Formation of nuclear stress granules involves HSF2 and ... · K562 cells (Sistonen et al., 1992). A...

IntroductionCells have developed a range of processes to increase survivaland adaptation in response to different forms of stress. One ofthese events is the induced expression of heat-shock genescoding for heat-shock proteins (Hsps). Hsps have an importantrole in cellular homeostasis and protection through their well-recognized properties as molecular chaperones (Bukau andHorwich, 1998). The expression of the heat-shock genes isprimarily regulated at transcriptional level by specific DNA-binding proteins called heat-shock factors (HSFs), which bindto the heat-shock promoter element (HSE) (Pirkkala et al.,2001). Four members of this family, HSF1-HSF4, have beenidentified in vertebrates (Rabindran et al., 1991; Sarge et al.,1991; Schuetz et al., 1991; Nakai and Morimoto, 1993; Nakaiet al., 1997), which has raised questions about specific oroverlapping functions among the distinct HSFs.

HSF1 is the most studied heat-shock transcription factor andis the classical HSF, which responds to elevated temperaturesand other forms of protein damaging stress (Pirkkala et al.,2001). In addition, HSF1 has been shown to be involved inextra-embryonic development and female fertility in mice(Xiao et al., 1999; Christians et al., 2000). Upon stress, HSF1is rapidly converted from a monomer to a trimer, is induciblyphosphorylated and concentrated in the nucleus to activateheat-shock gene transcription (Baler et al., 1993; Rabindran etal., 1993; Sarge et al., 1993). In addition to HSF1, chickenHSF3 is activated by similar but more severe stressors (Nakaiand Morimoto, 1993; Tanabe et al., 1997). In HSF3-deficient

cells the formation of HSF1 trimers is hampered and Hspexpression is reduced upon heat shock (Tanabe et al., 1998).The interdependency between HSF1 and HSF3 in avian cellsis so far the only example of cross-talk between different HSFs.However, no physical interaction has been reported betweenthese two proteins.

A characteristic feature of cellular stress in human cells isthe organization of HSF1 into specific subnuclear structures,termed stress granules. These irregularly shaped granules havebeen described to form under various stress conditions,including exposure to heat, cadmium, azetidine andproteasome inhibitors (Cotto et al., 1997; Jolly et al., 1997;Jolly et al., 1999; Holmberg et al., 2000). Stress-induced HSF1granules have been found in all investigated primary andtransformed human cells but not in rodent cells (Sarge et al.,1993; Mivechi et al., 1994; Cotto et al., 1997; Jolly et al.,1999). The appearance of stress granules correlates positivelywith the inducible phosphorylation and transcriptional activityof HSF1 (Sarge et al., 1993; Cotto et al., 1997; Jolly et al.,1999; Holmberg et al., 2000). Furthermore, HSF1 dissociatesfrom the stress granules and relocalizes diffusely in the cellduring attenuation and recovery from stress; upon subsequentexposure to stress, the granules reappear at the same sites (Jollyet al., 1999). The stress granules have not been shown torepresent other previously described nuclear structures (Cottoet al., 1997). In addition to HSF1, several RNA bindingproteins such as heterogeneous nuclear ribonucleoprotein(hnRNP) HAP (hnRNP A1 interacting protein), hnRNP M,

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The heat-shock response is characterized by the activationof heat-shock transcription factor 1 (HSF1), followed byincreased expression of heat-shock proteins (Hsps). Thestress-induced subnuclear compartmentalization of HSF1into nuclear stress granules has been suggested to be animportant control step in the regulation of stress responseand cellular homeostasis in human cells. In this study,we demonstrate that the less-well characterized HSF2interacts physically with HSF1 and is a novel stress-responsive component of the stress granules. Based onanalysis of our deletion mutants, HSF2 influences to thelocalization of HSF1 in stress granules. Moreover, our

results indicate that the stress granules are dynamicstructures and suggest that they might be regulated in anHsp70-dependent manner. The reversible localization ofHsp70 in the nucleoli strictly coincides with the presence ofHSF1 in stress granules and is dramatically suppressedin thermotolerant cells. We propose that the regulatedsubcellular distribution of Hsp70 is an importantregulatory mechanism of HSF1-mediated heat shockresponse.

Key words: Heat-shock response, Hsf, Hsp70, Stress granule

Summary

Formation of nuclear stress granules involves HSF2and coincides with the nucleolar localization of Hsp70Tero-Pekka Alastalo 1,*, Maria Hellesuo 1,*, Anton Sandqvist 1,2, Ville Hietakangas 1, Marko Kallio 1,‡

and Lea Sistonen 1,3,§

1Turku Centre for Biotechnology, University of Turku, Åbo Akademi University, BioCity, PO Box 123, FIN-20521 Turku, Finland2Department of Biochemistry and Pharmacy, Åbo Akademi University, Turku, Finland3Department 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])

Accepted 16 May 2003Journal of Cell Science 116, 3557-3570 © 2003 The Company of Biologists Ltddoi:10.1242/jcs.00671

Research Article

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Src-activated during mitosis (Sam68) and certain SR (serine-arginine) splicing factors have been identified in stress granules(Weighardt et al., 1999; Denegri et al., 2001). Recently, stressgranules were revealed to associate with human chromosome9q11-q12, corresponding to a large block of heterochromatincomposed primarily of satellite III repeats adjacent to thecentromere (Jolly et al., 2002). Furthermore, Denegri et al.(Denegri et al., 2002) have reported that, in addition tochromosome 9, chromosomes 12 and 15 also containnucleation sites for stress granules. The functional significanceof stress granules is still unknown.

