Phosphorylated Intrinsically Disordered Region of FACT Masks Its Nucleosomal DNA Binding Elements
2009; Elsevier BV; Volume: 284; Issue: 36 Linguagem: Inglês
10.1074/jbc.m109.001958
ISSN1083-351X
AutoresYasuo Tsunaka, Junko Toga, Hiroto Yamaguchi, Shin‐ichi Tate, Susumu Hirose, Kosuke Morikawa,
Tópico(s)DNA Repair Mechanisms
ResumoFACT is a heterodimer of SPT16 and SSRP1, which each contain several conserved regions in the primary structure. The interaction of FACT with nucleosomes induces chromatin remodeling through the combinatorial action of its distinct functional protein regions. However, there is little mechanistic insight into how these regions cooperatively contribute to FACT functions, particularly regarding the recognition of nucleosomal DNA. Here, we report the identification of novel phosphorylation sites of Drosophila melanogaster FACT (dFACT) expressed in Sf9 cells. These sites are densely concentrated in the acidic intrinsically disordered (ID) region of the SSRP1 subunit and control nucleosomal DNA binding by dFACT. This region and the adjacent segment of the HMG domain form weak electrostatic intramolecular interactions, which is reinforced by the phosphorylation, thereby blocking DNA binding competitively. Importantly, this control mechanism appears to support rapid chromatin transactions during early embryogenesis through the dephosphorylation of some sites in the maternally transmitted dSSRP1. FACT is a heterodimer of SPT16 and SSRP1, which each contain several conserved regions in the primary structure. The interaction of FACT with nucleosomes induces chromatin remodeling through the combinatorial action of its distinct functional protein regions. However, there is little mechanistic insight into how these regions cooperatively contribute to FACT functions, particularly regarding the recognition of nucleosomal DNA. Here, we report the identification of novel phosphorylation sites of Drosophila melanogaster FACT (dFACT) expressed in Sf9 cells. These sites are densely concentrated in the acidic intrinsically disordered (ID) region of the SSRP1 subunit and control nucleosomal DNA binding by dFACT. This region and the adjacent segment of the HMG domain form weak electrostatic intramolecular interactions, which is reinforced by the phosphorylation, thereby blocking DNA binding competitively. Importantly, this control mechanism appears to support rapid chromatin transactions during early embryogenesis through the dephosphorylation of some sites in the maternally transmitted dSSRP1. FACT (facilitates chromatin transcription), 2The abbreviations used are: FACTfacilitates chromatin transcriptionAPasealkaline phosphataseIDintrinsically disorderedCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidEMSAelectrophoretic mobility shift assayHMGhigh-mobility groupAFMatomic force microscopyWTwild type. an evolutionarily conserved protein in eukaryotes, is a heterodimer consisting of structure-specific recognition protein-1 (SSRP1) and SPT16 with a larger molecular mass than SSRP1 (1.Orphanides G. Wu W.H. Lane W.S. Hampsey M. Reinberg D. 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Orphanides G. Studitsky V.M. Reinberg D. Science. 2003; 301: 1090-1093Crossref PubMed Scopus (647) Google Scholar). The FACT subunits also display a range of physical and genetic interactions with other factors (2.Reinberg D. Sims 3rd, R.J. J. Biol. Chem. 2006; 281: 23297-23301Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 3.Heo K. Kim H. Choi S.H. Choi J. Kim K. Gu J. Lieber M.R. Yang A.S. An W. Mol. Cell. 2008; 30: 86-97Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 6.Tan B.C. Chien C.T. Hirose S. Lee S.C. EMBO J. 2006; 25: 3975-3985Crossref PubMed Scopus (132) Google Scholar, 11.Shimojima T. Okada M. Nakayama T. Ueda H. Okawa K. Iwamatsu A. Handa H. Hirose S. Genes Dev. 2003; 17: 1605-1616Crossref PubMed Scopus (97) Google Scholar, 13.VanDemark A.P. Blanksma M. Ferris E. Heroux A. Hill C.P. Formosa T. Mol. Cell. 2006; 22: 