Identification of Ewing’s sarcoma protein as a G-quadruplex DNA- and RNA-binding protein Kentaro Takahama1,*, Katsuhito Kino2,*, Shigeki Arai3, Riki Kurokawa3 and Takanori Oyoshi1 Department of Chemistry, Faculty of Science, Shizuoka University, Japan Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Kagawa, Japan Division of Gene Structure and Function, Saitama Medical University Research Center for Genomic Medicine, Japan Keywords Ewing’s sarcoma; G-quadruplex DNA; G-quadruplex RNA; RGG motif; RNA-binding protein Correspondence T Oyoshi, Department of Chemistry, Faculty of Science, Graduate School of Science, Shizuoka University, 836 Oya, Suruga, Shizuoka 422-8529, Japan Fax: +81 54 237 3384 Tel: +81 54 238 4760 E-mail: stohyos@ipc.shizuoka.ac.jp *These authors contributed equally to this work (Received 27 August 2010, revised 23 December 2010, accepted 13 January 2011) The Ewing’s sarcoma (EWS) oncogene contains an N-terminal transcription activation domain and a C-terminal RNA-binding domain Although the EWS activation domain is a potent transactivation domain that is required for the oncogenic activity of several EWS fusion proteins, the normal role of intact EWS is poorly characterized because little is known about its nucleic acid recognition specificity Here we show that the Arg-Gly-Gly (RGG) domain of the C-terminal in EWS binds to the G-rich single-stranded DNA and RNA fold in the G-quadruplex structure Furthermore, inhibition of DNA polymerase on a template containing a human telomere sequence in the presence of RGG occurs in an RGG concentration-dependent manner by the formation of a stabilized G-quadruplex DNA–RGG complex In addition, mutated RGG containing Lys residues replacing Arg residues at specific Arg-Gly-Gly sites and RGG containing Arg methylated by protein arginine N-methyltransferase decrease the binding ability of EWS to G-quadruplex DNA and RNA These findings suggest that the RGG of EWS binds to G-quadruplex DNA and RNA via the Arg residues in it doi:10.1111/j.1742-4658.2011.08020.x Introduction The current knowledge of Ewing’s sarcoma (EWS) derives primarily from studies of a group of dominant oncogenes that arise due to chromosomal translocations in which EWS is fused to a variety of cellular transcription factors [1–3] EWS fusion proteins are very potent transcription activators that depend on the EWS N-terminal domain and a C-terminal DNA-binding domain contributed by the fusion partner [4–9] EWS ⁄ ATF1 is a potent constitutive activator of ATFdependent promoters [10] The EWS N-terminal binds directly to the RNA polymerase II subunit hsRPB7 and this interaction is thought to be important for transactivation [11] In contrast to EWS fusion proteins, however, the normal function and the nucleic acid-binding properties of EWS remain poorly characterized EWS belongs to a family that includes the closely related proteins translocated in liposarcoma and the TATA-binding protein-associated factor 15 which are involved in several aspects of gene expression [12–15] This protein family contains the transcriptional activation domain in the N-terminal region and the RNA-binding domain Abbreviations dsHtelo, human telomere duplex DNA; EAD, Ewing’s sarcoma activation domain; EMSA, electrophoretic mobility shift assay; ETS, external transcribed spacer; EWS, Ewing’s sarcoma; FMRP, fragile X mental retardation protein; GST, glutathione S-transferase; Htelo, human telomere DNA; mut Htelo, mutated human telomere; mut rHtelo, mutated human telomere RNA; PRMT3, protein arginine N-methyltransferase 3; RBD, RNA-binding domain; rHtelo, human telomere RNA; RRM, RNA recognition motif; ZnF, zinc finger 988 FEBS Journal 278 (2011) 988–998 ª 2011 The Authors Journal compilation ª 2011 FEBS K Takahama et al Results and Discussion Several DNA-binding proteins that bind to G-quadruplex DNA have been investigated in vitro [28–36] Hanakahi et al [26] reported that the four RBD and the Arg-Gly-Gly repeats of nucleolin, which