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Novel testis-specific protein that interacts with heat shock factor 2
Gene 214 (1998) 139–146
Novel testis-specific protein that interacts with heat shock factor 2 Tadahiko Yoshima 1, Takashi Yura, Hideki Yanagi * HSP Research Institute, Kyoto Research Park, Kyoto 600-8813, Japan Received 4 February 1998; accepted 13 April 1998; Received by J. Wild
Abstract Although heat shock factor 2 (HSF2) binds to heat shock element (HSE ) constitutively during differentiation, development and spermatogenesis, little is known about the nature and mechanism of transcriptional control of heat shock genes by HSF2. Using the yeast two-hybrid system, we screened a human testis cDNA library for proteins that can associate with HSF2 by the yeast two-hybrid system, and isolated clones encoding a novel protein, designated HSF2-binding protein (HSF2BP), that associates with HSF2 in vitro and in vivo and is specifically synthesized in testis. The interaction seemed to occur between the trimerization domain of HSF2 and the N-terminal hydrophilic region of HSF2BP that comprises two leucine zipper motifs. HSF2BP may therefore be involved in modulating HSF2 activation in testis. © 1998 Elsevier Science B.V. All rights reserved. Keywords: HSF; Two-hybrid; Leucine zipper; Pull-down; Protein–protein interaction
1. Introduction Heat shock factor 2 (HSF2) is a member of the vertebrates HSF family that includes HSF1, HSF2, HSF3 and HSF4 (Rabindran et al., 1991; Schuetz et al., 1991; Nakai and Morimoto, 1993; Nakai et al., 1997) and binds to heat shock elements (HSEs) located at the promoter regions of genes encoding heat shock proteins (HSPs). HSF1 and HSF3 respond to a heat shock stress and other environmental stresses and induce transcription of HSP-encoding genes (Baler et al., 1993; Sarge et al., 1993; Nakai et al., 1995). Unlike HSF1 and HSF3, HSF2 is not activated by such stresses but is thought to play an important role during differentiation and development. HSF4 is believed to be involved in negative regulation of other HSFs binding to DNA * Corresponding author. Tel: +81 75 315 8685; Fax: +81 75 315 8659; e-mail: [email protected] 1 Present address: Sumitomo Pharmaceuticals Research Center, 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554-0022, Japan. Abbreviations: Aa, amino acid(s); bGal, b-galactosidase; EC, embryonal carcinoma; GAL4-BD, GAL4 DNA binding domain; GST, glutathione S-transferase; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HSE, heat shock element; HSF, heat shock factor; HSF2BP, HSF2 binding protein; HSP, heat shock protein; LZ, leucine zipper; nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamidegel electrophoresis; PBS, phosphate-buffered saline; PBST, PBS containing 0.1% Tween 20; SDS, sodium dodecyl sulfate . 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 20 8 - X
because of the absence of a transcriptional activation domain (Nakai et al., 1997). Although there have been several findings that suggest HSF2 activation during differentiation and development, the way in which HSF2 contributes to transcriptional regulation of heat shock genes remains unresolved. Sistonen et al. (1992) first reported that hemin-induced HSE binding of HSF2 in human K562 erythroleukemia cells was accompanied by transcriptional induction of the hsp70 gene. In spermatogenic cells of mouse testis, HSF2 was found to bind constitutively to HSE and appeared to be correlated with testisspecific expression of hsp70.2 (Sarge et al., 1994). However, constitutive HSE-binding activity found in mouse embryonal carcinoma ( EC ) cell extracts was composed predominantly of HSF2, whereas the HSE region of hsp70 promoter was not occupied by HSF2 (Murphy et al., 1994). Furthermore, zygotic expression of hsp70 and HSE-binding activity of HSF2 were detected during mouse embryonal development (Bensaude et al., 1983; Rallu et al., 1997), but no obvious correlation was found between expression patterns of major HSPs and of HSF2 in embryos (Bevilacqua et al., 1997; Rallu et al., 1997). In the case of HSF1, induction of DNA binding of the HSF1 trimer is not sufficient to activate transcription of hsp genes (Jurivich et al., 1992; Zuo et al., 1995), and additional regulatory step(s) or pathway(s) are
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required for transition from a transcriptionally inactive trimer to active forms. The transactivation function of HSF1 is now known to be modulated by constitutive phosphorylation and stress-induced hyperphosphorylation (Chu et al., 1996; Knauf et al., 1996; Kline and Morimoto, 1997; Xia and Voellmy, 1997). Although such modifications have not been reported for HSF2, it seemed likely that some unknown factors are involved in modulating the activity of HSF2. We report here on a novel cDNA encoding a 334-aa polypeptide HSF2BP that is specifically expressed in testis. The physical interaction between HSF2 and HSF2BP was demonstrated both in vitro and in vivo.
