Vol. 11, No. 1

MOLECULAR AND CELLULAR BIOLOGY, Jan. 1991, p. 281-288 0270-7306/91/010281-08$02.00/0 Copyright © 1991, American Society for Microbiology

Regulation of Heat Shock Factor in Schizosaccharomyces pombe More Closely Resembles Regulation in Mammals than in Saccharomyces cerevisiae GREGORY J. GALLO, THOMAS J. SCHUETZ, AND ROBERT E. KINGSTON* Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114, and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 Received 10 August 1990/Accepted 10 October 1990

The heat shock response appears to be universal. All eucaryotes studied encode a protein, heat shock factor (HSF), that is believed to regulate transcription of heat shock genes. This protein binds to a regulatory sequence, the heat shock element, that is absolutely conserved among eucaryotes. We report here the identification of HSF in the fission yeast Schizosaccharomyces pombe. HSF binding was not observed in extracts from normally growing S. pombe (28C) but was detected in increasing amounts as the temperature of heat shock increased between 39 and 45°C. This regulation is in contrast to that observed in Saccharomyces cerevisiae, in which HSF binding is detectable at both normal and heat shock temperatures. The S. pombe factor bound specifically to the heat shock element, as judged by methylation interference and DNase I protection analysis. The induction of S. pombe HSF was not inhibited by cycloheximide, suggesting that induction occurs posttranslationally, and the induced factor was shown to be phosphorylated. S. pombe HSF was purified to near homogeneity and was shown to have an apparent mobility of approximately 108 kDa. Since heat-induced DNA binding by HSF had previously been demonstrated only in metazoans, the conservation of heat-induced DNA binding by HSF among S. pombe and metazoans suggests that this mode of regulation is evolutionarily ancient.

Phosphorylation of human HSF is also induced by heat (10), suggesting that this modification might be widespread. The absolute conservation of the HSE in all eucaryotes suggests that the HSE and the factors that bind to it are derived from a common progenitor. The differences in the regulation of HSF in S. cerevisiae and metazoans therefore indicate that there has been an evolutionary divergence in certain aspects of the heat shock response. The observed heat inducibility of DNA binding by HSF in Drosophila and human cells may reflect a change in the regulation of the heat shock response that is limited to metazoans. Alternatively, inducible binding of HSF might be the norm in most eucaryotes, with constitutive binding by HSF in S. cerevisiae reflecting an exception. Schizosaccharomyces pombe is slightly more closely related to S. cerevisiae than to metazoans (20). Charaterizing the induction of HSF in S. pombe therefore is of interest in terms of the evolution of the heat shock response. Understanding whether regulation of HSF in S. pombe resembles that in metazoans is also of practical interest. A complete characterization of the mechanism of HSF induction in Drosophila and human cells is problematic because of the difficulties in performing homologous recombination in these organisms. When clones for these genes become available, it will be difficult to analyze the effects of mutations in vivo because of the concomitant expression of the endogenous HSF. Because of its easily manipulated genome, S. cerevisiae is a useful organism for the study of regulatory mechanisms; unfortunately, the constitutive binding of HSF prohibits the use of S. cerevisiae in the study of HSF induction. S. pombe resembles higher eucaryotes in some regulatory mechanisms such as transcription start site utilization (19a) and pre-mRNA splicing (8). The HSE from the human hsp70 promoter has been found to be regulated similarly in S. pombe and humans; that is, it plays no role in

The response of cells to heat has been well characterized for a wide variety of organisms (see references 3, 12, 13, and 18 for reviews). In particular, the induction of a 70-kDa heat shock protein gene (hsp70) has been analyzed in detail in Saccharomyces cerevisiae, Drosophila, and human cells. The hsp70 promoters in each of these organisms contain a common transcription regulatory element termed the heat shock element (HSE) that confers heat inducibility on hsp70 as well as other heat shock genes. The HSE was originally defined by the 14-bp consensus sequence, CnnGAAnnT TCnnG (18). Subsequent analyses of numerous HSEs have more precisely defined the HSE as an arrangement of a variable number of inverted nGAAn motifs (1, 30). The protein that binds to the HSE has been termed heat shock factor (HSF) and has been identified in S. cerevisiae (in which it has been cloned [21, 23, 24, 26]), Drosophila melanogaster (17, 27, 29), human cells (5, 9, 10, 14, 16, 31), and other eucaryotic cells (31). It is currently believed that a trimer of HSF is required for efficient binding in S. cerevisiae and D. melanogaster (19, 22). It appears that three nGAAn repeats constitute a single binding site for this HSF complex. Despite the apparent absolute conservation of the heat shock element in S. cerevisiae, Drosophila, and human cells, the regulation of the heat shock response varies in these species. All three organisms contain heat shock factor, but the Drosophila and human HSFs are induced to bind the HSE upon heat shock, while S. cerevisiae HSF exhibits constitutive binding at normal growth temperatures (9, 21, 27, 31). Activation of hsp70 transcription in S. cerevisiae under heat shock conditions is postulated to be dependent on a temperature-induced phosphorylation of HSF (21, 24).