is involved in transcription, rRNA processing and ribosome assembly, can bind to G-quadruplex DNA formed from the external transcribed spacer region of human rDNA, ETS-1 We performed an EMSA of EWS and ETS-1 to investigate the ability of EWS to bind to G-quadruplex DNA (Fig 1A, Table 1) Recombinant EWS, which contains RBD in the C-terminal region comprising RRM, ZnF and three RGG (RGG1, RGG2 and RGG3) for binding to nucleic B – – – EWS – ssDNA S Htelo dsHtelo EWS – EWS ssDNA L ETS-1 EWS A EWS (RBD) in the C-terminal region as multiple domains involved in nucleic acid–protein interactions: an RNA recognition motif (RRM) flanked by two regions in Arg-Gly-Gly repeats (RGG) and a C2C2 zinc finger (ZnF) with an RGG domain in the C-terminal [16,17] They bind to RNA as well as single- and doublestranded DNA [18–20] In the case of EWS, the C-terminal amino acids that constitute RGG specifically bind to poly G and poly U RNA in vitro [8] On the other hand, Hume et al [15] suggested that EWS binds to the proximal-element DNA of the macrophage-specific promoter of the CSF-1 receptor gene The RGG domain, initially identified as a singlestranded RNA-binding motif in hnRNP U, is reported to be the G-quadruplex RNA-binding motif in the fragile X mental retardation protein (FMRP) and the G-quadruplex DNA-binding motif in nucleolin [21– 27] The RGG domain of FMRP, which is an RNAbinding protein involved in nerve cell differentiation, interacts with the G-quartet forming RNA [22–25] In addition, the RBD and the RGG domain of nucleolin, a DNA-binding protein contributing to the transcription of ribosomal RNA, bind to the G-quadruplex forming ribosomal DNA [26] Moreover, nucleolin binds to the c-myc G-quadruplex DNA with high affinity in vitro [27] Little is known, however, about the DNA and RNA recognition specificity of EWS, which contains three RGG domains To gain further insight into the nucleic acid–EWS interaction, we performed an electrophoretic mobility shift assay (EMSA) with EWS and several G-quadruplex or single- or double-stranded DNA and RNA Here, we show that EWS specifically targets G-quadruplex DNA and RNA in vitro We also determined that the specificity of G-quadruplex recognition depends on the guanidinium group of the Arg in the RGG domain in the C-terminal of EWS Identification of Ewing’s sarcoma protein Fig Affinity of EWS for binding to G-quadruplex DNA (A) EMSA was performed with EWS (lanes and 4) and 32P-labeled ETS-1 (lanes and 4) or ssDNA L (lanes and 2) (B) EMSA was performed with EWS (lanes 2, and 6) and 32P-labeled Htelo (lanes and 4), dsHtelo (lanes and 6) or ssDNA S (lanes and 2) The structures of DNAs used as probes are indicated above each lane The DNA–protein complexes were resolved by 6% PAGE and visualized by autoradiography acids, was expressed in Escherichia coli as proteins fused to glutathione S-transferase (GST) and purified using glutathione agarose 32P-labeled ETS-1 was first incubated for 24 h in 100 mm KCl to allow for quadruplex formation and then with GST-tag-digested EWS for h at room temperature The EWS–DNA complexes were resolved by 6% PAGE and visualized by autoradiography Binding analyses revealed that EWS binds to the G-quadruplex formed from the ETS-1, but not to the control single-stranded DNA Similar results were obtained with a human telomere DNA (Htelo) in the presence of 100 mm K+ (Fig 1B, Table 1) The results demonstrated that EWS binds to Htelo, but not to human telomere duplex DNA (dsHtelo), in the presence of K+ The results of previous studies indicated that Htelo in a K+ ion-containing solution exists as an equilibrium G-quadruplex formation of some antiparallel form of the hybrid parallel ⁄ antiparallel (3 + 1) form together with the parallel propeller form and the basket type [37–42] These findings indicate that EWS binds to G-quadruplex DNAs formed from different synthetic oligonucleotides and thus appears to recognize the G-quadruplex DNA structure independently