MINIREVIEW The heat shock factor family and adaptation to proteotoxic stress Mitsuaki Fujimoto and Akira Nakai Yamaguchi University School of Medicine, Ube, Japan 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, MinamiKogushi 1-1-1, Ube 755-8505, Japan Fax: 81 836 22 2315 Tel: 81 836 22 2214 E-mail: anakai@yamaguchi-u.ac.jp (Received 10 May 2010, revised 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 folding 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 eukaryotes 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 Caenorhabditis elegans, Drosophila and mouse models of protein-misfolding disorders, by inducing the expression of heat shock genes Recently, numerous 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 substantial 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 necessary 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 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 Escherichia 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) composed 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 negatively regulated by HSPs into a trimer that binds to 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 M Fujimoto and A Nakai the HSE with high affinity, and the bound HSF1 rapidly 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 vertebrates, 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 developmental processes such as gametogenesis and neurogenesis, 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 avianspecific factor In this minireview, we summarize the evolution of the HSF gene family and HSF-mediated mechanisms of cellular adaptation to stress in vertebrates 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 Saccharomyces cerevisiae, Drosophila and human cells [3] Antibodies 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 isolated by screening a library with oligonucleotide probes derived from HSF peptide sequencing In mammals, 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 isolated by cross-hybridization with a mouse HSF1 cDNA probe [23] Furthermore, another HSF gene, HSF4, was isolated from human and mouse cells by Evolution and function of the HSF family the screening of human and mouse cDNA libraries with a chicken HSF3 cDNA probe [24,25], but a mammalian 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 comparison 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 genome, this genomic segment is likely to be an HSF3 pseudogene as no transcript was identified [31] Furthermore, 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%, respectively) 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 FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS 4113 Evolution and function of the HSF family M Fujimoto and A Nakai HSF1 Chicken Chr.2 DGAT1 SCRT1 HSF1 Mouse Chr.15 Human Chr.8 HSF1 BOP1 DGAT1 BOP1 SCRT1 HSF1 10 kb Chicken Chr.3 HSF2 SERINC1 HSF2 Mouse Chr.10 HSF2 Human Chr Chr.6 SERINC1 HSF2 SERINC1 20 kb Chicken Chr.4 Vsig4 HSF3 HEPH 10 kb Vsig4 HSF3 Mouse Chr.X HSF3 HEPH 100 kb Vsig4 Human Chr.X HSF3 HEPH 100 kb Chicken Chr.11 HSF4 Mouse Chr.8 Human Chr.16 TRADD FBXL8 TRADD FBXL8 HSF4 NoL3 HSF4 HSF4 NoL3 N L3 10 kb 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 exons 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 4114 Fig 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 chromosome 2, which is present in three copies in DT40 cells [42] The gray box in human chromosome X is probably an HSF3 pseudogene responsible for the heat-inducible HSE-binding activity In mammalian cells, HSF1 remains an inert monomer 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 expression of HSP110, HSP90a, HSP90b and HSP40 were not induced at all in chicken HSF3-null cells [11] These observations imply that duplicated HSF genes FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS M Fujimoto and A Nakai Evolution and function of the HSF family DBD hHSF1 Human hHSF2 hHSF4 mHSF1 mHSF2 Mouse mHSF3 mHSF4 cHSF1 HSF1 cHSF2 Chicken cHSF3 cHSF4 HR-A/B 16 120 130 X HRC 203 70 49 112 119 192 76 39 123 130 203 100 99 120 130 203 18 19 16 23 28 79 70 49 112 119 192 18 53 33 114 121 194 16 18 10 76 18 39 122 129 30 92 125 135 208 67 47 126 133 206 22 60 17 121 129 DmHSF 59 47 CeHSF1 202 90 691 31 194 210 275 ScHSF 671 46 173 hHSFY1 564 30 58 150 161 232 53 85 82 67 346 371 408 491 376 24 31 46 27 390 415 453 421 25 38 22 19 364 389 426 467 395 30 23 28 393 424 510 53 122 130 203 18 31 21 395 426 492 58 49 84 Y 96 97 83 380 405 442 525 411 46 22 24 359 384 421 535 390 15 24 355 380 492 202 92 21 DHR 384 409 446 529 415 46 22 25 360 385 422 536 391 34 24 396 427 492 32 276 347 424 833 31 80 hHSFX1 198 401 20 hHSF5 37 155 423 37 14 129 596 Fig 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 evolved differently in mammalian and avian species (Fig 4) As the amino acid sequence of HSF1 is highly conserved 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 deficient 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 functional 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 Alternatively, 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], indicating that both chicken and mouse HSF3 are heatresponsive factors Furthermore, overexpression of FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS 4115 Evolution and function of the HSF family M Fujimoto and A Nakai hHSFY1 hHSFX1 hHSF5 mHSF3 674 cHSF3 991 HSF3 cHSF2 mHSF2 1000 1000 602 cHSF4 1000 1000 mHSF4 1000 839 HSF2 hHSF2 HSF4 hHSF4 1000 cHSF1 mHSF1 1000 908 1000 HSF1 hHSF1 DmHSF 684 CeHSF1 ScHSF 0.05 Fig 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 program TREEVIEW [109] The bar represents 0.05 substitutions per site Amino acid sequences of the HSF family were shown previously [28,31] 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 examining a DNA-binding transcription factor required for the activation of the GAL genes in response to galactose (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 downstream 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 conserved 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 4116 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 (BRG1), a component of switch ⁄ sucrose nonfermenting (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 FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS M Fujimoto and A Nakai Evolution and function of the HSF family Mouse cell HSF1 Non-WGD HSF1 2RWGD HSF4 HSP induction High expression in the lens HSF2 