Skip to main content
Log in

Condensin I and II behaviour in interphase nuclei and cells undergoing premature chromosome condensation

  • Published:
Chromosome Research Aims and scope Submit manuscript

Abstract

Condensin is an integral component of the mitotic chromosome condensation machinery, which ensures orderly segregation of chromosomes during cell division. In metazoans, condensin exists as two complexes, condensin I and II. It is not yet clear what roles these complexes may play outside mitosis, and so we have examined their behaviour both in normal interphase and in premature chromosome condensation (PCC). We find that a small fraction of condensin I is retained in interphase nuclei, and our data suggests that this interphase nuclear condensin I is active in both gene regulation and chromosome condensation. Furthermore, live cell imaging demonstrates condensin II dramatically increases on G1 nuclei following completion of mitosis. Our PCC studies show condensins I and II and topoisomerase II localise to the chromosome axis in G1-PCC and G2/M-PCC, while KIF4 binding is altered. Individually, condensins I and II are dispensable for PCC. However, when both are knocked out, G1-PCC chromatids are less well structured. Our results define new roles for the condensins during interphase and provide new information about the mechanism of PCC.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

3D FISH:

Three-dimensional fluorescent in situ hybridization

Cal A:

Calyculin A

CENP-O:

Centromere protein O

Dox:

Doxycycline

DE:

Differential expression

EdU:

5-Ethynyl-2'-deoxyuridine

IF:

Immunofluorescence

NEBD:

Nuclear envelope breakdown

PCC:

Premature chromosome condensation

PFA:

Paraformaldehyde

SBP:

Streptavidin-binding peptide

Topo IIα:

Topoisomerase IIα

References

  • Bakhrebah M, Zhang T, Mann JR, Kalitsis P, Hudson DF (2015) Disruption of a conserved CAP-D3 threonine alters condensin loading on mitotic chromosomes leading to chromosome hypercondensation. J Biol Chem 290:6156–67

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Barrett T, Wilhite SE, Ledoux P, Evangelista C, Kim IF, Tomashevsky M, Marshall KA, Phillippy KH, Sherman PM, Holko M, Yefanov A, Lee H, Zhang N, Robertson CL, Serova N, Davis S, Soboleva A (2013) NCBI GEO: archive for functional genomics data sets—update. Nucleic Acids Res 41:D991–5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bauer CR, Hartl TA, Bosco G (2012) Condensin II promotes the formation of chromosome territories by inducing axial compaction of polyploid interphase chromosomes. PLoS Genet 8, e1002873

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bazile F, St-Pierre J, D'Amours D (2010) Three-step model for condensin activation during mitotic chromosome condensation. Cell Cycle 9:3243–55

    Article  CAS  PubMed  Google Scholar 

  • Belmont AS (2006) Mitotic chromosome structure and condensation. Curr Opin Cell Biol 18:632–8

    Article  CAS  PubMed  Google Scholar 

  • Berkopec A (2007) HyperQuick algorithm for discrete hypergeometric distribution. J Discret Algorithm 5:341–347

    Article  Google Scholar 

  • Buerstedde JM, Takeda S (1991) Increased ratio of targeted to random integration after transfection of chicken B cell lines. Cell 67:179–88

    Article  CAS  PubMed  Google Scholar 

  • Crasta K, Ganem NJ, Dagher R, Lantermann AB, Ivanova EV, Pan Y, Nezi L, Protopopov A, Chowdhury D, Pellman D (2012) DNA breaks and chromosome pulverization from errors in mitosis. Nature 482:53–8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cremer M, Grasser F, Lanctot C, Muller S, Neusser M, Zinner R, Solovei I, Cremer T (2008) Multicolor 3D fluorescence in situ hybridization for imaging interphase chromosomes. Methods Mol Biol 463:205–39

    Article  CAS  PubMed  Google Scholar 

  • Cremer M, Weierich C, Solovei I (2005) Multicolour 3D-FISH in vertebrate cells (PROT23) [Online]. http://www.epigenesys.eu/en/protocols/fluorescence-microscopy/181-multicolour-3d-fish-in-vertebrate-cells

