Heat shock factor-independent heat control of transcription of the CTT1 gene encoding the cytosolic catalase T of Saccharomyces cerevisiae.

Transcription of the Saccharomyces cerevisiae CTT1 gene encoding the cytosolic catalase T has been previously shown to be derepressed by nutrient stress. To investigate whether expression of this gene is also affected by other types of stress, the influence of heat shock on CTT1 expression was studied. The results obtained show that expression of the gene is low at 23 degrees C and is induced rapidly at 37 degrees C. By deletion analysis, a promoter element necessary for high level induction by heat shock was located between base pairs -340 and -364 upstream of the translation start codon. This region was demonstrated to be sufficient for heat shock control by placing it upstream of a S. cerevisiae LEU2-lacZ fusion gene. Mutagenesis of the region showed that the response to heat shock is not mediated by a sequence similar to canonical heat shock elements, but by DNA elements also involved in nutrient control of transcription. Catalase T appears to have a function in protecting yeast cells against oxidative damage under stress conditions. Catalase T-containing strains are less sensitive to exposure to 50 degrees C ("lethal heat shock") than isogenic catalase T-deficient mutants, and catalase T-containing strains pretreated by incubation at 37 degrees C are less sensitive to H2O2 than pretreated catalase-deficient mutants.

alase A is induced by fatty acids and appears to have a function connected to peroxisomal fatty acid P-oxidation (9).
The CTTl gene is under negative control by cAMP (10). In S. cereuisiae, this second messenger is involved in intracellular signaling of nutrient levels (11). Thus, catalase T levels are low while cells are grown on complete medium and rise during nutrient starvation. A similar response has been reported for several heat shock proteins of S. cereuisiae (12)(13)(14)(15). The combination of positive control by heat shock and negative cAMP control might therefore be characteristic for a class of stress proteins needed by yeast cells under conditions of stress caused by heat and/or by nutrient starvation.
We have tested therefore whether catalase T belongs to this class of proteins. Our results described in this paper show that CTTl transcription is positively controlled by heat shock via a heat shock transcription factor-independent mechanism. This finding suggests that the cytosolic catalase encoded by this gene is mainly needed when oxidative stress caused by hydrogen peroxide is combined with nutrient starvation or heat stress. Data obtained in the course of this investigation are consistent with such a function.

EXPERIMENTAL PROCEDURES
Yeast Strains, Media, Growth Conditions-The S. cerevisiae strains used in this study are listed in Table I. Strains were routinely grown on YPD medium (16) a t 30 "C. In heat shock experiments, cells were grown at 23 "C to an optical density at 600 nm of 3, and cultures were subsequently shifted to 37 "C for the time periods indicated for individual experiments.
"Lethal Heat Shock" Treatment, H202 Resistance-To test resistance of cells under a "lethal heat shock" regime, logarithmic cultures were grown to an optical density a t 600 nm of 1; stationary phase cultures were grown for 2 days. Cells were suspended in fresh medium at an optical density of 1, heated to 50 "C for 20 min, and plated after serial dilutions to assay for survivors. To test for H,02 resistance, logarithmic cultures grown at 23 "C were divided, one-half of each culture was subjected to heat shock, and heat-shocked and non-heatshocked cells were suspended a t a density of lo5 cells/ml in various concentrations of H202 in phosphate buffer, pH 6.8. Cells were incubated for 15 min, and serial dilutions were subsequently plated to assay for survivors.
Plasmids, Yeast Transformation-Plasmid pTB3 used for single copy integration of a CTTl-lac2 fusion gene into the chromosomal URA3 locus and its derivatives hearing deletions in the region upstream of the CTTl gene are described elsewhere (17). The CTTl portion of the CTTl-lac2 fusion genes used in this study extends to base pair +390 of CTTl (10). The yeast integration vector pLS9 (18) bearing a LEUZ-lac2 fusion gene lacking the UAS region of the LEU2 gene and containing a unique EcoRI site suitable for integration of upstream fragments of other genes was donated by M. Carlson (Department of Genetics and Development, Columbia University, New York). The insertion of CTTl upstream elements or synthetic oligonucleotides into this vector is described elsewhere (17). A plasmid containing a synthetic heat shock element, which was used as one of the D L S~ inserts (construct AW3: insert sequence:

