2925Journal of Cell Science 110, 2925-2934 (1997)Printed in Great Britain © The Company of Biologists Limited 1997JCS9684

HSF1 granules: a novel stress-induced nuclear compartment of human cells

José J. Cotto, Susan G. Fox and Richard I. Morimoto*

Department of Biochemistry, Molecular Biology, and Cell Biology, Rice Institute for Biomedical Research, Northwestern University,Evanston, IL 60208, USA*Author for correspondence (e-mail: r-morimoto@nwu.edu)

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Heat shock factor 1 (HSF1) is the ubiquitous stress-respon-sive transcriptional activator which is essential for theinducible transcription of genes encoding heat shockproteins and molecular chaperones. HSF1 localizes withinthe nucleus of cells exposed to heat shock, heavy metals, andamino acid analogues, to form large, irregularly shaped,brightly staining granules which are not detected duringattenuation of the heat shock response or when cells arereturned to their normal growth conditions. The kinetics ofdetection of HSF1 granules parallels the transient inductionof heat shock gene transcription. HSF1 granules are also

detected using an HSF1-Flag epitope tagged protein or achimeric HSF1-green fluorescent protein which reveals thatthese nuclear structures are stress-induced and can bedetected in living cells. The spatial organization of HSF1granules in nuclei of stressed cells reveals that they are novenuclear structures which are stress-dependent and providesevidence that the nucleus undergoes dynamic reorganiza-tion in response to stress.

Key words: Subnuclear structure, Heat shock, Transcription, Stresresponse

SUMMARY

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INTRODUCTION

The cellular response to adverse environmental and phylogical conditions such as heat shock, exposure to amino analogs, heavy metals, oxidative stress, anti-inflammatdrugs, and arachidonic acid leads to the rapid and transient vation of genes encoding heat shock proteins (hsps) molecular chaperones (Lindquist, 1986; Morimoto et al., 191994; Jurivich et al., 1992). Stress-induced transcriptionregulated by a family of heat shock transcription factors (HSIn vertebrates, four members of the HSF gene family (HSFs4) have been characterized (Rabindran et al., 1991; Sarge e1991; Schuetz et al., 1991; Nakai and Morimoto, 1993; Naet al., 1997). The co-expression of multiple HSFs and chaterization of regulatory conditions has revealed that differemembers of the HSF family mediate the response to distforms of cellular stress. Consistent with this, HSF1 respoto the classical inducer of the heat shock response, HSFactivated during embryogenesis, spermatogenesis erythroid differentiation, HSF3 functions as a high temperatactivator, and HSF4 has properties of a negative regulatoheat shock gene expression (Sistonen et al., 1992, 1994; Set al., 1993, 1994; Nakai et al., 1995, 1997).

Under normal conditions of cell growth, HSF1 is maintainin an inert non-DNA binding state which undergoes reversioligomerization to a DNA binding competent trimer in stresscells (reviewed by Morimoto et al., 1994 and Wu, 1995). Twdistinct mechanisms involving negative regulatory domaand constitutive phosphorylation at serine residues participto maintain HSF1 in its inert state (Green et al., 1995; Shal., 1995; Kline and Morimoto, 1997; Knauf et al., 1996). Hothe stress signal is transduced and results in the de-repre

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of the inert monomer remains unresolved, however, HSFtrimers appear within minutes of activation and can be detectbound to DNA shortly thereafter. The rapid induction of thheat shock response suggests that the relocalization of HSmust involve a dynamic process. Yet, unlike most proteinwhich either translocate constitutively to the nucleus or exhiba constant pattern of localization, the mechanisms involvinHSF1 are likely to be distinct. Previous studies on thimmunolocalization of the HSFs have detected the appearanof granules which seem to correlate with activation (Sarge al., 1993; Nakai et al., 1993, 1995; Nakai et al., 1997). In thstudy we examine the cell biological properties of HSF1 usina collection of monoclonal antibodies which specifically detethe appearance of HSF1 granules within the nucleus of strescells. The kinetics of induction of HSF1 granules by heat shoand other stresses which lead to heat shock gene transcripstrongly indicates a role in the stress response.

MATERIALS AND METHODS

Cell cultureHuman HeLa, A431 and HOS cells were grown in Dulbeccomodified Eagle’s medium (DMEM) with 5% fetal calf serum (FCS)Primary epithelial and fibroblast cells were grown in DMEM with10% FCS supplemented with essential and non-essential amino acvitamins, and buffered with 1 M Hepes, pH 7.4. HeLa S3 cells wegrown in Joklik’s medium with 5% calf serum. Cell growth, heashock conditions and exposure to heavy metals, amino acid analand anti-inflammatory drugs were as described before (Mosser et 1988; Jurivich et al., 1992; Sarge et al., 1993).

