A Mammalian Chromatin Remodeling Complex with Similarities to the Yeast INO80 Complex
2005; Elsevier BV; Volume: 280; Issue: 50 Linguagem: Inglês
10.1074/jbc.m509128200
ISSN1083-351X
AutoresJingji Jin, Yong Cai, Tingting Yao, Aaron J. Gottschalk, Laurence Florens, Selene K. Swanson, José L. Gutiérrez, Michael K. Coleman, Jerry L. Workman, Arcady Mushegian, Michael P. Washburn, Ronald Conaway, Joan Conaway,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe mammalian Tip49a and Tip49b proteins belong to an evolutionarily conserved family of AAA+ ATPases. In Saccharomyces cerevisiae, orthologs of Tip49a and Tip49b, called Rvb1 and Rvb2, respectively, are subunits of two distinct ATP-dependent chromatin remodeling complexes, SWR1 and INO80. We recently demonstrated that the mammalian Tip49a and Tip49b proteins are integral subunits of a chromatin remodeling complex bearing striking similarities to the S. cerevisiae SWR1 complex (Cai, Y., Jin, J., Florens, L., Swanson, S. K., Kusch, T., Li, B., Workman, J. L., Washburn, M. P., Conaway, R. C., and Conaway, J. W. (2005) J. Biol. Chem. 280, 13665–13670). In this report, we identify a new mammalian Tip49a- and Tip49b-containing ATP-dependent chromatin remodeling complex, which includes orthologs of 8 of the 15 subunits of the S. cerevisiae INO80 chromatin remodeling complex as well as at least five additional subunits unique to the human INO80 (hINO80) complex. Finally, we demonstrate that, similar to the yeast INO80 complex, the hINO80 complex exhibits DNA- and nucleosome-activated ATPase activity and catalyzes ATP-dependent nucleosome sliding. The mammalian Tip49a and Tip49b proteins belong to an evolutionarily conserved family of AAA+ ATPases. In Saccharomyces cerevisiae, orthologs of Tip49a and Tip49b, called Rvb1 and Rvb2, respectively, are subunits of two distinct ATP-dependent chromatin remodeling complexes, SWR1 and INO80. We recently demonstrated that the mammalian Tip49a and Tip49b proteins are integral subunits of a chromatin remodeling complex bearing striking similarities to the S. cerevisiae SWR1 complex (Cai, Y., Jin, J., Florens, L., Swanson, S. K., Kusch, T., Li, B., Workman, J. L., Washburn, M. P., Conaway, R. C., and Conaway, J. W. (2005) J. Biol. Chem. 280, 13665–13670). In this report, we identify a new mammalian Tip49a- and Tip49b-containing ATP-dependent chromatin remodeling complex, which includes orthologs of 8 of the 15 subunits of the S. cerevisiae INO80 chromatin remodeling complex as well as at least five additional subunits unique to the human INO80 (hINO80) complex. Finally, we demonstrate that, similar to the yeast INO80 complex, the hINO80 complex exhibits DNA- and nucleosome-activated ATPase activity and catalyzes ATP-dependent nucleosome sliding. The related mammalian Tip49a and Tip49b proteins are members of a family of AAA+ (associated with various cellular activities) ATPases with roles in DNA repair, recombination, and transcriptional regulation (1Neuwald A.F. Aravind L. Spouge J.L. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar, 2Caruthers J.M. McKay D. Curr. Opin. Struct. Biol. 2002; 12: 123-133Crossref PubMed Scopus (455) Google Scholar). In Saccharomyces cerevisiae, the Tip49a and Tip49b proteins (also known as Rvb1 and Rvb2) participate in chromatin remodeling as subunits of the multiprotein SWR1 and INO80 ATP-dependent chromatin remodeling complexes (3Mizuguchi G. Shen X. Landry J. Wu W.H. Sen S. Wu C. Science. 2004; 303: 343-348Crossref PubMed Scopus (1003) Google Scholar, 4Krogan N.J. Baetz K. Keogh M.C. Datta N. Sawa C. Kwok T.C. Thompson N.J. Davey M.G. Pootoolal J. Hughes T.R. Emili A. Buratowski S. Hieter P. Greenblatt J.F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13513-13518Crossref PubMed Scopus (202) Google Scholar, 5Kobor M.S. Venkatasubrahmanyam S. Meneghini M.D. Gin J.W. Jennings J.L. Link A.J. Madhani H.D. Rine J. PLoS Biol. 2004. 2004; : 2/E131Google Scholar). The SWR1 complex remodels chromatin by catalyzing ATP-dependent replacement of H2A-H2B histone dimers in nucleosomes by dimers containing histone variant Htz1 (referred to as H2AZ in mammalian cells) (3Mizuguchi G. Shen X. Landry J. Wu W.H. Sen S. Wu C. Science. 