The autoregulation of the heat-shock response is facilitatedby a direct interaction between HSF1 and molecularchaperones, such as Hsp70 and Hsp90 with their co-chaperones. This has been established in several biochemical,genetic and cell physiological studies (Abravaya et al., 1992;Baler et al., 1992; Kim et al., 1995; Ali et al., 1998; Shi et al.,1998; Zou et al., 1998; Bharadwaj et al., 1999; Morimoto,2002). Under normal physiological conditions, HSF1 exists ina repressed state associated with molecular chaperones. Uponstress, it is rapidly released from the complex, trimerizedand hyperphosphorylated, and acquires DNA-binding andtranscriptional activity. When exposed to continuous moderateheat stress, the equilibrium is shifted back to the monomeric,dephosphorylated and Hsp-bound state. The downregulationof HSF1 activity during prolonged stress is referred to asattenuation of the heat-shock response, and leads to thesuppression of HSF1 reactivation capacity upon subsequentstress. A similar repression of HSF1 activity is observedwhen cells recover from stressful conditions. This acquiredadaptation of cells to stress stimuli is called thermotolerance(Morimoto, 2002).

Biochemical characterization of HSF2 has revealed that,unlike HSF1, which undergoes a monomer-to-trimer transition,HSF2 is mainly converted from a dimer to a trimer uponactivation, and regulation of HSF2 appears not to includephosphorylation (Sarge et al., 1993; Sistonen et al., 1994). Theregulation of HSF1 and HSF2 expression varies dramatically.In contrast to stably and constitutively expressed HSF1, theHSF2 expression levels are regulated both transcriptionallyand by mRNA stabilization (Pirkkala et al., 1999). Rapidaccumulation of HSF2 protein by downregulation of theubiquitin proteolytic pathway has provided evidence thatHSF2 is a labile protein (Mathew et al., 1998; Pirkkala et al.,2000). The regulation of HSF2 has been linked to certaindevelopment- and differentiation-related processes, such asgametogenesis and pre- and postimplantation development ofmouse embryos (Mezger et al., 1994; Sarge et al., 1994; Ralluet al., 1997; Alastalo et al., 1998; Eriksson et al., 2000).Furthermore, the human HSF2 DNA-binding activity isinduced during hemin-mediated erythroid differentiation ofK562 cells (Sistonen et al., 1992). A recent study by Kallio etal. (Kallio et al., 2002) revealed that the disruption of mousehsf2 gene leads to brain abnormalities and defects ingametogenesis in both genders. No correlation between HSF2expression and activity with Hsp expression has been obtainedduring differentiation-related processes, indicating existence ofother HSF2 target genes (Rallu et al., 1997; Alastalo et al.,1998; Kallio et al., 2002).

A few reports have proposed that HSF2 is involved in theregulation of the stress response. According to Sheldon and

Kingston (Sheldon and Kingston, 1993), HSF2 localizes in thenucleus and in granule-type structures in HeLa cells exposedto heat stress. Furthermore, Mathew et al. (Mathew et al., 2001)have reported that, in murine fibroblasts, HSF2 has thebiochemical properties of a temperature-sensitive proteinbecause, upon heat shock, HSF2 is localized to the perinuclearregion, its solubility is decreased and DNA-binding activity isdiminished. This intriguing difference between human andmurine cells has also been observed with HSF1, because thestress-induced subnuclear compartmentalization of HSF1 hasbeen detected only in human cells (Sarge et al., 1993; Denegriet al., 2002). Altogether, direct evidence of HSF2, human ormurine, being a physiological transcriptional regulator of heatshock genes is missing.

Despite the characterization of several biochemicalproperties of HSF1 and HSF2, the actual stress sensors and thesignaling pathways regulating these two factors have remainedobscure. In this study, we provide evidence that HSF1 andHSF2 form a physically interacting complex and co-localize inthe dynamically regulated nuclear stress granules, suggestinga novel role for HSF2. We also show data suggesting that theassociation of HSF1 and HSF2 with stress granules might beregulated by Hsp70, and that the association is severelyimpaired in thermotolerant cells. Taken together, our resultssuggest that the regulation of the subcellular distribution ofHsp70 contributes to the regulation of HSF1-mediated heat-shock responses.

Materials and MethodsCell culture and experimental treatmentsHuman K562 erythroleukemia cells and HeLa cervical carcinomacells were maintained in RPMI-1640 medium and Dulbecco’smodified Eagle’s medium, respectively, supplemented with 10% fetalcalf serum (FCS), 2 mM L-glutamine and antibiotics (penicillin andstreptomycin) in a humidified 5% CO2 atmosphere at 37°C. Forexperimental treatments, K562 cells were seeded at 5×106 cells andHeLa cells at 3×106 per 10-cm-diameter plate. Heat shocks wereperformed at 42°C in a water bath. Hemin (Aldrich) was added to afinal concentration of 40 µM and cells were incubated at 37°C for theindicated time periods. Recovery periods were at 37°C.

Construction of plasmids and a stable cell lineHuman HSF1 with C-terminal Myc tags was constructed by PCR andcloned into EcoRI and HindIII sites in the pcDNA3.1(–)MycHis Avector (Invitrogen) in frame with the MycHis tag (Holmberg et al.,

Journal of Cell Science 116 (17)

Fig. 1. Heat-induced localization of HSF1 and HSF2 in nucleargranules. Fluorescence micrographs of HSF1 and HSF2 localizationin untreated (C) and 1-hour heat-shocked (HS) HeLa cells, usingpolyclonal antibodies against HSF1 (left) and HSF2 (right). Thephase contrast and DAPI-stained DNA of the same fields are alsoshown. (B) Cells were untreated (C), heat-shocked for 30 minutes to6 hours (30′-6h) or, after 1 hour of heat shock, allowed to recover for1 or 3 hours (HS+1R, HS+3R). (C) The graph represents the means ±SEM of three independent experiments in which the number ofgranule positive cells was analyzed by counting nine groups of 50cells. (D) The HSF DNA-binding activity and HSF1hyperphosphorylation were analyzed by gel mobility shift assay withradiolabeled HSE (top) and by western blotting (bottom),respectively. The arrow indicates the inducibly phosphorylatedHSF1. Scale bars, 5 µm.

