It remains a big challenge in modern cell biology to determine the mechanism by which a discrete set of rod-shaped chromosomes is assembled from an amorphous mass of interphase chromatin. Recent studies start to shed new lights on how this process is actively supported by a class of multiprotein complexes called condensins. In vertebrate cells, two different condensin complexes make distinct mechanistic contributions to determining the shape and integrity of mitotic chromosomes

Hirano,  T.

Cold Spring Harbor Laboratory Institutional Repository

©2003 Landes Bioscience. Not for Distribution[Cell Cycle 3:1, 26-28; January 2004]; ©2004 Landes Bioscience26 Cell Cycle 2004; Vol. 3 Issue 1Tatsuya HiranoCold Spring Harbor Laboratory; One Bungtown Road; Cold Spring Harbor, New YorkUSACorrespondence to: Tatsuya Hirano; Cold Spring Harbor Laboratory, One BungtownRoad; Cold Spring Harbor, New York 11724 USA;Tel.: 516.367.8370; Fax:516.367.8815; Email: hirano@cshl.orgReceived 10/17/03; Accepted 10/20/03Previously published online as a Cell Cycle E-publication at:http://www.landesbioscience.com/journals/cc/tocnew31.php?volume=3&Issue=1KEY WORDSchromosome condensation, chromosome scaf-fold, condensin I, condensin II, SMC proteinsExtra Views  Chromosome Shaping by Two CondensinsABSTRACTIt remains a big challenge in modern cell biology to determine the mechanism bywhich a discrete set of rod-shaped chromosomes is assembled from an amorphous massof interphase chromatin. Recent studies start to shed new lights on how this process isactively supported by a class of multiprotein complexes called condensins. In vertebratecells, two different condensin complexes make distinct mechanistic contributions todetermining the shape and integrity of mitotic chromosomes.Mitotic chromosome assembly is a critical preparatory process to ensure the faithfuland synchronous separation of sister chromatids at the onset of anaphase.1,2 Stronglyimplicated in this process is condensin, a multiprotein complex that was originally identifiedand characterized in Xenopus egg cell-free extracts.3,4 Purified condensin has the ability tointroduce positive superhelical tension into DNA in an ATP hydrolysis-dependentmanner, and this activity is activated by mitosis-specific phosphorylation mediated bycdc2.5 Genetic studies in other model organisms have shown that condensin function isrequired for proper segregation of chromosomes during mitosis.6-8 While these findingssupport the idea that condensin is a key (and perhaps the major) component responsiblefor the formation of mitotic chromosomes, the exact mechanism by which condensinorganizes mitotic chromosomes and facilitates their segregation remains elusive.One of the fundamental obstacles to progress in the field is that we still know very littleabout how chromatin fibers are organized within mitotic chromosomes.2,9 Inevitably, thecurrent definition of chromosome “condensation” is relatively vague. Some researchers usethis word very narrowly to mean a step(s) in which an extended DNA molecule is convertedinto a compact structure regardless of its final shape (or functional relevance). However,mitotic chromosome condensation more often refers to the structural reorganization ofchromosomes that occurs during mitosis. The goal of this complex process is to activelyuntangle and refold duplicated genomes so that they can be readily separated at the onsetof anaphase. With these functional considerations in mind, it would be helpful to conceptu-ally dissect the assembly of mitotic chromosomes into three distinct steps (Fig. 1A, 1–4).First, catenations of DNA strands from different chromosomes are removed, therebyallowing them to be converted into discrete units (individualization).10 Second, the fibersare folded and organized into rod-shaped structures (shaping or shape-determination).11Finally, the two chromatids within each chromosome are resolved so that they becomecytologically distinguishable from each other (resolution).12 It is important to emphasizethat these three steps do not occur independently and must be functionally and mech-anistically coordinated with each other. Here I will discuss accumulating lines of