from a Yeast hsp70 Promoter - Europe PMC

MOLECULAR AND CELLULAR BIOLOGY, Aug. 1988, p. 3423-3431 0270-7306/88/083423-09$02.00/0 Copyright © 1988, American Society for Microbiology

Vol. 8, No. 8

Isolation of Mutations That Act in trans To Alter Expression from a Yeast hsp70 Promoter R. CRAIG FINDLY,1* HOSSAIN ALAVI,1 AND TERRY PLATT2 Department of Genetics, University of Georgia, Athens, Georgia 30602,1 and Department of Biochemistry, University of Rochester Medical Center, Rochester, New York 146422 Received 28 December 1987/Accepted 16 May 1988

Transcription of SSAI (formerly YGIOO), a member of the hsp70 gene family in Saccharomyces cerevisiae, increases dramatically upon heat shock. An expression vector in which the promoter of SSA1 is fused to the Escherichia coli galactokinase gene (galK) was constructed and transformed into a galactokinase-deficient yeast strain. The transformants grew on galactose at 23°C, but increased expression of the SSAl-galK fusion gene inhibited growth of cells on galactose at 37°C. Selection for survivors under nonpermissive conditions yielded a class of mutants, termed HSR (for heat shock regulation), which showed reduced levels of expression of the hsp70-galK gene fusion as determined by measurement of galactokinase activity. Similar effects on (galactosidase activity were obtained when an SSAI-lacZ fusion vector was introduced into the mutants, suggesting action in trans through the SSAI promoter. Analysis of Northern (RNA) blots demonstrated that the reduction in expression was a result of decreased mRNA levels for the fusion gene. In addition, mRNA levels of the endogenous SSAI gene are reduced in an HSR mutant. Genetic analysis has shown that these mutations act in trans and affect both transcription from the SSAI promoter and turnover of the fusion transcript. These are the first trans-acting mutations known to affect directly the transcriptional regulation and transcript stability of heat shock genes in eucaryotes. The heat shock response is an apparently universal cellular response to a rapid temperature increase, and similar responses are activated in both eucaryotes and procaryotes. In eucaryotes, the response is induced by a sublethal temperature jump of 5 to 15°C. Induction results in rapid and abundant synthesis, from genes that were previously either silent or expressed at low levels, of both a small number of mRNA species and their translation products, the heat shock proteins. The major and evolutionarily most conserved heat shock protein, HSP70, is found in all eucaryotes and is 50% homologous with the dnaK protein of Escherichia coli (1). Heat shock proteins are also synthesized in response to other stresses, including treatment with ethanol or amino acid analogs, release from anoxia, treatment with uncouplers of oxidative phosphorylation, and some viral infections. How the response is triggered, what the precise functions of the heat shock proteins are, and how the protective aspects of the response (thermotolerance) are integrated with cell physiology are not well understood (7, 9, 12, 21, 28). Induction of the response occurs immediately upon temperature jump and is primarily due to activation of heat shock genes at the transcriptional level (13, 32, 48). Heat shock-induced genes have been best characterized in Drosophila melanogaster and Saccharomyces cerevisiae. DNA sequence analysis of the promoter regions of Drosophila heat shock genes revealed a conserved consensus sequence (C--GAA--TTC--G) which is found in multiple copies distributed over several hundred base pairs of a sequence upstream of the transcription start site (17, 19, 22). Deletion analysis demonstrated that this cis-acting sequence is essential for transcriptional regulation of the response (2, 38, 39). This conserved sequence, the heat shock element (HSE), is common to all heat shock genes in eucaryotes. In S. cerevisiae, the hsp70 gene family contains eight genes, and one of the three heat-inducible hsp70 genes *

(SSAI, SSA3, and SSA4), SSAI (formerly YGIOO [8]), also displays a low basal level of expression (4, 8, 46). This gene contains multiple upstream HSEs, and deletion analysis revealed that a proximal promoter element is responsible for most heat-inducible expression of SSAJ. Sequences flanking the upstream activation site are involved in regulating the basal level of expression of this gene, which suggests that negative regulation is also involved in controlling expression of SSAI (46). In E. coli, transcription of heat shock genes is controlled by the product of the htpR gene, which encodes the ur32 protein (14, 49). However, the mechanisms that control induction and regulate expression of heat shock genes in eucaryotes are not known. trans-Acting proteins that bind the HSE have been identified and partially purified from D. melanogaster and S. cerevisiae. These proteins, called heat shock activator protein (51-54) or heat shock transcription factor (36, 50), are found in uninduced cells and upon heat shock are converted to an activated form which presumably interacts with other transcription factors to stimulate RNA polymerase II transcription of heat shock genes (36). Howevcr, the exact relationship between the activator protein and the transcription factor remains to be defined. Protein syn.hesis following heat shock is not required to activate this binding activity (24, 54). In addition, although the yeast protein binds the HSEs in the Drosophila hsp70 promoter, it does not activate transcription of the gene in vitro (50) and may require heat-induced phosphorylation to activate transcription (47). We propose that a regulatory pathway for the heat shock response exists that is functionally integrated with other aspects of cell physiology. This heat shock regulatory system must sense an insult, trigger the response after some threshold is reached, and regulate the magnitude of the response as a function of the severity of the stress. It must recognize both temperature jumps, which lead to rapid induction, and other inducers that result in slower induction

Corresponding author. 3423

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FINDLY ET AL.