3559Interplay between HSF1 and HSF2

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2001). Mouse HSF1 with N-terminal Flag tags was a kind gift fromR. I. Morimoto (Northwestern University, IL, USA). Mouse HSF2-αand HSF2-β with N-terminal Flag tags have been described previously(Pirkkala et al., 2000). Expression vectors encoding mouse HSF2-αand HSF2-β with C-terminal Myc tags were constructed by PCR andcloned into the EcoRI site in the pcDNA3.1(–) MycHis B vector(Invitrogen) in frame with the MycHis tag. The mouse HSF2-βdeletion mutants (Fig. 4A) were constructed by PCR and cloned intothe EcoRI and EcoRV sites in frame with the N-terminal Flag tag inpFLAG-CMV-2 (Kodak). All PCR-amplified products weresequenced to exclude the possibility of second site mutagenesis.HSF1-Myc-His stably overexpressing cell line (4H9) was generatedby electroporating 30 µg of HSF1-Myc-His to K562 cells as describedbelow. After 2 days of recovery, neomycin-resistant cells wereselected by growing the cells in medium containing 500 µg ml–1 G418

(Life Technologies) for 2 weeks. Neomycin-resistant single cellclones were picked out after serial dilutions and screened for HSF1-Myc-His expression by western blotting. Cells were routinely grownin the presence of 500 µg ml–1 G418.

TransfectionsK562 and HeLa cells were transfected by electroporation (975 µF, 220V) using a Bio-Rad Gene Pulser electroporator. For this procedure,5×106 cells were washed, resuspended in 0.4 ml of Optimem (Gibco-BRL) and placed in a 0.4-cm-gap electroporation cuvette (BTX).Plasmid DNA (30 µg) was added and, after a brief incubation at roomtemperature, the cells were subjected to a single electric pulse.Thereafter, cells were cultured at 37°C for 40 hours prior to theindicated experimental treatments.

Indirect immunofluorescenceand confocal microscopyFor immunofluorescence analysis,HeLa cells growing on coverslipswere washed with PBS andsimultaneously fixed andpermeabilized for 15 minutes in0.5% Tween 20 in PBS containing3% paraformaldehyde, or the cellswere fixed for 15 minutes in 4°Cmethanol-acetone (1:1). After threewashes with PBS, cells wereincubated for 1 hour with blockingsolution (20% boiled normal goatserum in PBS). Rabbit anti-HSF1(Holmberg et al., 2000), rat anti-HSF1 (Neomarkers), rabbit anti-HSF2 (Sarge et al., 1993), mouseanti-Hsp70 (SPA-810; StressGen),mouse anti-Flag M2 (Sigma) ormouse anti-Myc (Sigma) antibody(1:500 dilutions) were added for 1hour. After washes with PBS, thebound primary antibodies weredetected using goat anti-mouseantibodies (1:400 dilution for 1 hour;


Fig. 2. Co-localization of HSF1 andHSF2 in stress granules. (A) HSF1and HSF2 co-localize to the stressgranules upon heat shock (HS).Monoclonal antibodies againstHSF1 were used to detectendogenous HSF1 and polyclonalHSF2 antibodies to detectendogenous HSF2 by doublestaining of HeLa cells analyzed byimmunofluorescence microscopy.(B) The epitope-tagged HSF1 andHSF2 constructs were transientlytransfected into HeLa cells. Anti-Myc antibody was used to detectthe ectopic HSFs (green) andpolyclonal antibodies against HSF1and HSF2 were used to detectendogenous HSFs (red). Yellowindicates co-localization in themerged image (Merge). Scale bars,5 µm.


Alexa 488, Molecular Probes), Cy3-conjugated donkey anti-rabbitantibodies (1:400 dilution for 1 hour; Jackson ImmunoResearchLaboratories), goat anti-rat antibodies (1:400 dilution for 1 hour; Alexa568, Molecular Probes) or donkey anti-rabbit antibodies (1:400dilution for 1 hour; Alexa 488, Molecular Probes). The DNA wasstained with DAPI (4′,6-diamidino-2-phenylindol, Sigma) for 2minutes before final washes and mounting with Vectashield (VectorLaboratories). The cells were analyzed and photographed using a LeicaDMR fluorescence microscope equipped with a digital HamamatsuORCA CCD camera or Leica TCS40 confocal laser scanningmicroscope using the SCANware 4.2a program. Images were furtherprocessed using Adobe Photoshop and CorelDraw software.

Immunoprecipitation and immunoblotting assaysFor in vivo coimmunoprecipitation experiments, transientlytransfected cells were lysed in 200 µl of lysis buffer [25 mMHEPES, 100 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 20 mMβ-glycerophosphate, 20 mM para-nitrophenyl phosphate, 100 µMortovanadate, 0.5 mM phenylmethylsulfonyl fluoride, 1 mMdithiotreitol, 1× complete mini protease inhibitor cocktail (RocheDiagnostics)] supplemented with 20 mM N-ethylmaleimide,followed by centrifugation for 25 minutes at 15,000 g at 4°C. Afterprotein extraction, 200-500 µg total cell protein was preincubated