evidencethat condensin plays important roles in all of these crucial steps of mitotic chromosomeassembly.The first indication that condensin is required for proper chromosome assembly wasprovided by the Xenopus egg cell-free system.3 In the simplest assay of this system, spermchromatin was incubated directly with a metaphase (CSF-arrested) extract withoutpreceding DNA replication to produce individual, rod-shaped chromosomes with a singlechromatid (i.e., there is no resolution step in this assay). Preincubation of the extract withan antibody against SMC4, a core ATPase subunit of condensin, produced a mass ofentangled, prophase-like thin chromatin fibers. Complete depletion of condensin subunitsexhibited a severer phenotype, resulting in the formation of a fuzzy, interphase-likechromatin mass (Fig. 1A, 5).4 Similar results were obtained under a more physiologicalcondition where chromosomes with sister chromatids were assembled after a single roundof DNA replication.13,14 These results show that condensin is required for both theindividualization and shaping steps. Addition of the same antibody to the extract after thewww.landesbioscience.com Cell Cycle 27assembly reaction was completed led to progressive disruption of therod-shaped chromosomes and converted them into randomly coiled,chromatin balls.3 While the interpretation of the antibodyblockingexperiments may not be straightforward (for instance, steric inhibitionby antibody binding cannot be excluded), this result suggests thatcondensin function is also required for the structural maintenance ofrod-shaped chromosomes. The apparent density of chromatin withinthe observed rod and ball structures was not so different. Therefore,an important role of condensin is to actively organize a chromatinfiber into a rod-shaped chromosome rather than to simply pack itinto a random structure.Early genetic studies in yeast utilizing in-situ hybridization alsosupported the idea that condensin is required for proper organizationof mitotic chromosomes.6,7 More recent genetic studies in highereukaryotic cells have allowed researchers to visualize the morphology ofchromosomes after depletion of SMC core subunits of condensin.15-17In Drosophila15 and chicken DT40 cells17 with a greatly reducedlevel of condensin, individual chromosomes were formed, but theywere abnormally fat and showed no discernible sister chromatids(Fig. 1B, 4). The length distribution of these chromosomes wereapparently normal, leading the authors to conclude that condensinfunction is important for the resolution step but may not berequired for the axial compaction of chromosomes. These studiesraise several interesting questions. First, why are the phenotypesobserved in vivo seemingly less drastic than those observed in theXenopus egg extracts? One possible explanation is that chromosomesare assembled from templates that have different degrees of DNAentanglement (or catenation) in the two experimental systems. Insomatic interphase nuclei, chromosomes are partitioned into specific“territories” in a largely nonoverlapping fashion,18 implying that thecatenations between different chromosomes have already beendissolved before entering mitosis (Fig. 1B, 1). In contrast, DNAmolecules within sperm chromatin (and in early embryonic nuclei)would be heavily catenated with each other (Fig. 1A, 1). If true,depletion of condensin in the cell-free system would emphasize itsrequirement for the individualization step (Fig. 1A, 5) whereasdepletion in the somatic cells would stress its participation in theshaping and resolution steps (Fig. 1B, 4). The second question iswhether axial and lateral compactions are regulated by differentfactors and whether condensin is only responsible for the latter.Given the currently limited knowledge of the chromosome foldingmechanism, more detailed structural analyses of the condensin-depleted chromosomes would be required before answering thisquestion. Importantly, however, an elegant assay involving alternateexposures of chromosomes to buffers containing Mg2+ or EDTAshowed that the structural integrity of chicken DT40 chromosomesis severely compromised in the absence of condensin.17A recent paper by Ono et