of the response. To isolate mutations affecting the regulatory pathway, we have begun a genetic analysis of the heat shock response in S. cerevisiae. This approach should eventually identify the components involved in controlling this cellular stress response, including factors that cannot be readily identified by DNA-binding or biochemical assays. transActing mutations that result in constitutive expression of heat shock genes in D. melanogaster have been recently isolated by using a similar genetic approach but a different selection system (37). We describe here a genetic selection designed to identify mutations in genes of the heat shock regulatory pathway. Since the trans-acting factors that bind the HSE, and presumably other regulatory factors, are present in unstressed cells, it is possible that they have multiple functions and that complete inactivation of these proteins would be lethal. Consequently, we developed a selection system in which cells with reduced levels of expression of the heat shock response are isolated. Differential growth on galactose is used as the basis of the selection. As previously shown for other genes in E. coli and S. cerevisiae, selection for growth on galactose can serve as a general positive or negative selection for isolating mutations affecting gene expression (15, 42, 43). An expression vector (pHSG) was constructed in which the promoter from the yeast hsp7O gene, SSAI, is used to express the E. coli galactokinase gene, galK. When expressed, galK complements null mutations in the yeast galactokinase gene (GALl) and permits growth of S. cerevisiae on galactose as the sole carbon source (43). However, excess accumulation of the metabolic product of galactokinase, galactose-1-phosphate, is toxic (10, 40). When S. cerevisiae transformed with the hsp7O-galK expression vector is grown under nonpermissive conditions on galactose, cell stasis or death results. Mutants that survive this negative selection, when compared with the initial wild-type cells, have reduced levels of galactokinase activity following heat shock. We show here that decreased expression of the SSAJ gene fusion following the temperature jump is due to transacting mutations that can either decrease the transcriptional response of the SSAI promoter or increase the posttranscriptional turnover of the fusion transcript. These represent the first class of mutations described in eucaryotes which have been shown to affect, in trans, transcriptional regulation of the heat shock response and stability of heat shock transcripts. We have designated them as HSR (heat shock regulation) mutants.

MATERIALS AND METHODS Construction of plasmids. The S. cerevisiae-E. coli shuttle vector pYSK9-10 was a generous gift of J. Gorman (42). The vector contains an E. coli replication origin and Ampr gene from pBR322 and the TRPI gene, ARSI replication origin, and CEN3 element from S. cerevisiae. The E. coli galactokinase gene (galK) is joined in frame as a translational fusion with the yeast CYCI gene and its 3'-flanking sequences (43). A 1.3-kilobase (kb) fragment of the yeast hsp7O gene, SSAI from the EcoRI site at -1200 to the RsaI site at +90 (amino acid 10), was modified by ligation of a BamHI linker to the RsaI site (46). This fragment, made blunt ended at the EcoRI site by treatment with the Klenow fragment of DNA polymerase I, was ligated into the HpaI and BamHI sites in the linker of pYSK9-10. This results in translational fusion of SSAI at codon 10 through an 18-base-pair linker with codon 5 of galK (see Fig. 1).

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The shuttle vector pZDO-2 has already been described (46). It contains the same EcoRI-BamHI fragment of SSAI ligated into the EcoRI-BamHI site of pMC2010 (5), resulting in translational fusion of codon 10 of SSAI to codon 8 of the lacZ gene. For Northern (RNA) hybridizations, gel-purified fragments of these plasmids were labeled with [32P]dATP by nick translation (30). The probes spanned the EcoRI-BssHII fragment from the galK gene of pHSG and the BamHI-SacI fragment from the lacZ gene of pZDO-2. For detection of transcripts from both the endogenous cellular gene SSAI and the SSAJ-lacZ gene fusion, a 0.6-kb SalI-BamHI fragment of pZDO-2 was used as a probe. This restriction fragment includes sequences for the first 10 amino acids and the 60-base leader of the SSAI transcript (46). Transformation of E. coli HB101 and plasmid DNA isolation from E. coli were done by standard methods. Plasmid DNAs were isolated from S. cerevisiae by suspending cells in 100 mM NaCI-10 mM Tris hydrochloride (pH 8)-i mM EDTA-0.1% sodium dodecyl sulfate. An equal volume of glass beads (0.5-mm diameter) was added, cells were lysed by vortexing, and the sample was extracted with phenol. After centrifugation, the aqueous phase was reextracted with phenol-chloroform and nucleic acids were precipitated with ethanol. Strains, media, and transformation of S. cerevisiae. YM126 (a gall-152 ura3-52 trpl-289) was used for selection of heat shock mutants (20). It contains a deletion in the GAL1 coding sequence at the 5' end of the gene and cannot grow on galactose. Yeast cells were grown on standard media (44). Spheroplasts were transformed as described by Hinnen et al. (16). To select transformants and for growth of strains containing pHSG and pZDO-2, cells were grown on synthetic complete medium lacking tryptophan (SC-trp). Cells in culture were grown to an optical density at 650 nm of between 0.1 and 1.0 for all experiments (in some instances, cell counts were used instead of optical density at 650 nm to determine cell density). HSR mutants were selected on SC-trp-2% galactose plates by isolating single colonies that appeared after 4 to 5 days of growth at 37°C. Cells were tested for growth on galactose on SC-trp plates containing 2% galactose by using galactose containing less than 0.01% glucose (Sigma Chemical Co.). Heat shock and labeling in vivo. The heat shock response was induced by shifting cell cultures from a 23°C water bath to a 55°C water bath for 15 s and then to a 37°C shaking water bath. Cells were kept at 37°C until collected. The shift caused the temperature of the culture to increase to 37°C in less than 1 min. For labeling with [3H]uridine (New England Nuclear Corp.; 49.7 Ci/mmol), cells were grown to an optical density at 650 nm of 0.2 to 0.4 in synthetic minimal/medium (SD) with 0.1% yeast extract (44) and labeled for 10 min with 50 ,uCi of [3H]uridine per ml at 23 or 37°C. Total cell RNA was prepared from 2-ml cultures. RNA isolation and hybridization. Cells were collected by centrifugation, washed in sterile water, and suspended (0.2 ml/107 cells) in 0.5 M NaCI-0.2 M Tris hydrochloride (pH 7.5-10 mM EDTA-1% sodium dodecyl sulfate. To this was added 0.4 g of glass beads and 0.2 ml of phenol-chloroformisoamyl alcohol (25:24:1), and cells were lysed by vortexing for 4 min. The aqueous phase was separated by centrifugation and reextracted with phenol-chloroform, and nucleic acids were precipitated with ethanol. For Northern analysis of RNAs, 5 to 10 p,g of total cell RNA (prestained with ethidium bromide) was separated by electrophoresis on 1.2% agarose gels as described by Rave et

