Determination of the consensus binding sequence for the purified embryonic heat shock factor 2 Martine Manuel 1, * , †, Murielle Rallu 1, * , ‡, Marie-The ´ re ` se Loones 1 , Vincenzo Zimarino 2 , Vale ´ rie Mezger 1 and Michel Morange 1 Laboratoire de Biologie Mole ´ culaire du Stress, Unite ´ de Ge ´ ne ´ tique Mole ´ culaire UMR8541, Ecole Normale Supe ´ rieure, Paris, France; 2 DIBIT, San Raffaele Scientific Institute, Milan, Italy Heat shock transcription factors (HSFs) are characterized by their ability, upon activation, to bind to heat shock response elements (HSE) present in the promoter of their target genes. HSE are composed of inverted repeats of the pentamer nGAAm. In this study, we compare the embryonic HSF2 protein, purified from F9 embryonal carcinoma cells tumor, and the in vitro synthesized HSF2. We show that the context of HSF2 synthesis influences its thermosensitivity and DNA-binding properties. Therefore, we determined the consensus binding sequence for the purified embryonic HSF2 by the technique of systematic evolution of ligands by exponential enrichment (SELEX). We show that embryonic HSF2 prefers sites containing three or four nGAAm inverted pentamers and that its optimal binding sequence contains the 8-mer palindromic core 5¢-TTCTAGAA-3¢. The consensus binding sequence for the embryonic HSF2 will be very helpful to identify new targets for this factor, during developmental and differentiation processes. Keywords: heat shock transcription factor-2; protein purifi- cation; cooperativity; SELEX; consensus binding sequence. Heat shock factor 2 (HSF2) belongs to the vertebrate heat shock factor family that also includes HSF1, HSF3 and HSF4 [1–5]. The members of the HSF family are defined by their ability to specifically bind the regulatory sequence heat shock element (HSE) [6]. Located in the regulatory regions of heat shock genes, HSE consists of the inverted repeat of a basal element nGAAm [7]. Two inverted repeats are sufficient for Drosophila HSF binding, but optimal binding is obtained with three repeats [8]. In agreement with this observation, the activated form of HSFs has been demon- strated to be a trimer in yeast [9], in Drosophila [10], in human [11,12] or in mouse [13]. The HSE-binding activity of heat shock factors is not constitutive, but induced by various stresses, by differentiation or developmental pro- cesses. HSF1 and HSF3 are activated by stresses that elicit the so-called Ôheat shock responseÕ and induce the tran- scription of heat shock genes. HSF1 corresponds to the paradigm member of the family and is the functional homolog, for its function in the heat shock response, of the unique HSF found in yeast and Drosophila. Avian HSF3 is activated by more severe stresses than HSF1, but is also required for an optimal response to stress [14,15]. Indeed, avian cells expressing HSF1, but in which the HSF3 gene has been disrupted, exhibit a diminished response to stress, even at mild heat shock temperatures [14]. Athough heterotrimers were never detected, HSFs may interact with each other in a more complex way. HSF4 is an exception and constitutively binds DNA as a trimer in the absence of stress. Its expression is regulated in a tissue-specific manner [5,16]. The Hsf4 gene generates both an activator or a repressor of heat shock genes by alternative splicing; the tissue-specificity of the two forms may create a modulation of expression of hsps in the different tissues. In contrast to HSF1 and HSF3, HSF2 is not activated in response to heat shock or other cellular stresses. It is found in a trimeric DNA-binding form during hemin-induced differentiation of the human erythroleukemia cells K562, in mouse embryonal carcinoma (EC) cells, and during mouse embryogenesis and spermatogenesis. During the differenti- ation of K562 cells, HSF2 is converted from an inert dimeric form to a DNA-binding trimer that is able to induce the transcription of Hsp70 gene [17–19]. In this system, it seems that although HSF1 and HSF2 are activated by distinct signals, they also induce a similar profile of heat shock gene transcription [17,18]. It was therefore suggested that in mammalian cells, HSF1 was responsible for heat shock gene induction upon stress, while HSF2 was responsible for the high spontaneous expression of heat shock genes, which is observed in the absence of stress in EC cells, and during mouse embryogenesis and spermatogenesis. However, an accumulation of data shows that the contribution of HSF2 to the transcriptional regulation of heat shock genes remains unclear. Indeed, athough HSF2 Correspondence to M. Morange, Laboratoire de Biologie Mole ´ culaire du Stress, Unite ´ de Ge ´ ne ´ tique Mole ´ culaire UMR8541, Ecole Normale Supe ´ rieure, 46 rue d’Ulm, 75230 Paris cedex 05, France. Fax: + 33 1 44 32 39 41, Tel.: + 33 1 44 32 39 46, E-mail: morange@wotan.ens.fr Abbreviations: HSF, heat shock transcription factor; HSE, heat shock response elements; SELEX, systematic evolution of ligands by exponential enrichment; EC, embryonal carcinoma; in vitro synthesized, i.v.s. *Note: these authors contributed equally to this work. Present address: Department of Biomedical Sciences, University of Edinburgh, UK. àPresent address: Developmental Genetics Program, Skirball Institute for Biomolecular Medicine, NYU Medical Center, New York, USA. (Received 5 December 2001, revised 28 February 2002, accepted 5 April 2002) Eur. J. Biochem. 