2004; 303: 343-348Crossref PubMed Scopus (1003) Google Scholar). In addition to Tip49a and Tip49b, the SWR1 complex includes the SNF2 family helicase Swr1, actin-related proteins Arp4 and Arp6, YEATS domain family member Yaf9, bromodomain protein Bdf1, and additional proteins Swc3–Swc7, which are of unknown function (3Mizuguchi G. Shen X. Landry J. Wu W.H. Sen S. Wu C. Science. 2004; 303: 343-348Crossref PubMed Scopus (1003) Google Scholar, 4Krogan N.J. Baetz K. Keogh M.C. Datta N. Sawa C. Kwok T.C. Thompson N.J. Davey M.G. Pootoolal J. Hughes T.R. Emili A. Buratowski S. Hieter P. Greenblatt J.F. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 13513-13518Crossref PubMed Scopus (202) Google Scholar, 5Kobor M.S. Venkatasubrahmanyam S. Meneghini M.D. Gin J.W. Jennings J.L. Link A.J. Madhani H.D. Rine J. PLoS Biol. 2004. 2004; : 2/E131Google Scholar). The INO80 complex catalyzes ATP-dependent sliding of nucleosomes along DNA and, based on genetic and other evidence, may be involved in the repair of DNA double strand breaks and in transcriptional regulation (6Shen X. Mizuguchi G. Hamiche A. Wu C. Nature. 2000; 406: 541-544Crossref PubMed Scopus (667) Google Scholar, 7Shen X. Hua X. Ranallo R. Wei-Hua W. Wu C. Science. 2002; 299: 112-114Crossref PubMed Scopus (292) Google Scholar, 8Steger D.J. Haswell E.S. Miller A.L. Wente S.R. O'Shea E.K. Science. 2003; 299: 114-116Crossref PubMed Scopus (315) Google Scholar, 9Fritsch O. Benvenuto G. Bowler C. Molinier J. Hohn B. Mol. Cell. 2004; 16: 479-485Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 10Ohdate H. Lim C.R. Kokubo T. Matsubara K. Kimata Y. Kohno K. J. Biol. Chem. 2003; 278: 14647-14656Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 11van Attikum H. Fritsch O. Hohn B. Gasser S.M. Cell. 2004; 119: 777-788Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar, 12Morrison A.J. Highland J. Krogan N.J. Arbel-Eden A. Greenblatt J.F. Haber J.E. Shen X. Cell. 2004; 119: 767-775Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar). The INO80 complex includes Tip49a and Tip49b, the SNF2 family helicase Ino80, actin-related proteins Arp4, Arp5, and Arp8, YEATS domain family member Taf14, HMG (high mobility group) domain protein Nhp10, and six additional proteins designated Ies1–Ies6 (6Shen X. Mizuguchi G. Hamiche A. Wu C. Nature. 2000; 406: 541-544Crossref PubMed Scopus (667) Google Scholar, 13Shen X. Ranallo R. Choi E. Wu C. Mol. Cell. 2003; 12: 147-155Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). Thus, the SWR1 and INO80 complexes share three proteins (Tip49a, Tip49b, and Arp4) and contain additional homologous components. In addition, each of the two complexes has a number of unique subunits. The orthologs of the Tip49a and Tip49b AAA+ ATPases also play roles in chromatin remodeling in higher eukaryotes. Tip49a and Tip49b are subunits of the mammalian and Drosophila melanogaster TRRAP-TIP60 histone acetyltransferase (HAT) 3The abbreviations used are: HAThistone acetyltransferaseATPγSadenosine 5′-O-(thiotriphosphate)HEKhuman embryonic kidneyhINO80human INO80-like proteinHPLChigh pressure liquid chromatographyMudPITmultidimensional protein identification technologyNFRKBnuclear factor related to κB-binding proteinORFopen reading frameTafTATA-binding protein-associated factorTip49a and Tip49bTATA-binding protein interacting 49-kDa proteins a and b. complexes (14Cai Y. Jin J. Tomomori-Sato C. Sato S. Sorokina I. Parmely T.J. Conaway R.C. Conaway J.W. J. Biol. Chem. 2003; 278: 42733-42736Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 15Doyon Y. Selleck W. Lane W.S. Cote J. Mol. Cell. Biol. 2004; 24: 1884-1896Crossref PubMed Scopus (445) Google Scholar, 16Ikura T. Ogryzko V. Gigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar, 17Kusch T. Florens L. Macdonald W.H. Swanson S.K. Glaser R.L. Yates J.R. Abmayr S.M. Washburn M.P. Workman J.L. Science. 