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hsp70 TRANSCRIPTION MUTANTS

al. (41) and transferred to nitrocellulose filters. RNA immobilized on filters was hybridized with 32P-labeled DNA probes in 50% formamide-4x SSC (lx SSC is 0.15 M NaCI plus 15 mM sodium citrate; pH 7.0S5x Denhardt solution0.1 M Tris hydrochloride (pH 7.8)-0.5% sodium dodecyl sulfate-0.1% sodium pyrophosphate-50 p.g of denatured calf thymus DNA per ml (hybridization buffer) for 18 to 30 h at 42 to 45°C. Filters were washed sequentially in 2x SSC, 0.1x SSC and 0.1% sodium dodecyl sulfate at 42°C for 2 h and again in 2x SSC. Autoradiographs of the Northern blots were scanned with a Beckman DU-6 spectrophotometer, and areas under the peaks were used to calculate the relative induction values. Pulse-labeled RNAs were hybridized simultaneously with two single-stranded M13 mp7 probes that contained a 1.8-kb HincII fragment of the lacZ gene in opposite orientations (a generous gift of R. Gourse). Single-stranded M13 DNA was prepared as described by Messing (33) and was loaded onto 24-mm-diameter nitrocellulose filters (2 ,ug per filter). RNA from each time point was suspended in 0.9 ml of hybridization buffer and hybridized with two filters. One filter, as a control for background hybridization, contained M13N07003 DNA, and the other filter contained M13N07004 DNA, which hybridized with the labeled RNA. Filter hybridizations were done at 45°C for 48 to 60 h. Filters were washed in 2x SSC, incubated in 2x SSC-10 ,ug of pancreatic RNase per ml for 30 min at 37°C, rinsed in 2x SSC, dried, and counted. Enzyme assays. Galactokinase activity was measured as previously described (15, 43), with the following modifications. Cells from 1 ml of culture were collected by centrifugation, suspended in 0.5 ml of 40% dimethyl sulfoxide at 4°C by vortexing, incubated at 30°C for 30 min and 4°C for 15 min, collected by centrifugation, washed twice with and then suspended in 0.5 ml of 50 mM Tris (pH 7.8). To assay galactokinase activity, 0.05 ml of the dimethyl sulfoxidetreated cells was mixed with 0.05 ml of the assay mixture containing [14C]galactose, incubated at 30°C for 20 min, and spotted onto DE81 filter disks (Whatman Inc.) which were then washed extensively with water. Radioactivity in duplicate samples from each reaction was determined, and background counts (reaction mixture without dimethyl sulfoxidetreated cells) were subtracted. ,-Galactosidase activity was determined as previously described (11, 34). All reactions were performed on four samples, and the average value was calculated. To

use

RESULTS differential growth on galactose

as a

selection

system, it was necessary to construct a vector in which a heat shock promoter is used to express the galK gene. The structure of the shuttle vector pHSG is shown in Fig. 1. It is derived from pYSK9-10 and contains both yeast and E. coli origins of replication and selectable markers and the yeast CEN3 element, which maintains the vector as a stable,

low-copy-number plasmid (6, 42, 43). Thus, the numbers of hsp7O genes are similar in pHSG-transformed and wild-type cells. The coding region of the E. coli galK gene is joined at its 3' end with coding and flanking sequences of yeast CYCI to allow correct termination in S. cerevisiae. At the 5' end, the galK gene is joined at amino acid 5 as a translational fusion through an 18-base-pair linker sequence to the codon for amino acid 10 of the S. cerevisiae hsp7O gene, SSAI. The SSAI insert ranges from an RsaI site at +90 to an EcoRI site 1.2 kb upstream and includes the promoter region for this gene (46).

3425

A

CYCY

'~galK

pHSG 9.0 kb

pBR 322

HSP 70

(SSA1)

CEN 3

B SSA1

galK

linker

TTA GGTiGGG GAT CCC GAA TTC CAA GAA AAA leu - gly- gly - asp - pro - glu - phe - gin -glu - lys 5 9 10 6

FIG. 1. Galactokinase expression vector pHSG. (A) Upstream flanking sequences, promoter, and 5' coding sequences to amino acid 10 of a yeast hsp7O gene, SSA1, joined as a translational fusion to amino acid 5 of the E. coli galK gene. The transcript of the SSAI-galK gene fusion is shown as a wavy line. (B) Sequence of the junction of SSAI with galK.

