Quantitative Analysis of Chromosome Condensation in Fission Yeast

ABSTRACT Chromosomes undergo extensive conformational rearrangements in preparation for their segregation during cell divisions. Insights into the molecular mechanisms behind this still poorly understood condensation process require the development of new approaches to quantitatively assess chromosome formation in vivo. In this study, we present a live-cell microscopy-based chromosome condensation assay in the fission yeast Schizosaccharomyces pombe. By automatically tracking the three-dimensional distance changes between fluorescently marked chromosome loci at high temporal and spatial resolution, we analyze chromosome condensation during mitosis and meiosis and deduct defined parameters to describe condensation dynamics. We demonstrate that this method can determine the contributions of condensin, topoisomerase II, and Aurora kinase to mitotic chromosome condensation. We furthermore show that the assay can identify proteins required for mitotic chromosome formation de novo by isolating mutants in condensin, DNA polymerase ε, and F-box DNA helicase I that are specifically defective in pro-/metaphase condensation. Thus, the chromosome condensation assay provides a direct and sensitive system for the discovery and characterization of components of the chromosome condensation machinery in a genetically tractable eukaryote.

[1]  M. Yanagida,et al.  Fission yeast cut3 and cut14, members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis. , 1994, The EMBO journal.

[2]  T. Toda,et al.  Role of the Schizosaccharomyces pombe F-Box DNA Helicase in Processing Recombination Intermediates , 2005, Molecular and Cellular Biology.

[3]  B. Turner,et al.  Antibodies to defined histone epitopes reveal variations in chromatin conformation and underacetylation of centric heterochromatin in human metaphase chromosomes , 1992, Chromosoma.

[4]  K. Nasmyth,et al.  Cohesins: Chromosomal Proteins that Prevent Premature Separation of Sister Chromatids , 1997, Cell.

[5]  J. J. Ward,et al.  Condensins Promote Chromosome Recoiling during Early Anaphase to Complete Sister Chromatid Separation , 2010, Developmental cell.

[6]  M. Yanagida,et al.  Condensin phosphorylated by the Aurora-B-like kinase Ark1 is continuously required until telophase in a mode distinct from Top2 , 2011, Journal of Cell Science.

[7]  D. D'Amours,et al.  Three-step model for condensin activation during mitotic chromosome condensation , 2010, Cell cycle.

[8]  Yoshinori Watanabe,et al.  Repositioning of aurora B promoted by chiasmata ensures sister chromatid mono-orientation in meiosis I. , 2011, Developmental cell.

[9]  K. Maundrell,et al.  TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. , 1993, Gene.

[10]  M. Yanagida,et al.  Identification of seven new cut genes involved in Schizosaccharomyces pombe mitosis. , 1993, Journal of cell science.

[11]  J. Ellenberg,et al.  Measuring structural dynamics of chromosomes in living cells by fluorescence microscopy. , 2007, Methods.

[12]  T. Toda,et al.  The NDA3 gene of fission yeast encodes β-tubulin: A cold-sensitive nda3 mutation reversibly blocks spindle formation and chromosome movement in mitosis , 1984, Cell.

[13]  E. Verdin,et al.  Regulation of global acetylation in mitosis through loss of histone acetyltransferases and deacetylases from chromatin. , 2001, The Journal of biological chemistry.

[14]  L. Magnaghi-Jaulin,et al.  Histone deacetylase activity is necessary for chromosome condensation during meiotic maturation in Xenopus laevis , 2006, Chromosome Research.

[15]  M. Botchan,et al.  Orc mutants arrest in metaphase with abnormally condensed chromosomes. , 2001, Development.

[16]  A. Lamond,et al.  Condensin and Repo-Man–PP1 co-operate in the regulation of chromosome architecture during mitosis , 2006, Nature Cell Biology.

[17]  U. K. Laemmli,et al.  Metaphase chromosome structure. Involvement of topoisomerase II. , 1986, Journal of molecular biology.

[18]  J. Ellenberg,et al.  Distinct functions of condensin I and II in mitotic chromosome assembly , 2004, Journal of Cell Science.

[19]  G. Goshima,et al.  Fission yeast living mitosis visualized by GFP-tagged gene products. , 2001, Micron.

[20]  K. Oegema,et al.  Molecular analysis of mitotic chromosome condensation using a quantitative time-resolved fluorescence microscopy assay , 2006, Proceedings of the National Academy of Sciences.

[21]  D. J. Clarke,et al.  In vivo analysis of chromosome condensation in Saccharomyces cerevisiae. , 2006, Molecular biology of the cell.

[22]  Steven J. M. Jones,et al.  High quality SNP calling using Illumina data at shallow coverage , 2010, Bioinform..

[23]  W. Earnshaw,et al.  Condensin: Architect of mitotic chromosomes , 2009, Chromosome Research.

