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The DNA-binding domain of yeast Hsf1 regulates both DNA-binding and transcriptional activities
BBRC Biochemical and Biophysical Research Communications 346 (2006) 1324–1329 www.elsevier.com/locate/ybbrc
The DNA-binding domain of yeast Hsf1 regulates both DNA-binding and transcriptional activities Ayako Yamamoto, Hiroshi Sakurai
*
Division of Health Sciences, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 920-0942, Japan Received 8 June 2006 Available online 19 June 2006
Abstract The heat shock transcription factor (HSF) is a key regulator of the heat shock response. In Saccharomyces cerevisiae, the transcription activating ability of Hsf1 is repressed by its DNA-binding domain, but the detailed mechanism by which the inhibitory function is relieved in response to stress remains unknown. In this study, we isolated and characterized three hsf1 mutants with temperature-sensitive mutations in the DNA-binding domain. Two mutations inhibited DNA-binding activity, leading to decreased expression of target genes. The third mutation caused transcriptional defects without affecting DNA binding, and its suppressor mutation was located in a region important for sensing heat shock. These results indicate that the DNA-binding domain regulates both the DNA-binding and transcriptional activities of Hsf1, and suggest that these functions are located within discrete regions of the DNA-binding domain. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Heat shock transcription factor; Heat shock element; DNA-binding domain; Winged helix–turn–helix motif; Temperature-sensitive mutation; Intragenic suppressor mutation
In eukaryotes, the heat shock response is regulated by the heat shock transcription factor (HSF). This protein contains a helix–turn–helix DNA-binding domain (DBD) and a hydrophobic repeat region that is required for homotrimer formation. Homotrimeric HSF binds to the heat shock element (HSE), a conserved regulatory DNA sequence comprising at least three contiguous inverted repeats of 5 0 -nGAAn-3 0 upstream of the heat shock genes [1–3]. In the yeast Saccharomyces cerevisiae, HSF is encoded by the essential gene HSF1. Mutations in this gene cause defects in cell wall integrity, spindle pole body duplication, and cell cycle progression [4–7]. The target genes of Hsf1 encode a variety of proteins that function in protein folding and maturation, energy generation, carbohydrate metabolism, and cell wall organization [8,9]. Hsf1 binds to and activates transcription via the ‘‘perfect’’ type of HSE
*
Corresponding author. Fax: +81 76 234 4369. E-mail address: [email protected] (H. Sakurai).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.057
described above, as well as the ‘‘discontinuous’’ type of HSE containing one (gap type) or two (step type) gaps between the repeating units [9–11]. In mammalian cells, the predominant HSF isoform (HSF1) responds to various stresses by forming a homotrimer that translocates from the cytoplasm to the nucleus, where it binds to the HSE and becomes transcriptionally competent upon stress-induced phosphorylation [2]. Unlike its mammalian counterpart, S. cerevisiae Hsf1 is able to bind to the HSE in both a constitutive and inducible manner [8,12]. The Hsf1 activation domain possesses constitutive activating ability, which is regulated negatively by the DBD under normal physiological conditions [13,14]. Hsf1 becomes hyperphosphorylated and transcriptionally competent under thermal and oxidative stress, resulting in the transcription of target genes [15–17]. It has been suggested that it is the heat shock-induced phosphorylation that mitigates the negative regulation effected by the DBD [18]. In order to explore the role played by the Hsf1 DBD, we isolated and characterized temperature-sensitive DBD mutants and intragenic suppressor mutations. Our results
A. Yamamoto, H. Sakurai / Biochemical and Biophysical Research Communications 346 (2006) 1324–1329
demonstrate that the DBD is important for binding DNA and for regulation of activator function. Together with previous mutation analyses, our results suggest that regulation of activation is modulated by discrete regions in the DBD. Materials and methods Yeast strains and media. Saccharomyces cerevisiae strains used in this study are listed in Table 1. Strain HS126 contains a null mutation of HSF1 in the chromosome and a wild-type copy on a URA3-marked centromeric plasmid (YCp-URA3-HSF1) [17]. Cells containing hsf1 mutations were constructed from HS126 using plasmid shuffling [9,17]. Rich glucose (YPD), enriched synthetic glucose (ESD), and synthetic glucose (SD) media were prepared as described previously [9]. Isolation of temperature-sensitive hsf1 mutations. Temperature-sensitive hsf1 mutations were isolated