Antibodies and indirect immunofluorescenceThe subcellular localization of HSF1 was determined using a panel

2926

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J. J. Cotto, S. G. Fox and R. I. Morimoto

anti-HSF1 rat monoclonal antibodies (10H8, 10H4 and 4B4) generafrom rat hybridoma cell lines using purified recombinant mouse HSas the antigen. The monoclonal antibodies were characterizedspecificity to HSF1 by ELISA and western blot analysis using purifirecombinant mouse HSF1 and total cell extracts from mouse and overtebrates (see Table 1). The epitope recognized by each monocantibody was determined by western blot analysis of a collectionmouse HSF1 deletion mutants (Shi et al., 1995; Kline and Morimo1997, see Fig. 1). The subcellular localization of the antigens recnized by each antibody was determined by indirect immunofluescence. The specificity of each antibody for the control monomnon-DNA binding form of HSF1 and the active trimer was determinby immunoprecipitation from extracts of control and heat shocked cand by the use of the antibodies for antibody upshift assays. The reof the characterization of each antibody are summarized in Table

Antibodies that recognized other nuclear structures included momonoclonal anti-splicing factor SC-35 antibody, from Sigma (Cata# S-4045), human autoimmune anti-kinetochore antibodies and aNuMA, monoclonal anti-nuclear lamin A and B antibody (provided bDr Robert Goldman, Northwestern University Medical School), anp80-coilin rabbit polyclonal antibody (provided by Dr Angus LamonUniversity of Dundee and Dr Edward Chen, The Scripps ReseaInstitute), anti-fibrillarin (provided by Dr David Spector, Cold SprinHarbor) and anti-PML monoclonal antibody (provided by Dr Luitzede Jong, University of Amsterdam). The antiserum titer was establisby sequential dilution to a range of 1:100 to 1:300 before use. Horadish peroxidase (HRP)-conjugated goat anti-rat IgG was obtaifrom Pierce (catalog #31475G) and goat anti-rabbit IgG antibody wobtained from Promega (catalog # W4011). Texas Red and fluores(FITC)-conjugated goat anti-rat IgG (catalog # 712-095-153, 712-0153), goat anti-rabbit IgG (catalog # 711-075-152) and goat anti-moIgG antibodies (catalog # 715-095-151) were obtained from JackImmunoresearch and mouse anti-bromo-uridine triphosphate (BrUmonoclonal antibody was obtained from Sigma (catalog # B2531).

For immunofluorescence analysis, adherent cells on coverslips wwashed in 1× PBS, fixed for 10 minutes with 2% paraformaldehyde 1× PBS at room temperature, washed twice with 1× PBS, and per-meabilized with 0.1% Triton X-100. The permeabilized cells wewashed twice with PBS and incubated for 1 hour with a blockisolution consisting of 1% bovine serum albumin (BSA) in PBS at rootemperature prior to incubation with antibodies. Fixed cells weincubated for 1 hour at 37°C with either rat monoclonal or rabbit poclonal anti-HSF1 antibody at dilutions of 1:100 and 1:300, resptively. To study the co-localization of HSF1 granules with other knownuclear structures, a mix of rat monoclonal anti-HSF1(1:100) weither anti-Brdu (1:100), anti-SC-35 (1:100), anti-kinetochore (1:20anti-p80 coilin (1:100), anti-fibrillarin (1:1), anti-lamin A and B (1:00)anti-NuMA (1:100) and anti-PML (1:5) was prepared and cells weincubated for 1 hour at 37°C prior to detection. The antibodies wdetected using Texas Red or FITC-conjugated goat anti-rat and anti-mouse and the staining pattern was analyzed by conventionalfluorescence microscopy on a Zeiss Axiophot microscope or by la

Table 1. Summary of the imunological charac

Western blot Imm

Antibody Epitope* 37°C 42°C 37°C

10H8 378-395 +†(1,2,3,4) +†(1,2,3,4) +†(110H4 295-378 +†(1,3,4) +†(1,3,4) −

4B4 425-439 +†(1,2,3,4) +†(1,2,3,4) +†(1

*Numbers correspond to the minimal amino acid region containing the erecombinant mouse HSF1 deletion mutants (Shi et al., 1995; Kline and M

†HSF1 species reactivity: 1, human; 2, monkey; 3, rat; 4, mouse.‡Not applicable.

tedF1 foredtherlonal ofto,og-or-ericedellssults 1.use

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confocal microscopy. To establish the spatial organization of HSgranules and the relationship of HSF1 granules with other nucstructures, Z-sections of the stained cells were collected in a conlaser scanning microscope (Zeiss) and the data were analyzed witprogram NIH Image, Vers. 1.60 for Macintosh.