2004; 306: 2084-2087Crossref PubMed Scopus (555) Google Scholar). In addition to Tip49a and Tip49b, the TRRAP-TIP60 complex includes ATM/phosphatidylinositol 3-kinase family member TRRAP, the SNF2 family p400 or Domino helicase, actin-related protein Arp4, bromodomain-containing protein BRD8, the enhancer of polycomb (EPC) and/or enhancer of polycomb-like (EPC-like) protein, inhibitor of growth 3 (ING3), DNA methyltransferase 1-associated protein (DMAP1), MRG15 and/or the related MRGX protein, the MRGBP protein, and TIP60, a HAT belonging to the MYST family. Characterization of the activities associated with the higher eukaryotic TRRAP-TIP60 complex revealed that it possesses HAT activity similar to that of the S. cerevisiae NuA4 HAT complex, which acetylates histones H2A and H4 (reviewed in Ref. 18Doyon Y. Cote J. Curr. Opin. Genet. Dev. 2004; 14: 147-154Crossref PubMed Scopus (291) Google Scholar). The human and D. melanogaster TRRAP-TIP60 complexes were found to play critical roles in double-stranded DNA break repair (16Ikura T. Ogryzko V. Gigoriev M. Groisman R. Wang J. Horikoshi M. Scully R. Qin J. Nakatani Y. Cell. 2000; 102: 463-473Abstract Full Text Full Text PDF PubMed Scopus (876) Google Scholar, 17Kusch T. Florens L. Macdonald W.H. Swanson S.K. Glaser R.L. Yates J.R. Abmayr S.M. Washburn M.P. Workman J.L. Science. 2004; 306: 2084-2087Crossref PubMed Scopus (555) Google Scholar). Notably, the D. melanogaster TRRAP-TIP60 complex is capable of acetylating nucleosomal phospho-H2Av and replacing it with unmodified H2Av, indicating that in flies this single complex performs functions closely related to those of the yeast NuA4 HAT and SWR1 histone exchange complexes (17Kusch T. Florens L. Macdonald W.H. Swanson S.K. Glaser R.L. Yates J.R. Abmayr S.M. Washburn M.P. Workman J.L. Science. 2004; 306: 2084-2087Crossref PubMed Scopus (555) Google Scholar). histone acetyltransferase adenosine 5′-O-(thiotriphosphate) human embryonic kidney human INO80-like protein high pressure liquid chromatography multidimensional protein identification technology nuclear factor related to κB-binding protein open reading frame TATA-binding protein-associated factor TATA-binding protein interacting 49-kDa proteins a and b. We recently identified a new mammalian Tip49a- and Tip49b-containing ATP-dependent chromatin remodeling complex that bears striking similarity to the S. cerevisiae SWR1 complex (19Cai Y. Jin J. Florence L. Swanson S.K. Kusch T. Li B. Workman J.L. Washburn M.P. Conaway R.C. Conaway J.W. J. Biol. Chem. 2005; 280: 13665-13670Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Purification of this complex revealed that it includes the SNF2 family and SWR1-related SRCAP helicase, as well as orthologs of most of the known subunits of the S. cerevisiae SWR1 complex. In the course of our characterization of the structure and function of the SRCAP complex, we identified an additional mammalian Tip49a- and Tip49b-containing chromatin remodeling complex. Here we describe the properties of this new chromatin remodeling complex, which includes orthologs of 8 of the 15 subunits of the S. cerevisiae INO80 chromatin remodeling complex as well as at least 5 additional subunits unique to the human INO80 (hINO80) complex. Generation and Growth of Mammalian Cell Lines—Full-length cDNAs encoding the human Tip49a, Tip49b, Arp8, PAPA-1 (hIes2), C18orf37 (hIes6), Amida, and FLJ90652 proteins or a fragment of FLJ20309 (residues 106–544) were obtained from the American Type Culture Collection, subcloned with FLAG tags into pcDNA5/FRT, and introduced into HEK293/FRT cells using the Invitrogen Flp-in system. Full-length cDNAs encoding the human PAPA-1 and C18orf37 proteins were subcloned with FLAG tags into pcDNA3.1 and introduced into HeLa S3 cells. Parental and stably transformed HEK293/FRT and HeLa S3 cells were maintained in Dulbecco's modified Eagle's medium with 5% glucose and 10% fetal bovine serum. For large scale cultures, HeLa cells were grown in spinner culture in Joklik medium with 5% calf serum. Anti-FLAG Agarose Chromatography—Whole cell extracts were prepared from HEK293/FRT cells as follows. Cells were grown to 70–80% confluence in four to five 15-cm dishes. Cells were washed in dishes with phosphate-buffered saline and then lysed by resuspension in buffer (1 ml/dish) containing 40 mm Hepes-NaOH (pH 7.9), 0.45 m NaCl, 1.5 mm MgCl2, 10% glycerol, 1 mm dithiothreitol, and 0.2% Triton X-100. The resulting suspension was transferred to centrifuge tubes and incubated with rotation at 4 °C for 30 min. The cell lysate was centrifuged