of the sequence context We further investigated the region of EWS that contributes to the G-quadruplex binding specificity by FEBS Journal 278 (2011) 988–998 ª 2011 The Authors Journal compilation ª 2011 FEBS 989 Identification of Ewing’s sarcoma protein A 287 K Takahama et al 347 469 501 544 656 RBD RGG1 EAD EWS RRM RGG2 ZnF RGG3 EAD RGG1 RRM-RGG2-ZnF C D RGG3 – – F E RGG3 Htelo mut Htelo RGG3 – EAD RGG1 B RRM-RGG2-ZnF RGG3 RGG3 dsHtelo – RGG3 RGG3 Htelo – – 1x 10x 100x Full length Pausing product Primer comparing the behavior of various mutant recombinant proteins, i.e the EWS activation domain (EAD), RGG1, RRM–RGG2–ZnF and RGG3, with regard to Htelo (Fig 2A) RGG3 interacted with Htelo in EMSA, whereas the proteins containing EAD, RGG1 and RRM–RGG2–ZnF did not bind to Htelo (Fig 2B) The RGG domain in FMRP has a closely spaced Arg-Gly-Gly repeat, which is necessary for G-quadruplex structure binding [23,24] RGG3, containing 12 RGG repeats, of EWS binds to the 990 – 1x 10x 100x Fig Structural features of EWS and DNA-binding specificities of RGG3 (A) Schematic representation of the deletion mutants constructed to map the Htelo-binding specificity of each one of the EWS AD (residues 1–287); RGG (288–347); RRM (residues 348–469); RGG (residues 450–501); ZnF (residues 502–544); RGG (residues 545– 656) (B) DNA-binding activities of EAD (lane 2), RGG1 (lane 3), RRM–RGG2–ZnF (lane 4) and RGG3 (lane 5) EMSA was performed with these proteins and 32P-labeled Htelo (C) EMSA was performed with RGG3 (lanes and 4) and 32P-labeled Htelo (lanes and 2) or mut Htelo (lanes and 4) (D, E) Binding competition assay, assaying binding of RGG3 to 32P-labeled Htelo in the presence of unlabeled dsHtelo (D) or Htelo (E) at the indicated molar ratios of unlabeled ⁄ labeled DNA The DNA–protein complexes were resolved by 6% PAGE and visualized by autoradiography (F) DNA polymerase arrest assays Primer extension reactions were performed with rTaq DNA polymerase The primer, full-length primer extension products and DNA polymerase arrest products are indicated by arrows Extension through the template after incubation in increasing concentrations of RGG3 The concentrations of RGG3 were lM (lane 1), 0.2 lM (lane 2), 0.5 lM (lane 3) and lM (lane 4) G-quadruplex structure, whereas RGG1, containing six fewer RGG repeats than RGG3, does not (Table 2) Additional binding studies demonstrated that recombinant RGG3 does not bind to dsHtelo or single-stranded DNA (Fig S1, Table 1) These studies revealed that RGG3 of EWS binds mainly to the G-quadruplex To test whether formation of the G-quadruplex is necessary for RGG3 binding, we assayed the binding of RGG3 with Htelo in the presence of K+ or Li+ FEBS Journal 278 (2011) 988–998 ª 2011 The Authors Journal compilation ª 2011 FEBS K Takahama et al Identification of Ewing’s sarcoma protein Table Sequence of oligonucleotides used in EMSA and CD spectroscopy Oligonucleotides were diluted to 0.5 mM (base concentration) in 50 mM Tris ⁄ HCl (pH 7.5) in the presence of 100 mM KCl or 100 mM LiCl, as specified Duplex annealing or quadruplex formation was performed by heating samples to 95 °C on a thermal heating block and cooling to °C at a rate of °CỈmin–1 Name Sequence ssDNAS ssDNA L ETS-1 Htelo dsHtelo mut Htelo rHtelo mut rHtelo d(CATTCCCACCGGGACCACCAC) d(CATTCCCACCGGGACCACCACCATTCCCACCGGGACCACCAC) d(TCTCTCGGTGGCCGGGGCTCGTCGGGGTTTTGGGTCCGTCC) d[ AGGG(TTAGGG) ] d[ AGGG(TTAGGG) ]⁄ d[CCCTAA) CCCT] ( 3 d[ AGGG(TTAGTG) TTAGGGJ r (UUAGGG) r[ UUAGGG(UUAGUG) UUAGGG] Table Amino acid sequences of RGG1 and RGG3 RGG1 RGG3 PGENRSMSGPDNRGRGRGGFDRGGMSRGGRGGGRGGMG SAGERGGFNKPGGPMDEGPDLDLGPPVDP APKPEGFLPPPFPPPGGDRGRGGPGGMRGGRGGLMDRGGP GGMFRGGRGGDRGGFRGGRGMDRGGFGGGRRGGPGGPP GPLMEQMGGRRGGRGGPGKMDKGEHRQERRDRPY Figures S1 and S2 show that RGG3 binding to Htelo in the presence of Li+, which did not form the G-quadruplex as confirmed by CD spectroscopy, was blocked To further test whether RGG3 bound to Htelo folds into a G-quadruplex, we