Formation of Nuclear Stress Granules Involves HSF2 and Coincides with the Nucleolar Localization of Hsp70

Research Article 3557 Formation of nuclear stress granules involves HSF2 and coincides with the nucleolar localization of Hsp70

Tero-Pekka Alastalo1,*, Maria Hellesuo1,*, Anton Sandqvist1,2, Ville Hietakangas1, Marko Kallio1,‡ and Lea Sistonen1,3,§ 1Turku Centre for Biotechnology, University of Turku, Åbo Akademi University, BioCity, PO Box 123, FIN-20521 Turku, Finland 2Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland 3Department of Biology, Åbo Akademi University, Turku, Finland *These authors contributed equally to this work ‡Present address: Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK-73104, USA. §Author for correspondence (e-mail: [email protected].ﬁ)

Summary The heat-shock response is characterized by the activation results indicate that the stress granules are dynamic of heat-shock transcription factor 1 (HSF1), followed by structures and suggest that they might be regulated in an increased expression of heat-shock proteins (Hsps). The Hsp70-dependent manner. The reversible localization of stress-induced subnuclear compartmentalization of HSF1 Hsp70 in the nucleoli strictly coincides with the presence of into nuclear stress granules has been suggested to be an HSF1 in stress granules and is dramatically suppressed important control step in the regulation of stress response in thermotolerant cells. We propose that the regulated and cellular homeostasis in human cells. In this study, subcellular distribution of Hsp70 is an important we demonstrate that the less-well characterized HSF2 regulatory mechanism of HSF1-mediated heat shock interacts physically with HSF1 and is a novel stress- response. responsive component of the stress granules. Based on analysis of our deletion mutants, HSF2 inﬂuences to the localization of HSF1 in stress granules. Moreover, our Key words: Heat-shock response, Hsf, Hsp70, Stress granule