with slurry of protein-G/Sepharose (Amersham Pharmacia Biotech)in TEG buffer (20 mM Tris-HCl pH 7.5, 1 mM EDTA, 10%glycerol) containing 150 mM NaCl and 0.1% Triton X-100 for 30minutes at 4°C followed by a brief centrifugation. The preclearedcellular lysate was incubated with anti-HSF1 (Neomarkers), anti-HSF2 (Neomarkers), anti-Flag or anti-Myc antibodies at roomtemperature for 30 minutes under rotation, after which 40 µl of a50% slurry of protein-G/Sepharose was added to the reactionmixture and incubated for 12 hours at 4°C under rotation. Aftercentrifugation, the Sepharose beads were washed with supplementedTEG buffer and the immunoprecipitated proteins were run on 8%SDS-polyacrylamide gel electrophoresis (SDS-PAGE) andtransferred to nitrocellulose filter (Protran Nitrocellulose; Schleicher& Schuell) for immunoblotting. For detection of protein input andexpression levels, 12 µg of protein was analyzed by SDS-PAGE asdescribed above. HSF1 was detected by polyclonal antibodiesspecific to mouse and human HSF1 (Sarge et al., 1993; Holmberget al., 2000), HSF2 by polyclonal antibodies specific to mouse HSF2(Sarge et al., 1993), the inducible form of Hsp70 by 4g4 (AffinityBioreagents), Hsc70 by SPA-815 (StressGen). Horseradishperoxidase (HRP)-conjugated secondary antibodies were purchasedfrom Promega and Amersham. The blots were developed with anenhanced chemiluminescence method (ECL; Amersham PharmaciaBiotech).

Fig. 3. Coimmunoprecipitation of HSF1 and HSF2. (A) Coimmunoprecipitation of HSF2 with transiently (left) or stably (right) overexpressedMyc-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 wereused as an antibody control. Cells were either left untreated (C) or heat shocked for 1 hour (HS). (C) The dynamics of the interacting complexwas 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). Thebottom 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.

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Cells (5×106 each of 4H9 and K562) were labeled with 0.25 mCiml–1 of TRAN35S Label (ICN) for 16 hours. Cells were lysed in 1ml 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)antibodies was performed as above. Immunoprecipitated proteinswere detected from the gels by autoradiography. To identify the bands,1×109 4H9 and K562 cells were lysed and immunoprecipitated asabove. The gels were silver stained (O’Connell and Stults, 1997)

and the bands were identified using standard matrix-assisted laserdesorption ionization mass spectrometry.

Gel mobility shift assayWhole cell extracts were prepared from experimentally treated cellsas previously described (Mosser et al., 1988) and incubated (12 µgprotein) with a 32P-labeled oligonucleotide representing the proximalHSE of the human hsp70promoter. The protein-DNA complexes were


Fig. 4. The oligomerization domainHR-A/B is essential for the physicalinteraction between HSF1 andHSF2. (A) Schematic presentationof HSF2 deletion mutants. TheDNA-binding domain (DBD), N-terminal oligomerization domain(HR-A/B) and C-terminal leucinezipper (HR-C) are indicated.(B) Anti-Myc antibodies were usedto immunoprecipitate HSF1, andantibodies against HSF1 and HSF2were used for western blotting. Anempty Myc vector was used in themock transfections. The expressionlevels were analyzed by westernblotting (WB), as indicated. Arrowsand asterisks indicate the induciblyphosphorylated HSF1 and theendogenous HSF2, respectively.Molecular weights (kDa) areshown.


analyzed on a native 4% polyacrylamide gel as described previously(Mosser et al., 1988).

ResultsSubnuclear organization of HSF1 and HSF2 in granule-like structuresThe activation of HSF1 upon stress is associated with a seriesof biochemical changes, including oligomerization andphosphorylation, which correlate with the subnuclearorganization of HSF1 in chromatin-associated stress granules(Pirkkala et al., 2001; Jolly et al., 2002). Upon exposure to heatshock, we found that another member of the HSF family,HSF2, also formed brightly staining nuclear granules similarto HSF1, as detected by immunofluorescence microscopy (Fig.1A). Interestingly, the morphology and organization pattern ofboth HSF1 and HSF2 granules varied dramatically dependingon duration of heat stress (Fig. 1B). Both endogenous HSF1and HSF2 localized in small and barely detectable granulesafter only 5 minutes of heat shock (data not shown), and theintensity and size increased until 1 hour (Fig. 1B). At 2 hoursof treatment, the granules began to mature into ring-likestructures. These structures were most clearly detected at 2-6hours of continuous heat shock, corresponding to the

attenuation of the heat-shock response (Fig. 1B). At the sametime as the staining experiment, samples were collected for gelmobility shift and western blotting analyses to compare themorphology of HSF1 and HSF2 granules to the DNA-bindingactivity and phosphorylation of HSF1. During 2-6 hours of heattreatment, the maturation of heat-shock granules into ring-likestructures preceded the dissociation of HSF1 and HSF2 fromthe granules and the loss of HSF1 DNA-binding activity (Fig.1D). In addition to the morphological analyses, the number ofgranule-positive cells during the course of heat shock wasdetermined (Fig. 1C). Already at 30 minutes of heat shock, thenumber of granule-positive cells had increased prominentlyand, at 1-2 hours, almost 100% of cells contained granules.After 2 hours, the number of granule-positive cells began todecline (Fig. 1C). The dephosphorylation of HSF1 correlatedtemporally with the dissociation of HSF1 from the granules(Fig. 1B,D). A dramatic change in the number and morphologyof both HSF1 and HSF2 granules were detected after a 1 hourrecovery from heat stress and, after 3 hours, hardly anygranules were found and both HSF1 and HSF2 were diffuselydistributed (Fig. 1B,C). Interestingly, the number andmorphology of HSF1 and HSF2 granules were almostidentical, suggesting that they correspond to the same nuclearstructure.