2.3. Western blotting with yeast lysates Yeast cell extracts were prepared using glass beads, and proteins (20 mg) were separated by 8% SDS–PAGE, and blotted on to nitrocellulose membranes (Amersham, Bucks, UK ). The membranes were treated with 5% skim milk in PBS containing 0.1% Tween 20 (PBST ) and with polyclonal rabbit antibodies against chicken HSF2 (aHSF2a) (kindly provided by A. Nakai). After washing with PBST, the membranes were treated with horseradish peroxidase-conjugated goat F(ab∞) anti-rabbit 2 antibody (Biosource, Camarillo, CA), washed with PBST, and detected using ECL reagent (Amersham). 2.4. Cloning of full-length cDNA for HSF2BP
2. Materials and methods 2.1. Plasmid constructions Expression plasmids for truncated forms of HSF2 or HSF1 fused with the GAL4 DNA-binding domain (GAL4-BD) were constructed from pGBT9 and pM (Clontech, Palo Alto, CA) as ‘baits’ for yeast and mammalian two-hybrid assays, respectively ( Yoshima et al., 1997). Glutathione S-transferase (GST ) fusion constructs of HSF2 or HSF1 and reporter plasmids pGLG4E5 and pRLSV40 for dual luciferase assay have also been described ( Yoshima et al., 1997). pVP16-HSF2BP that expresses HSF2BP fused to the VP16 activation domain was constructed by inserting HSF2BP cDNA into pVP16 (Clontech). For in-vitro translation of HSF2BP and its truncated forms, the corresponding regions of HSF2BP cDNA were inserted into pcDNA3.1HisA, B, or C (Invitrogen, Carlsbad, CA). 2.2. Yeast strains and two-hybrid screenings HF7c (MATa, ura3–52, his3–200, lys2–801, trp1–901, ade2–101, leu2–3,112, gal4–542, gal80–538, LYS:: GAL1–HIS3, URA3::(GAL4 17mers)3-CYC1-lacZ) used for two-hybrid screenings and a human testis cDNA library constructed with pGAD10 were obtained from Clontech. The transformation of yeast cells by the lithium-acetate method and the two-hybrid assay were described previously ( Yoshima et al., 1997). In brief, HF7c cells were transformed with a plasmid expressing HSF2 bait and with a human cDNA plasmid library, His+ transformants were selected and tested for bgalactosidase ( bGal ) activities. Plasmid DNAs were isolated from His+ bGal+ clones, and the nt sequence of the 5∞ ends of the cDNA inserts was determined by a DNA autosequencer PRISM 377 (Perkin Elmer, Foster City, CA).
A human testis cDNA library (lgt11 containing 1×106 independent clones, Clontech) was screened by plaque hybridization using a 32P-labeled cDNA fragment of HSF2BP obtained by the present screening as a probe. The longest clone obtained was used for nt sequencing. 2.5. Cell culture K562 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in 5% CO at 37°C. 2 Transfections were carried out by the lipofection method using Transfectam@ (Promega, Madison, W1), and transfected cells were cultured for 48 h and subjected to a Dual-Luciferase Reporter Assay (Promega). F9 cells were cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum in 5% CO at 37°C. 2 2.6. Northern blot analysis RNA was extracted from 60 mM hemin-treated or untreated K562 cells and F9 cells using Isogen (Nippon Gene, Tokyo, Japan), and poly(A)+ RNA was selected by Oligotex-dT30 ( Takara, Kyoto, Japan). Poly(A)+ RNA of human testis was obtained from Clontech. These RNAs were separated on 1% agarose gel containing formaldehyde and transferred to a nylon membrane (Du Pont). RNA blots of multiple human tissues were purchased from Clontech (MTN blot and MTNII blot). The membranes were treated with 32P-labeled cDNA probes for HSF2BP, HSF2, or glyceraldehyde3-phosphate dehydrogenase (G3PDH) (Clontech), washed in 0.2× SSPE containing 0.1% SDS at 42°C, followed by autoradiography. 2.7. GST pull-down assay GST and GST–HSF fusion proteins were purified, and immobilized on glutathione-Sepharose CL4B (Pharmacia, Uppsala, Sweden). HSF2BP and its trun-
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cated forms were translated in vitro using the TNT translation kit (Promega). Five microliters of the reaction mixture were incubated with immobilized GST or GST–HSF overnight at 4°C. Beads were washed with NETN buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40), and bound proteins were analyzed by SDS–PAGE, processed for autoradiography, and quantified by an imaging analyzer BAS2000 (Fuji, Tokyo, Japan).
3. Results 3.1. cDNA cloning of HSF2BP To search for proteins that can associate with HSF2 in testis, we screened a human testis cDNA library by using yeast two-hybrid system. HSF2 deletion mutants HSF2N96 or HSF2N96C328 fused to GAL4-BD were used as ‘baits’ for screening, because HSF2N96 was expected to fish out proteins that could interact with HSF2 that binds DNA but is transcriptionally inactive, whereas HSF2N96C328 was expected to find proteins that could interact with an activated form of HSF2 ( Yoshima et al., 1997). The expression of these two fusion proteins in yeast was confirmed by Western blot analysis ( Fig. 1). The pair of yeast strains were transformed with a pGAD10-based human testis cDNA library (2.5×106 independent clones). Altogether, 786 His+ bGal+ clones were obtained by screening 6.8×107 clones using HSF2N96 as a bait ( Table 1). In contrast, only nine clones were isolated from a screening of 5.0×107 clones using HSF2N96C328 as a bait, suggesting that many of the clones obtained with HSF2N96 indeed encode proteins that would specifically interact with HSF2N96. The nt sequences were determined for the 5∞ ends of 91 clones obtained with HSF2N96 and all nine clones obtained with HSF2N96C328. Among the 100 clones sequenced, nucleoporin p62 was the only known protein encoded, when the clones that appeared merely once were excluded. Most significantly, about half of the clones obtained with HSF2N96 and HSF2N96C328 were shown to encode part of the same protein, named HSF2-binding protein (HSF2BP) (Table 1). Accordingly, lgt11 human
Fig. 1. Human HSF2 used as baits for two-hybrid screening. (A) Schematic representation of human HSF2. DBD, DNA binding domain; HR-A/B, hydrophobic repeat for trimer formation (trimerization domain); HR-C, C-terminal hydrophobic repeat for negative regulation. Two truncated HSF2 mutants, HSF2N96 (aa 96 to C-terminus) and HSF2N96C328 (aa 96–328), were fused to GAL4-BD. (B) Western blot analysis of the GAL4-BD-HSF2 fusion proteins expressed in yeast. Yeast cell lysates (20 mg proteins) were subjected to 8% SDS–PAGE and analyzed by Western blotting, using anti-HSF2 serum and ECL reagent.
testis cDNA library (2.6×105 clones) was screened by plague hybridization, using one of the HSF2BP clones as a probe. This screening yielded 19 positive clones including alternatively spliced isoforms of HSF2BP, and a full-length cDNA for HSF2BP. The cloned cDNA was 1916 bp long and contained an open reading flame with 334 aa (M 37 644) (Fig. 2A). The N-terminal hydrophilic r region of the protein is predicted to form an a-helical structure containing two leucine-zipper motifs, whereas the C-terminal half is largely hydrophobic and forms bsheets (Fig. 2B). The nt database search indicated that
Table 1 Summary of two-hybrid screenings HSF2 baits
Screeneda
His+b
Gal+c
Sequencedd
HSF2BPe
p62f
N96C328 N96
5.0×107 6.8×107
14 1487
9 786
9 91
4 41
1 6
aTotal number of clones screened by two-hybrid assay using each HSF2 bait. bThe number of His+ clones obtained by selection on His− plates containing 3-aminotriazole. cThe number of bGal+ clones among His+ clones tested. dThe number of sequenced clones among bGal positives. e,fThe number of clones encoding HSF2BP and p62, respectively, among the sequenced clones.