CTGCACGAATTCGTGC-AGGTCGACTSTAGAAGCTTCTAGA
G G A T C C C C G G G T A C C-GAGCTCG3'; canonical heat shock elements (19) are underlined), was donated by P. Sorger (Department of Microbiology and Immunology, University of California, San Francisco).
Linear DNA fragments produced by cutting the plasmids a t a unique NcoI site within their URA3 gene were used for yeast transformation (20). Genomic DNA of transformants was analyzed by hybridization according to Southern (21), and single copy integrants were used for further analysis. Oligonucleotides used were synthesized by G. Schaffner, Institute of Molecular Pathology, Vienna.
Enzyme Activities of Crude Cell Extracts-@-Galactosidase activity of extracts prepared by breakage of cells with glass beads (22) was assayed spectrophotometrically using o-nitrophenyl-@-D-galactoside as substrate (23). Under the conditions used, @-galactosidase was detectable down to a level of 0.1 nmol of substrate hydrolyzed per min per mg of protein. Catalase activity was assayed spectrophotometrically by following disappearance of H 2 0 2 at 240 nm (24). Protein concentrations of extracts were assayed as described by Bradford (25). All values reported are averages of at least three independent experiments.
Gel Retardation Assay-Gel retradation experiments using purified heat shock transcription factor kindly donated by P. Sorger were carried out as described by Sorger and Pelham (27).

Expression of the CTTl Gene Is Induced by Heat S h c k -
To investigate whether the CTTl gene is under control by heat shock, its expression was examined at the mRNA level, by assaying the dependence of catalase T activity on heat treatment of cells, and by following the expression of a CTTl-lac2 fusion gene during heat shock. Fig. 1 demonstrates that the level of mRNA transcribed from the endogenous CTTl gene is significantly enhanced when yeast cells grown at 23 "C are exposed to a mild heat shock (37 "C). In agreement with this observation, 5-10-fold induction of catalase T activity and of @-galactosidase produced by expression of a CTTl-lac2 fusion gene by heat shock is observed (Fig. 2). In contrast to catalase T, the peroxisomal catalase A is not induced by heat shock.
Localization of CTTl Upstream Region Mediating Heat Shock Induction-The upstream region of the CTTl gene contains no elements conforming well to the canonical heat shock consensus (5'CNNGAANNTTCNNGS') (19) or to the sequences more recently defined as functional heat shock elements of Drosophila melanogaster (three repeats of GAA  from strain A2-200 grown at 23 "C (lane I ) and from cells heat shocked at 37 "C for 50 min ( l a n e 2) was probed with a CTT1 EcoRI fragment (+896 to +2014) (41) and an actin (ACTI) gene fragment after electrophoretic separation and transfer to nylon membranes. Cells of strain GA74-1A (CTTl'ctal ura3::CTTl-lacZ) and of strain DCTl-4B (cttl CTAI') were grown at 23 "C to logarithmic growth phase and transferred to 37 "C (0 min). Samples were taken, and specific catalase T (full circles) and @-galactosidase activity (triangles) was assayed in extracts from strain GA74-1A, catalase A activity (open circles) in extracts of strain DCT1-4B. modules in alternating orientation punctuated by two bases of lower conservation) (28, 29). A set of internal deletions (17) covering the region previously shown to be important for activity of the CTTl promoter (30) was therefore tested for heat shock element activity. Preliminary experiments had shown that the CTTl promoter has only low basal activity at 23 "C (see also Fig. 1) and that minimal heat stress, which is provided during routine growth of cells at 30 "C, is necessary for its induction under all physiological conditions tested. It was possible therefore to limit the analysis for heat shock element activity to those regions, which had previously been shown to contain positive promoter elements (17,30). When corresponding deletions are tested for their effect on heat shock control of a CTTl-lac2 fusion gene (Table 11), it is evident that all those deletions causing a pronounced decrease in heat shock control (7-10-fold) lack a DNA sequence between base pairs -340 and -364. This region should therefore contain or at least overlap with an element important for heat shock control. To test whether the region containing the element defined by deletion analysis is sufficient for heat shock control, promoter fragments or synthetic oligonucleo- Heat shock control of expression of CTTl-lac2 fusion genes Cells of strain WS17-5D were transformed with CTTI-lac2 fusion genes (single copy chromosomal integrants) with wild type promoter or with the deletions indicated. They were grown a t 23 "C to an optical density a t 600 nm of 3. One half of the culture was heat shocked at 37 "C for 60 min, and the other half was kept a t 23 "C. @-Galactosidase (nmoles of o-nitrophenyl-@-D-galactoside hydrolyzed per min per mg of protein (23)