analyzed the binding between RGG3 and a mutated human telomere (mut Htelo) that replaces G with T at positions and 15, which destabilized the G-quadruplex formation, as confirmed by CD and UV spectroscopy (Figs 2C, S2, Table 1) The analysis showed that RGG3 binds to the folded Htelo G-quadruplex, but not to unfolded mut Htelo despite containing one TTAGGG sequence Furthermore, competitive experiments performed in the presence of cold competitor Htelo or dsHtelo showed that Htelo effectively competed for binding, whereas dsHtelo had no effect, even at a 100-fold molar excess (Fig 2D, E) These findings suggest that RGG3 binds to G-quadruplex DNA with structure specificity Having found that EWS binds to G-quadruplex conformations and not to single- and double-stranded conformations by RGG3, we aimed to determine whether RGG3 of EWS modulates the formation or unwinding of Htelo G-quadruplex DNA To determine whether the RGG3 binding affected the stability of the G-quadruplex structure of Htelo, we performed a polymerase stop assay as described previously [43] The 32 P-labeled 25-mer primer annealed to the 3¢ end of the template and could be extended by a DNA polymerase upon the addition of the dNTPs If complete extension of the primer occurred, a full-length 76-mer product would be formed Factors that promote and stabilize intramolecular G-quadruplex formation, however, led to a specific pausing site on the template This assay showed that the stopping site corresponded to the base located 3¢ to the first guanine base involved in G-quadruplex formation (Fig 2F, lane 1) Moreover, as the RGG3 protein concentration increased, the full-length 76-mer product decreased, and the stopping site product increased (Fig 2F, lanes 2–4) Thus, these results indicate that RGG3 binds to and stabilizes the folded G-quadruplex formation To test whether RGG3 contributes not only to the G-quadruplex DNA binding, but also to G-quadruplex RNA binding, we assayed the binding of RGG3 with a human telomere RNA (rHtelo) in the presence of K+, which exists as a G-quadruplex formation of the parallel propeller form [44] Figure 3(A, B) shows that RGG3 bound to rHtelo in the presence of K+, whereas binding between RGG3 and a mutated human telomere RNA (mut rHtelo) that replaces G with T at positions and 15 destabilized the G-quadruplex formation, as confirmed by CD and UV spectroscopy (Fig S2, Table 1) Furthermore, competitive experiments performed in the presence of cold competitor rHtelo or mut rHtelo showed that rHtelo effectively competed for binding, whereas the mut rHtelo had no effect, even at a 100-fold molar excess (Fig 3C, D) These findings suggest that RGG3 also binds to G-quadruplex RNA with structure specificity To elucidate the ability of RGG3 to bind the G-quadruplex, various concentrations of RGG3 were incubated with 5¢ 32P-labeled Htelo or rHtelo in a K+ solution As the RGG3 concentration increased, the free DNA or RNA decreased, and the higher molecular weight complex increased (Fig 4) The mobility shift data were fitted to a hyperbolic equation to give a Kd of 13 ± nm (Htelo) and 10 ± nm (rHtelo) In comparison with RGG3, the full-length EWS and RBD containing RGG3 bound to Htelo with FEBS Journal 278 (2011) 988–998 ª 2011 The Authors Journal compilation ª 2011 FEBS 991 Identification of Ewing’s sarcoma protein K Takahama et al B A – D C RGG3 rHtelo – – – RGG3 – RGG3 – RGG3 Htelo rHtelo RGG3 mut rHtelo rHtelo 1x 10x 100x RGG3 mut rHtelo – – 1x 10x 100x Fig Protein–nucleic acid complexes (A) EMSA was performed with RGG3 (lanes and 4) and 32P-labeled Htelo (lanes and 2) or rHtelo (lanes and 4) (B) EMSA was performed with RGG3 (lanes and 4) and 32P-labeled rHtelo (lanes and 2) or mut rHtelo (lanes and 4) (C, D) Binding competition assay assaying binding of RGG3 to 32P-labeled rHtelo in the presence of unlabeled rHtelo (C) or mut rHtelo (D) at the indicated molar ratios of unlabeled ⁄ labeled DNA The DNA–protein complexes were resolved