Proteomics Analysis with a Nano Random Forest Approach Reveals Novel Functional Interactions Regulated by SMC Complexes on Mitotic Chromosomes
2016; Elsevier BV; Volume: 15; Issue: 8 Linguagem: Inglês
10.1074/mcp.m116.057885
ISSN1535-9484
AutoresShinya Ohta, Luis Fernando Montaño-Gutierrez, Flávia de Lima Alves, Hiromi Ogawa, Toramoto Iyo, Nobuko Sato, Ciaran G. Morrison, Shunichi Takeda, Damien F. Hudson, Juri Rappsilber, William C. Earnshaw,
Tópico(s)RNA and protein synthesis mechanisms
ResumoPackaging of DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although the structure and composition of mitotic chromosomes have been studied for over 30 years, these aspects are yet to be fully elucidated. Here, we used stable isotope labeling with amino acids in cell culture to compare the proteomes of mitotic chromosomes isolated from cell lines harboring conditional knockouts of members of the condensin (SMC2, CAP-H, CAP-D3), cohesin (Scc1/Rad21), and SMC5/6 (SMC5) complexes. Our analysis revealed that these complexes associate with chromosomes independently of each other, with the SMC5/6 complex showing no significant dependence on any other chromosomal proteins during mitosis. To identify subtle relationships between chromosomal proteins, we employed a nano Random Forest (nanoRF) approach to detect protein complexes and the relationships between them. Our nanoRF results suggested that as few as 113 of 5058 detected chromosomal proteins are functionally linked to chromosome structure and segregation. Furthermore, nanoRF data revealed 23 proteins that were not previously suspected to have functional interactions with complexes playing important roles in mitosis. Subsequent small-interfering-RNA-based validation and localization tracking by green fluorescent protein-tagging highlighted novel candidates that might play significant roles in mitotic progression. Packaging of DNA into condensed chromosomes during mitosis is essential for the faithful segregation of the genome into daughter nuclei. Although the structure and composition of mitotic chromosomes have been studied for over 30 years, these aspects are yet to be fully elucidated. Here, we used stable isotope labeling with amino acids in cell culture to compare the proteomes of mitotic chromosomes isolated from cell lines harboring conditional knockouts of members of the condensin (SMC2, CAP-H, CAP-D3), cohesin (Scc1/Rad21), and SMC5/6 (SMC5) complexes. Our analysis revealed that these complexes associate with chromosomes independently of each other, with the SMC5/6 complex showing no significant dependence on any other chromosomal proteins during mitosis. To identify subtle relationships between chromosomal proteins, we employed a nano Random Forest (nanoRF) approach to detect protein complexes and the relationships between them. Our nanoRF results suggested that as few as 113 of 5058 detected chromosomal proteins are functionally linked to chromosome structure and segregation. Furthermore, nanoRF data revealed 23 proteins that were not previously suspected to have functional interactions with complexes playing important roles in mitosis. Subsequent small-interfering-RNA-based validation and localization tracking by green fluorescent protein-tagging highlighted novel candidates that might play significant roles in mitotic progression. Mitotic chromosome condensation, regulated sister chromatid cohesion, and chromosome interactions with the spindle are crucial to ensuring appropriate genome segregation during mitosis and meiosis. The first 2 of these events require the activity of protein complexes containing structural maintenance of chromosomes (SMC) 1The abbreviations used are:SMCstructural maintenance of chromosomesACO1aconitase 1ADPadenosine diphosphateATPadenosine triphosphateAUCarea under the curveBSAbovine serum albuminCCANconstitutive centromere-associated networkCCDC99coiled-coil domain-containing protein 99CPCchromosome passenger complexDMEMDulbecco's Modified Eagle's MediumFBSfetal bovine serumGFPgreen-fluorescent proteinKMNKnl-1/Mis12/Ndc80 complexesMCCMatthews correlation coefficientMCMmini-chromosome maintenanceMSmass spectrometryORCorigin-recognition complexPBSphosphate-buffered salinePIpropidium iodidePPP2R2Aserine/threonine-protein phosphatase 2A, regulatory subunit B, alpha isoformppmparts per millionpre-RCprereplication complexPTPN6tyrosine-protein