Divergent Regulation of the Evolutionarily Closely Related Promoters of the Saccharomyces cerevisiae STA2 and MUC1 Genes

J Bacteriol. 1999 October; 181(20): 6497–6508.

PMCID: PMC103787

Divergent Regulation of the Evolutionarily Closely Related Promoters of the Saccharomyces cerevisiae STA2 and MUC1 Genes

Marco Gagiano, Dewald Van Dyk, Florian F. Bauer, Marius G. Lambrechts, and Isak S. Pretorius^*

Institute for Wine Biotechnology and Department of Microbiology, University of Stellenbosch, Stellenbosch ZA-7600, South Africa

^*Corresponding author. Mailing address: Institute for Wine Biotechnology, University of Stellenbosch, Stellenbosch ZA-7600, South Africa. Phone: 27-21-8084730. Fax: 27-21-8083771. E-mail: isp/at/maties.sun.ac.za.

Received April 26, 1999; Accepted August 3, 1999.

This article has been cited by other articles in PMC.

Abstract

The 5′ upstream regions of the Saccharomyces cerevisiae glucoamylase-encoding genes STA1 to -3 and of the MUC1 (or FLO11) gene, which is critical for pseudohyphal development, invasive growth, and flocculation, are almost identical, and the genes are coregulated to a large extent. Besides representing the largest yeast promoters identified to date, these regions are of particular interest from both a functional and an evolutionary point of view. Transcription of the genes indeed seems to be dependent on numerous transcription factors which integrate the information of a complex network of signaling pathways, while the very limited sequence differences between them should allow the study of promoter evolution on a molecular level. To investigate the transcriptional regulation, we compared the transcription levels conferred by the STA2 and MUC1 promoters under various growth conditions. Our data show that transcription of both genes responded similarly to most environmental signals but also indicated significant divergence in some aspects. We identified distinct areas within the promoters that show specific responses to the activating effect of Flo8p, Msn1p (or Mss10p, Fup1p, or Phd2p), and Mss11p as well as to carbon catabolite repression. We also identified the STA10 repressive effect as the absence of Flo8p, a transcriptional activator of flocculation genes in S. cerevisiae.

The STA1, STA2, and STA3 genes encode extracellular glucoamylase isozymes which enable Saccharomyces cerevisiae cells to utilize starch as a carbon source (reviewed in references 38, 40, and 50). The three genes have nearly identical sequences, and are located on chromosomes II (STA2), IV (STA1), and XIV (STA3). All three members of the STA gene family are located in subtelomeric positions, similar to the FLO (reviewed in reference 48), SUC (reviewed in reference 15), and MAL (reviewed in reference 32) gene families, which probably evolved through genomic duplications and chromosomal rearrangements. The 5′ upstream region of STA1 and STA2 (the nucleotide sequence of STA3 has not previously been determined) is almost identical to that of MUC1, which encodes a large membrane-bound, mucin-like protein that plays an important role in the processes of invasive growth, pseudohyphal development, and flocculation (8, 23, 27, 28). The homology extends over more than 3,500 bp upstream of the ATG start codon and includes the first 60 bp of the open reading frame (ORF) encoding a secretion signal sequence (52). With the exception of a few single nucleotide dissimilarities, the only significant differences between the promoters of STA2 and MUC1 are two inserts of 20 and 64 bp in the MUC1 promoter, which are absent from the STA2 promoter (23). These inserts stretch from nucleotides −1333 to −1313 and −933 to −869, respectively. This very limited sequence divergence between the STA and MUC1 promoter regions suggests a recent origin of the STA genes. The STA genes probably evolved through a recombination and sequence duplication process between the promoter and signal sequence of MUC1 and the ORF of the SGA1 gene that encodes a sporulation-specific intracellular glucoamylase. MUC1 and SGA1 are located on the right and left arms of chromosome IX, respectively (59, 60). Besides the strong sequence conservation between these genes, other arguments in favor of a recent origin of the STA genes and of the proposed molecular mechanism are (i) the subtelomeric position of the STA genes compared to the more central position of both MUC1 and SGA1; (ii) the presence of STA genes in only some S. cerevisiae strains, compared to the general presence of MUC1 (4, 59, 60) and SGA1 (59, 60) in all S. cerevisiae strains investigated so far; and (iii) the existence of homologous repeated sequences on either side of the proposed junctions (59, 60).

Analyses of the upstream areas of STA1 (1, 46), STA2 (22), and MUC1 (44) demonstrated that elements at distances of up to 2,800 bp from the translation start codon (ATG) are involved in the transcriptional control of the respective genes, therefore representing the largest S. cerevisiae promoters identified to date (7, 44). The STA and MUC1 upstream regions are therefore of particular interest from both an evolutionary and a functional point of view.

The extent of the promoter homology would suggest that genes involved in starch metabolism and pseudohyphal differentiation or invasive growth are coregulated to a large extent, and experimental data so far have supported this hypothesis. Lambrechts et al. (23, 24) and Gagiano et al. (8) showed that two transcriptional regulators, Msn1p and Mss11p, strongly induce transcription of both the STA2 and MUC1 genes when present on multiple-copy plasmids. Conversely, Δmsn1 or Δmss11 strains show strongly reduced transcription of these genes. Furthermore, Lo and Dranginis (28) demonstrated that MUC1 is regulated by Ste12p, a transcription factor responsible for both pheromone-specific (reviewed in reference 21) and, in combination with the TEA (or ATTS) family transcription factor Tec1p, filamentation-specific (9, 30) gene regulation. Gagiano et al. (8) presented evidence that the same factor regulates the STA genes in a similar way.

Other regulatory factors have so far only been associated with regulation of MUC1 or STA1, STA2, and STA3 independently. Recent data suggest that the transcription of MUC1 might be specifically regulated by a network of signal transduction pathways which control invasive growth and pseudohyphal differentiation (8, 28, 44). This network combines inputs from at least three interacting signal transduction modules, including (i) the filamentation-specific mitogen-activated protein (MAP) kinase cascade (25, 31, 42), (ii) the cyclic AMP (cAMP) and cAMP-dependent kinase (29, 43), and (iii) the cyclin-dependent kinase Cdc28p (6). In addition to the result mentioned above that MUC1 was subjected to MAP kinase-dependent regulation by Ste12p and Tec1p, the gene was shown to be regulated by cAMP levels, a regulation that occurs via Flo8p (44), a transcription factor initially identified for its role in flocculation (18). The gene was also shown to be negatively regulated by a suppressor of flocculation, Sfl1p, which interacts specifically with the yeast A kinase, Tpk2p, to repress MUC1 transcription in the absence of a cAMP signal (43).

Numerous data concerning the regulation of the STA genes have been published. Expression of STA1 to -3 is negatively regulated at several levels. Transcription is repressed on most readily metabolized carbon sources, including glucose, sucrose, maltose, and galactose (5, 17, 20, 41, 47). Carbon catabolite repression was reported to involve two separate pathways, of which one requires HXK2 and the other HAP2 (17). It was also reported that repression of STA2 does not require Mig1p, the common repressor of genes under carbon catabolite control. MUC1 was also shown to be repressed in medium containing glucose as a carbon source (8, 27), probably via the same mechanisms as STA1 and STA2. Transcription of STA1 to -3 is repressed in most, but not all, diploid strains of S. cerevisiae (5, 41). The mechanism through which repression occurs is not defined, since the removal of the putative a1-α2 repressor binding sites from the STA2 promoter does not relieve the repressive effect observed in diploid strains (22). In rich medium, MUC1 is also repressed in diploid strains, but under nitrogen starvation conditions it seems to be repressed more in haploid than diploid strains (28, 44).