  • Dewari PS, Bhargava P (2014) Genome-wide mapping of yeast histone chaperone anti-silencing function 1 reveals its role in condensin binding with chromatin. PLoS ONE 9, e108652

    Article  PubMed  PubMed Central  Google Scholar 

  • Earnshaw WC, Ratrie H 3rd, Stetten G (1989) Visualization of centromere proteins CENP-B and CENP-C on a stable dicentric chromosome in cytological spreads. Chromosoma 98:1–12

    Article  CAS  PubMed  Google Scholar 

  • Edgar R, Domrachev M, Lash AE (2002) Gene Expression Omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30:207–10

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • EL Achkar E, Gerbault-Seureau M, Muleris M, Dutrillaux B, Debatisse M (2005) Premature condensation induces breaks at the interface of early and late replicating chromosome bands bearing common fragile sites. Proc Natl Acad Sci U S A 102:18069–74

    Article  PubMed  PubMed Central  Google Scholar 

  • Fagerholm AE, Habrant D, Koskinen AM (2010) Calyculins and related marine natural products as serine-threonine protein phosphatase PP1 and PP2A inhibitors and total syntheses of calyculin A, B, and C. Mar Drugs 8:122–72

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Franklin R, Sale JE (2006) 2D cell cycle analysis. Subcell Biochem 40:405–8

    PubMed  Google Scholar 

  • Freeman L, Aragon-Alcaide L, Strunnikov A (2000) The condensin complex governs chromosome condensation and mitotic transmission of rDNA. J Cell Biol 149:811–24

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Frosi Y, Haering CH (2015) Control of chromosome interactions by condensin complexes. Curr Opin Cell Biol 34:94–100

    Article  CAS  PubMed  Google Scholar 

  • Gallego-Paez LM, Tanaka H, Bando M, Takahashi M, Nozaki N, Nakato R, Shirahige K, Hirota T (2014) Smc5/6-mediated regulation of replication progression contributes to chromosome assembly during mitosis in human cells. Mol Biol Cell 25:302–17

    Article  PubMed  PubMed Central  Google Scholar 

  • George CM, Bozler J, Nguyen HQ, Bosco G (2014) Condensins are required for maintenance of nuclear architecture. Cells 3:865–82

    Article  PubMed  PubMed Central  Google Scholar 

  • Gerlich D, Hirota T, Koch B, Peters JM, Ellenberg J (2006) Condensin I stabilizes chromosomes mechanically through a dynamic interaction in live cells. Curr Biol 16:333–44

    Article  CAS  PubMed  Google Scholar 

  • Gotoh E (2009) Drug-induced premature chromosome condensation (PCC) protocols: cytogenetic approaches in mitotic chromosome and interphase chromatin. Methods Mol Biol 523:83–92

    Article  CAS  PubMed  Google Scholar 

  • Gotoh E, Durante M (2006) Chromosome condensation outside of mitosis: mechanisms and new tools. J Cell Physiol 209:297–304

    Article  CAS  PubMed  Google Scholar 

  • Green LC, Kalitsis P, Chang TM, Cipetic M, Kim JH, Marshall O, Turnbull L, Whitchurch CB, Vagnarelli P, Samejima K, Earnshaw WC, Choo KH, Hudson DF (2012) Contrasting roles of condensin I and condensin II in mitotic chromosome formation. J Cell Sci 125:1591–604

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hirano T (2014) Condensins and the evolution of torsion-mediated genome organization. Trends Cell Biol 24:727–33

    Article  CAS  PubMed  Google Scholar 

  • Hirano T, Kobayashi R, Hirano M (1997) Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barren protein. Cell 89:511–21

    Article  CAS  PubMed  Google Scholar 

  • Hirota T, Gerlich D, Koch B, Ellenberg J, Peters JM (2004) Distinct functions of condensin I and II in mitotic chromosome assembly. J Cell Sci 117:6435–45

