©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Essential Transcription Factor, Mcm1, Is a Downstream Target of Sln1, a Yeast Two-component Regulator (*)

Guoying Yu (1), Robert J. Deschenes (2), Jan S. Fassler (1)(§)

From the (1) Genetics Ph.D. Program and Department of Biological Sciences and the (2) Biochemistry Department, University of Iowa, Iowa City, Iowa 52242

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In a search for mutants exhibiting altered activity of the yeast transcription factor, Mcm1, we have identified the SLN1 gene, whose product is highly related to bacterial two-component sensor-regulator proteins. sln1 alleles identified in our screen increased Mcm1p-mediated transcriptional activation, while deletion of the SLN1 locus severely reduced Mcm1p activity. Our data establish that Mcm1p is a downstream target of the Sln1 signaling pathway. Yeast Sln1p was recently shown to be involved in osmoregulation and to depend on the Hog1 MAP kinase (Maeda, T., Wurgler-Murphy, S., and Saito, H. (1994) Nature 369, 242-245). We show that SLN1-mediated regulation of Mcm1p activity is independent of the Hog1 MAP kinase, and suggest that the role of SLN1 is not restricted to osmoregulation.


INTRODUCTION

Prokaryotic organisms use a simple modular strategy for response and adaptation to their physical and chemical environments. In its simplest form, two components, a sensor and a response regulator, are combined to sense and transmit information using a high energy phosphohistidine intermediate (1) . The two-component regulatory system was recently demonstrated in eukaryotes by the isolation of genes in Arabidopsis thaliana (2) and Saccharomyces cerevisiae (3, 4, 5) encoding proteins containing many of the expected consensus features. We show here that one of the downstream targets of the yeast Sln1p two-component pathway is the transcription factor, Mcm1.

Mcm1 is a constitutively expressed DNA-binding protein that is essential for yeast viability (6) . The optimum site for Mcm1p binding (P site) consists of a 16-base pair perfect palindrome (7, 8, 9) . At low affinity (partially degenerate) binding sites, Mcm1p activity is both amplified and regulated by interactions with accessory proteins that are thought to act by altering the conformation of Mcm1p (10) . In contrast, at high affinity (palindromic) P sites, Mcm1p binds in an active conformation in the apparent absence of additional proteins (10, 11) .

The N-terminal 97 amino acids of Mcm1 carry the DNA binding, dimerization, and protein interaction functions of the protein (12, 13, 14) . This so-called ``MADS box'' domain is 70% identical to an equivalent domain of human serum response factor (SRF).() Like Mcm1, SRF activity is regulated and modified by its interactions with accessory proteins (15) . In addition, SRF-mediated activation is responsive to growth factor signals via kinase-based signal transduction cascades. To investigate the possibility that Mcm1p activity is modulated in response to extracellular signals, mutations leading to altered activity were sought. We previously found that mutations in the SPT13 ( GAL11) gene increased Mcm1-mediated activation from a high affinity (palindromic) P site (16) . Additional negative regulators of palindromic P site activity were sought, and one gene, NRP2, identified in this screen, is the subject of this report.


EXPERIMENTAL PROCEDURES

Yeast Strains and Media

All yeast strains used in this work (Table I) are from our strain collection or were constructed for this study except RC634 (17) . The media were prepared as described by Sherman et al. (18) and included synthetic complete medium (SC) lacking one or more specific amino acids ( e.g. SC-uracil) and rich medium (YPD). Plates for the detection of -galactosidase contained 50 µg of 5-bromo-4-chloro-3-indolyl - D-galactopyranoside (X-gal)/ml and were prepared as described by Larson et al. (19) . All yeast strains were grown at 30 °C.

EMS Mutagenesis

JF819 carrying the pGY48 reporter (below) was grown to saturation in SC-uracil. Cells were washed and resuspended in 0.1 M sodium phosphate, pH 7.0, prior to incubation with EMS (0.29% v/v) for 20 min at 30 °C. Following inactivation of the EMS, cells were diluted with 0.1 M phosphate buffer and plated on SC-uracil media at 30 °C. Colonies arose at roughly 40% the expected numbers. Mutants were selected on the basis of their blue phenotype following replica plating to SC-uracil media containing 50 µg/ml X-gal.