Introduction cells the formation of HSF1 trimers is hampered and Hsp Cells have developed a range of processes to increase survival expression is reduced upon heat shock (Tanabe et al., 1998). and adaptation in response to different forms of stress. One of The interdependency between HSF1 and HSF3 in avian cells these events is the induced expression of heat-shock genes is so far the only example of cross-talk between different HSFs. coding for heat-shock proteins (Hsps). Hsps have an important However, no physical interaction has been reported between role in cellular homeostasis and protection through their well- these two proteins. recognized properties as molecular chaperones (Bukau and A characteristic feature of cellular stress in human cells is Horwich, 1998). The expression of the heat-shock genes is the organization of HSF1 into specific subnuclear structures, primarily regulated at transcriptional level by specific DNA- termed stress granules. These irregularly shaped granules have binding proteins called heat-shock factors (HSFs), which bind been described to form under various stress conditions, to the heat-shock promoter element (HSE) (Pirkkala et al., including exposure to heat, cadmium, azetidine and 2001). Four members of this family, HSF1-HSF4, have been proteasome inhibitors (Cotto et al., 1997; Jolly et al., 1997; identified in vertebrates (Rabindran et al., 1991; Sarge et al., Jolly et al., 1999; Holmberg et al., 2000). Stress-induced HSF1 1991; Schuetz et al., 1991; Nakai and Morimoto, 1993; Nakai granules have been found in all investigated primary and et al., 1997), which has raised questions about specific or transformed human cells but not in rodent cells (Sarge et al., overlapping functions among the distinct HSFs. 1993; Mivechi et al., 1994; Cotto et al., 1997; Jolly et al., HSF1 is the most studied heat-shock transcription factor and 1999). The appearance of stress granules correlates positively is the classical HSF, which responds to elevated temperatures with the inducible phosphorylation and transcriptional activity and other forms of protein damaging stress (Pirkkala et al., of HSF1 (Sarge et al., 1993; Cotto et al., 1997; Jolly et al., 2001). In addition, HSF1 has been shown to be involved in 1999; Holmberg et al., 2000). Furthermore, HSF1 dissociates extra-embryonic development and female fertility in mice from the stress granules and relocalizes diffusely in the cell (Xiao et al., 1999; Christians et al., 2000). Upon stress, HSF1 during attenuation and recovery from stress; upon subsequent is rapidly converted from a monomer to a trimer, is inducibly exposure to stress, the granules reappear at the same sites (Jolly phosphorylated and concentrated in the nucleus to activate et al., 1999). The stress granules have not been shown to heat-shock gene transcription (Baler et al., 1993; Rabindran et represent other previously described nuclear structures (Cotto al., 1993; Sarge et al., 1993). In addition to HSF1, chicken et al., 1997). In addition to HSF1, several RNA binding HSF3 is activated by similar but more severe stressors (Nakai proteins such as heterogeneous nuclear ribonucleoprotein and Morimoto, 1993; Tanabe et al., 1997). In HSF3-deficient (hnRNP) HAP (hnRNP A1 interacting protein), hnRNP M, 3558 Journal of Cell Science 116 (17) Src-activated during mitosis (Sam68) and certain SR (serine- Kingston (Sheldon and Kingston, 1993), HSF2 localizes in the arginine) splicing factors have been identified in stress granules nucleus and in granule-type structures in HeLa cells exposed (Weighardt et al., 1999; Denegri et al., 2001). Recently, stress to heat stress. Furthermore, Mathew et al. (Mathew et al., 2001) granules were revealed to associate with human chromosome have reported that, in murine fibroblasts, HSF2 has the 9q11-q12, corresponding to a large block of heterochromatin biochemical properties of a temperature-sensitive protein composed primarily of satellite III repeats adjacent to the because, upon heat shock, HSF2 is localized to the perinuclear centromere (Jolly et al., 2002). Furthermore, Denegri et al. region, its solubility is decreased and DNA-binding activity is (Denegri et al., 2002) have reported that, in addition to diminished. This intriguing difference between human and chromosome 9, chromosomes 12 and 15 also contain murine cells has also been observed with HSF1, because the nucleation sites for stress granules. The functional significance stress-induced subnuclear compartmentalization of HSF1 has of stress granules is still unknown. been detected only in human cells (Sarge et al., 1993; Denegri The autoregulation of the heat-shock response is facilitated et al., 2002). Altogether, direct evidence of HSF2, human or by a direct interaction between HSF1 and molecular murine, being a physiological transcriptional regulator of heat chaperones, such as Hsp70 and Hsp90 with their co- shock genes is missing. chaperones. This has been established in several biochemical, Despite the characterization of several biochemical genetic and cell physiological studies (Abravaya et al., 1992; properties of HSF1 and HSF2, the actual stress sensors and the Baler et al., 1992; Kim et al., 1995; Ali et al., 1998; Shi et al., signaling pathways regulating these two factors have remained 1998; Zou et al., 1998; Bharadwaj et al., 1999; Morimoto, obscure. In this study, we provide evidence that HSF1 and 2002). Under normal physiological conditions, HSF1 exists in HSF2 form a physically interacting complex and co-localize in a repressed state associated with molecular chaperones. Upon the dynamically regulated nuclear stress granules, suggesting stress, it is rapidly released from the complex, trimerized a novel role for HSF2. We also show data suggesting that the and hyperphosphorylated, and acquires DNA-binding and association of HSF1 and HSF2 with stress granules might be transcriptional activity. When exposed to continuous moderate regulated by Hsp70, and that the association is severely heat stress, the equilibrium is shifted back to the monomeric, impaired in thermotolerant cells. Taken together, our results dephosphorylated and Hsp-bound state. The downregulation suggest that the regulation of the subcellular distribution of of HSF1 activity during prolonged stress is referred to as Hsp70 contributes to the regulation of HSF1-mediated heat- attenuation of the heat-shock response, and leads to the shock responses. suppression of HSF1 reactivation capacity upon subsequent stress. A similar repression of HSF1 activity is observed when cells recover from stressful conditions. This acquired Materials and Methods adaptation of cells to stress stimuli is called thermotolerance Cell culture and experimental treatments (Morimoto, 2002). Human K562 erythroleukemia cells and HeLa cervical carcinoma Biochemical characterization of HSF2 has revealed that, cells were maintained in RPMI-1640 medium and Dulbecco’s unlike HSF1, which undergoes a monomer-to-trimer transition, modified Eagle’s medium, respectively, supplemented with 10% fetal HSF2 is mainly converted from a dimer to a trimer upon calf serum (FCS), 2 mM L-glutamine and antibiotics (penicillin and streptomycin) in a humidified 5% CO2 atmosphere at 37°C. For activation, and regulation of HSF2 appears not to include × 6 phosphorylation (Sarge et al., 1993; Sistonen et al., 1994). The experimental treatments, K562 cells were seeded at 5 10 cells and HeLa cells at 3×106 per 10-cm-diameter plate. Heat shocks were regulation of HSF1 and HSF2 expression varies dramatically. performed at 42°C in a water bath. Hemin (Aldrich) was added to a In contrast to stably and constitutively expressed HSF1, the final concentration of 40 µM and cells were incubated at 37°C for the HSF2 expression levels are regulated both transcriptionally indicated time periods. Recovery periods were at 37°C. and by mRNA stabilization (Pirkkala et al., 1999). Rapid accumulation of HSF2 protein by downregulation of the ubiquitin proteolytic pathway has provided evidence that Construction of plasmids and a stable cell line HSF2 is a labile protein (Mathew et al., 1998; Pirkkala et al., Human HSF1 with C-terminal Myc tags was constructed by PCR and 2000). The regulation of HSF2 has been linked to certain cloned into EcoRI and HindIII sites in the pcDNA3.1(–)MycHis A development- and differentiation-related processes, such as vector (Invitrogen) in frame with the MycHis tag (Holmberg et al., gametogenesis and pre- and postimplantation development of mouse embryos (Mezger et al., 1994; Sarge et al., 1994; Rallu Fig. 1. Heat-induced localization of HSF1 and HSF2 in nuclear et al., 1997; Alastalo et al., 1998; Eriksson et al., 2000). granules. Fluorescence micrographs of HSF1 and HSF2 localization Furthermore, the human HSF2 DNA-binding activity is in untreated (C) and 1-hour heat-shocked (HS) HeLa cells, using induced during hemin-mediated erythroid differentiation of polyclonal antibodies against HSF1 (left) and HSF2 (right). The K562 cells (Sistonen et al., 1992). A recent study by Kallio et phase contrast and DAPI-stained DNA of the same fields are also al. (Kallio et al., 2002) revealed that the disruption of mouse shown. (B) Cells were untreated (C), heat-shocked for 30 minutes to hsf2 gene leads to brain abnormalities and defects in 6 hours (30′-6h) or, after 1 hour of heat shock, allowed to recover for gametogenesis in both genders. No correlation between HSF2 1 or 3 hours (HS+1R, HS+3R). (C) The graph represents the means ± expression and activity with Hsp expression has been obtained SEM of three independent experiments in which the number of granule positive cells was analyzed by counting nine groups of 50 during differentiation-related processes, indicating existence of cells. (D) The HSF DNA-binding activity and HSF1 other HSF2 target genes (Rallu et al., 1997; Alastalo et al., hyperphosphorylation were analyzed by gel mobility shift assay with 1998; Kallio et al., 2002). radiolabeled HSE (top) and by western blotting (bottom), A few reports have proposed that HSF2 is involved in the respectively. The arrow indicates the inducibly phosphorylated regulation of the stress response. According to Sheldon and HSF1. Scale bars, 5 µm. Interplay between HSF1 and HSF2 3559 3560 Journal of Cell Science 116 (17)