363-374Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), suggesting that FACT directs several different functions by interacting with multiple complexes. facilitates chromatin transcription alkaline phosphatase intrinsically disordered 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid electrophoretic mobility shift assay high-mobility group atomic force microscopy wild type. At the molecular level, FACT initially binds nucleosomes and/or nucleosomal DNA, and then destabilizes the interactions between the H2A/H2B dimers and the H3/H4 tetramer within nucleosomes (2.Reinberg D. Sims 3rd, R.J. J. Biol. Chem. 2006; 281: 23297-23301Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). Therefore, most studies have so far focused on interactions between FACT and histones. For example, it has been previously reported that the C-terminal region of human SPT16 (hSPT16) directly binds to H2A/H2B dimers (12.Belotserkovskaya R. Oh S. Bondarenko V.A. Orphanides G. Studitsky V.M. Reinberg D. Science. 2003; 301: 1090-1093Crossref PubMed Scopus (647) Google Scholar). A recent study has revealed that the N-terminal amino peptidase-like domain of Schizosaccharomyces pombe SPT16 associates with the H3/H4 histones, suggesting that this SPT16 may contribute to binding, eviction, and/or deposition of all histones (14.Stuwe T. Hothorn M. Lejeune E. Rybin V. Bortfeld M. Scheffzek K. Ladurner A.G. Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 8884-8889Crossref PubMed Scopus (102) Google Scholar). However, it remains unclear how the FACT protein interacts with the nucleosomal DNA at the initial step of chromatin remodeling, although several studies have reported that the high-mobility group (HMG) box domain of SSRP1 binds to DNA nonspecifically or by recognizing specific structures of DNA (15.Yarnell A.T. Oh S. Reinberg D. Lippard S.J. J. Biol. Chem. 2001; 276: 25736-25741Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 16.Bruhn S.L. Pil P.M. Essigmann J.M. Housman D.E. Lippard S.J. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 2307-2311Crossref PubMed Scopus (235) Google Scholar, 17.Gariglio M. Ying G.G. Hertel L. Gaboli M. Clerc R.G. Landolfo S. Exp. Cell Res. 1997; 236: 472-481Crossref PubMed Scopus (29) Google Scholar). The heterodimeric FACT complex consists of several distinct structural domains and intrinsically disordered (ID) regions (Fig. 1A). The functional aspects of these folded domains or unstructured regions have not been clarified yet. The smaller subunit, SSRP1, is categorized as a member of the HMG family (16.Bruhn S.L. Pil P.M. Essigmann J.M. Housman D.E. Lippard S.J. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 2307-2311Crossref PubMed Scopus (235) Google Scholar), and is essential for cell (18.Schlesinger M.B. Formosa T. Genetics. 2000; 155: 1593-1606Crossref PubMed Google Scholar) and animal (19.Cao S. Bendall H. Hicks G.G. Nashabi A. Sakano H. Shinkai Y. Gariglio M. Oltz E.M. Ruley H.E. Mol. Cell. Biol. 2003; 23: 5301-5307Crossref PubMed Scopus (60) Google Scholar) viability. This protein contains two structural domains, a structure-specific recognition (SSRC) motif (amino acids 186–436 in Drosophila) and an HMG-box domain (amino acids 555–624 in Drosophila). In yeast, the bipartite SSRP1 analog consists of Pob3 and Nhp6 (20.Brewster N.K. Johnston G.C. Singer R.A. Mol. Cell. Biol. 2001; 21: 3491-3502Crossref PubMed Scopus (103) Google Scholar). Further sequence analysis revealed that the other regions could be distinguished as four evolutionarily conserved regions: an N-terminal region (amino acids 1–186 in Drosophila), an acidic ID region (amino acids 437–518 in Drosophila) with limited homology to nucleolin, an HMG-flanking basic ID segment (amino acids 519–554 in Drosophila), and a mixed charge ID region at the extreme C terminus (amino acids 625–723 in Drosophila). SSRP1 initially acts on the nucleosomal DNA in the chromatin remodeling process through the combinatorial action of its distinct functional protein regions, such as the