Nuclease treatmentCells permeabilized with 0.05% Triton X-100 were treated with eithRNAse A (100 mg/ml) or DNAse I (5 units/30 ml of RNAse freDNAse) from Boehringer Mannheim at 37°C for pre-determintimes and washed with PBS prior to fixation and immunostaining

Cell extraction and biochemical analysisThe procedure for in situ sequential fractionation of heat shocked was performed as described (He et al., 1990; Bissoto et al., 1995)some variations. Briefly, heat shocked (42°C) HeLa S3 cells wextracted in suspension with centrifugation steps (600 g, 3 minutes)between treatments. Supernatants were collected for SDS-PAGEglycerol gradient analysis. Cells were first extracted in low ionic bu(20 mM Hepes, pH 7.9, 25% glycerol, 0.42 M NaCl, 1.5 mM MgC0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) for 5 minutes at 4°CAfter a brief wash with 1× PBS, cells were extracted with cytoskeletobuffer (10 mM Pipes, pH 6.8, 100 mM NaCl, 300 mM sucrose, 3 mMgCl2, 1 mM EGTA, 0.5% Triton X-100, 4 mM vanayl ribosidcomplex, 1 mM PMSF) for 3 minutes at 4°C. After washing with ×PBS, DNA was digested with 25 units/ml DNAse I for 30 minutes25°C in digestion buffer (essentially the same as cytoskeleton buffe50 mM NaCl and 0.05% Triton X-100). Chromatin was then removby three 10 minute washes with 0.25 mM ammonium sulfatedigestion buffer to yield the nuclear matrix intermediate filamestructure (He et al., 1990). An additional high salt treatment was appby washing the nuclear matrix with 2 M NaCl in digestion buffer. Aslast step the pellets were boiled in SDS sample buffer for SDS-PAanalysis.

Construction and expression of HSF1-Flag and HSF1-GFPfusion proteinsTo generate the carboxy-terminal epitope tagged mHSF1-Flag fuprotein, an oligonucleotide encoding an eight amino acid peptideDYKDDDDK-c) recognized by the monoclonal antibody Anti•FlaM2 (IBI Flag System, Kodak) was cloned at the 3′ end of the mouseHSF1 cDNA (Sarge et al., 1991) in the eukaryotic expression vepCDNA3 (InvitroGen), and verified by sequencing across the regThe construct corresponding to HSF1-green fluorescent pro(HSF1-GFP) fusion protein was made using the eukaryotic expresvector pEGFP-N1 (Clontech) to clone the green fluorescent pro(GFP) coding sequence to the 3′ end of mouse HSF1 cDNA.

For the expression of the fusion proteins, HeLa cells at a denof 40 to 50% confluence were transfected with 20 mg of DNA percm plate. Plasmid DNA was combined with 250 mM CaCl2 in a 500ml final volume. After chilling on ice, the DNA-Ca2+ solution wasadded dropwise to 500 ml of 2× Hepes (N-2-hydroxyethylpiperazine-

teristic of HSF1 specific monoclonal antibodies

unoprecipitation Supershift assay Immunofluorescence

42°C 37°C 42°C 37°C 42°C

,4) +†(1,4) N/A‡ +†(1,4) +†(1,2,3,4) +†(1,2,3,4)− N/A +†(1,4) +†(1,2,3,4) +†(1,2,3,4)

,4) +†(1,4) N/A +†(1,4) +†(1,2,3,4) +†(1,2,3,4)

pitopes for anti-HSF1 monoclonal antibodies as detected by western blot analysis oforimoto, 1997).

2927HSF1 granules

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N′-2-ethanesulfonic acid)-buffered saline (pH 7.06; 2× HeBes = 280mM NaCl, 1.5 mM Na2HPO4, 50 mM Hepes). After 20 minutes aroom temperature the precipitate was added to the cells dropwiseallowed to settle on the cells for 6 to 8 hours at 37°C. The plates wthen removed from the incubator and washed twice with 10 ml of×phosphate-buffered saline (PBS)-1 mM ethylene glycol-bis(aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA) and replacewith fresh medium for 48 hours before analysis.

Gel mobility shift assay and western blot analysis of HSF1 HSF1 DNA-binding activity was analyzed using the gel mobility shassay as described previously (Mosser et al., 1988) using a 32P-labeleddouble-stranded synthetic HSE containing four inverted nGArepeats (Sarge et al., 1991). Western blot analysis was perforusing whole cell extracts and rat monoclonal anti-HSF1 antibo(10H8). The immune complexes were analyzed using the Edetection system (Amersham).