Plasmids

The reporter plasmid, pGY48, a derivative of plasmid pLG670Z (20) , carries a CYC1-lacZ fusion gene lacking the CYC1 UAS. A 16-base pair palindromic Mcm1 binding (P) site oligonucleotide was cloned into the UAS position of the CYC1 promoter to create pGY48 (16) . pLG669Z carries a CYC1- lacZ fusion gene that contains the intact CYC1 UAS (20) . The 20B plasmid carries a GAL1-lacZ fusion gene in which the upstream regulatory sequences -473 to -632 of GAL1 have been deleted (21) . As a result, reporter gene expression is not glucose-repressible.

Various SLN1 subclones were generated from the SLN1 library plasmid (pGY111). pGY112 was generated by subcloning the 2.8-kb BamHI fragment from pGY111 into the BamHI-linearized LEU2 2-µm shuttle vector, pRS425 (Stratagene). pGY113 was constructed by religation of pGY111 in the absence of the 2.8-kb BamHI fragment. pGY121 consists of the 3.5-kb pGY111 PstI fragment in PstI-linearized pRS425. pGY140 was constructed by subcloning the 4.3-kb pGY111 ClaI fragment into pGEM5Zf(+) (Promega) to generate pGY129 and then reisolating that fragment with EagI ends for subcloning into the EagI site of pRS425. To construct pGY151, the 6.7-kb pGY111 BbuI fragment was first subcloned into BbuI-digested pGEM5Zf(+) to generate pGY138. Next, the 5-kb pGY138 EcoRV fragment was subcloned into EcoRV-cut pGEM5Zf(+) generating pGY144, and finally, a 5-kb pGY144 EagI fragment was subcloned into EagI-linearized pRS424 (Stratagene), a TRP1 2-µm shuttle vector. The insert sequences in pGY151 extend from the BbuI site to the EcoRV site in SLN1 (Fig. 1). The SLN1 integrating vector, pGY141 was constructed by inserting a 2.2-kb XhoI- SalI fragment containing the LEU2 gene into the polylinker SalI site of pGY138.


Figure 1: Restriction map and complementation analysis of the nrp2 complementing clone (pGY111) containing the SLN1 gene. Yeast sequences are shown as solid and vector sequences as dotted lines.



A marked deletion of the SLN1 gene was constructed by removal of an internal 2.1-kb HpaI fragment from plasmid pGY138, followed by insertion of a 2.2-kb XhoI- SalI LEU2 fragment or a 0.85-kb EcoRI- BglII TRPI fragment at the point of the deletion to generate plasmids pGY143 and pGY148. The disrupted SLN1 gene was isolated on a 7.5-kb (pGY143) or a 6.2-kb (pGY148) BbuI restriction fragment and used to transform a wild type diploid generated by mating strains JF819 and JF1220. GY61 and GY67 are haploid progeny of the disrupted sln1::LEU2 diploid. JF1350-1355 are haploid progeny of the sln1::TRP1 disrupted diploid.

The PTP2 plasmid, pGY153, was constructed by subcloning a 5-kb BamHI fragment from pRSYP2 (22) into the 2-µm TRP1 pRS424 vector (Stratagene). JF1361 and JF1360 were generated by transformation of JF819 and JF1359 with a 5.1-kb BamHI fragment isolated from pGEM-YP2:: LEU2 (22) . Putative PTP2 disruptions were confirmed by Southern hybridization analysis.

The hog1-::TRP1 disruption vector, pGY150, was constructed from pJB30 (23) by deletion of an internal 0.4-kb EcoRI fragment, and replacement with a 0.85-kb BglII- EcoRI TRP1 fragment. A 3.0-kb BamHI- ClaI fragment was isolated and introduced into JF1331 to generate JF1362 and into the nrp2-1 mutant, JF1359, to generate GY91. hog1- transformants were distinguished by their failure to grow on solid media containing 0.9 M NaCl (23) .

Plasmid YIp5- mcm1-DEQ (12) was used in the two-step transformation of the wild type strain JF820 to generate the mcm1 truncation mutant (JF1335) used as an Mcm1-deficient control in the halo and Northern (RNA) hybridization experiments.