HMG domain. It is thus crucial to clarify the molecular mechanism by which SSRP1 interacts with the nucleosomal DNA. Furthermore, while several studies have reported the phosphorylation of SSRP1 (8.Li Y. Keller D.M. Scott J.D. Lu H. J. Biol. Chem. 2005; 280: 11869-11875Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 21.Krohn N.M. Stemmer C. Fojan P. Grimm R. Grasser K.D. J. Biol. Chem. 2003; 278: 12710-12715Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 22.Keller D.M. Lu H. J. Biol. Chem. 2002; 277: 50206-50213Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar, 23.Keller D.M. Zeng X. Wang Y. Zhang Q.H. Kapoor M. Shu H. Goodman R. Lozano G. Zhao Y. Lu H. Mol. Cell. 2001; 7: 283-292Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar), it remains unclear how the phosphorylation of SSRP1 affects DNA binding. The ID regions of FACT are particularly intriguing. For instance, the C termini of SSRP1 and SPT16 consist of separate segments abundant in positive and negative charges (Fig. 1A). In fact, it has been demonstrated that the ID region of SPT16 is essential for the mRNA transcriptional elongation dictated by FACT (12.Belotserkovskaya R. Oh S. Bondarenko V.A. Orphanides G. Studitsky V.M. Reinberg D. Science. 2003; 301: 1090-1093Crossref PubMed Scopus (647) Google Scholar). These disordered regions lack well-defined three-dimensional structures, but in some proteins they fold into ordered conformations upon binding to their extrinsic targets (24.Dyson H.J. Wright P.E. Nat. Rev. Mol. Cell Biol. 2005; 6: 197-208Crossref PubMed Scopus (3038) Google Scholar, 25.Dunker A.K. Lawson J.D. Brown C.J. Williams R.M. Romero P. Oh J.S. Oldfield C.J. Campen A.M. Ratliff C.M. Hipps K.W. Ausio J. Nissen M.S. Reeves R. Kang C. Kissinger C.R. Bailey R.W. Griswold M.D. Chiu W. Garner E.C. Obradovic Z. J. Mol. Graph Model. 2001; 19: 26-59Crossref PubMed Scopus (1853) Google Scholar, 26.Liu J. Perumal N.B. Oldfield C.J. Su E.W. Uversky V.N. Dunker A.K. Biochemistry. 2006; 45: 6873-6888Crossref PubMed Scopus (527) Google Scholar). Recently, we have successfully visualized the ID regions of Drosophila melanogaster FACT (dFACT) in solution by high speed atomic force microscopy (AFM) (27.Miyagi A. Tsunaka Y. Uchihashi T. Mayanagi K. Hirose S. Morikawa K. Ando T. Chemphyschem. 2008; 9: 1859-1866Crossref PubMed Scopus (86) Google Scholar). However, the dynamic and functional behaviors of the FACT ID regions remain unknown at the molecular level. Using the dFACT proteins expressed in Sf9 cells, we investigated how the phosphorylation of dSSRP1 (D. melanogaster SSRP1) induces the inhibition of DNA binding. Mutational analyses demonstrated that the phosphorylation sites regulating the nucleosomal DNA binding activity are concentrated in the acidic ID region of dSSRP1. This acidic ID region forms an intramolecular interaction with both the HMG domain and the basic ID segment. Notably, phosphorylation of the acidic ID region markedly strengthens this interaction, thereby blocking the DNA binding to the HMG domain. The physiological significance of these findings is highlighted by our observation that some phosphorylation sites in the maternally transmitted dSSRP1 were dephosphorylated immediately after fertilization to accommodate rapid chromatin transactions during early embryogenesis. We used the same dFACT cDNA as in our previous study (11.Shimojima T. Okada M. Nakayama T. Ueda H. Okawa K. Iwamatsu A. Handa H. Hirose S. Genes Dev. 2003; 17: 1605-1616Crossref PubMed Scopus (97) Google Scholar). To obtain the His-tagged DNA sequences, the cDNAs encoding dSPT16 and dSSRP1 were cloned into the NdeI and XhoI sites of pColdI (Takara-bio). To construct the plasmid for dSSRP1 and dSPT16 co-expression, the His-tagged dSPT16 DNA was ligated into the EcoRI and SalI sites downstream of the PH promoter in the pFastBacDual plasmid (Invitrogen), and then the dSSRP1 DNA was cloned into the XhoI and SphI sites under the control of the p10 promoter in the same plasmid. His-tagged