*

Corresponding author. 281

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basal expression but confers high levels of heat inducibility (19a). A genetic analysis of HSF in S. pombe might therefore reveal mechanisms used during the heat shock response by metazoans. Before starting such an analysis, however, it is first necessary to characterize biochemically the induction of HSF in S. pombe. We present evidence here that S. pombe has an HSF that is induced to specifically bind the HSE subsequent to heat stress. The S. pombe HSF is present in cells prior to heat shock and is induced posttranslationally in the presence of elevated temperatures. We have purified the S. pombe HSF to near homogeneity by affinity chromatography and have shown that this protein has an apparent molecular size of approximately 108 kDa. The purified HSF is shown to be phosphorylated. The regulation of HSF in S. pombe is therefore similar to the regulation in metazoans with regard to the inducibility of DNA binding. S. pombe thus provides a useful system for genetic analysis of the heat shock response.

MATERIALS AND METHODS Strains and media. The S. pombe protease-deficient strain SP128 (h- leul) was used for all experiments. This strain was originally characterized in the laboratory of Mitsuhiro Yanagida (University of Kyoto, Kyoto, Japan) and was obtained from the laboratory of David Beach (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Cells were maintained on a shaker in YE medium (0.5% yeast extract, 2.0% dextrose) at 28°C. Cells for extract preparation were grown to approximately 107 cells per ml at 28°C and either maintained at 28°C or shifted to heat shock temperatures (39 and 43°C) for the times indicated in the figure legends. Cells inhibited with cycloheximide were grown in SD medium (0.17% yeast nitrogen base without amino acids, 0.5% ammonium sulfate, 2.0% dextrose) supplemented with 300 jig of L-leucine per ml. Cycloheximide (final concentration of 100 ,ug/ml) was added to the cells 30 min before the shift to heat shock temperatures. Extract preparation. Whole-cell extracts were prepared by using a modification of the procedure described by Sorger and Pelham (23). Normal and heat-shocked cells were immediately placed on ice and subsequently centrifuged at 4°C to pellet the cells. Cells were washed twice with ice-cold 1 x phosphate-buffered saline, pelleted, and resuspended at 2 x 109 cells per ml of breakage buffer (10% glycerol, 200 mM Tris [pH 8.0], 10 mM MgCI2, 1 mM phenylmethylsulfonyl fluoride [PMSF], 20 ,ug of leupeptin per ml, 40 ,ug of aprotinin per ml). Frozen beads were prepared by freezing the cell suspension dropwise in liquid N2. Frozen beads were ground to a fine powder in a mortar and pestle cooled in liquid N2 or in a Mini-Mate chopper (Cuisinart) for 1 min at high speed. The powder was thawed rapidly at room temperature and stirred gently on ice for 30 min after the addition of ammonium sulfate to a final concentration of 0.4 M. Cellular debris was removed by centrifugation at 47,000 rpm in Beckman type 70 Ti rotor for 1 h. The supernatant was precipitated by the addition of ammonium sulfate (0.35 g/ml), collected by centrifugation, and resuspended at 109 cells per 0.3 ml in dialysis buffer (20 mM HEPES [N-2hydroxyethylpiperazine-N'2-ethanesulfonic acid; pH 7.9], 20% glycerol, 0.1 M KCI, 0.2 mM EDTA, 0.5 mM dithiothreitol [DTT], 0.5 mM PMSF). The extract was dialyzed against two changes (100 volumes each) of dialysis buffer for 2 to 5 h.

MOL. CELL. BIOL.

Mobility shift gel electrophoresis. The 32P-labeled 40-bp synthetic HSE oligonucleotide (5'-AATTGCGAAACCCCTG GAATATTCCCGACCTGGCAGCCTC-3'; underlined motifs identify the human hsp7O promoter HSE) used as a probe has been described elsewhere, as have binding and electrophoresis conditions (9). The amount of S. pombe extract used in binding reactions was always based on equivalent cell numbers because the absolute protein concentrations are affected by the intensity and duration of heat shock. In general, binding reaction mixtures contained between 10 and 20 ng of S. pombe extract. Specific competition assays were performed by coincubating the probe and either a 20- or 50-fold molar excess of nonlabeled competitor DNA with the extract. The competitors included nonlabeled probe, a D. melanogaster HSE (6), a synthetic dimer of the HSE with the sequence 5'-GGTCGACTGGAATAlTCCGGAATAiLC CGGGATCCGAGCC-3' (the nGAAn motifs are underlined); a mutant dimer of HSE with the sequence 5'-GGTC-

