Tài liệu Báo cáo khoa học: The heat shock factor family and adaptation to proteotoxic stress pdf

MINIREVIEW
The heat shock factor family and adaptation to
proteotoxic stress
Mitsuaki Fujimoto and Akira Nakai
Yamaguchi University School of Medicine, Ube, Japan
Introduction
All living organisms respond to elevated temperatures
by producing a set of highly conserved proteins,
known as heat shock proteins (HSP) [1]. This response
is called the heat shock response, and is a universal
mechanism of protection against proteotoxic stress,
including heat shock and oxidative stress. In Escheri-
chia coli, heat shock genes are under the control of a
specific transcription factor, r32, which directs the
core RNA polymerase to promoters [2]. In eukaryotes,
the heat shock response is regulated mainly at the level
of transcription by heat shock factors (HSFs) [3]. Heat
shock genes, such as HSP110, HSP90, HSP70, HSP40
and HSP27, contain heat shock elements (HSEs) com-
posed of at least three inverted repeats of the highly
conserved consensus sequence nGAAn in the proximal
promoter region [4]. Here we call them ‘classical heat
shock genes’, which encode major HSPs or molecular
chaperones. Heat shock triggers the conversion of an
HSF1 monomer in a metazoan species that is nega-
tively regulated by HSPs into a trimer that binds to
Keywords
evolution; heat shock; protein homeostasis;
protein-misfolding disorder; transcription
factor; vertebrate
Correspondence

Akira Nakai, Department of Biochemistry
and Molecular Biology, Yamaguchi
University School of Medicine, Minami-
Kogushi 1-1-1, Ube 755-8505, Japan
Fax: 81 836 22 2315
Tel: 81 836 22 2214
E-mail:
(Received 10 May 2010, revised 7 July
2010, accepted 23 July 2010)
doi:10.1111/j.1742-4658.2010.07827.x
The heat shock response was originally characterized as the induction of a
set of major heat shock proteins encoded by heat shock genes. Because
heat shock proteins act as molecular chaperones that facilitate protein fold-
ing and suppress protein aggregation, this response plays a major role in
maintaining protein homeostasis. The heat shock response is regulated
mainly at the level of transcription by heat shock factors (HSFs) in eukary-
otes. HSF1 is a master regulator of the heat shock genes in mammalian
cells, as is HSF3 in avian cells. HSFs play a significant role in suppressing
protein misfolding in cells and in ameliorating the progression of Caenor-
habditis elegans, Drosophila and mouse models of protein-misfolding
disorders, by inducing the expression of heat shock genes. Recently, numer-
ous HSF target genes were identified, such as the classical heat shock genes
and other heat-inducible genes, called nonclassical heat shock genes in this
study. Importance of the expression of the nonclassical heat shock genes
was evidenced by the fact that mouse HSF3 and chicken HSF1 play a sub-
stantial role in the protection of cells from heat shock without inducing
classical heat shock genes. Furthermore, HSF2 and HSF4, as well as
HSF1, shown to have roles in development, were also revealed to be neces-
sary for the expression of certain nonclassical heat shock genes. Thus, the
heat shock response regulated by the HSF family should consist of the

induction of classical as well as of nonclassical heat shock genes, both of
which might be required to maintain protein homeostasis.
Abbreviations
BRG1, brahma-related gene 1; DAF-16, abnormal dauer formation 16; HR, hydrophobic heptad repeat; HSE, heat shock element; HSF, heat
shock factor; HSP, heat shock protein; MEF, mouse embryonic fibroblast; polyQ, polyglutamine.
4112 FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS
the HSE with high affinity, and the bound HSF1 rap-
idly induces a robust activation of the classical heat
shock genes [5,6].
There is a single gene encoding HSF in yeast, in
Caenorhabditis elegans and in Drosophila. HSF is
required not only for the heat shock response, but also
for cell growth and differentiation in yeast [7]. In verte-
brates, there are multiple HSF genes, which encode
members of the HSF family (HSF1–4). In mammals,
as in yeast and Drosophila, the HSF1 is required for
the heat shock response, whereas HSF3 is required for
this response in avian species [8,9]. Both mouse HSF1
and chicken HSF3 are necessary for thermotolerance,
at least through the expression of classical heat shock
genes [10,11]. In addition to their role in the heat
shock response, mouse HSFs are critical in develop-
mental processes such as gametogenesis and neuro-
genesis, in the maintenance of sensory and ciliated
tissues, and in immune responses [12–14]. HSF1- and
HSF3-mediated mechanisms of cellular adaptation to
heat shock have been analyzed in detail in chicken
cells, and were considered specific to chicken cells as it
was believed until recently that HSF3 was an avian-
specific factor. In this minireview, we summarize the