deletion constructs of dSSRP1 were generated in pColdI (Δ625-dSSRP1 and Mid-dSSRP1; Fig. 1A) or pET28a (AB-HMG, LB-HMG, SB-HMG, and AID, Fig. 1A). The His-tagged Mid-dSSRP1 construct was also cloned into the XhoI and SphI sites under the control of the p10 promoter in pFastBacDual. To obtain the Ser/Thr to Ala mutants and the Δ624-dSSRP1 deletion, site-directed mutagenesis of the dFACT proteins was performed using the QuikChange site-directed mutagenesis method (Stratagene). To produce the Sf9-AB-HMG and Sf9-AID proteins, the protease site insertions within the Mid-dSSRP1 protein (thrombin and factor Xa sites inserted between amino acid residues 433–434 and 518–519, respectively) were also performed, using the same mutagenesis method. Competent Escherichia coli DH10BAC cells (Invitrogen), transformed by these plasmids, were used to generate recombinant bacmids. The bacmid-cellfection complex was transfected into Sf9 cells. The Sf9 insect cells (ovary-derived cells from Spodoptera frugiperda) were propagated and maintained at 27 °C in Sf-900 II serum-free medium (Invitrogen). The baculovirus was collected at 72-h postinfection. The dFACT proteins were expressed at 27 °C for 48 h in Sf9 cells, which were infected at a density of 2 × 106 cells/cm2 with the baculovirus including the dFACT cDNAs. The infected cells were collected by centrifugation and were suspended in lysis buffer containing 0.15 m NaCl, 20 mm Tris-HCl, pH 8.5, 0.1% Nonidet P-40, and a protease inhibitor mixture (Nacalai-Tesque). After clarification of the lysate by two rounds of centrifugation at 15,000 rpm for 30 min, the supernatant was loaded onto a Histrap column (GE Healthcare). After washing the column with buffer A (20 mm Tris-HCl, pH8.5, 5 mm 2-mercaptoethanol) containing 20 mm imidazole and 0.6 m NaCl, the bound proteins were eluted with buffer A containing 0.5 m imidazole and 0.15 m NaCl. The proteins were then applied to a Hitrap Q exchange column (GE Healthcare), which was eluted with a concentration gradient of 0.15–1 m sodium chloride in buffer A. Peak fractions containing the FACT complex were dialyzed against buffer A containing 0.15 m NaCl, and then were purified on a Hitrap Heparin column (GE Healthcare) by elution with a concentration gradient of 0.15–1 m sodium chloride in buffer A. The expression and purification of dFACT proteins from E. coli have been described previously (27.Miyagi A. Tsunaka Y. Uchihashi T. Mayanagi K. Hirose S. Morikawa K. Ando T. Chemphyschem. 2008; 9: 1859-1866Crossref PubMed Scopus (86) Google Scholar, 28.Kasai N. Tsunaka Y. Ohki I. Hirose S. Morikawa K. Tate S. J. Biomol. NMR. 2005; 32: 83-88Crossref PubMed Scopus (15) Google Scholar). The purification of the Sf9-AID and Sf9-AB-HMG proteins was impossible due to their very low expression levels in the baculovirus insect cells. Thus, we expressed the Sf9-Mid-dSSRP1 protein with two protease sites (a thrombin site between amino acid residues 433 and 434, and a factor Xa site between amino acid residues 518 and 519) in Sf9 cells, and then treated them with thrombin and factor Xa to generate these desired proteins. First, the purified Sf9-Mid-dSSRP1 protein with two protease sites was cleaved with thrombin for 16 h at 4 °C. The resultant protein mixture was applied to a Histrap column equilibrated in buffer A containing 0.15 m NaCl, and the flow-through fraction contained the desired Sf9-AB-HMG protein. The Sf9-AB-HMG protein was further purified on a Hitrap Q exchange column by elution with a concentration gradient of 0.15–1 m sodium chloride in buffer A. Next, this Sf9-AB-HMG protein was cleaved with factor Xa for 16 h at 4 °C during dialysis against buffer A containing 0.15 m NaCl. The resultant protein mixture was applied to a Hitrap Q exchange column equilibrated in buffer A containing 0.15 m NaCl. The desired Sf9-AID protein was eluted with a concentration gradient of 0.15–1 m sodium chloride in buffer A. All of the purified proteins were dialyzed against buffer A containing 0.15 m NaCl. We