2001). Mouse HSF1 with N-terminal Flag tags was a kind gift from (Life Technologies) for 2 weeks. Neomycin-resistant single cell R. I. Morimoto (Northwestern University, IL, USA). Mouse HSF2-α clones were picked out after serial dilutions and screened for HSF1- and HSF2-β with N-terminal Flag tags have been described previously Myc-His expression by western blotting. Cells were routinely grown (Pirkkala et al., 2000). Expression vectors encoding mouse HSF2-α in the presence of 500 µg ml–1 G418. and HSF2-β with C-terminal Myc tags were constructed by PCR and cloned into the EcoRI site in the pcDNA3.1(–) MycHis B vector (Invitrogen) in frame with the MycHis tag. The mouse HSF2-β Transfections deletion mutants (Fig. 4A) were constructed by PCR and cloned into K562 and HeLa cells were transfected by electroporation (975 µF, 220 the EcoRI and EcoRV sites in frame with the N-terminal Flag tag in V) using a Bio-Rad Gene Pulser electroporator. For this procedure, pFLAG-CMV-2 (Kodak). All PCR-ampliﬁed products were 5×106 cells were washed, resuspended in 0.4 ml of Optimem (Gibco- sequenced to exclude the possibility of second site mutagenesis. BRL) and placed in a 0.4-cm-gap electroporation cuvette (BTX). HSF1-Myc-His stably overexpressing cell line (4H9) was generated Plasmid DNA (30 µg) was added and, after a brief incubation at room by electroporating 30 µg of HSF1-Myc-His to K562 cells as described temperature, the cells were subjected to a single electric pulse. below. After 2 days of recovery, neomycin-resistant cells were Thereafter, cells were cultured at 37°C for 40 hours prior to the selected by growing the cells in medium containing 500 µg ml–1 G418 indicated experimental treatments.

Indirect immunofluorescence and confocal microscopy For immunofluorescence analysis, HeLa cells growing on coverslips were washed with PBS and simultaneously fixed and permeabilized for 15 minutes in 0.5% Tween 20 in PBS containing 3% paraformaldehyde, or the cells were fixed for 15 minutes in 4°C methanol-acetone (1:1). After three washes with PBS, cells were incubated for 1 hour with blocking solution (20% boiled normal goat serum in PBS). Rabbit anti-HSF1 (Holmberg et al., 2000), rat anti- HSF1 (Neomarkers), rabbit anti- HSF2 (Sarge et al., 1993), mouse anti-Hsp70 (SPA-810; StressGen), mouse anti-Flag M2 (Sigma) or mouse anti-Myc (Sigma) antibody (1:500 dilutions) were added for 1 hour. After washes with PBS, the bound primary antibodies were detected using goat anti-mouse antibodies (1:400 dilution for 1 hour;

Fig. 2. Co-localization of HSF1 and HSF2 in stress granules. (A) HSF1 and HSF2 co-localize to the stress granules upon heat shock (HS). Monoclonal antibodies against HSF1 were used to detect endogenous HSF1 and polyclonal HSF2 antibodies to detect endogenous HSF2 by double staining of HeLa cells analyzed by immunoﬂuorescence microscopy. (B) The epitope-tagged HSF1 and HSF2 constructs were transiently transfected into HeLa cells. Anti- Myc antibody was used to detect the ectopic HSFs (green) and polyclonal antibodies against HSF1 and HSF2 were used to detect endogenous HSFs (red). Yellow indicates co-localization in the merged image (Merge). Scale bars, 5 µm. Interplay between HSF1 and HSF2 3561

Fig. 3. Coimmunoprecipitation of HSF1 and HSF2. (A) Coimmunoprecipitation of HSF2 with transiently (left) or stably (right) overexpressed Myc-tagged HSF1 (4H9) using monoclonal anti-Myc antibodies (IP). The lower blot shows the expression levels of HSF1 and HSF2 (WB). (B) Exogenous HSF1 was coimmunoprecipitated with endogenous HSF2 using monoclonal HSF2 antibodies (IP). Anti-Flag antibodies were used as an antibody control. Cells were either left untreated (C) or heat shocked for 1 hour (HS). (C) The dynamics of the interacting complex was analyzed by coimmunoprecipitation of endogenous HSF2 with endogenous HSF1 using monoclonal HSF1 antibodies in HeLa cells heat- shocked for indicated times (IP). Thermotolerance was acquired at 1 or 3 hours of recovery after a 1-hour heat shock (HS+1R, HS+3R). The bottom blots show the expression levels of HSF1 and HSF2 (WB). Hsc70 was an equal loading control. HSF2-α and -β isoforms are indicated. Arrows and asterisks mark the inducibly phosphorylated HSF1 and the endogenous HSF2, respectively.

Alexa 488, Molecular Probes), Cy3-conjugated donkey anti-rabbit with slurry of protein-G/Sepharose (Amersham Pharmacia Biotech) antibodies (1:400 dilution for 1 hour; Jackson ImmunoResearch in TEG buffer (20 mM Tris-HCl pH 7.5, 1 mM EDTA, 10% Laboratories), goat anti-rat antibodies (1:400 dilution for 1 hour; Alexa glycerol) containing 150 mM NaCl and 0.1% Triton X-100 for 30 568, Molecular Probes) or donkey anti-rabbit antibodies (1:400 minutes at 4°C followed by a brief centrifugation. The precleared dilution for 1 hour; Alexa 488, Molecular Probes). The DNA was cellular lysate was incubated with anti-HSF1 (Neomarkers), anti- stained with DAPI (4′,6-diamidino-2-phenylindol, Sigma) for 2 HSF2 (Neomarkers), anti-Flag or anti-Myc antibodies at room minutes before final washes and mounting with Vectashield (Vector temperature for 30 minutes under rotation, after which 40 µl of a Laboratories). The cells were analyzed and photographed using a Leica 50% slurry of protein-G/Sepharose was added to the reaction DMR fluorescence microscope equipped with a digital Hamamatsu mixture and incubated for 12 hours at 4°C under rotation. After ORCA CCD camera or Leica TCS40 confocal laser scanning centrifugation, the Sepharose beads were washed with supplemented microscope using the SCANware 4.2a program. Images were further TEG buffer and the immunoprecipitated proteins were run on 8% processed using Adobe Photoshop and CorelDraw software. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose filter (Protran Nitrocellulose; Schleicher & Schuell) for immunoblotting. For detection of protein input and Immunoprecipitation and immunoblotting assays expression levels, 12 µg of protein was analyzed by SDS-PAGE as For in vivo coimmunoprecipitation experiments, transiently described above. HSF1 was detected by polyclonal antibodies transfected cells were lysed in 200 µl of lysis buffer [25 mM specific to mouse and human HSF1 (Sarge et al., 1993; Holmberg HEPES, 100 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 20 mM et al., 2000), HSF2 by polyclonal antibodies specific to mouse HSF2 β-glycerophosphate, 20 mM para-nitrophenyl phosphate, 100 µM (Sarge et al., 1993), the inducible form of Hsp70 by 4g4 (Affinity ortovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM Bioreagents), Hsc70 by SPA-815 (StressGen). Horseradish dithiotreitol, 1× complete mini protease inhibitor cocktail (Roche peroxidase (HRP)-conjugated secondary antibodies were purchased Diagnostics)] supplemented with 20 mM N-ethylmaleimide, from Promega and Amersham. The blots were developed with an followed by centrifugation for 25 minutes at 15,000 g at 4°C. After enhanced chemiluminescence method (ECL; Amersham Pharmacia protein extraction, 200-500 µg total cell protein was preincubated Biotech). 3562 Journal of Cell Science 116 (17)

