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Analysis of HSF4 Binding Regions Reveals Its Necessity for Gene Regulation during Development and Heat Shock Response in Mouse Lenses

2008; Elsevier BV; Volume: 283; Issue: 44 Linguagem: Inglês

10.1074/jbc.m804629200

ISSN

1083-351X

Autores

Mitsuaki Fujimoto, Koji Oshima, Toyohide Shinkawa, Bei Bei Wang, Sachiye Inouye, Naoki Hayashida, Ryosuke Takii, Akira Nakai,

Tópico(s)

Bee Products Chemical Analysis

Resumo

Heat shock transcription factors (HSFs) regulate gene expression in response to heat shock and in physiological conditions. In mammals, HSF1 is required for heat-mediated induction of classic heat shock genes; however, we do not know the molecular mechanisms by which HSF4 regulates gene expression or the biological consequences of its binding to chromatin. Here, we identified that HSF4 binds to various genomic regions, including the introns and distal parts of protein-coding genes in vivo in mouse lenses, and a substantial numbers of the regions were also occupied by HSF1 and HSF2. HSF4 regulated expression of some genes at a developmental stage when HSF1 and HSF2 expression decreased. Although HSF4 binding did not affect expression of many genes, it induces demethylated status of histone H3K9 on the binding regions. Unexpectedly, a lot of HSF4 targets were induced by heat shock treatment, and HSF4 is required for induction of a set of non-classic heat shock genes in response to heat shock, in part by facilitating HSF1 binding through chromatin modification. These results suggest novel mechanisms of gene regulation controlled by HSF4 in non-classic heat shock response and in lens development. Heat shock transcription factors (HSFs) regulate gene expression in response to heat shock and in physiological conditions. In mammals, HSF1 is required for heat-mediated induction of classic heat shock genes; however, we do not know the molecular mechanisms by which HSF4 regulates gene expression or the biological consequences of its binding to chromatin. Here, we identified that HSF4 binds to various genomic regions, including the introns and distal parts of protein-coding genes in vivo in mouse lenses, and a substantial numbers of the regions were also occupied by HSF1 and HSF2. HSF4 regulated expression of some genes at a developmental stage when HSF1 and HSF2 expression decreased. Although HSF4 binding did not affect expression of many genes, it induces demethylated status of histone H3K9 on the binding regions. Unexpectedly, a lot of HSF4 targets were induced by heat shock treatment, and HSF4 is required for induction of a set of non-classic heat shock genes in response to heat shock, in part by facilitating HSF1 binding through chromatin modification. These results suggest novel mechanisms of gene regulation controlled by HSF4 in non-classic heat shock response and in lens development. Heat shock response is characterized by induction of a set of heat shock proteins (Hsps) 3The abbreviations used are: Hsp, heat shock protein; ChIP, chromatin immunoprecipitation; HSF, heat shock transcription factor; HSE, heat shock element; PBS, phosphate-buffered saline; GST, glutathione S-transferase; RT, reverse transcription. and is a fundamental adaptive response that maintains protein homeostasis (1Parsell D.A. Lindquist S. Annu. Rev. Genet. 1993; 27: 437-496Crossref PubMed Scopus (1881) Google Scholar, 2Westerheide S.D. Morimoto R.I. J. Biol. Chem. 2005; 280: 33097-33100Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 3Balch W.E. Morimoto R.I. Dillin A. Kelly J.W. Science. 2008; 319: 916-919Crossref PubMed Scopus (1760) Google Scholar). This response is regulated mostly at the level of transcription by heat shock transcription factors (HSF1–4) (4Morimoto R.I. Genes Dev. 1998; 12: 3788-3796Crossref PubMed Scopus (1536) Google Scholar). 