by 6% PAGE and visualized by autoradiography Kd = 30 ± and 14 ± nm, respectively (Fig S3) The EAD domain therefore inhibited the high-affinity Htelo binding of RGG3 and RBD The ability of RGG3 and RBD to repress transcription activation by EAD raised the possibility that RGG3 and RBD block the interaction between the EAD and RNA polymerase II subunit [11,45] The interaction between the EAD and RGG3 might inhibit the high-affinity Htelo binding of RGG3 992 To gain further insight into the induction of G-quadruplex formations by RGG3 of EWS, we performed a CD spectroscopic analysis that was conducted with Htelo in the presence of various amounts of RGG3 The CD spectrum of Htelo, a hybrid (3 + 1) form, showed a strong positive band at 290 nm and a negative band at around 235 nm, whereas the addition of ratio excess of RGG3 led to an increase in ellipticity and shifted the spectrum from a strong positive band to 265 nm (Fig 5), which is characteristic of the parallel form and consistent with the results of previous CD studies [40–42] These data indicate that RGG3 binds to the Htelo G-quadruplex and changes the hybrid (3 + 1) G-quadruplex formation of Htelo Moreover, it may provide a model showing the change from the hybrid (3 + 1) G-quadruplex to the parallel form with the association of RGG3 Incubation of rHtelo with RGG3 did not alter the G-quadruplex RNA, however, as demonstrated by CD spectrum analysis (data not shown) Rajpurohit et al [46] reported that binding of the recombinant hnRNP A1 protein to single-stranded nucleic acid is reduced upon enzyme methylation of Arg To evaluate the role of Arg in RGG3 on G-quadruplex DNA recognition, we performed EMSA using Htelo with RGG3 methylated by protein arginine N-methyltransferase (PRMT3) (Fig 6) In vitro methylation of the recombinant EWS with PRMT3 showed that PRMT3 is responsible for the asymmetric dimethylations of specific Arg in the RGG region [47] In our study, the methylation of the RGG3 by PRMT3 with [3H]AdoMet as a methyl donor was monitored with a liquid scintillation counter (Fig S4) RGG3-methylated Arg did not bind to Htelo (Fig 6, lane 6), whereas PRMT3 and AdoMet did not inhibit the Htelo binding of RGG3 (Fig 6, lanes 1–4) Similarly, RGG3-methylated Arg did not bind to rHtelo, whereas PRMT3 and AdoMet did not inhibit the rHtelo binding of RGG3 (Fig S4) These results indicate that enzyme methylation of Arg reduces the binding of RGG3 to G-quadruplex DNA or RNA Previous results demonstrated that nine Arg are potential methylation sites within RGG3 that react with PRMT3 [47] We next created mutated RGG3 to precisely define the residues within RGG3 that bind to G-quadruplex DNA (Fig 6B) Simultaneous substitution of Arg by Lys in two (KGG3-2) Arg within RGG3 reduced G-quadruplex DNA binding and in six (KGG3-6) and four Arg (KGG3-4) within RGG3, eliminated G-quadruplex DNA binding despite the basic nature of the Lys side-chain (Fig 6C) Similarly, KGG3-2, KGG3-4 and KGG3-6 reduced G-quadruplex RNA binding (Fig S5) These findings indicate FEBS Journal 278 (2011) 988–998 ª 2011 The Authors Journal compilation ª 2011 FEBS K Takahama et al Identification of Ewing’s sarcoma protein A B Parallel form 50 100 150 200 250 RGG3 concentration (nM) Hybrid (3 + 1) form Molar ellipticity (106 · mdeg · M–1 · cm–1) –2 240 280 300 260 Wavelength (nm) 16 32 64 125 250 0.8 0.6 0.4 0.2 220 320 Fig CD of Htelo in the presence of various amounts of RGG3 Titration of Htelo with RGG3 (1.2, 1.0, 0.8, 0.6, 0.4, 0.2, 0.1 and equiv of RGG3) in 100 mM KCl and 50 mM Tris ⁄ HCl (pH 7.5) The concentration of DNA was 0.2 mM base concentration Line colors: black = equiv.; blue = 0.1 equiv.; cyan = 0.2 equiv.; green = 0.4 equiv.; light green = 0.6 equiv.; yellow = 0.8 equiv.; orange = 1.0 equiv.; red = 1.2 equiv that Arg between amino acids 589 and 597 within RGG3 are important for the binding of RGG3 to G-quadruplex DNA and RNA In ssDNA and ssRNA RGG3 (nM) Fraction of RNA bound Fig Binding