GACTGTAATATTACGTAATAHTACGGGATC-

CGAGCC-3' (mutated residues are shown in bold), the CAAT element (9), and the ATF element (25). Bands were visualized by autoradiography. Methylation interference and DNase I protection assays. The probes used for both methylation interference and DNase I protection assays correspond to the sequences of the human hsp7O promoter between -132 and -75 (7). The top strand was 32p labeled by using T4 kinase, and the bottom strand was 32p labeled by using the Klenow fragment of DNA polymerase. Methylation interference assays were performed as described by Gilman et al. (4). Probes were methylated with dimethyl sulfate, incubated with 43°C S. pombe extract, and electrophoresed as described above. HSF-specific bands and nonbound probe bands were visualized by autoradiography, excised, and eluted. The isolated DNAs were cleaved at methylated G residues with piperidine and analyzed on a 10% sequencing gel. Interfering methylated residues were identified by comparing the bound and free lanes after autoradiography. DNase I protection assays were performed by incubation of affinity-purified S. pombe HSF with the probes described above and subsequent digestion with 0.8 U of DNase I. Cleavage products were analyzed on a 10% sequencing gel, and protected regions were identified by autoradiography. Denaturation and renaturation of S. pombe HSF. Renaturation of S. pombe HSF after denaturation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) was performed as described previously (2) except that protein bands were not visualized by KCl staining. A 6-mg sample of crude 43°C S. pombe extract was electrophoresed on a 10% SDS-polyacrylamide gel. The proteins were excised beginning at the resolving gel in a 1-cm gel slice and subsequently in 0.5-cm gel slices. The protein was electroeluted from the slices, acetone precipitated, and renatured exactly as described previously (2). The renatured fractions were assayed for S. pombe HSF by mobility shift gel electrophoresis. The apparent molecular weight ranges of HSF-containing fractions were verified by silver staining (15) after SDS-PAGE. Purification of S. pombe HSF. A 350-mg sample of heat shock S. pombe extract was brought to a final volume of 120 ml in load buffer A (20 mM HEPES [pH 7.9], 20% glycerol, 0.05 M KCI, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 1 jig of pepstatin A per ml, 1 ,ug of aprotinin per ml, 1 ,ug of leupeptin per ml, 0.5 mM benzamidine, 0.2% Nonidet P-40 [final concentrations]) and applied to a 50-ml packed-volume single-stranded DNA-cellulose column (Pharmacia) at a flow

S. pombe HEAT SHOCK FACTOR

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rate of 2 column volumes per h. The column was subsequently washed with 4 column volumes of the load buffer. HSF was eluted with 3 column volumes of the load buffer adjusted to 0.2 M KCI and collected in 10-ml fractions. Fractions were assayed for HSF activity by mobility shift gel electrophoresis, and the HSF-containing fractions were assayed for KCl concentration by conductivity and protein concentration by the Bradford assay (Bio-Rad). The HSFcontaining fractions were combined and adjusted to 0.3 M KC1-1 mM MgCl2-20 mM N-octylglucoside. The DNA affinity column was prepared exactly as described by Wu et al. (28), using the synthetic oligonucleotide 5'-CTAGAAGCTT-3' described by Sorger and Pelham (23). Hybridization and ligation of this oligonucleotide results in a sequence consisting entirely of nGAAn repeats. A 0.5-ml HSE affinity column was equilibrated in load buffer B (20 mM HEPES [pH 7.9], 20% glycerol, 0.3 M KCI, 1 mM MgCl2, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, 1 pLg of pepstatin A per ml, 1 ,ug of aprotinin per ml, 1 ,ug of leupeptin per ml, 0.5 mM benzamidine, 0.2% Nonidet P-40, 20 mM N-octylglucoside, 0.1 mg of hemoglobin per ml). The combined HSF fractions (see above) were applied to the HSE affinity column at a flow rate of 2 column volumes per h. The column was washed with 2 ml of load buffer B adjusted to 0.5 M KCl; HSF was eluted with load buffer B adjusted to 2.0 M KCl-20 mM MgCl2 and collected in 0.35-ml fractions. HSF-containing fractions were identified by mobility shift gel electrophoresis and subsequently combined and dialyzed against load buffer B. The dialyzed fractions were reapplied to the HSE affinity column and treated as described above. The peak fraction of HSF activity was dialyzed against dialysis buffer (see above) and was visualized by l1o PAGE with silver staining. The yield and fold purification for each column were calculated from the quantitation of HSF binding activity in mobility shift gel electrophoresis assays, using a Betascope 603 blot analyzer (Betagen Corp.). Phosphatase treatment and UV cross-linking of HSF. Approximately 20 ng of affinity-purified by S. pombe HSF was treated with 2 U of calf intestinal phosphatase (CIP) in the absence or presence of 33 ,uM ammonium molybdate, a phosphatase inhibitor, for 30 min at 37°C. The phosphatasetreated extract was analyzed by mobility shift gel electrophoresis. Phosphatase-treated factor was also used in binding reactions with a bromodeoxyuridine-substituted, 32p_ labeled HSE probe for UV cross-linking as described previously (10). Briefly, HSF-probe complexes were separated from free probe on a 1% low-melting-temperature agarose gel, irradiated with UV light for 15 min, and exposed to film. HSF-specific bands were excised, boiled in sample buffer, and analyzed on an 8% SDS-polyacrylamide gel. The cross-linked HSF-probe complexes were visualized by auto-

radiography. RESULTS S. pombe exhibits a heat-inducible HSE binding activity. Whole-cell extracts were prepared from S. pombe grown at the normal temperature (28°C) or shifted to heat shock temperatures (39 and 43°C). These extracts were analyzed for HSE binding activity in the gel mobility shift assay (Fig. 1). The labeled oligonucleotide used as a probe in this assay corresponds to the wild-type human hsp7O HSE (see Materials and Methods). This HSE contains two consensus nGAAn motifs and a third imperfect motif. This HSE has

