Differential recognition of heat shock elements by
members of the heat shock transcription factor family
Noritaka Yamamoto
1
, Yukiko Takemori
1
, Mayumi Sakurai
2
, Kazuhisa Sugiyama
2
and Hiroshi Sakurai
1
1 Department of Clinical Laboratory Science, Kanazawa University Graduate School of Medical Science, Japan
2 Department of Ophthalmology and Visual Science, Kanazawa University Graduate School of Medical Science, Japan
Heat shock transcription factor (HSF), a protein that
is evolutionarily conserved from yeast to humans, is a
major regulator of heat shock protein (HSP) expres-
sion. Many HSPs function as molecular chaperones
that aid the folding of damaged proteins, and
increased accumulation of HSPs is essential for sur-
vival of cells exposed to protein-damaging stresses,
including heat shock. The structure of HSF comprises
a conserved DNA-binding domain (DBD), which
binds to the 5 bp sequence nGAAn, and two hydro-
phobic repeat (HR) regions (HR-A and HR-B), which
are necessary for homotrimer formation. Trimeric
HSF recognizes a heat shock element (HSE) compris-
ing at least three inverted repeats of the 5 bp unit
[1,2].
Biochemical and genetic evidence indicates that HSF
regulates the expression of genes encoding proteins

uous HSEs rather than discontinuous HSEs, and heat shock of HeLa cells
caused expression of reporter genes containing continuous HSEs. HSF2,
whose function is implicated in neuronal specification and spermatogenesis,
exhibited a slightly higher binding affinity to discontinuous HSEs than did
HSF1. HSF4, a protein required for ocular lens development, efficiently
recognized discontinuous HSEs in a trimerization-dependent manner.
Among four human c-crystallin genes encoding structural proteins of the
lens, heat-induced HSF1 preferred HSEs on the cA-crystallin and cB-crys-
tallin promoters, whereas HSF4 preferred HSE on the cC-crystallin
promoter. These results suggest that the HSE architecture is an important
determinant of which HSF members regulate genes in diverse cellular
processes.
Abbreviations
DBD, DNA-binding domain; EGS, ethylene glycol bis-(succinimidylsuccinate); hHSF, human heat shock transcription factor; HR, hydrophobic
repeat; HSE, heat shock element; HSF, heat shock transcription factor; HSP, heat shock protein; mHSF, mouse heat shock transcription
factor; SD, standard deviation; yHSF, Saccharomyces cerevisiae heat shock transcription factor.
1962 FEBS Journal 276 (2009) 1962–1974 ª 2009 The Authors Journal compilation ª 2009 FEBS
carbohydrate metabolism, and maintenance of cell
integrity [6–8]. yHSF binds to and regulates gene
expression via HSEs comprising variously arranged
nGAAn units: a continuous perfect-type HSE, con-
sisting of consecutive inverted repeats of the nGAAn
unit (nTTCnnGAAnnTTCn); and a discontinuous
gap-type or step-type HSE, which contains one
insertion [nTTCnnGAAn(5 bp)nGAAn] or two inser-
tions [nTTCn(5 bp)nTTCn(5 bp)nTTCn], respectively,
between the nGAAn units [9,10]. Schizosaccharo-
myces pombe HSF is also able to recognize these
various HSE types [10].
In mammalian cells, three related HSF proteins,