affinity of RGG3 to Htelo or rHtelo The DNA or RNA concentration was fixed at nM, whereas the concentration of RNase-treated RGG3 added to the binding reaction was varied, as shown above each lane The equilibrium-binding curve was obtained by calculating the fraction of Htelo (A) or rHtelo (B) bound at varying RGG3 concentrations Kd was determined by fitting to the equation (see Materials and methods) The DNA–protein complexes were resolved by 6% PAGE and visualized by autoradiography Fraction of DNA bound RGG3 (nM) 0.8 0.6 0.4 0.2 0 16 32 64 125 250 50 100 150 200 250 RGG3 concentration (nM) recognition, the methylation of Arg in a peptide or a protein does not affect the binding strength [25,48,49] Methylated RGG3 of EWS inhibited G-quadruplex Htelo binding, but was able to bind mut Htelo (Fig S6) These findings indicate the importance of the guanidinium group of the Arg in RGG3 for binding to the G-quadruplex In conclusion, EWS appears to be a DNA- and RNA-binding protein that recognizes the G-quadruplex structure It remains unclear, however, whether the role of EWS in transcription or other functions is determined by its ability to target a specific DNA and RNA structure Rossow & Janknecht [50] reported that overexpression of EWS in RK13 and AKR cells leads to the activation of the c-fos, Xvent-2 and ErbB2 promoters, indicating that EWS functions as a transcriptional cofactor EWS, however, has not been reported to bind to double-stranded DNA in these promoters The c-fos and ErbB2 promoters contain G-rich sequences that could potentially form G-quadruplex structures [51,52] On the basis of a combination of in silico and experimental approaches, Verma et al [53,54] reported an enriched sequence with the potential to adopt the G-quadruplex motifs near transcription start sites These findings suggest that G-quadruplex motif-mediated regulation is a more common mode of transcription control On the other hand, Dejardin & Kingston [55] purified human telomeric chromatin using proteomics of isolated FEBS Journal 278 (2011) 988–998 ª 2011 The Authors Journal compilation ª 2011 FEBS 993 Identification of Ewing’s sarcoma protein K Takahama et al PRMT3 + + – RGG3 – + – 545 + – + – RGG3 + + C 656 + – 587 RGG3 KGG3-2 KGG3-4 KGG3-6 KGG3-4 KGG3-6 – + + + KGG3-2 – RGG3 B A AdoMet 610 MF RGG RGG D RGG F RGG RGMD RGG F MF KGG KGG D RGG F RGG RGMD RGG F MF KGG KGG D KGG F KGG RGMD RGG F MF KGG KGG D KGG F KGG KGMD KGG F Fig Identification of significant residues at RGG3 for G-quadruplex binding ability (A) Ability of RGG3 to bind to Htelo in the presence (+) or in the absence ()) of PRMT3 or AdoMet RGG3 (lanes 2, and 6) was incubated with (lanes 1, 2, and 6) or without (lanes and 4) PRMT3 in a potassium buffer with (lanes 3–6) or without (lanes and 2) AdoMet (B) Schematic illustration of amino acids 587–610 within RGG3 (residues 545–656) The point mutations are shown in bold (C) EMSA of RGG3 (lane 2), KGG3-2 (lane 3), KGG3-4 (lane 4) and KGG36 (lane 5) using Htelo The DNA–protein complexes were resolved by 6% PAGE and visualized by autoradiography chromatin segments and identified that the protein translocated in liposarcoma, which is related to EWS as a subgroup within the RNP family of RNA-binding proteins containing RRM and RGG domains, binds to telomeres Further studies are required to identify the role of EWS and the possible function of such G-quadruplex structures in genomic DNA Materials and methods Preparation, expression and purification of GST fusion proteins The EWS cDNA was cloned into the pGEX6P-1 vector between the EcoRI and XhoI sites for expression as an N-terminal GST fusion protein (pGEX–EWS) pGEX–EAD, pGEX–RGG1, pGEX–RRM–RGG2–ZnF and pGEX– RGG3 vectors contain a PCR encoding EWS amino acids 1– 287, 288–347, 348–544 and 545–656, respectively, cloned in pGEX6P-1 using the following sets of primers: EWS forward d(CGG AAT TCA TGG CGT CCA CGG ATT ACA G) and EWS reverse d(CGC TCG AGT CAC TAG TAG GGC CGA TCT CTG C), for