Most laboratory strains of S. cerevisiae contain an undefined repressor, STA10, which reduces transcription of the STA1 to -3 genes at least 20-fold (37, 41). It was reported that the repressive effect of STA10 results from interaction between two unlinked genes, IST1 and IST2 (34), but this was not confirmed. The negative effects of several other genes (i.e., INH1 [58], SGL1 [36], and SNS1 and MSS1 [1]) on the transcription of the STA genes have also been reported, but the relationships between these negatively acting genes and the repressive effect of STA10 remain to be determined.

Transcription of STA1 to -3 is subject to the repressive effect of chromatin on promoters, since SUD1, a component of a global chromatin-associated repressor of promoter activity, was shown to act on the STA1 promoter (57). Furthermore, transcription of STA1 to -3 also requires the presence of components of the SWI-SNF global activation complex (13, 20, 33, 61–63), which associates with the RNA polymerase holoenzyme at specific promoters and relieves the repressive effect of chromatin on transcription (19, 55).

cis-acting promoter elements in several regions within the STA1 (1, 46), STA2 (22), and MUC1 (44) promoters were shown to be required for transcriptional regulation. Two areas hosting upstream activating sequences (UASs) (UAS1 between nucleotides −1390 and −1074 and UAS2 between nucleotides −1940 and −1815), as well as three upstream repression sequences (URSs), were identified in the STA2 promoter (22). URS1 was found to reside in the area between nucleotides −1390 and −1074, which also hosts UAS1. URS2 was identified between nucleotides −1650 and −1390 and URS3 upstream of position −2457. Similar regions were defined for the STA1 promoter (1, 46). A recent, more systematic, analysis of the MUC1 promoter (44) revealed a vast array of regulatory elements which confer the regulation of several nutritional and cell-type signals on MUC1 expression levels. In good agreement with the previous studies of the highly homologous STA1 and STA2 promoters, four areas required for the activation of MUC1 and nine areas required for the repression thereof were identified. The transcriptional activator encoded by FLO8 was found to exert its activating effect through a 200-bp sequence stretching from nucleotides −1200 to −1000 in the upstream region of MUC1 (44).

The 5′ upstream areas of MUC1, STA1, and STA2 are predicted to contain a single small ORF, YIR020c, of unknown function, situated from nucleotides −1285 to −882 in the upstream region of MUC1. YIR020c lies in an area identified and experimentally defined as a regulatory region for STA1, STA2, and MUC1, and other regulatory regions were shown to exist upstream of this ORF (1, 22, 44, 46). Its occurrence therefore does not affect conclusions regarding the transcriptional regulation of STA1, STA2, or MUC1, independently of whether this ORF encodes a functional protein or not.

The homologous sequences from nucleotides −1390 to −1074 of the STA2 promoter and from nucleotides −1479 to −1136 of the MUC1 promoter are of particular interest since they (i) have previously been identified as areas hosting a UAS as well as a URS (22), (ii) confer increased levels of activity from a far-upstream position, and (iii) include one of the two significant differences between the upstream areas of MUC1 and STA1 to -3 (a sequence of 20 bp that is deleted in the STA2 promoter). The region might therefore contain an evolutionarily significant molecular change explaining differences in the regulation of STA1 to -3 and MUC1.

In this paper, we compare expression levels conferred by the full MUC1 and STA2 promoters on reporter gene expression. We furthermore present a detailed analysis of the promoter region from nucleotides −1390 to −1074 of STA2 and the corresponding area of MUC1, from nucleotides −1479 to −1136. We show that these regions of MUC1 and STA2 confer both similar and divergent regulation and contain sequences involved in general repression as well as areas for (i) activation by the transcriptional activators encoded by MSN1 and MSS11, (ii) activation by the transcriptional activator encoded by FLO8, (iii) carbon catabolite repression, and (iv) diploid repression. Our data indicate that differences in expression levels observed between MUC1 and STA2 are largely due to the two deletions of 20 and 64 bp that have occurred in the STA promoters. We also show that the repressive effect identified as STA10 in most laboratory S. cerevisiae strains is due to the absence of the FLO8-encoded transcriptional activator. Epistatic analysis furthermore suggests that FLO8 requires or is situated upstream of MSS11, but acts independently of MSN1.

MATERIALS AND METHODS

Strains, growth media, and genetic methods.

The S. cerevisiae strains used in this study, along with the relevant genotypes, are listed in Table Table1.1. Transformation of S. cerevisiae cells was carried out by the lithium acetate procedure (3). The one-step gene replacement method (3) was used to disrupt the FLO8 loci with the flo8::URA3 cassette, pΔflo8, in the genomes of strains ISP15 and ISP20 to generate strains ISP15Δflo8 and ISP20Δflo8, respectively. Successful disruptions of the FLO8 loci in these strains were verified by Southern blot analysis and confirmed by PCR analysis. The URA3 marker of strains ISP15Δflo8 and ISP15Δmsn1 was regenerated through transformations with the ura3::Kan^r disruption cassette, pΔura3::kan, and selected on medium containing 125 mg of kanamycin per ml and 1 mg of 5-fluoroorotic acid per ml. S. cerevisiae FY23 (56) is isogenic to the S288C genetic background, and L5366 (25) and L5366h (8) are isogenic to the Σ1278b genetic background. Strain JM2508 does not contain any of the STA1 to -3 genes and is from the culture collection of the late Julius Marmur.

TABLE 1

Yeast strains used in this

study

Unless specified differently, yeast cells were grown at 30°C in synthetic media containing a 0.67% yeast nitrogen base without amino acids (Difco Laboratories, Detroit, Mich.), supplemented with the required amino acids and 2% glucose for SCD medium, 3% glycerol and 3% ethanol for SCGE medium, and 2% cornstarch or potato starch (Sigma Chemical, St. Louis, Mo.) for SCS medium. SLAD medium, used for induction of invasive growth and pseudohyphae, was prepared as described previously (11). Solid media contained 2% agar (Difco Laboratories). SPD medium contained 0.17% yeast nitrogen base without (NH₄)₂SO₄ and without amino acids (Difco Laboratories), 2% glucose, and 0.1% filter-sterilized proline as the sole nitrogen source.

Escherichia coli DH5α (Gibco BRL/Life Technologies, Rockville, Md.) was used for propagation of all plasmids and was grown in Luria-Bertani (LB) broth at 37°C. All E. coli transformations and isolation of DNA were done according to the methods of Sambrook et al. (45).

Construction of plasmids.

FLO8 was isolated as a 3,252-bp SphI-EcoRV fragment from plasmid pF415-1 (18) and ligated to plasmids YEplac112 and YEplac181 (10), digested with SphI and SmaI, to generate plasmids YEplac112-FLO8 and YEplac181-FLO8. YEplac112-FLO8 was subsequently used to construct pΔflo8, a cassette for disrupting the FLO8 locus. In order to do this, a 760-bp PstI-BglII fragment, comprising the translational start codon (ATG) and a large part of the FLO8 ORF, was removed and replaced with a 1,084-bp NsiI-BamHI fragment containing the URA3 marker, isolated from plasmid pJJ242 (16).