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Fig. 2. Primary sequence of HSF2BP and prediction of secondary structures. (A) Upper line shows the 1916 nt of HSF2BP cDNA, and the second line shows the deduced aa sequence (bold). Asterisks and open rectangles show heptad repeats of hydrophobic aa. The DNA database Accession No. is AB007131. (B) a-helix, b-sheet and b-turn regions of HSF2BP were predicted according to the method of Chou–Fasman (Chou, 1990), and hydropathy was predicted by the method of Kyte and Doolittle (1982).
the HSF2BP gene was located on human chromosome 21q22.3, on which the Unverricht–Lundborg type of progressive myoclonus epilepsy (EPM1) and autoimmune polyglandular disease type I (APECED) have been mapped (Aaltonen et al., 1994; Lehesjoki et al., 1994).
3.2. Testis-specific expression of HSF2BP When the expression of HSF2BP in various human tissues was examined by Northern blotting, a transcript of approximately 1.9 kb was uniquely detected in testis ( Fig. 3). This seemed to be of interest because HSF2
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Fig. 3. Northern blot analysis. MTN blots (Clontech), containing 2 mg of poly(A)+ RNA from 16 different human tissues ( lanes 1–16), and a nylon membrane, onto which 1 mg of poly(A)+ RNA from K562 cells, F9 cells and human testis were blotted ( lanes 17–20), were hybridized with 32P-labeled HSF2BP, HSF2, and G3PDH cDNA probes.
itself was highly expressed in testis (Sarge et al., 1994; Fig. 3). In view of the constitutive binding to HSE of HSF2 in mouse testis, which is correlated with the testisspecific expression of HSP70.2 (Sarge et al., 1994), HSF2BP might well be involved in regulation of hsp70.2. Although HSE binding of HSF2 observed in F9 EC cells (Murphy et al., 1994) suggested a possible participation of HSF2BP in modulating HSF2 activation, we failed to detect any HSF2BP mRNA in the F9 cells under the conditions used (Fig. 3). Furthermore, HSF2BP mRNA was not detected in K562 cells in which hemin-induced HSE binding of HSF2 was reported previously (Sistonen et al., 1992). 3.3. Interaction of HSF2 with HSF2BP in vitro To confirm the direct interaction of HSF2BP with HSF2, a GST pull-down assay was performed, in which
GST and GST–HSF2 deletion proteins synthesized in E. coli ( Yoshima et al., 1997) were immobilized on glutathione-Sepharose beads and incubated with 35S-Met-labeled HSF2BP translated in vitro. As depicted in Fig. 4, HSF2BP showed significant binding to both GST-HSF2N96 (7.9% of input) and GSTHSF2N96C328 (9.3% of input), but very little binding to HSF2N199C328 lacking the trimerization domain (2.4% of input). These results suggested that the interaction is mediated by the trimerization domain of HSF2, consistent with the finding that both HSF2 constructs (N96 and N96C328) could fish out HSF2BP. The interaction of HSF2BP and a similar set of truncated forms of HSF1 were next examined using GST-HSF1N101 containing HSF1 lacking the DNA binding domain, GST-HSF1N101C318 with the trimerization and heat shock-responsive domains, and GSTHSF1N202C318 with only the heat shock-responsive
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Fig. 4. Interaction of HSF2BP with HSF2 and HSF1 in vitro. GST ( lane 2) and GST-HSFs ( lanes 3–8) synthesized in E. coli were immobilized on glutathione-Sepharose beads, and the GST-pull down assay was performed by incubating the beads with 35S-Met-labeled, in-vitro translated HSF2BP ( lane 1). Bound proteins were analyzed on SDS–PAGE and visualized by autoradiography. The slightly different mobilities of labeled-HSF2BP in different lanes result from interference by non-labeled proteins, such as GST-HSF2N96. The amounts of bound HSF2BP were quantified and shown as percentages to the input.