evolution of the HSF gene family and HSF-mediated
mechanisms of cellular adaptation to stress in verte-
brates by comparing mammalian and avian cells, and
also review HSF-mediated mechanisms of adaptation
to pathological states related to protein misfolding.
Evolution of the vertebrate HSF gene
family
An HSF protein that binds to the HSEs in the HSP
genes was purified from heat shock-induced Saccharo-
myces cerevisiae, Drosophila and human cells [3]. Anti-
bodies against HSF were used to isolate a single copy
of the S. cerevisiae HSF gene [15–17]. Thereafter, a
single HSF gene was isolated from another budding
yeast Kluyveromyces lactis, and from the fission yeast
Schizosaccharomyces pombe by cross-hybridization
[18,19]. A single copy of the Drosophila HSF was iso-
lated by screening a library with oligonucleotide
probes derived from HSF peptide sequencing. In mam-
mals, human HSF1 and a second HSF gene, HSF2,
were isolated by screening a library with degenerate
oligonucleotide probes [20,21], and the mouse HSF1
and HSF2 genes were isolated by cross-hybridization
with a human HSF1 cDNA probe [22]. In chicken,
HSF1, HSF2 and a third HSF gene, HSF3, were iso-
lated by cross-hybridization with a mouse HSF1
cDNA probe [23]. Furthermore, another HSF gene,
HSF4, was isolated from human and mouse cells by
the screening of human and mouse cDNA libraries
with a chicken HSF3 cDNA probe [24,25], but a mam-
malian orthologue of the chicken HSF3 gene was not

identified. Therefore, HSF3 was considered specific to
avian species, and HSF4 was considered specific to
mammalian species [5,8,9,12].
Although human and mouse genome sequences have
become available [26,27], no HSF3-related sequence
was identified in silico from the genome database [28].
However, analysis of the chicken genome enabled com-
parison of the syntenic regions [29], where the same
genes occur in a similar order along the chromosomes
of different organisms [30]. For example, HSF2 was
flanked by the SERINCI gene in human, mouse and
chicken orthologous segments (Fig. 1) [31]. Likewise,
the chicken HSF3 gene was located between Vsig4 and
HEPH on chromosome 4, and orthologous segments
containing the two genes were found on the human
and mouse X chromosome. Sequencing of a region
between the two genes revealed the mouse HSF3 gene.
Although sequences related to HSF3 were also
observed in an orthologous region of the human gen-
ome, this genomic segment is likely to be an HSF3
pseudogene as no transcript was identified [31]. Fur-
thermore, HSF4 was located in a region between the
TRADD–FBXL8 genes and the NoL3 gene in the
human and mouse genomes, and chicken HSF4 was
identified in an orthologous segment [31].
Comparison of the predicted amino acids of four
members of the vertebrate HSF family revealed that
sequences of the DNA-binding and trimerization
[hydrophobic heptad repeat (HR)-A ⁄ B] domains are
well conserved (Fig. 2) [31]. The identity of the amino

acid sequence in the DNA-binding domain of mouse
HSF1 was much lower for mouse HSF3 (53%) than
for mouse HSF2 or HSF4 (70% and 76%, respec-
tively). Furthermore, the amino acid sequence of the
DNA-binding domain of mouse HSF3 was only 60%
homologous to that of chicken HSF3, whereas the
sequences of mouse HSF1, HSF2 and HSF4 were
much more identical to the corresponding domains of
chicken HSF1, HSF2 and HSF4 (92%, 86% and 71%
identity, respectively). Moreover, a phylogenetic tree,
which was generated from full-length amino acid
sequences of the HSF family, showed the relatedness
of mouse HSF3 with chicken HSF3 to be much
weaker than that of mouse HSF1 with chicken HSF1,
that of mouse HSF2 with chicken HSF2, or even that
of mouse HSF4 with chicken HSF4 (Fig. 3). These
estimations indicate that the nucleic acid sequences of
HSF3 diverged most quickly during evolution, whereas
those of HSF1 and HSF2 were similarly conserved.
The phylogenetic tree also demonstrates the amino
M. Fujimoto and A. Nakai Evolution and function of the HSF family
FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS 4113
acid sequence of HSF1 to be most closely related with
that of HSF4 among the HSF family, and the amino
acid sequence of HSF2 to be most closely related with
that of HSF3. These findings are consistent with the
assertion that two rounds of whole-genome duplication
occurred in the vertebrate lineage (Fig. 4) [32–34].
Alignment of the human and chicken HSF genes with
the mouse HSF gene showed that sequences of the ex-