as described previously [9]. In brief, random nucleotide alterations were introduced into HSF1 by PCR amplification in the presence of 0.1 mM MnCl2. Strain HS134 was transformed with a library of hsf1 mutations and transformants were replica-plated onto ESD medium containing 5-fluoroortic acid, to lose the resident YCp-URA3HSF1. Plates were incubated at 38 °C. Temperature-sensitive hsf1 mutations were identified in plasmids pAY5, pAY8, and pAY9, which contained substitutions of asparagine to tyrosine at amino acid (aa) 222 (N222Y), histidine to arginine at aa 220 (H220R), and phenylalanine to serine at aa 256 (F256S), respectively. The substitution R206S was introduced into hsf1 alleles of pAY5 and pAY8 via site-directed mutagenesis, generating pAY20 and pAY21, respectively [9]. Isolation of intragenic mutations suppressing the hsf1-F256S mutation. Random nucleotide alterations were introduced into hsf1-F256S as described above and the amplified fragments were cloned into a LEU2marked centromeric vector. The mutant library was used to transform strain TH2670 (Open Biosystems, Co.), in which expression of HSF1 was under control of the tetO promoter [19]. Transformants were grown on SD medium containing 20 lg/ml histidine, 20 lg/ml methionine, and 10 lg/ml doxycycline at 38 °C. One hsf1-F256S suppressor allele contained the additional substitutions V210A, S305C, and T401A, whereas another contained V210A and T88A. The substitution V210A was introduced into hsf1 alleles as above. Gel retardation analysis. A fragment encoding the N-terminal 583 aa of Hsf1 was cloned into pGEX-6P1, expressed in Escherichia coli, and purified, as described previously [17]. The polypeptide was incubated with 0.15 ng of 32P-labeled oligonucleotides in 12 ll of buffer containing 20 mM Hepes–KOH (pH 7.6), 50 mM potassium acetate, 2 mM EDTA, 0.05% Nonidet P-40, 8.3 lg/ml poly(dI–dC), and 6.7% glycerol. Samples were separated by electrophoresis on a 4% polyacrylamide gel in 25 mM
Table 1 Yeast strains used in this study Strains
Genotype (plasmid)
HS126
MATa ade2 his3 leu2 trp1 ura3 can1 hsf1::HIS3 YCp-URA3-HSF1 (pSK906) LEU2::SSA4-lacZ of HS126 YCp-TRP1-HSF1 (pK157) of HS126 YCp-TRP1-hsf1-N222Y (pAY5) of HS126 YCp-TRP1-hsf1-H220R (pAY8) of HS126 YCp-TRP1-hsf1-F256S (pAY9) of HS126 YCp-TRP1-hsf1-R206S (pAY19) of HS126 YCp-TRP1-hsf1-R206S-N222Y (pAY20) of HS126 YCp-TRP1-hsf1-R206S-H220R (pAY21) of HS126 YCp-TRP1-hsf1-V210A (pAY73) of HS126 YCp-TRP1-hsf1-V210A-F256S (pAY74) of HS126 YCp-TRP1-hsf1-V210A-N222Y (pAY77) of HS126 YCp-TRP1-hsf1-V210A-H220R (pAY78) of HS126 his3-1 leu2-0 met15-0 URA3::CMV-tTA kanR-tetO-TATA-HSF1
HS134 HS170T YAY5-1 YAY8-1 YAY9-1 YAY19 YAY20 YAY21 YAY73 YAY74 YAY77 YAY78 TH2670
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Tris–glycine (pH 8.8) and subjected to phosphorimaging. The nucleotide sequences of the oligonucleotide probes are shown in Fig. 1C. RNA analysis. Cells grown in YPD medium at 28 °C were heatshocked at 39 °C, and total RNA prepared from the cells was analyzed by reverse transcription (RT)-PCR, as described previously [17]. The amounts of PCR products were compared following normalization using ACT1 mRNA (encoding actin) as a control.
Results Isolation of temperature-sensitive hsf1 mutations We isolated three temperature-sensitive hsf1 mutations that supported cell growth at 28 °C but not at 38 °C (Fig. 1A and B). The winged helix–turn–helix DBD comprises three helices, four b-sheets, and a flexible loop (termed the wing); mutations H220R and N222Y are located in the turn region and F256S in the fourth b-sheet. These HSF residues are highly conserved among organisms. Residues H220 and N222 are more than 40% solvent accessible, whereas F256 is less than 20% solvent accessible and anchors the b-sheet to the hydrophobic core of the DBD [20,21]. In the HSF–HSE co-crystal, the side chains of H220 and N222 form water-mediated hydrogen bonds with the phosphate backbone of the DNA; the peptide bond backbone of these residues is involved in protein– protein interactions of the DBD dimer interface [22]. Binding of mutant Hsf1 proteins to the HSE The effects of mutations on the Hsf1–HSE interaction were analyzed by gel retardation assays using various HSE oligonucleotides and recombinant Hsf1 polypeptides that contained the DBD and hydrophobic repeat region (Fig. 1C and D). Wild-type Hsf1 formed protein–DNA complexes with oligonucleotides containing the perfecttype HSE (three or four nGAAn repeats, i.e., HSE3P and HSE4Ptt, respectively), gap-type HSE (HSEgap), and step-type HSE (HSEstep) (Fig. 1D, lanes 2–4). Since two Hsf1 trimers bind cooperatively in the interaction between Hsf1 and HSE4Ptt [18], the binding affinity of that interaction is about 10-fold higher than between Hsf1 and HSE3P, HSEgap, or HSEstep. The mutants Hsf1-N222Y and Hsf1-H220R exhibited 10-fold lower affinity than wild type for all HSEs investigated (lanes 5–7 and 8–10, respectively). These results are consistent with crystallographic analysis [22] and indicate that residues H220 and N222 are involved in Hsf1–HSE interactions. In contrast, the mutation F256S did not influence binding of Hsf1 to the various HSEs (lanes 11–13). Transcription of Hsf1 target genes in hsf1-N222Y and hsf1-H220R cells To evaluate whether the decrease in DNA-binding affinity affects heat-induced transcription, the mRNA levels of Hsf1 target genes in hsf1-N222Y and hsf1-H220R cells were analyzed by RT-PCR (Fig. 2). A temperature shift from 28