domain. When GST-HSF1 protein-immobilized glutathione beads were incubated with 35S-labeled HSF2BP (Fig. 4, lanes 6–8), HSF2BP was bound to GSTHSF1N101 (8.5% of input) but not to GSTHSF1N202C318. The binding to GST-HSF1N101C318 (3.5% of input) seemed to be barely significant. The levels of GST-HSF1 and GST-HSF2 derivatives used in this assay were comparable as shown previously ( Yoshima et al., 1997). These results suggested that HSF2BP can bind to HSF1 as well as to HSF2 in vitro with similar affinity, although the binding profiles were slightly different. Four deletion derivatives (BP-C150, BP-C115, BP-C73 and BP-N115) of HSF2BP were then constructed ( Fig. 5A), translated in vitro with 35S-Met (Fig. 5B), and subjected to a pull-down assay with GSTHSF2N96C328. When compared with intact HSF2BP (12.9% bound), increased (BP-C150, 22.3% bound) or decreased binding (BP-C115, 5.7% bound) was observed (Fig. 5C, lanes 1–9), whereas little or no binding was detected with BP-C73 or BP-N115 ( lanes 10–15). Thus, the N-terminal half of HSF2BP appeared to be necessary and sufficient for its interaction with HSF2. Since the N-terminal a-helical domain of HSF2BP contains two LZ motifs (aa 21–42 and 50–71) (Fig. 2A), the interaction might occur between these motifs and the HR-A/B region (trimerization domain) of HSF2 or HSF1. The inability of BP-C73 to bind to HSF2 despite the presence of the two LZs suggests the involvement
Fig. 5. Deletion analysis of HSF2BP for the activity to interact with HSF2. (A) Truncated forms of HSF2BP tested are schematically shown. (B) Intact and four truncated forms of HSF2BP were in-vitro translated in the presence of 35S-methionine, analyzed by SDS–PAGE, and autoradiographed. Molecular weight markers are indicated on the right (kDa). The N-terminal hydrophilic region is delineated by stippled sections. (C ) Intact and truncated HSF2BPs were subjected to pull-down assay with immobilized GST or GST-HSF2N96C328. Bound proteins were analyzed by SDS–PAGE and visualized by autoradiography. The percentages of bound HSF2BPs are shown. Lanes 1, 4, 7, 10 and 13, input (one-fifth aliquots of incubated mixture were loaded ); lanes 2, 5, 8, 11 and 14, bound to GST; lanes 3, 6, 9, 12 and 15, bound to GST-HSF2N96C328.
of the predicted third or fourth a-helical domain ( Fig. 2B) for interaction. 3.4. Two-hybrid assays for HSF2BP–HSF2 interaction in mammalian cells To examine the interaction of HSF2BP with HSF2 or HSF1 in vivo, K562 cells were transfected with plasmids, each producing the HSF2 or HSF1 deletion derivatives
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Fig. 6. Two-hybrid assay for HSF2BP–HSF2 interaction in K562 cells. 1×106 cells of K562 were transfected with 1.5 mg each of the HSF expression plasmid, 1.5 mg of VP16 or VP16-HSF2BP expression plasmid, 0.5 mg of pGLG4E5 luciferase reporter plasmid and 0.2 mg of pRLSV40 reference plasmid. After 48 h, cells were harvested and subjected to dual luciferase assay. Activities of firefly luciferase were normalized with those of Renilla luciferase, and fold induction obtained by cotransfection with pVP16-HSF2BP vs. pVP16 for each HSF construct is presented as the mean±SD (bars), based on four independent experiments.
fused to the yeast GAL4-BD and HSF2BP fused to the VP16 activation domain, together with the pGLG4E5 luciferase reporter plasmid (Fig. 6). Simultaneous synthesis of GAL4-HSF2N96C328 with VP16-HSF2BP activated the reporter by sixfold over the control simultaneously synthesized with VP16, suggesting that HSF2BP can interact with HSF2 in mammalian cells. No significant interaction of HSF2N96 with HSF2BP was detected by this assay, though many HSF2BP clones were obtained from the yeast two-hybrid screening with HSF2N96 ( Table 1). Neither of the HSF1 constructs tested interacted with HSF2BP under these conditions.
4. Discussion We identified a novel human protein specifically synthesized in testis, HSF2BP, that can interact with HSF2. The interaction seems to occur between the trimerization domain (HR-A/B) of HSF2 containing heptad repeats and the N-terminal hydrophilic region of HSF2BP containing two LZ motifs ( Figs. 4 and 5). The specificity of this interaction is supported by the following grounds:
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(1) Known LZ-containing proteins were not isolated in the yeast two-hybrid screening except for nucleoporin p62 ( Yoshima et al., 1997), although almost half the sequenced bGal positive clones encoded HSF2BP ( Table 1). (2) Despite the structural similarity HSF1 was unable to interact with HSF2BP in K562 cells ( Fig. 6). Nucleoporin p62, a major component of nuclear pore complex, was previously isolated from K562 cell cDNA libraries by the yeast two-hybrid screening using the same baits ( Yoshima et al., 1997). Seven clones encoding p62 were also obtained from the present screening of the human testis library ( Table 1), although mRNA for p62 had not been detected in rat testis (Starr et al., 1990). The fact that we found clones for such rare species of mRNA provides a certain assurance for the validity of the present screening for interaction with HSF2 in testis. The failure to isolate HSF2 itself through its trimerization domain is probably explained by the conformation of bait HSF2 used. Since HSF2N96 as well as HSF2N96C328 lacking the HR-C region, when expressed in yeast cells, presumably forms trimers (Liu et al., 1997), it is unlikely that these baits can interact further with HSF2. We have demonstrated that HSF2BP interacts with HSF2N96C328 by the GST pull-down assay in vitro and by the two-hybrid assay in mammalian cells, consistent with the results of the yeast two-hybrid screening. However, no in-vivo interaction was detected with HSF2N96 or HSF1N101, though their interaction was confirmed in vitro. Since HSF2N96 was able to fish out HSF2BP efficiently in the yeast screening, HSF2N96 synthesized in yeast probably assumes the conformation that would permit DNA binding as described above, whereas in K562 cells, it may promote an intramolecular coiled-coil interaction that prevents access of HSF2BP to the trimerization domain of HSF2. It seems likely, therefore, that HSF2BP interacts with the DNA-binding trimer form of HSF2. It would be of great interest to see whether HSF2BP is found as part of complex involving HSE bound trimer form of HSF2 in testis. The histological distribution of both HSF2BP and HSF2 in the same developmental or spermatogenetic phase should also help to clarify the potential role of HSF2BP in modulating the HSF2 activity. The reason for the lack of interaction between HSF2BP and HSF1 in K562 cells remains to be resolved: some unknown factor associating with HSF1 or posttranslational modification of HSF1, such as phosphorylation, might be involved.
Acknowledgement We thank Akira Nakai for generously providing antibodies to HSF2, Hiderou Yoshida for constant discus-
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sions, Masako Nakayama, Mayumi Ueda and Hideaki Kanazawa for technical assistance.