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Chem. 2007; 282: 23240-23252Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Inversely, HSF1 also induces cell death by up-regulating a proapoptotic gene in response to stress in some cells such as male germ cells (16Nakai A. Suzuki M. Tanabe M. EMBO J. 2000; 19: 1545-1554Crossref PubMed Scopus (151) Google Scholar, 17Hayashida N. Inouye S. Fujimoto M. Tanaka Y. Izu H. Takaki E. Ichikawa H. Rho J. Nakai A. EMBO J. 2006; 25: 4773-4783Crossref PubMed Scopus (86) Google Scholar). Even in physiological conditions, all three HSFs, including HSF2 and HSF4, must regulate gene expression (18Pirkkala L. Nykanen P. Sistonen L. FASEB J. 2001; 15: 1118-1131Crossref PubMed Scopus (825) Google Scholar). HSF2 is highly expressed in early developmental stages and stays mostly a dimer (19Rallu M. Loones M. Lallemand Y. Morimoto R. Morange M. Mezger V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2392-2397Crossref PubMed Scopus (109) Google Scholar, 20Sistonen L. Sarge K.D. Morimoto R.I. Mol. Cell Biol. 1994; 14: 2087-2099Crossref PubMed Scopus (212) Google Scholar). Although we do not know what triggers HSF2 activation, it is converted to an active HSE-binding trimer when erythroleukemia cells are differentiated (21Sistonen L. Sarge K.D. Phillips B. Abravaya K. Morimoto R.I. Mol. Cell Biol. 1992; 12: 4104-4111Crossref PubMed Scopus (237) Google Scholar). HSF4 is highly expressed in the lens (22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (193) Google Scholar, 23Somasundaram T. Bhat S.P. J. Biol. Chem. 2004; 279: 44497-44503Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar), and basically remains as an HSE-binding trimer, because it uniquely lacks an inhibitory domain of trimerization (24Nakai A. Tanabe M. Kawazoe Y. Inazawa J. Morimoto R.I. Nagata K. Mol. 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Chem. 2007; 282: 37285-37292Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 35Takaki E. Fujimoto M. Sugahara K. Nakahari T. Yonemura S. Tanaka Y. Hayashida N. Inouye S. Takemoto T. Yamashita H. Nakai A. J. Biol. Chem. 2006; 281: 4931-4937Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), and in immune response (36Inouye S. Izu H. Takaki E. Suzuki H. Shirai M. Yokota Y. Ichikawa H. Fujimoto M. Nakai A. J. Biol. Chem. 2004; 279: 38701-38709Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 37Zheng H. Li Z. J. Immunol. 2004; 173: 5929-5933Crossref PubMed Scopus (45) Google Scholar). Furthermore, HSF1 plays major roles in lifespan (11Hsu A.L. Murphy C.T. Kenyon C. Science. 2003; 300: 1142-1145Crossref PubMed Scopus (1126) Google Scholar, 12Morley J.F. Morimoto R.I. Mol. Biol. Cell. 2004; 15: 657-664Crossref PubMed Scopus (540) Google Scholar) and in progression and maintenance of cancer (38Min J.N. Huang L. Zimonjic D.B. Moskophidis D. Mivechi N.F. Oncogene. 2007; 26: 5086-5097Crossref PubMed Scopus (126) Google Scholar, 39Dai C. Whitesell L. Rogers A.B. Lindquist S. Cell. 2007; 130: 1005-1018Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). It has been revealed that, in these physiological and pathological processes, HSFs not only maintain protein homeostasis by regulating constitutive expression of Hsps but are also involved in cell growth and differentiation by regulating expression of genes such as IL-6, FGFs, LIF, and p35 (9Inouye S. Katsuki K. Izu H. Fujimoto M. Sugahara K. Yamada S. Shinkai Y. Oka Y. Katoh K. Nakai A. Mol. Cell Biol. 2003; 23: 5882-5895Crossref PubMed Scopus (68) Google Scholar, 22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (193) Google Scholar, 31Chang Y. Ostling P. Akerfelt M. Trouillet D. Rallu M. Gitton Y. El Fatimy R. Fardeau V. Le Crom S. Morange M. Sistonen L. Mezger V. Genes Dev. 2006; 20: 836-847Crossref PubMed Scopus (71) Google Scholar, 35Takaki E. Fujimoto M. Sugahara K. Nakahari T. Yonemura S. Tanaka Y. Hayashida N. Inouye S. Takemoto T. Yamashita H. Nakai A. J. Biol. Chem. 2006; 281: 4931-4937Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 40Inouye S. Fujimoto M. Nakamura T. Takaki E. Hayashida N. Hai T. Nakai A. J. Biol. Chem. 2007; 282: 33210-33217Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). However, all reports have analyzed a limited set of classic heat shock genes and some development-related genes. We do not know how HSF2 and HSF4, as well as HSF1, regulate gene