used the same fly stocks as in the previous study (11.Shimojima T. Okada M. Nakayama T. Ueda H. Okawa K. Iwamatsu A. Handa H. Hirose S. Genes Dev. 2003; 17: 1605-1616Crossref PubMed Scopus (97) Google Scholar). Embryos 0–1, 1–3, and 0–3 h after egg laying (AEL) were collected from a population reared at 25 °C, and were dechorionated. Ovaries were extirpated from the abdominal segment of adult female flies from the same population in cold buffer, containing 130 mm NaCl, 5 mm KCl, 2 mm CaCl2, and 10 mm HEPES, pH 6.0. The ovaries and embryos were homogenized in cytosolic extract buffer, containing 50 mm NaCl, 20 mm Tris-HCl, pH 7.9, 3 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 5% glycerol, 0.3% Nonidet P-40, and a protease inhibitor mixture (Sigma), and after centrifugation, the supernatant was saved as the cytosolic extract. The nuclear pellet was then treated with nuclear extract buffer (the same composition as cytosolic extract buffer, except for 0.5 m NaCl) to yield the nuclear extract. dFACT proteins (300 pmol) were incubated with 15 units of calf intestine alkaline phosphatase (Takara-bio), in reaction mixtures containing 50 mm Tris-HCl, pH 9.0, and 1 mm MgCl2, at 20 °C for 2 h. The purified dFACT heterodimers and the extracts from ovaries and embryos were denatured with urea buffer (8 m urea, 20 mm Tris-HCl, pH 8.5, 100 mm NaCl). These samples were fractioned by 5% native-PAGE in Tris-glycine buffer, and then were detected on Western blot with anti-dSSRP1 antibodies. We used the same anti-dSSRP1 antibodies as in the previous study (11.Shimojima T. Okada M. Nakayama T. Ueda H. Okawa K. Iwamatsu A. Handa H. Hirose S. Genes Dev. 2003; 17: 1605-1616Crossref PubMed Scopus (97) Google Scholar). Proteins (20 μg) were loaded onto 4–7 IPG strips (GE Healthcare) in rehydration solution (8 m urea, 2% CHAPS, 20 mm dithiothreitol, 0.5% 4–7 IPG buffer (GE Healthcare)). Iso-electric focusing was performed using IPG strips in Ettan IPGphor II system (GE Healthcare). After first dimension run was completed, IPG strips were transferred onto gels and subjected to 10% SDS-PAGE. The samples were detected by CBB stain. Recombinant human full-length histone proteins were produced in E. coli and purified as reported previously (29.Tsunaka Y. Kajimura N. Tate S. Morikawa K. Nucleic Acids Res. 2005; 33: 3424-3434Crossref PubMed Scopus (114) Google Scholar). Mononucleosomes were reconstructed from histones and a 146 bp or a 263 bp DNA with the strongest positioning sequences (30.Luger K. Mäder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6929) Google Scholar, 31.Lowary P.T. Widom J. J. Mol. Biol. 1998; 276: 19-42Crossref PubMed Scopus (1212) Google Scholar). The 263-bp DNA fragment containing the nucleosome positioning sequence 601 was prepared by PCR from pGEM3Z-601 (kindly provided by J. Widom) (31.Lowary P.T. Widom J. J. Mol. Biol. 1998; 276: 19-42Crossref PubMed Scopus (1212) Google Scholar). The 146-bp palindromic DNA fragment (30.Luger K. Mäder A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6929) Google Scholar), derived from a human α-satellite region, was constructed and purified as described previously (32.Dyer P.N. Edayathumangalam R.S. White C.L. Bao Y. Chakravarthy S. Muthurajan U.M. Luger K. Methods Enzymol. 2004; 375: 23-44Crossref PubMed Scopus (540) Google Scholar). Mononucleosomes were assembled by a salt dialysis method using these core histones and the 146-bp or 263-bp DNA fragments and were purified by chromatography on a Mini Q column, as described previously (29.Tsunaka Y. Kajimura N. Tate S. Morikawa K. Nucleic Acids Res. 2005; 33: 3424-3434Crossref PubMed Scopus (114) Google Scholar). Purified mononucleosomes were incubated with the FACT proteins, in reaction mixtures containing 20 mm Tris-HCl, pH 8.0, 0.1 mm EDTA, 150 mm NaCl, and 5 mm 2-mercaptoethanol at 20 °C for 5 min. The samples containing the full-length dSSRP1 were electrophoresed at 4 °C on a 0.7% agarose gel in 0.5× TBE, and then were visualized by SYBR Gold nucleic