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 withradiolabeled HSE (left panel). Antibody perturbations with Flag and Myc antibodies (1:10) were used to detect the presence of HSF1 or HSF2in the DNA-binding complex, respectively (right panel). A hemin-treated sample was used as a control. An empty Myc vector was used in themock transfections. (B) Flag antibodies were used to immunoprecipitate HSF1 from the same lysates as in panel A. Western blotting wasperformed as in Fig. 3. The expression levels are presented in the right hand panel (WB). Arrows and asterisks indicate the induciblyphosphorylated HSF1 and the endogenous HSF2, respectively.

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Fig. 6.HSF2 affects thelocalization of HSF1 in stressgranules. (A-C) HeLa cellswere transiently transfectedwith Flag-tagged HSF2 andconstructs shown in Fig. 4A.Cells were subjected to a 1-hour heat shock (HS) or leftuntreated (C), andimmunodetected with anti-Flag antibodies for HSF2deletion mutants (green) andwith polyclonal HSF1antibodies for the endogenousHSF1 (red). Colocalization isshown in the merge lane(yellow) and DNA is stainedwith DAPI (blue).(D) Granule formation wasanalyzed by counting 50 Flag-positive cells from threeindependent experiments(total 150 cells), and the graphrepresents the means ± s.e.m.Scale bars, 5 µm.


Co-localization of HSF1 and HSF2 in the nuclear stressgranulesThe heat-induced localization of HSF1 and HSF2 in granulesdid not occur randomly, but a co-localization of these twoendogenous factors was detected after a 1-hour heat shock, asshown by double staining (Fig. 2A). To ensure that finding theHSF2 stress granules was not a result of cross-reactivitybetween the antibodies against HSF1 and HSF2, we constructedboth N- and C-terminally Flag- and Myc-tagged HSF1 andHSF2 plasmids. Upon transient expression in HeLa cells, thesubcellular localization of ectopic HSF1 and HSF2 wasanalyzed. Immunofluorescence analyses with monoclonalantibodies against Flag and Myc epitopes showed a similarnuclear pattern (Fig. 2B) to the experiments conducted with theantibodies against HSF1 and HSF2 (Fig. 1A,B, Fig. 2A). Theco-localization between HSF1 and HSF2 was further confirmedby confocal microscopy (data not shown). Double-stainingexperiments were also performed as in Fig. 1B to study the co-localization of HSF1 and HSF2 at different phases of granuledevelopment. In these studies, co-localization of HSF1 andHSF2 was observed from the compartmentalization to thedissociation of these factors from stress granules (data notshown). It is worth noticing that, although there was prominentco-localization of HSF1 and HSF2 in the same subnuclearstructures, some heterogeneity occurred (i.e. stress granuleswere detected in which HSF1 but not HSF2 was present).

Dynamic interaction between HSF1 and HSF2The dynamic localization of HSF1 with HSF2 in stressgranules upon heat shock provided new evidence for HSF2functioning as a heat-responsive factor, and for the stress-induced regulation of HSF1 and HSF2 being closely related.This finding prompted us to investigate whether a similar heat-induced subnuclear distribution of HSF1 and HSF2 could becaused by their physical interaction. Myc-tagged HSF1 wastransiently or stably expressed in K562 cells and theendogenous HSF2 was coimmunoprecipitated with both

transiently (Fig. 3A, left) and stably (Fig. 3A,right) overexpressed HSF1. Furthermore,monoclonal anti-HSF2 antibodies were used toimmunoprecipitate the endogenous HSF2, andwestern blotting showed that the overexpressedHSF1 coimmunoprecipitated with HSF2 (Fig.3B).

To analyze the dynamics of the interactionduring the course of heat-shock response andrecovery periods, endogenous HSF1 from HeLacells was immunoprecipitated with monoclonalanti-HSF1 antibody. The immunoprecipitatedcomplexes were analyzed by western blotting, anddynamic interaction of HSF1 and HSF2-α and -βisoforms (Pirkkala et al., 2001) was observed atdifferent time points (Fig. 3C). At controltemperature and heat shock up to 30 minutes,HSF2-β was the major HSF2 isoform in thecomplex, whereas the amount of HSF2-α beganto increase from 30 minutes to 1 hour and similaramounts of both isoforms were detected in thecomplex. After 1 hour of heat treatment, the levelsof HSF2-β in the complex rapidly declined and,

at 2-4 hours of continuous heat shock, HSF2-α was the majorHSF2 isoform interacting with HSF1. Upon prolonged heatshock (>4 hours) and recovery, the amount of HSF2-β beganto increase, restoring the ratio of the HSF2 isoforms observedat control temperature (Fig. 3C, Fig. 8B). The changes in theinteraction stoichiometry between HSF1 and HSF2 isoforms,as shown in immunoprecipitation samples (Fig. 3C, top), werereflected also in western blotting of the lysates (Fig. 3C,bottom), indicating altered solubility of the HSF2 isoformsduring different phases of the continuous heat shock.

After 3 hours of recovery from a 1-hour heat shock, whencells acquire thermotolerance, the interaction between HSF1and HSF2 was downregulated (Fig. 3C, Fig. 8B). In contrastto the dynamic regulation of HSF2 isoforms detected both inthe lysates and protein complex, the downregulation of HSF1-HSF2 interaction seen after recovery from stress cannot beexplained by changes in solubility. This suggests that there aremodifications in the HSF-associated protein complex duringthermotolerance and further confirms the dynamic nature ofHSF1-HSF2 interaction.

