Condensins: Organizing and Segregating the Genome

Condensins: Organizing and Review Segregating the Genome

Tatsuya Hirano and mechanisms behind this scene have largely remained elusive. During the past decade, however, a substantial body of evidence has accumulated that a Condensins are multi-subunit protein complexes that class of multiprotein complexes known as condensins play a central role in mitotic chromosome assembly is one of the key components that directly regulates and segregation. The complexes contain ‘structural chromosome architecture and dynamics. The chief maintenance of chromosomes’ (SMC) ATPase sub- function of condensins is to assemble and segregate units, and induce DNA supercoiling and looping in an chromosomes during mitosis and meiosis, though ATP-hydrolysis-dependent manner in vitro. Vertebrate recent studies suggest that they also play much cells have two different condensin complexes, con- broader roles in the expression and maintenance of densins I and II, each containing a unique set of reg- the genome. In this review, I will summarize recent ulatory subunits. Condensin II participates in an early progress in our understanding of the structure and stage of chromosome condensation within the function of condensins, and discuss unresolved prob- prophase nucleus. Condensin I gains access to chro- lems and future directions in the field. mosomes only after the nuclear envelope breaks down, and collaborates with condensin II to assemble Subunit Composition and Molecular Architecture of metaphase chromosomes with fully resolved sister Condensins chromatids. The complexes also play critical roles in The canonical condensin complex, now referred to as meiotic chromosome segregation and in interphase condensin I, was originally identified as a major processes such as gene repression and checkpoint protein component required for the establishment and responses. In bacterial cells, ancestral forms of con- maintenance of mitotic chromosomes in cell-free densins control chromosome dynamics. Dissecting extracts of Xenopus laevis eggs [1,2]. More recently, a the diverse functions of condensins is likely to be second condensin complex, condensin II, has been central to our understanding of genome organization, found in vertebrate cells [3,4]. Condensins I and II stability and evolution. have the same pair of core subunits but distinct sets of regulatory subunits. The two core subunits, CAP- E/SMC2 and CAP-C/SMC4, belong to a large family of chromosomal ATPases known as the ‘structural main- Introduction tenance of chromosomes’ (SMC) family. The In this era of genomics, a huge amount of sequence SMC2–SMC4 heterodimer adopts a V-shaped struc- information is accumulating every day, and the ture, characteristic of all SMC proteins, with an ATP- genome content of many species is being uncovered binding cassette (ABC) ATPase domain at the distal at an extraordinary pace. One of the important chal- end of each arm (Figure 1A,i) [5,6]. Like other SMC lenges in post-genomics research is to understand proteins, it is predicted that two ATP molecules are how the genome is organized into three-dimensional sandwiched between the ATP-binding ‘head’ domains chromatin structure within the cell. It is also important [7,8], and that ATP binding and hydrolysis modulate to determine how such structure is remodeled and engagement and disengagement of the head modified during the duplication, expression and domains, respectively (Figure 1A,ii) [9,10]. How the inheritance of the genome. Cytologically, the most mechanochemical cycle of the SMC subunits is used striking transformation of chromatin structure is to reconfigure DNA and chromatin structure is under observed during mitosis when the cell partitions its active investigation (see below). Note that the SMC duplicated genome into daughter cells. Upon disas- heterodimer is a huge molecule: each arm, composed sembly of the cell nucleus at the onset of mitosis, an of an anti-parallel coiled coil, is about 50 nm long, a amorphous mass of chromatin is converted into a length equivalent to that of 150 base pairs of double- discrete set of rod-shaped chromosomes in which stranded DNA. the two duplicated ‘sister’ chromatids are juxtaposed The two different condensin complexes are distin- along their length. This process, referred to as chro- guished by their unique sets of non-SMC subunits. mosome assembly or condensation, is an essential Among the three regulatory subunits of each complex, prerequisite of the faithful segregation of sister chro- two — CAP-D2 and CAP-G in condensin I, and CAP- matids at the subsequent stage of mitosis D3 and CAP-G2 in condensin II — contain HEAT (anaphase). repeats, a highly degenerate repeat motif implicated in Although the sudden appearance of thread-like protein–protein interactions [11]. The fifth subunits — chromosomes in early mitosis has fascinated cell biol- CAP-H in condensin I and CAP-H2 in condensin II — ogists for more than 100 years, the molecular players belong to the kleisin family of proteins [12]. The non- SMC subunits of condensin I form a subcomplex that Cold Spring Harbor Laboratory, One Bungtown Road, P.O. binds to the head domain(s) of the SMC heterodimer Box 100, Cold Spring Harbor, New York 11724, USA. (Figure 1B,i) [6,13]. It has been shown that the non- E-mail: [email protected] SMC subunits regulate chromosomal targeting of the Review R266