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A H1
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tcgacTTCtaGAAgcTTCcaGAAattagtgctactcga HSE3P
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tcgacTTCtaGAAgctagcaGAAattagtgctactcga HSEstep
tcgacTTCtactagcTTCcactaatTTCtgctactcga
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Fig. 1. Characterization of temperature-sensitive hsf1 mutations. (A) Positions of amino acid substitutions. Amino acid sequence of the Hsf1 DBD (aa 171–259), with residues conserved among eukaryotic HSFs shown in black. Secondary structural elements of the DBD are shown above the sequence, where rectangles and arrows indicate helices (H1–H3) and b-sheets (S1–S4), respectively. Positions of the temperature-sensitive (H220R, N222Y, and F256S) and suppressor (R206S and V210A) mutations are indicated. (B) Growth of mutant cells at normal and elevated temperatures. Wild-type HSF1 (WT), hsf1-H220R (H220R), hsf1-N222Y (N222Y), and hsf1-F256S (F256S) cells were streaked onto YPD medium and incubated at either 28 or 38 °C for 2 days. (C) Sequences of HSE oligonucleotides used for gel retardation analysis. The GAA units are indicated in bold uppercase with arrows. HSE4Ptt and HSE3P contain four or three continuous inverted repeats of the unit, respectively. HSEgap and HSEstep contain one or two gaps between the repeating units, respectively. (D) Binding of mutant Hsf1 polypeptides to various HSEs. The oligonucleotides HSE4Ptt, HSE3P, HSEgap, and HSEstep were 5 0 -endlabeled with 32P. Probes were incubated without (lane 1) or with increasing amounts (8.3, 25, and 75 ng) of wild-type (WT) and mutant (N222Y, H220R, or F256S) Hsf1 polypeptides. Samples were separated by electrophoresis on polyacrylamide gels and subjected to phosphorimaging.
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Fig. 2. Heat-induced transcription in cells containing hsf1-N222Y and hsf1-H220R mutations. HSF1 (WT) and hsf1 mutant cells were grown at 39 °C for 0, 15, or 30 min. Total RNA from each sample was subjected to RT-PCR analysis. The genes targeted by Hsf1 are classified according to the structure of their HSEs: 4P and 3P, four or three continuous inverted repeats of nGAAn, respectively; gap and step types [18]. The gene ACT1 (encoding actin) was used as a control.
to 39 °C caused HSF1 cells to accumulate transcripts from genes containing HSEs with four (BTN2 and HSP78) or three (HSP60 and SPI1) continuous inverted repeats of
nGAAn (3P or 4P type, respectively), as well as the gap (CPR6 and CUP1) and step (SGT2 and SSA3) HSE types (lanes 1–3). In hsf1-N222Y cells, transcriptional activation was inhibited for genes containing the 3P-, gap-, and steptype HSEs (lanes 4–6). The hsf1-H220R mutation also inhibited the heat shock response for the same set of genes (lanes 10–12). A normal level of activation was observed for genes containing the 4P-type HSE; this may be ascribed to its higher intrinsic binding affinity (see above). Therefore, in the N222Y and H220R mutations, there appears to be a correlation between DNA-binding and transcriptional defects. Phenotypes associated with the N222Y mutation are suppressed by the R206S substitution The substitution of arginine to serine at residue 206 (R206S) stimulates binding of Hsf1 to the HSE and causes constitutive expression of HSE-containing reporter genes [23]. This residue is directly involved in the DBD dimer interface [22]. Introduction of the R206S substitution into hsf1-N222Y cells (hsf1-R206S-N222Y) resulted in the recovery of normal growth at 38 °C (Fig. 3A). The Hsf1-
A. Yamamoto, H. Sakurai / Biochemical and Biophysical Research Communications 346 (2006) 1324–1329
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Fig. 3. Effect of combining the R206S substitution with the N222Y and H220R mutations. (A) Growth of mutant cells at elevated temperatures. HSF1 (WT) and hsf1 mutant cells were streaked onto YPD medium and incubated at 35 or 38 °C for 2 days. (B) Binding of various Hsf1 polypeptides to HSEs. Wild-type (WT) and mutant polypeptides were used for gel retardation analysis as described for Fig. 1D.