YCplac33-STA2 was constructed by inserting an XhoI-EcoRV fragment from plasmid pSPSTA2 (22) into the unique SalI-SmaI sites of YCplac33 (10). A 953-bp DraIII-XbaI fragment containing the entire UAS1 region and the area downstream thereof was removed from the promoter region of STA2 of plasmid YCplac33-STA2 and replaced with the corresponding area from the MUC1 promoter, a 1,045-bp DraIII-XbaI fragment isolated from plasmid pMUU (23). This generated YCplac33-PMUC1-STA2, a plasmid almost identical to YCplac33-STA2, the only difference being the presence of the two MUC1 promoter inserts of 20 and 64 bp.

A 1,675-bp XhoI-SnaBI fragment containing MSN1 was obtained from the plasmid pMS2A (24) and cloned into the unique SalI and SmaI sites of plasmid YEplac181 (10) to generate YEplac181-MSN1. A 3,326-bp EcoRI fragment containing MSS11 was derived from plasmid pMSS11-g (53) and cloned into the unique EcoRI site of plasmid YEplac181 to generate plasmid YEplac181-MSS11. A construct for regenerating the URA3 marker in strains ISP15Δflo8 and ISP15Δmsn1 was made by ligating a 1,586-bp EcoRV-PvuII fragment, containing the kanamycin resistance marker from plasmid pUG6, into plasmid pJJ242, of which a 248-bp EcoRV-StuI fragment was deleted from URA3.

The construction of plasmids with sequentially deleted promoter fragments upstream of lacZ is shown in Fig. Fig.1.1. The sequences of all of the primers used for these and other constructions are listed in Table Table2.2. The forward primers contain SalI sites and the reverse primers contain XhoI sites, so that, when used in combination during PCRs, these primers yield fragments with 5′ SalI and 3′ XhoI restriction sites. Primers FP3, FP11, and FP12 were used together with primer RP10 to amplify PCR fragments M3-10, M11-10, and M12-10 from the MUC1 promoter, with pMUU (23) as a template. The 20-bp insert, present in the MUC1 promoter but absent from STA2, occurs in the area between primers FP12 and FP13. The rest of the MUC1 UAS1 area is identical to that of STA2. Primers FP3, FP11, FP12, FP13, FP14, and FP15 were used together with RP10 to generate PCR fragments S3-10, S11-10, S12-10, 13-10, 14-10, and 15-10, with YCplac33-STA2 as a template. Expand High Fidelity polymerase, obtained from Roche Diagnostics (Randburg, South Africa), was used for all PCRs. Primers F-M20 and R-M20 were hybridized to generate fragment M20, the 20-bp MUC1 promoter insert, and primers F-M64 and R-M64 were hybridized to generate fragment M64, the 64-bp MUC1 promoter insert. These primers were designed to generate SalI- and XhoI-compatible single-stranded overhangs after pairwise annealing.

FIG. 1

Construction of a series of plasmids containing sequential deletions of the STA2 and MUC1 UAS1 area, upstream of the lacZ reporter gene in plasmid pHP41. The positions of UAS1 and UAS2 relative to the translation initiation codon (ATG) of the STA2 and (more ...)

TABLE 2

List of primers used to generate deletion fragments and lacZ fusions of the STA2 and MUC1

promoters

Plasmids pHP41 (35), pLG670-Z (12), and pLGΔ312 (54) contain the CYC1 promoter, fused in frame to the lacZ reporter gene. The CYC1 promoters present in pHP41 and pLG670-Z were modified in that the UASs were removed to yield low expression levels of lacZ, which makes it possible to identify sequences conferring activation. Plasmid pLGΔ312 contains the wild-type UAS, which results in high levels of lacZ expression, thereby making it possible to identify sequences conferring repression. The XhoI site in the linker of pHP41 is not unique; therefore, the plasmid was partially digested with XhoI, purified, and subsequently digested with SalI. Plasmid pLG670-Z was digested with both SalI and XhoI, and plasmid pLGΔ312 was digested with only XhoI. The PCR amplification products were digested with SalI and XhoI and subsequently ligated to pHP41, pLG670-Z, and pLGΔ312.

To generate plasmids containing the MUC1 and STA2 promoters fused in frame to the lacZ reporter gene, a forward primer, PMUC1-FX, was used in combination with primers PMUC1-RB and PSTA2-RB to amplify a 472-bp fragment containing the ATG and first 9 bp of the lacZ ORF fused to the first 460 bp of the MUC1 and STA2 promoters, respectively. The BamHI site in the lacZ ORF and the XbaI site that occurs around position −460 in both the MUC1 and STA2 promoters were used to clone these fragments into the unique BamHI and XbaI sites of plasmid pHP41. The rest of the MUC1 upstream region was inserted as a 3,257-bp AvrII-XbaI fragment, isolated from plasmid pMUU, and the rest of the STA2 promoter was inserted as a 3,173-bp AvrII-XbaI fragment, isolated from pSPSTA2, into the XbaI site of the plasmids with the 460-bp MUC1 and STA2 promoters fused to lacZ, generating plasmids pPMUC1-lacZ and pPSTA2-lacZ, respectively. To delete the UAS1 areas from these plasmids, a partial BamHI digestion was done, followed by complete digestion with EagI. The 360-bp STA2 UAS1 region and 380-bp MUC1 UAS1 region were removed, and the ends were filled in by using Klenow enzyme and subsequently religated to generate plasmids pPMUC1ΔUAS1-lacZ and pPSTA2ΔUAS1-lacZ.

All plasmids constructed were sequenced to verify that no mutations occurred during the PCR amplification of the promoter fragments and that the constructs were in the correct orientation. All of the constructs are listed in Table Table3.3. Enzymes for DNA modification and restriction digestions were obtained from Roche Diagnostics. All DNA manipulations were done according to the methods of Sambrook et al. (45).

TABLE 3

Plasmids and constructs used in this

study

Sequencing of the STA2 and STA3 promoters.

To sequence the 5′ upstream region of STA3, a series of nine primers were synthesized, covering the entire promoter area and first part of the STA3 ORF. The primers were designed from the available sequences of STA1 and STA2. Plasmid pSTA3-6-4 (59) was used as a template to determine the nucleotide sequence.

The sequence of the STA2 gene, upstream of position −2500, was also determined to establish how far the homology between the STA genes and MUC1 extends. For this purpose, a single reverse primer was designed from the STA2 sequence, and plasmid YCplac33-STA2 was used as a template for determination of the nucleotide sequence. From the sequence obtained, an additional primer was made and again used with YCplac33-STA2 as a template.

β-Galactosidase assays.