269, 2527–2537 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02917.x displays a strong DNA-binding activity in EC cells [20,21], the HSE region of Hsp70 promoter was found unoccupied by HSF2 [21]. Controversial data suggest that, in contrast to what was observed in mouse, HSF2 does not display any DNA-binding activity at any stage of the rat seminiferous epithelial cycle and that HSF2 expression does not correlate with any HSP expression pattern [22]. No correlation is found during pre- or post-implantation embryogenesis between the expression patterns of major HSPs and HSF2 profiles [23]. Even in the case of the K562 cell system, where the HSE sites were found occupied in vivo by HSF2 during hemin-induced differentiation [18], other data suggest a role of HSF1 and not HSF2 in the hemin-induced transcription of Hsp70 gene [24], re-addressing the respective role of the two factors in Hsp70 expression during differentiation. Therefore, the role of HSF2 during differentiation and development is likely distinct from a simple inducer of heat shock genes in nonstress conditions, in differentiation or developmental situations. Its role is still not unravelled and its targets as a transcription factor unknown. Studies performed on recombinant HSF1 and HSF2, produced in E. coli, using random oligonucleotide selection have shown that they display slightly distinct preferences, although both factors bind to the 5¢-nGAAm-3¢ basal motif [25]. Recombinant HSF2, in contrast to HSF1, does not bind to HSE in a cooperative manner. We purified HSF2 from F9 mouse embryonal carcinoma tumors and analyzed its DNA-binding properties at various temperatures in comparison with in vitro synthesized (i.v.s.) HSF2 protein, produced in reticulocyte lysates. This study demonstrates that the DNA-binding properties of the purified HSF2 are different from those of the i.v.s. HSF2. This suggests that HSF2 function is highly sensitive to the environment in which it is synthesized. We therefore decided to determine the consensus binding sequence for the purified embryonic factor, by a SELEX assay using a semirandom oligonucle- otides library. We found that the embryonic factor requires at least three 5¢-nGAAm-3¢ motifs and that its optimal binding sequence contains a palindromic 8-mer core 5¢-TTCTAGAA-3¢. This result is in contrast to what was found for the recombinant HSF2. MATERIALS AND METHODS Oligonucleotides The oligonucleotides used in this study are shown in Table 1. Embryonal carcinoma (EC) cell culture, acquisition of tumors in 129 mice and purification of HSF factors F9 EC cells were grown and extracts were prepared as previously described [20]. F9 tumor cells were obtained by subcutaneous injection of 2 · 10 6 F9 cells in 5-week-old syngenic mice (strain 129). Tumors were allowed to grow for about 2 weeks. After cervical dislocation, the tumors were rapidly dissected and immediately frozen in dry ice until use for extraction. Appropriate measures were taken to minim- ize animals pain or discomfort, in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). For HSF2 protein purification, 26 g of crude material (18 tumors) were extracted with 300 mL of extraction buffer (10 m M Hepes pH 7.9, 0.4 M NaCl, 0.1 M EGTA, 0.5 m M dithiothreitol, 5% glycerol, 0.5 m M phenyl- methanesulfonyl fluoride supplemented with 1 lgÆmL )1 pepstatin and 1 lgÆmL )1 aprotinin). Whole-cell extracts were clarified by centrifugation for 30 min at 100 000 g and the supernatants were stored at )80 °C. The final protein concentration of the extracts averaged 5.8 mgÆmL )1 . The complete purification of HSF2 protein was performed by adaptation of a three-step protocol previously described by Wu et al.[26]. (a) Whole-cell F9 tumor extracts were applied on an heparine-sepharose column (CL-6B, Pharmacia), washed with 300 mL of equilibration buffer (0.15 M NaCl, 20 m M Hepes pH 7.9, 0.1 m M EGTA, 10% glycerol, 0.5 m M PMSF, 1 lgÆmL )1 pepstatin and 1 lgÆmL )1 aprotinin). Bound proteins were eluted with a linear salt gradient from 0.15 to 1.5 M NaCl. Fractions were analyzed by electromobility shift assay (EMSA) and those containing an HSE-binding activity (0.2 to 0.6 M NaCl) were pooled. The yield and purification factors were calculated for this column and were found to be equal to 87% and 5.6, respectively. (b) A DNA-affinity resin was prepared by coupling HSE sequences to a CNBr-activated sepharose (CL-4B; Pharmacia Biotech), according to Kanodaga and Tjian [27]. The synthetic HSE oligonucleotide CTAGAAGCTT, similar to that of Sorger & Pelham [28], was annealed with itself in order to form double-stranded molecules with protruding ends, which were subsequently ligated. This resulted in the formation of polymers of about 100–200 bp, as estimated by agarose gels, that were linked to the resin. Fractions containing the HSE-binding activity were pooled and adjusted, by dilution, to 0.35 M NaCl, 26 m M Hepes pH 7.6, 20% glycerol, 0.3 m M dithiothrei- tol, 0.5 m M phenylmethanesulfonyl fluoride. The diluted fractions were incubated overnight at 4 °C, under gentle agitation, in the presence of resin, protease inhibitors (1 lgÆmL )1 pepstatin and aprotinin) as well as 1.5 lgÆmL )1 poly(dI-dC).poly(dI-dC) to avoid nonspecific interactions. After extensive washing with 0.2 M NaCl equilibration buffer (26 m M Hepes pH 7.6, 20% glycerol, 0.1% NP40, 0.1 m M EGTA, 0.3 m M dithiothreitol, Table 1. Oligonucleotide sequences used in this study. HSE2 5¢-TCGACAGATCTCCTAGAACGTTCTAGA AGCTTCGAGAGGATTC-3¢ 2U519m 5¢-CAGAATCTTCTCGATAGTTAGG-3¢ SHVAL 5¢-CTAGAACGTTCTAGAAGCTTCGAGA-3¢ SHVAL-SPZ 5¢-CTAGAACGTTCTAGAGAGTTTCCAG-3¢ NOG 5¢-CTAGAACGTTCTAGGGGGGGGGG-3¢ NOA 5¢-CTAGAACGTTCTAAAAAAAAAAA-3¢ MTH 5¢-CTAGAACGTTCTAAAAATTTCCAG-3¢ MCL 5¢-CTAGAACGTTCTAAAAAATTTCCAG-3¢ SHC 5¢-CTAGAACGTTCTAGAGAGAGAGAGA-3¢ JUL 5¢-CTAGAACGTTCTAGAACGTTCTCA-3¢ Deg-sb 5¢-CACGTGCGCTGGTACN 3 GAANNTTC N 14 GGCTATCGACTGGCG-3¢ CL39 5¢-ATGGAACATTCTAGAACCTTCTCTT-3¢ CL83 5¢-AGAGAACATTCTAGAACATGGGTAC-3¢ 83woTA 5¢-AGAGAACATTCACGAACATGGGTAC-3¢ 39woGAA 5¢-ATGCACCATTCTAGAACCTTCTCTT-3¢ 83woGAA 5¢-AGACACCATTCTAGAACATGGGTAC-3¢ 2528 M. Manuel et al. (Eur. J. Biochem. 