R206S-N222Y polypeptide exhibited near wild-type binding affinity to the 4P-, 3P-, and gap-type HSEs. However, the double mutant continued to demonstrate a 5-fold lower affinity than wild-type to the step-type HSE, since the substitution R206S caused inhibition of binding to HSEstep (Fig. 3B, lanes 5–10). In hsf1-R206S-N222Y cells, heat-induced transcription was restored for genes containing the 4P-, 3P-, and gap-type HSEs, but not the step-type HSE (Fig. 2, lanes 7–9). Therefore, the results from HSE typespecific binding and transcriptional defects appear to be in agreement. The N222Y mutation impairs binding of Hsf1 to the HSE, thereby inhibiting the heat shock response and cell growth at elevated temperatures. In contrast, the phenotypic changes associated with hsf1-H220R were exacerbated by the additional R206S substitution and the double mutants hsf1-R206S-H220R failed to grow at 35 °C (Fig. 3A). The binding affinities of Hsf1-R206SH220R to the various HSEs were more than 10-fold lower than for the wild-type control (Fig. 3B, lanes 11–13). In addition, the heat shock response of hsf1-R206S-H220R cells was inhibited significantly for all of the genes tested (Fig. 2, lanes 13–15). Although both H220R and N222Y mutations are located in the turn region and inhibit Hsf1 DNA binding, the additional R206S substitution results in completely different effects. The F256S mutation negatively regulates activator function of Hsf1 The F256S mutation did not effect a significant change in the binding of Hsf1 to the various HSEs (see Fig. 1D). However, it is known to inhibit heat-induced transcription of target genes [9]. Stress-induced phosphorylation of Hsf1
correlates with the acquisition of activating ability and hyperphosphorylated Hsf1 migrates more slowly than the hypophosphorylated form on denaturing polyacrylamide gels [15–17]. As determined by immunoblot analysis, the retarded migration of Hsf1 containing the mutations F256S, H220R, and N222Y suggested that heat-induced phosphorylation of mutant proteins was unaffected in yeast cells (data not shown). To explore the effects of the F256S mutation, we isolated intragenic mutations that suppressed the temperature-sensitive growth defect of hsf1-F256S. A V210A substitution was common to two suppressor alleles, and introduction of this substitution to hsf1-F256S enabled hsf1-V210AF256S cells to grow at 38 °C (Fig. 4A). Note that the V210A substitution failed to suppress the temperature-sensitive growth phenotypes associated with the DNA-binding defects of the H220R and N222Y mutations (data not shown). With the exception of the 4P type (BTN2 and HSP78) of HSE, heat-induced transcription of genes containing the other HSE types was inhibited in hsf1-F256S cells (Fig. 4B, lanes 4–6). The V210A substitution by itself exhibited only a marginal effect on activation of genes analyzed (lanes 7–9), although a previous report showed a slight inhibition of the heat shock response by the substitution [24]. The transcriptional defect caused by the F256S mutation was partially restored by the V210A substitution and in hsf1-V210A-F256S cells the heat-induced mRNA levels of HSP60, SPI1, and SGT2 were similar to those of wild-type cells (lanes 10–12). The reason why activation of CPR6, CUP1, and SSA3 was lower than the HSF1 control is unknown. The residue V210 is located in the second helix of the DBD (see Fig. 1A) and it has been proposed that this helix plays an important role in sensing thermal
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Fig. 4. Characterization of R256S and V210A mutations. (A) Growth of hsf1-R256S cells containing the intragenic V210A substitution. HSF1 (WT) and hsf1 mutant cells were streaked onto YPD medium and incubated at 28 or 38 °C for 2 days. (B) mRNA levels in hsf1-R256S cells containing the intragenic V210A substitution. Total RNA was prepared from HSF1 (WT) and hsf1 mutant cells and subjected to RT-PCR analysis, as described for Fig. 2.
step inhibited by the mutation. The F256 residue is located in the fourth sheet, which is connected to the third sheet by the wing. It has been demonstrated that a lysine to alanine substitution at residue 237 (K237A) in the third sheet does not affect DNA binding, but does result in constitutively active transcription of an HSE-containing reporter gene [24], and several mutations located in the third sheet and wing region enable Hsf1 to activate transcription independently of heat-induced phosphorylation [18]. Unlike other winged helix–turn–helix proteins, the wing of Hsf1 does not contact DNA [22]. Based on these observations, we suggest that the C-terminal region of the DBD (including the third sheet, wing, and fourth sheet) is involved in negative regulation of activator function. Consistent with this proposal, the transcriptional defect associated with F256S is suppressed by the V210A substitution, is located in the second helix, which is important for sensing heat shock. Thus, the Hsf1 DBD may be dissected functionally into two distinct regions: the turn and third helix mediate binding to the HSE; and the second helix and third sheet–wing– fourth sheet region regulate the transcriptional activating ability of Hsf1. Acknowledgments
stress [25]. We suggest that the F256S mutation enhances the negative regulatory role played by the DBD and that this effect is restrained by the substitution of V210A in the putative heat-sensing helix.