pGEX–EWS; EAD forward d(CGG AAT TCA TGG CGT CCA CGG ATT ACA G) and EAD reverse d(CGC TCG AGT CAT CCG GAA AAT CCT CCA GAC T), for pGEX–EAD; RGG1 forward d(CGG AAT TCC CAG GAG AGA ACC GGA GCA T) and RGG1 reverse d(CGC TCG AGT CAA TCA AGA TCT GGT CCT TCA TCC ATG G), for pGEX–RGG1; RRM– RGG2–ZnF forward d(CGG AAT TCC TAG GCC CAC CTG TAG ATC C) and RRM–RGG2–ZnF reverse d(CGC TCG AGT CAC TTA CAC TGG TTG CAC TCT GTT CTC C), for pGEX–RRM–RGG2–ZnF; and RGG3 forward d(CGG AAT TCG CCC CAA AGC CTG AAG GCT T) and RGG3 reverse d(CGC TCG AGT CAC TAG TAG GGC CGA TCT CTG C), for pGEX–RGG3 pGEX– 994 KGG3-2, pGEX–KGG3-4 and pGEX–KGG3-6 were obtained by replacing Arg with Lys in pGEX–RGG3 using a KOD -Plus- mutagenesis kit (Toyobo, Japan) To construct pGEX–KGG3-2, PCR was performed with pGEX–RGG3 as a template and the following primers: KGG3-2 forward d(AAA GGT GGC AAA GGT GGA GAC AGA GGT GGC TT) and KGG3-2 reverse d(GAA CAT TCC ACC GGG ACC ACC AC) pGEX–KGG3-4 was generated by PCR using pGEX–KGG2 as a template and the following primers: KGG3-4 forward d(AGA CAA AGG TGG CTT CAA AGG TGG CCG) and KGG3-4 reverse d(CCA CCT TTG CCA CCT TTG AAC A) PCR was conducted with pGEX–KGG3-4 as a template and the following primers: KGG3-6 forward d(GGC AAA GGC ATG GAC AAA GGT GGC TTT GG) and KGG3-6 reverse d(ACC TTT GAA GCC ACC TTT GTC TCC ACC), for pGEX–KGG36 All reactions were performed according to the manufacturer’s protocol for the KOD-Plus- mutagenesis kit (Toyobo) Escherichia coli strain BL21 (DE3) pLysScompetent cells were transformed with the vectors, and the transformants were grown at 37 °C in a Luria Bertani medium containing ampicillin (0.1 mgỈmL)1) Protein expression was induced at A600 = 0.6 with 0.1 mm isopropyl b-d-1-thiogalactopyranoside The cells were then grown for an additional 16 h at 25 °C and harvested by centrifugation (6400 g for 20 min) Pellets were resuspended in buffer A (100 mm Tris ⁄ HCl pH 7.5, 150 mm NaCl, mm EDTA acid and mm dithiothreitol) and lysed by sonication (model UR-20P, Tomy Seiko, Tokyo, Japan) at °C The supernatants containing the expressed proteins were centrifuged for 15 at 16 200 g at °C, and the proteins were then purified by glutathione agarose (Sigma, St Louis, MO, USA) The supernatant and glutathione agarose were incubated with gentle mixing for h at °C; resin was washed with buffer A at °C Proteins were eluted with buffer B (50 mm Tris ⁄ HCl pH 9.5, 20 mm reduced glutathione and mm dithiothreitol) Buffer B of the elution was exchanged FEBS Journal 278 (2011) 988–998 ª 2011 The Authors Journal compilation ª 2011 FEBS K Takahama et al with buffer C (50 mm Tris ⁄ HCl pH 7.5, 100 mm KCl and mm dithiothreitol) or buffer D (50 mm Tris ⁄ HCl pH 7.5, 100 mm LiCl and mm dithiothreitol) by dialysis The protein concentrations were determined using the BCA Protein Assay Kit (Thermo Scientific, USA) For all experiments, GST-tag was digested according to the manufacturer’s instructions (GE Healthcare, Precision Protease, Little Chalfont, UK), and 20 nmol of each protein was incubated with 20 lg RNase A (Nippon Gene, Tokyo, Japan) at °C for 16 h before use EMSA Labeled oligonucleotides were diluted to 0.2 mm (base concentration) in 50 mm Tris ⁄ HCl (pH 7.5) in the presence of 100 mm KCl or 100 mm LiCl, as specified Duplex annealing or quadruplex formation was performed by heating samples to 95 °C on a thermal heating block and cooling to °C at a rate of °CỈmin)1 Binding reactions were performed in a final volume of 20 lL using 100 fmol of the labeled oligonucleotide and a varying concentration (0– 2.5 lm) of purified proteins in a binding buffer (50 mm Tris ⁄ HCl pH 7.5, 0.5 mm EDTA, 0.5 mm dithiothreitol, 0.1 mgỈmL)1 bovine serum albumin, lgỈmL)1 calf thymus DNA and 100 mm KCl or 100 mm LiCl) After the samples were incubated for h at 25 °C, they were loaded on a 6% polyacrylamide (acrylamide ⁄ bisacrylamide = 19 : 1) nondenaturing gel; 0.5· TBE with 20 mm KCl was used, both in the gel and as the electrophoresis