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FIG. 1. Identification of an HSE-specific binding protein in whole-cell extracts prepared from heat-treated S. pombe. Shown are mobility shift gel electrophoresis assays using a labeled probe corresponding to the human hsp7O gene HSE (Materials and Methods): migration of probe without added protein is shown in lane 1 (P). (A) Binding of proteins in extracts prepared from S. pombe cells grown at the normal temperature (28°C; lane 2) or cells subjected to heat shock temperatures (39 and 43°C; lanes 3 and 4). Band H appears only in extracts from heat-treated cells. (B) competition of binding of 43°C extracts to the HSE probe with a variety of specific (HSE, Dros. [Drosophila], dimer; lanes 6 to 11) and nonspecific (mut [mutant dimer], CAAT, ATF; lanes 12 to 17) competitor DNAs (Materials and Methods). The first and second lanes for each competitor correspond to 20- and 50-fold molar excesses of competitor relative to probe DNA. Lanes 5 and 18 have no added competitor DNA.

been shown to regulate heat-inducible transcription in S. pombe (19a). Proteins that bound to the probe were apparent in 28, 39, and 43°C extracts (Fig. 1A, lanes 2 to 4); however, a more slowly migrating activity, not observed in 28°C extracts, was seen faintly in 39°C extracts and as a distinct band in 43°C extracts (lane 4, band H). To determine the binding specificity of band H, competitions were performed by using a variety of nonlabeled competitor DNA fragments. Band H was effectively competed for by all HSE-containing fragments (Fig. 1B, lanes 6 to 11) but not by a mutant HSE (lanes 12 and 13) or other transcription elements (lanes 14 to 17). These data indicate that there is a heat-inducible HSEspecific binding activity present in S. pombe grown at 43°C. The predominant band in all extracts (X; Fig. 1) was effec-

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284

*.

tively competed for by both HSE-containing fragments and an oligonucleotide containing the CAAT element (Fig. 1B, lanes 6 to 13), suggesting that it is not specific for the HSE. The CAAT competitor has no homology to the HSE consensus, but sequence analysis of the probe and CAAT DNA fragments reveals a region of homology (7 of 9 bp) that may contain the binding site for the band X factor. Because band X activity did not appear to be specific for the HSE, it is unlikely that this binding activity is a precursor to band H. Furthermore, the amount of band X did not appreciably decrease as the amount of band H increased, again inconsistent with a precursor-product relationship. Finally, phosphatase treatment did not shift band H to band X (see below) in a manner analogous to results of Sorger et al. (21) for S. cerevisiae HSF. Overall, these data suggest that S. pombe has an HSEspecific binding activity that is induced following heat shock. This activity will subsequently be referred to as HSF. The exact specificity of S. pombe HSF binding is addressed below. S. pombe HSF interacts specifically with the HSE. To characterize the binding specificity of HSF in more detail, methylation interference and footprinting assays were used. The probe used for these assays corresponds to human hsp7O sequences from -132 to +75, which includes the HSE centered at -100. Methylation interference assays were conducted by incubating methylated probe with crude 43°C S. pombe extract prior to gel mobility shift electrophoresis. The free and HSF-bound probe were isolated, cleaved, and analyzed. Methylated residues that interfere with HSF binding are identified as underrepresented bands in lanes B (bound) of Fig. 2A. The results demonstrate that residues methylated in the nGAAn motifs of the HSE interfered with binding. Interestingly, there appeared to be some interference by methylated residues in an nGAAn motif located between -114 and -112 (labeled U in Fig. 2C). The human HSE is defined as two inverted nGAAn motifs (labeled I and II in Fig. 2C) and one degenerate half-site (nGACn; labeled D in Fig. 2C); however, the U motif maintains the proper orientation (with one missing nGAAn motif) with the I and II nGAAn motifs of the HSE. It has been shown previously that the nGAAn motifs need not be contiguous for HSE function (1); thus, it appears that the S. pombe HSF can bind the intact but noncontiguous U motif that appears not to be utilized in human cells (9). It is unclear whether the S. pombe HSF interacts with the D motif, since it is protected

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G-residue cleavages of probe DNA (D), HSF-bound probe (B), and nonbound probe (F) are shown for both strands. Underrepresented bands in the B lanes (relative to the D and F lanes) are indicated with arrowheads to identify methylated G residues that interfere with HSF binding. Aberrantly cleaved A residues that are underrepresented in the B lanes are also seen but are not indicated by arrows. (B) Results of DNase I cleavage of labeled probes (described above) bound to either 0 (-), 18, or 36 ng of purified S. pombe HSF. The M lane for each probe is methylated probe DNA cleaved at G residues. Brackets indicate the boundaries of DNase I cleavage protection. (C) Summary of the data presented in panels A and B. The DNA sequence is that of the human hsp7O gene HSE; bars between the strands labeled I and 11 identify the nGAAn motifs of the HSE. The bar labeled D represents a degenerate nGAAn motif (GAC instead of GAA), and U represents an upstream nGAAn motif that is in the proper orientation for HSF binding (see Results). Arrows and brackets correspond to those depicted in panels A and B, respectively; dots represent methylated A residues that interfere with HSF binding (data not shown).