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Novel testis-specific protein that interacts with heat shock factor 2 Tadahiko Yoshima 1, Takashi Yura, Hideki Yanagi * HSP Research Institute, Kyoto Research Park, Kyoto 600-8813, Japan Received 4 February 1998; accepted 13 April 1998; Received by J. Wild
Abstract Although heat shock factor 2 (HSF2) binds to heat shock element (HSE ) constitutively during differentiation, development and spermatogenesis, little is known about the nature and mechanism of transcriptional control of heat shock genes by HSF2. Using the yeast two-hybrid system, we screened a human testis cDNA library for proteins that can associate with HSF2 by the yeast two-hybrid system, and isolated clones encoding a novel protein, designated HSF2-binding protein (HSF2BP), that associates with HSF2 in vitro and in vivo and is specifically synthesized in testis. The interaction seemed to occur between the trimerization domain of HSF2 and the N-terminal hydrophilic region of HSF2BP that comprises two leucine zipper motifs. HSF2BP may therefore be involved in modulating HSF2 activation in testis. © 1998 Elsevier Science B.V. All rights reserved. Keywords: HSF; Two-hybrid; Leucine zipper; Pull-down; Protein–protein interaction
1. Introduction Heat shock factor 2 (HSF2) is a member of the vertebrates HSF family that includes HSF1, HSF2, HSF3 and HSF4 (Rabindran et al., 1991; Schuetz et al., 1991; Nakai and Morimoto, 1993; Nakai et al., 1997) and binds to heat shock elements (HSEs) located at the promoter regions of genes encoding heat shock proteins (HSPs). HSF1 and HSF3 respond to a heat shock stress and other environmental stresses and induce transcription of HSP-encoding genes (Baler et al., 1993; Sarge et al., 1993; Nakai et al., 1995). Unlike HSF1 and HSF3, HSF2 is not activated by such stresses but is thought to play an important role during differentiation and development. HSF4 is believed to be involved in negative regulation of other HSFs binding to DNA * Corresponding author. Tel: +81 75 315 8685; Fax: +81 75 315 8659; e-mail: [email protected] 1 Present address: Sumitomo Pharmaceuticals Research Center, 3-1-98 Kasugade-naka, Konohana-ku, Osaka 554-0022, Japan. Abbreviations: Aa, amino acid(s); bGal, b-galactosidase; EC, embryonal carcinoma; GAL4-BD, GAL4 DNA binding domain; GST, glutathione S-transferase; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; HSE, heat shock element; HSF, heat shock factor; HSF2BP, HSF2 binding protein; HSP, heat shock protein; LZ, leucine zipper; nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamidegel electrophoresis; PBS, phosphate-buffered saline; PBST, PBS containing 0.1% Tween 20; SDS, sodium dodecyl sulfate . 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0 3 7 8 -1 1 1 9 ( 9 8 ) 0 0 20 8 - X
because of the absence of a transcriptional activation domain (Nakai et al., 1997). Although there have been several findings that suggest HSF2 activation during differentiation and development, the way in which HSF2 contributes to transcriptional regulation of heat shock genes remains unresolved. Sistonen et al. (1992) first reported that hemin-induced HSE binding of HSF2 in human K562 erythroleukemia cells was accompanied by transcriptional induction of the hsp70 gene. In spermatogenic cells of mouse testis, HSF2 was found to bind constitutively to HSE and appeared to be correlated with testisspecific expression of hsp70.2 (Sarge et al., 1994). However, constitutive HSE-binding activity found in mouse embryonal carcinoma ( EC ) cell extracts was composed predominantly of HSF2, whereas the HSE region of hsp70 promoter was not occupied by HSF2 (Murphy et al., 1994). Furthermore, zygotic expression of hsp70 and HSE-binding activity of HSF2 were detected during mouse embryonal development (Bensaude et al., 1983; Rallu et al., 1997), but no obvious correlation was found between expression patterns of major HSPs and of HSF2 in embryos (Bevilacqua et al., 1997; Rallu et al., 1997). In the case of HSF1, induction of DNA binding of the HSF1 trimer is not sufficient to activate transcription of hsp genes (Jurivich et al., 1992; Zuo et al., 1995), and additional regulatory step(s) or pathway(s) are
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required for transition from a transcriptionally inactive trimer to active forms. The transactivation function of HSF1 is now known to be modulated by constitutive phosphorylation and stress-induced hyperphosphorylation (Chu et al., 1996; Knauf et al., 1996; Kline and Morimoto, 1997; Xia and Voellmy, 1997). Although such modifications have not been reported for HSF2, it seemed likely that some unknown factors are involved in modulating the activity of HSF2. We report here on a novel cDNA encoding a 334-aa polypeptide HSF2BP that is specifically expressed in testis. The physical interaction between HSF2 and HSF2BP was demonstrated both in vitro and in vivo.