expression in physiological conditions, or the relationship between regulation of HSF1-mediated heat shock response and regulation of genes by HSFs during development. To answer these questions, it is necessary to identify HSF-target genes comprehensively and to analyze regulation of these genes. The lens is composed of only two cell types, epithelial cells and fiber cells (41McAvoy J.W. Chamberlain C.G. de Iongh R.U. Hales A.M. Lovicu F.J. Eye. 1999; 13: 425-437Crossref PubMed Scopus (219) Google Scholar). HSF4, as well as HSF1 and HSF2, is expressed in both cell types at early stage of lens development and is required for normal cell growth and differentiation of the two cells (22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (193) Google Scholar). Therefore, the lens tissue is suitable for comprehensive identification of HSF4-target genes and their analysis. Such analysis revealed unexpected roles of HSF4 in the regulation of gene expression during development and in the regulation of heat shock-mediated gene expression. Cell Culture and Adenovirus Infection—Two lenses from a 2-day-old wild-type or HSF4-null mouse (22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (193) Google Scholar) were treated with 0.5 ml of 0.1% trypsin/phosphate-buffered saline (PBS) at 37 °C for 30 min, and incubated for more 30 min after being added with 0.5 ml of 1% collagenase/PBS. The lens cells were dissected by using a vortex, collected by centrifugation, and maintained in a 48 well plate at 37 °C in 5% CO2 in Dulbecco's modified eagle medium containing 10% fetal bovine serum. Immortalized wild-type LEWd2 and HSF4-null LE4Nd2 cells were established by maintaining the cells for 24 h with a retroviral vector expressing a large T antigen of SV40 (42Li L.P. Schlag P.M. Blankenstein T. Hum. Gene Ther. 1997; 8: 1695-1700Crossref PubMed Scopus (27) Google Scholar), which was produced by using PLAT-E packaging cells (43Morita S. Kojima T. Kitamura T. Gene Ther. 2000; 7: 1063-1066Crossref PubMed Scopus (1361) Google Scholar). To overexpress HSF4 in cultured lens cells, we generated adenovirus expressing human HSF4b isoform (Ad-hHSF4) by inserting an XbaI/KpnI fragment of pGEM7-hHSF4b (25Tanabe M. Sasai N. Nagata K. Liu X.D. Liu P.C. Thiele D.J. Nakai A. J. Biol. Chem. 1999; 274: 27845-27856Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar) into a pShuttle vector (Clontech) as described previously (13Fujimoto M. Takaki E. Hayashi T. Kitaura Y. Tanaka Y. Inouye S. Nakai A. J. Biol. Chem. 2005; 280: 34908-34916Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Immortalized lens cells were infected with Ad-hHSF4 at a titer of 2 ∼ 10 × 105 plaque-forming units/ml. At 24 h after infection of viruses, cells were treated with heat shock and then harvested for analysis. Western Blot Analysis—To examine protein levels of HSFs, the lenses were homogenized in Nonidet P-40 lysis buffer (150 mm NaCl, 1.0% Nonidet P-40, 50 mm Tris (pH 8.0), 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml leupeptin, 1 μg/ml pepstatin) by sonication and centrifuged at 12,000 × g for 10 min (22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (193) Google Scholar). Aliquots containing 300 μg of protein were loaded on SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blotted with α-mHSF4t (25Tanabe M. Sasai N. Nagata K. Liu X.D. Liu P.C. Thiele D.J. Nakai A. J. Biol. Chem. 1999; 274: 27845-27856Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar). To detect HSF1 and HSF2, we used an antiserum α-mHSF1J that was raised against recombinant mouse HSF1 (amino acids 130–503) fused to GST, and α-mHSF2–4 that was raised against recombinant mouse HSF2 (amino acids 107–517) fused to GST. To detect Hsps and crystallins, aliquots containing 20 μg of protein were subjected to Western blot analysis (22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (193) Google Scholar). Gel Shift Assay and Gel Filtration—The lenses were dissected and stored at -80 °C until use. They were homogenized in buffer C by using a Dounce homogenizer, frozen, and then thawed (22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (193) Google