al. (2003)19 has reported the mostdramatic demonstration of the shape-determining role of condensin.The authors first showed that vertebrate cells have a second condensincomplex (condensin II) in addition to the canonical condensincomplex (henceforth referred to as condensin I). Condensin IIshares the same pair of SMC core subunits with condensin I butcontains a unique set of three non-SMC subunits. Depletion of theSMC core subunits by means of RNA interference (RNAi) in HeLacells produced cloud-like chromosomes with a very fuzzy appearance(Fig. 2, “fuzzy”), reminiscent of chicken DT40 chromosomes depletedof SMC2.17 Most strikingly, depletion of condensin I- or condensinII-specific subunits resulted in a distinct, highly characteristic defectin chromosome morphology (Fig. 2, “swollen” or “curly” chromo-somes, respectively). While the two complexes were equally abundantin HeLa cell nuclear extracts, the relative abundance of condensin Iand condensin II in Xenopus egg extracts was ~5:1. Consistently,CHROMOSOME SHAPING BY TWO CONDENSINSFigure 1. Conceptual pathways of mitotic chromosome assembly. (A) In spermchromatin or embryonic nuclei, chromatin fibers are heavily entangled witheach other (1). First, the entanglement between different fibers must beremoved, allowing them to be converted into discrete units (2; individual-ization). Second, the chromatin fibers are folded and organized intorod-shaped structures (3; shaping). Finally, two chromatids within a chro-mosome become cytologically distinguishable from each other (4; resolu-tion). In the absence of condensins, the initial individualization step wouldbe compromised, resulting in the formation of an interphase-like chromatinmass (5). (B) In somatic nuclei, chromatin fibers have already been individ-ualized into their “territories”(1). Therefore, depletion of condensins wouldcause defects in the shaping and resolutions steps (4). Note that the threesteps depicted in this conceptual pathway are mechanistically coupled anddo not necessarily occur independently of each other.Figure 2. Schematic view of chromosome morphologies observed afterdepletion of condensin subunits from HeLa cells.19 The chromosomal DNAis shown in blue, and the chromosome axis stained with anti-SMC2 is shownin magenta. Chromosomes were well organized in control cells containingboth condensins I and II (“normal”). Depletion of condensin I- or condensinII-specific subunits produced “swollen” or “curly” chromosomes, respectively.Simultaneous depletion of both condensins resulted in the formation ofcloud-like chromosomes with a “fuzzy” appearance.ABCHROMOSOME SHAPING BY TWO CONDENSINS28 Cell Cycle 2004; Vol. 3 Issue 1condensin I function was predominant in the extracts, but depletionof condensin II resulted in the formation of curly or wavy chromo-somes similar to those observed in condensin II-depleted HeLa cells.Condensins I and II displayed distinct distributions along the axis ofchromosomes assembled in vivo and in vitro. These results showconvincingly that condensins I and II make different mechanisticcontributions to directly determining the shape of vertebratechromosomes.Taken together with the previous antibody blocking experimentsin vitro,3 it is most likely that condensins play dual roles inshape-determination and shape-maintenance of mitotic chromosomes.Both roles of condensins could be supported by their participationin the dynamic assembly of the chromosome scaffold,11,17 asub-chromosomal structure that constitutes the central axis ofchromatids. Indeed, a biochemically-defined scaffold fraction wasfound to be destabilized after depletion of SMC2, and other proteinssuch as topoisomerase II could no longer be recovered into thisfraction.17 It should be emphasized that current models postulatethat the “scaffold” is a highly dynamic, mitosis-specific structure,11,17and is not a static filamentous structure that is already present in theinterphase nucleus.In this short article, I have first made an attempt to reconcile theapparent discrepancy in the phenotypes observed after condensin-depletion in vivo and in vitro. I have then discussed most recentevidence that vertebrate cells have two different