[24]  R. Durbin,et al.  Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes , 2010, Nature.

[25]  Yoshinori Watanabe,et al.  Condensin association with histone H2A shapes mitotic chromosomes , 2011, Nature.

[26]  U. K. Laemmli,et al.  A two-step scaffolding model for mitotic chromosome assembly. , 2003, Developmental cell.

[27]  A. Strunnikov,et al.  Unreplicated DNA in mitosis precludes condensin binding and chromosome condensation in S. cerevisiae. , 2008, Frontiers in bioscience : a journal and virtual library.

[28]  A. Murray,et al.  Dynamics of centromeres during metaphase-anaphase transition in fission yeast: Dis1 is implicated in force balance in metaphase bipolar spindle. , 1998, Molecular biology of the cell.

[29]  T. Uemura,et al.  Isolation and characterization of Schizosaccharomyces pombe cutmutants that block nuclear division but not cytokinesis , 1986, The EMBO journal.

[30]  Yoshinori Watanabe,et al.  Kinetochore geometry defined by cohesion within the centromere , 2009, Nature.

[31]  P. Sunnerhagen,et al.  Inhibition of type I histone deacetylase increases resistance of checkpoint-deficient cells to genotoxic agents through mitotic delay , 2009, Molecular Cancer Therapeutics.

[32]  S. Forsburg,et al.  The Schizosaccharomyces pombe aurora-related kinase Ark1 interacts with the inner centromere protein Pic1 and mediates chromosome segregation and cytokinesis. , 2002, Molecular biology of the cell.

[33]  W. Earnshaw,et al.  RNAi analysis reveals an unexpected role for topoisomerase II in chromosome arm congression to a metaphase plate , 2003, Journal of Cell Science.

[34]  I. Hagan,et al.  The use of cell division cycle mutants to investigate the control of microtubule distribution in the fission yeast Schizosaccharomyces pombe. , 1988, Journal of cell science.

[35]  T. Uemura,et al.  DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe , 1987, Cell.

[36]  R. Kobayashi,et al.  Condensins, Chromosome Condensation Protein Complexes Containing XCAP-C, XCAP-E and a Xenopus Homolog of the Drosophila Barren Protein , 1997, Cell.

[37]  K. Maundrell.  Thiamine-repressible expression vectors pREP and pRIP for fission yeast. , 1993, Gene.

[38]  J. Swedlow,et al.  How to be a mitotic chromosome , 2011, Chromosome Research.

[39]  R. Gassmann,et al.  Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes. , 2003, Developmental cell.

[40]  J. Ellenberg,et al.  Condensin I Stabilizes Chromosomes Mechanically through a Dynamic Interaction in Live Cells , 2006, Current Biology.

[41]  J. Peters,et al.  The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore–microtubule attachment and in maintaining the spindle assembly checkpoint , 2003, The Journal of cell biology.

[42]  H. Araki,et al.  DNA polymerase ε encoded by cdc20+ is required for chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe , 1998, Genes to cells : devoted to molecular & cellular mechanisms.

[43]  C. Sunkel,et al.  The Condensin I Subunit Barren/CAP-H Is Essential for the Structural Integrity of Centromeric Heterochromatin during Mitosis , 2005, Molecular and Cellular Biology.

[44]  C. Sunkel,et al.  A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis , 2001, Current Biology.

[45]  Kim Nasmyth,et al.  The Polo‐like kinase Cdc5p and the WD‐repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae , 1998, The EMBO journal.

[46]  J. Mccusker,et al.  Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae , 1999, Yeast.

[47]  S. Cotterill,et al.  Drosophila CAP-D2 is required for condensin complex stability and resolution of sister chromatids , 2005, Journal of Cell Science.

[48]  J. Ellenberg,et al.  Maximal chromosome compaction occurs by axial shortening in anaphase and depends on Aurora kinase , 2007, Nature Cell Biology.

[49]  J. Boeke,et al.  Efficient targeted integration at leu1-32 and ura4-294 in Schizosaccharomyces pombe. , 1994, Genetics.

[50]  W. Earnshaw,et al.  Topoisomerase II is a structural component of mitotic chromosome scaffolds , 1985, The Journal of cell biology.

[51]  Sue A. Krause,et al.  Aberrant replication timing induces defective chromosome condensation in Drosophila ORC2 mutants , 2000, Current Biology.

[52]  C. Sunkel,et al.  Condensin-dependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis , 2003, Journal of Cell Science.

[53]  Andrew W. Murray,et al.  GFP tagging of budding yeast chromosomes reveals that protein–protein interactions can mediate sister chromatid cohesion , 1996, Current Biology.

[54]  Wilhelm Burger,et al.  Digital Image Processing - An Algorithmic Introduction using Java , 2016, Texts in Computer Science.