    Article  CAS  PubMed  Google Scholar 

  • Hittelman WN, Menegaz SD, Mccredie KB, Keating MJ (1984) Premature chromosome condensation studies in human leukemia: 5. Prediction of early relapse. Blood 64:1067–73

    CAS  PubMed  Google Scholar 

  • Hori T, Suzuki Y, Solovei I, Saitoh Y, Hutchison N, Ikeda JE, Macgregor H, Mizuno S (1996) Characterization of DNA sequences constituting the terminal heterochromatin of the chicken Z chromosome. Chromosom Res 4:411–26

    Article  CAS  Google Scholar 

  • Huang K, Jia J, Wu C, Yao M, Li M, Jin J, Jiang C, Cai Y, Pei D, Pan G, Yao H (2013) Ribosomal RNA gene transcription mediated by the master genome regulator protein CCCTC-binding factor (CTCF) is negatively regulated by the condensin complex. J Biol Chem 288:26067–77

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hudson DF, Marshall KM, Earnshaw WC (2009) Condensin: architect of mitotic chromosomes. Chromosom Res 17:131–44

    Article  CAS  Google Scholar 

  • Hudson DF, Ohta S, Freisinger T, Macisaac F, Sennels L, Alves F, Lai F, Kerr A, Rappsilber J, Earnshaw WC, Spread PSSM (2008) Molecular and genetic analysis of condensin function in vertebrate cells. Mol Biol Cell 19:3070–9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hudson DF, Vagnarelli P, Gassmann R, Earnshaw WC (2003) Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes. Dev Cell 5:323–36

    Article  CAS  PubMed  Google Scholar 

  • Ishihara H, Martin BL, Brautigan DL, Karaki H, Ozaki H, Kato Y, Fusetani N, Watabe S, Hashimoto K, Uemura D et al (1989) Calyculin A and okadaic acid: inhibitors of protein phosphatase activity. Biochem Biophys Res Commun 159:871–7

    Article  CAS  PubMed  Google Scholar 

  • Johnson RT, Rao PN (1970) Mammalian cell fusion: induction of premature chromosome condensation in interphase nuclei. Nature 226:717–22

    Article  CAS  PubMed  Google Scholar 

  • Kato Y, Fusetani N, Matsunaga S, Hashimoto K (1988) Calyculins, potent antitumour metabolites from the marine sponge Discodermia calyx: biological activities. Drugs Exp Clin Res 14:723–8

    CAS  PubMed  Google Scholar 

  • Kim JH, Zhang T, Wong NC, Davidson N, Maksimovic J, Oshlack A, Earnshaw WC, Kalitsis P, Hudson DF (2013) Condensin I associates with structural and gene regulatory regions in vertebrate chromosomes. Nat Commun 4:2537

    PubMed  PubMed Central  Google Scholar 

  • Kranz AL, Jiao CY, Winterkorn LH, Albritton SE, Kramer M, Ercan S (2013) Genome-wide analysis of condensin binding in Caenorhabditis elegans. Genome Biol 14:R112

    Article  PubMed  PubMed Central  Google Scholar 

  • Kwon MS, Hori T, Okada M, Fukagawa T (2007) CENP-C is involved in chromosome segregation, mitotic checkpoint function, and kinetochore assembly. Mol Biol Cell 18:2155–68

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–9

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lau AC, Nabeshima K, Csankovszki G (2014) The C. elegans dosage compensation complex mediates interphase X chromosome compaction. Epigenetics Chromatin 7:31

    Article  PubMed  PubMed Central  Google Scholar 

  • Li W, Hu Y, Oh S, Ma Q, Merkurjev D, Song X, Zhou X, Liu Z, Tanasa B, He X, Chen AY, Ohgi K, Zhang J, Liu W, Rosenfeld MG (2015) Condensin I and II complexes license full estrogen receptor alpha-dependent enhancer activation. Mol Cell 59:188–202

    Article  CAS  PubMed  Google Scholar 

  • Liang Z, Zickler D, Prentiss M, Chang FS, Witz G, Maeshima K, Kleckner N (2015) Chromosomes progress to metaphase in multiple discrete steps via global compaction/expansion cycles. Cell 161:1124–37

