Vol. 11, No. 3
MOLECULAR AND CELLULAR BIOLOGY, Mar. 1991, p. 1232-1238 0270-7306/91/031232-07$02.00/0 Copyright X 1991, American Society for Microbiology
Heat Shock Transcription Factor Activates Transcription of the Yeast Metallothionein Gene PHILIPPE SILAR, GERALDINE BUTLER, AND DENNIS J. THIELE* Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606 Received 12 September 1990/Accepted 3 December 1990
In the yeast Saccharomyces cerevisiae, transcription of the metaliothionein gene CUP) is induced by copper and silver. Strains with a complete deletion of the ACE) gene, the copper-dependent activator of CUP) transcription, are hypersensitive to copper. These strains have a low but significant basal level of CUP) transcription. To identify genes which mediate basal transcription of CUP) or which activate CUP) in response to other stimuli, we isolated an extragenic suppressor of an acel deletion. We demonstrate that a single amino acid substitution in the heat shock transcription factor (HSF) DNA-binding domain dramatically enhances CUP) transcription while reducing transcription of the SSA3 gene, a member of the yeast hsp7O gene family. These results indicate that yeast metallothionein transcription is under HSF control and that metallothionein biosynthesis is important in response to heat shock stress. Furthermore, our results suggest that HSF may modulate the magnitude of individual heat shock gene transcription by subtle differences in its interaction with heat shock elements and that a single-amino-acid change can dramatically alter the activity of the factor for different target genes.
Metallothioneins (MTs) are small, cysteine-rich, metalbinding proteins involved in heavy-metal homeostasis and detoxification (reviewed in reference 10). In higher eukaryotes, MTs are encoded by a multigenic family and their synthesis is controlled at the transcriptional level by several stimuli including heavy metals (zinc, cadmium, copper), glucocorticoids, phorbol esters and interleukins. Higher eukaryotic MT promoters have a correspondingly complex arrangement of distinct and interdigitated cis-acting DNA sequences through which these transcriptional responses are mediated. The multitude of effectors of MT transcription in these systems suggests that a number of physiological conditions require increased MT biosynthesis. The complexity of these systems has also hindered studies of MT transcriptional control due to individual stimuli. The baker's yeast Saccharomyces cerevisiae contains a single MT gene, designated CUP], which shares many of the basic features of MT genes in higher eukaryotes (6). Genetic studies have clearly demonstrated that the CUP) gene is essential to prevent copper toxicity (11). Copper and silver are the only reported effectors of CUP) transcriptional induction (9). Metal-inducible transcription of CUP) is mediated through the interaction of the metal-activated DNAbinding protein, ACE1 (CUP2), with cis-acting metal-responsive sequences localized in the CUP) promoter region and called UASCup, (CUP) upstream activation sequences [8, 13, 14, 24, 25, 27]). Yeast strains harboring either a point mutation in the ACE) gene (4, 24) or a complete deletion of the ACE] gene (5) are hypersensitive to copper as a result of a lack of copper-inducible transcription of CUP). To identify other genes involved in transcriptional activation of CUP) in response to other environmental stimuli, we isolated a strain bearing a mutation which suppresses the requirement for ACE) in the activation of CUP) transcription. In this report, we present the isolation and characterization of one such suppressor, which is a mutant form of the heat shock transcription factor, HSF (22, 29). *
Corresponding author.
MATERIALS AND METHODS
Strains and growth conditions. The yeast strains used are as follows: DTY60 (MATot his6 leu2-3,112 LEU2::YipCL ura3-52 CUPJR-3 aceJ-A225 HSF) (5), DTY68 (MATot his6 leu2-3,112 LEU2::YipCL ura3-52 CUPJR-3 ace)-A&225 ADS) for the original ADS isolate, see text below), DTY69 (MATot his6 leu2-3,112 ade LEU2::YipCL ura3-52 CUPIR-3 acelA225 HSF) and DTY90 (MATa his6 leu2-3,112 LEU2::YipCL ura3-52 CUPIR-3 aceJ-A225 ADS) (see text). HSF refers to the wild-type heat shock transcription factor gene, and ADS refers to the mutant allele of the heat shock factor described in this report. The acel-A225 allele is a null allele (5). Strain DTY60 fails to grow on media containing copper sulfate at a concentration higher than 15 ,uM. All yeast strains were grown in rich (YPD) medium or synthetic complete (SC) medium lacking nutrients as specified (20) at 30°C except during heat shock induction experiments. Heat shock inductions were carried out as follows. Cells were grown at 23°C to an optical density of 0.6 and then rapidly shifted to 39°C for the times indicated in the figure legends. Copper was added in the form of copper sulfate. 5-Bromo-4-chloro-3-indolyl-P-Dgalactopyranoside (X-Gal) plates were prepared as described previously (15). Escherichia coli DHSa F' and XL-1 blue were used in all DNA manipulations and were grown under standard conditions (15). DNA manipulation and analysis. Standard protocols were used for all DNA manipulations and cell transformations (15). For the recovery of the ADS allele, DNA from S. cerevisiae DTY68 was isolated (20) and subsequently partially digested with Sau3AI and ligated into the BamHI site of shuttle vector pEMBL Ye24 (2). pEMBL Ye24 is a high-copy (2,um) vector that carries the URA3 marker. The ligation reaction was directly transformed into S. cerevisiae DTY60, and URA+ transformants were selected. The bank was estimated to contain three haploid genome equivalents on the basis of the average insert size and the number of independent transformants obtained. DNA fragments were subcloned into Bluescript vectors (Stratagene) and sequenced by using the Sequenase kit 1232
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(United States Biochemical Corp.). The universal primer as well as heat shock factor gene-specific primers were used. RNA and protein analysis. Primer extension reactions were carried out as described previously (23). Gel retardation experiments were performed as follows. Proteins were extracted from yeast strains as described previously (7). DNA probes were end labeled with Klenow fragment of DNA polymerase I and [a-32]dATP or [a-32P]dCTP. Binding assays were performed by mixing 25 ,Lg of protein from the extract and 0.5 ng of labeled probe (5,000 dpm) in 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 8.0)-30 mM KCI-2 mM dithiothreitol-10% glycerol. After a 10-min incubation at room temperature, samples were analyzed by electrophoresis on a 5% native polyacrylamide gel. Gels were exposed to Kodak XAR-5 film overnight at -70°C with intensifying screens. Antiserum neutralization DNA-binding assays were carried out by preincubating extracts with either rabbit polyclonal antiHSF or preimmune serum (a kind gift of J. Jose Bonner) for 5 min at room temperature and then adding the radioactive probe fragment. After a 10-min incubation at room temperature, the binding reactions were analyzed by gel electrophoresis as described above. RESULTS Isolation and characterization of the ADS mutation. To isolate and identify yeast strains capable of activating CUP) transcription in the absence of ACE1, we used a positive selection scheme. Strain DTY60 contains the ura3-52 mutation, three copies of the CUP) gene, a CUPI-lacZ fusion gene integrated at leu2, and a compete deletion of the ACE] gene (5). CUP) transcription is not significantly inducible by copper ions in this background; therefore, this strain is unable to grow on media containing more than 15 ,uM copper sulfate. Spontaneous suppressors of the copper-sensitive phenotype were isolated by plating approximately 1010 cells from strain DTY60 on SC medium