ons are well conserved, whereas those of introns are
not [31], suggesting that four duplicated HSF genes
have been conserved during evolution under selective
pressure, except for human HSF3.
Expression of classical heat shock
genes induced by two heat-responsive
HSFs
After the identification of mammalian HSF1 and
HSF2 [20–22], and of chicken HSF1, HSF2 and HSF3
[23], research was conducted to reveal the factors
responsible for the heat-inducible HSE-binding activ-
ity. In mammalian cells, HSF1 remains an inert mono-
mer in unstressed cells and forms a trimer that binds
to the HSE in response to heat shock [35,36], whereas
the HSE-binding activity of HSF2 is induced during
hemin-induced differentiation of erythroleukemia
cells and is constitutively high during early mouse
development [37–39]. In chicken cells, both an HSF1
monomer and an HSF3 dimer were converted to
homotrimers that bind to the HSE under heat shock
[40]. The disruption of HSF genes in mouse embryonic
fibroblasts (MEFs) clearly demonstrated that mouse
HSF1 is required for the expression of classical heat
shock genes [10], whereas mouse HSF2 is not [41].
Unexpectedly, disruption of chicken HSF3 in chicken
B-lymphocyte DT40 cells resulted in a severe reduction
in the inducible expression of HSP70, and the expres-
sion of HSP110, HSP90a, HSP90b and HSP40 were
not induced at all in chicken HSF3-null cells [11].
These observations imply that duplicated HSF genes

Mouse Chr.15
DGAT1
HSF1
BOP1
SCRT1
Chicken Chr.2
HSF1
HSF1
10 kb
Human Chr.8
Mouse Chr.15
DGAT1
HSF1BOP1
SCRT1
HSF1
BOP1
SERINC1
HSF2
HSF2 SERINC1
Human Chr 6
Mouse Chr.10
Chicken Chr.3
HSF2
SERINC1
HSF2
SERINC1
Chicken Chr.4
HSF3
HEPH
Vsig4

10 kb
20 kb
HSF2 SERINC1
Human Chr.
6
HSF3
Human Chr.X
HEPH
Vsig4
Mouse Chr.X
HSF3
Vsig4
100 kb
100 kb
HSF3
HEPH
Chicken Chr.11
TRADD FBXL8
HSF4
NL3
HSF4
Mouse Chr.8
NoL3TRADD FBXL8
HSF4
Human Chr.16
N
o
L3
HSF4
10 kb

Fig. 1. Comparative genomic analysis of
orthologous segments containing vertebrate
HSF genes. The location of each segment is
as follows: human Chr.8 q24.3 and mouse
Chr.15 D3 for HSF1; human Chr.6 q22.31,
mouse Chr.10 B4 and chicken 63.95–
63.98 Mb for HSF2; human Chr. X q12,
mouse Chr. X B4 and chicken 0.252–
0.265 Mb for HSF3; and human Chr.16
q22.1, mouse Chr.8 D3 and chicken 2.44–
2.45 Mb for HSF4. A genomic sequence
corresponding to chicken HSF1 cDNA has
not yet been identified. Arrows indicate the
5¢ to 3¢ orientation of each gene. The
chicken HSF1 gene is located on chromo-
some 2, which is present in three copies in
DT40 cells [42]. The gray box in human
chromosome X is probably an HSF3
pseudogene.
Evolution and function of the HSF family M. Fujimoto and A. Nakai
4114 FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS
evolved differently in mammalian and avian species
(Fig. 4).
As the amino acid sequence of HSF1 is highly con-
served in mammalian and chicken cells, the functional
difference was examined in more detail. In fact,
chicken HSF1 is dispensable for the expression of the
classical heat shock genes in DT40 cells [42], and the
ectopic expression of chicken HSF1 in MEF cells defi-
cient in mouse HSF1 does not restore the inducible

expression of the classical heat shock genes [28].
Thus, chicken HSF1 lacks the ability to induce the
expression of classical heat shock genes, whereas
mouse HSF1 is a master regulator of these genes.
Interestingly, the amino-terminal region of chicken
HSF1 containing an alanine-rich sequence and the
DNA-binding domain is sufficient to cause the func-
tional difference between the two orthologues [28]. As
chicken HSF1 can bind to the HSE, its amino-terminal
domain might inhibit exposure of the activation
domain to basal transcriptional machinery. Alterna-
tively, the corresponding domain of mouse HSF1, but
not that of chicken HSF1, could recruit components
required for gene activation.
Recent identification of mouse HSF3 enabled us to
examine the functional difference of HSF3 in mouse
and chicken cells [31]. In cells exposed to heat shock,
mouse HSF3 fused to green fluorescent protein moved
into the nucleus, similarly to chicken HSF3 [40], indi-
cating that both chicken and mouse HSF3 are heat-
responsive factors. Furthermore, overexpression of
DBD HR-A/B HR-
C
DHR
hHSF1
hHSF2
5291 16 120 130 203 384 409
415
446
5361 8 112 119 192 360 385