RESULTS

HSF1 localizes to discrete nuclear granules uponheat shockUpon exposure of HeLa cells to heat shock, HSF1 relocalito form brightly staining nuclear foci or granules which wedetected using polyclonal antisera (Sarge et al., 1993).further characterize these granules, we prepared a collectiorat monoclonal antibodies (10H8, 10H4 and 4B4) that speically recognized HSF1 in cells from mouse, human and otvertebrate species. Although the epitopes for each antibmapped to different regions of HSF1 (see Fig. 1 and Tableboth the inactive and active trimeric form of HSF1 can detected by various immunological assays including, immuprecipitation and gel mobility shift-supershift assays (data shown, see Table 1). Indirect immunofluorescence analusing any of these monoclonal antibodies in heat shocked c

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395 503

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Fig. 1.Schematic representation of various mouse HSF1 deletionmutants and a summary of the pattern of recognition detected byanti-HSF1 monoclonal antibodies 10H8, 10H4 and 4B4. E. coliwhole cell extracts expressing the recombinant mouse HSF1 delemutants were analyzed by western blot and the minimal peptideregions recognized by the different antibodies are indicated.Conserved structural motifs corresponding to DNA binding domai(DNB) and heptad repeats A,B and C (HR A,B and C) are shown.

t andere

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revealed the presence of HSF1 granules for which antib10H8 gave typical results (Fig. 2B).

To examine whether the immunofluorescence stainpattern detected by these monoclonal antibodies was eitheconsequence of how the cells were prepared for indirimmunofluorescence or an unusual feature of these antibreagents, we constructed a Flag epitope-tagged mouse Hgene (mHSF1-Flag) and a green fluorescent protein (GFHSF1 chimeric gene for transient expression studies in Hecells. The endogenous and heterologous HSF1 proteins wdetected by double-label immunofluorescence using rat moclonal antibody 10H8 and the anti-Flag antiserum or tintrinsic fluorescence of GFP, respectively. The mHSF1-Fprotein (Fig. 3B) co-localizes with human HSF1 (Fig. 3A) aa general diffuse nuclear staining pattern under control cditions (Fig. 3C). Upon heat shock, mHSF1-Flag (Fig. 3localizes to the same granules detected with the monocloantibodies which recognize human HSF1 (Fig. 3D and Similar results of co-localization were observed using HSFGFP (Fig. 4). Whereas GFP alone is distributed in a diffupattern throughout the cell under control (Fig. 4A) and heshocked conditions (Fig. 4B), mHSF1-GFP is primarinuclear in control cells (Fig. 4C) and upon heat shock relocizes to form HSF1 granules (Fig. 4D). These results estabthat the heat shock induced formation of HSF1 granules aspecific property of HSF1, they can be detected with an epittag and in living stressed cells.

We next examined whether HSF1 granules are found in othuman tissue culture cell lines. Primary fibroblasts and epitlial cells were exposed to various heat shock temperaturesexamined by indirect immunofluorescence. Brightly stainiHSF1 granules were readily detected upon exposure of eiprimary cell to heat shock with the majority of cells contaiing two large foci and occasional smaller speckles (Fig. 5B aD). The optimal temperatures required to detect HSF1 granuin primary human cells was higher (43-45°C) than requiredtransformed human cells (see companion paper in this isJolly et al., 1997). Examination of the HSF1 staining pattein two other human transformed cell lines, HOS (hyperdiploosteosarcoma cell line) and A431 (hypotetraploid epidermcarcinoma cell line) revealed that HOS cells have an averof 4 to 5 granules per nucleus (Fig. 5F) and HeLa (hyptetraploid cervical carcinoma cell line) and A-431 cells conta

tion

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Fig. 2.HSF1 nuclear granules are formed upon heat shock in HeLacells. Cells cultured at 37°C or heat shock at 42°C were subjected toimmunofluorescence analysis using anti-HSF1 rat monoclonalantibody 10H8 (A and B). Bar, 5 µm.

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Fig. 3. Immunofluorescence analysis ofHeLa cells transfected with mouse HSF1-Flag expression vector. Transfected HeLacells were cultured at 37°C and stained withanti-HSF1 (A) or anti-FLAG (B) antibodies.(C) Co-localization of HSF1 and mHSF1-FLAG. (D-F) Cells incubated at 42°C heatshock and treated as in A-C. Bar, 5 µm.

Fig. 4. mHSF1-GFP fusion protein localizes to nuclear granules inheat shocked HeLa cells. HeLa cells at 37°C or heat shocked at 42°Cwere transfected with the GFP vector (A and B) or the mHSF1-GFPvector (C,D) and visualized by confocal microscopy. Bar, 5 µm.