at 40,000 rpm for 60 min at 4 °C in a 70.1 Ti rotor (Beckman-Coulter). The resulting supernatant was subjected to anti-FLAG agarose chromatography. Nuclear extracts were prepared from HeLa S3 cells according to the method of Dignam et al. (20Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9164) Google Scholar). Whole cell or nuclear extracts were adjusted to 0.3 m NaCl and 0.2% Triton X-100 and centrifuged at 40,000 rpm for 30 min at 4 °C in a Ti-45 rotor. Supernatants were then mixed with anti-FLAG (M2) agarose beads (Sigma) in a ratio of 100 μl of packed beads/6 ml of supernatant and gently rocked for 4 h at 4 °C. The beads were washed three times with a 50-fold excess of buffer containing 40 mm Hepes-NaOH (pH 7.9), 0.25 m NaCl, 0.2% Triton X-100, and 10% glycerol and once with the same buffer containing 0.1 m NaCl. Proteins were eluted from the beads twice by incubation for 30 min on a rotator at 4 °C with 100 μl of 40 mm Hepes-NaOH (pH 7.9), 0.1 m NaCl, 0.1 mm EDTA, 10% glycerol, and 0.2 mg/ml FLAG peptide (Sigma). Glycerol was omitted from the elution buffer for samples to be analyzed by mass spectrometry. ATPase Assays—Reaction mixtures of 20 μl contained 50 mm Hepes-NaOH (pH 7.6), 70 mm NaCl, 5 mm MgCl2, 0.5 mm EGTA, 0.1 mm EDTA, 10% glycerol, 0.02% Nonidet P-40, 0.2 mm dithiothreitol, 100 μg/ml bovine serum albumin, 40 μm ATP, 0.2 μCi of [α-32P]ATP (400 Ci/mmol, Amersham Biosciences). Where indicated, reaction mixtures contained the hINO80 complex purified from HeLa cells expressing FLAG-hIes2 (PAPA-1) and 150 ng of mononucleosomes or long oligonucleosomes prepared from HeLa cells as described (21Owen-Hughes T. Utley R.T. Steger D.J. West J.M. John S. Cote J. Havas K.M. Workman J.L. Methods Mol. Biol. 1999; 119: 319-331PubMed Google Scholar). After incubation at 37 °C for 30 min, reactions were stopped by the addition of 2 μl of 20 mm EDTA (pH 8.0). A 2-μl aliquot of each reaction mixture was spotted onto a cellulose polyethyleneimine TLC plate (JT Baker). The plate was then developed with 0.375 m potassium phosphate (pH 3.5). Reaction products were detected and quantitated using a Typhoon phosphorimaging device (GE Healthcare). Nucleosome Remodeling Assays—A 216-bp DNA fragment (dSH-A) was generated by PCR from pGUB-dSH in the presence of [α-32P]dCTP. pGUB-dSH was generated by deleting the SalI to HindIII fragment of pGUB (22Juan L.J. Utley R.T. Vignali M. Bohm L. Workman J.L. J. Biol. Chem. 1997; 272: 3635-3640Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Mononucleosomes were reconstituted on this labeled DNA fragment by dilution transfer from HeLa long oligonucleosomes. ∼3 μg of nucleosomes was mixed with ∼1 pmol of 32P-labeled DNA fragment in 25 μl of buffer containing 1.0 m NaCl, 10 mm Tris-HCl (pH 8), 1 mm EDTA (pH 8.0), 0.5 mm phenylmethylsulfonyl fluoride, and 5 mm dithiothreitol. After 30 min at 30 °C, the mixture was sequentially adjusted to 0.8, 0.6, and 0.4 m NaCl by dilution with 10 mm Tris-HCl (pH 8), 1 mm EDTA (pH 8.0), 0.5 mm phenylmethylsulfonyl fluoride, and 5 mm dithiothreitol, with a 30-min incubation at 30 °C between each dilution. Final dilutions to 0.2 and 0.1 m NaCl were made using the same buffer plus 0.1% Nonidet P-40, 20% glycerol, and 200 μg/ml bovine serum albumin. After reconstitution, the mononucleosomes were stored in 30-μl aliquots at -20 °C. hINO80 complex purified from HeLa cells expressing FLAG-PAPA-1 (hIes2) was incubated at 37 °C with 2.5 μl of reconstituted mononucleosomes (∼0.01 pmol of labeled mononucleosome, ∼0.25 pmol of unlabeled oligonucleosomes) in buffer containing 20 mm Hepes-NaOH (pH 7.9), 50 mm NaCl, 4.5 mm MgCl2, 2 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 45 μg/ml bovine serum albumin, 10% glycerol, 0.02% Triton X-100, 0.02% Nonidet P-40, and 1 mm ATP. After a 30-min incubation, 0.5 μg of HeLa cell long oligonucleosomes (21Owen-Hughes T. Utley R.T. Steger D.J. West J.M. John S. Cote J. Havas K.M. Workman J.L. Methods Mol. Biol. 1999; 119: 319-331PubMed Google Scholar) and 0.75 μg of salmon sperm DNA (which had been sonicated, boiled, and quick-chilled) were added, and reactions were incubated for an additional 30 min at 37 °C to remove DNA- or nucleosome-binding proteins