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Sciences, Sports and Culture to H.S. References
Discussion We have identified two types of temperature-sensitive hsf1 mutations in the DBD. The H220R and N222Y mutations are located in the turn region and inhibit both DNAbinding and transcriptional activation. It has been shown that a histidine to alanine substitution at residue 220 (H220A) results in an inhibition of constitutive transcription of an HSE-containing reporter gene [24]. In contrast to N222Y, substitution to alanine (N222A) stimulates both activities [24] and although acting in opposition, these results indicate that residues H220 and N222 are important for recognition of the HSE. In addition, both the DNAbinding and transcriptional defects of Hsf1-N222Y are restored partially by the R206S substitution, which is located in the DBD–DBD interface. Mutations in the third helix, which is responsible for DNA recognition, cause severe defects in DNA binding and cell viability [26,27]. Our molecular genetic analysis supports previous structural analyses and indicates that the turn region and presumably the DBD–DBD interface are critical for protein–DNA interactions. The F256S mutation inhibited heat-induced transcription, without altering DNA-binding affinity. Since Hsf1F256S is hyperphosphorylated in response to heat shock, this modification would appear to precede the regulatory
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The DNA-binding domain of yeast Hsf1 regulates both DNA-binding and transcriptional activities Ayako Yamamoto, Hiroshi Sakurai
*
Division of Health Sciences, Graduate School of Medical Science, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 920-0942, Japan Received 8 June 2006 Available online 19 June 2006
Abstract The heat shock transcription factor (HSF) is a key regulator of the heat shock response. In Saccharomyces cerevisiae, the transcription activating ability of Hsf1 is repressed by its DNA-binding domain, but the detailed mechanism by which the inhibitory function is relieved in response to stress remains unknown. In this study, we isolated and characterized three hsf1 mutants with temperature-sensitive mutations in the DNA-binding domain. Two mutations inhibited DNA-binding activity, leading to decreased expression of target genes. The third mutation caused transcriptional defects without affecting DNA binding, and its suppressor mutation was located in a region important for sensing heat shock. These results indicate that the DNA-binding domain regulates both the DNA-binding and transcriptional activities of Hsf1, and suggest that these functions are located within discrete regions of the DNA-binding domain. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Heat shock transcription factor; Heat shock element; DNA-binding domain; Winged helix–turn–helix motif; Temperature-sensitive mutation; Intragenic suppressor mutation
In eukaryotes, the heat shock response is regulated by the heat shock transcription factor (HSF). This protein contains a helix–turn–helix DNA-binding domain (DBD) and a hydrophobic repeat region that is required for homotrimer formation. Homotrimeric HSF binds to the heat shock element (HSE), a conserved regulatory DNA sequence comprising at least three contiguous inverted repeats of 5 0 -nGAAn-3 0 upstream of the heat shock genes [1–3]. In the yeast Saccharomyces cerevisiae, HSF is encoded by the essential gene HSF1. Mutations in this gene cause defects in cell wall integrity, spindle pole body duplication, and cell cycle progression [4–7]. The target genes of Hsf1 encode a variety of proteins that function in protein folding and maturation, energy generation, carbohydrate metabolism, and cell wall organization [8,9]. Hsf1 binds to and activates transcription via the ‘‘perfect’’ type of HSE
*
Corresponding author. Fax: +81 76 234 4369. E-mail address: [email protected] (H. Sakurai).
0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.06.057
described above, as well as the ‘‘discontinuous’’ type of HSE containing one (gap type) or two (step type) gaps between the repeating units [9–11]. In mammalian cells, the predominant HSF isoform (HSF1) responds to various stresses by forming a homotrimer that translocates from the cytoplasm to the nucleus, where it binds to the HSE and becomes transcriptionally competent upon stress-induced phosphorylation [2]. Unlike its mammalian counterpart, S. cerevisiae Hsf1 is able to bind to the HSE in both a constitutive and inducible manner [8,12]. The Hsf1 activation domain possesses constitutive activating ability, which is regulated negatively by the DBD under normal physiological conditions [13,14]. Hsf1 becomes hyperphosphorylated and transcriptionally competent under thermal and oxidative stress, resulting in the transcription of target genes [15–17]. It has been suggested that it is the heat shock-induced phosphorylation that mitigates the negative regulation effected by the DBD [18]. In order to explore the role played by the Hsf1 DBD, we isolated and characterized temperature-sensitive DBD mutants and intragenic suppressor mutations. Our results
A. Yamamoto, H. Sakurai / Biochemical and Biophysical Research Communications 346 (2006) 1324–1329
