[Cell Cycle 3:5, 538-540; May 2004]; ©2004 Landes Bioscience
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Could Condensin Scaffold the Mitotic Chromosome?
3Faculdade de Ciências da Saúde; Universidade Fernando Pessoa; Porto, Portugal
*Correspondence to: Claudio E. Sunkel; Instituto de Biologia Molecular e Celular; Rua do Campo Alegre 823; 4150-180 Porto, Portugal; Tel: +351.22.6074900; Fax: +351.22.6099157; Email:
[email protected] Received 03/01/04; Accepted 03/05/04
More than a century has passed since Walter Flemming first showed that during mitosis the nuclear contents did not just disappear but were transformed into well-defined structures, the mitotic chromosomes. In recent years, significant progress has been made in the identification and functional characterization of several molecules required for the process by which individual DNA fragments faithfully reproduce a higher order structural organization every cell cycle. However, the process still remains mechanistically poorly understood. Current models suggest that interphase chromatin is converted into chromosomes during early stages of mitosis by the activity of condensin, a multiprotein complex that was initially identified and characterized in Xenopus egg extracts.1 Condensin is made up of two core subunits (SMC2 and SMC4) and three non-SMC subunits.2 In vitro, condensin has the ability to bind and modify the topology of DNA in an ATP-dependent manner,2,3 an activity that can be stimulated by mitotic specific phosphorylation of the complex by cdk1.4 Depletion of condensin from Xenopus egg extracts showed that the complex was required not only to promote but also to maintain chromosome condensation.1,5 Also, genetic analysis of condensin subunits in fission and budding yeast showed that mutant cells had abnormal chromatin condensation but nevertheless attempted to segregate their chromosomes resulting in the characteristic cut phenotype.6,7 Thus, a model has developed which views chromosome condensation as a process that starts late in G2 when the raising activity of mitotic-cdks activates condensins which in turn promote and maintain a higher order chromatin organization. Concomitantly, inactivation of cdks during mitotic exit would lead to inactivation of condensin and to a release of the condensed state of chromatin. Thus, the major questions regarding chromosome condensation at present would involve working out the detailed mechanism by which condensin packs the chromatin into a defined structure with reproducible landmarks every cell cycle. However, recent in vivo functional studies in a number of higher eukaryotes with welldefined chromosome morphology have raised questions about the role of condensin in chromosome condensation. Initially, genetic analysis of the Drosophila gene encoding the SMC4 core subunit of condensin8 demonstrated that in cells carrying mutant alleles with reduced or undetectable levels of SMC4, mitotic chromosomes do show a substantial degree of organization. Typically these can shorten their longitudinal axis just like chromosomes of normal cells, but sister chromatids are never clearly resolved at the arms. Nevertheless, the centromeres disjoin and during anaphase are pulled towards the spindle poles leading to severe chromatin bridges, chromosome breakage and apoptosis. Subsequently, studies in C.elegans9 showed that depletion of SMC4 by RNAi did not appear to affect chromosome condensation during metaphase. However, severe chromosome segregation defects were observed in mitosis and meiosis II associated with extensive chromatin bridges. Moreover, it was shown that depletion of ScII/SMC2 in chicken DT40 cells using a conditional knockout10 caused a delay but did not compromise chromosome condensation during mitosis. Similarly to the results in Drosophila, sister chromatid resolution was severely compromised and most cells in anaphase showed chromatin bridges. These data,8-10 together with the results obtained after genetic analysis in yeasts6,7 demonstrate that, when
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SMC, topoisomerase II, condensin, chromosomes, mitosis, scaffold
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This manuscript has been published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=828
One of the most remarkable and yet poorly understood events during the cell cycle is how dispersed chromatin fragments are transformed into chromosomes every time cells undergo mitosis. It has been postulated that mitotic chromosomes might contain an axial scaffold that is involved in condensation but its molecules and structure have remained elusive. Recent data suggests that the condensin complex might indeed be an essential part of the scaffold that provides a platform for other proteins to localize and promote different aspects of chromosome condensation.