VOL.

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S. pombe HEAT SHOCK FACTOR 0

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________________________ - Free FIG. 3. Time course of S. pombe HSF activation during 43°C heat shock. Mobility shift gel electrophoresis was performed by using a labeled HSE probe (see Fig. 1) incubated with 28°C extract (0) and extracts from cells incubated at 43°C for 15, 30, 60, and 120 min prior to extract preparation. H represents the S. pombe HSF.

from DNase I cleavage while the results of methylation interference indicate only a potential weak interaction at this site (Fig. 2C). The involvement of three nGAAn motifs in S. pombe HSF binding is consistent with the current models for the HSE in which the optimal HSE is composed of three or multiples of three nGAAn motifs (1, 30). DNase footprint analyses with crude 43°C S. pombe extract resulted in only weak protection of the HSE from DNase cleavage (data not shown). In an effort to improve DNase protection, subsequent assays utilized purified S. pombe HSF (see below). Purified HSF protected a region of DNA surrounding the HSE from DNase digestion (Fig. 2B). DNase protection extended beyond the GAA between -114 to -112, further suggesting interaction of S. pombe HSF with this site. The results of methylation interference and DNase footprint assays are summarized in Fig. 2C. The results of gel mobility shift competitions, methylation interference, and DNase footprints strongly argue that band H (Fig. 1) is a heat-inducible S. pombe HSF. The regulation of this factor in response to heat is further analyzed below. S. pombe HSF is induced posttranstionally. S. pombe cells grown at 28°C were brought to 43°C, and extracts were prepared at 15, 30, 60, and 120 min after the temperature shift. HSF binding activity was apparent within 15 min of the shift to heat shock temperatures, raising the possibility that the factor is not synthesized de novo (Fig. 3). To test this possibility, cells were heat shocked in the presence of the protein synthesis inhibitor cycloheximide. S. pombe HSF binding activity could be induced in the presence of cycloheximide (Fig. 4). The levels of cycloheximide used effectively inhibited protein synthesis, as shown by [35S]methionine labeling of aliquots of the cells used for extract preparation (data not shown). These results suggest that inducible DNA binding by S. pombe HSF occurs through posttranslational modification of preexisting molecules rather than a de novo synthesis of binding-capable molecules. Purification of S. pombe HSF. Prior to chromatographic purification of S. pombe HSF, an approximate size for the factor was determined by separating crude heat shock extract on an SDS-polyacrylamide gel and renaturing HSF binding activity. S. pombe crude extract was excised in 0.5-cm gel fragments from an SDS-polyacrylamide gel. After recovery of the protein from each fragment, the samples

-Free

FIG. 4. Activation of S. pombe HSF in the absence and presence of the protein synthesis inhibitor cycloheximide. Mobility shift gel electrophoresis was performed as for Fig. 3, using either 2 or 4 RI of extract prepared from equivalent numbers of cells grown in the absence (- cyc) or of cycloheximide (+ cyc) presence at 28 or 43°C. Cycloheximide was added to 100 ,ug/ml 30 min before a 30-min incubation at 43°C. H identifies the S. pombe HSF.

were denatured and subsequently renatured. The renatured protein fractions were analyzed for HSE binding activity by the gel mobility shift assay (Fig. 5). The recovered activity was present in fractions 3 and 4; these fractions correspond to molecular size ranges 135 to 114 and 122 to 105 kDa, respectively, suggesting that S. pombe HSF has an apparent mobility of between 135 and 105 kDa. The results of purification support this conclusion. HSF from crude S. pombe extract was purified to near homogeneity by conventional affinity chromatography. Extract was first passed over a single-stranded DNA-cellulose column at 0.05 M KCl, a concentration that retains HSF. The S. pombe HSF was eluted with 0.2 M KCI. HSF-

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FIG. 5. Denaturation and renaturation of S. pombe HSF from crude 43°C extract. Crude 43°C S. pombe extract isolated as specific fractions from an SDS-polyacrylamide gel were denatured and renatured before use in mobility shift electrophoresis. Lane E is crude extract used as starting material. Fractions 1 to 8 progress from high to low molecular weight. Fractions 3 and 4 contain HSF (H) and correspond to apparent mobility ranges of 135 to 114 and 122 to 105 kDa, as determined by analyzing the protein by silver staining an SDS-polyacrylamide gel.

