. Taking into account that the repressor complex and the DNA context are the same for MAT
and MATa/
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. 5
a) whereas no
-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
and MATa/
cells.
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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
isw2 mutants, we first analysed the effect of the isw2 mutation on cellular morphology. The isw2 mutant strain exhibited a MAT
-specific morphological defect (Fig. 6
a) that is not prevented by the presence of sorbitol in the medium (not shown). Moreover, the isw2 mutant showed MAT
-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
-specific constitutive activation of the mating pathway, and cell type-independent constitutive activation of the cell integrity pathway.
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DISCUSSION |
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Autocrine signalling causes constitutive activation of the pheromone signalling pathway in MAT itc1 mutants
In this work we present evidence that MAT 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
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
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
itc1 mutant produces and secretes a-factor by measuring its ability to inhibit growth of a MAT
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
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 itc1 and homozygous diploid MATa/
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 itc1 and MATa/
itc1/itc1 mutant cells. Expression of BAR1 in the context of an
cell type provides a feasible explanation for the low amount of
-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
itc1 mutant may degrade
-pheromone and interfere with the establishment of an adequate
-factor gradient, thus explaining the reported mating defect in liquid medium. By the same token, the low amount of
-pheromone might explain why, even though the Ste2p receptor is inappropriately expressed in the MAT
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 doseresponse relationship for mating pheromone-induced cell cycle inhibition. The fact that the MAT
itc1 mutant does not secrete wild-type levels of a-pheromone might thus explain the observed lack of growth arrest.
A role for the Itc1pIsw2p 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 Isw2pItc1p 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 Isw2pItc1p complex. It is known from structural studies that chromatin around the asg operator is well organized in MAT
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 Itc1pIsw2p complex in the maintenance of the chromatin structure around the asg operator in MAT
cells (Fig. 7
).
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In summary, our analysis shows that in the MAT 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
and MATa/
mutant cells. Our work points for the first time to the involvement of the Isw2pItc1p complex in the transcriptional regulation of a group of genes that are constitutively repressed in a cell type-dependent manner.
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ACKNOWLEDGEMENTS |
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Received 31 July 2002;
revised 22 October 2002;
accepted 30 October 2002.