269) Ó FEBS 2002 0.5 m M phenylmethanesulfonyl fluoride, 1 lgÆmL )1 pepst- atin and aprotinin), proteins specifically bound to the resin were eluted by steps of increasing NaCl concentration (0.3 to 2 M ). Fractions were analysed by EMSA and those containing HSE-binding activity (0.6 to 1.6 M NaCl) were pooled, concentrated 20-fold and analyzed by SDS/ PAGE. Silver staining of the gel revealed the presence of several bands. (c) Therefore, active fractions were subjected to a second DNA-affinity chromatography. Fractions from the first affinity chromatography were brought a second time to 0.35 M NaCl, incubated with the HSE-affinity resin and eluted exactly as before. Elution of HSE-binding activity occured between 0.6 and 1.7 M NaCl. The active fractions were pooled and used for gel-shift assay. During the two successive steps of DNA-affinity chro- matography, fractions were collected in silanized tubes to prevent sticking on plastic walls. Total yields in HSE- binding activity and protein amount were estimated and allowed to calculate a total purification factor equal to 3000. SDS/PAGE and Western-blot analysis Fractions containing HSF2 protein were pooled and loaded on G25 sephadex (NAP columns; Pharmacia Biotech) in order to discard most of the salts. Eluted material from the Sephadex columns was then lyophilized and resuspended in water so that the final volume was 100-fold less than at the beginning. Three quarters of this concentrated material was loaded on a 10% polyacrylamide gel and revealed by silver staining in parallel with known concentrations of BSA to estimate the amounts of purified protein. The last quarter was used for Western blotting after transfer to a nitrocel- lulose filter. HSF2 polyclonal antibodies were used at 1 : 2500 dilution as previously described [23]. Detection was performed using the ECL peroxydase detection system (Amersham). Electromobility shift assays (EMSA) Binding reactions were performed as described previously [20]. Both strands of the DNA template were 32 P end- labeled using T4 polynucleotide kinase and [c- 32 P]ATP. Fourteen microliters of extracts containing 10–20 lgof proteins from crude extracts, 0.7 ng of pure HSF2 protein or 3 lLofin vitro translated proteins were mixed with 9 lL of binding solution [0.2 ng of 32 P-labeled double-stranded DNA template, 4 lg of double stranded polydI-dC, 9% (w/ v) Ficoll, 44 m M Hepes pH 7.6, 2.2 m M MgCl 2 and 88 m M KCl]. In competition experiments, 20 ng of unlabeled double stranded DNA template were added to the binding solution. The reaction mixtures were loaded on a 4% acrylamide gel (acrylamide/bisacrylamide, 29 : 1, w/w) in 0.25 · Tris/borate/EDTA buffer. Analysis of HSF2 thermosensitivity properties HSF2 factors (i.v.s. or embryonic) were incubated at a moderate (37 °C) or high temperature (44 or 45 °C) and samples were taken at increasing periods of time, brought to room temperature and subjected to the binding reaction in presence of the oligonucleotide HSE2. Samples were then immediately loaded on the migrating gel. Quantification of the signal in the specific retarded complexes was performed using a Bas1000 Imager (Fuji) after 1 h exposure. Arbitrary values measured at distinct incubation times were standard- ized to the initial value. Multiple probes band shift assay Synthetic oligonucleotides, containing an increasing num- ber of the conserved 5 bp units nGAAm (organized in contiguous arrays where each unit is inverted relative to the immediately flanking one) and their complementary strands were obtained from Genset (Paris, France). The same oligonucleotides as those described by Xiao et al. [29] were used, where n and m are A and T, respectively, for GAA and TTC. Flanking sequences, added to this core region in order to limit self-annealing, were identical to those present at the ends of the oligonucleotide used for affinity chromatography. According to the number of repeats, oligonucleotides were named Rep2, Rep3, Rep4, Rep5 and Rep6. Binding reactions were performed as described above, except that the binding solution contained a total amount 0.2 ng of 32 P end-labeled double-stranded oligonucleotides corresponding to a mixture of Rep2, Rep3, Rep4, Rep5 and Rep6 at the same concentration. Protein extracts and range of protein amounts used to perform these experi- ments were as follows: 0.7 ng of HSF2 protein purified to homogeneity (supplemented with 200 lgofBSA),and 3 lL of recombinant HSF2 protein expressed in reticulo- cyte lysates. Binding reactions were performed at room temperature during increasing periods of time ranging from 0.5 min to 3 h and were followed by pore exclusion limit electrophoresis. Samples were loaded on a 3–10% gradient acrylamide gel (acrylamide/bisacrylamide, 29 : 1, w/w) and migration was performed for 6 h at 350 V in 0.25 · Tris/borate/EDTA buffer, until the complexes reached a position in the gel preventing their migration. The position of specific complexes was detected by direct autoradiography. Bands containing the specific complexes as well as free DNA were cut out of the gel and oligonucleotides present in these gel slices were eluted overnight in distilled water at 37 °C. Samples were extracted once in phenol-chloroform and once in chloroform, then concentrated in speed-vacuum apparatus. They were then directly resuspended in the sequencing loading buffer and analyzed on a denaturing 10% polyacrylamide gel in 1x TBE. The relative amounts of the different oligonucleotides contained in each band were quantified as previously described. SELEX assay The SELEX procedure was performed according to a strategy described previously [30]. Preparation of a random sequence library The 55-mer oligonucleotides Deg-sb (5¢-CACGTGCGC TGGTACN 3 GAAN 2 TTCN 14 GGCTATCGACTGGCG- 3¢), containing two inverted trimers GAA and 19 random nucleotides, and two PCR primers: P1, corresponding to the first (top strand) 15 bases, and P2, complementary to the last (bottom strand) 15 bases, were manufactured by Ó FEBS 2002 Determining the optimal binding sequence for HSF2 (Eur. J. Biochem. 