containing 150 ,uM copper sulfate. After a 1-week incubation at 30°C, 29 copperresistant isolates were recovered. To select suppressors which were trans-activators of CUP) expression, we patched copper-resistant isolates onto SC medium containing X-Gal, the chromogenic substrate for 3-galactosidase. One isolate, denoted DTY68, which was resistant to up to 300 ,uM copper sulfate and which expressed a high level of ,-galactosidase from the chromosomal CUPI-lacZ fusion, was selected for further study. The suppressor mutation in DTY68 was designated ADS (ace) deletion suppressor). The steady-state levels of CUP) mRNA were analyzed by primer extension in the wild-type starting strain DTY60 and strain DTY68 harboring the ADS mutation. The CUP) mRNA level was elevated in DTY68 at least 10-fold, as estimated by laser densitometry, over the level in strain DTY60 (Fig. 1A). The levels of the internal control RNA, derived from the alcohol dehydrogenase gene (ADHI), were similar in both strains. The steady-state level of CUP) mRNA in strain DTY68 was not further increased by exposure of these cells to several different concentrations of copper sulfate (data not shown). To determine whether the ADS mutation affects the binding of yeast proteins to the CUP) promoter, we carried out mobility shift assays with extracts prepared from strains DTY60 and DTY68. A large promoter fragment from -241 to +37 was used as a probe in these experiments. Extracts from DTY68 promoted the formation of a large heterogeneous complex with the CUP) promoter (Fig. 1B). This
HEAT SHOCK ACTIVATES CUP] TRANSCRIPTION
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FIG. 1. Effect of the ADS mutation on CUPI transcription. (A) Total RNA (15 ,ug) from either strain DTY60 (WT) or strain DTY68 (ADS mutant) was used in extension reactions with radiolabeled primers complementary to CUP] and ADHI mRNA. The amount of mRNA in each reaction does not vary, as shown by the control ADHI extension product. CUP) mRNA levels are dramatically increased in the ADS mutant (lane ADS) compared with the wild type (lane WT). (B) Proteins from strains DTY60 (WT) and DTY68 (ADS) were mixed with a radiolabeled CUPI promoter probe (from positions -241 to +37 numbered with respect to the transcription start) and analyzed by electrophoresis on a 5% polyacrylamide gel. Extract from the ADS mutant strain promotes the formation of a large heterogeneous complex (lane ADS). This complex is barely detectable with extract from the wild-type strain (lane WT). Free probe (without extract) is shown as a control (lane probe). Although the nature of the minor band in the free-probe lane is unknown, this appears upon repeated polyacrylamide gel purification of the probe fragment and is believed to be an altered form of the free probe. Abbreviations: c, complex; p, free probe.
complex could not be detected with extracts from strain DTY60 under these conditions. The ADS suppressor mutation was genetically analyzed by crossing to the tester strain DTY69. ADS is a semidominant mutation, since the heterozygous diploid strain has an intermediate level of copper resistance (50 ,uM) and exhibits ,-galactosidase activity between the haploid wild-type DTY69 and the haploid ADS mutant DTY68. Analysis of spores from 13 complete tetrads revealed that the copper resistance phenotype segregated 2:2 through meiosis. Cosegregation of the copper resistance, the high level of P-galactosidase activity, and DNA-protein complex formation were observed in all tetrads analyzed. These data indicate that the ADS mutation resides in a single nuclear locus and that this mutation activates expression from the CUP) promoter in trans. Isolation and analysis of the ADS allele. Based on the semidominant nature of the ADS mutation, a genomic bank
derived from the ADS mutant strain (DTY68) was constructed in the high-copy vector pEMBL Ye24 (2) and transformed into the wild-type copper-sensitive strain, DTY60. One copper-resistant transformant expressing a high level of ,-galactosidase was obtained. The plasmid retrieved from this transformant contained an insert of
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approximately 3 kb (Fig. 2A, pADSb). Strain DTY60 transformed with this plasmid was resistant to copper concentrations up to 300 ,uM. To analyze the ADS allele, the nucleotide sequence of the pADSb insert was determined. A search of the GenBank and EMBL data bases revealed that this insert represents the promoter and 590 amino-terminal codons (out of a total of 833) of the heat shock transcription factor (HSF), a transcription factor involved in the activation of the yeast heat shock gene families (22, 29). To verify the authenticity of the cloned gene, we integrated a URA3 marker adjacent to the wild-type HSF locus and analyzed the linkage with the ADS allele. Analysis of 10 complete tetrads revealed no recombination between URA3 and ADS, demonstrating that the ADS mutation is closely linked to the HSF locus. The complete wild-type allele from DTY60 and the ADS allele from DTY68 were recovered by integration via homologous recombination and excision (31) and used for the experiments described below. To determine whether the ADS allele product was a component of the DNA-protein complex observed in DTY68 extracts (Fig. 1B), we carried out mobility shift assays with extracts from strain DTY60 transformed with pADS8. In the mobility shift assays, the protein-DNA complex formed with the extract from strain DTY60 transformed with pADS8 migrated faster than the complex formed with extract from strain DTY68 (Fig. 2B), as expected from the size difference between the truncated protein encoded by the pADSB insert and the full-length protein encoded by the chromosomal ADS allele. This strongly suggests that the protein encoded by the ADS allele is present in the DNA-protein complex observed in Fig. 1B. The sequence of the pADS5 insert revealed a single-base change between the ADS allele and the published wild-type sequences of the HSF gene (22, 29). This mutation changes a valine codon to an alanine codon in the DNA-binding domain of HSF (29) at position 203 (Fig. 2A). It also destroys a MaeIII site present in the DNA sequence of the wild-type HSF gene (22, 29) as determined by genomic DNA blotting experiments (data not shown). The valine-to-alanine mutation is not a polymorphism since it is not present in the wild-type gene isolated from DTY60. Furthermore, transformation of strain DTY60 with a linear DNA fragment from the ADS allele encompassing this mutation (from BamHI to the distal NruI site in Fig. 2A) allowed the recovery of chromosomal recombinant isolates exhibiting all ADS phenotypes described above. One of these transformant clones, DTY90, was used in the experiments described below. Effects of the wild-type and ADS alleles on the CUP) promoter. To determine whether the wild-type HSF gene plays a role in regulation of CUP) transcription, the entire ADS mutant and wild-type HSF gene were separately introduced into strain DTY60 on a high-copy vector. Under standard growth conditions at 30°C, both constructs conferred an increased resistance to copper (Fig. 2A). The wild-type HSF gene allowed growth of this strain on medium containing 100 ,uM CuS04, and the ADS allele allowed growth on up to 500 puM CUSO4. These values represent approximately 6- and 33-fold increases in copper resistance conferred by multiple copies of HSF and ADS, respectively. This result indicates that both the wild-type and ADS mutant alleles activate transcription from the CUP) promoter. Mobility shift experiments with the CUP) promoter fragment confirmed this hypothesis (Fig. 2C). As demonstrated in Fig. 1B, we observed no detectable complex with the CUP) promoter fragment when using extracts from the wild-type strain (lane WT); however, when using extracts from the
MOL. CELL. BIOL.