demonstrate that the DBD is important for binding DNA and for regulation of activator function. Together with previous mutation analyses, our results suggest that regulation of activation is modulated by discrete regions in the DBD. Materials and methods Yeast strains and media. Saccharomyces cerevisiae strains used in this study are listed in Table 1. Strain HS126 contains a null mutation of HSF1 in the chromosome and a wild-type copy on a URA3-marked centromeric plasmid (YCp-URA3-HSF1) [17]. Cells containing hsf1 mutations were constructed from HS126 using plasmid shuffling [9,17]. Rich glucose (YPD), enriched synthetic glucose (ESD), and synthetic glucose (SD) media were prepared as described previously [9]. Isolation of temperature-sensitive hsf1 mutations. Temperature-sensitive hsf1 mutations were isolated as described previously [9]. In brief, random nucleotide alterations were introduced into HSF1 by PCR amplification in the presence of 0.1 mM MnCl2. Strain HS134 was transformed with a library of hsf1 mutations and transformants were replica-plated onto ESD medium containing 5-fluoroortic acid, to lose the resident YCp-URA3HSF1. Plates were incubated at 38 °C. Temperature-sensitive hsf1 mutations were identified in plasmids pAY5, pAY8, and pAY9, which contained substitutions of asparagine to tyrosine at amino acid (aa) 222 (N222Y), histidine to arginine at aa 220 (H220R), and phenylalanine to serine at aa 256 (F256S), respectively. The substitution R206S was introduced into hsf1 alleles of pAY5 and pAY8 via site-directed mutagenesis, generating pAY20 and pAY21, respectively [9]. Isolation of intragenic mutations suppressing the hsf1-F256S mutation. Random nucleotide alterations were introduced into hsf1-F256S as described above and the amplified fragments were cloned into a LEU2marked centromeric vector. The mutant library was used to transform strain TH2670 (Open Biosystems, Co.), in which expression of HSF1 was under control of the tetO promoter [19]. Transformants were grown on SD medium containing 20 lg/ml histidine, 20 lg/ml methionine, and 10 lg/ml doxycycline at 38 °C. One hsf1-F256S suppressor allele contained the additional substitutions V210A, S305C, and T401A, whereas another contained V210A and T88A. The substitution V210A was introduced into hsf1 alleles as above. Gel retardation analysis. A fragment encoding the N-terminal 583 aa of Hsf1 was cloned into pGEX-6P1, expressed in Escherichia coli, and purified, as described previously [17]. The polypeptide was incubated with 0.15 ng of 32P-labeled oligonucleotides in 12 ll of buffer containing 20 mM Hepes–KOH (pH 7.6), 50 mM potassium acetate, 2 mM EDTA, 0.05% Nonidet P-40, 8.3 lg/ml poly(dI–dC), and 6.7% glycerol. Samples were separated by electrophoresis on a 4% polyacrylamide gel in 25 mM
Table 1 Yeast strains used in this study Strains
Genotype (plasmid)
HS126
MATa ade2 his3 leu2 trp1 ura3 can1 hsf1::HIS3 YCp-URA3-HSF1 (pSK906) LEU2::SSA4-lacZ of HS126 YCp-TRP1-HSF1 (pK157) of HS126 YCp-TRP1-hsf1-N222Y (pAY5) of HS126 YCp-TRP1-hsf1-H220R (pAY8) of HS126 YCp-TRP1-hsf1-F256S (pAY9) of HS126 YCp-TRP1-hsf1-R206S (pAY19) of HS126 YCp-TRP1-hsf1-R206S-N222Y (pAY20) of HS126 YCp-TRP1-hsf1-R206S-H220R (pAY21) of HS126 YCp-TRP1-hsf1-V210A (pAY73) of HS126 YCp-TRP1-hsf1-V210A-F256S (pAY74) of HS126 YCp-TRP1-hsf1-V210A-N222Y (pAY77) of HS126 YCp-TRP1-hsf1-V210A-H220R (pAY78) of HS126 his3-1 leu2-0 met15-0 URA3::CMV-tTA kanR-tetO-TATA-HSF1
HS134 HS170T YAY5-1 YAY8-1 YAY9-1 YAY19 YAY20 YAY21 YAY73 YAY74 YAY77 YAY78 TH2670
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Tris–glycine (pH 8.8) and subjected to phosphorimaging. The nucleotide sequences of the oligonucleotide probes are shown in Fig. 1C. RNA analysis. Cells grown in YPD medium at 28 °C were heatshocked at 39 °C, and total RNA prepared from the cells was analyzed by reverse transcription (RT)-PCR, as described previously [17]. The amounts of PCR products were compared following normalization using ACT1 mRNA (encoding actin) as a control.
Results Isolation of temperature-sensitive hsf1 mutations We isolated three temperature-sensitive hsf1 mutations that supported cell growth at 28 °C but not at 38 °C (Fig. 1A and B). The winged helix–turn–helix DBD comprises three helices, four b-sheets, and a flexible loop (termed the wing); mutations H220R and N222Y are located in the turn region and F256S in the fourth b-sheet. These HSF residues are highly conserved among organisms. Residues H220 and N222 are more than 40% solvent accessible, whereas F256 is less than 20% solvent accessible and anchors the b-sheet to the hydrophobic core of the DBD [20,21]. In the HSF–HSE co-crystal, the side chains of H220 and N222 form water-mediated hydrogen bonds with the phosphate backbone of the DNA; the peptide bond backbone of these residues is involved in protein– protein interactions of the DBD dimer interface [22]. Binding of mutant Hsf1 proteins to the HSE The effects of mutations on the Hsf1–HSE interaction were analyzed by gel retardation assays using various HSE oligonucleotides and recombinant Hsf1 polypeptides that contained the DBD and hydrophobic repeat region (Fig. 1C and D). Wild-type