391
422
649407232225
Human
XY
mHSF1
mHSF2
18 2446 224970
hHSF4
76
100
39
6999
28
79
34
97
24
83
mHSF3
mHSF4
HSF1
16 24153353
30 21313976
58 678592
92
82
Mouse
cHSF1
cHSF2

cHSF3
58 678592
92
82
1342
4647 7
2
76
25 193849 2206
Chicken
cHSF4
30 23
285384
CeHSF1
DmHSF
53
59
58
31
30
hHSFY1
31
ScHSF
CeHSF1
53
31
46
32
hHSFX1
20

hHSF5
37
4921 19 123 130 203 396 427
5251
16
120 130 203 380 405
411
442
5351 8 112 119 192 359 384
390
421
4921 10 114 121 194 355
380
4921 18 122 129 202 395 426
4911 21 125 135 208 346 371
376
408
564
1 22 126 133 206 390 415
421
453
4671 17 121 129 202 364 389
395
426
5101 18 122 130 203 393 424
147 150
691161 232
1 90 194 671210 275
1173
833

276 347
424
1 80 198 401
1 37 155 423
1 14 129 596
Fig. 2. Members of the HSF superfamily. Diagrammatic representation of vertebrate and nonvertebrate HSF family members and of human
HSF-related gene products. The percentage identity between human HSF1 and each HSF was established using the computer program
GENETYX-MAC. The number of amino acids of each HSF is shown at the amino-terminal end. c, chicken; Ce, Caenorhabditis elegans ; DHR,
downstream of HR-C; DBD, DNA-binding domain; DHR, downstream of HR-C; Dm, Drosophila melanogaster; h, human; HR, hydrophobic
heptad repeat; m, mouse; Region X, a region upstream of the HR-C domain; Region Y, a C-terminal region downstream of the HR-C-like
domain; Sc, Saccharomyces cerevisiae. hHSF1 (hHSF1-a) [20]; hHSF2 (hHSF2-a) [21]; hHSF4 (hHSF4b) [24]; mHSF1 (hHSF1-a) and mHSF2
(hHSF2-a) [35]; mHSF3 (mHSF3a) [31]; mHSF4 (mHSF4b) [25]; cHSF1, cHSF2 and cHSF3 [23]; cHSF4 (cHSF4b) [31]; DmHSF [Wu 1990];
CeHSF1 (Swiss-P accession no. Q9XW45); ScHSF [Pelham; Parker 1988]; hHSFY1 (SP accession no. Q96LI6) [106,107]; hHSFX1 ⁄ LW-1 (SP
accession no. Q9UBD0); hHSF5 (SP accession no. Q4G112). The hatched box indicates an HR-C-like domain, in which hydrophobic amino
acids are not well conserved. The DBD domain of HSF family members is conserved with one region in hHSFY1, hHSFX1 and hHSF5 that
may not bind to the HSE.
M. Fujimoto and A. Nakai Evolution and function of the HSF family
FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS 4115
chicken HSF3 in HSF1-null MEF cells induced the
constitutive and heat-induced expression of classical
heat shock genes. In marked contrast, overexpression
of mouse HSF3 in the same cells did not affect the
expression of classical heat shock genes at all, even
after heat shock. Therefore, mouse HSF3 lacks the
ability to induce the expression of classical heat shock
genes, whereas chicken HSF3 is a master regulator.
Why does mouse HSF3 fail to induce the expression
of classical heat shock genes? It was revealed, by exam-
ining a DNA-binding transcription factor required for
the activation of the GAL genes in response to galac-

tose (GAL4) site-directed luciferase activity, that mouse
HSF3 has strong potential to induce transcription [31].
Deletion analysis showed that the activation domain of
mouse HSF3 is located in its C-terminal region down-
stream of the HR-C-like domain (region Y) whereas
that of chicken HSF3 is located upstream of the HR-C
domain (region X) (Fig. 2). The amino acid sequence
of the activation domain of mouse HSF3 is not con-
served in chicken HSF3, which is consistent with a
functional divergence of the activation domain during
evolution. Domains of mouse HSF3 were swapped with
the corresponding domains of chicken HSF3, and the
chimeras possessing the chicken HSF3 activation
domain induced the expression of the classical heat
shock genes in response to heat shock. In contrast, the
chimeras possessing only the mouse HSF3 activation
domain did not induce their expression. Furthermore,
the C-terminal activation domain of human HSF1
[43–45] was swapped with the mouse HSF3 activation
domain, and the resultant protein did not induce gene
expression in response to heat shock. These results
indicate that the activation domain of mouse HSF3
does not have the potential to activate the classical
heat shock genes.
Human HSF1 recruits brahma-related gene 1
(BRG1), a component of switch ⁄ sucrose nonferment-
ing (SWI ⁄ SNF) chromatin remodeling complexes, to
the HSP70 promoter through direct interaction [46],
and expression of an HSF1 mutant, which cannot
interact with BRG1, did not restore the induction of

HSP70 mRNA expression in HSF1-null MEF cells
during heat shock [47,48]. It was revealed that mouse
HSF3 does not bind to BRG1, or recruit BRG1 to the
HSP70 promoter [31], whereas chicken HSF3 does
bind to and recruit BRG1. These observations indicate
that mouse HSF3 does not induce the expression of
the classical heat shock genes, at least in part because
of its inability to interact with BRG1.
HSF-mediated adaptation to thermal
stress
Heat shock induces both apoptotic and necrotic cell
death, but the pathways of cell death and the factors
hHSF5
hHSFY1
hHSFX1
mHSF3
cHSF3
mHSF2
cHSF2
674
991
1000
1000
1000
1000
602
1000
1000
1000
1000