ing transcription via discontinuous gap-type and step-
type HSEs, indicating that hHSF1 recognizes HSEs in
a different way from yHSF [10]. In this study, we ana-
lyzed in vitro binding of hHSF1, hHSF2 and hHSF4
to various HSE types and characterized S . cerevisiae
and HeLa cells expressing hHSFs. Our results show
that the members of the hHSF family differentially
recognize HSEs, and suggest that the regulated expres-
sion of different hHSF target genes is dependent upon
the architecture of the HSE.
Results
Human HSF1, HSF2 and HSF4 exhibit differential
binding specificities for various HSE types
in vitro
Interactions between hHSFs and various HSE types
were investigated using electrophoretic mobility shift
assays with in vitro-synthesized polypeptides and oligo-
nucleotide probes (Fig. 1A). Protein–DNA complexes
were formed when binding reactions were carried out
using hHSF1-programmed transcription ⁄ translation
mixtures, but not in control reaction mixtures that
A
B
Fig. 1. Binding of hHSFs to various HSE types in vitro. (A) Nucleo-
tide sequences of four model HSEs. The GAA and inverted TTC
sequences are indicated in bold upper-case letters with arrows.
These HSE oligonucleotides were used as DNA probes for electro-
phoretic mobility shift assays and as cis-acting sequences for HSE–
SV40p–LUC reporters. (B) Electrophoretic mobility shift assay of
hHSFs. Typical results obtained using in vitro-synthesized hHSF1

amounts were threefold and 20-fold lower, respectively,
than that of hHSF1–HSE3P. This demonstrates that
hHSF1 preferentially binds to continuous HSEs. Note
that the interaction of hHSF1 with HSEs was
stimulated without changing HSE specificity when the
binding reaction was carried out at 43 °C rather than
37 °C (Fig. 1B), as reported for binding of mouse HSF
(mHSF)1 to an HSE containing four continuous,
inverted nGAAn repeats [31].
When hHSF2 polypeptide was incubated with perfect-
type HSEs, the electrophoretic mobility and the amount
of hHSF2–HSE4Ptt were almost the same as those of
hHSF2–HSE3P (Fig. 1B), indicating that a single
hHSF2 trimer binds to HSE4Ptt. Gap-type and step-
type HSEs were recognized by hHSF2, although the
binding affinity for HSEstep was threefold lower than
that for HSE3P. The amount of hHSF2 polypeptide
used in the assay was fourfold lower than that of hHSF1
polypeptide, and the addition of fourfold more hHSF2
polypeptide to the reaction mixture caused an increase in
the amount of hHSF2–HSE complexes without chang-
ing HSE specificity (Fig. S1). Although it is unknown
whether all polypeptides synthesized are active for
binding, hHSF2 appears to have a higher binding affin-
ity for at least discontinuous HSEs than does hHSF1.
Human HSF4 was observed to bind as a single tri-
mer to 4Ptt-type HSE, as judged from the amount and
mobility of the complex (Fig. 1B). Notably, the
amount of complex formed with hHSF4 and HSEgap
or HSEstep was more than 70% that of hHSF4–

the heat-induced transcription of all target genes
analyzed was appropriately regulated, with the excep-
tion of transcriptional activation via step-type HSEs,
which was slightly lower in hHSF2-expressing cells than
in yHSF-expressing cells. hHSF4 was also able to
compensate for yHSF in the regulation of target gene
expression; however, mRNA levels were slightly
reduced in hHSF4 cells as compared to yHSF cells. The
low mRNA levels may be due to the relatively weak
transcriptional activity [19,23] and ⁄ or the low-level
expression of hHSF4 (Fig. 2B). Unlike trimerization-
prone hHSF1 derivatives, which fail to activate
transcription of genes containing gap-type, step-type or
DR-type HSEs in yeast cells [10], hHSF2 and hHSF4
activate transcription of various target genes, and thus
support cell viability at elevated temperatures.
Heat-induced expression of reporter genes
containing various HSE types in HeLa cells
The transcriptional activity of various HSE types in
mammalian cells was analyzed using reporter genes con-
taining HSE oligonucleotides positioned upstream of
the SV40 promoter–luciferase gene fusion (HSE–
SV40p–LUC). In HeLa cells, insertion of HSEs in the
reporter gene did not significantly affect the basal
expression level under normal culture conditions
(Fig. 3A). This suggests that endogenous hHSFs are
not involved in the expression, although it is possible
that they bind to HSEs of reporter genes without affect-
ing the expression. When cells were heat-shocked at
43 °C for 1 h and allowed to recover at 37 °C for 5 h,