    Article  CAS  PubMed  Google Scholar 

  • Maeshima K, Imai R, Tamura S, Nozaki T (2014) Chromatin as dynamic 10-nm fibers. Chromosoma 123:225–37

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Maeshima K, Laemmli UK (2003) A two-step scaffolding model for mitotic chromosome assembly. Dev Cell 4:467–80

    Article  CAS  PubMed  Google Scholar 

  • Mayburd AL, Martlinez A, Sackett D, Liu H, Shih J, Tauler J, Avis I, Mulshine JL (2006) Ingenuity network-assisted transcription profiling: identification of a new pharmacologic mechanism for MK886. Clin Cancer Res 12:1820–7

    Article  CAS  PubMed  Google Scholar 

  • Moser SC, Swedlow JR (2011) How to be a mitotic chromosome. Chromosom Res 19:307–19

    Article  Google Scholar 

  • Nakazawa N, Sajiki K, Xu X, Villar-Briones A, Arakawa O, Yanagida M (2015) RNA pol II transcript abundance controls condensin accumulation at mitotically up-regulated and heat-shock-inducible genes in fission yeast. Genes Cells 20:481–99

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nishimura K, Fukagawa T, Takisawa H, Kakimoto T, Kanemaki M (2009) An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat Methods 6:917–22

    Article  CAS  PubMed  Google Scholar 

  • Ohta S, Bukowski-Wills JC, Sanchez-Pulido L, Alves Fde L, Wood L, Chen ZA, Platani M, Fischer L, Hudson DF, Ponting CP, Fukagawa T, Earnshaw WC, Rappsilber J (2010) The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics. Cell 142:810–21

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ono T, Fang Y, Spector DL, Hirano T (2004) Spatial and temporal regulation of condensins I and II in mitotic chromosome assembly in human cells. Mol Biol Cell 15:3296–308

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ono T, Losada A, Hirano M, Myers MP, Neuwald AF, Hirano T (2003) Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell 115:109–21

    Article  CAS  PubMed  Google Scholar 

  • Ono T, Yamashita D, Hirano T (2013) Condensin II initiates sister chromatid resolution during S phase. J Cell Biol 200:429–41

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Paulson JR (1989) Scaffold morphology in histone-depleted HeLa metaphase chromosomes. Chromosoma 97:289–95

    Article  CAS  PubMed  Google Scholar 

  • Paulson JR (2007) Inactivation of Cdk1/Cyclin B in metaphase-arrested mouse FT210 cells induces exit from mitosis without chromosome segregation or cytokinesis and allows passage through another cell cycle. Chromosoma 116:215–25

    Article  CAS  PubMed  Google Scholar 

  • Paulson JR, Laemmli UK (1977) The structure of histone-depleted metaphase chromosomes. Cell 12:817–28

    Article  CAS  PubMed  Google Scholar 

  • Paulson JR, Mause E (2013) Calyculin A induces prematurely condensed chromosomes without histone H1 phosphorylation in mammalian G1-phase cells. Adv Biol Chem 3:8

    Article  Google Scholar 

  • Paulson JR, Patzlaff JS, Vallis AJ (1996) Evidence that the endogenous histone H1 phosphatase in HeLa mitotic chromosomes is protein phosphatase 1, not protein phosphatase 2A. J Cell Sci 109(Pt 6):1437–47

    CAS  PubMed  Google Scholar 

  • Pospisil P, Iyer LK, Adelstein SJ, Kassis AI (2006) A combined approach to data mining of textual and structured data to identify cancer-related targets. BMC Bioinforma 7:354

    Article  Google Scholar 

  • Pruitt KD, Tatusova T, Brown GR, Maglott DR (2012) NCBI Reference Sequences (RefSeq): current status, new features and genome annotation policy. Nucleic Acids Res 40:D130–5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ravi M, Nivedita K, Pai GM (2012) Chromatin condensation dynamics and implications of induced premature chromosome condensation. Biochimie