HSF1-FlagHSF1

Con

trol

Hea

t S

hock

an average of 7 granules per nucleus (Fig. 5H and J). number of HSF1 granules per cell is relatively constant in aparticular cell line with fewer granules being detected primary cells than in transformed cell lines. These results athose of Jolly et al. (1997) suggest a possible relationsbetween the number of granules and chromosomal ploidy.

The kinetics of HSF1 granule formation parallels theactivation of HSF1 in stressed cellsThe activation of HSF1 is associated with a series of rapioccurring events including oligomerization of the non-DNbinding monomer to the DNA binding trimer, inducible serinphosphorylation, and transcriptional induction of heat shogenes (Sorger and Pelham, 1988; Baler et al., 1993; Sargal., 1993; Cotto et al., 1996). During continuous exposureheat shock, HSF1 activity attenuates as reflected by the lostranscriptional activity, dephosphorylation, and conversiontrimers to monomers (Abravaya et al., 1991a,b; Sarge et1993). Therefore, we examined whether the appearanceHSF1 granules is temporally associated with its activity atranscriptional activator.

Within 30 minutes of heat shock, HSF1 granules rangingsize from 0.5 to 1.5 µm were detected in 80-90% of HeLa cell(Fig. 6A), and by 60 minutes of heat shock, 95% of the ceexhibited brightly staining HSF1 granules. Up through twhours of heat shock, HSF1 granules were ubiquitous incells. Analysis of the size distribution of the granules in Hecells heat shocked at 42°C for 2 hours reveals that they cadescribed as two populations of which 60% of the foci corspond to smaller (0.5 to 1.5 µm) brightly staining granules orspeckles and the remaining are larger (1.5 to 2.5 µm) clusteredor ring-like granular structures (Fig. 7A). The majority (55%of the cells contained an average of 7 HSF1 granules, althothere was substantial cell-to-cell variation (Fig. 7B). Durincontinuous exposure to heat shock, both the fluoresceintensity and the numbers of HSF1 foci increased. Compson to the level of HSF1 DNA binding activity (Fig. 6Breveals that the appearance of HSF1 granules correl

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closely with both the acquisition of HSF1-DNA bindinactivity and the inducibly phosphorylated state of HSF1 (F6B and C). After 2 hours of continuous heat shock, the fractof cells which exhibit HSF1 granules rapidly declines to 5of the population; likewise HSF1 DNA binding attenuates ais dephosphorylated to the control state (Fig. 6B and C).

The activation of HSF1 is a multi-step process which involvthe stable appearance of intermediate states (Jurivich et1992; Lee et al., 1995; Cotto et al., 1996). To examine whethe appearance of HSF1 granules reflects the formation of HDNA binding trimers alone or requires complete activation

2929HSF1 granules

Fig. 5.Subcellular localization of HSF1 in various primary and transformed human cells. Cells either at control or 42°C heat shock conditionswere analyzed by immunofluorescence using rat monoclonal antibody 10H8. Human primary fibroblasts (A,B), epithelial cells (C,D), humanHOS cells (E,F), HeLa (G,H), and A431 (I,J) cells were examined. Bar, 5 µm.

Fig. 6.Kinetics of HSF1 granule formation and comparison with DNA binding activity and inducible phosphorylation during heat shock.(A) Immunofluorescence analysis in HeLa cells prior to heat shock (0 minutes) or after incubation at 42°C for 30, 60, 120, 180 and 240 minutesusing HSF1 rat monoclonal antibody 10H8. (B) Kinetics of HSF1 DNA binding activity measured using gel mobility shift analysis, and (C)western blot analysis of whole cell extracts from samples indicated in B. The slower mobility of HSF1 in heat shocked cells is due to inducibleserine phosphorylation. Bar, 5 µm.

2930

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Fig. 7.Quantitative analysis of the number and size of HSF1granules in HeLa cells. (A) The average diameter was calculated 200 granules using the program NIH image. In HeLa cells, HSF1granules can be divided in two sub-populations based on theiraverage size; small granules (~0.5 to 1.6 µm) and large granules(~1.6 to 3 µm). (B) The number of HSF1 granules was establishedby analysis of 100 nuclei. The mean number of HSF1 granules/ce6.8±2.4.

the transcriptionally competent trimer state, HeLa cells wexposed to three inducers of HSF1 activity. Sodium salicylinduces HSF1 trimers which are nuclear-localized, transctionally inert, and not-inducibly phosphorylated (Jurivich et a1992; Cotto et al., 1996), the amino acid analogue azetidinduces a transcriptionally active form of HSF1 which is ninducibly phosphorylated, and the heavy metal cadmiinduces a transcriptionally active and inducibly phosphorylaform of HSF1 (Sarge et al., 1993) (Fig. 8). Although eachthese conditions activate equivalent levels of HSF1 DNbinding activity (Fig. 8A), HSF1 granules were only detectin azetidine or cadmium treated cells and not in salicyltreated cells (Fig. 8B). These results reveal, that the appearof HSF1 granules is a reliable visual indicator of the transctional activity of HSF1 and that HSF1 granules are inducedother stresses which activate the heat shock response.

ereaterip-l.,ineot

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ditionally, these results reveal that HSF1 granules are not consequence of heat shock-induced aggregation of HSF1.