Hsf1 formed protein–DNA complexes with oligonucleotides containing the perfecttype HSE (three or four nGAAn repeats, i.e., HSE3P and HSE4Ptt, respectively), gap-type HSE (HSEgap), and step-type HSE (HSEstep) (Fig. 1D, lanes 2–4). Since two Hsf1 trimers bind cooperatively in the interaction between Hsf1 and HSE4Ptt [18], the binding affinity of that interaction is about 10-fold higher than between Hsf1 and HSE3P, HSEgap, or HSEstep. The mutants Hsf1-N222Y and Hsf1-H220R exhibited 10-fold lower affinity than wild type for all HSEs investigated (lanes 5–7 and 8–10, respectively). These results are consistent with crystallographic analysis [22] and indicate that residues H220 and N222 are involved in Hsf1–HSE interactions. In contrast, the mutation F256S did not influence binding of Hsf1 to the various HSEs (lanes 11–13). Transcription of Hsf1 target genes in hsf1-N222Y and hsf1-H220R cells To evaluate whether the decrease in DNA-binding affinity affects heat-induced transcription, the mRNA levels of Hsf1 target genes in hsf1-N222Y and hsf1-H220R cells were analyzed by RT-PCR (Fig. 2). A temperature shift from 28
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tcgacTTCtaGAAgcTTCcaGAAattagtgctactcga HSE3P
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tcgacTTCtaGAAgctagcaGAAattagtgctactcga HSEstep
tcgacTTCtactagcTTCcactaatTTCtgctactcga
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Fig. 1. Characterization of temperature-sensitive hsf1 mutations. (A) Positions of amino acid substitutions. Amino acid sequence of the Hsf1 DBD (aa 171–259), with residues conserved among eukaryotic HSFs shown in black. Secondary structural elements of the DBD are shown above the sequence, where rectangles and arrows indicate helices (H1–H3) and b-sheets (S1–S4), respectively. Positions of the temperature-sensitive (H220R, N222Y, and F256S) and suppressor (R206S and V210A) mutations are indicated. (B) Growth of mutant cells at normal and elevated temperatures. Wild-type HSF1 (WT), hsf1-H220R (H220R), hsf1-N222Y (N222Y), and hsf1-F256S (F256S) cells were streaked onto YPD medium and incubated at either 28 or 38 °C for 2 days. (C) Sequences of HSE oligonucleotides used for gel retardation analysis. The GAA units are indicated in bold uppercase with arrows. HSE4Ptt and HSE3P contain four or three continuous inverted repeats of the unit, respectively. HSEgap and HSEstep contain one or two gaps between the repeating units, respectively. (D) Binding of mutant Hsf1 polypeptides to various HSEs. The oligonucleotides HSE4Ptt, HSE3P, HSEgap, and HSEstep were 5 0 -endlabeled with 32P. Probes were incubated without (lane 1) or with increasing amounts (8.3, 25, and 75 ng) of wild-type (WT) and mutant (N222Y, H220R, or F256S) Hsf1 polypeptides. Samples were separated by electrophoresis on polyacrylamide gels and subjected to phosphorimaging.
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Fig. 2. Heat-induced transcription in cells containing hsf1-N222Y and hsf1-H220R mutations. HSF1 (WT) and hsf1 mutant cells were grown at 39 °C for 0, 15, or 30 min. Total RNA from each sample was subjected to RT-PCR analysis. The genes targeted by Hsf1 are classified according to the structure of their HSEs: 4P and 3P, four or three continuous inverted repeats of nGAAn, respectively; gap and step types [18]. The gene ACT1 (encoding actin) was used as a control.
to 39 °C caused HSF1 cells to accumulate transcripts from genes containing HSEs with four (BTN2 and HSP78) or three (HSP60 and SPI1) continuous inverted repeats of
nGAAn (3P or 4P type, respectively), as well as the gap (CPR6 and CUP1) and step (SGT2 and SSA3) HSE types (lanes 1–3). In hsf1-N222Y cells, transcriptional activation was inhibited for genes containing the 3P-, gap-, and steptype HSEs (lanes 4–6). The hsf1-H220R mutation also inhibited the heat shock response for the same set of genes (lanes 10–12). A normal level of activation was observed for genes containing the 4P-type HSE; this may be ascribed to its higher intrinsic binding affinity (see above). Therefore, in the N222Y and H220R mutations, there appears to be a correlation between DNA-binding and transcriptional defects. Phenotypes associated with the N222Y mutation are suppressed by the R206S substitution The substitution of arginine to serine at residue 206 (R206S) stimulates binding of Hsf1 to the HSE and causes constitutive expression of HSE-containing reporter genes [23]. This residue is directly involved in the DBD dimer interface [22]. Introduction of the R206S substitution into hsf1-N222Y cells (hsf1-R206S-N222Y) resulted in the recovery of normal growth at 38 °C (Fig. 3A). The Hsf1-
A. Yamamoto, H. Sakurai / Biochemical and Biophysical Research Communications 346 (2006) 1324–1329
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Fig. 3. Effect of combining the R206S substitution with the N222Y and H220R mutations. (A) Growth of mutant cells at elevated temperatures. HSF1 (WT) and hsf1 mutant cells were streaked onto YPD medium and incubated at 35 or 38 °C for 2 days. (B) Binding of various Hsf1 polypeptides to HSEs. Wild-type (WT) and mutant polypeptides were used for gel retardation analysis as described for Fig. 1D.