Fig. 4. The oligomerization domain HR-A/B is essential for the physical interaction between HSF1 and HSF2. (A) Schematic presentation of HSF2 deletion mutants. The DNA-binding domain (DBD), N- terminal oligomerization domain (HR-A/B) and C-terminal leucine zipper (HR-C) are indicated. (B) Anti-Myc antibodies were used to immunoprecipitate HSF1, and antibodies against HSF1 and HSF2 were used for western blotting. An empty Myc vector was used in the mock transfections. The expression levels were analyzed by western blotting (WB), as indicated. Arrows and asterisks indicate the inducibly phosphorylated HSF1 and the endogenous HSF2, respectively. Molecular weights (kDa) are shown.

Cells (5×106 each of 4H9 and K562) were labeled with 0.25 mCi and the bands were identiﬁed using standard matrix-assisted laser ml–1 of TRAN35S Label (ICN) for 16 hours. Cells were lysed in 1 desorption ionization mass spectrometry. ml of modiﬁed RIPA buffer (50 mM TRIS pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% deoxycholate, 1 mM EDTA, 100 µM orthovanadate, 2 mM NaF) and the immunoprecipitation with anti-c-Myc (Sigma) Gel mobility shift assay antibodies was performed as above. Immunoprecipitated proteins Whole cell extracts were prepared from experimentally treated cells were detected from the gels by autoradiography. To identify the bands, as previously described (Mosser et al., 1988) and incubated (12 µg 1×109 4H9 and K562 cells were lysed and immunoprecipitated as protein) with a 32P-labeled oligonucleotide representing the proximal above. The gels were silver stained (O’Connell and Stults, 1997) HSE of the human hsp70 promoter. The protein-DNA complexes were Interplay between HSF1 and HSF2 3563

Fig. 5. HSF1 is the major component of the heat-induced HSE-binding complex. (A) Cells were subjected to different periods of heat shock (HS; 1-6 h) or left untreated (C). The HSF DNA-binding activity in the whole cell extracts was analyzed by gel mobility shift assay with radiolabeled HSE (left panel). Antibody perturbations with Flag and Myc antibodies (1:10) were used to detect the presence of HSF1 or HSF2 in the DNA-binding complex, respectively (right panel). A hemin-treated sample was used as a control. An empty Myc vector was used in the mock transfections. (B) Flag antibodies were used to immunoprecipitate HSF1 from the same lysates as in panel A. Western blotting was performed as in Fig. 3. The expression levels are presented in the right hand panel (WB). Arrows and asterisks indicate the inducibly phosphorylated HSF1 and the endogenous HSF2, respectively. analyzed on a native 4% polyacrylamide gel as described previously attenuation of the heat-shock response (Fig. 1B). At the same (Mosser et al., 1988). time as the staining experiment, samples were collected for gel mobility shift and western blotting analyses to compare the morphology of HSF1 and HSF2 granules to the DNA-binding Results activity and phosphorylation of HSF1. During 2-6 hours of heat Subnuclear organization of HSF1 and HSF2 in granule- treatment, the maturation of heat-shock granules into ring-like like structures structures preceded the dissociation of HSF1 and HSF2 from The activation of HSF1 upon stress is associated with a series the granules and the loss of HSF1 DNA-binding activity (Fig. of biochemical changes, including oligomerization and 1D). In addition to the morphological analyses, the number of phosphorylation, which correlate with the subnuclear granule-positive cells during the course of heat shock was organization of HSF1 in chromatin-associated stress granules determined (Fig. 1C). Already at 30 minutes of heat shock, the (Pirkkala et al., 2001; Jolly et al., 2002). Upon exposure to heat number of granule-positive cells had increased prominently shock, we found that another member of the HSF family, and, at 1-2 hours, almost 100% of cells contained granules. HSF2, also formed brightly staining nuclear granules similar After 2 hours, the number of granule-positive cells began to to HSF1, as detected by immunoﬂuorescence microscopy (Fig. decline (Fig. 1C). The dephosphorylation of HSF1 correlated 1A). Interestingly, the morphology and organization pattern of temporally with the dissociation of HSF1 from the granules both HSF1 and HSF2 granules varied dramatically depending (Fig. 1B,D). A dramatic change in the number and morphology on duration of heat stress (Fig. 1B). Both endogenous HSF1 of both HSF1 and HSF2 granules were detected after a 1 hour and HSF2 localized in small and barely detectable granules recovery from heat stress and, after 3 hours, hardly any after only 5 minutes of heat shock (data not shown), and the granules were found and both HSF1 and HSF2 were diffusely intensity and size increased until 1 hour (Fig. 1B). At 2 hours distributed (Fig. 1B,C). Interestingly, the number and of treatment, the granules began to mature into ring-like morphology of HSF1 and HSF2 granules were almost structures. These structures were most clearly detected at 2-6 identical, suggesting that they correspond to the same nuclear hours of continuous heat shock, corresponding to the structure. 3564 Journal of Cell Science 116 (17)