Oligomerization domain is essential for the interactionbetween HSF1 and HSF2Different members of the HSF family share a similar structure,consisting of a leucine-rich oligomerization domain (HR-A/B)adjacent to the N-terminal DNA-binding domain, and anotherleucine-rich region near the C-terminus (HR-C) (Rabindran etal., 1991; Sarge et al., 1991; Schuetz et al., 1991). To examinewhether the interaction of HSF1 and HSF2 depended on anintact oligomerization domain, we constructed a series of Flag-tagged HSF2 deletion mutants (Fig. 4A). The Myc-taggedHSF1 was transiently expressed with these HSF2 mutants inK562 cells, and HSF1 was immunoprecipitated with anti-Mycantibody, and the presence of HSF2 in the complex wasanalyzed. As shown in Fig. 4B, HSF2 mutants lacking HR-A/B(HSF2 203-517 and HSF2 ∆126-201) did not form complexeswith HSF1.

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HSF2 is not found in the protein complex binding to theHSE upon heat shockConsidering that HSF2 interacts with HSF1, we wanted toinvestigate whether the HSF1-HSF2 heterocomplex could bindin vitro to the HSE of human hsp70 promoter. Flag-tagged HSF1and Myc-tagged HSF2 were transiently expressed in K562 cellsand a strong heat-induced DNA-binding activity was detected inthe lysates (Fig. 5A, left). The spontaneous DNA-bindingactivity in untreated samples was due to overproduction of HSFs.To analyze the DNA-binding complexes, we performed antibodyperturbation assays with anti-Flag and anti-Myc antibodies todetect the presence of HSF1 or HSF2 molecules, respectively.As shown in Fig. 5A (right), the anti-Flag (HSF1) antibodysupershifted the heat-induced DNA-binding complexescompletely, whereas the anti-Myc (HSF2) antibody had no effecton the complex. Because hemin induces the DNA-bindingactivity of HSF2 (Pirkkala et al., 1999), a hemin-treated sampleexpressing Myc-tagged HSF2 was included as a positive controlfor HSF2 DNA-binding activity and anti-Myc antibody. Theexistence of HSF1-HSF2 complex in the same lysates wasdetermined by coimmunoprecipitation (Fig. 5B).

To exclude the possibility that the C-terminal Myc-epitopein the HSF2 fusion protein was being masked in the heat-induced DNA-binding complex and could therefore not bedetected, we repeated the above mentioned experiments usingC-terminally Myc-tagged HSF1 and N-terminally Flag-taggedHSF2. The results were identical to those in Fig. 5, with onlyHSF1 being detected in the HSE-binding complex (data notshown). These results were also confirmed by overexpressingHSF2 alone, and no HSF2 was detected in the heat-inducedHSE-binding complex (data not shown).

HSF2 influences the localization of HSF1 in nuclearstress granulesBased on the obtained results, the HSF1-HSF2 complexformation could have significance in other steps of the stress-regulated signaling pathways unrelated directly to the HSE-binding and heat-shock gene expression. The appearance ofstress-induced nuclear granules has been shown to coincidewith HSF1 activation (Cotto et al., 1997; Jolly et al., 1999;Holmberg et al., 2000), so we investigated whether theHSF1-HSF2 interaction affects the subnuclearcompartmentalization of HSF1. For this purpose, the Flag-tagged HSF2 deletion mutants (Fig. 3A) were transientlytransfected into HeLa cells and double staining wasperformed with anti-Flag and anti-HSF1 antibodies. Asshown in Fig. 6A, the full-length HSF2 co-localized withHSF1 during heat shock. Unexpectedly, the DNA-binding-domain deletion mutant (HSF2 108-517), which was able tointeract physically with HSF1 (Fig. 4B), did not localize inthe granules, and also the translocation of HSF1 into thegranules was severely impaired (Fig. 6A,D). The HR-A/Boligomerization domain deletion mutants (HSF2 203-517 andHSF2 ∆126-201) could not interact with HSF1 (Fig. 4B) butdid not translocate into the granules; instead, these cellsdisplayed normal localization of HSF1 in the granules (Fig.6B,D). The C-terminal deletion mutant (HSF2 1-391), whichwas capable of interacting with HSF1 (Fig. 4B), localizedspontaneously to granules and, intriguingly, HSF1 showedclear co-localization with this HSF2 mutant at both control

and elevated temperatures (Fig. 6C,D). Taken together,the immunofluorescence analyses, combined with thecoimmunoprecipitation results (Fig. 4B), indicate that HSF2could affect the translocation of HSF1 into the nuclear stressgranules, because either induction or prevention of HSF1subnuclear compartmentalization was observed depending onwhich HSF2 mutant was used.

Localization of HSF1 and HSF2 in stress granules issuppressed in thermotolerant cellsIn addition to co-localization of HSF1 and HSF2 in the samedynamically regulated protein complex, we observed profoundchanges in subnuclear compartmentalization and HSF1-HSF2interaction during attenuation and recovery of the heat-shockresponse. To address the question of how thermotoleranceaffects the localization of HSF1 and HSF2 in stress granules,we induced thermotolerance by exposing HeLa cells to a 1-hour heat shock, after which the cells were allowed to recoverat normal temperature for 3 hours. During recovery, HSF1 andHSF2 dissociated from the granules and both proteins werepartly translocated into the cytosol (Fig. 7A). After a secondheat shock of 30 minutes or 1 hour, a decrease in granule-positive cells was detected, because almost 100% of primarilyheat-shocked cells, but less than 15% of thermotolerant cells,exposed to a 1-hour heat shock contained HSF-positivegranules (Fig. 7B). Because HSF1 and HSF2 behavedidentically, only HSF1 was used as a marker for nuclear stressgranules in the following experiments.