1000
839
908
0.05
684
HSF3
HSF2
HSF4
HSF1
hHSF2
cHSF4
mHSF4
hHSF4
ScHSF
mHSF1
hHSF1
cHSF1
CeHSF1
DmHSF
Fig. 3. The phylogenetic tree generated in CLUSTAL W [108] for members of the HSF family. Gaps were excluded from all phylogenetic
analyses. The numerals represent bootstrap values (1000 bootstrap replicates were performed). The unrooted tree was drawn using the pro-
gram TREEVIEW [109]. The bar represents 0.05 substitutions per site. Amino acid sequences of the HSF family were shown previously
[28,31].
Evolution and function of the HSF family M. Fujimoto and A. Nakai
4116 FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS
that are primarily impaired are not clear, as heat
shock causes various types of stress, including proteo-
toxic stress. Cells in different states of metabolism and
in different stages of differentiation may be induced to
die by different mechanisms, and there must be vari-

ous targets of extremely high temperatures that induce
cell death. Therefore, HSPs should not be the only
proteins that protect against cell death. Nevertheless,
HSPs are recognized as major players in the protec-
tion of cells from heat shock, especially from proteo-
toxic stress [1,2]. As the expression of a set of HSPs is
regulated by HSFs, HSFs should be involved in the
protection of cells from heat shock or proteotoxic
stress [49].
It is well established that cells pretreated with suble-
thal heat shock can survive lethal heat shock. This
phenomenon is called induced thermotolerance, and is
regulated by mouse HSF1 and chicken HSF3 through
the activation of the heat shock genes [10,11]. HSPs
prevent the denaturation and aggregation of cellular
proteins, and support their renaturation when the cells
are recovering [1]. At the same time, HSPs inhibit sev-
eral molecules, such as apoptotic peptidase activating
factor 1 (Apaf-1) and cytochrome c, which are
involved in mitochondria-mediated apoptotic pathways
[50].
Both HSF1 and HSF3 complementarily regulate the
constitutive expression of some HSPs in normally
growing chicken DT40 cells [11,42]. In these cells, a
lack of the two factors resulted in increased sensitivity
to a single exposure to high temperature because of
reduced Hsp90a expression, and the cell cycle is
blocked at the G
2
phase [42]. A similar phenotype was

observed in yeast S. cerevisiae harbouring a mutant
HSF [51,52]. Mouse HSF1 also regulates the expres-
sion of some HSPs, including Hsp90, in various mouse
tissues [53–56]. Therefore, HSFs could be involved in
determining a temperature at which cells can survive
by regulating the constitutive expression of HSPs such
as Hsp90.
Curiously, in chicken cells, HSF1 induced only very
low levels of expression of the classical heat shock
genes, but had a significant effect on the protection of
cells from heat shock [28]. This effect was not medi-
ated through the induction of classical heat shock
genes or regulation of the constitutive expression of
heat shock genes such as Hsp90. HSF1-null MEF cells,
which lack induced expression of the classical heat
shock genes, are more sensitive to high temperatures
than wild-type cells. Remarkably, overexpression of
chicken HSF1 in the HSF1-null MEF cells restored
resistance to heat shock [28]. Moreover, mouse HSF3
HSF1
2RWGD
Mouse cell
HSF1
Non-WGD
HSP induction
High expression
HSF2
HSF4
HSF3
Ancestor cell

in the lens
Polyploid
Chicken cell
HSF3
HSF4
HSF4
HSF2
HSF1
HSF1
> 310 Myr ago
High expression
in the lens
HSF3
HSP induction
HSF2
Fig. 4. A model to explain the evolution of HSF genes. Two rounds of whole-genome duplication (WGD) may have occurred in vertebrate
ancestral cells more than 440 million years ago (Ma) [110,111], which resulted in polyploidization. Thereafter, avian and mammalian cells
evolved differently from an ancestral cell 310 Ma. The expression and function of the four HSF genes were conserved or diverged during
evolution [112]. For example, during mammalian evolution, HSF1 retained the ability to induce the expression of heat shock proteins,
whereas it lost this function during avian evolution. Instead, avian HSF3 retained the function. Expression of the HSF4 gene increased in the
lens during both avian and mammalian evolutions (M. Fujimoto and A. Nakai, unpublished). Diamonds, circles and triangles represent regula-
tory regions driving expression in different tissues.
M. Fujimoto and A. Nakai Evolution and function of the HSF family
FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS 4117
was able to protect HSF1-null MEF cells from heat
shock without inducing the expression of the classical
heat shock genes [31]. These observations indicate that
chicken HSF1 and mouse HSF3 protect cells from
thermal stress by regulating the expression of heat-
inducible genes other than classical heat shock genes.