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TABLE 1. Purification of S. pombe HSF Vol

Protein

Protein source

(ml)

(mg/ml)

Total protein (mg)

tfmol/pl)"

Activity

Sp act (fmol/mg)b

Starting material Single-stranded DNA-cellulose Affinity 1 Affinity 2 Overall

120 20 2.0 0.35

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350 8.6 NA 2.3 x 10-3

0.10 0.34 0.68 0.45

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containing fractions were brought to 0.3 M KCI and loaded onto an HSE-DNA affinity column. After a 0.5 M KCI wash, HSF was eluted with 2.0 M KCI-20 mM MgCl2. The HSF-containing fractions were combined, dialyzed to 0.3 M KCI, reapplied to the affinity column, and eluted as described above. This procedure resulted in approximately a 1,900-fold purification of HSF with a 1.4% yield (Table 1). The peak HSF affinity fraction as identified by mobility shift gel electrophoresis was analyzed by SDS-PAGE (Fig. 6). The predominant band observed by silver stain was a diffuse band centered at 108 kDa. Minor contaminants (the most obvious of which was 70 kDa) were visible; however, several lines of evidence suggest that the 108-kDa band was S. pombe HSF. The size of the diffuse 108-kDa band is consistent with the estimated size of S. pombe HSF determined by denaturation-renaturation assays (see above) and UV cross-linking (see below). Furthermore, the intensity of the 108-kDa band correlates with the amount of binding activity present in both HSF-containing peak and shoulder fractions (data not shown). The diffuse nature of the HSF band in silver stain gels was consistently observed in several purifications. This may be the result of limited proteolysis despite the presence of protease inhibitors (see Materials and Methods) or may be representative of different degrees

69,000d

Yield (%)

Fold purification

57 20 12 1.4

22 NA 87 1,900

of posttranslational modifications. Both S. cerevisiae and human HSF have been shown to be phosphorylated (10, 21, 23, 24, 31). This phosphorylation has been postulated to be central to HSF-mediated stimulation of transcription (see Discussion); therefore, the phosphorylation state of S. pombe HSF was assayed. S. pombe HSF is modified by phosphorylation. Since affinity-purified S. pombe HSF yielded a discrete band in the gel mobility shift assay, this preparation was used to determine the effect of phosphatase on the mobility of HSF in gel shift assays and SDS-PAGE. Purified S. pombe HSF was treated with CIP in the absence and presence of the phosphatase inhibitor ammonium molybdate. The results of gel mobility shift assays showed that the mobility of the HSE-HSF complex was not significantly altered by phosphatase treatment (Fig. 7A). This result is consistent with those observed for human HSF indicating that phosphatase does not significantly affect HSE binding (3a, 10). To assess the phosphorylation state of S. pombe HSF more specifically, protein was treated with phosphatase and incubated with a bromodeoxyuridine-substituted, 32P-labeled HSE probe before electrophoresis and irradiation of the gel with UV light. The cross-linked protein was subsequently analyzed by SDS-PAGE (Fig. 7B). The HSF treated

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FIG. 7. Effect of CIP on purified S. pombe HSF. (A) Mobility shift gel electrophoresis of purified HSF (H), purified HSF treated with CIP (C), and purified HSF treated with CIP in the presence of the phosphatase inhibitor ammonium molybdate (C/A). (B) SDSpolyacrylamide gel of purified HSF treated as described above after UV cross-linking to a bromodeoxyuridine-substituted, 32P-labeled probe (Materials and Methods). The relative mobilities (by comparison with protein standards) are 128 kDa for untreated HSF (H), 121 kDa for phosphatase-plus-ammonium molybdate-treated HSF (C/ A), and 116 kDa for phosphatase-treated HSF (C).

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with CIP in the absence of inhibitor showed an increased mobility relative to the control reaction performed in the presence of inhibitor (Fig. 7B, lanes C versus C/A). The apparent molecular sizes of these bands are 116 and 121 kDa, respectively. The increase in the apparent molecular sizes of these bands relative to purified HSF (108 kDa; Fig. 6) is most likely due to a contribution from the cross-linked probe DNA. The increase in mobility of S. pombe HSF upon CIP treatment verifies the presence of phosphate modification of this protein. Note that the mobility of the HSF treated with CIP plus phosphatase inhibitor (121 kDa) was greater than that of untreated HSF (128 kDa). The reason for this difference is unknown at present. These data demonstrate that S. pombe HSF is phosphorylated. DISCUSSION The HSE and HSF have been identified as common components of the heat shock response in S. cerevisiae, Drosophila, and human cells. To date, however, studies of the heat shock response in S. pombe have been limited. The absolute conservation of the HSE sequence across all studied eucaryotes strongly suggested that this sequence would bind a regulatory factor in S. pombe. We demonstrate here that S. pombe indeed has a heat-induced protein with all of the characteristics of HSF. We further show that S. pombe HSF has characteristics that more closely resemble metazoan HSFs than S. cerevisiae HSF. The amount of HSF detectable in S. pombe increases with increasing temperature. Maximal HSF activity is obtained at heat shock temperatures of between 43 and 45°C, but significantly lower levels of HSF activity are seen at 39 and 41°C (Fig. 1 and data not shown). The gradual effect of temperature upon the levels of HSF may be indicative of a carefully regulated induction of HSF binding capability. Consistent with this suggestion is the observation that levels of transcription from the human hsp7O promoter in S. pombe gradually increase with increasing temperatures of heat shock between 37 and 43°C (19a). This transcriptional induction depends on the same HSE that we used here to identify S. pombe HSF and presumably results from HSF binding. It has been shown that higher eucaryotes also express intermediate levels of HSF at moderate heat shock temperatures

(31).