  • Robinson MD, Mccarthy DJ, Smyth GK (2010) edgeR: a bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–40

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Saitoh N, Goldberg IG, Wood ER, Earnshaw WC (1994) ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure. J Cell Biol 127:303–18

    Article  CAS  PubMed  Google Scholar 

  • Samejima K, Ogawa H, Cooke CA, Hudson DF, Macisaac F, Ribeiro SA, Vagnarelli P, Cardinale S, Kerr A, Lai F, Ruchaud S, Yue Z, Earnshaw WC (2008) A promoter-hijack strategy for conditional shutdown of multiply spliced essential cell cycle genes. Proc Natl Acad Sci U S A 105:2457–62

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Samejima K, Samejima I, Vagnarelli P, Ogawa H, Vargiu G, Kelly DA, De Lima Alves F, Kerr A, Green LC, Hudson DF, Ohta S, Cooke CA, Farr CJ, Rappsilber J, Earnshaw WC (2012) Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase II alpha. J Cell Biol 199:755–70

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–82

    Article  CAS  PubMed  Google Scholar 

  • Shintomi K, Takahashi TS, Hirano T (2015) Reconstitution of mitotic chromatids with a minimum set of purified factors. Nat Cell Biol 17:1014–23

    Article  CAS  PubMed  Google Scholar 

  • Smith HF, Roberts MA, Nguyen HQ, Peterson M, Hartl TA, Wang XJ, Klebba JE, Rogers GC, Bosco G (2013) Maintenance of interphase chromosome compaction and homolog pairing in Drosophila is regulated by the condensin cap-h2 and its partner Mrg15. Genetics 195:127–46

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Stevens JB, Abdallah BY, Regan SM, Liu G, Bremer SW, Ye CJ, Heng HH (2010) Comparison of mitotic cell death by chromosome fragmentation to premature chromosome condensation. Mol Cytogenet 3:20

    Article  PubMed  PubMed Central  Google Scholar 

  • Sutani T, Sakata T, Nakato R, Masuda K, Ishibashi M, Yamashita D, Suzuki Y, Hirano T, Bando M, Shirahige K (2015) Condensin targets and reduces unwound DNA structures associated with transcription in mitotic chromosome condensation. Nat Commun 6:7815

    Article  PubMed  PubMed Central  Google Scholar 

  • Sutani T, Yuasa T, Tomonaga T, Dohmae N, Takio K, Yanagida M (1999) Fission yeast condensin complex: essential roles of non-SMC subunits for condensation and Cdc2 phosphorylation of Cut3/SMC4. Genes Dev 13:2271–83

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Szklarczyk D, Franceschini A, Kuhn M, Simonovic M, Roth A, Minguez P, Doerks T, Stark M, Muller J, Bork P, Jensen LJ, VON Mering C (2011) The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res 39:D561–8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vagnarelli P (2012) Mitotic chromosome condensation in vertebrates. Exp Cell Res 318:1435–41

    Article  CAS  PubMed  Google Scholar 

  • Vagnarelli P, Hudson DF, Ribeiro SA, Trinkle-Mulcahy L, Spence JM, Lai F, Farr CJ, Lamond AI, Earnshaw WC (2006) Condensin and Repo-Man-PP1 co-operate in the regulation of chromosome architecture during mitosis. Nat Cell Biol 8:1133–42

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wallace HA, Klebba JE, Kusch T, Rogers GC, Bosco G (2015) Condensin II regulates interphase chromatin organization through the Mrg-binding motif of Cap-H2. G3 (Bethesda) 5:803–17

    Article  CAS  Google Scholar 

  • Wang BD, Eyre D, Basrai M, Lichten M, Strunnikov A (2005) Condensin binding at distinct and specific chromosomal sites in the Saccharomyces cerevisiae genome. Mol Cell Biol 25:7216–25

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang YD, Caldwell RB (2006) Isolation of nuclear and cytoplasmic proteins from DT40 cells. Subcell Biochem 40:439–40