After transformation, at least three colonies of each transformation were grown overnight in 10 ml of selective SCD medium. From each overnight culture, 10-ml SCD, SCGE, SLAD, and SPD cultures were inoculated to an optical density at 600 nm (OD₆₀₀) of 0.1 and incubated to grow for four to five generations at 30°C to an OD₆₀₀ of ~1.0. To obtain postdiauxic- shift cultures, SCD cultures were incubated for longer periods until they had reached an OD₆₀₀ of >3.0. The effect of osmotic shock on expression levels was determined in 10-ml selective SCD cultures that were grown to an OD₆₀₀ of 1.0. Sterile NaCl was added to a final concentration of 0.7 M, after which the cultures were incubated at 30°C for 1 h. The effect of heat shock was determined in 10-ml selective SCD cultures grown to an OD₆₀₀ of 1.0 and placed at 42°C for 1 h. β-Galactosidase assays were done according to the method of Ausubel et al. (3). Margins of error were calculated for each set of assays and were usually less than 7.5% and never higher than 15%.

Invasive growth and pseudohyphal development assays.

Three colonies from a transformation were inoculated into SCD medium and grown to an OD₆₀₀ of 1.0. To assess the ability of these yeast cells to grow invasively into the agar, 10 μl of this liquid culture suspension was spotted onto SLAD, SCS, SCGE, and SCD agar plates. Plates were incubated at 30°C and investigated for invasive growth at intervals of 2 days. Yeast colonies were washed off the surface of the agar by rubbing the surface of the plates with a gloved finger under running water. Cells that grow invasively into the agar cannot be washed off and are clearly seen below the surface of the agar.

Plates were photographed both before and after the washing process. After washing off the cells, each of the colonies was investigated for elongated cells or filaments under the ×10 magnification of a light microscope (Nikon Optiphot-2), and photographs of cells below the agar surface were taken with an Intellicam 2 (Matrox Electronics Inc.).

Plate assays to determine starch utilization.

The STA2 gene encodes an extracellular glucoamylase which hydrolyzes starch by liberating glucose molecules from the nonreducing end of the starch molecule (50). The presence of the STA2 gene therefore enables most yeast strains to grow on starch as the sole carbon source. On plates containing starch as a carbon source (SCS), a clear zone is formed around such starch-degrading colonies, and the size of the colony, as well as the diameter of the zone, is indicative of the amount of glucoamylase secreted (39, 59). The levels of expression of STA2 in yeast strains were therefore determined by the size of the colonies and the clear zone around each of the colonies on SCS plates.

Yeast cells were grown in a 10-ml SCD culture until it reached an OD of 1.0. Of these cultures, 10 μl was spotted onto each of the different starch plates. Plates were incubated at 30°C for 4 to 6 days, after which they were placed at 4°C for 2 days to allow for the starch to precipitate. This precipitation of unutilized starch results in a clear zone around the colony where secreted glucoamylase has hydrolyzed the starch.

Sequence analysis and homology searches.

Homology searches in the yeast genome subdivision of GenBank were done with BLAST software (2). Sequence fragment assembly and individual alignments between the STA genes and MUC1 were done with the OMIGA v1.1 package (Oxford Molecular Ltd.).

Nucleotide sequence accession number.

A 2,779-bp sequence comprising the STA3 promoter and the first part of the ORF was submitted to the GenBank database and assigned accession no. U95022. A 1,462-bp sequence comprising the far upstream region of the STA2 promoter was submitted to the GenBank database and assigned accession no. AF169185.

RESULTS

Similar and divergent regulation of STA2 and MUC1.

To determine the extent of the coregulation between MUC1 and STA2, we determined the β-galactosidase activity of the MUC1 and STA2 promoters fused to the lacZ reporter gene with plasmids pPMUC1-lacZ and pPSTA2-lacZ, respectively, under different growth conditions as well as in the presence of multiple copies of the transcriptional activators FLO8, MSN1, and MSS11. The results for these assays are given in Tables Tables44 and and5.5.

TABLE 4

Levels of expression from MUC1 and STA2 promoters in S. cerevisiae

strains^a

TABLE 5

Effect of multiple copies of FLO8, MSN1 and MSS11 on expression levels of MUC1 and STA2 wild-type promoters, as well as promoters from which the UAS1 areas were deleted, fused to the lacZ reporter gene on a centromeric plasmid in S. cerevisiae

ISP20 (more ...)

The data show that reporter gene expression levels observed for both pPMUC1-lacZ and pPSTA2-lacZ were low under most conditions, similar to those reported for genes transcribed at low levels, e.g., PHIS3-lacZ (3). MUC1 promoter-dependent expression levels were, however, consistently lower than STA2 promoter-dependent levels.

The data indicate that in haploid strains, both pPMUC1-lacZ and pPSTA2-lacZ are repressed in rich glucose medium, are derepressed in glycerol-ethanol medium, and can be induced by multiple copies of FLO8, MSN1, and MSS11. In the haploid Σ1278b strain (Table (Table4),4), pPSTA2-lacZ has 13.6-fold higher expression levels when grown in glycerol-ethanol (SCGE) medium than on medium containing glucose as the carbon source (SCD). In the same strain and under the same conditions, expression levels of the pPMUC1-lacZ construct increased threefold. Interestingly, this increase is nearly completely absent in diploid strains, where pPSTA2-lacZ expression was only increased twofold, and no increase at all was observed for pPMUC1-lacZ. A 2.6-fold increase in expression levels of pPSTA2-lacZ was also seen in the postdiauxic-shift SCD cultures, where most of the glucose has been utilized. Again, this postdiauxic shift induction could not be observed for the pPMUC1-lacZ construct. These data are in good agreement with previous reports on the transcriptional activity of either STA2 or MUC1, determined by Northern blot analysis. Transcription of MUC1 was reported to be repressed in rich medium (27, 28, 44) or medium containing glucose as the carbon source (8), whereas STA1 and STA2 were reported to be repressed in all media containing readily metabolized carbon sources such as glucose (5, 8, 17, 20, 41, 47).

Despite the high homology between the STA2 and MUC1 promoters, pPMUC1-lacZ responds differently to some of the growth conditions. It is, in particular, activated in medium containing limiting amounts of (NH₄)₂SO₄ as a nitrogen source (SLAD), in which a twofold increase in expression levels of pPMUC1-lacZ was observed, whereas no such increase was observed with pPSTA2-lacZ. Another clear difference can be seen in the response to multiple copies of FLO8. Whereas the STA2 promoter is strongly induced in both glucose and glycerol-ethanol media, the MUC1 promoter was only activated in medium containing glucose. The data show that both promoters do not respond to osmotic shock (NaCl) or heat shock (42°C) conditions and are not induced by poor nitrogen sources like proline (SPD).

The effect of the genetic background on the expression levels of the two genes can be observed when comparing the levels of expression of pPMUC1-lacZ and pPSTA2-lacZ of the wild-type ISP20 strain in SCD and SCGE media (Table (Table5)5) to that of the Σ1278b haploid strain, L5366h, under the same conditions (Table (Table4).4). Whereas levels of expression in SCGE medium were 13.6-fold higher than on SCD medium for pPSTA2-lacZ in the Σ1278b haploid strain, only a 3.7-fold difference was observed for ISP20. A similar effect was seen for pPMUC1-lacZ where expression levels in SCGE medium were 2.9-fold higher than in SCD medium in the Σ1278b haploid strain but only 1.5-fold higher in ISP20. The general tendencies with regard to repression and activation, however, were always the same.

The STA10 repressive effect in S288C-derived strains is due to a mutation in FLO8.