Probes for Northern hybridization analysis included: a 1.6-kb EcoRI fragment containing MF1 isolated from plasmid p69A (24) , a 3.8-kb EcoRI fragment containing the ACT1 gene isolated from plasmid pACT1 (F. Winston), and a 2-kb NdeI fragment that includes the MCM1 coding region isolated from plasmid pGA1761 (25) .

Halo Assay

The halo assay was as described previously (26) except that synthetic media was used to permit growth of the sln1- strains. The tester lawn was RC634 ( MATa sst1-3) (17) .

RNA Isolation and Northern Hybridization Analysis

Cells were grown to a concentration of 1-2 10/ml in SC-uracil media. RNA was prepared by the method of Carlson and Botstein (27) . Electrophoresis, blotting and hybridization were performed as described previously (28) . P-Labeled probes were prepared using random primers (29, 30) .

-Galactosidase Assays

-Galactosidase assays were performed using cleared lysates (31) as described previously (32) . Strains were cultured in SC-uracil media except in the salt induction experiments, overnight cultures grown in SC-uracil were diluted into rich media (YPD) and grown for two to three doublings to a final density of 1 10/ml. Sodium chloride was added as a solid to a final concentration of 0.9 M. At the indicated times, 10-ml aliquots were removed and -galactosidase assays performed.

Western Analysis

Strains were harvested in exponential growth at the times indicated following the addition of solid NaCl to 0.9 M. 50 µg of each extract, prepared as described previously (23) , was subjected to 10% SDS-polyacrylamide gel electrophoresis, blotted to nitrocellulose, and probed with anti-phosphotyrosine antibody (1:2000 dilution, 4G10; Upstate Biotechnology Inc.). Immune complexes were visualized by ECL chemiluminescence (Amersham Corp.).


RESULTS AND DISCUSSION

To identify new genes that modulate the transcriptional activity of Mcm1p, cells carrying a plasmid-borne, CYC1-lacZ fusion gene directed by a high affinity P site (UAS(P)- lacZ) (pGY48) were subject to EMS mutagenesis. A wild type strain carrying the UAS(P)- lacZ reporter has a pale blue phenotype on media containing the chromogenic -galactosidase substrate, X-gal. Following mutagenesis, colonies were identified that exhibited increased blue color on X-gal media and increased activity in liquid -galactosidase assays. Mutants with increased reporter gene activity were designated NRP, which stands for Negative Regulators of P site activity. One complementation group, NRP2, consisting of three alleles, is the subject of this report. Each of the three nrp2 mutations increased activity of the UAS(P)- lacZ reporter approximately 5-fold (Table II). The nrp2 mutations increased the activity of the closely related (UAS( CYC1)- lacZ) reporter only 1.1-1.7 fold; hence, the large effect of the nrp2 mutations appears to depend on the UAS(P) element (). The activity of the unrelated (UAS( GAL1)- lacZ) reporter was decreased slightly () in two of the nrp2 strains.

Since the increased UAS(P)- lacZ activity of the nrp2 mutants was a recessive phenotype ( nrp2-1/NRP2, ), the gene was cloned from a centromere based genomic library (P. Hieter) by complementation of the defect leading to the X-gal phenotype. Among 10,000 transformants, a single plasmid, pGY111, was identified that rescued the blue colony phenotype of the nrp2-2 mutation. pGY111 also complemented the X-gal phenotype of the nrp2-1 and nrp2-3 mutants. The complementing gene was localized to a 5-kb BbuI- EcoRV fragment within the 10-kb insert of pGY111 by testing the complementing activity of subclones and deletions (Fig. 1). The DNA sequence in the vicinity of the BamHI site in the BbuI- EcoRV complementing clone was determined and found to be identical to a region of the previously isolated SLN1 gene (5) .

Evidence that the X-gal phenotype of nrp2 mutants was due to mutation of the SLN1 gene was provided by the mutant phenotype of an nrp2-1/sln1- diploid (). Linkage between SLN1 and nrp2 was also tested. The SLN1 integrating plasmid, pGY141, was linearized at the unique BamHI site within the SLN1 gene and Leuintegrants of an nrp2-3 diploid were isolated. Diploid integrants exhibited a white phenotype on X-gal plates, and following sporulation, the mutant (blue on X-gal plates) phenotype segregated 2:2 in dissected tetrads. A white Leuspore colony was then mated with JF819 ( SLN1). The absence of the mutant phenotype among the progeny of this diploid (0/44) was consistent with linkage between the SLN1 and nrp2 loci.