Yeast strain YM126 contains a partial deletion of the yeast galactokinase gene (gall) and cannot utilize galactose as a carbon source unless the strain is transformed with a functional galactokinase gene. YM126 cells were transformed with pHSG (pHSG/126), and galactokinase activity was determined before and after heat shock. At 230C, a low level of galactokinase activity was detected, and this activity rapidly increased following heat shock (Table 1). As a control, cells were transformed with the vector pYSK9-10, which lacks the SSAI insert (42). galK activity was not detected in these cells. Expression of pHSG is transcriptionally regulated (Fig. 2). At 23°C, a low level of transcription was observed, but following a shift to 37°C, transcription of the hsp7O-galK gene abruptly increased. Maximum mRNA levels were detected after 10 min, and the intensity of the signal decreased in value at later times. RNA isolated from YM126 cells not transformed with pHSG did not hybridize with this probe (data not shown). Gels were photographed before transfer, and comparison of the intensities of the rRNA bands in the various samples verified that similar amounts of TABLE 1. Induction of galactokinase activity by heat shock Vector, temp (°C), and time (min)

'4C cpm/106 cells'

Induction (37°C/23°C)

pYSK9-10 (no promoter) 23; 0 37;40

63 42

pHSG 23; 0 37; 15 37; 30 37; 60 37;90

198 582

1,141 1,228 1,849

" Values for the pHSG vector are averages of three experiments.

1.0 2.9 5.8 6.2 9.3

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C-)

FIG. 2. Induction of transcription of pHSG by heat shock. Northern blot of whole-cell RNA (5 jig per lane) hybridized with the galK probe. RNA was isolated from pHSG-transformed YM126 cells at 23°C or after a shift to 37°C. The transcript has an estimated size of 1.8 kb when compared with 32P-labeled lambda HindIlI markers (not shown). Lanes: 1, 23°C; 2 to 6, 37°C heat shock for 10, 20, 30, 45, or 60 min, respectively. The figure was overexposed to show the 23°C time point.

(.0

0 1

1000

ClC-)

0

RNA had been loaded on each lane. It was not feasible to use a second gene probe as an internal hybridization standard because transcription of other polymerase II genes decreases during heat shock, eliminating them as stable steadystate transcripts for comparison (13, 27). Isolation of heat shock regulatory mutants. To determine the temperature range at which pHSG/126 cells could grow on galactose, cells were transferred to SC-trp plates containing either glucose or galactose as a carbon source. On glucose plates, pHSG/126 cells grew at 23, 30, or 37°C, but on galactose plates they did not grow at 37°C. The most likely explanation for this pattern of differential growth is that increased transcription of the SSAJ-galK fusion gene at 37°C results in an increase in the steady-state level of galactokinase. For cells growing on galactose, this could lead to accumulation of toxic levels of galactose-1-phosphate. The cells grow well on glucose at 37°C, which rules out other possible temperature-sensitive effects. This hypothesis was tested by measuring the level of galactokinase activity in pHSG/126 cells in which the temperature was raised gradually to 30 or 37°C, since this more accurately reflects growth conditions on plates. A culture at 23°C was split into three, and one flask was kept at 23°C. The others were shifted slowly to 30 or 37°C, and after 4 h galactokinase activity was measured. Compared with the 23°C value, galactokinase activity, corrected for cell density, increased 1.5-fold at 30°C and 2.2-fold at 37°C (data not shown). This suggests that an increase in galactokinase activity of only two- to threefold over the 23°C level is toxic. This implied that cells with reduced galactokinase levels should be able to grow at 37°C on galactose. Spontaneous mutants were selected by culturing pHSG/126 cells on galactose at 37°C and isolating cells that survived and grew. From approximately 106 cells, 16 mutants were isolated. However, 106 represents a minimal estimate of cells screened, since this selection is probably not immediately toxic and the cells may undergo several additional divisions before growth ceases. These mutants were restreaked onto galactose plates and tested for growth at 23, 30, and 37°C. In all cases, they could grow at the three temperatures, although they varied in their rates of growth at 37°C. Three mutants carrying hsr2, hsr3, and hsr5 were picked for further study. To determine whether the mutations affected host functions or were cis acting and segregated with the pHSG vector, the HSR mutants were cured of their plasmids by growth under nonselective conditions and then retransformed with pHSG. All three still grew at 37°C. In addition, the plasmid vectors from each of the three HSR mutants were isolated and transformed into E. coli, and the plasmids were reisolated and again used to transform YM126 cells. In

0

30

60

90

120

Minutes After Temperature Shift FIG. 3. Heat shock induction of the SSAI-galK gene in YM126 and HSR mutants. Galactokinase activity is expressed as 14C counts per minute per 106 cells. Cells were shifted from 23 to 37°C at 0 min, and galactokinase activity was determined at 0 min (23°C) and at 15, 30, 45, 60, 90, and 120 min after the temperature shift. The points are averages of two experiments. Symbols: O, YM126; O, lIsr2: A,