Fig. 6. HSF2 affects the localization of HSF1 in stress granules. (A-C) HeLa cells were transiently transfected with Flag-tagged HSF2 and constructs shown in Fig. 4A. Cells were subjected to a 1- hour heat shock (HS) or left untreated (C), and immunodetected with anti- Flag antibodies for HSF2 deletion mutants (green) and with polyclonal HSF1 antibodies for the endogenous HSF1 (red). Colocalization is shown in the merge lane (yellow) and DNA is stained with DAPI (blue). (D) Granule formation was analyzed by counting 50 Flag- positive cells from three independent experiments (total 150 cells), and the graph represents the means ± s.e.m. Scale bars, 5 µm. Interplay between HSF1 and HSF2 3565 transiently (Fig. 3A, left) and stably (Fig. 3A, right) overexpressed HSF1. Furthermore, monoclonal anti-HSF2 antibodies were used to immunoprecipitate the endogenous HSF2, and western blotting showed that the overexpressed HSF1 coimmunoprecipitated with HSF2 (Fig. 3B). To analyze the dynamics of the interaction during the course of heat-shock response and recovery periods, endogenous HSF1 from HeLa cells was immunoprecipitated with monoclonal anti-HSF1 antibody. The immunoprecipitated complexes were analyzed by western blotting, and dynamic interaction of HSF1 and HSF2-α and -β isoforms (Pirkkala et al., 2001) was observed at different time points (Fig. 3C). At control temperature and heat shock up to 30 minutes, HSF2-β was the major HSF2 isoform in the complex, whereas the amount of HSF2-α began to increase from 30 minutes to 1 hour and similar amounts of both isoforms were detected in the complex. After 1 hour of heat treatment, the levels of HSF2-β in the complex rapidly declined and, Co-localization of HSF1 and HSF2 in the nuclear stress at 2-4 hours of continuous heat shock, HSF2-α was the major granules HSF2 isoform interacting with HSF1. Upon prolonged heat The heat-induced localization of HSF1 and HSF2 in granules shock (>4 hours) and recovery, the amount of HSF2-β began did not occur randomly, but a co-localization of these two to increase, restoring the ratio of the HSF2 isoforms observed endogenous factors was detected after a 1-hour heat shock, as at control temperature (Fig. 3C, Fig. 8B). The changes in the shown by double staining (Fig. 2A). To ensure that finding the interaction stoichiometry between HSF1 and HSF2 isoforms, HSF2 stress granules was not a result of cross-reactivity as shown in immunoprecipitation samples (Fig. 3C, top), were between the antibodies against HSF1 and HSF2, we constructed reflected also in western blotting of the lysates (Fig. 3C, both N- and C-terminally Flag- and Myc-tagged HSF1 and bottom), indicating altered solubility of the HSF2 isoforms HSF2 plasmids. Upon transient expression in HeLa cells, the during different phases of the continuous heat shock. subcellular localization of ectopic HSF1 and HSF2 was After 3 hours of recovery from a 1-hour heat shock, when analyzed. Immunofluorescence analyses with monoclonal cells acquire thermotolerance, the interaction between HSF1 antibodies against Flag and Myc epitopes showed a similar and HSF2 was downregulated (Fig. 3C, Fig. 8B). In contrast nuclear pattern (Fig. 2B) to the experiments conducted with the to the dynamic regulation of HSF2 isoforms detected both in antibodies against HSF1 and HSF2 (Fig. 1A,B, Fig. 2A). The the lysates and protein complex, the downregulation of HSF1- co-localization between HSF1 and HSF2 was further confirmed HSF2 interaction seen after recovery from stress cannot be by confocal microscopy (data not shown). Double-staining explained by changes in solubility. This suggests that there are experiments were also performed as in Fig. 1B to study the co- modifications in the HSF-associated protein complex during localization of HSF1 and HSF2 at different phases of granule thermotolerance and further confirms the dynamic nature of development. In these studies, co-localization of HSF1 and HSF1-HSF2 interaction. HSF2 was observed from the compartmentalization to the dissociation of these factors from stress granules (data not shown). It is worth noticing that, although there was prominent Oligomerization domain is essential for the interaction co-localization of HSF1 and HSF2 in the same subnuclear between HSF1 and HSF2 structures, some heterogeneity occurred (i.e. stress granules Different members of the HSF family share a similar structure, were detected in which HSF1 but not HSF2 was present). consisting of a leucine-rich oligomerization domain (HR-A/B) adjacent to the N-terminal DNA-binding domain, and another leucine-rich region near the C-terminus (HR-C) (Rabindran et Dynamic interaction between HSF1 and HSF2 al., 1991; Sarge et al., 1991; Schuetz et al., 1991). To examine The dynamic localization of HSF1 with HSF2 in stress whether the interaction of HSF1 and HSF2 depended on an granules upon heat shock provided new evidence for HSF2 intact oligomerization domain, we constructed a series of Flag- functioning as a heat-responsive factor, and for the stress- tagged HSF2 deletion mutants (Fig. 4A). The Myc-tagged induced regulation of HSF1 and HSF2 being closely related. HSF1 was transiently expressed with these HSF2 mutants in This finding prompted us to investigate whether a similar heat- K562 cells, and HSF1 was immunoprecipitated with anti-Myc induced subnuclear distribution of HSF1 and HSF2 could be antibody, and the presence of HSF2 in the complex was caused by their physical interaction. Myc-tagged HSF1 was analyzed. As shown in Fig. 4B, HSF2 mutants lacking HR-A/B transiently or stably expressed in K562 cells and the (HSF2 203-517 and HSF2 ∆126-201) did not form complexes endogenous HSF2 was coimmunoprecipitated with both with HSF1. 