2.3. Western blotting with yeast lysates Yeast cell extracts were prepared using glass beads, and proteins (20 mg) were separated by 8% SDS–PAGE, and blotted on to nitrocellulose membranes (Amersham, Bucks, UK ). The membranes were treated with 5% skim milk in PBS containing 0.1% Tween 20 (PBST ) and with polyclonal rabbit antibodies against chicken HSF2 (aHSF2a) (kindly provided by A. Nakai). After washing with PBST, the membranes were treated with horseradish peroxidase-conjugated goat F(ab∞) anti-rabbit 2 antibody (Biosource, Camarillo, CA), washed with PBST, and detected using ECL reagent (Amersham). 2.4. Cloning of full-length cDNA for HSF2BP
2. Materials and methods 2.1. Plasmid constructions Expression plasmids for truncated forms of HSF2 or HSF1 fused with the GAL4 DNA-binding domain (GAL4-BD) were constructed from pGBT9 and pM (Clontech, Palo Alto, CA) as ‘baits’ for yeast and mammalian two-hybrid assays, respectively ( Yoshima et al., 1997). Glutathione S-transferase (GST ) fusion constructs of HSF2 or HSF1 and reporter plasmids pGLG4E5 and pRLSV40 for dual luciferase assay have also been described ( Yoshima et al., 1997). pVP16-HSF2BP that expresses HSF2BP fused to the VP16 activation domain was constructed by inserting HSF2BP cDNA into pVP16 (Clontech). For in-vitro translation of HSF2BP and its truncated forms, the corresponding regions of HSF2BP cDNA were inserted into pcDNA3.1HisA, B, or C (Invitrogen, Carlsbad, CA). 2.2. Yeast strains and two-hybrid screenings HF7c (MATa, ura3–52, his3–200, lys2–801, trp1–901, ade2–101, leu2–3,112, gal4–542, gal80–538, LYS:: GAL1–HIS3, URA3::(GAL4 17mers)3-CYC1-lacZ) used for two-hybrid screenings and a human testis cDNA library constructed with pGAD10 were obtained from Clontech. The transformation of yeast cells by the lithium-acetate method and the two-hybrid assay were described previously ( Yoshima et al., 1997). In brief, HF7c cells were transformed with a plasmid expressing HSF2 bait and with a human cDNA plasmid library, His+ transformants were selected and tested for bgalactosidase ( bGal ) activities. Plasmid DNAs were isolated from His+ bGal+ clones, and the nt sequence of the 5∞ ends of the cDNA inserts was determined by a DNA autosequencer PRISM 377 (Perkin Elmer, Foster City, CA).
A human testis cDNA library (lgt11 containing 1×106 independent clones, Clontech) was screened by plaque hybridization using a 32P-labeled cDNA fragment of HSF2BP obtained by the present screening as a probe. The longest clone obtained was used for nt sequencing. 2.5. Cell culture K562 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum in 5% CO at 37°C. 2 Transfections were carried out by the lipofection method using Transfectam@ (Promega, Madison, W1), and transfected cells were cultured for 48 h and subjected to a Dual-Luciferase Reporter Assay (Promega). F9 cells were cultured in Dulbecco’s Modified Eagle Medium containing 10% fetal bovine serum in 5% CO at 37°C. 2 2.6. Northern blot analysis RNA was extracted from 60 mM hemin-treated or untreated K562 cells and F9 cells using Isogen (Nippon Gene, Tokyo, Japan), and poly(A)+ RNA was selected by Oligotex-dT30 ( Takara, Kyoto, Japan). Poly(A)+ RNA of human testis was obtained from Clontech. These RNAs were separated on 1% agarose gel containing formaldehyde and transferred to a nylon membrane (Du Pont). RNA blots of multiple human tissues were purchased from Clontech (MTN blot and MTNII blot). The membranes were treated with 32P-labeled cDNA probes for HSF2BP, HSF2, or glyceraldehyde3-phosphate dehydrogenase (G3PDH) (Clontech), washed in 0.2× SSPE containing 0.1% SDS at 42°C, followed by autoradiography. 2.7. GST pull-down assay GST and GST–HSF fusion proteins were purified, and immobilized on glutathione-Sepharose CL4B (Pharmacia, Uppsala, Sweden). HSF2BP and its trun-
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cated forms were translated in vitro using the TNT translation kit (Promega). Five microliters of the reaction mixture were incubated with immobilized GST or GST–HSF overnight at 4°C. Beads were washed with NETN buffer (20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40), and bound proteins were analyzed by SDS–PAGE, processed for autoradiography, and quantified by an imaging analyzer BAS2000 (Fuji, Tokyo, Japan).
3. Results 3.1. cDNA cloning of HSF2BP To search for proteins that can associate with HSF2 in testis, we screened a human testis cDNA library by using yeast two-hybrid system. HSF2 deletion mutants HSF2N96 or HSF2N96C328 fused to GAL4-BD were used as ‘baits’ for screening, because HSF2N96 was expected to fish out proteins that could interact with HSF2 that binds DNA but is transcriptionally inactive, whereas HSF2N96C328 was expected to find proteins that could interact with an activated form of HSF2 ( Yoshima et al., 1997). The expression of these two fusion proteins in yeast was confirmed by Western blot analysis ( Fig. 1). The pair of yeast strains were transformed with a pGAD10-based human testis cDNA library (2.5×106 independent clones). Altogether, 786 His+ bGal+ clones were obtained by screening 6.8×107 clones using HSF2N96 as a bait ( Table 1). In contrast, only nine clones were isolated from a screening of 5.0×107 clones using HSF2N96C328 as a bait, suggesting that many of the clones obtained with HSF2N96 indeed encode proteins that would specifically interact with HSF2N96. The nt sequences were determined for the 5∞ ends of 91 clones obtained with HSF2N96 and all nine clones obtained with HSF2N96C328. Among the 100 clones sequenced, nucleoporin p62 was the only known protein encoded, when the clones that appeared merely once were excluded. Most significantly, about half of the clones obtained with HSF2N96 and HSF2N96C328 were shown to encode part of the same protein, named HSF2-binding protein (HSF2BP) (Table 1). Accordingly, lgt11 human
Fig. 1. Human HSF2 used as baits for two-hybrid screening. (A) Schematic representation of human HSF2. DBD, DNA binding domain; HR-A/B, hydrophobic repeat for trimer formation (trimerization domain); HR-C, C-terminal hydrophobic repeat for negative regulation. Two truncated HSF2 mutants, HSF2N96 (aa 96 to C-terminus) and HSF2N96C328 (aa 96–328), were fused to GAL4-BD. (B) Western blot analysis of the GAL4-BD-HSF2 fusion proteins expressed in yeast. Yeast cell lysates (20 mg proteins) were subjected to 8% SDS–PAGE and analyzed by Western blotting, using anti-HSF2 serum and ECL reagent.