269) 2529 Eurobio (Les Ulis, France). A random sequence library, Sel0, was generated by a primer extension reaction carried out with Deg-sb as template and the (bottom) primer P2. 800 pmol of Deg-sb, annealed to a mix of 1600 pmol of cold P2 and 80 pmol of radiolabeled P2, were extended with 100 U of Klenow fragment in a 200-lL Klenow reaction mixture. The extended products were purified on a 12% acrylamide gel. Selection and amplification of sequences that bind HSF2 Sel0 (450 ng in 90 lL of binding solution) was mixed with 30 mL of pooled elution fractions of purified embryonic HSF2 ( 7 ng) and 1 mg of BSA. The reaction mixture was incubated 15 min at room temperature and loaded on a 4% acrylamide gel. After migration, the wet gel was wrapped with Saran and exposed to X-ray film. The gel region harboring HSF2-Sel0 complexes was localized by comparison with the electrophoretic mobility of the HSF2-radiolabeled Shvalspz complex that was loaded on the adjacent control lane. An appropriate gel slice was excised (large enough to take into account the smeary binding pattern displayed by the purified HSF2 protein) and soaked overnight at 37 °C in elution buffer (0.3 M NaCl, 1 m M EDTA, 0.1% SDS). The eluted DNA was purified on a Sephadex G-25 column (NAP-25 column, Pharmacia Biotech) and concentrated to a volume of 50 lL in water. A 5-lL sample was added to a PCR mixture together with 75 pmol of primer P1, 75 pmol of primer P2 and 2.5 U of Tfl DNA polymerase (Promega) in a final volume of 100 lL containing 20 m M Tris/acetate (pH 9), 10 m M ammonium sulfate, 75 m M potassium acetate, 0.05% Tween 20, 1.25 m M MgSO 4 ,and75l M of each dNTP. Eight such reaction mixtures were set up. The samples were heated for 1 min at 94 °C (hot start). For each 35 cycles of PCR, samples were denatured at 94 °C for 30 s, annealed at 46 °C for 30 s, and extended at 75 °C for 15 s. All eight reaction mixtures were pooled and the DNA was purified on a 12% acrylamide gel. About 50–100 ng of DNA was used for the next cycle of the SELEX procedure. Cloning of the products of selection DNA amplified from the last cycle of selection was rendered blunt-ended using T4 DNA polymerase and inserted at the EcoRV site of pBluescript (pKS+, Stratagene). Sequencing of the products of selection After each round of selection, the amplified selected DNA was sequenced as follows using the T7-sequencing kit from Pharmacia Biotech with the following modifi- cations to take into account the short size of the sequences. P2 (10 ng) was end-labeled with [c- 32 P]ATP and annealed to 10 ng of selected DNA in a 14-lL volume containing 0.15 M Tris/HCl (pH 7.6), 15 m M MgCl 2 and 23 m M dithiothreitol. The mix was boiled for 5 min and left on ice for 10 min 4 U of T7 DNA polymerase in 2 lL of dilution buffer [20 m M Tris/HCl (pH 7.5), 5 m M dithiothreitol, 100 lgÆmL )1 BSA and 5% glycerol], 4 lLof33m M NaCl and 1 lLof100m M MnCl 2 , 150 m M sodium isocitrate were added to the annealing mix on ice. 4.5 lL of this mixture were added to 2.5 lL of each of the four ddNTP Mix-Short solutions [840 l M each dN 1 TP, dN 2 TP, dN 3 TP; 93.5 l M dN 4 TP; 14 l M ddN 4 TP; 40 m M Tris/HCl (pH 7.6) and 50 m M NaCl]. The reaction mix was incubated at 37 °Cfor 20 min The sequences were analysed on a 10% denaturing acrylamide gel. Individual Sel6 clones were sequenced using the T7 sequencing kit from Pharmacia Biotech and the T7 primer according to the manufacturer’s instructions. RESULTS Purification of mHSF2 protein from EC cells Sufficient starting amounts for the purification of HSF2 protein were obtained from tumors of F9 embryonal carcinoma cells. These tumors were produced by injection of F9 cells, in which HSF2 is highly expressed, under the skin of syngenic mice. We verified that extracts produced from tumor cells displayed an HSE-binding activity similar to that of extracts from in vitro cultivated F9 cells (data not shown), showing that mouse or tissue manipulations did not uncover any stress-inducible activity (due to HSF1 protein). The complete procedure for HSF2 purification combined heparin and DNA affinity chromatographies [26]. HSF2 protein elution profile was monitored by the presence of an HSE-binding activity in gel-shift assay (at room tempera- ture). The first step of this purification procedure (i.e. the heparine–sepharose chromatography) led to the separation of HSF2 protein from 80% of the proteins present in crude extracts. The following steps consisted of two HSE-affinity chromatographies (see Materials and methods). After the first one, HSF2 protein was separated from most of the remaining proteins but a few of them were still co-eluted with it. Therefore, HSF2-containing fractions were reloaded on the same column in order to obtain a pure protein. Analysis on SDS/PAGE