forms oligomers and binds to HSEs at physiological
temperatures [33]. Consistent with the results of heat
shock response, hHSF1–VP16 activated constitutive
expression of SV40p–LUC reporters containing contin-
uous HSEs, but the levels of activation for reporters
containing discontinuous HSEs were less than twofold
(Fig. 3C). The reporter gene expression in the presence
of hHSF2–VP16 was similar in pattern to that
observed in the presence of hHSF1–VP16, except that
HSEgap–SV40p–LUC expression was activated three-
fold. In contrast, hHSF4–VP16 was a potent activator
of reporter genes containing gap-type and step-type
HSEs. The HSE type-specific differences in transcrip-
tion of these reporters were consistent with the in vitro
binding affinity of each hHSF and HSE type, suggest-
ing that hHSF1, hHSF2 and hHSF4 differentially
recognize various HSEs in mammalian cells.
yHSF
hHSF4
45 15 0 45 15 0 45 15 0 45 15 0
hHSF2
45 15 0
YCp YEp YCp YEp
C
HSP42
HSP78
Gap
3P
4P
control

39
o
C (min)
15 0 15 0
50
70
yHSF
hHSF2
28
o
C
YCp
YEp
hHSF4
YEp
YCp
hHSF4
YCp YEp
15 0 15 0
NS
Fig. 2. Characterization of yeast cells expressing hHSF2 and
hHSF4. (A) Growth of hHSF cells. Cells of strains HS170T (YCp-
yHSF), YYT49 (YCp-hHSF2), YYT42 (YEp-hHSF2), YYT50 (YCp-
hHSF4) and YYT17 (YEp-hHSF4) were streaked onto YPD medium
and incubated at the indicated temperatures for 2 days. (B) Immu-
noblot analysis of hHSF proteins. Cells were grown in YPD medium
at 28 °C and heat-shocked at 39 °C for the indicated times.
Extracts of cells expressing hHSF2 (2 lg of protein) or hHSF4
(20 lg of protein) and recombinant hHSF proteins (not shown)
were subjected to immunoblot analysis with antibodies against

VP16) exhibited transcriptional activation via 3P-type
HSE, but failed to do so via gap-type or step-type
HSEs. An extended deletion construct leading to partial
removal of HR-A (hHSF4-n159–VP16) was abundantly
expressed but failed to activate transcription.
The roles of HR-A and HR-B were examined by
introducing amino acid substitutions. In hHSF4-
L140P–VP16 and hHSF4-I186P–VP16, a helix-destabi-
lizing residue (proline) was substituted for a hydro-
phobic residue (leucine or isoleucine) in HR-A and
HR-B, respectively. To analyze oligomer formation of
these hHSF4–VP16 derivatives, polypeptides synthe-
sized in vitro were subjected to chemical crosslinking
with ethylene glycol bis-(succinimidylsuccinate) (EGS)
(Fig. 4C). The band of approximately 220 kDa, which
corresponds to the size of a trimer, was detected by
treatment of wild-type hHSF4–VP16 with EGS. The
L140P and I186P substitutions appeared to inhibit tri-
mer formation, and most of the polypeptides migrated
at the position of a monomer (75 kDa). When an elec-
trophoretic mobility shift assay was conducted
(Fig. 4D), the substitution derivatives and 3P-type
HSE formed complexes exhibiting mobilities similar to
that of wild-type hHSF4–VP16 trimer–HSE3P complex
[this may be somewhat surprising; however, the com-
plex formation might be supported by DBD–DBD and
DBD–HSE3P interactions (see Discussion)]. However,
they exhibited reduced binding affinities for gap-type
and step-type HSEs. In HeLa cells, the L140P and
I186P substitutions in hHSF4–VP16 inhibited tran-