3566 Journal of Cell Science 116 (17) HSF2 is not found in the protein complex binding to the and elevated temperatures (Fig. 6C,D). Taken together, HSE upon heat shock the immunofluorescence analyses, combined with the Considering that HSF2 interacts with HSF1, we wanted to coimmunoprecipitation results (Fig. 4B), indicate that HSF2 investigate whether the HSF1-HSF2 heterocomplex could bind could affect the translocation of HSF1 into the nuclear stress in vitro to the HSE of human hsp70 promoter. Flag-tagged HSF1 granules, because either induction or prevention of HSF1 and Myc-tagged HSF2 were transiently expressed in K562 cells subnuclear compartmentalization was observed depending on and a strong heat-induced DNA-binding activity was detected in which HSF2 mutant was used. the lysates (Fig. 5A, left). The spontaneous DNA-binding activity in untreated samples was due to overproduction of HSFs. To analyze the DNA-binding complexes, we performed antibody Localization of HSF1 and HSF2 in stress granules is perturbation assays with anti-Flag and anti-Myc antibodies to suppressed in thermotolerant cells detect the presence of HSF1 or HSF2 molecules, respectively. In addition to co-localization of HSF1 and HSF2 in the same As shown in Fig. 5A (right), the anti-Flag (HSF1) antibody dynamically regulated protein complex, we observed profound supershifted the heat-induced DNA-binding complexes changes in subnuclear compartmentalization and HSF1-HSF2 completely, whereas the anti-Myc (HSF2) antibody had no effect interaction during attenuation and recovery of the heat-shock on the complex. Because hemin induces the DNA-binding response. To address the question of how thermotolerance activity of HSF2 (Pirkkala et al., 1999), a hemin-treated sample affects the localization of HSF1 and HSF2 in stress granules, expressing Myc-tagged HSF2 was included as a positive control we induced thermotolerance by exposing HeLa cells to a 1- for HSF2 DNA-binding activity and anti-Myc antibody. The hour heat shock, after which the cells were allowed to recover existence of HSF1-HSF2 complex in the same lysates was at normal temperature for 3 hours. During recovery, HSF1 and determined by coimmunoprecipitation (Fig. 5B). HSF2 dissociated from the granules and both proteins were To exclude the possibility that the C-terminal Myc-epitope partly translocated into the cytosol (Fig. 7A). After a second in the HSF2 fusion protein was being masked in the heat- heat shock of 30 minutes or 1 hour, a decrease in granule- induced DNA-binding complex and could therefore not be positive cells was detected, because almost 100% of primarily detected, we repeated the above mentioned experiments using heat-shocked cells, but less than 15% of thermotolerant cells, C-terminally Myc-tagged HSF1 and N-terminally Flag-tagged exposed to a 1-hour heat shock contained HSF-positive HSF2. The results were identical to those in Fig. 5, with only granules (Fig. 7B). Because HSF1 and HSF2 behaved HSF1 being detected in the HSE-binding complex (data not identically, only HSF1 was used as a marker for nuclear stress shown). These results were also confirmed by overexpressing granules in the following experiments. HSF2 alone, and no HSF2 was detected in the heat-induced HSE-binding complex (data not shown). Nucleolar localization of Hsp70 coincides with the localization of HSF1 in stress granules HSF2 influences the localization of HSF1 in nuclear One of the mechanisms regulating thermotolerance and stress granules attenuation of the heat shock response is the inhibition of HSF1 Based on the obtained results, the HSF1-HSF2 complex activity by molecular chaperones such as Hsp70 and Hsp90 formation could have significance in other steps of the stress- and their co-chaperones (Morimoto, 2002). However, the role regulated signaling pathways unrelated directly to the HSE- of these chaperones in the regulation of nuclear stress granules binding and heat-shock gene expression. The appearance of has not been elucidated. Using immunoprecipitation of stress-induced nuclear granules has been shown to coincide Myc-tagged HSF1 from stably transfected 4H9 cells after with HSF1 activation (Cotto et al., 1997; Jolly et al., 1999; methionine labeling followed by identification of the Holmberg et al., 2000), so we investigated whether the interacting proteins by mass spectrometry, we found Hsc70 and HSF1-HSF2 interaction affects the subnuclear Hsp70 forming a complex with HSF1 in a stoichiometric compartmentalization of HSF1. For this purpose, the Flag- manner (Fig. 8A). During attenuation of the heat-shock tagged HSF2 deletion mutants (Fig. 3A) were transiently response and in thermotolerant HeLa cells, dynamically transfected into HeLa cells and double staining was regulated complex formation was detected between HSF1 and performed with anti-Flag and anti-HSF1 antibodies. As Hsp70 (Fig. 8B). At the control temperature and upon heat shown in Fig. 6A, the full-length HSF2 co-localized with shock of less than 30 minutes, HSF1 was found in a complex HSF1 during heat shock. Unexpectedly, the DNA-binding- with Hsp70, whereas this complex disassembled after 30 domain deletion mutant (HSF2 108-517), which was able to minutes and was hardly detectable at 2 hours (Fig. 8B, data not interact physically with HSF1 (Fig. 4B), did not localize in shown). After 2 hours of continuous heat shock, the interaction the granules, and also the translocation of HSF1 into the began to increase. The interaction pattern with Hsp70 granules was severely impaired (Fig. 6A,D). The HR-A/B correlated well with the onset of attenuation seen previously in oligomerization domain deletion mutants (HSF2 203-517 and HSF1 DNA-binding activity and phosphorylation, as well as HSF2 ∆126-201) could not interact with HSF1 (Fig. 4B) but with the decline in granule-positive cells (Fig. 1). During did not translocate into the granules; instead, these cells recovery periods, complex formation between HSF1 and displayed normal localization of HSF1 in the granules (Fig. Hsp70 gradually increased (Fig. 8B). 6B,D). The C-terminal deletion mutant (HSF2 1-391), which Next, we investigated how the subcellular localization of was capable of interacting with HSF1 (Fig. 4B), localized Hsp70 corresponded to the interaction pattern seen in Fig. 8B spontaneously to granules and, intriguingly, HSF1 showed and to the heat-shock granule formation. Hsp70 and its co- clear co-localization with this HSF2 mutant at both control chaperones such as Hsp40 are rapidly translocated from the Interplay between HSF1 and HSF2 3567