phosphatase nonreceptor type 6RFRandom ForestROCreceiver operating characteristicRPMIRoswell Park Memorial InstituteSACspindle assembly checkpointSCXstrong cation-exchange chromatographySDS-PAGEsodium dodecyl sulfate-polyacrylamide gel electrophoresisSILACstable isotope labeling with amino acids in cell culturesiRNAsmall interfering RNASKAPsmall kinetochore-associated proteinSMARCA1SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 1VRK1vaccinia-related kinase 1. proteins. SMC proteins are large polypeptides that fold back upon themselves via a central hinge region, enabling the formation of a long, antiparallel coiled-coil domain (1.Barysz H. Kim J.H. Chen Z.A. Hudson D.F. Rappsilber J. Gerloff D.L. Earnshaw W.C. Three-dimensional topology of the SMC2/SMC4 subcomplex from chicken condensin I revealed by cross-linking and molecular modelling.Open Biol. 2015; 5: 150005Crossref PubMed Scopus (41) Google Scholar). ATP binding to a bipartite adenosine triphosphate (ATP)-binding cassette ATPase motif juxtaposes the N- and C-terminal globular domains of each SMC protein, forming a closed loop. A strap-like kleisin protein then holds the heads of both SMC proteins together. Although the exact role of the ATPase activity is unknown, it is essential for condensin function (2.Hudson D.F. Ohta S. Freisinger T. Macisaac F. Sennels L. Alves F. Lai F. Kerr A. Rappsilber J. Earnshaw W.C. Molecular and genetic analysis of condensin function in vertebrate cells.Mol. 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The SMC complexes can be divided into 3 groups. The first and most-studied SMC complex is cohesin, which contains SMC1, SMC3, the kleisin Scc1, and 1 of 3 auxiliary different subunits (SA1-SA3; Scc3 in budding yeast) (5.Losada A. Yokochi T. Kobayashi R. Hirano T. Identification and characterization of SA/Scc3p subunits in the Xenopus and human cohesin complexes.J. Cell Biol. 2000; 150: 405-416Crossref PubMed Scopus (258) Google Scholar, 6.Sumara I. Vorlaufer E. Gieffers C. Peters B.H. Peters J.M. Characterization of vertebrate cohesin complexes and their regulation in prophase.J. Cell Biol. 2000; 151: 749-762Crossref PubMed Scopus (336) Google Scholar, 7.Prieto I. Pezzi N. Buesa J.M. Kremer L. Barthelemy I. Carreiro C. Roncal F. Martinez A. Gomez L. Fernandez R. Martinez-A C. Barbero J.L. STAG2 and Rad21 mammalian mitotic cohesins are implicated in meiosis.EMBO Rep. 2002; 3: 543-550Crossref PubMed Scopus (75) Google Scholar). 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This analysis revealed that SMC complexes act independently of each other, with only weak dependence between condensin I and condensin II. Condensin depletions appeared to have the most profound effects on the mitotic chromosome proteome, and depletions of the SMC5/6 complex appeared to have the least impact, with the only significant changes occurring exclusively among members of that complex. This analysis revealed few structural links between cohesin and condensin, in contrast to suggestions of several published studies (14.Shintomi K. Hirano T. The relative ratio of condensin I to II determines chromosome shapes.Genes Dev. 2011; 25: 1464-1469Crossref PubMed Scopus (111) Google Scholar, 38.Hirota T. Gerlich D. Koch B. Ellenberg J. Peters J.-M. Distinct functions of condensin I and II in mitotic chromosome assembly.J. Cell Sci. 2004; 117: 6435-6445Crossref PubMed Scopus (270) Google Scholar). 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HeLa and U2OS cells in the exponential growth phase were seeded onto coverslips and grown overnight in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS at 37 °C in an atmosphere containing 5% CO2. DT40 cells were incubated with nocodazole for 13 h, resulting in a mitotic index of 70–90%. Mitotic chromosomes were isolated using a buffer system containing polyamines and ethylenediaminetetraacetic acid optimized for mitotic chromosome isolation from DT40 cells (35.Ohta S. Bukowski-Wills J.-C. Sanchez-Pulido L. Alves F. de L. Wood L. Chen Z.A. Platani M. Fischer L. Hudson D.F. Ponting C.P. Fukagawa T. Earnshaw W.C. Rappsilber J. The Protein Composition of Mitotic Chromosomes Determined Using Multiclassifier Combinatorial Proteomics.Cell. 2010; 142: 810-821Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar, 42.Lewis C.D. 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