Figure 1. Architecture of condensins A (i) (ii) and their related complexes. Hinge (A) SMC2 and SMC4 form the core of ATP the condensin complexes. Each SMC binding subunit folds intramolecularly by anti- N C N C Coiled-coil C N C N parallel coiled-coil interactions, and arm forms an ATP-binding head domain composed of its amino- and carboxy- ATP Engagement hydrolysis terminal sequences. A hinge–hinge interaction between SMC2 and SMC4 N NN C then produces a V-shaped heterodimer CCHead C N (i). Two ATP molecules (yellow) are SMC2 SMC4 sandwiched between two SMC head domains and induce their engagement. Disengagement of the two head B (i)(ii) (iii) (iv) domains is triggered upon hydrolysis of the ATP (ii). (B) Condensin I (i) and condensin II (ii) share the same pair of SMC2 and SMC4 as their core subunits. Each of the three non-SMC sub- SMC2 SMC4 SMC2 SMC4 SMC2 SMC4V SMC SMC units of condensin I has a distantly (DPY-27) related counterpart in those of condensin II. The CAP-D2, CAP-G, CAP-D3 and CAP-G2 subunits contain HEAT H H2 DPY-26 repeats, whereas the CAP-H and CAP- ScpA H2 subunits belong to the kleisin family of proteins. The nematode Caenorhab- D2 G D3 G2 DPY-28 ScpB ditis elegans has a specialized condensin-like complex which regulates Condensin I Condensin II Dosage Bacterial dosage compensation (iii). The core of compensation SMC complex this complex is a heterodimer of the complex authentic SMC2 subunit and an SMC4 variant called DPY-27. DPY-28 has kleisin HEAT HEAT repeats whereas DPY-26 has Current Biology kleisin motifs. The Bacillus subtilis SMC protein complex is composed of an SMC homodimer, a kleisin subunit (ScpA) and another small subunit called ScpB (iv).

SMC dimer in Xenopus egg extracts and modulate its organization (Figure 2A and B), the genome size of an ATPase and DNA-binding activities in vitro [14]. Con- organism is not always a good indicator to predict densin II is anticipated to have a similar architecture whether it possesses condensin II or not. For (Figure 1B,ii). example, all condensin II genes were found in the Eukaryotic cells have another SMC protein genome of the unicellular red algae Cyanidioschyzon complex, known as cohesin, that plays a central role merolae (Table 1) [16]: this compact genome is only in sister chromatid cohesion during mitosis and 16.5 Mb, a similar size to that of the yeast meiosis [15]. The core of the cohesin complex is a Saccharomyces cerevisiae. heterodimer of SMC1 and SMC3, and one of the two The apparent loss of condensin I in C. elegans and regulatory subunits (Scc1) belongs to the kleisin C. briggsae is puzzling, but may be related to their family. Despite their similar subunit composition, the unique, holocentric chromosome structure (Figure cohesin and condensin complexes display different 2C) [17,18]. Alternatively, an ancient condensin I arm conformations, as judged by electron complex may have lost its mitotic functions during microscopy, which may contribute to their specialized evolution of these nematodes and been modified and cellular and biochemical functions [6]. adapted to execute a specialized function in the dosage compensation that equalizes expression of X- Phylogeny of Condensin Subunits linked genes in the two sexes [19]. The core of the Phylogenic analysis of condensin subunits sheds dosage compensation complex (DCC) is composed intriguing light on the evolution of chromosome archi- of the SMC2 ortholog MIX-1 and an SMC4 variant tecture. All non-SMC subunits of condensin I are (DPY-27) that is unique to the worm. The complex highly conserved from yeast to humans, with notable also contains two non-SMC subunits, DPY-28 and exceptions of the nematodes Caenorhabditis elegans DPY-26, which have limited similarities to the CAP-D2 and C. briggsae (Table 1). The non-SMC subunits of and CAP-H subunits of condensin I, respectively condensin II are found in plants and vertebrates, but (Figure 1B,iii; Table 1). not in yeast or other fungi such as Aspergillus nidu- Finally, it is important to note that even bacterial cells lans and Neurospora crassa. While it is tempting to have condensin-like complexes. In Bacillus subtilis, for speculate that condensin II has evolved to provide example, an SMC homodimer associates with two non- large chromosomes with an additional level of SMC subunits, ScpA and ScpB, and participates in Current Biology R267

Table 1. Condensin subunits in model organisms.