HSF1 granules are novel sub-nuclear structuresTo determine whether HSF1 granules represent a novel nuccompartment or correspond to previously characterized snuclear structures, we used double immunofluorescence winumber of antibodies to nuclear antigens and analysis by laconfocal microscopy. The fluorescence labeling patterns of eantibody and 8 to 10 horizontal optical sections of each field wscanned from top to bottom of the cell and the results of stacimages are presented. The data in Fig. 9 represent the supeposed images of fluorescein (FITC) and Texas Red-coupsecondary antibody labeling of different primary antibodies. Fig. 9A, the sites of DNA replication were marked by incoporation of BrdU prior to heat shock and detected with anti-Brdantibody. No co-localization of HSF1 granules with sites of DNreplication were detected. Likewise, the immunofluorescenpattern of mitotic cells stained with anti-HSF1 and anti-kinetchore antibody (Fig. 9B), the anti-splicing factor SC-35 antibo(Fig. 9C), and the coiled body marker anti-p80-coilin antibod(Fig. 9D) did not reveal co-localization with HSF1 granules. this study, no heterogeneity was noticed in the appearance or mphology of the HSF1 granules during the cell cycle.

As previous studies have shown that the nucleolar morphogy is affected by heat shock (Welch and Suhan, 1985), investigated whether the HSF1 granules would correspondan accumulation of the factor into nucleoli. This assumptiwas strongly supported by the granular morphology of HSfoci which is similar to that of nucleoli detected with a markeof the dense fibrillar center (Roussel et al., 1993). HSF1 wdetected by immunofluorescence together with a marker of dense fibrillar center, the UBF cofactor (upstream bindinfactor) using an anti-UBF antibody (Roussel et al., 1993however, no co-distribution between HSF1 foci and nuclewas observed (Fig. 9E). Likewise, HSF1 granules do not clocalize with PML, a nuclear localized protein involved ipromyelocytic leukemia (Koken et al., 1994; Weis et al., 199or with nuclear matrix proteins, such as nuclear lamins (A B) and NuMA (nuclear matrix mitotic apparatus protein) whichave been shown to accumulate into nuclear domainsspecific points in the cell cycle (Moir et al., 1994) (data nshown). Overall, these results clearly demonstrated that HSaccumulates into discrete nuclear substructures which distinct from other previously described nuclear granules.

Co-localization experiments carried out in our laboratohave clearly shown that HSF1 granules do not contain other hshock transcription factors (data not shown). Indeed, in earstudies, HSF2, HSF3, and HSF4 have been visualizednuclear speckles, not granules (Sarge et al., 1993; SheldonKingston, 1993; Nakai et al., 1995, 1997). Furthermore, thespeckles are detected constitutively and do not correlate wgene activation and heat shock response. In contrast, the appance of HSF1 granules correlates with transformation of tfactor from the inert to the active state and with gene activati

Recently, an increasing number of transcription factoincluding the mineralocorticoid and glucocorticoid receptorthe haemopoietic factors GATA-1 and -3, and p53, have beshown to accumulate into nuclear domains (Jackson et 1994; van Steensel et al., 1995, 1996; Elefanty et al., 199Although we have not performed a comprehensive comparis

for

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2931HSF1 granules

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Fig. 8. Effects of differentstress conditions on theactivation of HSF1 and theformation of HSF1 granules.(A) Gel mobility shiftanalysis of HSF1 DNAbinding activity in wholecell extracts from HeLacells at control (37°C)conditions or treated with20 mM salicylate, 5 mMazetidine, 30 µM CdSO4,or 42°C heat shock.(B) Intracellular localizationof HSF1 in HeLa cellsexposed to conditionsindicated in A and stainedwith monoclonal anti-HSF1antibody. Bar, 5 µm. 30 µM CdSO4 Heat shock (42°C)5 mM azetidine20 mM salicylateControl

between HSF1 and each one of these transcription factHSF1 does not co-localize with members of the GATA fam(data not shown) thus ruling out a common mechanism compartmentalization of transcription factors.