that would alter mononucleosome electrophoretic mobility. The reaction products were then applied to 5% polyacrylamide gels (37.5:1 acrylamide:bis-acrylamide) gels in 0.5× Tris borate-EDTA (23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual, 2nd Ed. 1989; : B.23Google Scholar) and subjected to electrophoresis at 4 °C for 4.5 h at 200 V. Gels were dried and exposed to a storage phosphor screen overnight. Mass Spectrometry—Identification of proteins was accomplished as described (19Cai Y. Jin J. Florence L. Swanson S.K. Kusch T. Li B. Workman J.L. Washburn M.P. Conaway R.C. Conaway J.W. J. Biol. Chem. 2005; 280: 13665-13670Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) using a modification of the multidimensional protein identification technology (MudPIT) procedure of Washburn et al. (24Washburn M.P. Wolters D. Yates J.R. II I Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4099) Google Scholar). Briefly, trichloroacetic acid-precipitated proteins were reduced, alkylated, and digested with modified trypsin (Roche Applied Science). Peptide mixtures were applied to a three-phase microcapillary HPLC column (25McDonald W.H. Ohi R. Miyamoto D.T. Mitchison T.J. Yates J.R. Int. J. Mass Spectrom. 2002; 219: 245-251Crossref Scopus (269) Google Scholar) packed with 5 μm C18 reverse phase resin (Aqua, Phenomenex), followed by strong cation exchange resin (Partisphere SCX, Whatman) and then by 5 μm C18 reverse phase resin (Aqua, Phenomenex), and equilibrated in 5% acetonitrile, 0.1% formic acid (Buffer A). Peptides were sequentially eluted from the SCX resin to the reverse phase resin with six steps of increasing salt concentration. After each step, peptides were eluted from the reverse phase resin with a gradient of acetonitrile into a Deca-XP ion trap mass spectrometer equipped with a nano-liquid chromatography electrospray ionization source (ThermoFinnigan). The program 2-3 (26Sadygov R.G. Eng J. Durr E. Saraf A. McDonald H. MacCoss M.J. Yates J.R. II I J. Proteome Res. 2002; 1: 211-215Crossref PubMed Scopus (183) Google Scholar) was used to determine the charge state and to delete poor quality spectra. The SEQUEST algorithm (27Eng J.K. McCormick A.L. Yates J.R. II I J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5471) Google Scholar) was used to match tandem mass spectrometry spectra to human peptides extracted from the NCBI NR data base (27Eng J.K. McCormick A.L. Yates J.R. II I J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5471) Google Scholar,196 human protein sequences as of March 24, 2004). Spectra/peptide matches were only retained if they had a normalized difference in cross-correlation scores of at least 0.08 and a minimum cross-correlation score of 1.8 for +1, 2.5 for +2, and 3.5 for +3 spectra and if the peptides were at least 7 amino acids long. Human Tip49a and Tip49b Are Associated with a Human INO80-like Protein (hINO80)—As part of our characterization of the structures and functions of mammalian TRRAP-TIP60 and SRCAP chromatin remodeling complexes, we generated cell lines stably expressing either Tip49a or Tip49b with N-terminal FLAG epitope tags and then purified Tip49a- and Tip49b-associating proteins by anti-FLAG agarose immunoaffinity chromatography. As a control for the specificity of immunoaffinity purifications, extracts prepared from untransformed, parental cells were subjected to the same procedure. As shown in Fig. 1, anti-FLAG agarose eluates from FLAG-Tip49a- and FLAG-Tip49b-expressing cells appeared to include similar sets of proteins (compare lanes 1 and 2). To identify and compare FLAG-Tip49a- and FLAG-Tip49b-associating proteins, we took advantage of MudPIT (24Washburn M.P. Wolters D. Yates J.R. II I Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4099) Google Scholar, 28Wolters D. Washburn M.P. Yates J.R. Anal. Chem. 2001; 73: 5683-5690Crossref PubMed Scopus (1576) Google Scholar), a sensitive method for identifying proteins present in complex mixtures. In a Mud-PIT experiment, a mixture of proteins is first digested into peptides, which are then fractionated by two-dimensional strong cation exchange and reverse phase HPLC and analyzed by in-line tandem mass spectrometry. As summarized in Fig. 2, MudPIT