Nucleolar localization of Hsp70 coincides with thelocalization of HSF1 in stress granulesOne of the mechanisms regulating thermotolerance andattenuation of the heat shock response is the inhibition of HSF1activity by molecular chaperones such as Hsp70 and Hsp90and their co-chaperones (Morimoto, 2002). However, the roleof these chaperones in the regulation of nuclear stress granuleshas not been elucidated. Using immunoprecipitation ofMyc-tagged HSF1 from stably transfected 4H9 cells aftermethionine labeling followed by identification of theinteracting proteins by mass spectrometry, we found Hsc70 andHsp70 forming a complex with HSF1 in a stoichiometricmanner (Fig. 8A). During attenuation of the heat-shockresponse and in thermotolerant HeLa cells, dynamicallyregulated complex formation was detected between HSF1 andHsp70 (Fig. 8B). At the control temperature and upon heatshock of less than 30 minutes, HSF1 was found in a complexwith Hsp70, whereas this complex disassembled after 30minutes and was hardly detectable at 2 hours (Fig. 8B, data notshown). After 2 hours of continuous heat shock, the interactionbegan to increase. The interaction pattern with Hsp70correlated well with the onset of attenuation seen previously inHSF1 DNA-binding activity and phosphorylation, as well aswith the decline in granule-positive cells (Fig. 1). Duringrecovery periods, complex formation between HSF1 andHsp70 gradually increased (Fig. 8B).

Next, we investigated how the subcellular localization ofHsp70 corresponded to the interaction pattern seen in Fig. 8Band to the heat-shock granule formation. Hsp70 and its co-chaperones such as Hsp40 are rapidly translocated from the



cytosol to the nucleoli upon heat shock (Welch andFeramisco, 1984; Welch and Suhan, 1986; Hattori et al.,1993) but the significance of this rapid change in subcellulardistribution is poorly understood. The localization ofHSF1 and Hsp70 was investigated by double-staining

immunofluorescence microscopy and, atnormal temperature, HSF1 was foundboth in the cytosol and nucleus, whereasHsp70 was mainly cytosolic (Fig. 8C).Upon heat shock of less than 1 hour, theincreasing intensity and size of the stressgranules coincided with the ongoingtranslocation of Hsp70 into the nucleoli(data not shown). At 1 hour, HSF1 wasprimarily detected in mature nucleargranules and Hsp70 concentrated in thenucleoli (Fig. 8C). Interestingly, at thistime point, the interaction betweenHSF1 and Hsp70 was dramaticallydownregulated (Fig. 8A). During theattenuation of the heat-shock response (2-6 hours of continuous heat shock), bothHsp70 and HSF1 gradually dissociatedfrom nucleoli and the stress granules,respectively, and HSF1-Hsp70 complexformation increased (Fig. 8A,C, data notshown). In thermotolerant cells, HSF1and Hsp70 formed an interacting complexand were both mainly localized in thecytosol (HS+3R; Fig. 8A,C). Uponexposure of thermotolerant cells to asecond heat shock, a high level ofinteraction persisted and HSF1-positivegranules were detected only in cells inwhich Hsp70 was translocated intonucleoli (HS+3R+HS1h; Fig. 8C). Thenumber of cells containing HSF1 in stressgranules and Hsp70-positive nucleolicorresponded to the heat-shockedthermotolerant cells shown in Fig. 7B(data not shown). In these experiments,the nucleolar localization of Hsp70coincided with the downregulation of

HSF1-Hsp70 interaction and localization of HSF1 in nuclearstress granules. The reversible localization pattern of Hsp70into and out of the nucleoli appeared to be closely related tothe subcellular distribution of HSF1, and could therebycontribute to the regulation of HSF1 activity.

Fig. 7.The localization of HSF1 and HSF2 instress granules is suppressed in thermotolerantHeLa cells. (A) Fluorescence micrographs ofthe localization of HSF1 and HSF2 inuntreated (C), heat-shocked (30′ or 1h),thermotolerant (HS+3R) and heat-shockedthermotolerant (HS+3R+HS30′ orHS+3R+HS1h) cells. Proteins were visualizedusing monoclonal antibodies against HSF1(red) and polyclonal antibodies against HSF2(green). Co-localization is shown in the mergelane (yellow) and DNA is stained with DAPI(blue). Scale bars, 5 µm. (B) The graphrepresents the means ± s.e.m. of the threeindependent experiments in which the numberof granule-positive cells was analyzed bycounting nine groups of 50 cells.

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DiscussionThe physiological role of HSF1 in the regulation of the heat-shock response is well established but the possible interplaybetween HSF1 and other HSFs is largely unresolved. Although

Sheldon and Kingston (Sheldon and Kingston, 1993) earlierreported that the intracellular localization of human HSF2 isregulated by heat stress, the function of HSF2 has ever sincebeen solely linked to differentiation-related processes. In this


Fig. 8. Nucleolar localization of Hsp70 correlates with the activation anddeactivation of HSF1. (A) An autoradiograph image of proteins interacting withMyc-HSF1 in labeled 4H9 cells. Anti-Myc antibodies were used forimmunoprecipitation. K562 cells were used as a negative control. The two obtainedbands were detected also by silver staining and analyzed by matrix-assisted laserdesorption ionization mass spectrometry. (B) Coimmunoprecipitation of endogenousHSF2 and Hsp70 with endogenous HSF1 in heat-shocked HeLa cells. MonoclonalHSF1 antibodies were used for immunoprecipitations (IP). Cells were untreated (C)or heat shocked for 30 minutes to 6 hours (30′-6h). Thermotolerance was obtainedby 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′ orHS+3R+HS1h). The bottom blots (WB) show the expression levels of HSF1, HSF2and Hsp70. Hsc70 was a control for equal loading. Arrows indicate the induciblyphosphorylated HSF1. (C) Fluorescence micrographs showing the localization ofHSF1 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 isstained by DAPI (blue). Scale bar, 5 µm.


study, we provide evidence of human HSF2 being an integralmember of the cellular stress response pathway. Specifically,HSF2 is a novel component of the nuclear stress granules, andthe localization of HSF2 in the granules coincides temporallywith the localization and activation of HSF1. By analyzingboth HSF1 and HSF2 separately and together, we detected aclear maturation pattern in the formation of stress granules.The mechanisms behind the transformation from small clustersto single globular units, followed by the maturation into ring-like structures, are still obscure. However, the morphologicalchanges in the granules are temporally related to the alterationsin the DNA-binding activity of HSF1. Both acquisition andattenuation of DNA-binding activity strictly correlate with theassembly and disassembly, respectively, of the stress granules.