hsr3; 0, hsr5. this case, none of the cells could grow at 37°C on galactose, indicating that trans-acting mutations affecting host functions that regulate expression of an hsp7O gene, SSA1, had been isolated. To determine whether, as predicted, the HSR mutants showed lower levels of activity than do pHSG/126 cells, galactokinase activity was measured following a rapid temperature shift. At 23°C, all had similar levels of activity, but galactokinase activity in the HSR mutants was reduced relative to that of the wild type following a shift to 37°C (Fig. 3). After 120 min at 37°C, 11-fold induction was observed in the pHSG/126 cells, but the mutants strains showed inductions of only 3- to 4-fold. In the HSR mutants, both galactokinase activity and its relative level of induction were lower than in pHSG/126 cells. During the interval examined, galactokinase levels began to plateau after 90 to 120 min at 37°C. In contrast to the enzyme level, mRNA produced by the fusion gene in pHSG/126 cells reached a maximum level after 10 min at 37°C and then began to decrease in abundance (Fig. 2). In all of these experiments, cells were grown on glucose, not galactose, to reduce any effect of galactose metabolism on the measured activity. These data suggest that mutations that decrease expression of the hsp7O gene, SSAI, have been identified. PI-Galactosidase induction by SSAI. To confirm that these mutations act only to regulate expression of the hsp7O gene, a second expression vector, pZDO-2, was also used (46). In this vector, the same SSAI restriction fragment used to construct pHSG was inserted as a translational fusion to the E. coli lacZ gene in the yeast-E. coli shuttle vector pMC2010 (5). This resulted in the fusion of amino acid 10 of hsp7O to amino acid 8 of the ,-galactosidase gene (Fig. 4). In contrast to pHSG, this construct does not contain a specific yeast terminator sequence. Since this enzyme is not naturally found in S. cerevisiae, it measures the activity of the SSAI promoter but with no effect on cellular metabolism. This vector is present at a higher copy number than pHSG, since it contains the 2,um replication origin. The hsr2, hsr3, and hsr5 mutant strains, cured of pHSG, and the wild-type strain YM126 were transformed with pZDO-2. Cells were heat shocked by a rapid shift from 23 to


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A 1 2

3

5

4

6

in0W

7

8

9 10 11 12

0

B 1 2 3 4 5 6 7 8 9 10 11 12

.C

lac Z

1

FIG. 4. P-Galactosidase expression vector pZDO-2. The vector pZD0-2 contains the same SSA I promoter insert as pHSG, joined as a translational fusion to the E. coli lacZ gene. The transcript of the hsp7O-lacZ gene fusion is indicated by a wavy line.

37°C, and ,B-galactosidase activity was measured in cell extracts. The results were similar to those of the galactokinase assays (Fig. 5). In all cases, similar levels of Igalactosidase activity were measured at 23°C, but the activity in the HSR mutants after 4 h at 37°C was significantly lower than that in pZDO-2/126 cells. The relative induction of pHSG/126 was 20-fold after 240 min, but inductions of only 9-fold for hsr2, 3-fold for hsr3, and 7-fold for hsr5 were observed. The measured levels of enzymatic activity in the HSR mutants varied relative to each other in the two assays. In the galactokinase assay, hsr3 exhibited a higher level of activity than hsr2 or hsr5 but showed the least relative change when ,-galactosidase activity was measured. Although replica assays of the same culture were very consistent, measurements obtained from different cultures of the same cell lines were less consistent. This is probably due, in part, to the problem of exact reproducibility of the heat shock response, since small changes in the rate of the 6000

o

5000 -

,s

4000

O C.)/ o 3000

,,2000 1000 0

60 120 180 240 Minutes After Temperature Shift

0

FIG. 5. Heat shock induction of SSAI-lacZ gene in YM126 and HSR mutants. f3-Galactosidase activity before and following temperature shift of cells transformed with pZD0-2. Cells were shifted from 23 to 37°C at 0 min, and 0-galactosidase activity was determined at 0 min (23°C) and at 10, 30, 60, 90, 120, 180, and 240 min after the temperature shift. The points are averages of three experiments. Symbols: O, YM126; O, hsr2; A, hsr3; 0, hsr5.

2

3

4

5

6

7

8

9 10 11 12

Wwwi FIG. 6. Inducible expression of SSAI fusion mRNA in HSR mutants. Northern blots of whole-cell RNA isolated from control or heat-shocked cells. RNAs (5 ,g per lane) isolated from cells transformed with pHSG were run on a single gel, transferred, and hybridized with the galK probe. The autoradiogram was overexposed for 30 h to show expression at 23°C (A) or given a normal 14 h exposure (B). RNA was also isolated from cells transformed with pZDO-2 and hybridized with the lacZ probe (C). The transcript from lacZ has an estimated size of 3 kb. Lanes: 1 to 3, YM126; 4 to 6, hsr2; 7 to 9, hsr3; 10 to 12, hsrS; 1, 4, 7, and 10, control (23°C); 2, 5, 8, and 11, heat shock (37°C) for 10 min; 3, 6, 9, and 12, heat shock for 60 min.

temperature shift and the final temperature reached by the culture affect the extent of the response. It is also possible that plasmid copy number affects these measurements, since pZDO-2 is a 2,um-based vector and pHSG is a CEN3 vector. Nevertheless, it is clear that the HSR mutants showed significantly lower reporter enzyme activities than did YM126 cells following heat shock in either of two different assays. These results confirm the prediction that mutations acting to down regulate expression of heat shock genes could be isolated by galactokinase selection. These mutations act in trans to regulate expression of an hsp7O gene fusion and cause a severalfold decrease in expression of reporter activity following a rapid temperature shift. Northern analysis of HSR mutAnts. To determine whether the reduced expression of the heat shock fusion gene in the HSR mutants was a transcriptional effect, the abundance of the fusion gene mRNA was examined before and after heat shock. hsr2, hsr3, and hsr5 Mutants transformed with pHSG exhibited patterns of transcription different from that of YM126 (Fig. 6). At 23°C, galactokinase mRNA levels in the hsr2 and hsr3 mutants were similar to that of the wild type (Fig. 6, lanes 4 and 7 versus 1), but after 10 min at 37°C, these cells had lower levels of the transcript (lanes 5 and 8 versus 2). After 60 min at 37°C, the hsr2 and hsr3 mutants had barely detectable levels of this transcript, whereas pHSG/126 still showed a significant level of the transcript (lanes 6 and 9 versus 3). The fusion transcript was more abundant at 23°C in the hsr5 mutant than in pHSG/126 or the other two HSR mutants, and following a 10-min heat shock, the hsr5 (lane 11) and pHSG/126 (lane 2) mutants showed similar levels. However, as with the other two mutants, the hsr5 transcript was barely detectable after 60 min at 37°C (lane 12).