To assess whether nucleic acids are a component of Hgranules, heat shocked cells were permeabilized and incubwith either DNAse I or RNAse A (Fig. 10). Interestingly, thnumber, size, or distribution of HSF1 granules were naffected despite the substantial reduction in nuclear Dobserved in DNase I treated cells as detected by staining Hoechst dye (Fig. 10A). Likewise, RNAse A treatment did nhave an effect on HSF1 granules (Fig. 10B, a-c). These resreveal that the general features of the granules are not altby depolymerization of nucleic acids. However, given the ratively large size of the HSF1 granules, it is also possible their location within the nucleus may be unaffected evennucleic acids are an important structural component.

The possible association of HSF1 granules with the nucmatrix was examined using biochemical fractionation coditions known to extract the nuclear matrix (He et al., 199Bissoto et al., 1995). The majority of the HSF1 which wremoved by extraction with a low ionic strength buffer (Fi11, compare lanes 1 and 2) corresponds to trimeric HSF1;remaining HSF1 can be extracted with the non-ionic detergTriton X-100 (lane 4). HSF1 was not detected in the insolupellet corresponding to the nuclear matrix (lane 7). The eof extraction of HSF1 by low ionic buffer and relatively milnon-ionic detergents reveal that HSF1 is neither in an insolufraction nor associated specifically with the nuclear matr

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These results also suggest that HSF1 granules are labile readily disrupted even upon gentle lysis of the nucleus.

DISCUSSION

HSF1 granules represent a unique class of subcellular strtures which appear transiently in the nucleus of human cewhen heat shock genes are transcriptionally induced adisappear rapidly during attenuation of the heat shock respoas the transcription of heat shock genes diminishes to conlevels. These results establish HSF1 granules as a nodynamic feature of the heat shock response and underscthe potential for new information on the effects of stress nuclear structure.

The detection of HSF1 granules is likely to reflect thgeneral response to stress as exposure of human cells in cuto heat shock, cadmium and azetidine gave indistinguisharesults. Furthermore, these results rule out the possibility tHSF1 granules result from the aggregation of HSF1 at elevatemperatures, as other stresses which induce these granuleeffective at 37°C. Additionally, the effects of elevated temperatures and other stresses on the biochemical propertieHSF1 result in the oligomerization to a trimeric DNA bindinstate rather than association of HSF1 with the nuclear maor other high molecular sized nuclear structures. Anothobservation presented here which links the appearanceHSF1 granules with the transcriptional activity of heat shogenes is the absence of HSF1 granules in salicylate-trea

2932

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J. J. Cotto, S. G. Fox and R. I. Morimoto

Fig. 9.HSF1 does not co-localize with other known subnuclear structures. Immunofluorescence analysis of double-stained HeLa cells. Greenchannel represents HSF1 staining and red channel represents (A) DNA replication sites (anti-Brdu), (B) kinetochores, (C) splicing factor SC-35, (D) coiled body and (E) nucleolar dense fibrillar center staining. Bar, 5 µm.

cells. Although salicylate treatment induces HSF1 trimewhich exhibit complete DNA binding activity, this form oHSF1 is transcriptionally inert. In contrast, stress-inducesuch as heavy metals, amino acid analogues, and heat sresults in the fully active form of HSF1 which corresponds wstress-induced granules. Finally, the temporal link in the westablished kinetics of the heat shock response and the rrecovery during attenuation parallels precisely the transiappearance and disappearance of HSF1 granules. T

Fig. 10.Analysis of the effects ofnuclease treatment on HSF1granules. (A) Heat shocked HeLacells grown on coverslips werepermeabilized in 0.05% Triton X-100 and incubated in DNAse I for 0,15 or 30 minutes prior toimmunofluorescence analysis withHSF1 monoclonal antibody 10H8(a-c). Reduction of Hoechst staining(d-f) indicates digestion of DNA inthese cells. (B) Same as A, but cellswere treated with RNAse A prior toimmunofluorescence analysis (a-c).Treatment with RNAse did notaffect Hoechst staining (d-f).

rsfrs

hockithellapidentaken

together, these results reveal that HSF1 granules represenew component of the heat shock response in human cells. results and those presented in the accompanying paper rethat HSF1 granules are in all human cells. The relationsbetween the number of granules per nucleus and chromosoploidy noted here and by the accompanying paper (Jolly et 1997) is intriguing and suggests the existence of specific chmosomal targets for HSF1 foci.