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1Instituto de Biologia Molecular e Celular; 2Instituto de Ciências Biomédicas de Abel Salaz; Universidade do Porto; Porto, Portugal
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ABSTRACT
Paula A. Coelho1 Joana Queiroz-Machado1,3 Claudio E. Sunkel1,2,*
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COULD CONDENSIN SCAFFOLD THE MITOTIC CHROMOSOME?
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Figure 1. Schematic view of mitotic chromosome condensation and sister chromatid segregation, in the presence or absence of condensin. During chromosome condensation an axial structure that contains condensin, and other yet unknown components, as well as Topoisomerase II, is organized. At this stage cohesin concentrates between the unseparated sister chromatids. Shortening of the longitudinal axis and resolution of sister chromatids probably occurs simultaneously and segregation of sister chromatids takes place after proteolysis of cohesin subunits. In the absence of condensin, a defined axial structure is never organized, Topoisomerase II localizes diffusely throughout the chromatin, axial shortening does take place but sister chromatid resolution fails. However, sister chromatid segregation proceeds but chromosome arms show extensive chromatin bridges probably due to catenated DNA that Topoisomerase II failed to resolve at earlier stages.
showed that in the absence of condensin not only Topo II but also a whole group of polypeptides failed to be recovered in this fraction. Work in Drosophila23 has provided further evidence that depletion of the SMC4 protein causes mislocalization of Topo II. Rather than localizing to a defined central core within each sister chromatid, Topo II appeared dispersed all over the chromatin. Interestingly, in vitro studies also showed that chromatin-associated Topo II from SMC4-depleted cells was unable to decatenate kinetoplast DNA supporting previous studies in which the activity of Topo II was also compromised in the absence of Barren, a non-SMC subunit of condensin.24 All these data suggest that condensin might indeed be an essential scaffold component providing a structural framework for non-histone proteins to associate with chromatin and allow a specific higher order structure to form. However, recent studies have raised important issues regarding the organization and properties of a chromosome scaffold component and the role of condensin. Detailed immunolocalization of Topo IIα and condensin on human chromosomes25 has shown that these proteins are distributed in a barber pole-like configuration along the chromosomal axis, where regions rich in Topo II and condensin appear to alternate. Thus if condensin is required for the organization of the chromosome scaffold, then this structure is either discontinuous or is built with yet uncharacterised components that interact with condensin. Indeed, biophysical studies have shown that chromosome structure displays a remarkable elasticity that is sensitive to nuclease activity26 suggesting that the scaffold might be a discontinuous proteinaceous structure. Could Topo II together with condensin be involved in the organization of the scaffold? This is also unlikely because it has been shown that Topo II contributes little to the physical properties of the scaffold27 and turns over very rapidly on mitotic chromosomes making it an unlikely structural protein.28 Furthermore, it was shown that chromosomes depleted of Topo II by RNAi are able to organize into well-defined structures,29 that lost of condensin compromises Topo II chromatin localization10,23 and that in the absence of Topo II condensin appears normally localized29 (Queiroz-Machado J, Coelho P, Sunkel CE; unpublished results). Taking all these observations into consideration condensin does appear to play a central role in the proper organization of the mitotic