The Sln1 protein exhibits a high degree of similarity to both the sensor and response regulator modules of bacterial two component regulators (5) . In bacterial two-component regulation, signal transmission depends on autophosphorylation of a conserved histidine in the sensor and phosphotransfer to a conserved aspartate in the response regulator, the function of which is to relay the signal. Activity of the pathway depends on the half-life of the phosphoaspartate, which is determined by the relative rates of phosphotransfer and phosphohydrolysis carried out by the two modules (1, 33) .

To evaluate the phenotype of a SLN1 null mutation, a marked SLN1 gene disruption was constructed. Consistent with the phenotype previously described (5) , the sln1- mutant failed to grow or formed only microcolonies on YPD media, and grew slowly on synthetic complete media. The activity of the UAS(P)- lacZ reporter activity in sln1- strains measured under permissive growth conditions (SC-uracil, 30°C) was 10-fold less than in SLN1strains (). Although deletion of SLN1 decreased P site activity, the original recessive nrp2 activating alleles increased P site activity. One possible explanation for these phenotypes is that Sln1, like bacterial two-component systems (1) , harbors both positive and negative activities, and that the negative Sln1 function can be provided in trans. The reduced UAS(P)- lacZ reporter activity observed in sln1- mutants suggests that SLN1 is required for full P-mediated activation.

To confirm the effect of the sln1- mutation on P-mediated activation, expression of the Mcm1-regulated MF1 gene encoding mating pheromone was analyzed. Deletion of SLN1 caused a reduction in MF1 levels as seen by halo assay (Fig. 2 A) and by Northern (RNA) hybridization analysis (Fig. 2 B). In these experiments the mcm1DEQ (12) mutant was included as a negative control. The activity of the UAS(P)- lacZ reporter in this Mcm1 truncation mutant is 18% of that in a wild type strain (12) . Densitometric analysis indicated that MF1 levels were reduced in the sln1- mutants to 25% of wild type levels. The reduction in MF1 expression was a specific result of the sln1 defect and was not attributable to the slow growth of the sln1 mutant strain since expression of an Mcm1-independent gene, ACT1, was unaffected in the mutant (92.5% of wild type) (Fig. 2 B).


Figure 2:sln1- decreases Mcm1-activated expression of endogenous genes. A, halo assay. MAT strains were grown permissively on synthetic medium and then transferred to a lawn of MATa cells that are supersensitive to mating pheromone. The size of the zone of non-growth reflects MF gene transcript levels. B, Northern (RNA) hybridization. Equal amounts (15 µg) of total RNA from the indicated MAT strains were separated by formaldehyde-agarose gel electrophoresis. A single filter was hybridized to a P-labeled restriction fragments containing sequences from the MF1 gene (from plasmid pHK2; Ref. 24), the ACT1 gene from plasmid pACT1 (F. Winston), and the MCM1 gene from plasmid pGA1761 (25). Hybridization to 0.7-kb ( MF1), 1.3-kb ( ACT1), and several 1 to 1.6-kb ( MCM1) transcripts was detected as expected. Ethidium bromide staining of the same gel is shown to the right. MAT strains: wild type, JF820; sln1-::TRP1, JF1350; sln1-::LEU2, GY61 and GY67; mcm1-DEQ, JF1335 (12); nrp2-1, GY31.



The effect of sln1- on P-mediated activation was not due to reduced MCM1 message or protein levels. MCM1 transcript (Fig. 2 B) and protein (data not shown) levels were comparable in sln1- and SLN1strains. These results indicate that Sln1p is essential for full Mcm1p activity, and not for its expression. One way that P-mediated activation could be modulated involves changes in the affinity of the Mcm1 protein for its binding site; however, gel mobility shift experiments in which sln1- and SLN1extracts were incubated with a palindromic P site probe revealed no SLN1-dependent changes in the level of Mcm1p complex formed (data not shown).