Subunits Human Xenopus Arabidopsis Drosophila C. elegans C. merolae S. pombe S. cerevisiae (3,080 Mb)1 (125 Mb) (165 Mb) (100 Mb) (16.5 Mb) (13.8 Mb) (12.1 Mb)

Core (I & II) DCC2 SMC hCAP-E XCAP-E AtCAP-E1/E2 DmSMC2 MIX-1 MIX-1 CMG189C Cut14 Smc2

SMC hCAP-C XCAP-C AtCAP-C DmSMC4 SMC-4 (DPY-27) CME029C Cut3 Smc4

I-specific HEAT hCAP-D2 XCAP-D2 CAB72176 CG1911 – (DPY-28) CMR484C Cnd1 Ycs4

HEAT hCAP-G XCAP-G BAB08309 CG17054 – – CMS422C Cnd3 Ycs5/Ycg1 kleisin hCAP-H XCAP-H AAC25941 Barren – (DPY-26) CMF069C Cnd2 Brn1

II-specific HEAT hCAP-D3 XCAP-D3 At4g15890.1 CG31989 HCP-6 CM0236C – –

HEAT hCAP-G2 XCAP-G2 At1g64960.1 – F55C5.4 CMA089C – – kleisin hCAP-H2 XCAP-H2 At3g16730.1 CG14685 C29E4.2 CMI207C – –

1The genome size of each organism is shown in parentheses. 2The subunits of the C. elegans dosage compensation complex (DCC) are boxed. MIX-1 is the authentic SMC2 whereas DPY-27 is a variant of SMC4. Although DPY-28 and DPY-26 are mostly related to the CAP-D2 and CAP- H subunits, respectively, they cannot be considered their orthologs. proper organization and segregation of the genome assays. It will also be important to determine whether (Figure 1B,iv) [10,20]. a single condensin complex is capable of mediating these reactions or whether cooperative interactions Molecular Actions of Condensins of multiple condensin complexes may be essential. How does the uniquely designed, two-armed struc- Full reconstitution of condensins I and II from ture of condensins interact with DNA and manipulate their recombinant subunits will be crucial to further its conformation? Despite the exciting data that are dissect the action of these highly elaborated protein accumulating, the answer to this question remains machines. elusive. Purified SMC2–SMC4 dimers have the ability Recent studies of bacterial SMC protein complexes to convert complementary single-stranded DNAs into have shed new light on the basic mechanism of action a double-stranded DNA in an ATP-independent of SMC proteins in general. By constructing mutant manner [21]. This reannealing activity appears to be forms of B. subtilis SMC proteins that are defective in supported by dynamic protein–protein interactions its ATPase cycle at three different stages, it was as judged by atomic force electron microscopy [22]. shown that ATP-dependent engagement of the head A simple gel-shift assay also indicates a rather domains is indeed crucial for dynamic interactions complex, cooperative interaction between the SMC between SMC proteins and DNA [10]. The non-SMC dimer and double-stranded DNA [23]. No ATP- subunits, ScpA and ScpB, play both positive and neg- dependent activities have been detected for ative roles in the SMC–DNA interactions by suppress- SMC2–SMC4 dimers, except that they display a ing the ability of the SMC complex to hydrolyze ATP. weak ATPase activity [14,23]. In Escherichia coli, the MukBEF complex is thought The holocomplex of condensin I purified from to be the structural and functional homolog of the B. Xenopus egg extracts has the ability to induce subtilis SMC–ScpAB complex. The results of single- positive superhelical tension into double-stranded DNA-molecule experiments using optical tweezers DNA in an ATP-hydrolysis-dependent manner suggest that MukBEF compacts the DNA molecule by [24–26]. Importantly, the supercoiling activity is not assembling a flexible nucleoprotein filament in an ATP- supported by the SMC2–SMC4 heterodimer alone dependent manner [29]. ATP hydrolysis is not required [14]: it requires the non-SMC subunits and their phos- for this compaction reaction, a property significantly phorylation by the master mitotic kinase cyclin different from that of the eukaryotic condensin I B–Cdk1 [25,27]. A similar activity has been detected complex [28]. Moreover, no supercoiling activity has in a condensin fraction purified from C. elegans been detected so far in these bacterial SMC or Muk embryos, which is predicted to be composed of con- protein complexes. Thus, it is likely that the action of densin II [18]. Most recently, a single-DNA-molecule the bacterial counterparts is related, but not identical, nanomanipulation technique using magnetic tweez- to that of the eukaryotic condensin complexes; never- ers has shown that condensin I is able to physically theless, the simple composition and ease of purification compact DNA in an ATP-hydrolysis-dependent of the former will continue to make great contributions manner [28]. The compaction reaction occurs in a to unveiling the dynamic behavior of SMC proteins. highly dynamic and reversible fashion, possibly involving a looping mechanism. It remains to be clar- Cell-Cycle Regulation of Condensins ified how this activity detected in the single-DNA-mol- Spatial and temporal distribution of the condensin sub- ecule assay is mechanistically related with the units during the cell cycle appears to vary among dif- supercoiling activity revealed by the bulk biochemical ferent eukaryotes. For example, they are constitutively Review R268