ADS mutant strain, we observed a complex indistinguishable from that in Fig. 1B (lane ADS). We detected the large heterogeneous complex, previously observed with extracts from the ADS mutant strain DTY68, with extract from cells containing the wild-type allele of HSF placed on a high-copy vector (Fig. 2C, lane WT/HC). As expected, we detected a dramatically increased level of a complex with the same mobility when using extracts from a strain containing the ADS allele on a high-copy plasmid (Fig. 2C, lane ADS/HC). To determine whether CUP) expression is induced in response to heat shock, we performed primer extension experiments on RNA extracted from DTY60 and DTY90 cells before and after heat shock treatment (Fig. 2D). CUP) mRNA levels increased during the heat shock, with the maximum level reached approximately 20 min after the heat shock. Both the wild-type and ADS alleles were able to induce this heat shock response, with mRNA levels correspondingly greater in the ADS allele background. No changes in ADHI mRNA were observed in response to the heat shock treatment. Although the CUP) heat shock induction was not large, this induction is reproducible in several experiments. Furthermore, several previously characterized heat shock genes exhibit a small transcriptional stimulation in response to heat shock (28). Competition experiments with a gel retardation assay demonstrated that the large complex formed with extracts from cells containing the wild-type allele of HSF in a high-copy vector is inhibited by an oligonucleotide encompassing the two strong ACE)-binding sites in the CUP) promoter (8, 13, 14). However, an oligonucleotide adjacent to this binding site failed to compete for formation of this complex (Fig. 3A). Qualitatively similar results were obtained when extracts from the ADS mutant strains DTY68 and DTY90 were used in competition experiments (data not shown). These experiments demonstrate that CUP) is a heat shock-responsive gene recognized by HSF and that HSF binds within UASCupJ. Indeed, within the region of UASCup, covered by the competitor oligonucleotide, sequences similar (but not identical) to the consensus heat shock element (HSE) are present (Fig. 3B). However, another potential binding site can be postulated around position -170, because several GAA repeats can also be found in this region. Effect of the ADS mutation on heat shock gene expression. Heat shock genes are involved in a number of important cellular processes including thermotolerance, protein folding, translocation, and degradation (19). To determine the potential effect of the ADS mutation on the expression of genes important for growth at elevated temperatures, we compared the thermosensitivity of strains DTY60 and DTY90. These strains exhibited doubling times of 3.7 and 6.6 h, respectively, after a log phase shift from 23 to 39°C, suggesting that the mutant ADS protein may not efficiently activate transcription of one or more of the heat shock genes involved in optimal growth at 39°C. We then examined the effect of the ADS allele on the expression of the heat shock gene SSA3, a member of the hsp7O family known to be dramatically induced by heat shock (3, 28). Primer extension experiments (Fig. 4A) demonstrated that SSA3 is not fully induced in the ADS mutant background compared with the wild-type background. Indeed, we observed only a slight induction of SSA3 mRNA levels over the time course of heat shock stress with DTY90 cells compared with strain DTY60. These results indicate that the protein encoded by the ADS allele has a dramatically reduced ability to activate SSA3 transcription after heat shock stress. Because we mapped
HEAT SHOCK ACTIVATES CUP] TRANSCRIPTION
VOL. 11, 1991
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FIG. 2. Heat shock transcription factor activates CUP] expression. (A) Molecular organization of the ADS and wild-type alleles of the HSF gene (22, 29). The arrow indicates the transcription start. Abbreviations: B, BamHI; E, EcoRI; N, NruI; S, Sau3AI. Not all of the Sau3AI sites are shown; the size of the complete wild-type and ADS fragment is 3.95 kb. The clone pADSS is a 3' truncation of the ADS allele DNA fragment (retaining 1 kb of the promoter region and 590 amino-terminal codons of the coding sequence). The sequence analysis revealed a single nucleotide change, transforming a valine (GTC) to an alanine (GCC) at amino acid position 203 in the DNA-binding domain of the protein. Resistance levels of strain DTY60 containing the indicated inserts in the high-copy vector pEMBL Ye24 were measured by growth on SC containing a range of copper sulfate concentrations. Strain DTY60 transformed with the control plasmid pEMBL Ye24 failed to grow on media containing more than 15 ,uM copper sulfate. (B) Protein extracts from strains DTY60 (lane WT), DTY68 (lane ADS), and DTY60 transformed with pADSB (lane pADS8) were analyzed for the formation of a complex with the CUP] promoter as in Fig. 1B, except that only 12.5 ,ig of pADSB extract was added. The complex formed with extract from DTY60 transformed with pADSB (complex ci) migrated faster than the complex formed with DTY68 (complex c2). The origin of the other DNA-protein complexes seen in this experiment is unknown. Abbreviations: c, complex; p, free probe. (C) Proteins were extracted and analyzed as described in the legend to Fig. 1B. With a single genomic copy of a wild-type HSF gene, extract from strain DTY60 (lane WT) is unable to readily promote the formation of the large complex visible here with extract from strain DTY90, which contains the ADS allele as a single genomic copy (lane ADS). However, transformation of strain DTY60 with the HSF wild-type fragment on a high-copy vector (lane WT/HC) allows the detection of this complex. A corresponding increase in complex formation is observed in extracts from strain DTY60 transformed with the ADS allele in a high-copy vector (lane ADS/HC). (D) Strains DTY60 (WT) and DTY90 (ADS) were grown at 23°C (optical density at 650 nm, 0.6) and subjected to heat shock at 39°C for 10, 20, and 30 min. RNA was extracted at each time point and analyzed by primer extension as in Fig. 1A. In ADS or wild-type backgrounds, heat shock results in an increase in CUP] mRNA levels with no alteration in ADHI mRNA levels. Lanes: 0, before heat shock; 10, 20, and 30, time after heat shock (in minutes).
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the mutation in the ADS allele to a single-amino-acid substitution in the HSF DNA-binding domain, we carried out experiments to determine whether this reduction in SSA3 gene activation in the ADS background might be due to an alteration in the interaction of the ADS protein with the heat shock elements in the SSA3 promoter. Figure 4B shows the results of mobility shift assays with a fragment from the SSA3 promoter which encompasses functional HSEs (3). We observed several protein-DNA complexes, including a large complex detectable only in the strain containing the HSF or ADS allele on a high-copy vector (Fig. 4B, lanes hc/WT and hc/ADS). To confirm that this large complex was specifically due to binding of the HSF or ADS protein on the SSA3 promoter fragment, we preincubated the extract derived from the strain carrying HSF on a high-copy vector with anti-HSF antiserum prior to DNA binding. Under these conditions only the large complex disappeared (lane WT/I). The complex was present when the preimmune serum was used in the binding reaction (lane WT/P). Similar results were obtained with extracts from the strain bearing the ADS allele on a high-copy vector. These results clearly demonstrate that the large complex observed in these experiments is due to the binding of the HSF or ADS protein on the SSA3 promoter fragment. This complex is reduced with extracts from the ADS mutant (lane hc/ADS) compared with the wild-type allele (lane hc/WT). This correlates with the decreased transcriptional activation of the SSA3 gene observed in Fig. 4A. This suggests that the protein encoded by the ADS allele fails to fully activate SSA3 transcription as a result of a decreased ability to form stable complexes with the HSEs in the SSA3 promoter. Therefore, the wild-type and ADS proteins have opposite effects on CUP] and SSA3 transcription, owing, at least in part, to differences in DNAprotein interactions.
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FIG. 3. Identification of the HSF-binding region within the CUP] promoter. (A) Protein extract from strain DTY60 transformed with the wild-type HSF sequence on a high-copy vector was analyzed by mobility shift assays as in Fig. 2C (lane 1). The binding was also performed in the presence of 100 ng of unlabeled doublestranded oligonucleotide derived from the CUP] promoter sequence from -112 to -91 (lane 2) or from -141 to -107 (lane 3). Abbreviations: c, complex; p, free probe. (B) CUPJ promoter sequence from -210 to -91. Nucleotides previously observed to be protected from DNase I digestion by ACEI binding are underlined (bottom strand) and overlined (top strand) (8, 14). The locations of GAA repeats that are part of the HSF-binding site consensus are indicated by boldface letters.