after silver-staining showed one unique band of  70 kDa (Fig. 1A). This band was recognized by HSF2 antibodies (Fig. 1B) and comigrated with one protein product present in reticulocyte lysates expressing HSF2 protein. Thus, it appeared that HSF2 protein from F9 embryonic cells was purified to near homogeneity. The purification factor was estimated to be equal to 3000. The pure protein was stable at )70 °C and could sustain more than two cycles of freeze-thawing. However, gel shift assays with pure protein gave poor reproducible results, and we considered that, at these low protein concentrations, the rare molecules of HSF2 protein might stick on the tube walls, even when silanized. Therefore, we added 200 lgof BSA to each point of binding reaction and got a reprodu- cible stabilization of the purified HSF2 protein. We called HSF2 purified from F9 tumor cells Ôembryonic HSF2Õ. Conditions of binding and elution of HSF2 protein, in the affinity column, gave several informative results about its properties. Indeed, whereas binding conditions of HSF2 protein to the heparine–sepharose resin were similar to that of Drosophila HSF, conditions used for the HSE-DNA affinity chromatography were quite different. HSF2-con- taining fractions required a longer incubation time with the resin in order to bring the reaction to completion and the 2530 M. Manuel et al. (Eur. J. Biochem. 269) Ó FEBS 2002 ionic strength had to be increased to 0.35 M NaCl (in comparison to 0.25 M NaCl for Drosophila HSF). In fact, we showed that optimal binding to HSE sequences occurred at slightly higher NaCl concentrations for HSF2 protein (present in extracts from F9 control cells) than for HSF1 (present in extracts from F9 heat-shocked cells), the Drosophila HSF homolog, which could explain the discrep- ancy observed between HSF2 and Drosophila HSF in binding the resin (Fig. 2). Besides this differential sensitivity to ionic strength conditions, other components of the binding buffer did not differentially affect HSF2, except for MgCl 2 (optimal concentrations: 0 m M for HSF2, 1 m M for HSF1), which appeared slightly detrimental to HSF2 binding to DNA (data not shown). The purified embryonic HSF2 protein displays a different thermosensitivity than the i . v .s. factor I.v.s. HSF1 and HSF2 proteins display very distinct behaviors. HSF1 protein produced in reticulocyte lysates is active for DNA binding, provided that the extracts have first been heated. In contrast, HSF2 protein shows a constitutive HSE-binding activity but loses this activity upon heat treatment [3,32]. Therefore, it appeared that the DNA-binding activity of HSF2 protein was much more sensitive than that of HSF1 protein, at least when synthes- ized in vitro. Using electromobility shift assay (EMSA), we analyzed the thermosensitivity properties of the purified embryonic HSF2 in comparison with the i.v.s. factor, produced in reticulocyte lysates. I.v.s. or embryonic purified proteins were incubated at various temperatures before being subjected to EMSA. This experiment allowed to analyze the sensitivity properties of soluble HSF2 proteins, by measuring their remaining capacity to bind their target sequences after exposure to denaturating temperatures. The remaining ability of the factors to bind a consensus target was quantified and plotted as a function of time. The inactivation ratio of pure embryonic HSF2 protein was estimated to be about 20% after 20 min at 37 °Cand 80% after 20 min at 45 °C (Fig. 3). I.v.s. HSF2 protein was also denatured by incubation at 37 °Cor45°C(Fig.3).At high temperatures, the i.v.s. factor appeared to be signifi- cantly more rapidly inactivated than the embryonic factor. The inactivation of the i.v.s. protein observed at 37 °C occurred in a limited manner and, unexpectedly, was preceded by a transient phase of activation. Therefore, incubation of the i.v.s. HSF2 at a moderate temperature highly activated its DNA-binding abilities. This result was uppermost striking as HSF2 appeared to be quite sensitive to high temperature when synthesized in vitro [3]. Furthermore, the pure embryonic factor did not behave in the same way. Thus, HSF2 protein synthesized in the reticulocyte lysates displayed a specific ability to become further activated following a short exposure to a moderate temperature. This seemed not to be characteristic of the factor itself but rather of the conditions in which it had been produced. The purified i . v .s. and embryonic HSF2 proteins exhibit differences of cooperativity in DNA binding In order to look for the cooperativity of HSF2 binding to HSE sequences, we used the same methodology as that Fig. 2. Effect of ionic (NaCl) strength on Heat-Shock Factors 1 and 2 DNA binding activities. Whole cell extracts from control unshocked (F9C, corresponding to HSF2) or heat-shocked (F9HS, corresponding to HSF1) F9 cells were incubated with labeled HSE oligonucleotide under varying NaCl concentrations. After PHOSPHORIMAGER quanti- fication, data were reported as fractions of the maximal value. Extracts from heat-shocked cells (F9HS, HSF1) are plotted as circles; extracts from control cells (F9C, HSF2) are plotted as triangles. Fig. 1. Purification to homogeneity of HSF2 from F9 tumor extracts. Elution fractions from the first and second cycle of HSE-affinity col- umn (as well as HSF2 synthesized in reticulocyte lysates) were run on SDS/PAGE after 100-fold concentration. (A) Silver staining. The multiple bands observed above the 70 kDa i.v.s. HSF2 likely corres- pond to additional proteins present in reticulocyte lysates. The smear observed above the 70 kDa purified embryonic HSF2 is due to remaining salts. (B) Western blot analysis using the HSF2 antiserum at a 1 : 5000 concentration. Ó FEBS 2002 Determining the optimal binding sequence for HSF2 (Eur. J. Biochem. 