analyses of anti-FLAG agarose eluates from FLAG-Tip49a- and FLAG-Tip49b-expressing cells identified, in addition to the known subunits of the TRRAP-TIP60 and SRCAP complexes, a collection of proteins not previously found in either TRRAP-TIP60 or SRCAP preparations or in MudPIT control samples. Among these proteins was a previously uncharacterized SNF2 family helicase encoded by the KIAA1259 ORF. As suggested by analysis of symmetrical best matches in data base searches and by phylogenetic inference, the 1561-amino acid KIAA1259 protein is an apparent human ortholog of the S. cerevisiae Ino80 helicase (29Bakshi R. Prakash T. Dash D. Brahmachari V. Biochem. Biophys. Res. Commun. 2004; 320: 197-204Crossref PubMed Scopus (22) Google Scholar). 4A. Mushegian, unpublished observations. Also among these proteins were the actin-related proteins Arp5 and Arp8, each of which has a yeast ortholog found in the INO80 complex but not in the SWR1 complex (Fig. 2, lanes 1 and 2). Subunit Composition of the hINO80 Complex—To investigate the possibility that some or all of these proteins were subunits of a mammalian INO80 complex, we generated an HEK293/FRT cell line stably expressing Arp8 with an N-terminal FLAG tag. Extracts prepared from these cells were subjected to anti-FLAG agarose chromatography, and proteins present in anti-FLAG agarose eluates were identified by Mud-PIT. As shown in Fig. 2, Arp8 copurified with hIno80, Tip49a and Tip49b, Baf53a (Arp4), Arp5, and an additional seven proteins that were not present in either the TRRAP-TIP60 or SRCAP complexes. These proteins included the "Pim-1 kinase-associated protein-associated protein 1" (PAPA-1, GI 13775202) (30Kuroda T.S. Maita H. Tabata T. Taira T. Kitaura H. Ariga H. Iguchi-Ariga S.M. Gene (Amst.). 2004; 340: 83-89Crossref PubMed Scopus (10) Google Scholar), Amida (also known as TCF3(E2A) fusion partner in childhood leukemia, GI 7019371) (31Irie Y. Yamagata K. Gan Y. Miyamoto K. Do E. Kuo C.H. Taira E. Miki N. J. Biol. Chem. 2000; 275: 2647-2653Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 32Brambillasca F. Mosna G. Colombo M. Rivolta A. Caslini C. Minuzzo M. Giudici G. Mizzi L. Biondi A. Privitera E. Leukemia. 1999; 13: 369-375Crossref PubMed Scopus (30) Google Scholar), nuclear factor related to κB-binding protein (NFRKB, GI 23346420) (33Adams B.S. Leung K.Y. Hanley E.W. Nabel G.J. New Biol. 1991; 3: 1063-1073PubMed Google Scholar), microspherule protein 1 (MCRS1 or MSP58, GI 29893564) (34Ren Y. Busch R.K. Perlaky Y. Busch H. Eur. J. Biochem. 1998; 253: 734-742Crossref PubMed Scopus (57) Google Scholar), and previously uncharacterized proteins encoded by the FLJ90652 (GI 27734727), C18orf37 (GI 34916002), and FLJ20309 (GI 38488718) genes. To determine whether these proteins were present in the same complex, we generated additional HeLa and HEK293/FRT cell lines stably expressing full-length PAPA-1, C18orf37, Amida, or FLJ90652, all with N-terminal FLAG tags, or a C-terminally FLAG-tagged fragment of FLJ20309 (residues 106–544). As shown in Fig. 1 (compare lanes 4 and 5), anti-FLAG agarose eluates prepared from FLAG-PAPA-1 expressing HeLa cells and from FLAG-FLJ90652 expressing HEK293/FRT cells appeared to include similar sets of proteins. Furthermore, MudPIT analyses revealed that FLAG-tagged PAPA-1, C18orf37, Amida, FLJ20309, and FLJ90652 each copurified with the hINO80 helicase and the Tip49a, Tip49b, PAPA-1, C18orf37, Arp4, Arp5, Arp8, Amida, NFRKB, MCRS1, FLJ90652, and FLJ20309 proteins, which argues that they are all components of a multiprotein hINO80-containing complex (Fig. 2). Notably, unique subunits of the TRRAP-TIP60 or SRCAP complexes were not detected by MudPIT in any of these purified samples (Fig. 2). Sequence analysis suggests that several of these hINO80-associated proteins are previously unrecognized orthologs of subunits of the yeast INO80 complex. PAPA-1 is orthologous to the Ies2 subunit of the yeast INO80 complex, and we henceforth designate it hIes2. When hIes2 is used as a query in a PSI-BLAST search, the PAPA-1 ortholog from Arabidopsis passes the threshold of 0.001 at the fourth iteration followed by vertebrate co-orthologs at iterations 7–10. We note that although hIes2/PAPA-1 is annotated in the NCBI data base as high mobility group AT-hook 1-like 4, it does not appear to contain recognizable AT-hook or high mobility group DNA-binding domains. Instead, the N-terminal half of the protein consists of a predicted long helical region, whereas the C-terminal half is globular and contains several conserved cysteine residues (data not shown). The human C18orf37 protein is orthologous to the Ies6 subunit of the yeast INO80 complex. Comparison of protein family alignments using the pairwise Hidden Markov Model-based algorithm, HHsearch (35Soding J. Bioinformatics. 