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2

3

4

5

a

7

8

TABLE 2. Hybridization of pulse-labeled RNA from SSAI-lacZ gene fusion with lacZ gene % Hybridization'

Cells

FIG. 7. Reduction of inducible expression of endogenous SSA1 mRNA in the hsr3 mutant. Northern blot of whole-cell RNA (10 ,ug per lane) isolated from control or heat-shocked cells and hybridized with a probe complementary to both SSAI-lacZ and SSAI transcripts. Lanes: 1 to 4, pZDO-2/126; 5 to 8, hsr3; 1 and 5, control (23°C); 2 and 6, heat shock (37°C) for 10 min; 3 and 7, heat shock for 30 min; 4 and 8, heat shock for 60 min.

RNA was also isolated from pZDO-2-transformed cells, and the results obtained with the higher-copy-number vector were similar to those obtained with pHSG (Fig. 6). At 23°C, pZDO-2/126 cells and the three HSR mutants all exhibited low levels of transcription. After a shift to 37°C for 10 min, the pZDO-2/126 cells showed a major increase in lacZ mRNA levels, and similar increases were observed in the hsr2 and hsr5 mutants. In contrast, only a minor increase in this transcript was observed in the hsr3 mutant after a 10-min heat shock. The abundance of the fusion transcript decreased to levels similar to that of the 23°C control in the pZDO-2/126 cells following heat shock for 60 min. This differed from pHSG/126 cells, in which a substantial level of the transcript was still observed at 60 min. Although the reasons for this difference in transcript stability between the two vectors are not known, it may be because the lacZ gene in the pZDO-2 vector does not contain a yeast terminator, unlike the pHSG construct. Both the Northern analysis of transcriptional activity and the assays of enzyme activity were designed to detect only the products of the SSAJ gene fusions. The transcripts or hsp7O proteins derived from transcription of endogenous hsp7O genes or cognate genes are not monitored with these assays. To confirm that the HSR mutants affect transcriptional regulation of cellular hsp7O genes and not just the gene fusions, the transcription pattern of the endogenous SSAJ gene was also examined. The hsr3 mutant was used, since of the HSR mutants it exhibited the most difference from YM126. A Northern blot of RNA isolated from YM126 and hsr3 mutant cells transformed with pZDO-2 was hybridized under stringent conditions with a probe that anneals with transcripts from both the SSAJ-lacZ gene fusion and the endogenous SSAI gene. The probe included the region corresponding to the first 10 amino acids and the 60-base untranslated leader of SSAI plus additional noncoding upstream sequences. In addition, the probe can react with transcripts from other hsp7O genes, SSA2 in particular, since the coding regions for the first 10 amino acids of these hsp7O genes are highly conserved (M. R. Slater, Ph.D. thesis, University of Wisconsin, Madison, 1986). Two bands were visible (Fig. 7); the higher-molecular-weight band corresponds to the transcript from the SSAI-lacZ gene fusion, and the lower-molecular-weight band corresponds to the SSAI transcript. Comparison of the hybridization signals from the two transcripts revealed that the upper band was more intense, as expected, since it arose from a multicopy vector. Most importantly, Fig. 7 demonstrates that the transcription pattern of the endogenous SSAI gene is like that of the SSAI-lacZ gene fusion. In the hsr3 mutant after 10 min at 37°C, only slight increases in transcript abundance were seen for both the SSAJ and SSAI-lacZ transcripts compared with the significant increases observed in YM126. In the control

YM126 hsr2 mutant hsr3 mutant hsr5 mutant

37C heat shock for:

23°C pre-heat shock control'

10 min

30 min

60 min

0.10 0.06 0.03 0.08

0.61 0.59 0.09 0.47

0.09 0.12 0.07 0.07

0.15 0.11 0.04 0.07

a Values are averages of two experiments. Percentages of total counts in a 10-min pulse-label that hybridized with the lacZ gene are shown. The background counts retained on the filter containing noncomplementary probe DNA were subtracted from the counts resulting from hybridization to the lacZ gene. These values were then multiplied by 1.67, since the probe contains only 1.8 kb of the 3.0-kb lacZ gene (23). Incorporation of [3H4uridine during the 10-min label was as follows: control, 2 x 1io to 1.1 x 106 cpm; 10 min, 3 x 105 to 7 x 105 cpm; 30 min, 0.3 x 105 to 0.9 x 105 cpm; 60 min, 1 x 105 to 3 x 105 cpm. b Cells were labeled with 50 pLCi of [3H]uridine per ml for 10 min. Control cells were labeled for 10 min just before the shift to 37°C, and heat-shocked cells were labeled from 0 to 10, 20 to 30, and 50 to 60 min after the temperature shift.