269) 2531 described by Liu-Johnson et al.[33],andappliedto Drosophila HSF by Xiao et al. [29]. The strategy consists in measuring the affinity of the factors for a series of oligonucleotides containing an increasing number of the binding motif. Immediately following the addition of the different oligonucleotides, the factor recognizes equally well all the oligonucleotides but, as time proceeds and if the binding reaction is cooperative, the factor will bind more and more preferentially to the sequences that contain a higher number of motifs. To be able to estimate quantitatively the cooperativity of HSF2 binding to its sites, we measured, in the purified complexes, the ratio of the oligonucleotides (rep4–6) as compared to rep3. Both the i.v.s. and the purified embryonic HSF2 display a higher affinity for sequences containing a higher number of consensus trimers. In the case of the purified embryonic HSF2, the ratio rep6/rep3 reaches the plateau very quickly (15–20 min) (Fig. 4B). In contrast, in thecaseofthei.v.s. HSF2, the ratio rep6/rep3 still increases after 100 min (Fig. 4A). Therefore, the i.v.s. HSF2 displays a higher cooperativity than the purified embryonic HSF2. Embryonic HSF2 has a weak affinity for a good binding sequence selected by the recombinant factor Kroeger & Morimoto [25] had selected, from a random- sequenced DNA library, sequences that could bind the recombinant protein HSF2, synthesized in E. coli. Among the selected sequences, the oligonucleotide 2U519 was bound with a very good affinity. We compared, by EMSA, the affinity of the purified embryonic HSF2 for the oligonucleotide 2U519 and for the oligonucleotides Shval and Shvalspz. Shval was commonly used in the laboratory to detect HSE-binding activity in cell extracts. It was designed according to the description, at that time, of the basic heat shock response element; it contains four inverted nGAAm pentamers. Shvalspz is a modified version of Shval; it contains two inverted nGAAm pentamers followed by the weak HSE sequence present in the promoter of the putative target of HSF2 in spermato- genesis, the testis specific gene Hsp70.2. While the i.v.s. factor displayed, as expected, a better affinity for the oligonucleotide 2U519 compared to Shval, in contrast, the purified embryonic protein had a better affinity for Shval (Fig. 5). Strikingly, the purified embryonic factor could bind Shvalspz with a much higher affinity than 2U519 or Shval. Fig. 4. Comparison of the cooperativity properties of in vivo and i.v.s. HSF2. Proteins were incubated at room temperature with the mix of rep oligonucleotides during increasing periods of time. The binding mixture was then subjected to pore exclusion limit electrophoresis. Bands containing the specific complexes as well as free DNA were cut out of the gel and oligonucleotides present in these gel slices were eluted. The relative amounts of the different oligonucleotides con- tained in each band were quantified as previously described. The figure shows the ratios rep6/rep3, rep5/rep3 and rep4/rep3 in the whole complex, as a function of the incubation time. (A) I.v.s. HSF2. (B) purified embryonic HSF2. Fig. 3. Comparison of the thermosensitivity of in vivo and i.v.s. HSF2 at 37 °C and 45 °C. Proteins were incubated at 37 or 45 °Cduring increasing times and thereafter submitted to gel-shift assay. Signal amounts in the specific retarded complexes were quantified and then compared to the initial value. 2532 M. Manuel et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Determination of the consensus binding sequence for the purified embryonic mHSF2 by SELEX As we found that the recombinant and embryonic HSF2 proteins displayed significant differences in their DNA binding properties, we decided to determine the consensus binding sequence for the purified embryonic mHSF2 by the technique of SELEX. We followed the procedure described by Blackwell [30]. A semirandom-sequence oligonucleotide library, Deg-sb, was ordered from Eurobio. These 55-nucleotide sequences contain a central semirandom sequence, with 19 randomized nucleotides and two inverted GAA trimers, flanked by two 15 fixed nucleotide sequences for PCR amplification and sequencing. The two trimers in the central sequence are separated by two randomized nucleotides and they are located at 18 nucleotides from the 5¢ end of the sequence. The design of the semi-random sequence was based on the high affinity of the embryonic HSF2 observed for the oligonucleotide Shvalspz. The use of a semi-random DNA library, in which a skeleton of HSE site has been conserved, instead of a totally random DNA library, allows a faster enrichment in HSF2 binding sequences and the analysis of the selected sites using the pool sequencing assay [30]. A double-stranded DNA library, Sel0, was obtained from Degsb by a primer extension reaction. Sel0 sequences were incubated with the purified embryonic mHSF2 and the bound molecules, Sel1, were isolated by EMSA, amplified by PCR, sequenced as a pool and subjected to the next round of EMSA. After the second cycle of selection, the sequence of the pool of selected molecules revealed a significant enrichment in sequences containing a third GAA trimer separated from the two fixed trimers by a TA dinucleotide. This result was confirmed by the next rounds of selection (Fig. 6). The SELEX assay was stopped after six cycles of selection/amplification as no difference could be observed between the sequence of Sel4, Sel5 and Sel6. At this step the preferred binding sequence for embryonic HSF2 was 5¢-(A/G)(G/T)(A/G)GAA(C/T)(A/G)TTCTA GAAN (A/G)(A/T)-3¢ (top strand), as could be determined from the sequence of Sel6. Sel6 sequences were subcloned in pBluescript and 57 individual clones were sequenced (Fig. 7). Strikingly, almost all of the sequences contained a third GAA trimer, one of them containing a GAT instead of GAA, and 46 sequences displayed a TA dinucleotide between the second and the third GAA trimer (all of the 11 remaining sequences displaying either the T or the A). 