When compared to feral S. cerevisiae strains, most laboratory strains (e.g., S288C) exhibit a 20-fold reduction in STA1 to -3 expression (41). This phenomenon was believed to be due to the presence of a repressor, designated STA10 (37). It was, however, recently reported that most laboratory strains contain a point mutation in FLO8, a transcriptional activator of the flocculation genes, which renders these strains unable to flocculate, grow invasively, or form pseudohyphae (26). Due to the extensive homology between the STA2 and MUC1 promoter regions and since FLO8 was shown to be required for transcription of MUC1 (44), we investigated whether a genetic relationship between STA10 and FLO8 exists.

From Fig. Fig.2A,2A, it is evident that in the S288C genetic background, the STA10 repressive effect is due to the lack of the FLO8-encoded activator and is not due to the presence of a repressor. Strain FY23, isogenic to the S288C genetic background (56), was transformed with a centromeric plasmid, YCplac33, bearing STA2 and the centromeric vector YCplac22 without any insert. This strain is unable to utilize starch as the sole carbon source. The same strain, transformed with centromeric plasmids YCplac33-STA2 and YCplac22-FLO8, bearing STA2 and FLO8, respectively, was fully able to degrade starch. To verify the requirement of FLO8 for STA1 to -3 expression, FLO8 was deleted from the genomes of sta10 strains ISP15 and ISP20. As can be seen in Fig. Fig.2B,2B, the absence of FLO8 reduced the ability of these strains to utilize starch, resulting in a phenotype similar to that reported for STA10.

FIG. 2

(A) The S288C-derived STA10 strain, FY23, transformed with the centromeric plasmids YCplac22 and YCplac33 (Ctrl.), YCplac33 and YCplac22-FLO8 (FLO8), YCplac22 and YCplac33-STA2 (STA2), and YCplac22-FLO8 and YCplac33-STA2 (STA2 + FLO8) on plates (more ...)

Flo8p acts independently of Msn1p but upstream of Mss11p.

FLO8 is one of several transcriptional regulators required for the transcriptional activation of the STA1 to -3 genes and MUC1. The epistatic relationships between these transcriptional regulators revealed a complex network of signal transduction pathways that converge at the promoter of the MUC1 (8, 44) and STA1 to -3 (8) genes. To establish the epistatic relationship between FLO8 and other transcriptional regulators required for MUC1 and STA1 to -3 expression, MSN1 and MSS11, present on 2μm plasmids, were transformed into strains with deleted FLO8 loci, ISP15Δflo8 and ISP20Δflo8. A 2μm plasmid carrying FLO8 was also transformed into strains with deleted MSN1 and MSS11 loci. These strains were spotted onto SLAD (limited nitrogen source) and SCS (potato starch as carbon source) plates and scored for their ability to grow invasively into the agar and to utilize starch.

The results of the epistatic analysis on limited nitrogen SLAD medium can be seen in Fig. Fig.3.3. The wild-type strain was able to grow invasively into the agar. Multiple copies of FLO8, MSN1, and especially MSS11 significantly increased the invasive growth of the strain. Deletions of the FLO8, MSN1, and MSS11 loci, on the other hand, completely eliminated invasive growth. In strains with deleted FLO8 loci, multiple copies of MSN1 and MSS11 were able to restore invasive growth to higher-than-wild-type levels, with MSS11 being the more efficient gene. Similar results were obtained when multiple copies of FLO8 and MSS11 were transformed into strains with a deleted MSN1 locus. However, multiple copies of MSN1 or FLO8 were unable to restore invasive growth in a strain with a deleted MSS11 locus. The data indicate that (i) FLO8 and MSN1 act independently of each other when relaying the invasive growth signal and that (ii) Mss11p functions downstream of—or is required for activity by—both Msn1p and Flo8p. Similar results were obtained with strain ISP15 (data not shown). The epistatic analysis was also performed with respect to the ability to utilize starch as a carbon source, and the same conclusion was reached (data not shown).

FIG. 3

Determination of the epistatic relationships between MSN1, MSS11, and FLO8 on limited nitrogen (SLAD) medium in strain ISP20. Wild-type ISP20, ISP20Δflo8, ISP20Δmsn1, and ISP20Δmss11 were transformed with YEplac112 without insert (more ...)

Role of UAS1 in determining expression levels of STA2 and MUC1.

Deletions of the UAS1 area from the promoters of MUC1 and STA2 (Table (Table5)5) indicated that this area is required for glucose repression and transcriptional activation by MSS11, FLO8, and MSN1. The data show that multiple copies of MSN1 or MSS11 were still able to increase expression levels conferred by the MUC1 and STA2 promoters when UAS1 is deleted. This suggests that the corresponding gene products act through regulatory sequences both within and outside of UAS1. Interestingly, the same does not apply for multiple copies of FLO8, which are unable to induce reporter gene expression when UAS1 is deleted. Flo8p is therefore completely dependent on sequences within the UAS1 region to assert its effect on MUC1 and STA2 transcription.

The data furthermore show that UAS1 plays a significant role in glucose-dependent repression of the two promoters. In medium that contains glucose as the carbon source (SCD), the pPSTA2ΔUAS1-lacZ and PMUC1ΔUAS1-lacZ constructs exhibited 1.8- and 1.7-fold increases, respectively, in expression compared to the wild-type promoter. The UAS1 region therefore confers some glucose repression on the STA2 and MUC1 promoters.

However, both ΔUAS1 promoters no longer showed any significant increases between glucose (SCD) and glycerol-ethanol (SCGE) media, suggesting that glucose-dependent repression has been eliminated. In addition, the ΔUAS1 constructs failed to reach expression levels conferred by the wild-type promoter under derepressed conditions, indicating that sequences required for activation must have been deleted.

Compared to the wild-type strain, multiple copies of MSS11 resulted in a 4.3-fold increase in expression levels from the native MUC1 promoter and an 11.6-fold increase in those from the native STA2 promoter on SCD medium. The effect of multiple copies of MSS11 in SCGE medium was, however, more pronounced for the native MUC1 promoter, since a 6.2-fold increase in lacZ expression was observed, whereas a 3.3-fold increase in expression was observed for the native STA2 promoter under the same conditions. Expression levels of lacZ under control of the STA2 promoter were, however, always much higher than those observed for the MUC1 promoter.

In the presence of multiple copies of MSS11 on SCGE medium, deletion of the UAS1 area from the promoters of MUC1 and STA2 still results in increased promoter activity, but at levels which are 2.9- and 2.1-fold lower, respectively, than those of the wild-type promoter under the same conditions. This indicates that MSS11 exerts its activation effect in part via this area. In SCD medium, however, the opposite happens, since an increase in activity was observed for both the MUC1 and STA2 promoters. This again indicates that other areas are required for activation by MSS11. However, the elimination of the glucose repression exerted by the UAS1 region allows higher levels of activation by multiple copies of MSS11. Multiple copies of FLO8 have a more pronounced effect on expression levels of STA2 than MUC1. For the native STA2 promoter, an 18.8-fold increase in lacZ expression was observed in SCD medium, whereas only a 2-fold increase in lacZ expression levels was observed for the MUC1 promoter. In SCGE medium, multiple copies of FLO8 were able to significantly activate expression of the STA2-dependent reporter gene, but not of the MUC1 promoter-dependent reporter gene. Deletion of the UAS1 area from both the MUC1 or STA2 promoters resulted in a complete loss of FLO8-dependent activation.