mitotic progression. Cyclin A accumulates in the AB C nucleus during G2 phase and prophase, and is degraded in late prophase or early prometaphase (Figure 3B); cyclin B, however, is sequestered into the cytoplasm during G2 and enters the nucleus only after early prophase. Active cyclin B–Cdk1 then triggers nuclear envelope breakdown (NEBD), allowing condensin I to gain access to chromosomes. One possibility is that cyclin A–Cdk1 phosphorylates and Condensin I Condensin I Condensin II activates condensin II to initiate an early stage of Condensin II chromosome condensation within the prophase Yeasts Animals and plants Nematodes nucleus. Upon NEBD, cyclin B–Cdk1 phosphorylates (monocentric) (monocentric) (holocentric) and activates condensin I [25,27], and may further Current Biology phosphorylate condensin II (Figure 3C). This hypothe- sis, although admittedly highly speculative at present, explains nicely how the sequential activation of the Figure 2. Condensins and evolution of chromosome architecture. two major mitotic kinases might contribute to spatial (A) Yeasts (S. cerevisiae and S. pombe) and other fungi and temporal regulation of chromosome condensation (A. nidulans and N. crassa) have only condensin I (blue). The chromosome is shown in light green and the sister kineto- supported by the two condensation machineries [33]. chores in yellow. Microtubules are shown in gray lines. (B) Vertebrates and plants have both condensin I (blue) and Dynamic Distribution of Condensins During Mitosis condensin II (magenta). The two complexes apparently alter- A recent study [38] which combined light and electron nate along the arm and display specialized geometry at the microscopy suggests that, in mammalian tissue centromere/kinetochore region. (C) Nematodes (C. elegans culture cells, an early stage of chromosome conden- and C. briggsae) have condensin II (magenta), but apparently not condensin I. Unlike many other organisms in which each sation in prophase is mediated by successive coiling chromatid has a single kinetochore (monocentric), numerous of chromatin fibers: that is, hierarchical folding. kinetochores assemble along the entire length of each chro- SMC2, an SMC core subunit common to condensins matid in C. elegans (holocentric). The C. elegans condensin II I and II, associates first with peripheral regions of complex localizes to the centromere/kinetochore regions of chromosomes at this stage, and then progressively the metaphase chromosome. accumulates into the central axis of chromatids from prometaphase through metaphase. Notably, the relo- nuclear throughout the cell cycle in S. cerevisiae calization of SMC2 coincides with NEBD and the [30,31]; in the fission yeast Schizosaccharomyces recruitment of condensin I to chromosomes. These pombe, however, most condensin molecules are cyto- data suggest that structural changes of plasmic during interphase, and transported into the chromosomes observed before and after NEBD may nucleus during mitosis in a Cdk1-dependent manner be mechanistically distinct (Figure 3C). Condensin II [32]. In vertebrate cells, condensin II is predominantly could initiate the early stage of condensation by hier- nuclear, whereas condensin I is sequestered in the archical folding. Upon NEBD, condensin I cooperates cytoplasm during interphase (Figure 3A) [33,34]. Con- with condensin II to shape, resolve and stabilize chro- densin I gains access to the chromosomes only after mosomes by forming an ‘axial glue’ structure the nuclear envelope breaks down in prometaphase, within the chromatids. and the two complexes alternate along the chromatid Maeshima and Laemmli [39] propose a different axis by metaphase [3,33]. A simple prediction from this model in which an early stage of condensation is observation is that an early stage of chromosome con- driven by topoisomerase II-mediated axis formation densation within the prophase nucleus may primarily and is followed by the action of condensin subunits. be mediated by condensin II. Small-interfering RNA This model does not, however, take into account the (siRNA) experiments in HeLa cells indeed support this role of condensin II, which was discovered only notion: depletion of condensin II-specific subunits recently. In the future, real-time analyses of distribu- delays prophase condensation, whereas depletion of tion and dynamics of these chromosomal components condensin I-specific subunits does not [33,34]. Con- will help test and refine the currently existing models. sistently, depletion of an SMC subunit also affects The observations described above raise an prophase condensation in chicken DT40 cells [35]. interesting question about the ‘reversibility’ of chro- However, such a clear division of labor between the mosome condensation. In response to various types two condensin complexes may not be applicable to all of stress, for example, as a result of chromosome metazoans. For instance, in Drosophila, which appar- damage or microtubule disassembly, cells in early ently has no gene for the CAP-G2 subunit, mutations in prophase decondense their chromosomes and return the CAP-G subunit affect interphase gene to G2 phase, whereas such reversal of condensation expression [36,37]. does not occur in cells after late prophase [40]. How is the action of the two condensin complexes Recent studies [41,42] have suggested that this regulated during mitosis? In Xenopus egg extracts, ‘antephase’ checkpoint, activation of which requires Cdk1-dependent phosphorylation is at the heart of the p38 MAP kinase, monitors global chromatin topol- condensin regulation [2,27]. In somatic cells, two ogy before making the final commitment to mitosis major cyclin–Cdk complexes are known to regulate (Figure 3D). It seems plausible that the reversible Current Biology R269