    PubMed  Google Scholar 

  • Wilkins BJ, Rall NA, Ostwal Y, Kruitwagen T, Hiragami-Hamada K, Winkler M, Barral Y, Fischle W, Neumann H (2014) A cascade of histone modifications induces chromatin condensation in mitosis. Science 343:77–80

    Article  CAS  PubMed  Google Scholar 

  • Williams DM, Scott CD, Beck TM (1976) Premature chromosome condensation in human leukemia. Blood 47:687–93

    CAS  PubMed  Google Scholar 

  • Young MD, Wakefield MJ, Smyth GK, Oshlack A (2010) Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol 11:R14

    Article  PubMed  PubMed Central  Google Scholar 

  • Zhang CZ, Leibowitz ML, Pellman D (2013) Chromothripsis and beyond: rapid genome evolution from complex chromosomal rearrangements. Genes Dev 27:2513–30

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

The authors thank Dr. Matthew Burton for providing flow cytometry and microscopy technical support and Dr. Kathryn Marshall for careful reading of the manuscript.

This work was supported by NHMRC project grant GNT1030358 and GNT1047009 and by the Victorian Government’s Operational Infrastructure Support Program.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Damien F. Hudson.

Ethics declarations

Conflict of interest

The authors declare they have no conflict of interest.

Additional information

Responsible Editor: Tatsuo Fukagawa, Ph.D

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online Resource 1

A small pool of condensin I is retained in the interphase nuclei of chicken DT40 cells. a The slide in Fig. 2a was examined by confocal microscopy (Zeiss LSM780). Representative images are shown, demonstrating that some CAP-H-GFP-SBP is present in the interphase nuclei. Line profiles across the cell were plotted and are shown at the right. Scale bar represents 2 μm. b Asynchronous CAP-H-GFP-SBP cells were fixed with 4 % PFA and co-stained with mouse anti-SBP and rabbit anti-CAP-H antibodies. DNA was stained with DAPI and the cells were examined by confocal microscopy (Zeiss LSM 780). Line profiles across the cell were plotted and are shown at the right. Scale bar represents 10 μm. Inserts are higher magnification of selected cell. c Asynchronous CAP-H-GFP cells were fixed with PFA and stained with Lamin B1 antibodies. Optical sections (0.2 μm) were imaged and analysed by Imaris 8.1.2. Orthogonal (XY, XZ and YZ) views of CAP-H-GFP and Lamin B1 show clearly that CAP-H-GFP is present inside the nuclear envelope. Line profiles across the cell were plotted and are shown at the top right. Scale bar represents 5 μm. d Ponceau staining of the blot in Fig. 2d is shown as a loading control. (PDF 432 kb)

Online Resource 2

Determination of cell cycle stages in live cell imaging. a CAP-H-GFP H2B-RFP and CAP-D3-GFP H2B-RFP cells (n = 7) were subjected to live cell imaging for 15 h with 2-min intervals. Cell size during the timecourse was determined by measuring the cell diameter (μm) using the polygon function in SoftWoRx 4.1 and plotted as a function of time. CAP-H-GFP and CAP-D3-GFP cellular localisations were monitored and this is reflected in the background colour of the graph. Cell cycle stages were determined accordingly. b, c CAP-H-GFP H2B-RFP cells were subjected to live cell imaging for 15 h with 2-min intervals. Normalised CAP-H-GFP intensity was plotted with time according to the description in Fig. 1a, T = 0 min was set as a completion of anaphase. Note the level of CAP-H in the nucleus drops over time. Representative images in different cell cycle stages are shown in b. H2B-RFP intensity during the timecourses were normalised to anaphase single cell H2B-RFP intensity at T = 0 min, respectively. Normalised H2B-RFP intensity was plotted with time in c. Cell cycle stages were determined based on the change of normalised H2B-RFP intensity (i.e. double H2B) and DIC morphology. Error bars represent standard error of the mean (SEM), n = 15. (PDF 589 kb)