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the condensin complex is compromised in vivo, abnormal sister chromatid segregation is the only recurrent phenotype but that chromatin still organizes into structures that resemble chromosomes (Fig. 1). More recently, it was shown that a number of higher eukaryotes contain at least two condensin complexes.11 Accordingly, it might be argued that the chromosome condensation observed after depleting condensin in vivo could be the result of the newly identified condensin II complex. However, condensin fails to assemble12,13, 11 in the absence of core subunits and because both complexes share the same core subunits loss of SMC2 or SMC4 is likely to inactivate both. Since all in vivo functional studies referred above targeted core SMC subunits, it is reasonable to suggest that while condensin is clearly essential for the resolution of sister chromatids, its role as a major factor in overall chromosome condensation is still unclear. Several features of mitotic chromosomes, such as the existence of a fixed length and particular banding patterns have suggested that they fold to an invariable structure. Whatever the details of the mechanism, it is likely that the organization of mitotic chromosomes occurs by a deterministic process. Laemmli and co-workers were the first to propose the existence of a clearly defined scaffold within mitotic chromosomes.14-16 After nuclease digestion and salt extraction of isolated chromosomes, they were able to obtain a mostly insoluble protein fraction14-16 that was later shown to contain Topoisomerase II (Topo II) 17,18 and condensin.1,19 More surprisingly, electron microscope studies showed that isolated scaffolds could retain their overall structure.15,20,21 These observations lead to the hypotheses that the scaffold could underlay mitotic chromosome structure.22 Therefore, since condensin has been shown to be part of the scaffold fraction one might ask what happens to this structure in cells that have been depleted of condensin? Closer inspection of chromosomes assembled in the absence of condensin has provided some valuable insights. Hudson et al.,10 showed that although condensin-deficient isolated chromosomes from DT40 cells could condense, their structure was not stable. Normal chromosomes could be induced to undergo cycles of unfolding and folding, while condensin-deficient chromosomes once unfolded could not fold back into a defined structure suggesting that they had lost their “structural memory”. Also, isolation and biochemical analysis of chromosome scaffold-associated proteins
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References
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1. Hirano T, Mitchison TJ. A heterodimeric coiled-coil protein required for mitotic chromosome condensation in-vitro. Cell 1994; 79:449-58. 2. Kimura K,Hirano T. ATP-dependent positive supercoiling of DNA by 13S condensin: A biochemical implication for chromosome condensation. Cell 1997; 90:625-34. 3. Kimura K, Rybenkov VV, Crisona NJ, Hirano T, Cozzarelli NR. 13S condensin actively reconfigures DNA by introducing global positive writhe: Implications for chromosome condensation. Cell 1999; 98:239-48. 4. Kimura K, Hirano M, Kobayashi R, Hirano T. Phosphorylation and activation of 13S condensin by Cdc2 in vitro. Science 1998; 282:487-90. 5. Hirano T, Kobayashi R, Hirano M. Condensins, chromosome condensation protein complexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila barren protein. Cell 1997; 89:511-21. 6. Saka Y, Sutani T, Yamashita Y, Saitoh S, Takeuchi M, Nakaseko Y, Yanagida M. Fission yeast Cut3 and Cut14, members of a ubiquitous protein family, are required for chromosome condensation and segregation in mitosis. EMBO J 1994; 13:4938-52. 7. Strunnikov AV, Hogan E, Koshland D. Smc2, a Saccharomyces-Cerevisiae gene essential for chromosome segregation and condensation, defines a subgroup within the Smc family. Gene Dev 1995; 9:587-99. 8. Steffensen S, Coelho PA, Cobbe N, Vass S, Costa M, Hassan B, et al. A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis. Curr Biol 2001; 11:295-307. 9. Hagstrom KA, Holmes VF, Cozzarelli NR, Meyer BJ. C-elegans condensin promotes mitotic chromosome architecture, centromere organization, and sister chromatid segregation during mitosis and meiosis. Gene Dev 2002; 16:729-42. 