Multiple copies of MSN1, on the other hand, had a more pronounced effect on expression levels from both promoters in both SCD and SCGE media. In SCD medium, the wild-type MUC1 and STA2 promoters yielded 19.2- and 33-fold increases in activity, respectively, in the presence of multiple copies of MSN1 and 5.6- and 4.9-fold increases in activity in SCGE medium. Deletion of the UAS1 area from the promoters of STA2 and MUC1 resulted in reductions in expression levels in the presence of multiple copies of MSN1 in SCD medium. Compared to the levels of activity from the native promoters under the same conditions, a 5.2-fold decrease for the MUC1 promoter and a 4.7-fold decrease for the STA2 promoter were observed. In SCGE medium, however, multiple copies of MSN1 resulted in higher levels of expression from the STA2 and MUC1 promoters from which the UAS1 region was deleted than from the native promoters. Compared to the wild-type promoters under the same conditions, a 1.3-fold increase for the MUC1 promoter and a 1.8-fold increase for the STA2 promoter, from both of which the UAS1 area had been deleted, were observed.

Identification of regulatory regions within the UAS1 area.

Both a previous report (44) and the data presented in Table Table55 suggest that FLO8 confers regulation via a sequence within the UAS1 area. Our data (Table (Table5)5) furthermore show that Msn1p and Mss11p act in part via the same region. To better define this area, sequential deletions of UAS1 were generated through PCR amplification, by using the promoters of MUC1 and STA2 as templates. These fragments were introduced into the UAS-less CYC1 promoter fused to lacZ as a reporter gene on the centromeric vector pHP41 (Fig. (Fig.1).1). These constructs, as well as the vector without any insert as a control, were transformed into different genetic backgrounds, and the levels of β-galactosidase conferred by these fragments were determined.

To locate the sequences in UAS1 through which FLO8, MSS11, and MSN1 confer activity, we transformed the UAS1 sequential deletion constructs and the vector without any insert as a control into strains ISP15, ISP15Δflo8, ISP15Δmss11, and ISP15Δmsn1. The wild-type strain represents the expression levels conferred by single copies of FLO8, MSS11, and MSN1, and the deletion strains represent the absence of the respective factors. To determine the effect of multiple copies of FLO8, MSS11, and MSN1 on expression levels, we cotransformed the deletion constructs into the wild-type strain, ISP15, along with YEplac112-FLO8, YEplac112-MSS11, or YEplac112-MSN1, i.e., 2μm plasmids bearing FLO8, MSS11, and MSN1, respectively. The expression levels conferred by the deletion constructs in these strains are given in Table Table66 (FLO8), Table Table77 (MSS11), and Table Table88 (MSN1). From the data in these tables, it is clear that the UAS1 area, inserted in the CYC1 promoter upstream of the lacZ reporter gene, conferred largely similar regulation patterns in the full STA2 and MUC1 promoters, which is a confirmation of the results obtained with deletions of this area from the native promoters (Table (Table5).5). UAS1 is repressed in medium containing glucose as a carbon source, derepressed in medium containing glycerol and ethanol as carbon sources, and subject to activation by FLO8, MSS11, and MSN1.

TABLE 6

Identification of FLO8-responsive regions in the UAS1 area of STA2 and MUC1 in strain ISP15 in SCD

medium

TABLE 7

Identification of MSS11-responsive regions in the UAS1 area of STA2 and MUC1 in strain ISP15 in SCGE

medium

TABLE 8

Identification of MSN1-responsive regions in the UAS1 area of STA2 and MUC1 in strain ISP15 in SCD

medium

When compared to data obtained in the wild-type strain, the presence of multiple copies of FLO8 resulted in 1.9- and 2.9-fold increases in MUC1 (pHP41 + M3-10) and STA2 (pHP41 + S3-10) UAS1-dependent reporter gene activity, respectively. Deletion of FLO8, on the other hand, did not affect expression levels significantly. Similar patterns were observed for all of the deletion fragments, with the exception of the two shortest constructs (pHP41 + 14-10 and pHP41 + 15-10). In these cases, a nearly threefold reduction in reporter gene activity was observed in the Δflo8 strain, while the activation effects of multiple copies of FLO8 were maintained. These data suggest that Flo8p acts through a sequence in this fragment to activate the STA2 and MUC1 promoters.

Expression levels conferred by all of the UAS1 deletion fragments were higher in the presence of multiple copies of MSS11 and lower in the Δmss11 background (Table (Table7).7). These effects of the copy number of MSS11 were, however, more marked with the larger constructs than with the shorter constructs. However, as observed for FLO8, the smallest fragment, 15-10, still conferred a 1.3-fold increase in reporter gene expression when MSS11 was present in multiple copies and a 1.6-fold decrease in expression in a Δmss11 strain, suggesting that Mss11p also acts through a sequence in this area to confer activation of STA2 and MUC1. This is not surprising, since the epistatic analysis suggested that MSS11 was required for FLO8-dependent activation of invasive growth and of starch degradation, and MSS11 can be expected to affect expression from Flo8p-dependent promoter areas. The strong effect of MSS11 copy number on the full UAS1 promoter fragment suggests, however, that Mss11p exerts some activity on other regions within UAS1.

Expression levels conferred by all fragments were the highest in the presence of multiple copies of MSN1. As with FLO8, the smallest fragment, 15-10, still exhibited MSN1-dependent behavior, and only this fragment resulted in a significant decrease in activity in a strain with MSN1 deleted. Reporter gene activity with this construct resulted in a 4.1-fold increase in the presence of multiple copies of MSN1, whereas a 3.8-fold decrease was observed when MSN1 was deleted.

With the data shown in Tables Tables6,6, ,7,7, and and8,8, it is clear that a strong repressive element was present in all fragments, except fragment 15-10. The deletion of the area immediately upstream of 15-10, i.e., the area still present in 14-10 but removed from 15-10, resulted in the biggest increases in expression levels. This would suggest that a cis-acting element, conferring repression on UAS1, is present in the sequence immediately upstream of 15-10. Fragment 15-10 was, however, still susceptible to activation by Flo8p, Mss11p, and Msn1p, since strains transformed with multiple-copy plasmids bearing FLO8, MSS11, or MSN1 resulted in higher expression levels than the wild-type strain. Concomitantly, expression levels for fragment 15-10 were also lower in strains with deleted FLO8, MSS11, and MSN1 loci.

Fragments M12-10 and S12-10 exhibited low levels of activity under most conditions tested and in all genetic backgrounds investigated, except when FLO8, MSS11, or MSN1 was present in multiple copies. A Mig1p binding site present in this fragment might explain some of the observed decreases, e.g., such as in SCD medium. It was shown that Mig1p is not involved in repression of the STA genes (17), but at the large distance from the ORF in the native promoter context, the presence of this binding site might not be relevant. However, in the CYC1 promoter of the reporter plasmid, pHP41, this site is much closer to the ORF and might therefore become relevant. In this case, this result would be artifactual.

Effect of the small MUC1 promoter inserts on expression levels.