Online Resource 3

Complete depletion of CAP-H. a CAP-H KO cells were grown in the absence (CAP-HON) or presence (CAP-HOFF) of dox for 36 h in order to deplete CAP-H. Cells were collected for Western blotting to detect CAP-H and α-tubulin, and FACS analysis. b CAP-HON and CAP-HOFF (+ dox 36 h) cells were collected for Annexin V staining and subjected for FACS analysis. c CAP-HON and CAP-HOFF (+ dox 36 h) cells were collected and fixed with 4 % PFA. Immunofluorescent staining was used to visualise γ-H2AX (red). DNA was stained with DAPI (blue). CAP-HON cells treated with Adriamycin 6 h were used as a positive control for γ-H2AX staining. Representative images are shown. Three different experiments were performed. γ-H2AX foci were counted and scored as 0, 1, 2, 3, 4 and ≥5 foci from 100 cells in each experiment. The average percentage of cells with 0, 1, 2, 3, 4 and ≥5 γ-H2AX foci is shown in the right. (PDF 682 kb)

Online Resource 4

Functional studies of CAP-H in interphase. a Human homologs of 693 DT40 genes that are significantly misregulated in CAP-H KO were analysed with Ingenuity Pathway Analysis (IPA). The top five canonical pathways are shown at the left. The top five diseases and biological functions are shown at the right. b Significant DE genes in this study that are involved in the prostate cancer signalling pathway are shown in red. c 116 significantly DE genes with CAP-H enrichment were analysed with DAVID Bioinformatics Resources 6.7. The top nine GO terms are shown. (PDF 380 kb)

Online Resource 5

Heat map of 116 misregulated genes directly due to CAP-H binding removal. Heat map representing 116 DE genes with CAP-H enrichment in comparison between CAP-HON and CAP-HOFF samples displays significant gene expression change. Each gene is represented by a row of coloured boxes, and each replicate is represented by a column. Red indicates gene expression up-regulation, blue indicates gene expression down-regulation. (PDF 178 kb)

Online Resource 6

Condensins I and II in the G1-PCC cells. a G1 synchronised CAP-D3-GFP-SBP cells were treated with 50 nM Cal A for 1 h. Cells were collected, fixed with methanol/acetic acid (3:1) and co-stained with mouse anti-SBP and rabbit anti-CAP-H antibodies to detect CAP-D3-GFP-SBP (green) and CAP-H (red). DNA was stained with DAPI (blue). Representative G1-PCC chromosomes are shown. Scale bar represents 5 μm. b CAP-D3-GFP H2B-RFP cells were subjected to live cell imaging. NEBD times (min) were plotted according to cell sizes (cell diameter <9 μm or >9 μm) at t = 0 min with p = 6.72E-13 as described in Fig. 6a. Error bars represent 95 % confidence intervals (CI), n = 30. (PDF 149 kb)

Online Resource 7

Chromosome shattering in the PCC cells. a WT H2B-GFP cells with and without Cal A treatment were subjected to live cell imaging for 7 h at 2-min intervals. Live cell imaging was started within 7 min of Cal A addition. At t = 0 min, G1 cells (cell diameters <9 μm, based on Online Resource 2a), G2/M cells, S cells (cell diameters >9 μm, based on Online Resource 2a) and anaphase cells were selected for monitoring. b Live cell imaging using H2B-GFP of G1-PCC, S-PCC and anaphase-PCC cells. Note the shattering in the final frame for each cell. c, d CAP-H-GFP-SBP cells were treated as described in Fig. 4c, collected and fixed with PFA. Immunofluorescent staining was used to visualise (c) γ-H2AX (red) or (d) RAD51 (red). DNA was stained with DAPI (blue). CAP-H-GFP-SBP cells blocked with hydroxyurea (HU) for 16 h and then released were used as a positive control for RAD51 staining. Representative images are shown. (PDF 1332 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Zhang, T., Paulson, J.R., Bakhrebah, M. et al. Condensin I and II behaviour in interphase nuclei and cells undergoing premature chromosome condensation. Chromosome Res 24, 243–269 (2016). https://doi.org/10.1007/s10577-016-9519-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10577-016-9519-7

Keywords

Navigation