Figure 3. Cell cycle regulation of NEBD condensins I and II. (A) The ‘traditional’ classification of A G2 pro prometa meta ana telo mitosis and subcellular distributions of condensins I and II during the cell cycle. Nuclear envelope breakdown (NEBD), Condensin I Nucleus one of the key events in early mitosis, occurs at the transition from prophase Cytoplasm (pro) to prometaphase (prometa) in this classification. The distributions of con- Condensin II densin I and condensin II complexes are shown in blue and magenta, respectively. (B) Sequential activation of cyclin B A–Cdk1 and cyclin B–Cdk1 during Cdk Cyclin A-cdk1 Cyclin B-cdk1 mitosis. (C) Hypothesized activities of activities condensins I and II during mitosis. According to this model, condensin II is C responsible for prophase condensation, Condensin Condensin I which may involve reversible, hierarchi- activities Condensin II cal folding. Upon NEBD, condensin I gains access to chromosomes and Hierarchical folding Axis formation cooperates with condensin II to build Reversible Irreversible fully resolved sister chromatids. This D 1234 5 process accompanies the formation of the chromatid axis and cannot be Commitment reversed by DNA damage. (D) A classifi- to mitosis cation of mitotic stages proposed by Current Biology Pines and Rieder [85], which empha- sizes the ‘point of no return’ that makes the final commitment to mitosis. phase of condensation might correspond to the stage recruiting condensins to chromosomes, available lines of condensin II-mediated hierarchical folding, and that of evidence do not support this notion [45,46]. A recent condensin II may be an integral component of this paper [47] reports that a subfraction of condensin sub- surveillance mechanism. For instance, condensin II- units co-purifies with the DNA methyltransferase mediated conformational changes of chromosomes DNMT3B, which further interacts with a histone might be used as a strategy to monitor potential deacetylase (HDAC1). Although the functional rele- chromosome aberrations. If such aberrations are vance of these interactions remains to be determined, found, the checkpoint is activated and reverses the exploring potential crosstalk between condensins and action of condensin II possibly by down-regulating the epigenetic machineries will be an exciting future cyclin A–Cdk1 [43]. This checkpoint control is likely to direction in the field. be less stringent or even absent in early embryonic cells. Consistent with this notion, condensin II is much Condensins and Metaphase Chromosome less abundant, and its contribution to chromosome Architecture architecture is less prominent in early embryonic While early studies in Xenopus cell-free extracts [1,2] extracts than in somatic cells [3]. and yeast genetics [30,32,48–51] demonstrated that In metaphase chromosomes, condensins I and II condensin function is required for the establishment accumulate along the axis of sister chromatids, and maintenance of metaphase chromosome struc- possibly in an alternative fashion [3,33]. Biochemical ture, it remains controversial exactly how condensins experiments have shown that at least condensin I con- contribute to these processes [52,53]. For example, stitutes part of a fraction known as the chromosome individual masses of chromatin with a certain degree scaffold [35,39,44]. The two complexes can be tar- of compaction are observed in condensin mutants of geted to chromosomes independently of each other in Drosophila [36,54,55] and C. elegans [18] or in verte- both HeLa cells and Xenopus egg extracts [3,34]. brate cells depleted of condensin subunits [34,35], What, then, determines the differential distribution of leading to the suggestion that condensins may not be condensins I and II along the chromatids? It is possi- the sole factor required for chromatin compaction ble, but unlikely, that their distribution depends solely during mitosis. In this section, an attempt is made to on underlying DNA sequences. Given the observation clarify and discuss problems and difficulties that the that the relative abundance of condensins I and II is current field is faced with. different between the embryonic and somatic cells [3], First of all, it is clear that part of the controversy an epigenetic mechanism may exist that flexibly and stems from our lack of knowledge about how sister dynamically regulates the distribution of the two con- chromatid fibers are folded and organized to densin complexes along the arm. Currently, no specific assemble a metaphase chromosome [56]. This histone modification is known that affects targeting or problem leaves a large window for disparate interpre- function of condensins. While it has long been sus- tations of seemingly similar observations, and pro- pected that mitosis-specific phosphorylation of serine vides an opportunity to draw unconvincing 10 (and/or serine 28) of histone H3 might participate in conclusions from a limited amount of data. Figure 4 Review R270