15 sequences contained a fourth inverted GAA trimer at the right position, i.e. two nucleotides from the third trimer. A consensus binding sequence was determined by calculating, for each position of the central semi-random sequence, the percentage of selected molecules containing each of the four nucleotides (Fig. 8). This consensus sequence was composed of the first fixed GAA trimer, a 8-mer TTCTAGAA core, which was present in almost 100% of the selected sequences, and a fourth inverted GAA motif. It is noticeable that the preferred dinucleotide (top strand) preceding the first and third GAA trimers is TA in both cases, and the preferred dinucleotide (top strand) preceding the second and fourth trimers is C A / G . The affinity of the embryonic HSF2, present in F9 crude extracts, for each of the 57 selected sequences was tested by EMSA, and no significant differences were found between them. HSF2 could bind the sequences containing three trimers and those containing four trimers with comparable affinities, as determined by the quantification of the complexes (data not shown). Complementary results on the important features of the HSF2 binding site We had noticed the very good affinity of HSF2 for the oligonucleotide Shvalspz. In parallel with the SELEX assay, Fig. 6. Comparison of the sequences of Sel0 and Sel4. The semirandom sequence oligonucleotides before selection (Sel0) or after the fourth round of selection (Sel4) were sequenced as a pool. Fig. 5. Comparison of the affinity of the in vivo and i.v.s. HSF2 for the oligonucleotides 2U519, Shval and Shvalspz. 32 P-Radiolabeled oligo- nucleotides were incubated with i.v.s. or in vivo synthesized (embry- onic) HSF2 and the HSF2-DNA complexes (arrow) were visualized by EMSA and autoradiography. Ó FEBS 2002 Determining the optimal binding sequence for HSF2 (Eur. J. Biochem. 269) 2533 we studied, by EMSA experiments, which features of the oligonucleotide Shvalspz were crucial for the binding of HSF2. For that purpose, several double-stranded oligonu- cleotides were designed. In the oligonucleotides NOA and NOG, the third and fourth (top strand) imperfect GAA trimers of Shvalspz were replaced by a repetition of A, for NOA, or G, for NOG. In SHC, the third trimer was kept but the fourth imperfect trimer was replaced by a repetition of the dinucleotide GA. In MCL and MTH, the third trimer was replaced by a repetition of A. In MTH, the fourth trimer was placed at the right position according to the standard HSE sequence, i.e. seven nucleotides from the second trimer instead of eight nucleotides in MCL. In JUL, the 5¢ half (top strand) of Shvalspz, containing two perfect inverted GAA trimers, was repeated twice. The embryonic HSF2, present in E9.5 embryos or F9 cells crude extracts, could bind to JUL with a very good affinity (Fig. 9). The sequence of JUL, as well as the sequence of Shvalspz, are very close to the consensus binding sequence determined by the SELEX assay (they both contain the 8-mer core), which explains the very good affinity of HSF2 for those oligonucleotides. HSF2 could not bind to NOG (Fig. 9), NOA (not shown), MCL (Fig. 9) and MTH (not shown). HSF2 could bind to SHC but with a reduced affinity compared to Shvalspz (Fig. 9). These results suggest that three adjacent nGAAm pentamers are required for the binding of HSF2. Similar experiments were carried out with double-stran- ded oligonucleotides derived from two sequences selected by Fig. 7. Sequences of individual Sel6 clones. After the sixth round of selection, the selected oligonucleotides were subcloned in E. coli and individual clones were sequenced. Fig. 8. Consensus binding sequences for the purified embryonic HSF2 and for the recombinant HSF2. The consensus binding sequence for the purified embryonic mHSF2 was obtained from the sequences of individual Sel6 clones. 2534 M. Manuel et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the SELEX assay, CL39 and CL83. CL39 contains four GAA trimers while CL83 contains only three trimers. In the oligonucleotides 39woGAA and 83woGAA, the first GAA trimer (top strand) was replaced by a CAC trimer. In the oligonucleotide 83woTA, the TA dinucleotide between the second and third trimers (top strand) was replaced by the AC dinucleotide. HSF2 could bind to 39woGAA with a comparable affinity than to CL39 (Fig. 10A), but it could not bind to 83woGAA (Fig. 10B). This result confirms that the binding of HSF2 requires at least three nGAAm pentamers. HSF2 could bind to 83woTA but with a slightly reduced affinity compared to CL83 (Fig. 10B), confirming the importance of the dinucleotide TA. DISCUSSION In this article, we have compared the active HSF2 protein purified from EC cells (or present in E9.5 embryos or F9 cells crude extracts), called ÔembryonicÕ or Ôin vivo synthes- izedÕ, with the active HSF2 protein synthesized in vitro in reticulocyte lysates. The stability at various temperatures and cooperativity of HSF2 synthesized in these different