Ancestor cell HSF3 HSF1 Polyploid HSF4 HSF2 Chicken cell HSF3 HSF1 HSF4 > 310 Myr ago High expression in the lens HSF2 HSF3 HSP induction Fig 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 regulatory regions driving expression in different tissues that are primarily impaired are not clear, as heat shock causes various types of stress, including proteotoxic 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 various 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 protection of cells from heat shock, especially from proteotoxic 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 sublethal 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 several molecules, such as apoptotic peptidase activating factor (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 G2 phase [42] A similar phenotype was observed in yeast S cerevisiae harbouring a mutant HSF [51,52] Mouse HSF1 also regulates the expression 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 mediated 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 FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS 4117 Evolution and function of the HSF family M Fujimoto and A Nakai 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 heatinducible genes other than classical heat shock genes Expression of nonclassical heat shock genes induced by HSFs Although the heat shock response was originally characterized 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 Comprehensive 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 substantial 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 knockdown of mouse HSF3 completely abolished the induction 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 nonclassical genes, suggesting importance of the regulation of the nonclassical heat shock genes 4118 It is worth noting that HSF4 also regulates nonclassical 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, unexpectedly, 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 facilitating 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 vertebrate 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 HSE HSPs HSF3 HSE Protein homeostasis PDZK3 PROM2 Sat III etc ? Adaptation to proteotoxic stress Fig 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 interacting with HSF1 [6,113] Members of the HSF family coordinately bind to the less-conserved HSE located on and near numerous nonclassical heat shock genes, and greatly affect heat-induced expression of the genes including PDZK3, PROM2 and satellite III [31,63,66] HSF3 is not expressed in human cells FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS M Fujimoto and A Nakai 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 degradation, 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 development, 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 necessary 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 pathways and transcription factors display a striking dependence on the chaperone machinery [79,80] Furthermore, HSF1 expression is elevated in human cancer 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 proliferation and survival were examined First, human cervical epithelial HeLa cells stably expressing short hairpin Evolution and function of the HSF family 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] Unexpectedly, in addition to being required for carcinogenesis 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 common 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 HSEbinding 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 trimeric HSF1 is increased in cancer cells and may regulate 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 disease, Parkinson’s disease, amyotrophic lateral sclerosis, prion disease and polyglutamine diseases These diseases are characterized by conformational changes in diseasecausing proteins that results in misfolding and aggregation, and are therefore termed protein-misfolding disorders or protein conformational disorders [49,88] Polyglutamine (polyQ) diseases are caused by an expansion of CAG repeats, coding for glutamine, in their respective proteins Misfolding and aggregation of aggregation-prone polyQ proteins results in cellular FEBS Journal 277 (2010) 4112–4125 ª 2010 The Authors Journal compilation ª 2010 FEBS 4119 Evolution and function of the HSF family M Fujimoto and A Nakai 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 formation 16), which is a component of the insulin-like signalling pathway that regulates life span, also inhibits polyQ aggregates, indicating that aging and protein homeostasis 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 lesstoxic 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 ⁄ 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 muscle, heart, liver and pancreas, in mice [93] Ubiquitous overexpression of HSP70 in the R6 ⁄ Huntington’s model had no effect on the life span or neuronal phenotypes 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] Remarkably, overexpression of an active HSF1 in nonneuronal tissues in R6 ⁄ 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 neuronal 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 formation 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 ameliorating disease progression One therapeutic strategy for protein-misfolding disorders 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 4120 shown that treatment with arimoclomol, a co-inducer of HSPs through activating HSF1, delays disease progression 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 protein-misfolding disorders Conclusions and perspectives We have learned from the identification and characterization 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 inducing the expression of the classical heat shock genes It was revealed recently that there are numerous nonclassical heat shock genes, whose expression is induced during 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 observations suggest the importance of the regulation and function 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 protein-misfolding disorders more than HSPs and should be beneficial in identifying pathways involved in adaptation to proteotoxic stress Furthermore, these analyses would develop our understanding of the biological significance of the heat shock response Acknowledgements We thank members of our laboratory for discussions and Naoki 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