buffer Electrophoresis was performed at 10 VỈcm)1 for h at °C The gels were exposed in a phosphorimager cassette and imaged (Personal Molecular Imager FX; Bio-Rad, Hercules, CA, USA) Bands were quantified using imagequant software The data were plotted as u (1 fraction of free DNA) versus the protein concentration to determine the Kd, which is equal to the protein at which half of the free DNA is bound Kd were extracted by nonlinear regression using Microsoft Excel 2007 and the following equation: u = [P] ⁄ {Kd + [P]} DNA polymerase stop assay This assay was adapted from the method described by Han et al [43] The 25-mer primer was 5¢-labeled with 32 P, mixed with the 76-mer template DNA and annealed as described above The polymerase reaction was performed in a final volume of 20 lL using 20 fmol of the duplex and various amounts of purified RGG3 in a binding buffer (50 mm Tris ⁄ HCl pH 7.5, mm dithiothreitol, 100 lgỈmL)1 bovine serum albumin, lgỈmL)1 calf thymus DNA and 100 mm KCl) RGG3 was incubated with the duplex for h at room temperature The polymerase extension reaction was initiated by adding Taq polymerase, dNTP (1 mm each) and MgCl2 (10 mm) The reaction was incubated at 30 °C for 10 and then stopped by adding an equal volume of a stop buffer (95% formamide, Identification of Ewing’s sarcoma protein 10 mm EDTA, 10 mm NaOH, 0.1% bromophenol blue and 0.1% xylenecyanol) Extension products were separated on a 12% polyacrylamide (acrylamide ⁄ bisacrylamide = 19 : 1) gel; 1· TBE was used, both in the gel and as the electrophoresis buffer Electrophoresis was performed at 1500 V for h at °C, and gels were visualized on a phosphorimager CD spectroscopy CD spectra were recorded on a CD spectrometer model J-500A (Jasco) The CD spectra of Htelo (0.2 mm base concentration) and RGG3 (1.2, 1.0, 0.8, 0.6, 0.4, 0.2, 0.1, 0) equivalent to Htelo DNA (RGG3 ⁄ DNA) in 50 mm Tris ⁄ HCl (pH 7.5) and 100 mm KCl were recorded using a 0.2 cm path length cell at 25 °C The 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responds to CREB and AP-1 is developmentally regulated in vivo PLoS ONE 5, e11235 53 Verma A, Halder K, Halder R, Yadav VK, Rawal P, Thakur RK, Mohd F, Sharman A & Chowdhury S (2008) Genome-wide computational and expression analyses reveal G-quadruplex DNA motifs as conserved cis-regulatory elements in human and related species J Med Chem 51, 5641–5649 FEBS Journal 278 (2011) 988–998 ª 2011 The Authors Journal compilation ª 2011 FEBS 997 Identification of Ewing’s sarcoma protein K Takahama et al 54 Verma A, Yadav VK, Basundra R, Kumar A & Chowdhury S (2009) Evidence of genome-wide G4 DNA-mediated gene expression in human cancer cell Nucleic Acids Res 37, 4194–4204 55 Dejardin J & Kingston RE (2009) Purification of proteins associated with specific genomic loci Cell 136, 175–186 Supporting information The following supplementary material is available: Fig S1 Affinity of RGG3 for binding to a G-quadruplex DNA and RNA Fig S2 CD spectra of DNAs and RNAs Fig S3 Binding affinity of EWS and RBD to Htelo 998 Fig S4 In vitro arginine methylation of RGG3 by PRMT3 Fig S5 Identification of significant residues at RGG3 for rHtelo binding ability Fig S6 Ability of RGG3 and RGG3 methylated by PRMT3 to bind to G-quadruplex This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 278 (2011) 988–998 ª 2011 The Authors Journal compilation ª 2011 FEBS ... protein arginine methyltransferase and toward the Ewing sarcoma protein and a peptide Proteins 61, 164–175 48 Raman B, Guarnaccia C, Nadassy K, Zakhariev S, Pintar A, Zanuttin F, Frigyes D, Acatrinei... 4) and 32P-labeled ETS-1 (lanes and 4) or ssDNA L (lanes and 2) (B) EMSA was performed with EWS (lanes 2, and 6) and 32P-labeled Htelo (lanes and 4), dsHtelo (lanes and 6) or ssDNA S (lanes and. .. ACA G) and EWS reverse d(CGC TCG AGT CAC TAG TAG GGC CGA TCT CTG C), for pGEX–EWS; EAD forward d(CGG AAT TCA TGG CGT CCA CGG ATT ACA G) and EAD reverse d(CGC TCG AGT CAT CCG GAA AAT CCT CCA GAC