TABLE I11
Heat shock element activity of CTTl upstream fragments Cells of strain WS17-5D were transformed with plasmid pLS9 (18) or one of its derivatives (single copy chromosomal integrants) bearing the inserts indicated in the unique EcoRI site upstream of a LEU2-lac2 fusion gene. Transformants were grown a t 23 "C to an optical density a t 600 nm of 3. One half of the culture was heat shocked a t 37 "C for 60 min, and the other half was kept a t 23 "C. @-Galactosidase (nmoles of o-nitrophenyl-P-D-galactoside hydrolyzed per min per mg of protein (23)) was assayed in crude extracts. @-Galactosidase Gene activity of cells 37  tides with corresponding sequences were tested for their ability to confer heat shock control to the yeast LEU2 promoter lacking its own UAS element (Table 111). The results obtained show that upstream fragments containing the region deleted in TB350 have heat shock element activity. They are functional in both orientations, and two copies of the region are comparable in their activity to a synthetic canonical heat shock element tested in combination with the LEU2 promoter (AW3).
To localize the element mediating heat shock control more precisely, blocks of point mutations were introduced into construct AWlN (Fig. 3), and the mutant sequences were again tested for heat shock element activity. As shown in Table IV, mutation of the sequence similar to a canonical heat shock element (AWlN-13) has no significant effect on heat shock control. However, mutation of the sequence similar to the cAMP responsive elements of the SSA3 promoter (31) (consensus sequence: 5'TA/TAGGGAT3') significantly reduces response to heat shock (AWlN-11). A similar effect is observed with construct AWlN-14, but in this case, both basal promoter function and response to heat shock are affected. The sequence mutated in this construct (S'GTATTGTTTC3') is also found in the upstream region

FIG. 3. Mutations introduced into construct A W l N .
Identities to the cAMP responsive element of the yeast SSA3 gene (31) (consensus: 5'TA/TAGGGAT3'; solid line), to the canonical heat shock consensus (19) (5'CNNGAANNTTCNNG3'; broken line) or to a sequence of the upstream region of the yeast UBZ4 gene (32) (5'GTATTGTTTC3'; double line) are indicated by bold letters in the sequence of AWlN. Base positions given are those of the CTTl wildtype promoter. Only bases mutated are printed for constructs AWlN-11, AWlN-13, and AWlN-14.