Unlike MUC1, the STA1 to -3 genes are not present in the genomes of the S288C-derived laboratory strains that were used in the sequencing of the S. cerevisiae genome. Laboratories working on starch metabolism in S. cerevisiae therefore contributed the sequences of the STA1 and STA2 genes. STA3 is the only member of the STA gene family that had not been sequenced to date. To establish whether the promoter is identical to those of the other members of the family, the 5′ upstream region of STA3 was sequenced and compared to the available sequences of STA1 and STA2. The sequence proved to be identical to those of the STA1 and STA2 promoters, with the exception of some single nucleotide substitutions (data not shown).

Only 2,500 bp of the upstream regions of the STA2 gene had been sequenced to date. An additional 1,462 bp of the STA2 promoter, upstream of position −2500 relative to the STA2 ORF, was sequenced to see how far the homology between the upstream regions of MUC1 and STA2 stretches. An alignment of this sequence with the upstream sequence of MUC1 revealed that the homology extends over more than 3.9 kb. The 20- and 64-bp sequences found at nucleotides −1333 to −1313 and nucleotides −933 to −869 of the MUC1 promoter are not present in any of the STA1 to -3 upstream regions, and thorough BLAST homology searches (2) revealed that the sequences thereof do not have significant homology to any other submitted sequence. This suggests the possibility of a unique regulatory role for these inserts in the MUC1 promoter.

From the expression levels of the STA2 and MUC1 UAS1 deletion constructs given in Tables Tables6,6, ,7,7, and and8,8, it is evident that the presence of the 20-bp MUC1 promoter insert in constructs M3-10 and M11-10 resulted in decreases in expression levels. This reduction was reproducible in all strains and under most conditions tested (data not shown). The only other differences between the STA1 to -3 upstream regions and that of MUC1 exist around the TATA boxes. MUC1 has only one functional TATA box at position −100, whereas STA1 to -3 have two at positions −75 and −100 (51). To investigate whether these inserts are the major factors determining the decreased expression levels observed for the MUC1 upstream region and that these decreases are not contributed by any other dissimilarity between the two promoters, e.g., the use of different TATA boxes, we took advantage of the fact that the STA1 to -3 genes could be used as reporter genes in a glucoamylase plate assay. Plasmid YCplac33-STA2, bearing the wild-type STA2 gene under its native promoter, and plasmid YCplac33-PMUC1-STA2, which is identical but for the presence of the 20- and 64-bp MUC1 promoter inserts, were transformed into strain JM2508, which does not contain any of the STA1 to -3 genes in its genome. In addition, the different transcriptional activators of STA2, i.e., FLO8, MSN1, and MSS11, present on 2μm plasmids, were cotransformed along with YCplac33-STA2 and YCplac33-PMUC1-STA2 into strain JM2508. The different transformants were grown on SCD medium until they reached mid-log phase (OD₆₀₀ = 1.0), before 10 μl of these cell suspensions was spotted onto cornstarch plates (SCS). Expression levels of the STA2 gene are reflected in the size of the halos around the different colonies. In Fig. Fig.4A,4A, it is evident that the yeast strain containing only the plasmids YCplac33 and YEplac112 was unable to utilize starch, whereas the cells transformed with the wild-type STA2 gene were able to degrade starch efficiently. The presence of multiple copies of FLO8, MSN1, and MSS11 clearly resulted in increased production of glucoamylase when the STA2 gene was regulated by its native promoter. Figure Figure4B4B shows the expression levels of the different colonies of JM2508, transformed with a copy of the STA2 gene, which has the two MUC1 promoter inserts in its upstream region. The strain without STA2 was unable to degrade starch, as expected. Glucoamylase production from STA2 with the MUC1 promoter inserts in its upstream area, YCplac33-PMUC1-STA2, was almost undetectable. Only multiple copies of FLO8, MSN1, or MSS11 were able to overcome this repressive effect, resulting in visually detectable levels of expression of STA2, albeit at more reduced levels than those of strains bearing STA2 under regulation of its native promoter (Fig. (Fig.4A).4A). Interestingly, multiple copies of MSN1 and MSS11 were able to overcome the repressive effect conferred by the MUC1 promoter fragments much more efficiently than multiple copies of FLO8.

FIG. 4

(A) SCS plate (containing 2% cornstarch as a carbon source) to investigate the starch utilization of S. cerevisiae JM2508 transformed with plasmids YCplac33 and YEplac112 without any inserts (no. 1), YCplac33-STA2 and YEplac112 (no. 2), YCplac33- (more ...)

The levels of expression conferred by the 20- and 64-bp MUC1 promoter inserts alone were also determined. The two fragments were cloned into vectors pHP41, pLG670-Z, and pLGΔ312, and the effect on the levels of expression of lacZ in different strains and under different growth conditions was determined. Only in the low-copy-number plasmid pHP41 did these fragments confer the expected repressive effect. In the multiple-copy vectors pLG670-Z and pLGΔ312, the fragments seemed to confer activation rather than repression, since even on repressive SCD medium (Table (Table9),9), high levels of lacZ activity were observed. These high levels of activity were observed under all of the conditions tested, and at no stage was any specific regulation observed. These data could illustrate the unsuitability of multiple-copy plasmids in the functional analysis of promoter fragments. The large number of cis elements created by the use of multiple-copy plasmids could titrate out regulatory factors, leaving a percentage of a DNA sequence that would normally be subject to regulation in an unregulated state, thereby masking the true nature of the fragment.

TABLE 9

Expression levels of MUC1 promoter inserts in different plasmids on SCD medium in strain

ISP20

DISCUSSION

The MUC1 and STA1 to -3 promoters are of particular interest, since they (i) consist of evolutionarily closely related sequences, allowing the study of promoter evolution on a molecular level; (ii) represent the largest S. cerevisiae promoters identified to date; and (iii) might integrate the information transmitted by several separated signal transduction pathways to specifically result in an adaptive cellular differentiation process. Our results confirm previously published data suggesting that the expression of the MUC1 and the STA genes is indeed controlled by the complex interaction of a large number of factors which are regulated by several independent signaling pathways.

Our data regarding the transcriptional activity of the entire promoters reveal several general features. First, PMUC1-dependent reporter gene expression is very low under most conditions and is generally well below levels observed for the UAS-less reporter plasmid alone, indicating that the entire promoter has a repressive effect. The STA2 promoter, on the other hand, is in a less repressed state. Indeed, expression levels of the PSTA2-dependent reporter gene are consistently higher than those for the PMUC1-dependent reporter gene.

Second, the data show that overall variations in expression levels conferred by the entire promoters in a wild-type strain are much more important for the STA2 promoter than for the MUC1 promoter, even if the general regulation patterns are very similar. Since the STA genes encode extracellular glucoamylases and can therefore provide otherwise inaccessible nutrients, high expression levels and strong induction can obviously be advantageous to the cell. MUC1 expression, on the other hand, has to be more tightly controlled, since overexpression of the gene could result in profound physiological changes. MUC1 is essential for pseudohyphal differentiation and invasive growth, and both processes can be induced through overexpression of MUC1 from a heterologous promoter or, to a lesser degree, by multiple copies of the gene (8, 23, 28). From an evolutionary perspective, the changes between the two promoters have therefore allowed them to retain a similar regulation pattern, ensuring a coregulation of pseudohyphal differentiation and invasive growth with starch degradation, while allowing for much stronger production of glucoamylases.

The data show that the parameters which affect expression of both genes are (i) the presence or absence of a fermentable carbon source, (ii) the ploidy of the strain, and (iii) the presence or absence of several transcriptional regulators. As stated above, in all of these cases, we found that changes conferred by the STA2 promoter are generally more significant than those conferred by the MUC1 promoter.