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FIG. 4. Effect of the ADS mutation on transcription of SSA3. (A) The same RNA samples from the experiment described in the legend to Fig. 2D were analyzed by primer extension with a primer complementary to the SSA3 mRNA (+52 to +69) (3). After heat shock, the SSA3 RNA levels in DTY60 (wild type) increased with kinetics similar to those previously described (28). We observed the heterogeneous 5' termini characteristic of the SSA3 transcripts (3). In DTY90, the induction of SSA3 RNA is reduced. Lanes: 0, before heat shock; 10, 20, and 30, time after heat shock (in minutes). (B) The binding of protein from cells containing the HSF or ADS allele to the SSA3 promoter was analyzed by gel retardation. The probe used is a fragment of the SSA3 promoter (from -236 to -123) that contains the functional HSF-binding sites (3). No large complex is detected with either allele present in a single genomic copy (lanes WT and ADS). Densitometric analysis reveals that the complex formed with the extract from DTY60 transformed with the wild-type sequence on a high-copy vector (lane hc/WT) is at least three times stronger than that obtained with an equal amount of extract from DTY60 transformed with the ADS mutant on a high-copy vector (lane hc/ADS). In lane WT/P, extract from DTY60 harboring the HSF gene on pEMBL Ye24 was preincubated with 0.1 ,ul of rabbit preimmune serum, and in lane WT/I this extract was preincubated with 0.1 p.l of rabbit polyclonal anti-HSF antiserum prior to probe addition. All the mobility shift assays here and in Fig. 2C and 3A were carried out with the same extracts. The origins of the other DNA protein complexes seen in this experiment are unknown. Abbreviations: c, complex; p, free probe.
DISCUSSION In this study we found that the S. cerevisiae heat shock transcription factor HSF directly activates CUP] gene transcription. This was achieved by cloning a mutant form of HSF. Both the wild-type and the ADS mutant alleles regulate CUP] transcription in response to heat shock. The ADS mutant isolated shows a more stable binding to the CUP] promoter and promotes a higher basal level of CUP] transcription. An oligonucleotide that was derived from the CUP] promoter and was capable of competing for the binding of HSF to the CUP] promoter contains an element similar to the sequences shown to serve as HSF-binding sites (HSEs), which consist of GAA repeats in alternate orientation interspaced by 2 nucleotides (Fig. 3B) (1). However, the repeats in the CUP] promoter sequence are spaced by 3 and 4 nucleotides respectively. Another potential HSE is located within the CUP] promoter between positions -185 and -160. A previous analysis of the expression of a CUP] promoter-galK fusion gene showed that some mutations
HEAT SHOCK ACTIVATES CUPI TRANSCRIPTION
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within the sequence encompassed by the competitor oligonucleotide increased basal-level expression of galK (9). Because HSF is known to promote basal-level expression of some heat shock genes (16), the HSF protein could have a higher affinity for some of these mutagenized sequences, thereby promoting increased galK expression from the fusion gene. Because the competitor oligonucleotide encompasses two binding sites for the copper-activated DNAbinding protein ACE1, and because ACE1 does not bind to the CUP] promoter without copper in vivo (14), the possibility that HSF and ACE] act synergistically or competitively remains to be examined. The HSF gene is essential for yeast cell viability (22, 29); therefore, analysis of the action of HSF on CUP] transcription and its potential interactions with ACEJ will require precise mapping of HSF-binding site(s) on the CUP] promoter and a comprehensive promoter mutagenic analysis. Two previously observed properties of HSF might explain the data obtained with the ADS mutation. Trimerization of the HSF protein occurs in solution and when it binds to DNA (21). Competition between wild-type and ADS mutant proteins for the formation of this trimer would explain the semidominant nature of the ADS mutation. The temperature-dependent phosphorylation (22) of the high-molecularweight trimer could account for the heterogeneity of the large complex observed in the gel retardation experiments. The effects of the ADS allele on the expression of the SSA3 gene are opposite to the effects on the expression of the CUP] gene and directly correlate with the results of the DNA-binding experiments. Moreover, a strain carrying the ADS mutation shows a reduced growth rate at 39°C compared with a strain carrying the wild-type allele. These data are consistent with the possibility that the ADS protein has a lower affinity for the HSE sequences in the SSA3 promoter and some other heat shock gene promoters than the wildtype protein, thereby resulting in a corresponding decrease in transcription. We suggest that the conservative change from valine to alanine in the DNA-binding domain of HSF may reduce the affinity of the protein for some HSE sequences and increase its affinity for others. This could be directly tested by in vitro DNA-binding experiments with a variety of HSE variant sequences. Interestingly, we have demonstrated that a single conservative amino acid substitution in a transcription factor can have dramatic effects on activity both in vitro and in vivo. The induction of MT protein synthesis in tissues from whole animals in response to stresses such as heat, cold, exercise, or starvation has been suggested previously (17). Although HSEs are conserved among eukaryotes (18, 30), we did not detect sequences resembling HSEs in the promoter sequences from Drosophila melanogaster, rodent, or human MT genes (10). We suggest that induction of transcription of some of these genes as a result of these stresses could be indirect. These stimuli are known to increase circulating levels of glucocorticoid hormones, which activate MT transcription through a receptor-mediated pathway (12). The biological significance of the induction of MT during heat shock is yet not clear. MT could detoxify free radicals produced during heat shock (26) or trap ions released from metal-binding proteins denatured by elevated temperatures.
protocol for preparation of DNA-binding extracts, and T. Sosinowski for technical assistance. This work was supported by Public Health Service grant GM41840 from the National Institutes of Health. G.B. was supported by a University of Michigan Thurnau Molecular Genetics Postdoctoral Fellowship, and P.S. was supported by a University of Michigan Postdoctoral Fellowship from the Program in Protein Structure and Design. REFERENCES 1. Amin, J., J. Ananthan, and R. Voellmy. 1988. Key features of heat shock regulatory elements. Mol. Cell. Biol. 8:3761-3769. 2. Baldari, C., and G. Cesarini. 1982. Plasmids pEMBLY: new single-stranded shuttle vectors for the recovery and analysis of yeast DNA sequences. Gene 35:27-32. 3. Boorstein, W. R., and E. A. Craig. 1990. Transcriptional regu-
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10. 11. 12. 13. 14.