TABLE IV
Mutational analysis of heat shock control region of CTTl Cells of strain WS17-5D were transformed with plasmid AWlN or one of its mutant versions (see Fig. 3). Transformants were grown a t 23 "C to an optical density at 600 nm of 3. One half of the culture was heat-shocked a t 37 "C for 60 min, the other half was kept at 23 "C. @-Galactosidase (nmoles of o-nitrophenyl-P-D-galactoside hydrolyzed per minute per mg of protein (23) which has been reported to be under control by heat shock and cAMP (12). The response of the promoter to nitrogen limitation is also considerably reduced by both mutations affecting heat shock control.' Other mutations, which, together with AWlN-ll, AWlN-13, and AWlN-14 cover the entire region between base pairs -365 and -326, have no significant effect on heat shock control (data not shown). In gel retardation experiments, no binding in vitro of purified heat shock transcription factor to oligonucleotides covering the region corresponding to base pairs -382 to -325, and no competition of this sequence for binding of heat shock transcription factor with a canonical heat shock sequence was observed (Fig. 4). It can be concluded from these results that the two DNA sequences mutated in constructs AWlN-11 and AWlN-14 are important for heat shock control of the promoter, and that the sequence similar to a canonical heat shock element, which is mutated in construct AWlN-13, is not involved in control of CTTl transcription.
Functional Relevance of Heat Shock Induction of the Cytosolic Catalase T-Although it is generally assumed that catalase protects cells against hydrogen peroxide or perhaps other peroxides, the function of this well characterized enzyme is not really sufficiently understood in detail. There is even less information concerning the relevance of extraperoxisomal catalases. The finding that expression of the gene encoding catalase T is controlled by heat shock suggests a function of this protein under heat stress conditions. We have attempted to obtain evidence for such a function by testing the heat shock resistance of various yeast strains producing catalase T or lacking it because of a disruption of the CTTl gene. The results obtained are summarized in Table  V. The fact that stationary phase cells are much more resistant to heat shock C. Schuller and G. Marchler, unpublished results.

TABLE V
Resistance of catalase T-positive strains and of catalase T-deficient mutants to "lethal heat shock" Cells were grown to logarithmic or stationary phase, incubated a t 60 "C for 20 min, and plated to assay for survivors. Means k S.D. of three independent experiments are presented for the ratios of survivors in the pairs of isogenic CTTI' and cttl mutant strains. than logarithmic cells and that mutations in the RAS-CAMP nutrient signaling pathway cause alterations in heat resistance is well established (see e.g. Refs. 33,34). Further, it cannot be doubted that multiple factors, most of them not yet identified, contribute to heat resistance. The results presented in Table V establish the cellular catalase T level as one of these factors. In two different isogenic sets of strains, the cttl mutants, which lack catalase T , exhibited significantly lower heat resistance than catalase T-containing CTTI wild-type cells. This effect was observed in logarithmic and in stationary phase wild-type cells and in ras2 mutant strains. The only exception to this observation were the pair of sral (bcyl ) mutants tested, which lack the protein kinase A regulatory subunit. This latter result is also consistent with a role of catalase T in heat resistance since we have observed that sral mutants do not show heat shock induction of catalase T and that their catalase T level is similar to that of cttl mutants (17).
The observation that catalase T contributes to heat resistance of yeast cells provides a functional reason for its inducibility by heat shock. I t is likely that it is the function of catalase T to protect cells against oxidative damage by HeO?, which might be more dangerous to yeast during heat stress than at lower temperatures. The results summarized in Fig. 5 show that pretreatment of yeast cells by a mild heat shock (37 "C) for 30 min dramatically increases their resistance to H,02. This increase is much more pronounced in the catalasepositive strain which induces catalase T by the heat shock treatment than in the catalase-deficient mutant studied.
A similar result was also obtained when strain GA74-1A (CTTP c t a l ) was compared with the isogenic cttl disruption mutant GA74T-1A (data not shown).