testis cDNA library (2.6×105 clones) was screened by plague hybridization, using one of the HSF2BP clones as a probe. This screening yielded 19 positive clones including alternatively spliced isoforms of HSF2BP, and a full-length cDNA for HSF2BP. The cloned cDNA was 1916 bp long and contained an open reading flame with 334 aa (M 37 644) (Fig. 2A). The N-terminal hydrophilic r region of the protein is predicted to form an a-helical structure containing two leucine-zipper motifs, whereas the C-terminal half is largely hydrophobic and forms bsheets (Fig. 2B). The nt database search indicated that
Table 1 Summary of two-hybrid screenings HSF2 baits
Screeneda
His+b
Gal+c
Sequencedd
HSF2BPe
p62f
N96C328 N96
5.0×107 6.8×107
14 1487
9 786
9 91
4 41
1 6
aTotal number of clones screened by two-hybrid assay using each HSF2 bait. bThe number of His+ clones obtained by selection on His− plates containing 3-aminotriazole. cThe number of bGal+ clones among His+ clones tested. dThe number of sequenced clones among bGal positives. e,fThe number of clones encoding HSF2BP and p62, respectively, among the sequenced clones.
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Fig. 2. Primary sequence of HSF2BP and prediction of secondary structures. (A) Upper line shows the 1916 nt of HSF2BP cDNA, and the second line shows the deduced aa sequence (bold). Asterisks and open rectangles show heptad repeats of hydrophobic aa. The DNA database Accession No. is AB007131. (B) a-helix, b-sheet and b-turn regions of HSF2BP were predicted according to the method of Chou–Fasman (Chou, 1990), and hydropathy was predicted by the method of Kyte and Doolittle (1982).
the HSF2BP gene was located on human chromosome 21q22.3, on which the Unverricht–Lundborg type of progressive myoclonus epilepsy (EPM1) and autoimmune polyglandular disease type I (APECED) have been mapped (Aaltonen et al., 1994; Lehesjoki et al., 1994).
3.2. Testis-specific expression of HSF2BP When the expression of HSF2BP in various human tissues was examined by Northern blotting, a transcript of approximately 1.9 kb was uniquely detected in testis ( Fig. 3). This seemed to be of interest because HSF2
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Fig. 3. Northern blot analysis. MTN blots (Clontech), containing 2 mg of poly(A)+ RNA from 16 different human tissues ( lanes 1–16), and a nylon membrane, onto which 1 mg of poly(A)+ RNA from K562 cells, F9 cells and human testis were blotted ( lanes 17–20), were hybridized with 32P-labeled HSF2BP, HSF2, and G3PDH cDNA probes.
itself was highly expressed in testis (Sarge et al., 1994; Fig. 3). In view of the constitutive binding to HSE of HSF2 in mouse testis, which is correlated with the testisspecific expression of HSP70.2 (Sarge et al., 1994), HSF2BP might well be involved in regulation of hsp70.2. Although HSE binding of HSF2 observed in F9 EC cells (Murphy et al., 1994) suggested a possible participation of HSF2BP in modulating HSF2 activation, we failed to detect any HSF2BP mRNA in the F9 cells under the conditions used (Fig. 3). Furthermore, HSF2BP mRNA was not detected in K562 cells in which hemin-induced HSE binding of HSF2 was reported previously (Sistonen et al., 1992). 3.3. Interaction of HSF2 with HSF2BP in vitro To confirm the direct interaction of HSF2BP with HSF2, a GST pull-down assay was performed, in which
GST and GST–HSF2 deletion proteins synthesized in E. coli ( Yoshima et al., 1997) were immobilized on glutathione-Sepharose beads and incubated with 35S-Met-labeled HSF2BP translated in vitro. As depicted in Fig. 4, HSF2BP showed significant binding to both GST-HSF2N96 (7.9% of input) and GSTHSF2N96C328 (9.3% of input), but very little binding to HSF2N199C328 lacking the trimerization domain (2.4% of input). These results suggested that the interaction is mediated by the trimerization domain of HSF2, consistent with the finding that both HSF2 constructs (N96 and N96C328) could fish out HSF2BP. The interaction of HSF2BP and a similar set of truncated forms of HSF1 were next examined using GST-HSF1N101 containing HSF1 lacking the DNA binding domain, GST-HSF1N101C318 with the trimerization and heat shock-responsive domains, and GSTHSF1N202C318 with only the heat shock-responsive
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Fig. 4. Interaction of HSF2BP with HSF2 and HSF1 in vitro. GST ( lane 2) and GST-HSFs ( lanes 3–8) synthesized in E. coli were immobilized on glutathione-Sepharose beads, and the GST-pull down assay was performed by incubating the beads with 35S-Met-labeled, in-vitro translated HSF2BP ( lane 1). Bound proteins were analyzed on SDS–PAGE and visualized by autoradiography. The slightly different mobilities of labeled-HSF2BP in different lanes result from interference by non-labeled proteins, such as GST-HSF2N96. The amounts of bound HSF2BP were quantified and shown as percentages to the input.