15.
lation of SSA3, an HSP70 gene from Saccharomyces cerevisiae. Mol. Cell. Biol. 10:3262-3267. Buchman, C., P. Skroch, J. Welch, S. Fogel, and M. Karin. 1989. The CUP2 gene product, regulator of yeast metallothionein expression, is a copper-activated DNA-binding protein. Mol. Cell. Biol. 9:4091-4095. Butler, G., and D. J. Thiele. 1991. ACE2, an activator of yeast metallothionein expression which is homologous to SWIS. Mol. Cell. Biol. 11:476-485. Butt, T. R., and D. J. Ecker. 1987. Yeast metallothionein and applications in biotechnology. Microbiol. Rev. 51:351-364. Company, M., C. Adler, and B. Errede. 1988. Identification of a Tyl regulatory sequence responsive to STE7 and STE12. Mol. Cell. Biol. 8:2545-2554. Evans, C. F., D. R. Engelke, and D. J. Thiele. 1990. ACE1 transcription factor produced in Escherichia coli binds multiple regions within yeast metallothionein upstream activation sequences. Mol. Cell. Biol. 10:426-429. Furst, P., S. Hu, R. Hackett, and D. H. Hamer. 1988. Copper activates metallothionein gene transcription by altering the conformation of a specific DNA binding protein. Cell 55:705717. Hamer, D. 1986. Metallothionein. Annu. Rev. Biochem. 55:913951. Hamer, D. H., D. J. Thiele, and J. F. Lemontt. 1985. Function and autoregulation of yeast copperthionein. Science 228:685690. Hodges, J. R., M. T. Jones, and M. A. Stockham. 1962. Effect of emotion on blood corticotrophin and cortisol concentrations in man. Nature (London) 193:1187-1188. Hu, S., P. Furst, and D. Hamer. 1990. The DNA and Cu binding functions of ACE1 are interdigitated within a single domain. New Biologist 2:1-13. Huibregste, J. M., D. R. Engelke, and D. J. Thiele. 1989. Copper-induced binding of cellular factors to yeast metallothionein upstream activation sequences. Proc. Natl. Acad. Sci. USA 86:65-69. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
16. McDaniel, D., A. J. Caplan, M. S. Lee, C. C. Adams, B. R. Fishel, D. S. Gross, and W. T. Garrard. 1989. Basal-level
17. 18. 19. 20.
ACKNOWLEDGMENTS
We thank D. Engelke, D. Friedman, and M. Szczypka for critical reading of the manuscript, E. Craig for the SSA3 probe, J. Josd Bonner for preimmune and anti-HSF antiserum, B. Errede for the
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expression of the yeast HSP82 gene requires a heat shock regulatory element. Mol. Cell. Biol. 9:4789-4798. Oh, S. H., J. T. Deagen, P. D. Whanger, and P. H. Weswig. 1978. Biological function of metallothionein. V. Its induction in rats by various stresses. Am. J. Physiol. 234:E282-E285. Pelham, H. R. B. 1982. A regulatory upstream promoter element in the Drosophila hsp7O heat-shock gene. Cell 30:517-528. Schlesinger, M. J. 1990. Heat shock proteins. J. Biol. Chem. 265:12111-12114. Sherman, F., G. R. Fink, and J. B. Hicks. 1986. Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Sorger, P. K., and H. C. M. Nelson. 1989. Trimerization of a yeast transcriptional activator via a coiled-coil motif. Cell 59:807-813.
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22. Sorger, P. K., and H. R. B. Pelham. 1988. Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation. Cell 54:855-864. 23. Szczypka, M. S., and D. J. Thiele. 1989. A cysteine-rich nuclear protein activates yeast metallothionein gene transcription. Mol. Cell. Biol. 9:421-429. 24. Thiele, D. J. 1988. ACE] regulates expression of the Saccharomyces cerevisiae metallothionein gene. Mol. Cell. Biol. 8:27452752. 25. Thiele, D. J., and D. H. Hamer. 1986. Tandemly duplicated upstream control sequences mediate copper-induced transcription of the Saccharomyces cerevisiae copper-metallothionein gene. Mol. Cell. Biol. 6:1158-1163. 26. Thornalley, P. J., and M. Vasak. 1985. Possible role for metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim. Biophys. Acta 827:36 44.
MOL. CELL. BIOL. 27. Welch, J., S. Fogel, C. Buchman, and M. Karin. 1989. The CUP2 gene product regulates the expression of the CUP] gene, coding for yeast metallothionein. EMBO J. 8:255-260. 28. Werner-Washburne, M., J. Becker, J. Kosic-Smithers, and E. A. Craig. 1989. Yeast Hsp7O RNA levels vary in response to the physiological status of the cell. J. Bacteriol. 171:2680-2688. 29. Wiederrecht, G., D. Seto, and C. S. Parker. 1988. Isolation of the gene encoding the S. cerevisiae heat shock transcription factor. Cell 54:841-853. 30. Wiederrecht, G., D. J. Shuey, W. A. Kibbe, and C. S. Parker. 1987. The Saccharomyces and Drosophila heat shock transcription factors are identical in size and DNA binding properties. Cell 48:507-515. 31. Winston, F., F. Chumley, and G. R. Fink. 1983. Eviction and transplacement of mutant genes in yeast. Methods Enzymol. 101:211-227.
MOLECULAR AND CELLULAR BIOLOGY, Mar. 1991, p. 1232-1238 0270-7306/91/031232-07$02.00/0 Copyright X 1991, American Society for Microbiology
Heat Shock Transcription Factor Activates Transcription of the Yeast Metallothionein Gene PHILIPPE SILAR, GERALDINE BUTLER, AND DENNIS J. THIELE* Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606 Received 12 September 1990/Accepted 3 December 1990
In the yeast Saccharomyces cerevisiae, transcription of the metaliothionein gene CUP) is induced by copper and silver. Strains with a complete deletion of the ACE) gene, the copper-dependent activator of CUP) transcription, are hypersensitive to copper. These strains have a low but significant basal level of CUP) transcription. To identify genes which mediate basal transcription of CUP) or which activate CUP) in response to other stimuli, we isolated an extragenic suppressor of an acel deletion. We demonstrate that a single amino acid substitution in the heat shock transcription factor (HSF) DNA-binding domain dramatically enhances CUP) transcription while reducing transcription of the SSA3 gene, a member of the yeast hsp7O gene family. These results indicate that yeast metallothionein transcription is under HSF control and that metallothionein biosynthesis is important in response to heat shock stress. Furthermore, our results suggest that HSF may modulate the magnitude of individual heat shock gene transcription by subtle differences in its interaction with heat shock elements and that a single-amino-acid change can dramatically alter the activity of the factor for different target genes.
Metallothioneins (MTs) are small, cysteine-rich, metalbinding proteins involved in heavy-metal homeostasis and detoxification (reviewed in reference 10). In higher eukaryotes, MTs are encoded by a multigenic family and their synthesis is controlled at the transcriptional level by several stimuli including heavy metals (zinc, cadmium, copper), glucocorticoids, phorbol esters and interleukins. Higher eukaryotic MT promoters have a correspondingly complex arrangement of distinct and interdigitated cis-acting DNA sequences through which these transcriptional responses are mediated. The multitude of effectors of MT transcription in these systems suggests that a number of physiological conditions require increased MT biosynthesis. The complexity of these systems has also hindered studies of MT transcriptional control due to individual stimuli. The baker's yeast Saccharomyces cerevisiae contains a single MT gene, designated CUP], which shares many of the basic features of MT genes in higher eukaryotes (6). Genetic studies have clearly demonstrated that the CUP) gene is essential to prevent copper toxicity (11). Copper and silver are the only reported effectors of CUP) transcriptional induction (9). Metal-inducible transcription of CUP) is mediated through the interaction of the metal-activated DNAbinding protein, ACE1 (CUP2), with cis-acting metal-responsive sequences localized in the CUP) promoter region and called UASCup, (CUP) upstream activation sequences [8, 13, 14, 24, 25, 27]). Yeast strains harboring either a point mutation in the ACE) gene (4, 24) or a complete deletion of the ACE] gene (5) are hypersensitive to copper as a result of a lack of copper-inducible transcription of CUP). To identify other genes involved in transcriptional activation of CUP) in response to other environmental stimuli, we isolated a strain bearing a mutation which suppresses the requirement for ACE) in the activation of CUP) transcription. In this report, we present the isolation and characterization of one such suppressor, which is a mutant form of the heat shock transcription factor, HSF (22, 29). *
Corresponding author.