The STA2 gene seems to have retained most specific regulatory elements but has evolved a less attenuated or less repressed promoter. This could indicate that the sequences which are found in the MUC1 promoter, but which are deleted in the STA1 to -3 promoters, are required for general repression. Our data suggest that this is indeed the case, since (i) the two inserts reduce STA2 expression strongly when present upstream of this gene and (ii) the 20-bp insert has a repressive effect, as the analysis of the UAS1 region clearly demonstrates. Our data in addition show that this repression is specifically linked to the Flo8p transcriptional activator. Multiple copies of FLO8 indeed result in strong production of glucoamylases when the STA2 gene is controlled by its own promoter, but fail to do so when the two MUC1 promoter inserts are present. Multiple copies of MSN1 and MSS11 do not result in a similar difference between the two promoters but efficiently increase STA2 expression in the presence or absence of the inserts. The repressive effect of these sequences might therefore depend on directly or indirectly inhibiting the Flo8p-dependent regulation of MUC1.

The sequence does not seem to confer a repressive effect on its own, but its regulatory activity seems context specific. When tested in the pHP41 plasmid, both the M20 and the M64 inserts reduce transcription of the reporter gene. However, and surprisingly, both sequences confer activation to a reporter gene when tested in a different reporter plasmid. The strong activation observed in the case of the pLG670-Z plasmid might be due to the creation of a spurious activation sequence, even if this hypothesis is difficult to reconcile with the fact that the two insert sequences do not present any homologies. These results could nevertheless suggest that the two inserts are the target of a DNA-binding protein, whose binding could result in repression in the specific context of the MUC1 gene promoter.

Our study of the entire promoters confirms that MUC1 and STA2 respond similarly to the deletion or the presence of multiple copies of MSN1 and MSS11. However, and as suggested by the effect of the MUC1 promoter inserts on STA2 expression, the responses of the two genes to multiple copies and deletion of FLO8 differ. Multiple copies of FLO8 result in strongly increased expression of the STA2 gene in medium containing either glucose or glycerol-ethanol as a carbon source but induce MUC1 expression only in medium containing glucose as a carbon source. Rupp et al. (44) showed that Flo8p was required for the cAMP-dependent regulation of invasive and pseudohyphal growth. The only physiologically significant variation in intracellular cAMP concentration is observed when glucose is added to cells grown on nonfermentable carbon sources (reviewed in references 14 and 49), and data suggest that the main role of cAMP could be the sensing of fermentable sugars. Our data could therefore indicate that Flo8p is required only for MUC1 induction during growth on substrates containing fermentable sugars, as is the case on nitrogen-limited SLAD medium, which is the main or only medium used for the assessment of filamentation by most authors. Flo8p could interact with other factors to induce filamentation during nitrogen limitation, when glucose levels are still high but might not be required or act differently under other conditions.

The size of the promoter, coupled to the apparent complexity of the regulatory processes, renders detailed molecular analysis of the entire promoter a difficult task. For most promoter studies with yeast published so far, a reasonable correlation between mechanistically (i.e., the binding of a regulatory protein to a specific sequence) and physiologically (i.e., the resulting change in transcription levels) relevant data can easily be achieved. However, in the case of the MUC1 and the STA1 to -3 genes, data suggesting specific molecular interactions and regulatory events in a specific area of the promoters might not result in the expected and corresponding changes in the overall transcriptional activity of the genes. The activating or repressing effect expected after the binding of a transcription factor to a region within the promoter might frequently be masked and covered by other regulatory signals acting through other areas.

Physiologically, the only significant data are those that relate to the activity of the promoter as a whole. However, in order to establish mechanistically relevant data concerning, for example, cis-acting transcription factor binding sites, it is necessary to dissect the promoter by using smaller sequence fragments. For purposes of analysis, these fragments are placed in a new, very different sequence context (i.e., plasmid sequences), and the data obtained have to be interpreted carefully when considering the effects on the native promoters. For this reason, we have focused our investigation on a small section of the STA2 and MUC1 promoters that combines several of the interesting features of the entire, intact promoters within a relatively short stretch of DNA. Our data show that this area (i) confers transcriptional regulation from a far-upstream (>1,000 nucleotides) position in the context of the native promoter and (ii) regulates reporter gene expression very similarly to the entire promoters when analyzed on its own. More specifically, this area of the MUC1 and STA2 promoters indeed (i) confers a general repressive effect on reporter gene transcription under most conditions and (ii) contains sequences responsible for both specific activation and specific repression. Furthermore, the area contains one of the two significant changes between the MUC1 and STA2 promoters.

Our data clearly establish that this additional sequence contributes to the general repression or attenuation of the MUC1 promoter, giving a clear indication of a molecular rearrangement during promoter evolution. In addition, the sequence is a target of glucose repression. The three transcriptional regulators investigated during this study, Flo8p, Msn1p, and Mss11p, all act, at least in part, via UAS1 to activate transcription of MUC1 and STA2. The deletion analysis pinpoints the sequences within UAS1 which confer these regulations, and these short sequences can now be investigated further to establish the binding sites of the factors involved. Flo8p and Mss11p clearly act in the 80-bp region between nucleotides −1160 and −1070 in the STA2 promoter and nucleotides −1210 to −1130 in the MUC1 promoter.

We also identify the STA10 repressive effect as being due to a mutation in the gene encoding the transcriptional regulator Flo8p. Indeed, we clearly demonstrate that a single copy of FLO8 in an S288C genetic background allows production of an amount of glucoamylase similar to that observed in naturally occurring starch-degrading strains. FLO8 was shown to be required for invasive growth in S288C-derived strains (26), since transformation of these strains with a single copy of wild-type FLO8 restored the ability to invade the growth medium. W303, another commonly used laboratory strain, contains, in addition to a mutation in FLO8, mutations in other activators required for invasive growth and pseudohyphal differentiation (26) and is therefore unable to form pseudohyphae or grow invasively. In this strain, a single copy of FLO8 was also unable to restore glucoamylase expression from a plasmid-borne STA2 gene (data not shown), suggesting that the STA10 phenotype in W303 strains might be due to the requirement of FLO8 as well as other transcriptional activator(s). We also show that Flo8p requires Mss11p to induce both starch degradation and pseudohyphal differentiation and invasive growth. Since Mss11p is able to overcome mutations in FLO8, we suggest that Mss11p is situated downstream of Flo8p in a linear signal transduction cascade. However, Flo8p apparently acts independently of Msn1p, which is probably situated in a parallel pathway.

As expected for such a complex promoter and as discussed above, some of the data obtained for UAS1 do not correlate properly with those seen for the entire promoter. Most tendencies are, however, conserved, and the data are mechanistically significant. Only a combination of studies of all UAS and URS sequences of the MUC1 and STA promoters will allow us to reveal a complete picture of how transcription factors combine to result in either repression or activation.

ACKNOWLEDGMENTS

We thank T. Cooper for plasmid pHP41, J. H. Hegemann for plasmid pUG6, and I. Yamashita for plasmid pSTA3-6-4. This work was supported by grants from the National Research Foundation (NRF) and the South African wine industry (Winetech) to I.S.P.

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