Fig. 7. The localization of HSF1 and HSF2 in stress granules is suppressed in thermotolerant HeLa cells. (A) Fluorescence micrographs of the localization of HSF1 and HSF2 in untreated (C), heat-shocked (30′ or 1h), thermotolerant (HS+3R) and heat-shocked thermotolerant (HS+3R+HS30′ or HS+3R+HS1h) cells. Proteins were visualized using monoclonal antibodies against HSF1 (red) and polyclonal antibodies against HSF2 (green). Co-localization is shown in the merge lane (yellow) and DNA is stained with DAPI (blue). Scale bars, 5 µm. (B) The graph represents the means ± s.e.m. of the three independent experiments in which the number of granule-positive cells was analyzed by counting nine groups of 50 cells.

immunoﬂuorescence microscopy and, at normal temperature, HSF1 was found both in the cytosol and nucleus, whereas Hsp70 was mainly cytosolic (Fig. 8C). Upon heat shock of less than 1 hour, the increasing intensity and size of the stress granules coincided with the ongoing translocation of Hsp70 into the nucleoli (data not shown). At 1 hour, HSF1 was primarily detected in mature nuclear granules and Hsp70 concentrated in the nucleoli (Fig. 8C). Interestingly, at this time point, the interaction between HSF1 and Hsp70 was dramatically downregulated (Fig. 8A). During the attenuation of the heat-shock response (2- 6 hours of continuous heat shock), both Hsp70 and HSF1 gradually dissociated from nucleoli and the stress granules, respectively, and HSF1-Hsp70 complex formation increased (Fig. 8A,C, data not shown). In thermotolerant cells, HSF1 and Hsp70 formed an interacting complex and were both mainly localized in the cytosol (HS+3R; Fig. 8A,C). Upon exposure of thermotolerant cells to a second heat shock, a high level of interaction persisted and HSF1-positive granules were detected only in cells in which Hsp70 was translocated into nucleoli (HS+3R+HS1h; Fig. 8C). The number of cells containing HSF1 in stress granules and Hsp70-positive nucleoli corresponded to the heat-shocked thermotolerant cells shown in Fig. 7B (data not shown). In these experiments, the nucleolar localization of Hsp70 coincided with the downregulation of cytosol to the nucleoli upon heat shock (Welch and HSF1-Hsp70 interaction and localization of HSF1 in nuclear Feramisco, 1984; Welch and Suhan, 1986; Hattori et al., stress granules. The reversible localization pattern of Hsp70 1993) but the signiﬁcance of this rapid change in subcellular into and out of the nucleoli appeared to be closely related to distribution is poorly understood. The localization of the subcellular distribution of HSF1, and could thereby HSF1 and Hsp70 was investigated by double-staining contribute to the regulation of HSF1 activity. 3568 Journal of Cell Science 116 (17)

Fig. 8. Nucleolar localization of Hsp70 correlates with the activation and deactivation of HSF1. (A) An autoradiograph image of proteins interacting with Myc-HSF1 in labeled 4H9 cells. Anti-Myc antibodies were used for immunoprecipitation. K562 cells were used as a negative control. The two obtained bands were detected also by silver staining and analyzed by matrix-assisted laser desorption ionization mass spectrometry. (B) Coimmunoprecipitation of endogenous HSF2 and Hsp70 with endogenous HSF1 in heat-shocked HeLa cells. Monoclonal HSF1 antibodies were used for immunoprecipitations (IP). Cells were untreated (C) or heat shocked for 30 minutes to 6 hours (30′-6h). Thermotolerance was obtained by a 1-hour heat shock, followed by 1 or 3 hours of recovery (HS+1R or HS+3R). Thermotolerant cells were heat shocked for 30 minutes or 1 hour (HS+3R+HS30′ or HS+3R+HS1h). The bottom blots (WB) show the expression levels of HSF1, HSF2 and Hsp70. Hsc70 was a control for equal loading. Arrows indicate the inducibly phosphorylated HSF1. (C) Fluorescence micrographs showing the localization of HSF1 and Hsp70 in untreated (C), heat-shocked (HS) for 1 hour or 6 hours, thermotolerant (HS+3R), and heat-shocked thermotolerant HeLa cells (HS+3R+HS1h). Merge lane indicates possible co-localization (yellow) and DNA is stained by DAPI (blue). Scale bar, 5 µm.