condensin complexesthat make distinct contributions to mitotic chromosome architecture.The discovery of condensin II raises an exciting array of new questionsabout chromosome structure and evolution. What is the precisegeometry of the two condensin complexes within metaphasechromatids, and how do they functionally interact with each other?Are condensins I and II recruited to specific regions in asequence-specific manner? Is there any relationship between thedistribution of the two complexes and classical chromosome banding?Yeasts have condensin I but not condensin II, but why does condensinII apparently substitute for condensin I in C. elegans? Is this relatedto the unique, holocentric nature of C. elegans chromosomes? Futureinvestigation into these questions will undoubtedly enrich ourunderstanding of mitotic chromosome architecture and dynamics.References1. Nasmyth K. Segregating sister genomes: The molecular biology of chromosome separation.Science 2002; 297:559-65.2. Swedlow JR, Hirano T. The making of the mitotic chromosome: Modern insights into clas-sical questions. Mol Cell 2003; 11:557-69.3. Hirano T, Mitchison TJ. A heterodimeric coiled-coil protein required for mitotic chromo-some condensation in vitro. Cell 1994; 79:449-58.4. Hirano T, Kobayashi R, Hirano M. Condensins, chromosome condensation protein com-plexes containing XCAP-C, XCAP-E and a Xenopus homolog of the Drosophila Barrenprotein. Cell 1997; 89:511-21.5. Kimura K, Hirano M, Kobayashi R, Hirano T. Phosphorylation and activation of 13S con-densin by Cdc2 in vitro. Science 1998; 282:487-90.6. Saka Y, Sutani T, Yamashita Y, Saitoh S, Takeuchi M, Nakaseko Y, et al. Fission yeast cut3and cut14, members of a ubiquitous protein family, are required for chromosome conden-sation and segregation in mitosis. EMBO J 1994; 13:4938-52.7. Strunnikov AV, Hogan E, Koshland D. SMC2, a Saccharomyces cerevisiae gene essentialfor chromosome segregation and condensation, defines a subgroup within the SMC fami-ly. Genes Dev 1995; 9:587-99.8. Bhat MA, Philp AV, Glover DM, Bellen HJ. Chromatid segregation at anaphase requiresthe barren product, a novel chromosome associated protein that interacts with topoiso-merase II. Cell 1996; 87:1103-14.9. Koshland D, Strunnikov A. Mitotic chromosome condensation. Annu Rev Cell Dev Biol1996; 12:305-33.10. Gimenez-Abian JF, Clarke DJ, Devlin J, Gimenez-Abian MI, De la Torre C, Johnson RT,et al. Premiototic chromosome individualization in mammalian cells depends on topoiso-merase II activity. Chromosoma 2000; 109:235-344.11. Maeshima K, Laemmli UK. A two-step scaffolding model for mitotic chromosome assem-bly. Dev Cell 2003; 4:467-80.12. Losada A, Hirano M, Hirano T. Cohesin release is required for sister chromatid resolution,but not for condensin-mediated compaction, at the onset of mitosis. Genes & Dev 2002;16:3004-16.13. Cuvier O, Hirano T. A role of topoisomerase II in linking DNA replication to chromo-some condensation. J Cell Biol 2003; 160:645-55.14. Wignall SM, Deehan R, Maresca TJ, Heald R. The condensin complex is required forproper spindle assembly and chromosome segregation in Xenopus egg extracts. J Cell Biol2003; 161:1041-51.15. Steffensen S, Coelho PA, Cobbe N, Vass S, Costa M, Hassan B, et al. A role for DrosophilaSMC4 in the resolution of sister chromatids in mitosis. Curr Biol 2001; 11:295-307.16. Hagstrom KA, Holmes VF, Cozzarelli NR, Meyer BJ. C. elegans condensin promotesmitotic chromosome architecture, centromere organization, and sister chromatid segrega-tion during mitosis and meiosis. Genes Dev 2002; 16:729-42.17. Hudson DF, Vagnarelli P, Gassmann R, Earnshaw WC. Condensin is required for nonhis-tone protein assembly and structural integrity of vertebrate chromosomes. Dev Cell 2003;5:323-36.18. Cremer T, Cremer C. Chromosome territories, nuclear architecture and gene regulation inmammalian cells. Nat Rev Genet 2001; 2:292-301.19. Ono T, Losada A, Hirano M, Myers MP, Neuwald AF, Hirano T. Differential contributionsof condensin I and condensin II to mitotic chromosome architecture in vertebrate cells.Cell 2003; 115:109-21.

Chromosome shaping by two condensins

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