R206S-N222Y polypeptide exhibited near wild-type binding affinity to the 4P-, 3P-, and gap-type HSEs. However, the double mutant continued to demonstrate a 5-fold lower affinity than wild-type to the step-type HSE, since the substitution R206S caused inhibition of binding to HSEstep (Fig. 3B, lanes 5–10). In hsf1-R206S-N222Y cells, heat-induced transcription was restored for genes containing the 4P-, 3P-, and gap-type HSEs, but not the step-type HSE (Fig. 2, lanes 7–9). Therefore, the results from HSE typespecific binding and transcriptional defects appear to be in agreement. The N222Y mutation impairs binding of Hsf1 to the HSE, thereby inhibiting the heat shock response and cell growth at elevated temperatures. In contrast, the phenotypic changes associated with hsf1-H220R were exacerbated by the additional R206S substitution and the double mutants hsf1-R206S-H220R failed to grow at 35 °C (Fig. 3A). The binding affinities of Hsf1-R206SH220R to the various HSEs were more than 10-fold lower than for the wild-type control (Fig. 3B, lanes 11–13). In addition, the heat shock response of hsf1-R206S-H220R cells was inhibited significantly for all of the genes tested (Fig. 2, lanes 13–15). Although both H220R and N222Y mutations are located in the turn region and inhibit Hsf1 DNA binding, the additional R206S substitution results in completely different effects. The F256S mutation negatively regulates activator function of Hsf1 The F256S mutation did not effect a significant change in the binding of Hsf1 to the various HSEs (see Fig. 1D). However, it is known to inhibit heat-induced transcription of target genes [9]. Stress-induced phosphorylation of Hsf1
correlates with the acquisition of activating ability and hyperphosphorylated Hsf1 migrates more slowly than the hypophosphorylated form on denaturing polyacrylamide gels [15–17]. As determined by immunoblot analysis, the retarded migration of Hsf1 containing the mutations F256S, H220R, and N222Y suggested that heat-induced phosphorylation of mutant proteins was unaffected in yeast cells (data not shown). To explore the effects of the F256S mutation, we isolated intragenic mutations that suppressed the temperature-sensitive growth defect of hsf1-F256S. A V210A substitution was common to two suppressor alleles, and introduction of this substitution to hsf1-F256S enabled hsf1-V210AF256S cells to grow at 38 °C (Fig. 4A). Note that the V210A substitution failed to suppress the temperature-sensitive growth phenotypes associated with the DNA-binding defects of the H220R and N222Y mutations (data not shown). With the exception of the 4P type (BTN2 and HSP78) of HSE, heat-induced transcription of genes containing the other HSE types was inhibited in hsf1-F256S cells (Fig. 4B, lanes 4–6). The V210A substitution by itself exhibited only a marginal effect on activation of genes analyzed (lanes 7–9), although a previous report showed a slight inhibition of the heat shock response by the substitution [24]. The transcriptional defect caused by the F256S mutation was partially restored by the V210A substitution and in hsf1-V210A-F256S cells the heat-induced mRNA levels of HSP60, SPI1, and SGT2 were similar to those of wild-type cells (lanes 10–12). The reason why activation of CPR6, CUP1, and SSA3 was lower than the HSF1 control is unknown. The residue V210 is located in the second helix of the DBD (see Fig. 1A) and it has been proposed that this helix plays an important role in sensing thermal
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Fig. 4. Characterization of R256S and V210A mutations. (A) Growth of hsf1-R256S cells containing the intragenic V210A substitution. HSF1 (WT) and hsf1 mutant cells were streaked onto YPD medium and incubated at 28 or 38 °C for 2 days. (B) mRNA levels in hsf1-R256S cells containing the intragenic V210A substitution. Total RNA was prepared from HSF1 (WT) and hsf1 mutant cells and subjected to RT-PCR analysis, as described for Fig. 2.
step inhibited by the mutation. The F256 residue is located in the fourth sheet, which is connected to the third sheet by the wing. It has been demonstrated that a lysine to alanine substitution at residue 237 (K237A) in the third sheet does not affect DNA binding, but does result in constitutively active transcription of an HSE-containing reporter gene [24], and several mutations located in the third sheet and wing region enable Hsf1 to activate transcription independently of heat-induced phosphorylation [18]. Unlike other winged helix–turn–helix proteins, the wing of Hsf1 does not contact DNA [22]. Based on these observations, we suggest that the C-terminal region of the DBD (including the third sheet, wing, and fourth sheet) is involved in negative regulation of activator function. Consistent with this proposal, the transcriptional defect associated with F256S is suppressed by the V210A substitution, is located in the second helix, which is important for sensing heat shock. Thus, the Hsf1 DBD may be dissected functionally into two distinct regions: the turn and third helix mediate binding to the HSE; and the second helix and third sheet–wing– fourth sheet region regulate the transcriptional activating ability of Hsf1. Acknowledgments
stress [25]. We suggest that the F256S mutation enhances the negative regulatory role played by the DBD and that this effect is restrained by the substitution of V210A in the putative heat-sensing helix.
This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Sciences, Sports and Culture to H.S. References
Discussion We have identified two types of temperature-sensitive hsf1 mutations in the DBD. The H220R and N222Y mutations are located in the turn region and inhibit both DNAbinding and transcriptional activation. It has been shown that a histidine to alanine substitution at residue 220 (H220A) results in an inhibition of constitutive transcription of an HSE-containing reporter gene [24]. In contrast to N222Y, substitution to alanine (N222A) stimulates both activities [24] and although acting in opposition, these results indicate that residues H220 and N222 are important for recognition of the HSE. In addition, both the DNAbinding and transcriptional defects of Hsf1-N222Y are restored partially by the R206S substitution, which is located in the DBD–DBD interface. Mutations in the third helix, which is responsible for DNA recognition, cause severe defects in DNA binding and cell viability [26,27]. Our molecular genetic analysis supports previous structural analyses and indicates that the turn region and presumably the DBD–DBD interface are critical for protein–DNA interactions. The F256S mutation inhibited heat-induced transcription, without altering DNA-binding affinity. Since Hsf1F256S is hyperphosphorylated in response to heat shock, this modification would appear to precede the regulatory
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