The many unique features of HSF1 granules encouraged

2933HSF1 granules

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Fig. 11. Biochemical fractionation of heat shocked cells. (A) Heatshocked (42°C) HeLa S3 cell pellets (approximately 2×107

cells/pellet) were either directly solubilized with SDS-sample buffeto detect total amount of HSF1 in the cell (lane 1) or extractedsequentially with Buffer C (lane 2), a 1× PBS wash (lane 3), 0.5%Triton X-100 in cytoskeleton buffer (lane 4), DNAse (25 units/ml)and 0.25 M ammonium sulfate to remove chromatin (lane 5), a higsalt wash in 2 M NaCl (lane 6) and the insoluble material wassolubilized directly in SDS-sample buffer (lane 7). Extracted matewas dialyzed and lyophilized before solubilization in sample buffer(lanes 2-6). Extracts were analyzed directly by western blot analyusing monoclonal antibody 10H8.

to redouble our efforts to ensure that the granules coulddetected by other reagents or methodologies which did depend on indirect immunofluorescence or traditional formscell fixation. Two complementary approaches were describewhich an epitope tagged HSF1 allowed the detection of HSgranules via an antibody to a heterologous epitope and by dvisualization of HSF1 granules in living cells using an HSFGFP fusion protein. Thus, the detection of HSF1 granules isan artifact of either the polyclonal or the newly characterizmonoclonal antibodies. The ability to detect HSF1 granulesliving heat shocked cells by direct visualization of HSF1-Gchimeric protein also rules out possible artifacts inherent inprocedures employed for visualization and detection of the aHSF1 antibodies. Additionally, the latter result reveals that otproteins (e.g. GFP) can be translocated into HSF1 granules

Our knowledge on nuclear structure, while still limited, hgrown rapidly in recent years. Most nuclear functions includreplication, transcription, RNA splicing and RNA transport alocalized within the nucleus rather than distributed diffuse(reviewed by de Jong et al., 1990; van Driel et al., 1991, 19Moen et al., 1995). Among the most prominent sub-nuclstructures is the nucleolus which organizes events assocwith the transcription and processing of ribosomal RNA (Schand Benavente, 1990; Hernandez-Verdun, 1991). The strucand function of other nuclear domains are less well characized, in part because functional compartmentalization is apparent. Some nuclear domains for which a function has bsuggested include the small nuclear RNP clusters involvepre-mRNA processing (Piñol-Roma et al., 1989; Fu aManiatis, 1990; Spector, 1990; Lamond and Carmo-Fons1993b) and coiled bodies, which may play a role in spliceosoassembly or recycling and in the metabolism of small nucRNAs (Carmo-Fonseca et al., 1991; Lamond and CarmFonseca, 1993a; Bohmann et al., 1995). Analysis of the cellular distribution of certain transcription factors also revea non-homogeneous distribution within the nucleus (Jacksoal., 1994; van Steensel et al., 1995, 1996; Elefanty et al., 19Yet, for each of these factors, granules or speckles are contive and do not appear to correspond with other functioncharacterized sub-nuclear structures. Indeed, other heat s

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factors (HSF2, 3, 4) have been visualized as nuclear spec(Sarge et al., 1993; Sheldon and Kingston, 1993; Nakai et 1995, 1997). However, these speckles are present constitutiand do not correlate with gene activation and heat shoresponse, and they are distinct from the HSF1 granules. Onlthe case of HSF1 does the appearance of these granules corwith the activation of the factor from the inert to active states

Despite the cell biological analysis and biochemical charaterization presented in this study and the accompanying pa(Jolly et al., 1997), the role of HSF1 granules in the heat shoresponse remains enigmatic. Perhaps this is because analysis only encompasses our current expectations of a trtional role for HSF1 in the heat shock response. While thehas been substantial progress to understand the organizaand regulation of HSFs, only a few molecular targets for HSbinding, corresponding to the Hsp90 and Hsp70 genes, hbeen characterized. Others have shown that HSFs are asated with other chromosomal loci and suggested that HSmay have roles in the repression of non heat-shock geduring heat shock (Westwood et al., 1991; Cahill et al., 199Indeed, heat shock causes a complete arrest in the transtion of non-heat shock genes by mechanisms which have to be addressed.

We thank Dr Robert Goldman for the anti-lamin A and B monoclonal antibody and the NuMA autoantibody, Dr Angus Lamond fothe anti-coilin polyclonal antibody, Dr Edward Chen for the anti-p80coilin polyclonal antibody R288, Dr David Spector for anti-fibrillarinautoantibodies, and Dr Luitzen de Jong for the anti-PML monoclonantibody 5E10. We thank Dr Claire Vourc’h and Caroline Jolly fotheir valuable discussion during the course of this work and thcomments on the manuscript and Dr Robert Holmgren for advicemicroscopy. These studies were supported by a grant to R.I.M. frthe National Institutes of General Medical Sciences.

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(Accepted 1 October 1997)