Discussion Sheldon and Kingston (Sheldon and Kingston, 1993) earlier The physiological role of HSF1 in the regulation of the heat- reported that the intracellular localization of human HSF2 is shock response is well established but the possible interplay regulated by heat stress, the function of HSF2 has ever since between HSF1 and other HSFs is largely unresolved. Although been solely linked to differentiation-related processes. In this Interplay between HSF1 and HSF2 3569 study, we provide evidence of human HSF2 being an integral interaction with HSF1 and localization in the stress granules. member of the cellular stress response pathway. Specifically, Unexpectedly, the C-terminal deletion mutant (HSF2 1-396), HSF2 is a novel component of the nuclear stress granules, and which both interacted and co-localized with HSF1, was the localization of HSF2 in the granules coincides temporally spontaneously translocated into the stress granules with with the localization and activation of HSF1. By analyzing endogenous HSF1. This further emphasizes the interdependency both HSF1 and HSF2 separately and together, we detected a between HSF1 and HSF2 in their stress granule localization. clear maturation pattern in the formation of stress granules. A characteristic feature of the heat-shock response is The mechanisms behind the transformation from small clusters acquired upon recovery from stressful conditions. The to single globular units, followed by the maturation into ring- mechanisms regulating thermotolerance have been closely like structures, are still obscure. However, the morphological related to the inhibition of HSF1 by direct interaction between changes in the granules are temporally related to the alterations upregulated Hsp70 and its co-chaperones (Morimoto, 2002). in the DNA-binding activity of HSF1. Both acquisition and Owing to the changes in HSF1 regulation upon acquired attenuation of DNA-binding activity strictly correlate with the thermotolerance, we wanted to examine the localization of assembly and disassembly, respectively, of the stress granules. HSF1 and HSF2 in nuclear stress granules under these In addition to the co-localization of HSF1 and HSF2 in conditions. Our results demonstrate a suppression of HSF1 nuclear stress granules, we provide the first in vivo evidence and HSF2 localization in stress granules in heat-shocked of complex formation between HSF1 and HSF2. Our data thermotolerant cells, which correlates well with the inhibition reveal a regulated interaction between HSF1 and HSF2 of HSF1 activity during thermotolerance and suggests a role for isoforms during the course of heat shock, possibly caused by Hsps in the regulation of granule formation. We also show that regulated solubility of different HSF2 isoforms, as indicated by the translocation of Hsp70 into and out of the nucleoli coincides Mathew et al. (Mathew et al., 2001). We also show that with the localization of HSF1 to nuclear stress granules and the HSF1-HSF2 complex is efficiently disassembled in interaction with HSF1. The dissociation of the HSF1-Hsp70 thermotolerant cells, suggesting modifications in the properties complex during heat shock corresponds to the translocation of of the HSF1-interacting protein complex. Further experiments Hsp70 into nucleoli. Simultaneously, the DNA-binding activity are needed to reveal whether the downregulation of HSF1- and hyperphosphorylation of HSF1, and the number of granule- HSF2 interaction is involved in the suppression of HSF1 positive cells are at maximum. During the attenuation of HSF1 activity during thermotolerance, and how the HSF2 isoform activity, Hsp70 dissociates from the nucleoli, the complex composition reflects the overall heat shock response. By formation between Hsp70 and HSF1 increases, and HSF1 analyzing a set of HSF2 deletion mutants, we show that the relocates from the stress granules. Concomitant with the physical interaction with HSF1 requires an intact HR-A/B inhibition of HSF1 localization to granules in thermotolerant oligomerization domain of HSF2. It is still an open question cells exposed to a second heat shock, fewer cells are observed whether HSF1 and HSF2 form a heterodimer or heterotrimer, that contain Hsp70 in their nucleoli. Taken together, our or whether the interaction occurs between two homo- results show a strict correlation between the subnuclear oligomers. Considering our results and the structural similarity, compartmentalization of HSF1 and Hsp70 nucleolar it is likely that HSF1 and HSF2 form a hetero-oligomer. localization, and, because the dissociation of HSF1 from stress Despite the prominent interaction between HSF1 and HSF2, granules during attenuation also coincides the translocation of we failed to detect HSF2 in the heat-shock-induced HSE-binding Hsp70 out from the nucleoli, it is plausible that the nucleolar complex, which is in agreement with Mathew et al. (Mathew et localization of Hsp70 contributes to the regulation of HSF1. al., 2001). Because these observations indicate that heat stress does not activate HSF2 HSE-binding activity, the HSF1-HSF2 We thank K. M. Heiskanen, T. Katajamäki and H. Saarento for complex formation together with the subnuclear co-localization excellent technical assistance. We are grateful to L. Pirkkala for represents a previously uncharacterized step in the regulation suggestions in the initial phase of this study and valuable comments of the stress-signaling pathway in human cells. To address on the manuscript. P. Roos-Mattjus is also acknowledged for discussions and critical comments. This work was supported by the the question of whether HSF1-HSF2 interaction affects the Academy of Finland, the Sigrid Jusélius Foundation, the Wihuri localization of HSF1 in stress granules, we performed double- Foundation, the Finnish Cancer Organizations, and the Finnish staining experiments using the HSF2 deletion mutants. Strikingly, Cultural Foundation. T-PA, MH and VH were supported by the Turku expression of HSF2 mutant lacking the DNA-binding domain Graduate School of Biomedical Sciences. (HSF2 108-517) inhibited the recruitment of HSF1 into the stress granules, which could be due to the physical interaction between this mutant and HSF1. An attractive explanation is that the effect References caused by HSF2 108-517 would lead to a defect in the interaction Abravaya, K., Myers, M. P., Murphy, S. P. and Morimoto, R. I. (1992). between the chromosomal nucleation sites and the HSF1-HSF2 The human heat shock protein Hsp70 interacts with HSF, the transcription 108-517 heterocomplex. This is in good agreement with another factor that regulates heat shock gene expression. Genes Dev. 6, 1153-1164. study, in which the granule-forming-ability of HSF1 was shown Alastalo, T.-P., Lönnstrom, M., Leppä, S., Kaarniranta, K., Pelto-Huikko, M., Sistonen, L. and Parvinen, M. (1998). Stage-specific expression and to be strictly dependent on the DNA-binding and the cellular localization of the heat shock factor 2 isoforms in the rat oligomerization domains of HSF1 (Jolly et al., 2002). By seminiferous epithelium. Exp. Cell Res. 240, 16-27. contrast, neither of the deletion mutants lacking the HR-A/B Ali, A., Bharadwaj, S., O’Carroll, R. and Ovsenek, N. (1998). HSP90 oligomerization domain (HSF2 203-517, HSF2 ∆126-201) was interacts with and regulates the activity of heat shock factor 1 in Xenopus oocytes. Mol. Cell. Biol. 18, 4949-4960. able to form a complex with HSF1 and thereby affect the Baler, R., Welch, W. J. and Voellmy, R. (1992). Heat shock gene regulation compartmentalization of HSF1 in the stress granules. Therefore, by nascent polypeptides and denaturated proteins: Hsp70 as a potent the capability of HSF2 to oligomerize seems to be crucial for both autoregulatory factor. J. Cell Biol. 117, 1151-1159. 3570 Journal of Cell Science 116 (17)

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