The increased P site activity in the nrp2 mutants was not reflected in increased MF gene expression. In fact, MF1 expression was somewhat decreased in nrp2-1 strains. This may indicate that, while the imperfect P sites (PQ) (7) present in the promoters of -specific genes like MF1 are dependent on Sln1p for activity, PQ-mediated activation is insensitive to the special activating form of Sln1p encoded by the nrp2 mutants. The possibility that an nrp2-activated Mcm1p or Mcm1p complex has reduced affinity for PQ sites merits further investigation.

Although the physiological role of Sln1p is currently unclear, recent studies demonstrate that Sln1p function is mediated by the Hog1 MAP kinase in the osmosensing pathway and depends on the activity of protein tyrosine phosphatase ( PTP) genes (34) . To understand the basis of the effect of sln1 mutations on Mcm1p activation, we investigated the relationship of PTP2 and HOG1 to the activity of the UAS(P)- lacZ reporter. The growth phenotype of the sln1- mutation is suppressed by increased dosage of the PTP2 gene (34) . However, introduction of a high copy plasmid carrying the PTP2 gene only very weakly suppressed the nrp2-1 phenotype and the effect of ptp2- was equally modest (Table III). Hence, Ptp2 is unlikely to be a normal component of this pathway. Alternatively, the small effect of PTP2 dosage on UAS(P)- lacZ activity may be a reflection of the involvement of additional related protein phosphatase activities in the pathway.

The recent isolation of mutations in the HOG1 MAP kinase gene as suppressors of the growth defects of a sln1- mutant (3) led to the suggestion that phospho-Sln1p negatively regulates the yeast osmosensing pathway. The role of Hog1p in Sln1p-mediated activation of the UAS(P)- lacZ reporter was measured in wild type cells exposed to 0.9 M NaCl. Although the expected increase in Hog1p tyrosine phosphorylation was observed, no change in P reporter activity was found under these conditions (Fig. 3, ) (3, 23) . P site activity was also not significantly changed by deletion of the HOG1 gene in either SLN1() or nrp2-1 strains (data not shown). The fact that P-mediated activation is neither positively nor negatively regulated by HOG1 suggests that Sln1p may be separately involved in control of osmoregulation and in control of Mcm1p activity.

The identification of the well characterized Mcm1 transcription factor as a target of a two-component regulator reveals for the first time a specific transcriptional outcome of this newly recognized signaling pathway in yeast. We find that Sln1p-mediated regulation of P activity is independent of the osmosensing MAP kinase pathway, implying that Sln1p has multiple signaling functions. We anticipate that the analysis of additional nrp mutants will help to define the signal and refine our understanding of the post-translational events involved in signal transmission.

  
Table: Yeast strains used in this work


  
Table: Effect of NRP2 mutations on P-mediated activation of lacZ reporter gene

-Galactosidase activity was measured in unclarified or clarified glass bead supernatants. Numbers are the average of at least four trials on at least two different transformants. Normalized activity = (Miller units (mutant)/Miller units (wild type) 100. Standard deviations were less than 30% of the average.


  
Table: Effect of PTP2 on P site activation

Assay methods and calculations are described in the notes to Table II. Activities are the average of three or more trials.


  
Table: Effect of salt on UAS(P)-lacZ expression

Assay methods and calculations are described in the notes to Table II. All values were normalized to the activity of the wild type strain measured in the absence of salt.



FOOTNOTES

*
This work was supported by grants from the National Institutes of Health (to J. S. F. and R. J. D.), the American Cancer Society (to J. S. F.), and the Pardee Cancer Research Fund (to R. J. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: 138 Biology Bldg., University of Iowa, Iowa City, IA 52242. Tel.: 319-335-1542; Fax: 319-335-1069.

The abbreviations used are: SRF, serum response factor; X-gal, 5-bromo-4-chloro-3-indolyl - D-galactopyranoside; EMS, ethyl methanesulfonate; kb, kilobase pair(s); UAS, unactivated sequence; P, Mcm1 binding site.


ACKNOWLEDGEMENTS

We thank G. Gussin, D. Weeks and Lois Weisman for their critical reading of this manuscript; M. Gustin, I. Ota, A. Varshavsky, K.-L. Guan, C. Christ, and B. K. Tye for plasmids and strains; S. Eliason for experimental assistance; and members of the Fassler and Deschenes laboratories for helpful discussions.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.