Scholar). After centrifuging at 100,000 × g for 5 min at 4 °C, the supernatants were frozen in liquid nitrogen and stored at -80 °C. Aliquots containing 80 μg of proteins were subjected to gel shift assay using an ideal HSE oligonucleotide as a probe in the presence or absence of antiserum for each HSF, α-HSF1γ, α-HSF2d, or α-HSF4b (2.0 ml of 1: 10 diluted antiserum in PBS) (24Nakai A. Tanabe M. Kawazoe Y. Inazawa J. Morimoto R.I. Nagata K. Mol. Cell Biol. 1997; 17: 469-481Crossref PubMed Scopus (285) Google Scholar, 44Nakai A. Kawazoe Y. Tanabe M. Nagata K. Morimoto R.I. Mol. Cell Biol. 1995; 15: 5168-5178Google Scholar). Random oligonucleotide selection was performed essentially as described previously (45Kroeger P.E. Sarge K.D. Morimoto R.I. Mol. Cell Biol. 1993; 13: 3370-3383Crossref PubMed Scopus (96) Google Scholar). Recombinant GST-hHSF4 were induced in Escherichia coli by treating it with 0.4 mm isopropyl thiogalactosidase for 3 h, purified by using glutathione-Sepharose 4B (GE Healthcare Bio-Sciences Ltd.), and aliquots containing 2 μg of recombinant protein were subjected to gel shift assay. To compare the DNA binding activity of HSFs, aliquots containing equal amounts of recombinant GST-hHSF1, GST-hHSF2, and GST-hHSF4 were subjected to gel shift assay. Gels were dried and exposed to HR-HA30 film (Fujifilm) with an intensifying screen. The DNA-binding activities were quantified by using NIH Image. Gel-filtration analysis was performed as described previously (25Tanabe M. Sasai N. Nagata K. Liu X.D. Liu P.C. Thiele D.J. Nakai A. J. Biol. Chem. 1999; 274: 27845-27856Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 35Takaki E. Fujimoto M. Sugahara K. Nakahari T. Yonemura S. Tanaka Y. Hayashida N. Inouye S. Takemoto T. Yamashita H. Nakai A. J. Biol. Chem. 2006; 281: 4931-4937Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) by using the lens extracts in buffer C. Chromatin Immunoprecipitation—Immortalized lens epithelial LEW2d cells established from the lenses of a 2-day-old wild-type mouse in a 100-mm dish were treated with 10 ml of 1% formaldehyde/Dulbecco's modified eagle medium containing 10% fetal bovine serum at 37 °C for 10 min. After washing with PBS two times, the cell were suspended in 1 ml of PBS and centrifuged at 2000 rpm for 2 min. The pellet fractions were suspended in 200 ml of SDS-lysis buffer (1% SDS, 10 mm EDTA, 50 mm Tris-HCl (pH 8.1)), incubated at 4 °C for 10 min, sonicated, mixed with 4 μl of an rabbit antiserum for HSF4 (α-hHSF4–3 that was raised against recombinant human HSF4 fused to GST), and incubated at 4 °C for 16 h. Chromatin immunoprecipitation (ChIP) was performed using a ChIP assay kit (Upstate, New York) essentially according to the manufacturer's instructions. The precipitated DNA fragments were inserted into a pCR-Blunt II-TOPO vector (Invitrogen) after they were treated with T4 DNA polymerase. Sequencing reactions of the cloned DNA fragments were performed with an ALFexpress AutoRead sequencing kit, and the sequences were analyzed using an ALFexpress sequencer (GE Healthcare Bio-Sciences Ltd.) and mapped to the mouse genome using the BLAST algorithm. To confirm specific binding of HSF4 in LEWd2 cells, 35–40 cycles of PCR reactions were performed to amplify DNA using primers that hybridize to the end of each precipitated DNA (supplemental Table S1). Primers used to amplify Hsp70-1 promoter were described previously (46Ostling P. Björk J.K. Roos-Mattjus P. Mezger V. Sistonen L. J. Biol. Chem. 2007; 282: 7077-7086Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). ChIP analysis of the lens was performed exactly as reported previously (22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (193) Google Scholar), by using 12 lenses from E18.5 embryos, 8 lenses from 2-day-old mice, and 4 lenses from 3-week-old mice. Antibodies used for ChIP assay were a rabbit antiserum for HSF1 (α-cHSF1c) (40Inouye S. Fujimoto M. Nakamura T. Takaki E. Hayashida N. Hai T. Nakai A. J. Biol. Chem. 2007; 282: 33210-33217Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), for HSF2 (α-mHSF2–4), or for acetyl-histone H3 (Lys9) (#07-352, Upstate), a rabbit IgG for dimethyl-histone H3 (Lys9) (#07-212, Upstate), and a mouse IgG for phospho-RNA polymerase II (#05-623, Upstate). The amplified DNA was stained with ethidium bromide, photographed, scanned, and quantified by NIH Image. RT-PCR Analysis—RT-PCR analysis of mRNA levels of genes on and near the HSF4 binding regions was performed essentially as described previously (22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (193) Google Scholar), using specific primers (supplemental Table S2). The amplified DNA was stained with ethidium bromide and photographed using Epi-Light UV FA1100 (Aisin Cosmos R&D Co., Japan). Lens Organ Culture—Lenses isolated from 2-day-old wild-type, HSF4-null (22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (193) Google Scholar), and HSF1-null (9Inouye S. Katsuki K. Izu H. Fujimoto M. Sugahara K. Yamada S. Shinkai Y. Oka Y. Katoh K. Nakai A. Mol. Cell Biol. 2003; 23: 5882-5895Crossref PubMed Scopus (68) Google Scholar) mice were incubated in 35-mm dishes at 37 °C in 5% CO2 in 2 ml of serum-free medium 199 (Invitrogen) (47Baruch A. Greenbaum D. Levy E.T. Nielsen P.A. Gilula N.B. Kumar N.M. Bogyo M. J. Biol. Chem. 2001; 276: 28999-29006Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). To treat the lenses with heat shock, culture dishes were submerged in a water bath for 30 min at 37, 41, 42, 43, and 45 °C, and then lens extracts in an Nonidet P-40 lysis buffer were prepared for Western blot analysis. Alternatively, the heat-shocked lenses were recovered at 37 °C at 6 h, and total RNAs were isolated for RT-PCR analysis. Statistical Analysis—Significant values were determined by analyzing data with the Mann-Whitney U test using StatView version 4.5J for Macintosh (Abacus Concepts, Berkley, CA). A level of p < 0.05 was considered significant. Temporal Profiles of HSF Expression during Mouse Lens Development—We previously showed that HSF4 mRNA starts to be expressed at embryonic day 13.5 in both lens epithelial and fiber cells. Also, disruption of HSF4 gene results in abnormal expression of FGF genes in lens epithelial cells and of Hsp27 and γ-crystallin genes in fiber cells (22Fujimoto M. Izu H. Seki K. Fukuda K. Nishida T. Yamada S. Kato K. Yonemura S. Inouye S. Nakai A. EMBO J. 2004; 23: 4297-4306Crossref PubMed Scopus (193) Google Scholar). These results indicate that HSF4 plays major roles in both lens cell types during development and maintenance. Therefore, we examined the relative expression levels of HSF family members in whole mouse lenses in detail, and found that the level of HSF4 protein in the lenses was high even at embryonic day 15.5, reaching a peak at 2 days after birth. Then, it gradually decreases, but continues to be expressed at substantial levels even at 6 weeks (Fig. 1A). In marked contrast, levels of HSF1 and HSF2 proteins in the lenses were relatively high at embryonic days 15.5 and 18.5, but they decreased quickly after birth. Consequently, we detected a small amount of HSF1 and HSF2 proteins at 2 days after birth, but hardly detected them at 3 weeks (Fig. 1A). Similar expression profiles of HSFs in developing rat lenses were reported previously (23Somasundaram T. Bhat S.P. J. Biol. Chem. 2004; 279: 44497-44503Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). We next examined DNA-binding activity with gel shift assay using an consensus HSE probe and found that HSE-binding activity in the lenses was high even at embryonic day 15.5, reaching a peak at 2 days after birth, and then gradually decreasing. A supershift experiment using each specific antiserum demonstrated that HSE-binding activities in the lenses at embryonic day 18.5, at day 2 after birth, and at 3 weeks were mostly composed of HSF4 (Fig. 1B). To clarify why HSF1 and HSF2 do not have major HSE-binding activities in the lenses, we examined oligomeric forms by gel-filtration analysis. As we expected (24Nakai A. Tanabe M. Kawazoe Y. Inazawa J. Morimoto R.I. Nagata K. Mol. Cell Biol. 1997; 17: 469-481Crossref PubMed Scopus (285) Google Scholar, 25Tanabe M. Sasai