MATERIALS AND METHODS
Strains and growth conditions. The yeast strains used are as follows: DTY60 (MATot his6 leu2-3,112 LEU2::YipCL ura3-52 CUPJR-3 aceJ-A225 HSF) (5), DTY68 (MATot his6 leu2-3,112 LEU2::YipCL ura3-52 CUPJR-3 ace)-A&225 ADS) for the original ADS isolate, see text below), DTY69 (MATot his6 leu2-3,112 ade LEU2::YipCL ura3-52 CUPIR-3 acelA225 HSF) and DTY90 (MATa his6 leu2-3,112 LEU2::YipCL ura3-52 CUPIR-3 aceJ-A225 ADS) (see text). HSF refers to the wild-type heat shock transcription factor gene, and ADS refers to the mutant allele of the heat shock factor described in this report. The acel-A225 allele is a null allele (5). Strain DTY60 fails to grow on media containing copper sulfate at a concentration higher than 15 ,uM. All yeast strains were grown in rich (YPD) medium or synthetic complete (SC) medium lacking nutrients as specified (20) at 30°C except during heat shock induction experiments. Heat shock inductions were carried out as follows. Cells were grown at 23°C to an optical density of 0.6 and then rapidly shifted to 39°C for the times indicated in the figure legends. Copper was added in the form of copper sulfate. 5-Bromo-4-chloro-3-indolyl-P-Dgalactopyranoside (X-Gal) plates were prepared as described previously (15). Escherichia coli DHSa F' and XL-1 blue were used in all DNA manipulations and were grown under standard conditions (15). DNA manipulation and analysis. Standard protocols were used for all DNA manipulations and cell transformations (15). For the recovery of the ADS allele, DNA from S. cerevisiae DTY68 was isolated (20) and subsequently partially digested with Sau3AI and ligated into the BamHI site of shuttle vector pEMBL Ye24 (2). pEMBL Ye24 is a high-copy (2,um) vector that carries the URA3 marker. The ligation reaction was directly transformed into S. cerevisiae DTY60, and URA+ transformants were selected. The bank was estimated to contain three haploid genome equivalents on the basis of the average insert size and the number of independent transformants obtained. DNA fragments were subcloned into Bluescript vectors (Stratagene) and sequenced by using the Sequenase kit 1232
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(United States Biochemical Corp.). The universal primer as well as heat shock factor gene-specific primers were used. RNA and protein analysis. Primer extension reactions were carried out as described previously (23). Gel retardation experiments were performed as follows. Proteins were extracted from yeast strains as described previously (7). DNA probes were end labeled with Klenow fragment of DNA polymerase I and [a-32]dATP or [a-32P]dCTP. Binding assays were performed by mixing 25 ,Lg of protein from the extract and 0.5 ng of labeled probe (5,000 dpm) in 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 8.0)-30 mM KCI-2 mM dithiothreitol-10% glycerol. After a 10-min incubation at room temperature, samples were analyzed by electrophoresis on a 5% native polyacrylamide gel. Gels were exposed to Kodak XAR-5 film overnight at -70°C with intensifying screens. Antiserum neutralization DNA-binding assays were carried out by preincubating extracts with either rabbit polyclonal antiHSF or preimmune serum (a kind gift of J. Jose Bonner) for 5 min at room temperature and then adding the radioactive probe fragment. After a 10-min incubation at room temperature, the binding reactions were analyzed by gel electrophoresis as described above. RESULTS Isolation and characterization of the ADS mutation. To isolate and identify yeast strains capable of activating CUP) transcription in the absence of ACE1, we used a positive selection scheme. Strain DTY60 contains the ura3-52 mutation, three copies of the CUP) gene, a CUPI-lacZ fusion gene integrated at leu2, and a compete deletion of the ACE] gene (5). CUP) transcription is not significantly inducible by copper ions in this background; therefore, this strain is unable to grow on media containing more than 15 ,uM copper sulfate. Spontaneous suppressors of the copper-sensitive phenotype were isolated by plating approximately 1010 cells from strain DTY60 on SC medium containing 150 ,uM copper sulfate. After a 1-week incubation at 30°C, 29 copperresistant isolates were recovered. To select suppressors which were trans-activators of CUP) expression, we patched copper-resistant isolates onto SC medium containing X-Gal, the chromogenic substrate for 3-galactosidase. One isolate, denoted DTY68, which was resistant to up to 300 ,uM copper sulfate and which expressed a high level of ,-galactosidase from the chromosomal CUPI-lacZ fusion, was selected for further study. The suppressor mutation in DTY68 was designated ADS (ace) deletion suppressor). The steady-state levels of CUP) mRNA were analyzed by primer extension in the wild-type starting strain DTY60 and strain DTY68 harboring the ADS mutation. The CUP) mRNA level was elevated in DTY68 at least 10-fold, as estimated by laser densitometry, over the level in strain DTY60 (Fig. 1A). The levels of the internal control RNA, derived from the alcohol dehydrogenase gene (ADHI), were similar in both strains. The steady-state level of CUP) mRNA in strain DTY68 was not further increased by exposure of these cells to several different concentrations of copper sulfate (data not shown). To determine whether the ADS mutation affects the binding of yeast proteins to the CUP) promoter, we carried out mobility shift assays with extracts prepared from strains DTY60 and DTY68. A large promoter fragment from -241 to +37 was used as a probe in these experiments. Extracts from DTY68 promoted the formation of a large heterogeneous complex with the CUP) promoter (Fig. 1B). This
HEAT SHOCK ACTIVATES CUP] TRANSCRIPTION
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FIG. 1. Effect of the ADS mutation on CUPI transcription. (A) Total RNA (15 ,ug) from either strain DTY60 (WT) or strain DTY68 (ADS mutant) was used in extension reactions with radiolabeled primers complementary to CUP] and ADHI mRNA. The amount of mRNA in each reaction does not vary, as shown by the control ADHI extension product. CUP) mRNA levels are dramatically increased in the ADS mutant (lane ADS) compared with the wild type (lane WT). (B) Proteins from strains DTY60 (WT) and DTY68 (ADS) were mixed with a radiolabeled CUPI promoter probe (from positions -241 to +37 numbered with respect to the transcription start) and analyzed by electrophoresis on a 5% polyacrylamide gel. Extract from the ADS mutant strain promotes the formation of a large heterogeneous complex (lane ADS). This complex is barely detectable with extract from the wild-type strain (lane WT). Free probe (without extract) is shown as a control (lane probe). Although the nature of the minor band in the free-probe lane is unknown, this appears upon repeated polyacrylamide gel purification of the probe fragment and is believed to be an altered form of the free probe. Abbreviations: c, complex; p, free probe.
complex could not be detected with extracts from strain DTY60 under these conditions. The ADS suppressor mutation was genetically analyzed by crossing to the tester strain DTY69. ADS is a semidominant mutation, since the heterozygous diploid strain has an intermediate level of copper resistance (50 ,uM) and exhibits ,-galactosidase activity between the haploid wild-type DTY69 and the haploid ADS mutant DTY68. Analysis of spores from 13 complete tetrads revealed that the copper resistance phenotype segregated 2:2 through meiosis. Cosegregation of the copper resistance, the high level of P-galactosidase activity, and DNA-protein complex formation were observed in all tetrads analyzed. These data indicate that the ADS mutation resides in a single nuclear locus and that this mutation activates expression from the CUP) promoter in trans. Isolation and analysis of the ADS allele. Based on the semidominant nature of the ADS mutation, a genomic bank
derived from the ADS mutant strain (DTY68) was constructed in the high-copy vector pEMBL Ye24 (2) and transformed into the wild-type copper-sensitive strain, DTY60. One copper-resistant transformant expressing a high level of ,-galactosidase was obtained. The plasmid retrieved from this transformant contained an insert of
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SILAR ET AL.