*
*
*
*
*
*
*
*
**
NS
Fig. 3. Expression of artificial reporter genes containing various HSE types in HeLa cells. (A) Heat shock-induced expression. Cells were
transfected with DNA mixtures containing 100 ng of SV40p–LUC plasmid (none) or HSE–SV40p–LUC plasmids (4Ptt, 3P, Gap, and Step). For
heat shock experiment, cells were incubated at 43 °C for 1 h, and culture was continued at 37 °C for 5 h. Luciferase activity (fold activation)
was expressed relative to that of SV40p–LUC plasmid-transfected cells (control, left panel) or to that of cells grown at 37 °C (heat shock,
right panel). Each bar represents the mean ± standard deviation (SD) for at least five experiments. Asterisks indicate significant differences
(P < 0.01) as compared with SV40p–LUC control as determined by Student’s t-test. (B) Immunoblot analysis of hHSF–VP16 fusion proteins.
Cells were transfected with DNA mixtures containing 100 ng of reporter plasmid (lane –) and hHSF1–VP16 (10 ng), hHSF2–VP16 (100 ng) or
hHSF4–VP16 (10 ng) expression constructs. Extracts prepared from cells grown at 37 °C were subjected to immunoblotting using an anti-
body against VP16. The positions of molecular mass markers are shown on the left in kilodaltons. NS denotes nonspecific band. The experi-
ments were performed at least twice, and yielded similar results. (C) Constitutive expression in cells cotransfected with hHSF–VP16
plasmids. Transfection was carried out described for (B). Luciferase activity (fold activation) was expressed relative to that of cells
transfected with the reporter plasmid alone. Each bar represents the mean ± SD for at least five experiments. Asterisks indicate significant
differences (P < 0.01) as compared with SV40p–LUC control as determined by Student’s t-test.
HSE-type specific recognition by human HSFs N. Yamamoto et al.
1966 FEBS Journal 276 (2009) 1962–1974 ª 2009 The Authors Journal compilation ª 2009 FEBS
but not via 3P-type HSE (Fig. 4A). These results show
that trimerization facilitated by HR-A ⁄ B is obligatory
for binding of hHSF4 to discontinuous HSEs.
Differential expression of c-crystallin promoter–
luciferase reporter genes by heat-induced hHSF1

n159
n355
n217
n178
A
I186P
L140P
159
178
217
355
VP16
493
HR-A/BDBD
VP16
VP16
VP16
VP16
VP16
VP16
Fold activation
0
493
493
C
B
240
100
140
70

Fig. 4. Expression of reporter genes by hHSF4–VP16 derivatives in HeLa cells. (A) Expression in cells cotransfected with hHSF4–VP16 deriv-
atives. Structures of hHSF4–VP16 derivatives are shown on the left. The DBD and two HRs (HR-A ⁄ B) are shown. Numbers indicate amino
acid positions. Vertical bars show the positions of amino acid substitutions. Cells were transfected with DNA mixtures containing 100 ng of
reporter plasmid and 10 ng of the indicated hHSF4–VP16 derivatives. Luciferase activity (fold activation) was expressed relative to that of
cells transfected with the reporter plasmid alone. Each bar represents the mean ± SD for at least four experiments. Asterisks indicate signif-
icant differences (P < 0.01) as compared with hHSF4–VP16 control as determined by Student’s t-test. (B) Immunoblot analysis of hHSF4–
VP16 derivatives. Extracts were prepared from cells transfected as described for (A), and were subjected to immunoblot analysis. Positions
of molecular mass markers are shown on the left in kilodaltons. NS denotes nonspecific band. The experiments were performed at least
twice, and yielded similar results. (C) Chemical crosslinking analysis of hHSF4–VP16 derivatives. In vitro-synthesized polypeptides (4.0 ng)
were incubated without or with 1.0 and 3.0 m
M EGS, and were subjected to immunoblot analysis. Positions of molecular mass markers are
shown on the left in kilodaltons. Open and closed circles indicate the positions of hHSF4–VP16 monomers and trimers, respectively. The
experiments were performed at least twice, and yielded similar results. (D) Electrophoretic mobility shift assay of hHSF4–VP16 derivatives.
Typical results using in vitro-synthesized polypeptides (4.0 ng) are shown as described for Fig. 1B. Open arrowheads indicate the positions
of DNA fragments bound by a single hHSF4–VP16 trimer. The experiments were performed at least three times, and yielded similar results.
N. Yamamoto et al. HSE-type specific recognition by human HSFs
FEBS Journal 276 (2009) 1962–1974 ª 2009 The Authors Journal compilation ª 2009 FEBS 1967
A
B C
Fig. 5. Expression of c-crystallin promoter-luciferase reporter genes in HeLa cells. (A) Expression by heat-induced hHSF1 and by
cotransfected hHSF4–VP16. Structures of the c-crystallin promoter–luciferase reporter genes are shown on the left. Bars represent the
crystallin genes, and open boxes indicate the HSEs. The cC-crystallin promoter contains two HSEs, one at a distal (dHSE) position and one
at a proximal (pHSE) position. Numbers indicate nucleotide positions relative to the translation initiation site. HSE sequences are shown in
which the GAA and inverted TTC sequences are indicated by bold upper-case letters with numbers. The nucleotides alterations are shown
below the HSEs. For heat shock experiments, cells were transfected with DNA mixtures containing 200 ng of the indicated reporter
plasmid, and luciferase activity (fold activation) was determined as described for Fig. 3A. For cotransfection experiments, cells were
transfected with DNA mixtures containing 200 ng of the indicated reporter plasmid and 10 ng of hHSF4–VP16 expression construct.
Luciferase activity (fold activation) was determined as described for Fig. 3C. Each bar represents the mean ± SD for at least four
experiments. Asterisks indicate significant differences (P < 0.01) as compared with wild-type control as determined by Student’s t-test. (B)
Electrophoretic mobility shift assay of hHSF1 and hHSF4–VP16. Typical results obtained using in vitro-synthesized hHSF1 (4.8 ng) and