2005; 21: 951-960Crossref PubMed Scopus (1875) Google Scholar), indicates that both proteins contain a modified zinc ribbon, most closely related to the YL1 family of putative transcriptional activators (36Horikawa I. Tanaka H. Yuasa Y. Suzuki M. Oshimura M. Biochem. Biophys. Res. Commun. 1995; 208: 999-1007Crossref PubMed Scopus (17) Google Scholar, 37Horikawa I. Tanaka H. Yuasa Y. Suzuki M. Shimizu M. Oshimura M. Exp. Cell Res. 1995; 220: 11-17Crossref PubMed Scopus (10) Google Scholar). We previously identified the YL1 protein as a component of the mammalian TRRAP-TIP60 and SRCAP complexes (19Cai Y. Jin J. Florence L. Swanson S.K. Kusch T. Li B. Workman J.L. Washburn M.P. Conaway R.C. Conaway J.W. J. Biol. Chem. 2005; 280: 13665-13670Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). The five remaining proteins appear to be unique to the human INO80 complex. NFRKB is a large (more than 1300 amino acids) protein. The C-terminal half of NFRKB contains low complexity, mucin-like repeats. The N-terminal half of this protein is broadly conserved in metazoans (except for Caenorhabditis elegans), in plants, and in Giardia, but it appears to have no homologs in fungi. MCRS1/MSP58 contains a forkhead-associated (FHA) domain in its N terminus. Although there are FHA-like domains in fungi, the N-terminal 350 amino acids of MCRS1 represent a distinct domain, well conserved in metazoans, plants, and some protists, but not found in the available fungal genomes. The FLJ20309 protein has a characteristic pattern of conserved histidine and cysteine residues. Like the MCRS1 protein, FLJ20309 orthologs appear to be present in multicellular eukaryotes but not in fungi. Amida, or TCF3 (E2A) fusion partner in childhood leukemia, contains a putative DNA-binding domain of the b-ZIP type. The FLJ90652 protein has an N-terminal domain that is distantly related to b-ZIP domains. The hINO80 Complex Catalyzes ATP-dependent Nucleosome Sliding— Previous studies revealed that the S. cerevisiae INO80 complex possesses both DNA- and nucleosome-activated ATPase and ATP-dependent nucleosome sliding activities. To investigate the possibility that the mammalian INO80-related complex possesses similar activities, we assayed anti-FLAG agarose eluates from FLAG-hIes2-expressing HeLa cells for ATPase and nucleosome remodeling activities. As shown in Fig. 3, the hINO80 complex, immunopurified from FLAG-hIes2-expressing cells, catalyzed ATP hydrolysis in a reaction that was strongly dependent on the addition of DNA or nucleosomes. Although both mononucleosomes and oligonucleosomes stimulated ATPase more strongly than did DNA, free histone octamers had no effect on the reaction. To determine whether the hINO80 complex could catalyze ATP-dependent nucleosome mobilization, anti-FLAG agarose eluates from FLAG-hIes2 expressing HeLa cells were mixed with mononucleosomes reconstituted on a 32P-labeled, 216-base pair DNA fragment in the presence of ATP or a mixture of ATP and ATPγS, a potent inhibitor of many ATPases. At the conclusion of the reaction, HeLa oligonuclesomes and free DNA were added as competitor to remove DNA- or nucleosome-binding proteins that would alter mononucleosome electrophoretic mobility. Reaction products were then fractionated on native polyacrylamide gels. The electrophoretic mobility of mononucleosomes depends on the position of the nucleosome on the DNA. The mobility of a nucleosome positioned in the middle of the DNA fragment is slow, whereas nucleosomes positioned more laterally migrate more rapidly (38Meersseman G. Pennings S. Bradbury E.M. EMBO J. 1992; 11: 2951-2959Crossref PubMed