approximately 3 kb (Fig. 2A, pADSb). Strain DTY60 transformed with this plasmid was resistant to copper concentrations up to 300 ,uM. To analyze the ADS allele, the nucleotide sequence of the pADSb insert was determined. A search of the GenBank and EMBL data bases revealed that this insert represents the promoter and 590 amino-terminal codons (out of a total of 833) of the heat shock transcription factor (HSF), a transcription factor involved in the activation of the yeast heat shock gene families (22, 29). To verify the authenticity of the cloned gene, we integrated a URA3 marker adjacent to the wild-type HSF locus and analyzed the linkage with the ADS allele. Analysis of 10 complete tetrads revealed no recombination between URA3 and ADS, demonstrating that the ADS mutation is closely linked to the HSF locus. The complete wild-type allele from DTY60 and the ADS allele from DTY68 were recovered by integration via homologous recombination and excision (31) and used for the experiments described below. To determine whether the ADS allele product was a component of the DNA-protein complex observed in DTY68 extracts (Fig. 1B), we carried out mobility shift assays with extracts from strain DTY60 transformed with pADS8. In the mobility shift assays, the protein-DNA complex formed with the extract from strain DTY60 transformed with pADS8 migrated faster than the complex formed with extract from strain DTY68 (Fig. 2B), as expected from the size difference between the truncated protein encoded by the pADSB insert and the full-length protein encoded by the chromosomal ADS allele. This strongly suggests that the protein encoded by the ADS allele is present in the DNA-protein complex observed in Fig. 1B. The sequence of the pADS5 insert revealed a single-base change between the ADS allele and the published wild-type sequences of the HSF gene (22, 29). This mutation changes a valine codon to an alanine codon in the DNA-binding domain of HSF (29) at position 203 (Fig. 2A). It also destroys a MaeIII site present in the DNA sequence of the wild-type HSF gene (22, 29) as determined by genomic DNA blotting experiments (data not shown). The valine-to-alanine mutation is not a polymorphism since it is not present in the wild-type gene isolated from DTY60. Furthermore, transformation of strain DTY60 with a linear DNA fragment from the ADS allele encompassing this mutation (from BamHI to the distal NruI site in Fig. 2A) allowed the recovery of chromosomal recombinant isolates exhibiting all ADS phenotypes described above. One of these transformant clones, DTY90, was used in the experiments described below. Effects of the wild-type and ADS alleles on the CUP) promoter. To determine whether the wild-type HSF gene plays a role in regulation of CUP) transcription, the entire ADS mutant and wild-type HSF gene were separately introduced into strain DTY60 on a high-copy vector. Under standard growth conditions at 30°C, both constructs conferred an increased resistance to copper (Fig. 2A). The wild-type HSF gene allowed growth of this strain on medium containing 100 ,uM CuS04, and the ADS allele allowed growth on up to 500 puM CUSO4. These values represent approximately 6- and 33-fold increases in copper resistance conferred by multiple copies of HSF and ADS, respectively. This result indicates that both the wild-type and ADS mutant alleles activate transcription from the CUP) promoter. Mobility shift experiments with the CUP) promoter fragment confirmed this hypothesis (Fig. 2C). As demonstrated in Fig. 1B, we observed no detectable complex with the CUP) promoter fragment when using extracts from the wild-type strain (lane WT); however, when using extracts from the
MOL. CELL. BIOL.
ADS mutant strain, we observed a complex indistinguishable from that in Fig. 1B (lane ADS). We detected the large heterogeneous complex, previously observed with extracts from the ADS mutant strain DTY68, with extract from cells containing the wild-type allele of HSF placed on a high-copy vector (Fig. 2C, lane WT/HC). As expected, we detected a dramatically increased level of a complex with the same mobility when using extracts from a strain containing the ADS allele on a high-copy plasmid (Fig. 2C, lane ADS/HC). To determine whether CUP) expression is induced in response to heat shock, we performed primer extension experiments on RNA extracted from DTY60 and DTY90 cells before and after heat shock treatment (Fig. 2D). CUP) mRNA levels increased during the heat shock, with the maximum level reached approximately 20 min after the heat shock. Both the wild-type and ADS alleles were able to induce this heat shock response, with mRNA levels correspondingly greater in the ADS allele background. No changes in ADHI mRNA were observed in response to the heat shock treatment. Although the CUP) heat shock induction was not large, this induction is reproducible in several experiments. Furthermore, several previously characterized heat shock genes exhibit a small transcriptional stimulation in response to heat shock (28). Competition experiments with a gel retardation assay demonstrated that the large complex formed with extracts from cells containing the wild-type allele of HSF in a high-copy vector is inhibited by an oligonucleotide encompassing the two strong ACE)-binding sites in the CUP) promoter (8, 13, 14). However, an oligonucleotide adjacent to this binding site failed to compete for formation of this complex (Fig. 3A). Qualitatively similar results were obtained when extracts from the ADS mutant strains DTY68 and DTY90 were used in competition experiments (data not shown). These experiments demonstrate that CUP) is a heat shock-responsive gene recognized by HSF and that HSF binds within UASCupJ. Indeed, within the region of UASCup, covered by the competitor oligonucleotide, sequences similar (but not identical) to the consensus heat shock element (HSE) are present (Fig. 3B). However, another potential binding site can be postulated around position -170, because several GAA repeats can also be found in this region. Effect of the ADS mutation on heat shock gene expression. Heat shock genes are involved in a number of important cellular processes including thermotolerance, protein folding, translocation, and degradation (19). To determine the potential effect of the ADS mutation on the expression of genes important for growth at elevated temperatures, we compared the thermosensitivity of strains DTY60 and DTY90. These strains exhibited doubling times of 3.7 and 6.6 h, respectively, after a log phase shift from 23 to 39°C, suggesting that the mutant ADS protein may not efficiently activate transcription of one or more of the heat shock genes involved in optimal growth at 39°C. We then examined the effect of the ADS allele on the expression of the heat shock gene SSA3, a member of the hsp7O family known to be dramatically induced by heat shock (3, 28). Primer extension experiments (Fig. 4A) demonstrated that SSA3 is not fully induced in the ADS mutant background compared with the wild-type background. Indeed, we observed only a slight induction of SSA3 mRNA levels over the time course of heat shock stress with DTY90 cells compared with strain DTY60. These results indicate that the protein encoded by the ADS allele has a dramatically reduced ability to activate SSA3 transcription after heat shock stress. Because we mapped
HEAT SHOCK ACTIVATES CUP] TRANSCRIPTION
VOL. 11, 1991
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the mutation in the ADS allele to a single-amino-acid substitution in the HSF DNA-binding domain, we carried out experiments to determine whether this reduction in SSA3 gene activation in the ADS background might be due to an alteration in the interaction of the ADS protein with the heat shock elements in the SSA3 promoter. Figure 4B shows the results of mobility shift assays with a fragment from the SSA3 promoter which encompasses functional HSEs (3). We observed several protein-DNA complexes, including a large complex detectable only in the strain containing the HSF or ADS allele on a high-copy vector (Fig. 4B, lanes hc/WT and hc/ADS). To confirm that this large complex was specifically due to binding of the HSF or ADS protein on the SSA3 promoter fragment, we preincubated the extract derived from the strain carrying HSF on a high-copy vector with anti-HSF antiserum prior to DNA binding. Under these conditions only the large complex disappeared (lane WT/I). The complex was present when the preimmune serum was used in the binding reaction (lane WT/P). Similar results were obtained with extracts from the strain bearing the ADS allele on a high-copy vector. These results clearly demonstrate that the large complex observed in these experiments is due to the binding of the HSF or ADS protein on the SSA3 promoter fragment. This complex is reduced with extracts from the ADS mutant (lane hc/ADS) compared with the wild-type allele (lane hc/WT). This correlates with the decreased transcriptional activation of the SSA3 gene observed in Fig. 4A. This suggests that the protein encoded by the ADS allele fails to fully activate SSA3 transcription as a result of a decreased ability to form stable complexes with the HSEs in the SSA3 promoter. Therefore, the wild-type and ADS proteins have opposite effects on CUP] and SSA3 transcription, owing, at least in part, to differences in DNAprotein interactions.