Figure 4. Different types of chromo- ABC some ‘compaction’. (A–C) Three examples of hypothetical chromosome structure assembled Top view from a fixed volume of chromatin are viewed from top (first row) or side (second row). Each structure contains duplicated sister chromatids (shown in blue and magenta), the folding paths of which are shown in the third row. Side view Without any active folding mecha- Axial D nisms, the two chromatids would be lengthening converted into unresolved, random coils (A). One mechanism of ordered folding would produce a rod-shaped structure in which the two chromatids are folded together without being Chromatin resolved from each other (B). An addi- path tional mechanism would be required to make a ‘functional’ metaphase chromosome in which the two sister chro- Axial E Random coil matids are fully resolved and folded Ordered shortening separately (C). (D,E) Two representa- tive variations of the resolved chromo- Unresolved Resolved some with their volumes fixed. Axial Current Biology lengthening would create a longer and thinner chromosome (D), whereas axial shortening would produce a shorter and thicker chromosome (E). depicts three examples of hypothetical structures that chromatid resolution [54,55], but it remains to be fully can be produced from a fixed volume of chromatin. It established to what extent and how condensins is very important to distinguish between different participate in the ordered folding process. structures that may sometimes be considered equally The second problem, closely related to the first one, ‘compacted’. During mitosis, disassembly of the is the lack of reliable, quantitative assays to probe nuclear envelope allows release of chromatin fibers metaphase chromosome structure. It is well known from specific tethering within the interphase nucleus. that chromosome morphology is highly sensitive to a Without an active folding mechanism, this would number of chemical and physical treatments, and produce random coils (Figure 4A) rather than varies depending on different fixation and imaging extended chromatin fibers in the cell. Electrostatic techniques. At the same time, it is difficult to analyze interactions within the chromatin fibers and/or passive chromosome structure in great detail without aid of exclusion from the molecularly crowded cytoplasm some spreading techniques. In fact, the contribution may confer an additional level of compaction. This of condensins to metaphase chromosome architec- random compaction is clearly distinct from the con- ture can be demonstrated most convincingly when densation of mitotic chromosomes, which is a deter- chromosomes are subjected to hypotonic treatments ministic, shaping process rather than a simple linear [3,34,35]. Under such conditions, Ono et al. [3] compaction process [57,58]. showed that condensins I and II have distinct roles in It is also important to note that one of the chief determining the shape of metaphase chromosomes. functions of metaphase chromosome assembly is to On the other hand, Hudson et al. [35] devised a novel ‘resolve’ sister chromatids, an essential prerequisite assay in which chromosomes were exposed alter- for their rapid and synchronous separation at nately to a buffer containing MgCl2 or its chelator, and anaphase. In principle, one type of ordered folding demonstrated that the structural integrity of chromo- can produce rod-shaped chromosomes in which somes is severely compromised in the absence of juxtaposed sister chromatids are folded together SMC2. These results clearly underscore the funda- (Figure 4B), but these unresolved chromosomes are mental importance of condensins in shaping and unlikely to be segregated properly in anaphase. Only maintaining metaphase chromosome architecture. when fully resolved sister chromatids are constructed, The current approaches should, however, be comple- does the chromosomes become ‘functional’ — that is, mented by more quantitative and less invasive competent for anaphase segregation (Figure 4C). approaches in the future. Those may include live-cell Further variations of this structure include axial imaging analyses in which the density/volume of chro- lengthening and shortening, which accompany lateral matin is measured throughout the cell cycle or in contraction and expansion, respectively (Figure 4D,E). which the dynamics of specifically labeled chromoso- Thus, without knowing the actual folding path of the mal loci is followed in real time. chromatid fibers within a chromosome, it is often Finally, it should be emphasized that Xenopus cell- misleading to judge whether its assembly is normal or free extracts remain a very powerful experimental not. Currently, there is a clear consensus that system for studying mitotic chromosome architecture condensin function is essential for facilitating sister and dynamics. In general, much severer phenotypes Current Biology R271