reporter contained a 3P-like HSE (units 2, 3, and 4).
The m1 and m6 reporter gene alterations significantly,
but not completely, inhibited heat shock-induced
expression, although these reporters contained a
3P-like HSE. This result could be explained by a
model in which wild-type CRYGA promoter is bound
by two hHSF1 trimers; one trimer binding to units 1,
2, and 3, and the other binding to units 4 and 6.
Consistently, hHSF1–wild-type complex migrated more
slowly than hHSF1–m1 complex or hHSF1–m6 com-
plex in electrophoretic mobility shift assays (Fig. 5C).
Expression of m2 and m3 reporters was reduced to the
level of the m4 reporter, suggesting that gap-like and
step-like HSEs of these reporters are nonfunctional for
binding by hHSF1 (Fig. 5A). In hHSF4–VP16 cotrans-
fection experiments, unit 1 of the CRYGA HSE was
dispensable for activation (m1), although alterations of
other units, including unit 6, caused significant inhibi-
tion of activation (m2, m3, m4, and m6). Similar
results were obtained in electrophoretic mobility shift
assays (Fig. 5B). The observation that hHSF4–VP16
did not bind stably to sequences containing either
3P-like, gap-like or step-like HSEs might be explained
by the divergence of GAA-like sequences from the
canonical GAA sequence.
An alteration of either the distal or proximal HSE
of CRYGC–LUC caused inhibition of hHSF4–VP16-
induced expression, suggesting that both HSEs are
involved in hHSF4–VP16 binding (mdHSE and mp3,6)
(Fig. 5A). Notably, changing GAC to GAA in unit 3