(Fig. 7, lane 1) of YM126, the band whose mobility fell between the SSAJ-lacZ and SSAI transcripts presumably represents cross-hybridization with the transcript from another unidentified gene of the hsp7O gene family. These results demonstrate that the hsr3 mutation acts not only on the gene fusion but on the cellular SSAJ gene as well and affects regulation of this endogenous heat shock gene. The data demonstrate that the HSR mutants had altered levels of fusion gene transcripts after heat shock when compared with wild-type cells. The hsr2 and hsr5 mutants displayed little difference from the control in induction but appeared to degrade the fusion transcript more rapidly. The hsr3 mutant, in addition to displaying more rapid turnover of the transcript, like the other two mutants, also showed significantly less induction of both the fusion transcript and the endogenous SSAI gene after heat shock than did wildtype cells. These data demonstrate the relative abundance of the transcripts from the SSAJ gene fusions or the SSAI gene at these times but do not indicate whether the altered patterns observed in the HSR mutants relative to YM126 represent changes in rates of synthesis or degradation (or both) of these transcripts. Transcription rates and turnover. To examine these questions, pZD0-2-transformed wild-type and HSR mutant cells were pulse-labeled for 10 min with [3H]uridine before and at several times after a temperature shift to 37°C to determine the rate of mRNA synthesis from the hsp7O-p-galactosidase gene fusion. Total 3H-labeled RNA was isolated from these cells and hybridized with single-stranded M13 DNA probes containing the complementary strands of a 1.8-kb region of the lacZ gene. Incorporation of [3H]uridine by the HSR mutants and YM126 was similar, and none of the mutants showed significantly less incorporation. For each cell line, incorporation of [3H]uridine during the first 10 min at 37°C was similar to the value obtained at 23°C but then decreased for 20 to 30 min after the shift. After 50 to 60 min at 37°C, incorporation rose slightly but did not return to the original values. For YM126 cells, a sixfold increase in transcription of the fusion gene was observed in the first 10 min after a shift from 23 to 37°C, and 0.6% of pulse-labeled RNA represented transcripts from the hsp7O-lacZ gene (Table 2). Transcrip-

VOL. 8, 1988

tion from this gene then decreased to pre-heat shock rates at 30 and 60 min after heat shock, although the cells remained at 37°C. For pZDO-2-transformed YM126, the increase in transcription following heat shock, measured by pulse-labeling or on Northern blots, was similar. Northern blots of RNA isolated from pZDO-2/126 cells at 23°C and after 10 min at 37°C were hybridized with the 3-galactosidase gene probe, and the autoradiograms were then scanned with a densitometer to determine the relative changes in mRNA levels. For several different RNA preparations, this demonstrated, on average, a 10-fold increase in transcription levels (e.g. Fig. 6). This value agrees well with the sixfold increase observed by pulse-labeling. The HSR mutants fall into two classes when their transcription patterns are compared with that of YM126. In the first class (hsr2 and hsrS), the pattern resembles that of YM126. The second class (hsr3) shows the most difference, and the measured rates of synthesis are substantially lower than that of YM126. These data clearly indicate that, following induction by heat shock, there is a smaller increase in transcription from the fusion gene in the hsr3 mutant than in YM126. This decrease is similar to that observed with Northern blots (Fig. 6). In addition, the data suggest that the fusion transcript has an increased rate of turnover in HSR mutants. In the hsr2 and hsrS mutants, the rates of synthesis were similar to that of the wild-type. However, Northern blots of RNA isolated from the hsr2 and hsr5 mutants (Fig. 6) demonstrated that after 60 min at 37°C the transcript was less abundant than in pre-heat shock cells, whereas in YM126 the transcript was still present at levels significantly higher than in 23°C cells. In the hsr3 mutants which have a reduced level of transcription, the fusion transcript was also degraded more rapidly. In summary, these data demonstrate that in the HSR mutants regulatory mechanisms controlling both the level of transcription and the rate of mRNA turnover have been affected for the hsp7O gene fusion and the endogenous SSAI gene. DISCUSSION Among the least understood aspects of the heat shock response are the mechanisms by which transcription of heat shock genes is rapidly induced following a temperature jump. In bacteria, the htpR gene encodes c32, which is a specific transcription factor for heat shock genes (14, 49). Nevertheless, how cJ2 itself is induced or activated in response to stress is unclear. It appears to be an unstable protein whose half-life is transiently extended upon heat shock, but whether this is due to modification of the protein or regulation of specific proteases is not known (49). In eucaryotes, the heat shock activator protein or transcription factor that interacts with the HSE must be an integral part of the induction mechanism. These proteins are found in nonheat-shocked cells, and upon transcriptional activation of heat shock genes by temperature jump they become stably, although reversibly, activated (47, 50, 54). The extent, if any, to which these factors may be homologous with E. coli cr32 is not known. In eucaryotes, a major obstacle to our further understanding of the induction mechanisms has been the paucity of mutations affecting the response at the transcriptional level. If a regulatory pathway exists that controls the induction of the heat shock response, then it should be possible to identify and isolate mutations affecting induction of the response. We report here a selection that has allowed identification of transcriptional mutants in S. cerevisiae. The