Expression of nonclassical heat shock
genes induced by HSFs
Although the heat shock response was originally char-
acterized based on the expression of a limited number
of classical heat shock genes, HSF1 was recently
revealed to regulate the expression of numerous other
genes in the absence or presence of heat shock. Com-
prehensive analyses of HSF-binding regions in the
whole genome revealed that  3% of genes are direct
targets in heat-shocked cells in yeast and Drosophila
[57,58], and expression of the majority of the target
genes is induced during heat shock in unicellular yeast
[57]. Even in mammalian cells, HSF1 (similarly to
yeast and Drosophila HSF) binds to the promoters of
a great number of genes in the whole genome [59,60],
and about half of the target genes are expressed during
heat shock [59]. We now refer to these heat-inducible
genes that are different from the classical heat shock
genes as ‘nonclassical heat shock genes’. As already
discussed, chicken HSF1 and mouse HSF3 play a sub-
stantial role in the protection of cells from heat shock
[28,31], implying significance of the nonclassical heat
shock genes in this process.
To establish whether the expression of nonclassical
heat shock genes was induced by mouse HSF3 or
chicken HSF1, nonclassical heat shock genes were
identified in MEF cells [31]. Induction of one of the
nonclassical heat shock genes, the gene for the PSD-95 ⁄
Dlg-A ⁄ ZO-1 (PDZ) domain-containing protein
PDZK3 ⁄ PDZD2 ⁄ PAPIN (plakophilin-related armadillo

repeat protein-interacting protein) [61], decreased, but
was still observed in HSF1-null MEF cells during heat
shock [31]. Overexpression of mouse HSF3 or chicken
HSF1 in the HSF1-null MEF cells restored the marked
induction of expression of PDZK3, whereas knock-
down of mouse HSF3 completely abolished the induc-
tion. Induction of another nonclassical heat shock
gene, that for a membrane glycoprotein, prominin-2
(PROM2) [62], was abolished in HSF1-null MEF cells,
but was restored when mouse HSF3 or chicken HSF1
was overexpressed [31]. These observations clearly
demonstrated that evolutionally conserved mouse
HSF3 and chicken HSF1 uniquely regulate only non-
classical genes, suggesting importance of the regulation
of the nonclassical heat shock genes.
It is worth noting that HSF4 also regulates nonclas-
sical heat shock genes in lens cells although it is not a
heat-responsive factor. A set of HSF4-binding regions
was identified in lens cells, and the expression of genes
located in and near these regions was examined [63].
Interestingly, a great number of the genes (33%) were
expressed in response to heat shock, and, unexpect-
edly, the expression of these genes was not induced in
HSF1-null lens cells. Surprisingly, HSF4 was required
for the expression of half of the genes, in part by facil-
itating the binding of HSF1 to the promoters [63].
Moreover, the expression of satellite III repeat
sequences during heat shock was extensively studied
[64,65]. HSF1 is required for expression of the satellite
III gene during heat shock, but HSF2 also greatly

affects its expression, possibly by interacting with
HSF1 [66]. Taken together, all members of the verte-
brate HSF family are involved in the regulation of
gene expression during heat shock (Fig. 5).
HSF is essential in yeast and human
cancer cells
In the budding yeast S. cerevisiae, HSF is essential for
survival under normal conditions [15,16], consistent
with the notion that S. cerevisiae HSF is constitutively
Classical heat shock genes Nonclassical heat shock genes
HSF2
HSF1
HSF1
HSF4
HSF3
HSE
HSE
HSPs
PDZK3
PROM2
Sat III
etc
Protein homeostasis
?
Adaptation to proteotoxic stress
Fig. 5. Adaptation to proteotoxic stress by the HSF family in mice.
HSF1 remains mostly as an inert monomer in unstressed cells, and
is converted to an active trimer that binds to the HSE located in
the proximal promoter region of a limited number of classical heat
shock genes during heat shock, which results in induction of the

expression of HSPs. HSF2 may modulate this process by interact-
ing with HSF1 [6,113]. Members of the HSF family coordinately
bind to the less-conserved HSE located on and near numerous non-
classical heat shock genes, and greatly affect heat-induced expres-
sion of the genes including PDZK3, PROM2 and satellite III
[31,63,66]. HSF3 is not expressed in human cells.
Evolution and function of the HSF family M. Fujimoto and A. Nakai
4118 FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS
a trimer that binds to the HSE. However, the binding
of HSF to the promoter of a heat shock gene markedly
increased during heat shock in S. cerevisiae in vivo [67].
Furthermore, HSF was essential even in the fission
yeast S. pombe, in which HSF forms a trimer only
under stress, similar that observed for vertebrate HSF1
[19]. These observations implied that a balance of the
monomer and trimer HSF affects the amount of HSF
bound to the HSE in vivo, but even a small amount of
the trimer could regulate the gene expression, which is
required for survival under normal growth conditions
in unicellular yeasts. In fact, HSF binds to many target
genes in vivo, and their products have a broad range of
biological functions, including protein folding and deg-
radation, energy generation and protein trafficking
[57]. Human HSF2, but not HSF1, forms a trimer and
functionally complements the viability defect of yeast
cells lacking HSF, and both human HSF1 and HSF2
partially rescue the induction of heat-inducible genes,
which is associated with acquired thermotolerance [68].
These observations suggest that the roles of HSF
under normal growth conditions can be distinguished