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FIG. 4. Effect of the ADS mutation on transcription of SSA3. (A) The same RNA samples from the experiment described in the legend to Fig. 2D were analyzed by primer extension with a primer complementary to the SSA3 mRNA (+52 to +69) (3). After heat shock, the SSA3 RNA levels in DTY60 (wild type) increased with kinetics similar to those previously described (28). We observed the heterogeneous 5' termini characteristic of the SSA3 transcripts (3). In DTY90, the induction of SSA3 RNA is reduced. Lanes: 0, before heat shock; 10, 20, and 30, time after heat shock (in minutes). (B) The binding of protein from cells containing the HSF or ADS allele to the SSA3 promoter was analyzed by gel retardation. The probe used is a fragment of the SSA3 promoter (from -236 to -123) that contains the functional HSF-binding sites (3). No large complex is detected with either allele present in a single genomic copy (lanes WT and ADS). Densitometric analysis reveals that the complex formed with the extract from DTY60 transformed with the wild-type sequence on a high-copy vector (lane hc/WT) is at least three times stronger than that obtained with an equal amount of extract from DTY60 transformed with the ADS mutant on a high-copy vector (lane hc/ADS). In lane WT/P, extract from DTY60 harboring the HSF gene on pEMBL Ye24 was preincubated with 0.1 ,ul of rabbit preimmune serum, and in lane WT/I this extract was preincubated with 0.1 p.l of rabbit polyclonal anti-HSF antiserum prior to probe addition. All the mobility shift assays here and in Fig. 2C and 3A were carried out with the same extracts. The origins of the other DNA protein complexes seen in this experiment are unknown. Abbreviations: c, complex; p, free probe.
DISCUSSION In this study we found that the S. cerevisiae heat shock transcription factor HSF directly activates CUP] gene transcription. This was achieved by cloning a mutant form of HSF. Both the wild-type and the ADS mutant alleles regulate CUP] transcription in response to heat shock. The ADS mutant isolated shows a more stable binding to the CUP] promoter and promotes a higher basal level of CUP] transcription. An oligonucleotide that was derived from the CUP] promoter and was capable of competing for the binding of HSF to the CUP] promoter contains an element similar to the sequences shown to serve as HSF-binding sites (HSEs), which consist of GAA repeats in alternate orientation interspaced by 2 nucleotides (Fig. 3B) (1). However, the repeats in the CUP] promoter sequence are spaced by 3 and 4 nucleotides respectively. Another potential HSE is located within the CUP] promoter between positions -185 and -160. A previous analysis of the expression of a CUP] promoter-galK fusion gene showed that some mutations
HEAT SHOCK ACTIVATES CUPI TRANSCRIPTION
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within the sequence encompassed by the competitor oligonucleotide increased basal-level expression of galK (9). Because HSF is known to promote basal-level expression of some heat shock genes (16), the HSF protein could have a higher affinity for some of these mutagenized sequences, thereby promoting increased galK expression from the fusion gene. Because the competitor oligonucleotide encompasses two binding sites for the copper-activated DNAbinding protein ACE1, and because ACE1 does not bind to the CUP] promoter without copper in vivo (14), the possibility that HSF and ACE] act synergistically or competitively remains to be examined. The HSF gene is essential for yeast cell viability (22, 29); therefore, analysis of the action of HSF on CUP] transcription and its potential interactions with ACEJ will require precise mapping of HSF-binding site(s) on the CUP] promoter and a comprehensive promoter mutagenic analysis. Two previously observed properties of HSF might explain the data obtained with the ADS mutation. Trimerization of the HSF protein occurs in solution and when it binds to DNA (21). Competition between wild-type and ADS mutant proteins for the formation of this trimer would explain the semidominant nature of the ADS mutation. The temperature-dependent phosphorylation (22) of the high-molecularweight trimer could account for the heterogeneity of the large complex observed in the gel retardation experiments. The effects of the ADS allele on the expression of the SSA3 gene are opposite to the effects on the expression of the CUP] gene and directly correlate with the results of the DNA-binding experiments. Moreover, a strain carrying the ADS mutation shows a reduced growth rate at 39°C compared with a strain carrying the wild-type allele. These data are consistent with the possibility that the ADS protein has a lower affinity for the HSE sequences in the SSA3 promoter and some other heat shock gene promoters than the wildtype protein, thereby resulting in a corresponding decrease in transcription. We suggest that the conservative change from valine to alanine in the DNA-binding domain of HSF may reduce the affinity of the protein for some HSE sequences and increase its affinity for others. This could be directly tested by in vitro DNA-binding experiments with a variety of HSE variant sequences. Interestingly, we have demonstrated that a single conservative amino acid substitution in a transcription factor can have dramatic effects on activity both in vitro and in vivo. The induction of MT protein synthesis in tissues from whole animals in response to stresses such as heat, cold, exercise, or starvation has been suggested previously (17). Although HSEs are conserved among eukaryotes (18, 30), we did not detect sequences resembling HSEs in the promoter sequences from Drosophila melanogaster, rodent, or human MT genes (10). We suggest that induction of transcription of some of these genes as a result of these stresses could be indirect. These stimuli are known to increase circulating levels of glucocorticoid hormones, which activate MT transcription through a receptor-mediated pathway (12). The biological significance of the induction of MT during heat shock is yet not clear. MT could detoxify free radicals produced during heat shock (26) or trap ions released from metal-binding proteins denatured by elevated temperatures.
protocol for preparation of DNA-binding extracts, and T. Sosinowski for technical assistance. This work was supported by Public Health Service grant GM41840 from the National Institutes of Health. G.B. was supported by a University of Michigan Thurnau Molecular Genetics Postdoctoral Fellowship, and P.S. was supported by a University of Michigan Postdoctoral Fellowship from the Program in Protein Structure and Design. REFERENCES 1. Amin, J., J. Ananthan, and R. Voellmy. 1988. Key features of heat shock regulatory elements. Mol. Cell. Biol. 8:3761-3769. 2. Baldari, C., and G. Cesarini. 1982. Plasmids pEMBLY: new single-stranded shuttle vectors for the recovery and analysis of yeast DNA sequences. Gene 35:27-32. 3. Boorstein, W. R., and E. A. Craig. 1990. Transcriptional regu-
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ACKNOWLEDGMENTS
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