acid gel stain. Each band was quantified using the ImageJ v1.41o (United States National Institutes of Health). The Mid-dSSRP1 was fractionated by 5% PAGE in 0.2× TBE. A 20-base pair (bp) dsDNA fragment (5′-GCATAAATACGCATAAATAC-3′) (0.1 nmol) was incubated with an equimolar amount of the proteins for 5 min at 20 °C, in reaction mixtures containing 20 mm Tris-HCl, pH 8.0, 0.1 mm EDTA, 150 mm NaCl, and 5 mm 2-mercaptoethanol. AID proteins (0.1nmol) were also incubated with an equimolar amount of LB-HMG for 10 min at 20 °C in the same reaction mixtures. In the competitive assay, AID proteins (0.1nmol) were preincubated with an equimolar amount of LB-HMG for 10 min at 20 °C. The resultant complexes were titrated with 0.3, 1.0, 3.0, and 10.0-fold amounts of dsDNA, and then were incubated for 5 min at 20 °C under the same buffer conditions. The samples were fractionated at 4 °C by 15% native-PAGE in Tris-glycine buffer, and then were detected by CBB stain, EtBr stain, and ProQ-Diamond phosphoprotein stain. E. coli BL21 (DE3) cells, transformed by the SB-HMG protein expression plasmid, were cultured in M9 minimal medium containing 15NH4Cl as the sole nitrogen source to produce the uniformly 15N-labeled protein. The purified protein was dissolved in a solution, containing 5 mm Tris-HCl, pH 7.5, 50 mm KCl, and 10 mm MgCl2 in 95% H2O, 5% D2O. The AID protein was also dissolved in the same buffer solution, containing 5 mm Tris-HCl, pH 7.5, 50 mm KCl, and 10 mm MgCl2. The backbone 1H, 15N, 13Cα, 13Cβ, 13C′ resonances for the SB-HMG protein in the above solution were assigned using HNCO, HNCA, HN(CO)A, HNCACB, and CBCA(CO)NH triple resonance experiments performed with a 600MHz spectrometer, Bruker DMX600 (33.Cavanagh J. Fairbrother W.J. Palmer III, A.G. Skelton N.J. Protein NMR Spectroscopy. Academic Press, San Diego, CA1996: 411-528Google Scholar). A series of 1H-15N HSQC spectra were collected for samples with different molar ratios of SB-HMG to the AID protein. The molar ratios of the AID protein to SB-HMG were set to 0.1, 0.2, 0.5, 1.0, 1.5, and 2.0 versus the 0.1 mm SB-HMG concentration. The spectra were collected on a DMX750 spectrometer operating at 750 MHz for the 1H resonance frequency, at 25 °C. All NMR data were processed with the program NMRPipe (34.Delaglio F. Grzesiek S. Vuister G.W. Zhu G. Pfeifer J. Bax A. J. Biomol. NMR. 1995; 6: 277-293Crossref PubMed Scopus (11570) Google Scholar). Peak positions were elucidated by using contour simulation in the program PIPP (35.Garrett D.S. Powers R. Gronenborn A.M. Clore G.M. J. Magn. Reson. 1991; 95: 214-220Crossref Scopus (802) Google Scholar), which calculated the peak position as an average of centers of simulated contour circles for all displayed contour levels. The chemical shift differences were calculated in Hz units in Equation 1, Δδ=δ1Hbound−δ1Hfree×Fr1H2+δ15Nbound−δ15Nfree×Fr15N21/2(Eq. 1) where δ1Hbound and δ1Hfree are the 1H chemical shifts in ppm for the bound and ligand-free states, respectively. δ15Nbound and δ15Nfree denote the counterparts for the 15N chemical shifts. Fr(1H) and Fr(15N) were set to 749.93 and 75.99 in the present experiments performed on a 750 MHz spectrometer. When dSSRP1 and dSPT16 (D. melanogaster SPT16) were co-expressed in the baculovirus-Sf9 insect cell system and purified from a cytosolic fraction of Sf9 cells, we obtained the full-length dFACT as a 1:1 complex between dSSRP1 and dSPT16 (Sf9-dFACT, supplemental Fig. S1). The Sf9-dFACT bound only 36–46% to 146 bp and 263 bp mononucleosomes, even at 1 μm. (Fig. 2A, lanes 3 and 8). This could be due to phosphorylation of dFACT, as phosphorylation of hSSRP1 by casein kinase 2 (CK2) inhibited the nonspecific DNA binding activity of FACT (8.Li Y. Keller D.M. Scott J.D. Lu H. J. Biol. Chem. 2005; 280: 11869-11875Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). To examine this possibility, the Sf9-dFACT protein was treated with calf intestine alkaline phosphatase (APase), and then the dSSRP1, dissociated from the dFACT