Our demonstration that S. pombe has a heat-inducible HSF contradicts the conclusions of Zimarino et al. (31), who suggest that S. pombe has only a constitutively binding form of HSF. These investigators performed their heat shock at 39°C, a temperature at which we see only a faint HSF band by mobility shift gel electrophoresis (Fig. 1A, lane 3). The characterization of the constitutive HSE binding activity previously identified (31) was not extensive; it is therefore unclear whether the reported binding protein is entirely specific for the HSE. The only band we identify that behaves similarly to a constitutive binding protein is band X (Fig. 1), which we demonstrate not to be HSE specific. The similarity of heat-induced DNA binding by HSF in S. pombe and metazoans makes it interesting to compare other aspects of the HSFs and their regulation in these organisms. The apparent molecular size of S. pombe HSF is 108 kDa. This value is within the range of reported mobilities for HSF, which vary between 83 to 87 kDa for human HSF and 130 to 150 kDa for S. cerevisiae HSF (5, 23). The data show that S. pombe HSF can be induced to bind the HSE in the absence of protein synthesis, implying that HSF is not synthesized de novo upon heat shock. These data for S. pombe HSF

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activation are analogous to the posttranslational induction of human HSF (9, 10) and HSF from other higher eucaryotes (31, 32). We have shown that S. pombe HSF is phosphorylated. This phosphorylation is apparently not required for the maintenance of binding since factor treated with phosphatase can still bind the HSE. The S. cerevisiae and human HSFs are also phosphorylated (10, 21, 24). Although the role of HSF phosphorylation remains unclear, it has been suggested that phosphorylation might be required for stimulation of transcription (21, 24). We have presented evidence that the regulation of S. pombe HSF is similar to such regulation in higher eucaryotes. The identification of a heat-inducible HSF in fungi suggests that this mode of regulation is evolutionarily ancient. It would not be surprising if heat induction of HSF is a conserved regulatory mechanism among most eucaryotes, given the high degree of conservation in other aspects of the heat shock response. This conclusion raises the possibility that S. cerevisiae may have divergently evolved a constitutive DNA-binding form of HSF. It will be interesting to compare regulation of HSF in other eucaryotes such as plants and protists. The similarity in regulation of the heat shock response between S. pombe and metazoans is reminiscent of similarities in other cellular pathways between these organisms. For example, the mechanisms of cell cycle regulation in S. pombe and in humans share many characteristics. These have been exploited to help identify and characterize factors important for regulation of the cell cycle in humans (e.g., see reference 11). A similar approach may prove useful for analysis of the heat shock response. A genetic analysis of the heat shock response in S. pombe might therefore identify genes and pathways relevant to the regulation of heat shock in metazoans. ACKNOWLEDGMENTS We thank H. L. Prentice, J. L. Workman, I. C. A. Taylor, and J. S. Larson for helpful comments and critical reading of the manuscript. We thank Rachel Hyde for help in preparing the manuscript. G.J.G. was supported by National Research Service award 5 F32 GM12865-02 from the National Institute of General Medical Sciences. This work was supported by a grant from Hoechst AG.

REFERENCES 1. Amin, J., J. Ananthan, and R. Voellmy. 1988. Key features of heat shock regulatory elements. Mol. Cell. Biol. 8:3761-3769. 2. Bagchi, M. K., S. Y. Tsai, M.-J. Tsai, and B. W. O'Malley. 1987. Purification and characterization of chicken ovalbumin gene upstream promoter transcription factor from homologous oviduct cells. Mol. Cell. Biol. 7:4151-4158. 3. Craig, E. A. 1985. The heat shock response. Crit. Rev. Microbiol. 18:239-280. 3a.Gallo, G. J. Unpublished data. 4. Gilman, M., R. Wilson, and R. Weinberg. 1986. Multiple protein binding sites in the 5'-flanking region regulate c-fos expression. Mol. Cell. Biol. 6:4305-4316. 5. Goldenberg, C. J., Y. Luo, M. Fenna, R. Baler, R. Weinmann, and R. Voellmy. 1988. Purified human factor activates heat shock promoter in HeLa cell free transcription system. J. Biol. Chem. 263:19734-19739. 6. Holmgren, R., K. Livak, R. Morimoto, R. Freund, and M. Meselson. 1979. Studies of cloned sequences from four Drosophila heat shock loci. Cell 18:1359-1370. 7. Hunt, C., and R. I. Morimoto. 1985. Conserved features of eukaryotic hsp70 genes revealed by comparison with the nucleotide sequence of human hsp70. Proc. Natl. Acad. Sci. USA 82:6455-6459.