[55]  Angus I. Lamond,et al.  Quantitative analysis of chromatin compaction in living cells using FLIM–FRET , 2009, The Journal of cell biology.

[56]  T. Itoh,et al.  Identification of cis-acting sites for condensin loading onto budding yeast chromosomes. , 2008, Genes & development.

[57]  H. Hakonarson,et al.  ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data , 2010, Nucleic acids research.

[58]  A. Porter,et al.  Construction, characterization, and complementation of a conditional-lethal DNA topoisomerase IIalpha mutant human cell line. , 2004, Molecular biology of the cell.

[59]  G. Goshima,et al.  Bir1/Cut17 moving from chromosome to spindle upon the loss of cohesion is required for condensation, spindle elongation and repair , 2001, Genes to cells : devoted to molecular & cellular mechanisms.

[60]  M. Jantsch,et al.  The Aurora B Kinase AIR-2 Regulates Kinetochores during Mitosis and Is Required for Separation of Homologous Chromosomes during Meiosis , 2002, Current Biology.

[61]  V. Guacci,et al.  Chromosome condensation and sister chromatid pairing in budding yeast , 1994, The Journal of cell biology.

[62]  W. Earnshaw,et al.  Condensin I interacts with the PARP-1-XRCC1 complex and functions in DNA single-strand break repair. , 2006, Molecular cell.

[63]  Juri Rappsilber,et al.  The Protein Composition of Mitotic Chromosomes Determined Using Multiclassifier Combinatorial Proteomics , 2010, Cell.

[64]  W. Earnshaw,et al.  ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure , 1994, The Journal of cell biology.

[65]  R. Allshire,et al.  Kinetochore and heterochromatin domains of the fission yeast centromere , 2004, Chromosome Research.

[66]  T. Kunkel,et al.  The Major Roles of DNA Polymerases Epsilon and Delta at the Eukaryotic Replication Fork Are Evolutionarily Conserved , 2011, PLoS genetics.

[67]  Kevin W Eliceiri,et al.  NIH Image to ImageJ: 25 years of image analysis , 2012, Nature Methods.

[68]  S. Forsburg,et al.  A Screen for Schizosaccharomyces pombe Mutants Defective in Rereplication Identifies New Alleles of rad4+, cut9+ and psf2+ , 2005, Genetics.

[69]  S. Kawashima,et al.  Aurora controls sister kinetochore mono-orientation and homolog bi-orientation in meiosis-I , 2007, The EMBO journal.

[70]  F. M. Yeong,et al.  Identification of a Subunit of a Novel Kleisin-β/SMC Complex as a Potential Substrate of Protein Phosphatase 2A , 2003, Current Biology.

[71]  I. Hagan,et al.  S. pombe Aurora Kinase/Survivin Is Required for Chromosome Condensation and the Spindle Checkpoint Attachment Response , 2003, Current Biology.

[72]  A. Belmont.  Mitotic chromosome structure and condensation. , 2006, Current opinion in cell biology.

[73]  T. Mitchison,et al.  A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro , 1994, Cell.

[74]  T. Uemura,et al.  Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization. , 1984, The EMBO journal.

[75]  Yuda Fang,et al.  Spatial and temporal regulation of Condensins I and II in mitotic chromosome assembly in human cells. , 2004, Molecular biology of the cell.

[76]  I. Hagan,et al.  The S. pombe aurora-related kinase Ark1 associates with mitotic structures in a stage dependent manner and is required for chromosome segregation. , 2001, Journal of cell science.

[77]  T. Hirano.  Condensins: Organizing and Segregating the Genome , 2005, Current Biology.

[78]  D. Gerlich,et al.  Assays for mitotic chromosome condensation in live yeast and mammalian cells , 2009, Chromosome Research.

[79]  M. Yanagida,et al.  Dissection of the essential steps for condensin accumulation at kinetochores and rDNAs during fission yeast mitosis , 2008, The Journal of cell biology.

[80]  P. Nurse,et al.  Nuclear size control in fission yeast , 2007, The Journal of cell biology.

[81]  A. F. Neuwald,et al.  Differential Contributions of Condensin I and Condensin II to Mitotic Chromosome Architecture in Vertebrate Cells , 2003, Cell.

[82]  M. Yanagida.  Clearing the way for mitosis: is cohesin a target? , 2009, Nature Reviews Molecular Cell Biology.

[83]  Dongsup Kim,et al.  Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe , 2010, Nature Biotechnology.

[84]  P. Nurse,et al.  Schizosaccharomyces pombe cdc20+ encodes DNA polymerase epsilon and is required for chromosomal replication but not for the S phase checkpoint. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[85]  M. Whitby,et al.  A failure of meiotic chromosome segregation in a fbh1Δ mutant correlates with persistent Rad51-DNA associations , 2010, Nucleic acids research.