DISCUSSION
The results of this investigation demonstrate that transcription of the CTTl gene, which encodes the cytosolic catalase T of S. cereuisiae, is controlled by heat shock. This finding increases our understanding of the complex mode of regulation of this gene, which has previously been shown to be controlled by oxygen via heme and by nutrient availability via CAMP. The control of expression of CTTI is likely to reflect functional aspects. I t suggests that the cytosolic catalase of S. cereuisiae is needed when oxidative stress caused by oxygen metabolites is combined with other types of stress (heat, nutrient starvation). Results obtained in the course of this investigation are consistent with this conclusion. They demonstrate that catalase T is not only induced by heat shock, but also has a protective effect at high temperatures. It is obvious from our results as well as from previous information that catalase is only one of a number of factors important for heat stress protection. Our demonstration of a protective HZOZ (mM) FIG. 5. Hydrogen peroxide resistance of heat shocked and noninduced cells. Strains were grown a t 22 "C to logarithmic phase. A part of the cultures was subjected to heat shock at 37 "C for 30 min, and heat shocked and noninduced cells were suspended a t various concentrations of H202 in phosphate buffer, pH 6.8, a t a density of 10" cells/ml, incubated for 15 min a t 22 "C, and plated to assay for survivors. Closed symbols, cells kept a t 22 "C; open symbols, cells pretreated by heat shock; circles, strain A50 (cttl ctal); triangles, strain SP4 (CTTI'CTAI'). effect of a mild heat shock against toxic effects of hydrogen peroxide is consistent with the plausible role of catalase T as a peroxide scavenging agent under stress conditions. However, some questions connected to this function remain to be answered. One of these questions concerns the nature of the main cellular targets of hydrogen peroxide. It should be possible to identify these targets with the help of catalase null mutants of S. cerevisiae obtained by gene disruption.
Cross-protection by different types of stress is a phenomenon already known from prokaryotic systems. In Salmonella typhimurium, an overlapping set of proteins induced by oxidative stress and heat shock has been reported (35). Starvation-induced cross-protection against hydrogen peroxide has been observed in Escherichia coli (36). One of the catalases of E. coli, hydroperoxidase 11, the product of the katE gene, is induced at the level of transcription in cells entering stationary phase (37). In contrast to katE, katG, which encodes hydroperoxidase I, is induced by oxidative stress via the oxyR sensor (38).
The localization of the positive promoter element mediating heat shock control of CTTl should be an important basis for further insights into cellular mechanisms of stress protection. Our data show that heat shock induction of CTTl transcription is possible without involvement of heat shock transcription factor. A similar finding has recently been reported for the S. cerevisiae DDRA2 gene, which is activated by heat shock and by DNA damage (39). It is likely that heat shock control of DDRA2 and CTTl is mediated by the same factor(s) since the DDRA2 region reported to be sufficient for heat shock induction (39) contains sequences similar to both types of elements identified in this study. Coordinate control of expression of a catalase gene and of a gene transcribed in response to DNA damage is not surprising, since DNA damage is thought to be the main cause of toxicity of oxygen radicals (40).
Our results further demonstrate that induction of CTTl transcription by heat shock and by nitrogen starvation occurs via the same DNA elements. It is consistent with this notion that both effects have been shown to be antagonized by CAMP-dependent protein phosphorylation, and that no significant derepression of CTTl transcription by a low cAMP level was detected in cells grown at 23 "C (10,17). An element of the SSA3 gene encoding one of the HSP70 proteins of S. cerevisiae has recently been reported to be important for induction of transcription by nutrient limitation, and to be under negative control by cAMP (31), but a role of this element in heat shock control was not recognized in this study. This element is similar in sequence to one of the two CTTl regions important for nutrient and heat shock control (sequence 5'TAAGGG3'; see Fig. 3). The individual functions of the two subelements important for control by heat shock and by nutrients (CAMP) have to be investigated in further experiments. In principle, either both elements might be targets of heat shock and cAMP signals or one of the two sequences could be a nutrient (CAMP) control element and the other one a heat shock element. In the latter case, synergistic interaction of both elements would be necessary for full response to heat shock and nutrient levels. The fact that synergism between the separate heme (HAP1) control elements of the promoter with heat shock and nutrient control elements has been observed (17) demonstrates that this assumption is reasonable.
It appears likely that the combination of positive control by heat shock with negative cAMP control will be observed in a major subgroup of genes encoding stress proteins. Sequences similar to one of the elements identified in our study (5'TAAGGG3') are present in the upstream regions of all genes reported to be under combined control by these two mechanisms (UBI4 (12, 32) and HSPl2 (14) in addition to CTTl and SSA3). It should be pointed out, however, that, with the exception of DDRA2 and CTTl, the heat shock response of the genes mentioned is at least partly mediated by heat shock transcription factor. It remains to be investigated whether these newly discovered control elements respond to other types of stress, how the two sequences important for derepression by heat shock and nutrient stress interact, and which factors are involved in the integration of different types of signals like heat stress and nutrient availability.