Figure 5. Multilayered contribution of condensins to mitotic chromosome segregation. Chromosome (A) Condensins play critical roles in the A bridges resolution of sister chromatids in Sister metaphase. Defects in this process chromatid lead to the formation of chromosome resolution bridges in the subsequent anaphase. Kinetochore B (B) Condensins regulate proper assem- orientation bly of centromeric heterochromatin and Abnormal thereby contribute to determining the microtubule back-to-back orientation of sister kine- attachment tochores (yellow). When this process is rDNA C compromised, abnormal interactions organization between kinetochores and microtubules are observed. (C) Condensins rDNA segregation may have a specialized role in the orga- defects nization and segregation of repetitive DNAs. Condensin’s participation in the Current Biology segregation of rDNA (orange) has been described in S. cerevisiae. are observed in extracts immunochemically depleted of defect is observed in many different organisms, condensins than in somatic tissue culture cells treated including yeast [48,49], Drosophila [55,63] and with condensin siRNAs. When both condensins I and II chicken DT40 cells [35], and is likely to be a direct are depleted from the extracts, no individual chromo- consequence of poor resolution and/or abnormal somes are assembled, regardless of the presence or compaction of sister chromatids in the preceding absence of prior DNA replication [2,59]. It has also been metaphase. In these cells, the kinetochores appear to shown that single depletion of condensin I or II pro- function normally and attempt to pull unresolved duces a drastically distinct phenotype [3]. One poten- chromatids to opposite poles without success (Figure tial explanation for these ‘clean’ phenotypes may be 5A). In Xenopus egg extracts [59] or human tissue that whole condensin complexes, either condensin I or culture cells [33], however, severe defects in kineto- II or both, are depleted from Xenopus egg cell-free chore-microtubule interactions, indicative of extracts. In contrast, only a single subunit (or at most merotelic attachment, are frequently observed after two subunits) is depleted by RNA interference or by condensin depletion (Figure 5B). Merotelic attach- conditional knockdown in tissue culture experiments. ment does not activate the spindle checkpoint [64], The fate of other non-target subunits is not fully char- consistent with the observation that condensin deple- acterized in the latter studies, with an assumption that tion does not induce robust arrest at mitosis. depletion of one subunit compromises all functions Abnormal attachment of kinetochore microtubules executed by the whole complex. A difference in the is even more prominently observed in condensin- efficiency of depletion may also lead to variable sever- deficient embryos of C. elegans [17,18]. In holocentric ity of defective phenotypes, as exemplified in recent chromosomes of C. elegans, numerous kinetochores studies in C. elegans [18,60]. Another explanation is assemble along the entire length of each chromatid, that the two experimental systems use different start- forming two ‘lines’ on the outer surfaces of a ing materials for chromosome assembly, which may in metaphase chromosome. Condensin II localizes turn produce seemingly different terminal phenotypes, underneath these structures (Figure 2C), and does so as discussed elsewhere [52]. Furthermore, the possibil- in an Aurora B-dependent manner. At first glance, this ity cannot be ruled out that chromosome assembly in distribution of condensin II in the C. elegans chromo- embryonic cells depends solely on condensins, somes is very different from that in vertebrate whereas somatic cells use an additional factor(s) chromosomes. A recent study [33], however, shows besides condensins. In this sense, it is intriguing to note that, in human cells, a subpopulation of condensin II is that even the relative abundance and functional contri- enriched at or near the inner kinetochore plate (Figure butions of condensins I and II are different between 2B), and that the kinetochore-specific localization of embryonic and somatic cells [3]. Moreover, measure- condensin II, but not its distribution along the arm, is ments of the mechanical properties of chromosomes — under the control of Aurora B. Thus, monocentric and for example, the stiffness and elasticity — detect differ- holocentric chromosomes do share many properties ences between embryonic and somatic ones [61,62]. in common. Condensins are likely to contribute to Thus, developmental regulation of chromosome archi- proper assembly of centromeric chromatin in both tecture is another interesting subject of future research. types of chromosomes, which in turn plays a critical role in establishing the back-to-back orientation of Contributions of Condensins to Specific sister kinetochores. Chromosomal Domains Kinetochore orientation rDNA segregation One of the classical phenotypes observed in A number of recent papers [65–68] report that condensin-deficient cells is a massive amount of condensin’s recruitment and function are regulated by chromosome bridges in anaphase. This segregation a unique mechanism at the rDNA locus in S. cerevisiae. Review R272