N. Nagata K. Liu X.D. Liu P.C. Thiele D.J. Nakai A. J. Biol. Chem. 1999; 274: 27845-27856Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), a major population of HSF4 was an HSE-binding trimer, whereas HSF1 was a monomer and HSF2 could be a dimer in the lenses at embryonic day 18.5 (Fig. 1C). A substantial population of HSF1 forms a trimer in the lenses at 1 week, but the level of whole HSF1 protein was quite low at this developmental stage (Fig. 1A). Identification of HSF4 Binding Regions—To identity HSF4 binding regions in the lens tissue, we first performed ChIP analysis by using immortalized lens LEWd2 cells established from the lenses of 2-day-old wild-type mice to diminish false-positive clones. The precipitated DNA fragments were cloned into a plasmid vector, and their sizes were 120 bp to 1.6 kb (161 clones). Among the DNA fragments from 120 to 500 bp in size (125 clones, 78%), 100 fragments were sequenced and 71 sequences were mapped to the mouse genome using a BLAST search, although other 29 sequences were repetitive sequences or could not be determined. We generated primers specifically hybridized to the 71 DNA fragments (supplemental Table S1) and confirmed binding of HSF4 on the identified DNA in the lenses from 2-day-old mice by ChIP analysis (Fig. 2A). We concluded that HSF4 binds specifically to 58 of 71 DNA regions (82%), because a preimmune serum did not precipitate the DNA fragments and they were not precipitated in the HSF4-null lens. We failed to show binding of the other 13 DNA regions in the lens, but in the LEWd2 cells we were able to detect the binding of HSF4 to 6 of 13 DNA regions (total of 64 HSF4-binding DNA regions, 90%) (data not shown). We next determined the locations of HSF4 binding in 58 regions relative to gene coding sequences along the genome in the lens (Fig. 2B). It appeared that 31 regions (53%) located on the introns and exons of protein-coding genes and 23 regions (40%) were on distal parts of the genes. Among 31 regions on the introns and exons, HSF4 preferentially bound to the first intron (13 regions, 22%). Unexpectedly, a number of HSF4 binding regions on the promoter, which is a 5′-proximal region within 10 kb from each transcription start site, was quite low (3 regions, 5%). These results demonstrated the importance of genomic regions distinct from the promoter-proximal regions as HSF4 binding sites, and highlighted the introns of protein-coding gene and distal parts. Sequence Properties of HSF4 Binding Sites—To understand the molecular mechanisms of HSF cooperation on the HSF4 binding regions, we first examined sequences of HSF4 binding sites in vivo. We found 222 HSE-like sequences on the 58 HSF4 binding regions, which is composed of at least three inverted repeats of nGAAn pentameric units ("G" should be conserved) (48Xiao H. Lis J.T. Science. 1988; 239: 1139-1142Crossref PubMed Scopus (306) Google Scholar, 49Amin J. Ananthan J. Voellmy R. Mol. Cell Biol. 1988; 8: 3761-3769Crossref PubMed Scopus (434) Google Scholar). Among them, only six HSE-like sequences contained two to four perfect GAA sequences, but the others had none or only one GAA sequence (supplemental Fig. S1). To identify binding site sequences of HSF4 more clearly, we performed in vitro random oligonucleotide selection by using purified recombinant GST-HSF4 as described previously to identify binding sequences for HSF1 and HSF2 (50Kroeger P.E. Morimoto R.I. Mol. Cell. Biol. 1994; 14: 7592-7603Crossref PubMed Google Scholar). Sequence analysis of 108 HSF4-bound clones revealed that they contain HSE-like sequences (supplemental Fig. S2) for which conservation at each position was indicated as the height of the stack in a sequence logo generator, WebLogo (weblogo.berkeley.edu). We found that HSF4 binds to inverted repeats of nGnnn pentameric units (Fig. 3A). Ten clones (9%) were composed of only two inverted repeats but may use another distantly located unit (51Santoro N. Johansson N. Thiele D.J. Mol. Cell Biol. 1998; 18: 6340-6352Crossref PubMed Scopus (90) Google Scholar). Because the

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