from those under stress.
Among multicellular organisms, HSF-null Drosophila
was the first to be generated, although a single HSF
was required for oogenesis and early larval develop-
ment, indicating that HSF is dispensable for cell
growth and survival under normal conditions [69].
Subsequently, HSF1-, HSF2- and HSF4-deficient mice
were generated, indicating that these HSFs are also
dispensable [41,70–73]. Detailed analysis of these mice
demonstrated that meiosis was impaired in the absence
of HSF1 and HSF2 in female [53,74] and male [71,75–
77] germ cells. Neuronal differentiation and migration
were affected in the HSF2-null cerebral cortex [78].
Furthermore, the differentiation and maintenance of
sensory placodes required HSF1 and HSF4
[13,54,72,73]. Thus, members of the HSF family exert
essential activities in the absence of stress and are
required for the differentiation of many types of cells
during development [14]. However, they are not neces-
sary for cell proliferation and survival under normal
conditions in multicellular organisms.
Cancer cells proliferate and survive in different ways
from normal cells, and many of the signalling path-
ways and transcription factors display a striking
dependence on the chaperone machinery [79,80]. Fur-
thermore, HSF1 expression is elevated in human can-
cer cells [81,82], suggesting that cancer cells may be
dependent on the heat shock response. Therefore, the
effects of loss of HSF1 function on cancer cell prolifer-
ation and survival were examined. First, human cervi-

cal epithelial HeLa cells stably expressing short hairpin
RNA for HSF1 were generated (these cells show >
95% reduction in the HSF1 level), and were highly
sensitive to combined treatment with both elevated
temperature and anticancer reagents [83]. Then,
decreased lymphomagenesis in a p53-deficient mouse
model was shown in the absence of HSF1 [84]. Unex-
pectedly, in addition to being required for carcinogene-
sis in mice, HSF1 is required for proliferation and
survival in various human cancer cell lines, including
HeLa cells, but not in normal or immortalized cells
[85]. This observation suggests the possibility of com-
mon HSF-mediated mechanisms for cell proliferation
and survival in cancer cells and in yeast cells.
Is the HSF1 in cancer cells activated? As HSF1
expression, which is correlated with HSE-binding
activity [35], is elevated in human cancer cells [81,82],
the HSE-binding activity of HSF1 might be higher in
cancer cells than in normal cells. Even so, the HSE-
binding activity is robustly induced in response to heat
shock in cancer cells, such as HeLa cells, compared
with normal cells [35,36] or in fission yeast cells [19].
HSF1 is involved in regulating translation, ribosome
biogenesis and glucose metabolism in cancer cells [85],
and is also required for the expression of numerous
genes, including those for inflammatory cytokines,
chemokine-related genes and interferon-related genes,
even in normally growing primary cultures of MEF
cells [86]. Furthermore, the ability of HSF1 to form a
trimer is required for the gene expression [87]. Taken

together, only a little amount of HSF1 trimer regulates
the gene expression in normal cells, which is not
required for cell growth and survival. However, the tri-
meric HSF1 is increased in cancer cells and may regu-
late the expression of genes, which is indispensable for
cell growth and survival, as in fission yeast cells for
example [19].
Adaptation to misfolding-related
pathological conditions
An imbalance of protein homeostasis is associated with
aging and age-related pathological conditions such as
neurodegenerative disorders, including Alzheimer’s dis-
ease, Parkinson’s disease, amyotrophic lateral sclerosis,
prion disease and polyglutamine diseases. These diseases
are characterized by conformational changes in disease-
causing proteins that results in misfolding and aggrega-
tion, and are therefore termed protein-misfolding
disorders or protein conformational disorders [49,88].
Polyglutamine (polyQ) diseases are caused by an expan-
sion of CAG repeats, coding for glutamine, in their
respective proteins. Misfolding and aggregation of
aggregation-prone polyQ proteins results in cellular
M. Fujimoto and A. Nakai Evolution and function of the HSF family
FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS 4119
toxicity. Gain of HSF1 function significantly inhibits
the aggregation of polyQ protein and prolongs life span
in C. elegans models of polyQ diseases, whereas loss of
HSF1 function accelerates the aggregation of polyQ and
shortens life span [89,90]. It is considered that HSF1
function is mediated through the expression of HSPs