In addition to the co-localization of HSF1 and HSF2 innuclear stress granules, we provide the first in vivo evidenceof complex formation between HSF1 and HSF2. Our datareveal a regulated interaction between HSF1 and HSF2isoforms during the course of heat shock, possibly caused byregulated solubility of different HSF2 isoforms, as indicated byMathew et al. (Mathew et al., 2001). We also show thatthe HSF1-HSF2 complex is efficiently disassembled inthermotolerant cells, suggesting modifications in the propertiesof the HSF1-interacting protein complex. Further experimentsare needed to reveal whether the downregulation of HSF1-HSF2 interaction is involved in the suppression of HSF1activity during thermotolerance, and how the HSF2 isoformcomposition reflects the overall heat shock response. Byanalyzing a set of HSF2 deletion mutants, we show that thephysical interaction with HSF1 requires an intact HR-A/Boligomerization domain of HSF2. It is still an open questionwhether HSF1 and HSF2 form a heterodimer or heterotrimer,or whether the interaction occurs between two homo-oligomers. Considering our results and the structural similarity,it is likely that HSF1 and HSF2 form a hetero-oligomer.

Despite the prominent interaction between HSF1 and HSF2,we failed to detect HSF2 in the heat-shock-induced HSE-bindingcomplex, which is in agreement with Mathew et al. (Mathew etal., 2001). Because these observations indicate that heat stressdoes not activate HSF2 HSE-binding activity, the HSF1-HSF2complex formation together with the subnuclear co-localizationrepresents a previously uncharacterized step in the regulationof the stress-signaling pathway in human cells. To addressthe question of whether HSF1-HSF2 interaction affects thelocalization of HSF1 in stress granules, we performed double-staining experiments using the HSF2 deletion mutants. Strikingly,expression of HSF2 mutant lacking the DNA-binding domain(HSF2 108-517) inhibited the recruitment of HSF1 into the stressgranules, which could be due to the physical interaction betweenthis mutant and HSF1. An attractive explanation is that the effectcaused by HSF2 108-517 would lead to a defect in the interactionbetween the chromosomal nucleation sites and the HSF1-HSF2108-517 heterocomplex. This is in good agreement with anotherstudy, in which the granule-forming-ability of HSF1 was shownto be strictly dependent on the DNA-binding and theoligomerization domains of HSF1 (Jolly et al., 2002). Bycontrast, neither of the deletion mutants lacking the HR-A/Boligomerization domain (HSF2 203-517, HSF2 ∆126-201) wasable to form a complex with HSF1 and thereby affect thecompartmentalization of HSF1 in the stress granules. Therefore,the capability of HSF2 to oligomerize seems to be crucial for both

interaction with HSF1 and localization in the stress granules.Unexpectedly, the C-terminal deletion mutant (HSF2 1-396),which both interacted and co-localized with HSF1, wasspontaneously translocated into the stress granules withendogenous HSF1. This further emphasizes the interdependencybetween HSF1 and HSF2 in their stress granule localization.

A characteristic feature of the heat-shock response isacquired upon recovery from stressful conditions. Themechanisms regulating thermotolerance have been closelyrelated to the inhibition of HSF1 by direct interaction betweenupregulated Hsp70 and its co-chaperones (Morimoto, 2002).Owing to the changes in HSF1 regulation upon acquiredthermotolerance, we wanted to examine the localization ofHSF1 and HSF2 in nuclear stress granules under theseconditions. Our results demonstrate a suppression of HSF1and HSF2 localization in stress granules in heat-shockedthermotolerant cells, which correlates well with the inhibitionof HSF1 activity during thermotolerance and suggests a role forHsps in the regulation of granule formation. We also show thatthe translocation of Hsp70 into and out of the nucleoli coincideswith the localization of HSF1 to nuclear stress granules andinteraction with HSF1. The dissociation of the HSF1-Hsp70complex during heat shock corresponds to the translocation ofHsp70 into nucleoli. Simultaneously, the DNA-binding activityand hyperphosphorylation of HSF1, and the number of granule-positive cells are at maximum. During the attenuation of HSF1activity, Hsp70 dissociates from the nucleoli, the complexformation between Hsp70 and HSF1 increases, and HSF1relocates from the stress granules. Concomitant with theinhibition of HSF1 localization to granules in thermotolerantcells exposed to a second heat shock, fewer cells are observedthat contain Hsp70 in their nucleoli. Taken together, ourresults show a strict correlation between the subnuclearcompartmentalization of HSF1 and Hsp70 nucleolarlocalization, and, because the dissociation of HSF1 from stressgranules during attenuation also coincides the translocation ofHsp70 out from the nucleoli, it is plausible that the nucleolarlocalization of Hsp70 contributes to the regulation of HSF1.

We thank K. M. Heiskanen, T. Katajamäki and H. Saarento forexcellent technical assistance. We are grateful to L. Pirkkala forsuggestions in the initial phase of this study and valuable commentson the manuscript. P. Roos-Mattjus is also acknowledged fordiscussions and critical comments. This work was supported by theAcademy of Finland, the Sigrid Jusélius Foundation, the WihuriFoundation, the Finnish Cancer Organizations, and the FinnishCultural Foundation. T-PA, MH and VH were supported by the TurkuGraduate School of Biomedical Sciences.

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