complex with 8 m urea, was subjected to native-PAGE. The APase treatment caused the gel mobility of dSSRP1 to become slower, suggesting that APase had removed some of the negatively charged phosphate groups of dSSRP1 (Fig. 2B, lane 3 versus lane 4). The dFACT, dephosphorylated with APase, recovered the nucleosomal DNA-binding activity, as compared with the phosphorylated form (Fig. 2A, lanes 2 and 3 versus lanes 4 and 5; lanes 7 and 8 versus lanes 9 and 10). Therefore, we concluded that the phosphorylation of dFACT inhibits its binding to nucleosomal DNA. Next, we analyzed the competitive binding of the dephosphorylated dFACT between the nucleosome and dsDNA (supplemental Fig. S2). The nucleosome complex with dFACT was dissociated upon the addition of dsDNA, as revealed by the appearance of free nucleosomes (supplemental Fig. S2, lanes 3–5). These results demonstrate that the increased interaction is due to an increase in nonspecific DNA binding. In addition, the dephosphorylated dFACT more strongly interacts with nucleosomes that contain linker DNA, in agreement with the finding that HMG-box proteins bind to linker DNA. For example, the interaction with the 146-bp nucleosome, which completely lacks linker DNAs, required higher dFACT concentrations than that with the 263-bp nucleosome carrying linker DNAs (Fig. 2A, lanes 4 and 5 versus lane 9 and 10). However, this result is not consistent with the previous data that the linker DNA did not promote the binding of the yeast SPT16-Pob3-Nhp6A complex (36.Ruone S. Rhoades A.R. Formosa T. J. Biol. Chem. 2003; 278: 45288-45295Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). This discrepancy may be due to the fact that metazoan FACTs contain the HMG domain within the single polypeptide of dSSRP1, while the domain is on a separate polypeptide in the yeast FACT complex. The previous study has suggested that among the three residues identified as CK2 phosphorylation sites in vitro, the phosphorylation of serine 510 alone plays a crucial role in binding between hSSRP1 and DNA (8.Li Y. Keller D.M. Scott J.D. Lu H. J. Biol. Chem. 2005; 280: 11869-11875Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This led us to search for the functional phosphorylation sites in dSSRP1 responsible for inhibiting the nucleosomal DNA binding. An extensive sequence alignment around Ser-510 of hSSRP1 revealed 11 potential phosphorylation sites (Fig. 1B; red open and filled circles) of dSSRP1 as CK2 target sequences ((S/T)XX(D/E) or (S/T)(D/E)). To find the actual phosphorylation sites in living cells, mutational analyses were carried out using native-PAGE and Ser/Thr to Ala mutants in Mid-dSSRP1 (Fig. 1A), which consists of the SSRC motif, the acidic region, the following basic segment, and the HMG domain. These analyses would reveal phosphorylation sites, because mutations from Ser/Thr to Ala of actual sites result in slower band migration than that for the wild-type (Fig. 3A). For example, the Ala mutation of the actual phosphorylation site (S515A) resulted in slower migration of the band than those of the WT and the S526A mutant (Fig. 3A, left). Except for Ser-526 (Fig. 1B, red filled circle) in the basic segment, ten sites (Fig. 1B, red open circles) were found to be phosphorylated in Sf9 cells. The mutant protein, whose actual phosphorylation sites were entirely replaced by Ala (10SA-Mid-dSSRP1), exhibited almost the same gel mobility as the unmodified wild-type protein expressed in E. coli (Eco-WT-Mid-dSSRP1) (Fig. 3A, right). Various mutants, in which the ten actual sites were replaced to different degrees (1SA, 2SA, 3SA, 4SA, 5SA, 6SA, and 9SA-Mid-dSSRP1), exhibited band shifts, as they stepwise got closer to that of Eco-WT-Mid-dSSRP1 (Fig. 3A, right). To determine the phosphorylation sites more rigorously, we performed similar analyses using two-dimensional gel electrophoresis and Mid-dSSRP1 (supplemental Fig. S3). The spots of S506A and S506A/S515A mutants (Rf
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