domain. When GST-HSF1 protein-immobilized glutathione beads were incubated with 35S-labeled HSF2BP (Fig. 4, lanes 6–8), HSF2BP was bound to GSTHSF1N101 (8.5% of input) but not to GSTHSF1N202C318. The binding to GST-HSF1N101C318 (3.5% of input) seemed to be barely significant. The levels of GST-HSF1 and GST-HSF2 derivatives used in this assay were comparable as shown previously ( Yoshima et al., 1997). These results suggested that HSF2BP can bind to HSF1 as well as to HSF2 in vitro with similar affinity, although the binding profiles were slightly different. Four deletion derivatives (BP-C150, BP-C115, BP-C73 and BP-N115) of HSF2BP were then constructed ( Fig. 5A), translated in vitro with 35S-Met (Fig. 5B), and subjected to a pull-down assay with GSTHSF2N96C328. When compared with intact HSF2BP (12.9% bound), increased (BP-C150, 22.3% bound) or decreased binding (BP-C115, 5.7% bound) was observed (Fig. 5C, lanes 1–9), whereas little or no binding was detected with BP-C73 or BP-N115 ( lanes 10–15). Thus, the N-terminal half of HSF2BP appeared to be necessary and sufficient for its interaction with HSF2. Since the N-terminal a-helical domain of HSF2BP contains two LZ motifs (aa 21–42 and 50–71) (Fig. 2A), the interaction might occur between these motifs and the HR-A/B region (trimerization domain) of HSF2 or HSF1. The inability of BP-C73 to bind to HSF2 despite the presence of the two LZs suggests the involvement
Fig. 5. Deletion analysis of HSF2BP for the activity to interact with HSF2. (A) Truncated forms of HSF2BP tested are schematically shown. (B) Intact and four truncated forms of HSF2BP were in-vitro translated in the presence of 35S-methionine, analyzed by SDS–PAGE, and autoradiographed. Molecular weight markers are indicated on the right (kDa). The N-terminal hydrophilic region is delineated by stippled sections. (C ) Intact and truncated HSF2BPs were subjected to pull-down assay with immobilized GST or GST-HSF2N96C328. Bound proteins were analyzed by SDS–PAGE and visualized by autoradiography. The percentages of bound HSF2BPs are shown. Lanes 1, 4, 7, 10 and 13, input (one-fifth aliquots of incubated mixture were loaded ); lanes 2, 5, 8, 11 and 14, bound to GST; lanes 3, 6, 9, 12 and 15, bound to GST-HSF2N96C328.
of the predicted third or fourth a-helical domain ( Fig. 2B) for interaction. 3.4. Two-hybrid assays for HSF2BP–HSF2 interaction in mammalian cells To examine the interaction of HSF2BP with HSF2 or HSF1 in vivo, K562 cells were transfected with plasmids, each producing the HSF2 or HSF1 deletion derivatives
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Fig. 6. Two-hybrid assay for HSF2BP–HSF2 interaction in K562 cells. 1×106 cells of K562 were transfected with 1.5 mg each of the HSF expression plasmid, 1.5 mg of VP16 or VP16-HSF2BP expression plasmid, 0.5 mg of pGLG4E5 luciferase reporter plasmid and 0.2 mg of pRLSV40 reference plasmid. After 48 h, cells were harvested and subjected to dual luciferase assay. Activities of firefly luciferase were normalized with those of Renilla luciferase, and fold induction obtained by cotransfection with pVP16-HSF2BP vs. pVP16 for each HSF construct is presented as the mean±SD (bars), based on four independent experiments.
fused to the yeast GAL4-BD and HSF2BP fused to the VP16 activation domain, together with the pGLG4E5 luciferase reporter plasmid (Fig. 6). Simultaneous synthesis of GAL4-HSF2N96C328 with VP16-HSF2BP activated the reporter by sixfold over the control simultaneously synthesized with VP16, suggesting that HSF2BP can interact with HSF2 in mammalian cells. No significant interaction of HSF2N96 with HSF2BP was detected by this assay, though many HSF2BP clones were obtained from the yeast two-hybrid screening with HSF2N96 ( Table 1). Neither of the HSF1 constructs tested interacted with HSF2BP under these conditions.
4. Discussion We identified a novel human protein specifically synthesized in testis, HSF2BP, that can interact with HSF2. The interaction seems to occur between the trimerization domain (HR-A/B) of HSF2 containing heptad repeats and the N-terminal hydrophilic region of HSF2BP containing two LZ motifs ( Figs. 4 and 5). The specificity of this interaction is supported by the following grounds:
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(1) Known LZ-containing proteins were not isolated in the yeast two-hybrid screening except for nucleoporin p62 ( Yoshima et al., 1997), although almost half the sequenced bGal positive clones encoded HSF2BP ( Table 1). (2) Despite the structural similarity HSF1 was unable to interact with HSF2BP in K562 cells ( Fig. 6). Nucleoporin p62, a major component of nuclear pore complex, was previously isolated from K562 cell cDNA libraries by the yeast two-hybrid screening using the same baits ( Yoshima et al., 1997). Seven clones encoding p62 were also obtained from the present screening of the human testis library ( Table 1), although mRNA for p62 had not been detected in rat testis (Starr et al., 1990). The fact that we found clones for such rare species of mRNA provides a certain assurance for the validity of the present screening for interaction with HSF2 in testis. The failure to isolate HSF2 itself through its trimerization domain is probably explained by the conformation of bait HSF2 used. Since HSF2N96 as well as HSF2N96C328 lacking the HR-C region, when expressed in yeast cells, presumably forms trimers (Liu et al., 1997), it is unlikely that these baits can interact further with HSF2. We have demonstrated that HSF2BP interacts with HSF2N96C328 by the GST pull-down assay in vitro and by the two-hybrid assay in mammalian cells, consistent with the results of the yeast two-hybrid screening. However, no in-vivo interaction was detected with HSF2N96 or HSF1N101, though their interaction was confirmed in vitro. Since HSF2N96 was able to fish out HSF2BP efficiently in the yeast screening, HSF2N96 synthesized in yeast probably assumes the conformation that would permit DNA binding as described above, whereas in K562 cells, it may promote an intramolecular coiled-coil interaction that prevents access of HSF2BP to the trimerization domain of HSF2. It seems likely, therefore, that HSF2BP interacts with the DNA-binding trimer form of HSF2. It would be of great interest to see whether HSF2BP is found as part of complex involving HSE bound trimer form of HSF2 in testis. The histological distribution of both HSF2BP and HSF2 in the same developmental or spermatogenetic phase should also help to clarify the potential role of HSF2BP in modulating the HSF2 activity. The reason for the lack of interaction between HSF2BP and HSF1 in K562 cells remains to be resolved: some unknown factor associating with HSF1 or posttranslational modification of HSF1, such as phosphorylation, might be involved.
Acknowledgement We thank Akira Nakai for generously providing antibodies to HSF2, Hiderou Yoshida for constant discus-
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sions, Masako Nakayama, Mayumi Ueda and Hideaki Kanazawa for technical assistance.
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