10. Hudson DF, Vagnarelli P, Gassmann R, Earnshaw WC. Condensin is required for nonhistone protein assembly and structural integrity of vertebrate mitotic chromosomes. Dev Cell 2003; 5:323-36. 11. Ono T, Losada A, Hirano M, Myers MP, Neuwald AF, Hirano T. Differential contributions of condensin I and condensin II to mitotic chromosome architecture in vertebrate cells. Cell 2003; 115:109-21. 12. Kimura K, Hirano T. Dual roles of the 11S regulatory subcomplex in condensin functions. Proc Nat Acad Sci USA 2000; 97:11972-7. 13. Lavoie BD, Hogan E, Koshland D. In vivo dissection of the chromosome condensation machinery: reversibility of condensation distinguishes contributions of condensin and cohesin. J Cell Biol 2002; 156:805-15. 14. Adolph KW, Cheng SM, Paulson JR, Laemmli UK. Isolation of a protein scaffold from mitotic Hela-cell chromosomes. Proc Nat Acad Sci USA 1977; 74:4937-41. 15. Lewis CD, Laemmli UK. Higher-order metaphase chromosome structure—evidence for metalloprotein interactions. Cell 1982; 29:171-81. 16. Mirkovitch J, Gasser SM, Laemmli UK. Scaffold attachment of DNA loops in metaphase chromosomes. J Mol Biol 1988; 200:101-9. 17. Earnshaw WC, Halligan B, Cooke CA, Heck MMS, Liu LF. Topoisomerase-II is a structural component of mitotic chromosome scaffolds. J Cell Biol 1985; 100:1706-15. 18. Gasser SM, Laemmli UK. Cohabitation of scaffold binding regions with upstream enhancer elements of 3 developmentally regulated genes of Drosophila-Melanogaster. Cell 1986; 46:521-30. 19. Earnshaw WC, Laemmli UK. Architecture of metaphase chromosomes and chromosome scaffolds. J Cell Biol 1983; 96:84-93. 20. Adolph KW, Cheng SM, Laemmli UK. Role of nonhistone proteins in metaphase chromosome structure. Cell 1977; 12:805-16. 21. Mirkovitch J, Mirault ME, Laemmli UK. Organization of the higher-order chromatin loop —Specific DNA attachment sites on nuclear scaffold. Cell 1984; 39:223-32. 22. Laemmli UK. Levels of organization of the DNA in eukaryotic chromosomes. Pharmacol Rev 1978; 30:469-76. 23. Coelho PA, Queiroz-Machado J, Sunkel CE. Condensin-dependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis. J Cell Sci 2003; 116:4763-76. 24. Bhat MA, Philp AV, Glover DM, Bellen HJ. Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with topoisomerase II. Cell 1996; 87:1103-14.
25. Maeshima K, Laemmli UK. A two-step scaffolding model for mitotic chromosome assembly. Dev Cell 2003; 4:467-80. 26. Poirier MG, Marko JF. From the cover: Mitotic chromosomes are chromatin networks without a mechanically contiguous protein scaffold. PNAS 2002; 99:15393-7. 27. Almagro S, Riveline D, Hirano T, Houchmandzadeh B, Dimitrov S. The mitotic chromosome is an assembly of rigid elastic axes organized by structural maintenance of chromosomes (SMC) proteins and surrounded by a soft chromatin envelope. J Biol Chem 2004; 279:5118-26. 28. Tavormina PA, Come M-G, Hudson JR, Mo Y-Y, Beck WT, Gorbsky GJ. Rapid exchange of mammalian topoisomerase IIα at kinetochores and chromosome arms in mitosis. J Cell Biol 2002; 158:23-9. 29. Chang C-J, Goulding S, Earnshaw WC, Carmena M. RNAi analysis reveals an unexpected role for topoisomerase II in chromosome arm congression to a metaphase plate. J Cell Sci 2003; 116:4715-26.
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chromosome, however, one cannot yet clearly define its role. It is certainly important for the “coordination” of a scaffold component but probably we are still missing important additional factors. Also, either directly or indirectly, condensin provides a “platform” for the correct enzymatic activity of Topo II because sister chromatid arms cannot decatenate properly when condensin is depleted. Finally, condensin appears to provide a yet completely undefined “structural memory” upon which chromatin can fold into a defined and wellrecognized higher order structure. So, the question might not be whether condensin could scaffold the mitotic chromosome, because it is certainly intimately involved in the process but what are the other components that make up this remarkable structure.
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