contexts were analyzed and found different. Kroeger et al. [34] mentioned that the environment in which the HSF is synthesized determines its activation state. Indeed, in most cell lines or adult tissues, HSF2 is not constitutively active, while it is constitutively active when synthesized in E. coli or in reticulocyte lysates. But in our case, differences relate to a factor already active, either purified from F9 cells or synthesized in reticulocytes lysates. (a) HSE-binding properties at various temperatures are highly dependent on the context in which HSF2 was synthesized. The stability of the i.v.s. HSF2 and that of the embryonic purified HSF2 are markedly different. Progres- sive inactivation of HSE-binding activity, at 37 °C, is not observed for the i.v.s. HSF2 as it was for the protein purified from F9 cells. Instead, a significant stimulation of its DNA- binding activity is observed, as if the activation of HSF2 in reticulocyte lysates was incomplete and could be further achieved in the reaction buffer. This abnormal behavior is observed for short incubation times which explains that former results reported that incubation at 37 °Cfor60min had no effect on HSF2 DNA-binding activity [3]. (b) The i.v.s. HSF2 displays a higher cooperativity than the purified embryonic HSF2. The cooperativity displayed, in our hands, by the factor synthesized in vitro in reticulocyte lysates is in contrast with what was observed for an HSF2 factor synthesized in E. coli and subsequently purified [25]. Because of the differences observed between the HSF2 protein synthesized in vivo and the protein synthesized in reticulocyte lysates, we can conclude that the in vivo context of synthesis is very important to give HSF2 its properties. Therefore, we selected optimal binding sites for the purified embryonic HSF2, from a semi-random DNA library, and determined the consensus binding sequence for this embryonic factor. We found that the embryonic HSF2 (purified from, or present in crude extracts of F9 cells) preferred sites containing three or four nGAAm inverted pentamers and that it was unable to bind to sites containing only two adjacent pentamers. This is in contrast to what was previously found for a recombinant HSF2 produced in E. coli, which preferred sites containing two or three pentamers [25]. The consensus binding site for the embry- onic HSF2 is more stringent than the sites found for the recombinant factors HSF2 and HSF1, as it contains a 8-mer palindromic core TTCTAGAA that was very strongly selected, and as the other ÔNÕ positions of each pentamer are not random. The consensus binding site for the recombinant proteins HSF2 and HSF1 was determined by selecting sequences that could bind efficiently the factors from a totally random DNA library. In contrast, we used a semi- random DNA library for the SELEX assay to study the purified embryonic factor. This could be an explanation for the higher stringency found for the embryonic HSF2 consensus binding sequence, compared to what was described for the recombinant proteins, as the use of a semi-random DNA library, in which two inverted GAA trimers were fixed, allowed a much faster enrichment in sequences binding the transcription factor with a high affinity. However, the inability of the embryonic HSF2 to bind sequences containing only two nGAAm pentamers shows that this factor has a higher requirement for its binding site than the recombinant factor. Using the program TARGETFINDER [35], we identified several genes containing the consensus binding site for the embryonic HSF2 in their promoter. Those genes are Fig. 10. Comparison of the affinity of embryonic HSF2, present in F9 cells extracts, for the oligonucleotides CL39 and 39woGAA and for the oligonucleotides CL83, 83woGAA and 83woTA. 32 P-Radiolabeled oligonucleotides were incubated with F9 cells extracts and the HSF2– DNA complexes were visualized by EMSA and autoradiography. HSF2 binds CL83 and CL39 with a comparable affinity, as determined by the quantification of the complexes (not shown). The binding data presented in (A) and (B) are from separate gels. Fig. 9. Comparison of the affinity of embryonic HSF2, present in E9.5 embryos extracts, for the oligonucleotides Shvalspz, JUL, SHC, MCL and NOG. 32 P-Radiolabeled oligonucleotides were incubated with E9.5 embryo extracts and the HSF2–DNA complexes (arrow) were visualized by EMSA and autoradiography. Ó FEBS 2002 Determining the optimal binding sequence for HSF2 (Eur. J. Biochem. 269) 2535 currently being studied as genes potentially regulated by the transcription factor HSF2, taking advantage of our Hsf2 )/) mice [36]. We conclude from this study that the intrinsic properties of HSF2, illustrated by those of the purified embryonic factor, can be deeply modified by the cellular context in which it is synthesized. Therefore, HSF2 which is active for DNA-binding until midgestation (for a longer period in the brain) could have targets and transcriptional capacity different from one organ to another or at various times during the mouse development. The consensus binding site for the embryonic HSF2 will help us to identify new targets for this factor, that might be involved in developmental and differentiation processes. ACKNOWLEDGEMENTS We are grateful to Dr Agne ` s Delahodde and Dr Olivier Jean-Jean for the helpful advice about the SELEX assay. This work and M. 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Biochem. 26 9, 25 27 25 37 (20 02) Ó FEBS 20 02 doi:10.1046/j.14 32- 1033 .20 02. 029 17.x displays a strong DNA -binding activity in EC cells [20 ,21 ], the HSE region of