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8. Kaufer, N. F., V. Simanis, and P. Nurse. 1985. Fission yeast Schizosaccharomyces pombe correctly excises a mammalian RNA transcript intervening sequence. Nature (London) 318:7880. 9. Kingston, R. E., T. J. Schuetz, and Z. Larin. 1987. Heatinducible human factor that binds to a human hsp70 promoter. Mol. Cell. Biol. 7:1530-1534. 10. Larson, J. S., T. J. Schuetz, and R. E. Kingston. 1988. Activation in vitro of sequence specific DNA by a human regulatory factor. Nature (London) 335:372-375. 11. Lee, M. G., and P. Nurse. 1987. Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature (London) 327:31-35. 12. Lindquist, S. 1986. The heat shock response. Annu. Rev. Biochem. 55:1151-1191. 13. Lindquist, S., and E. A. Craig. 1988. The heat shock proteins. Annu. Rev. Genet. 22:631-677. 14. Morgan, W. D., G. T. Williams, R. I. Morimoto, J. Greene, R. E. Kingston, and R. Tjian. 1987. Two transcriptional activators, CCAAT-box-binding transcription factor and heat shock transcription factor, interact with a human hsp70 promoter. Mol. Cell. Biol. 7:1129-1138. 15. Morrissey, J. H. 1981. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal. Biochem. 117:307-310. 16. Mosser, D. D., N. G. Theodorakis, and R. I. Morimoto. 1988. Coordinate changes in heat shock element-binding activity and hsp70 transcription rates in human cells. Mol. Cell. Biol. 8:4736-4744. 17. Parker, C., and J. Topol. 1984. A Drosophila RNA polymerase II transcription factor for the heat shock gene binds to the regulatory site of an hsp 70 gene. Cell 37:273-283. 18. Pelham, H. R. B. 1985. Activation of heat-shock genes in eukaryotes. Trends Genet. 1:31-35. 19. Perisic, O., H. Xiao, and J. T. Lis. 1989. Stable binding of Drosophila heat shock factor to head-to-head and tail-to-tail repeats of a conserved 5 bp recognition unit. Cell 59:797-806. 19a.Prentice, H. L. Unpublished data. 20. Sipiczki, M. 1989. Taxonomy and phylogenesis, p. 431-452. In A. Nasim, P. Young, and B. F. Johnson (ed.), Molecular

MOL. CELL. BIOL.

21. 22.

23. 24. 25.

26. 27.

28.

29. 30. 31. 32.

biology of the fission yeast. Academic Press, Inc., San Diego, Calif. Sorger, P. K., M. J. Lewis, and H. R. B. Pelham. 1987. Heat shock factor is regulated differently in yeast and HeLa cells. Nature (London) 329:81-84. Sorger, P. K., and H. C. M. Nelson. 1989. Trimerization of a yeast transcriptional activator via a coiled-coil motif. Cell 59:807-813. Sorger, P. K., and H. R. B. Pelham. 1987. Purification and characterization of a heat-shock element binding protein from yeast. EMBO J. 6:3035-3041. 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. Taylor, I. C. A., and R. E. Kingston. 1990. Ela transactivation of human HSP70 gene promoter substitution mutants is independent of the composition of upstream and TATA elements. Mol. Cell. Biol. 10:176-183. Weiderrecht, G., D. Seto, and C. S. Parker. 1988. Isolation of the gene encoding S. cerevisiae heat shock transcription factor. Cell 54:841-853. Wu, C. 1984. Activating protein factor binds in vitro to upstream control sequences in heat-shock gene chromatin. Nature (London) 311:81-84. Wu, C., C. Tsai, and S. Wilson. 1988. Affinity chromatography of sequence-specific DNA-binding proteins, p. 67-74. In J. K. Setlow (ed.), Genetic engineering: principles and methods. Plenum Press, New York. Wu, C., S. Wilson, B. Walker, I. Dawid, T. Paisley, V. Zimarino, and H. Ueda. 1987. Purification and properties of Drosophila heat shock activator protein. Science 238:1247-1253. Xiao, H., and J. T. Lis. 1988. Germline transformation used to define key features of heat-shock response elements. Science 239:1139-1142. Zimarino, V., C. Tsai, and C. Wu. 1990. Complex modes of heat shock factor activation. Mol. Cell. Biol. 10:753-759. Zimarino, V., and C. Wu. 1987. Induction of sequence-specific binding of Drosophila heat shock activator proteins without protein synthesis. Nature (London) 327:727-730.

Regulation of heat shock factor in Schizosaccharomyces pombe more closely resembles regulation in mammals than in Saccharomyces cerevisiae.

The heat shock response appears to be universal. All eucaryotes studied encode a protein, heat shock factor (HSF), that is believed to regulate transc...
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