slightly activates transcription of CUP1 [23]. Recently,
Fujimoto et al. [34] have shown that mHSF4 is
required for induction of a set of genes in response to
heat shock, in part by facilitating mHSF1 binding.
Although these results implied a similarity between
hHSF1 and hHSF4 in HSE binding specificity, our
data show that hHSF4 exhibits a binding specificity
clearly distinguishable from that of hHSF1 and
hHSF2, and is able to recognize discontinuously posi-
tioned nGAAn units. We suggest that genes containing
discontinuous HSEs are preferred targets for hHSF4
but not for hHSF1 or hHSF2. Consistently, Fujimoto
et al. [34] identified genomic regions that are occupied
by only mHSF4, and showed that the HSF4 binding
consensus sequence is more ambiguous than that of
HSF1 and HSF2. Two hHSF4 isoforms, hHSF4a and
hHSF4b, share the same DBD and HR-A ⁄ B, but func-
tion as a repressor and activator, respectively [23–25].
Phosphorylation of HSF4b by extracellular signal-
related kinase leads to increased ability of hHSF4b to
bind DNA [35]. Therefore, genes containing discontin-
N. Yamamoto et al. HSE-type specific recognition by human HSFs
FEBS Journal 276 (2009) 1962–1974 ª 2009 The Authors Journal compilation ª 2009 FEBS 1969
uous HSEs are subject to positive and negative regula-
tion by phosphorylated and unphosphorylated hHSF4
isoforms.
The L140P and I186P substitutions in hHSF4–VP16
inhibited binding to gap-type and step-type HSEs but
not to 3P-type HSE. In mammalian HSF1, HR-A ⁄ B
interacts with the third HR region (HR-C) and main-

both hHSF1 and hHSF2 preferentially bind to contin-
uous HSEs in vitro and in HeLa cells. hHSF1 and
hHSF2 consistently share the same target genes as
judged by chromatin immunoprecipitation analysis
[43]. In binding to continuous HSEs, mHSF1 prefers
long arrays of the nGAAn unit, whereas mHSF2 pre-
fers short arrays [27]. These differences are related to
differences in the capability for cooperative interac-
tions of trimers [26,27], which was confirmed by our
electrophoretic mobility shift assay (Fig. 1B). The
wing region of the DBD facilitates interactions among
mHSF1 trimers [41]. We have shown that in vitro and
in HeLa cells, hHSF2 exhibits a slightly higher bind-
ing affinity for discontinuous HSEs than observed for
hHSF1, and that unlike hHSF1 [10], hHSF2 expressed
in yeast cells properly regulates gene expression via
atypical HSEs as well as discontinuous HSEs. In this
regard, it should be noted that the mouse p35 gene, a
specific target of mHSF2, contains a putative HSE
that diverges from the canonical HSE [17]. It was
recently reported that mammalian HSF1 and HSF2
form heterotrimers and that HSF2 modulates the
activity of stress-induced HSF1 in a gene-specific
manner [44,45]. Differences in cooperativity and HSE
specificity are likely to be important determinants of
the interaction between HSF1–HSF2 heterotrimers
and HSEs.
In mouse, expression of the c-crystallin gene family
in the lens is regulated by various transcription factors,
including HSF1 and HSF4 [21,22,46]. Our analysis of

plasmid pcDNA3.1(+) (Invitrogen, Carlsbad, CA, USA).
For expression of hHSFs in Escherichia coli, hHSF1,
hHSF2 and hHSF4b (amino acids 220–493) were cloned
into plasmid pGEX6P-1 (GE Healthcare, Piscataway, NJ,
USA). For expression in yeast cells, hHSF2 and hHSF4b
were inserted between the ADH1 promoter and terminator
HSE-type specific recognition by human HSFs N. Yamamoto et al.
1970 FEBS Journal 276 (2009) 1962–1974 ª 2009 The Authors Journal compilation ª 2009 FEBS
of low-copy-number plasmid pSK484 (YCp-TRP1–P
ADH1
–
T
ADH1
) [48] and high-copy-number plasmid pK346 (YEp-
LEU2–P
ADH1
–T
ADH1
) [10]. For expression of hHSF–VP16
fusion proteins in HeLa cells, hHSF1, hHSF2 and hHSF4b
were cloned into pK543, a derivative of pcDNA3.1(+) con-
taining an activation domain of herpes simplex virus VP16
(amino acids 413–490). Derivatives of hHSF4–VP16 were
created by using standard methods. The reporter gene
HSE–SV40p–LUC contained an HSE oligonucleotide (see
Fig. 1A) upstream of the SV40 promoter–firefly luciferase
gene fusion (SV40p–LUC) of pGL3-Promoter vector (Pro-
mega, Madison, WI, USA). The promoter regions of the
human cA-crystallin, cB-crystallin, cC-crystallin and cD-
crystallin genes were cloned upstream of the luciferase gene