3429

selection utilizes growth inhibition on galactose of yeast cells that carry a galactokinase gene fusion to the promoter for the hsp7O gene, SSAJ, and has yielded mutants that exhibit reduced induction of the fusion gene product. These down mutations appear to act in trans in two ways to control the abundance of the mRNA transcripts from SSAI. They can decrease transcription of the gene, and they may also act posttranscriptionally by increasing turnover of its mRNA. The mutants were initially selected on galactose, but after selection the cells were grown on glucose to eliminate any possible effects of galactose metabolism on expression of the fusion gene. Measurement of galactokinase activity in these cells before and after induction by temperature jump demonstrated that the HSR mutants displayed less activity of this enzyme than did wild-type cells. The lowered galactokinase activity resulted from reduced levels of its mRNA. Although enzyme activity remained stable in HSR mutants for at least 2 h, the fusion gene mRNA returned to preinduced levels within 30 to 60 min. To confirm that these mutations affected regulation of the promoter of the SSAJ gene and were neither promoter mutations nor related to galactose metabolism, the HSR mutants were cured of the pHSG vector and retransformed with a second vector, pZDO-2. This vector utilizes the same SSAJ promoter to express the E. coli lacZ gene. P-Galactosidase does not occur naturally in S. cerevisiae and acts only as a reporter enzyme. SSAI-lacZ mRNA levels and ,Bgalactosidase activity in the HSR mutants were similar to those determined for galK expression. These results demonstrated that the mutations acted in trans to regulate expression of an hsp7O gene SSAJ. Mutations affecting the heat shock response have been described in a variety of organisms. In D. melanogaster, mutants have been isolated that lower the temperature at which heat shock proteins are expressed (3) and which result in constitutive expression of the heat shock response in a tissue-specific manner (37). The Drosophila mutants that show constitutive expression may be affected at the transcriptional level, although this has not been demonstrated. Cells that fail to become thermotolerant have been isolated in Dictyostelium discoideum (29) and Tetrahymena thermophila (26). Finally, in S. cerevisiae several mutations affecting the synthesis of heat shock proteins and acquisition of thermotolerance have been isolated (18, 45). The HSR mutants reported here are the first mutations that have been shown directly to affect, in trans, the transcriptional expression of a heat shock gene in eucaryotic cells. The HSR mutants did not appear to be deleterious to the general viability of the cells, because the mutants grew on glucose as well as did the wild-type cells at 23 or 30°C or after heat shock at 37°C. This demonstrates that the HSR mutations are specific for the heat shock response and do not affect general transcription or cell viability. In the hsr3 mutant, it is clear that following heat shock, transcription of both the SSAI fusion gene carried on the vector and the endogenous SSAJ gene is coordinately down regulated when compared with the wild type. It is not possible on the basis of these data to eliminate an increase in turnover of the SSAI transcript as the cause, but we prefer the explanation that the hsr3 mutation acts to down regulate the transcriptional induction of the SSAI gene following induction of the heat shock response. This mutation presumably acts through the upstream HSE of the SSAI promoter, but other sequences contained within the large upstream region of the SSAJ promoter used in these experiments could also be involved. It is not known whether the mutations affect a single gene

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FINDLY ET AL.MOL. BIOL. MOL. CELL. CELL. BIOL.

or represent different complementation groups. The results obtained from the Northern blots and pulse-labeling experiments suggest that the mutants fall into two classes of altered transcription patterns, suggesting the possibility of several complementation groups. The hsr2 and hsr5 mutants display transcription patterns similar to that of YM126 but degrade the fusion transcript faster than does wild type. However, the hsr3 mutant exhibits both a lower initiation and a higher turnover rate than do wild-type cells. Preliminary results obtained from mating pHSG-transformed mutants with cells also carrying a gall deletion demonstrated that all of the diploids grew on galactose at 35 to 37°C. However, the wild-type diploid derived from pHSG/126 did not grow at 37°C on galactose, which suggests that some of these HSR mutations may be dominant (R.C.F., unpublished data). It is possible that some of these mutations affect the heat shock activator protein or transcription factors that have already been described (50, 53). However, they may also represent mutations in unrecognized regulatory factors. The changes in posttranscriptional regulation affect two transcripts, both of which are derived primarily from unrelated E. coli genes. However, the fusion transcripts share in common at their 5' ends the 60-nucleotide untranslated leader and the sequence encoding the first 10 amino acids of SSAI. It is likely that this region serves as the recognition site for controlling transcript stability. In D. melanogaster, the untranslated leader of hsp7O mRNA directs the selective translation of the mRNA during heat shock (25, 31). In addition, the 5' region of histone mRNAs functions to regulate their stability with respect to the cell cycle (35). The differences in phenotype exhibited by these mutants compared with wild-type YM126 cells should allow the regulatory genes to be cloned by using growth on galactose at 37°C for selection. Such analysis, coupled with the identification of regulatory mutants selected in this same manner but using other inducers of the response, should lead to a general understanding of the mechanisms that control induction of the heat shock response. ACKNOWLEDGMENTS We thank Michael R. Slater for the SSA1 promoter and pZDO-2 and S. V. Derbyshire for assistance with the construction of pHSG. This work was supported in part by grants from the National Science Foundation to R.C.F. (DCB-8504116) and T.P. (PCM8411035 and DCB-8542181). LITERATURE CITED 1. Bardwell, J. C. A., and E. A. Craig. 1984. Major heat shock gene of Drosophila and the Escherichia coli heat inducible dnaK gene are homologous. Proc. Natl. Acad. Sci. USA 79:525-529. 2. Bienz, M., and H. R. B. Pelham. 1986. Heat shock regulatory elements function as an inducible enhancer in the Xenopus hsp7O gene and when linked to a heterologous promoter. Cell 45:753-760. 3. Bonner, J. J., C. Parks, J. Parker-Thornburg, M. Martin, and H. R. B. Pelham. 1984. The use of promoter fusions in Drosophila genetics: isolation of mutations affecting the heat shock response. Cell 37:979-991. 4. Brazzell, C., and T. D. Ingolia. 1984. Stimuli that induce a yeast heat shock gene fused to P-galactosidase. Mol. Cell. Biol. 4: 2573-2579. 5. Casadaban, M. J., A. Martinez-Arias, S. K. Shapira, and J. Chou. 1983. ,-Galactosidase gene fusions for analyzing gene expression in Escherichia coli and yeast. Methods Enzymol. 100:293-308. 6. Clarke, L., and J. Carbon. 1980. Isolation of a yeast centromere and construction of functional small circular chromosomes. Nature (London) 287:504-509.

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