[49,88]. Interestingly, a forkhead box (FOXO) family
transcription factor, DAF-16 (abnormal dauer forma-
tion 16), which is a component of the insulin-like signal-
ling pathway that regulates life span, also inhibits polyQ
aggregates, indicating that aging and protein homeosta-
sis are highly related [89,90]. In a C. elegans model of
Alzheimer’s disease, HSF1 inhibited the formation of
toxic aggregates of an aggregation-prone peptide Ab
(1–42) whereas DAF-16 promoted the formation of less-
toxic high-molecular-weight aggregates [91]. Thus,
HSF1 and DAF-16 regulate distinct pathways that
reduce the toxicity of aggregation-prone proteins.
Among mouse polyQ models, the R6 ⁄ 2 polyQ model
has been extensively studied as it is transgenic only for
the 5¢ end of the human huntingtin gene carrying 115–
150 CAG repeat expansions [92]. The formation of
polyQ aggregates is observed not only in the brain but
also in nonneuronal tissues, including the skeletal mus-
cle, heart, liver and pancreas, in mice [93]. Ubiquitous
overexpression of HSP70 in the R6 ⁄ 2 Huntington’s
model had no effect on the life span or neuronal phe-
notypes of the mice and delayed aggregation only
slightly [94,95]. There is only one HSF1 transgenic
mouse model, in which an actively mutated HSF1 is
expressed in tissues such as the testis, skeletal muscle,
heart and stomach, but not in the brain [75]. Remark-
ably, overexpression of an active HSF1 in nonneuronal
tissues in R6 ⁄ 2 mice crossed with HSF1 transgenic
mice suppressed polyQ aggregates, at least in skeletal
muscle, and markedly extended the life span [96].

Inversely, HSF1 deficiency dramatically shortened the
life span of the prion disease model mice, in which
scrapie prions were inoculated [97], and even resulted
in impaired protein homeostasis of the untreated neu-
ronal cells in some genetic backgrounds [98]. These
observations imply significant beneficial effects of the
overexpression of an active HSF1 on the progression
of protein-misfolding disorders in mice. Interestingly,
mouse HSF3 and chicken HSF1 suppressed the forma-
tion of aggregates in a cellular polyQ model [28,31],
suggesting that the nonclassical heat shock genes as
well as classical heat shock genes play roles in amelio-
rating disease progression.
One therapeutic strategy for protein-misfolding dis-
orders such as polyQ disease would be to elevate the
levels of HSPs that assist normal protein folding and
prevent abnormal folding and aggregation [99]. It was
shown that treatment with arimoclomol, a co-inducer
of HSPs through activating HSF1, delays disease pro-
gression in the amyotrophic lateral sclerosis mouse
model, which overexpresses human mutant Cu ⁄ Zn
superoxide dismutase-1 [100]. HSF1-activating reagents,
17-allylamino-17-demethoxygeldanamycin (17-AAG) and
geranylgeranylacetone (GGA), also ameliorated disease
progression in a Drosophila model of spinocerebellar
ataxias [101] or in a mouse model of spinal and bulbar
muscular atrophy [102,103]. Furthermore, novel small
molecules that activate HSF1 were identified and
shown to ameliorate protein misfolding and toxicity of
polyQ aggregates [104,105]. Thus, activation of HSF1

is actually a promising therapeutic approach for pro-
tein-misfolding disorders.
Conclusions and perspectives
We have learned from the identification and character-
ization of HSF1 and HSF3 in chicken and mouse cells
that the function of these two heat-responsive factors
has diverged greatly during the evolution of vertebrates,
even though the nucleic acid sequence of each has been
well conserved. Importantly, chicken HSF1 and mouse
HSF3 are involved in protecting cells from heat shock
and in maintaining protein homeostasis without induc-
ing the expression of the classical heat shock genes. It
was revealed recently that there are numerous nonclas-
sical heat shock genes, whose expression is induced dur-
ing heat shock in various organisms. Remarkably,
chicken HSF1 and mouse HSF3, as well as mammalian
HSF2 and HSF4, play a role in inducing the expression
of only nonclassical heat shock genes. These observa-
tions suggest the importance of the regulation and func-
tion of the nonclassical heat shock genes. Analysis of
these new findings will help us to understand why the
activation of HSF1 suppresses the progression of pro-
tein-misfolding disorders more than HSPs and should
be beneficial in identifying pathways involved in adap-
tation to proteotoxic stress. Furthermore, these analy-
ses would develop our understanding of the biological
significance of the heat shock response.
Acknowledgements
We thank members of our laboratory for discussions
and Naoki Hayashida for comments on the manu-

script. This work was supported in part by Grants-in-
Aid for Scientific Research and on Priority Area-a
Nuclear System of DECODE, from the Ministry of
Education, Culture, Sports, Science and Technology,
Japan, and by the Yamaguchi University Research
Project on STRESS.
Evolution and function of the HSF family M. Fujimoto and A. Nakai
4120 FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS
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