HSE oligonucleotide for 20 min at 37 or 43 °C. The
samples were electrophoresed on a 3.5% polyacrylamide gel
at room temperature, and subjected to phosphorimaging as
described previously [9,10].
Oligomer formation of polypeptides was analyzed by
chemical crosslinking with EGS [9]. In vitro-synthesized
polypeptides (1.0–1.5 lL, 4.0 ng of protein) in 5 lLof
13 mm Tris ⁄ Cl (pH 7.6) and 100 mm NaCl were incubated
without or with 1.0 and 3.0 mm EGS for 20 min at room
temperature. The reaction was quenched by the addition of
glycine to 75 mm. Samples were subjected to SDS ⁄ PAGE
and immunoblot analysis using an antibody against VP16
(Abcam, Cambridge, UK).
Yeast strains, immunoblot analysis, and RT-PCR
Yeast strain HS126 (MATa ade2 his3 leu2 trp1 ura3 can1
hsf1::HIS3 YCp-URA3–yHSF) contains a null mutation of
the chromosomal yHSF gene and bears wild-type yHSF on
a URA3-marked centromeric plasmid [10]. For construction
of strains HS170T, YYT49, YYT42, YYT50, and YYT17,
HS126 was transformed respectively with YCp-TRP1–
yHSF, YCp-TRP1–P
ADH1
–hHSF2–T
ADH1
, YEp-LEU2–
P
ADH1
–hHSF2–T
ADH1
, YCp-TRP1–P

2
atmosphere. Cells grown in 12-well plates
were transfected using HilyMax (Dojindo Laboratories,
Kumamoto, Japan), with DNA mixtures including 100 or
200 ng of firefly luciferase reporter plasmid, 10 ng of pRL-
TK control plasmid containing the Renilla luciferase gene
driven by the HSV-TK promoter (Promega), 10 or 100 ng of
hHSF–VP16 expression plasmid, and sufficient carrier
pcDNA3.1(+) to bring the total amount of DNA to 1.6 lg.
Cells were cultured for 20–24 h following transfection, and
firefly and Renilla luciferase activities were measured using
the Dual-Luciferase Reporter Assay System (Promega) and a
luminometer (AB-2200-R; ATTO Co., Tokyo, Japan). The
Renilla luciferase activity of each sample was used to nor-
malize firefly luciferase for transfection efficiency.
The expression of hHSF–VP16 fusion proteins in trans-
fected cells was analyzed as follows. Cells were lysed in
buffer containing 50 mm Tris ⁄ Cl (pH 8.0), 150 mm NaCl,
1% Triton X-100, 0.5 mm phenylmethanesulfonyl fluoride,
and protease inhibitor cocktail (Nakarai Tesque, Kyoto,
N. Yamamoto et al. HSE-type specific recognition by human HSFs
FEBS Journal 276 (2009) 1962–1974 ª 2009 The Authors Journal compilation ª 2009 FEBS 1971
Japan). After centrifugation at 20 000 g for 10 min, the
supernatant (100 lg of protein) was subjected to
SDS ⁄ PAGE and immunoblot analysis using an antibody
against VP16 (Abcam).
Acknowledgements
We thank A. Nakai for providing the cDNA clones
and antibodies. This work was supported in part by
Grants-in-Aid for Scientific Research from the Minis-

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Supporting information
The following supplementary material is available:
Fig. S1. Electrophoretic mobility shift assay of hHSF–
HSE complexes.
Fig. S2. Electrophoretic mobility shift assay of hHSF–
VP16 polypeptides.
This supplementary material can be found in the
online version of this article.
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HSE-type specific recognition by human HSFs N. Yamamoto et al.
1974 FEBS Journal 276 (2009) 1962–1974 ª 2009 The Authors Journal compilation ª 2009 FEBS