Scopus (262) Google Scholar, 39Duband-Goulet I. Carot V. Ulyanov A.V. Douc-Rasy S. Prunell A. J. Mol. Biol. 1992; 224: 981-1001Crossref PubMed Scopus (34) Google Scholar). The reconstituted nucleosomes used in our experiments include a mixture of centrally located and lateral nucleosomes, with the majority of nucleosomes located at the DNA ends (Fig. 4A, lane 4, and B, lane 1). Upon the addition of increasing amounts of the purified hINO80 complex, nucleosomes were shifted to a more central position (Fig. 4A, lanes 5 and 6). Similar results were obtained when hINO80 complexes were immunopurified from cells expressing FLAG-Amida or FLAG-FLJ90652 (Fig. 4A, compare lane 11 with lanes 12 and 13). The change in nucleosome position catalyzed by the hINO80 complex depends upon ATP (Fig. 4B) and is inhibited by ATPγS. Thus, like the yeast INO80 complex, the hINO80 complex can support ATP-dependent nucleosome sliding. Notably, both the yeast and human INO80 complexes move nucleosomes from a lateral to a central position. Summary and Perspective—In this report we have exploited a Mud-PIT-based subtractive proteomics approach to identify and characterize a novel mammalian ATP-dependent chromatin remodeling complex that shares structural and functional properties with the S. cerevisiae INO80 complex (TABLE ONE). This new mammalian INO80-related chromatin remodeling complex contains the INO80-like, SNF2 family helicase encoded by the previously uncharacterized KIAA1259 ORF, as well as several additional proteins that appear to be orthologs of subunits of the yeast INO80 complex; these include the Tip49a and Tip49b AAA+ ATPases, the actin-related proteins Arp4, Arp5, and Arp8, hIes2 (PAPA-1), and hIes6 (C18orf37). Notably, the hINO80 complex contains five additional proteins that appear to lack yeast orthologs; these include the b-ZIP domain-containing Amida protein, the forkhead-associated domain-containing MCRS1 protein, the NFRKB protein, and proteins encoded by the FLJ90652 and FLJ20309 ORFs. Finally, we show that, despite these apparent differences in the subunit compositions of the yeast and human INO80 complexes, these complexes possess similar chromatin remodeling activities. Future studies investigating the contributions of the individual subunits of the hINO80 complex should provide more in-depth insights into the functional similarities and differences between the yeast and mammalian chromatin remodeling complexes.TABLE ONEComparison of the subunit compositions of the yeast INO80- and mammalian INO80-related chromatin remodeling complexesYeast INO80Human INO80Molecular function and domain structureComplexGI no.aGI, GenInfo Identifier, a unique identifier for protein or nucleic acid sequences in the NCBI Entrez protein and nucleotide data basesComplexGI no.aGI, GenInfo Identifier, a unique identifier for protein or nucleic acid sequences in the NCBI Entrez protein and nucleotide data basesIno806321289KIAA125933469139SNF2-like helicaseArp82492678Arp839812115Actin-related proteinArp51730738Arp531542680Actin-related proteinArp4728794BAF53a/Arp430089997Actin-related proteinRvb16320396TIP49a4506753AAA + ATPaseRvb26325021TIP49b5730023AAA + ATPaseIes26324114PAPA-113775202Helical, perhaps nonglobular, N-terminal half and mostly β-strand C-terminal halfIes66320791C18orf3734916002Modified zinc ribbon, distantly related to YL-1 family of putative transcription factorsAmida/TCF3 (E2A) fusion partner7019371b-ZIP-type, possibly DNA-binding, proteinFLJ9065227734727Conserved in metazoa (not C. elegans)NFRKB23346420Conserved in metazoa (not C. elegans), plants, Giardia; nonglobular mucin-like C-halfMCRS12384717FHAbFHA, forkhead-associated domain in the C terminusFLJ2030938488718Two copies of CxnHhhhhxnCxnC signature; orthologs in plants and metazoanTaf14461510YEATS domainNhp106320202HMGcHMG, high mobility group type II domain; binds kinked, looped, distorted, or four-way DNAIes114318508Ies36323081Ies46324763Ies56320938a GI, GenInfo Identifier, a unique identifier for protein or nucleic acid sequences in the NCBI Entrez protein and nucleotide data basesb FHA, forkhead-associatedc HMG, high mobility group Open table in a new tab
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