While most chromosomal regions are segregated upon required for meiotic (but not mitotic) chromosome cleavage of a cohesin subunit at the segregation [71], providing an additional level of metaphase/anaphase transition, rDNA segregation complexity to this problem. In vertebrates and plants, occurs in mid-anaphase and requires a pathway it will be of great importance to determine whether involving Cdc14, a protein phosphatase that is acti- condensins I and II are subjected to different, tempo- vated by the ‘fourteen early anaphase release’ (FEAR) ral and spatial regulation during meiosis, and whether network. Condensin is recruited to the rDNA locus in they play non-overlapping functions in meiotic anaphase in a Cdc14-dependent manner and mediates chromosome recombination and segregation. rDNA condensation and resolution (Figure 5C). As has been demonstrated in previous studies of Ipl1/Aurora kinase activity is required for the conden- cohesin [15], further characterization of condensins sation process, but not for resolution. during meiosis should not only facilitate our under- The molecular nature of the cohesin-independent standing of their meiosis-specific functions but also linkage that persists until mid-anaphase at the rDNA provide deeper insights into their basic functions locus is unknown. The primary candidate for such a during mitosis. It will be of particular interest to eluci- linkage is the catenation between sister chromatids, date the mechanism of condensin’s action in resolv- resolution of which requires a combined action of ing the recombination-dependent linkages of meiosis condensin and topoisomerase II (although one study I chromosomes [69], which may also occur during argues against this possibility [67]). Alternatively, con- mitosis at repetitive loci such as rDNA and telomeres densin function may be required to resolve inter- [67,72]. By analogy to cohesin [73–75], condensins repeat recombination in the rDNA array that would could also use a unique set of subunits to execute otherwise impede segregation [31]. Interestingly, yeast their specialized functions in meiotic cells. Although condensin subunits are subjected to multiple post- no such candidates have been found so far by bioin- translational modifications in anaphase. For example, formatic approaches, this may not be surprising the Ycg1/CAP-G subunit of condensin is phosphory- because even the similarity between the non-SMC lated in an Ipl1/Aurora-dependent manner [65], and subunits of condensins I and II is very limited [3]. A the Ycs4/CAP-D2 subunit is sumoylated in a Cdc14- combination of genetics and biochemistry may yet stimulated manner [67]. The functional significance of identify meiosis-specific condensin subunits. these modifications remains to be determined, however. The repetitive nature of rDNA in yeast may Condensins, Gene Regulation and Genome Stability offer an excellent model system for studying the Accumulating lines of evidence suggest that action of condensins in organisms with more complex condensins have important functions in chromosome genomes. regulation outside mitosis and meiosis. In S. cerevisiae, transcriptional silencing is altered in a locus- Condensins and Meiotic Chromosome Functions specific manner in condensin mutants [31]. For Given the fundamental functions of condensins in example, silencing is enhanced at the rDNA locus but mitotic chromosome assembly and segregation, it is reduced at telomeres, and this change is accompa- not surprising to find that condensins also play nied by relocalization of the silencing factor Sir2 from crucial roles in the structural and functional organiza- telomeres to the rDNA arrays [76]. In S. pombe, a tion of meiotic chromosomes. In S. cerevisiae, con- condensin mutant displays hypersensitivity to a treat- densin subunits localize to the axial core of ment that slows down DNA replication and fails to pachytene chromosomes and contribute to their axial activate the checkpoint kinase Cds1/Chk2 under such compaction and individualization [69]. Chromosomal a condition [77]. In Drosophila, condensin function is localization of Zip1, a component of the central required for transcriptional repression in centromere- element, is severely disturbed in condensin mutants, proximal heterochromatin [36], and its mutation resulting in improper assembly of the synaptonemal exacerbates the ‘rough eye’ phenotype that is induced complex. As a consequence, homologue pairing and by overexpression of the centromere-specific histone processing of double-strand breaks are perturbed in variant CENP-A [37]. Although global defects in chro- these mutant cells. Evidence is also available that matin structure may be sufficient to account for these condensin is required for the resolution of recombi- diverse phenotypes observed in the condensin nation-dependent linkages between homologues in mutants, more specific involvement of condensin sub- meiosis I and perhaps for the segregation of sister units in each event cannot be excluded. In verte- chromatids in meiosis II as well. brates, potential contribution of condensin II to the A requirement for condensin function in both structural and functional organization of interphase meiosis I and meiosis II is consistent with results from chromatin remains to be explored. Arabidopsis [70] and C. elegans [60]. Unlike in S. cere- An elegant series of genetic and biochemical visiae, in C. elegans the condensin subunits associate studies [19] has revealed that a condensin-like with chromosomes only after the exit from pachytene, complex regulates dosage compensation in C. and restructure them in preparation for the subse- elegans. The dosage compensation complex (DCC) is quent two meiotic divisions. The difference may be likely to be the outcome of an evolutionary adaptation explained by the fact that S. cerevisiae has condensin that arose to mediate chromosome-wide gene repres- I alone, whereas C. elegans contains condensin II sion in the nematode. A crucial question is how the only. However, the non-SMC components of the DCC is specifically targeted to both X chromosomes dosage compensation complex in C. elegans are also in XX animals and reduces the level of transcripts by Current Biology R273

half. Recent work [78] has identified cis-acting DNA address the evolution and developmental regulation of elements that recruit this complex to the X chromo- chromosome architecture from a completely new per- some and facilitate its spreading along the entire spective. It is anticipated that the structural and func- length of the chromosome. Intriguingly, the same tional characterization of condensins will continue to complex is also targeted to the sex-determining auto- provide new and surprising insights into chromosome somal gene her-1 and represses its transcription >20- biology and beyond. fold [79]. It is not known how the DCC is able to support the two different modes of gene repression: Acknowledgments the two-fold chromosome-wide repression and I thank members of the Hirano laboratory for critically reading twenty-fold gene-specific repression. the manuscript. I am also grateful to many colleagues in the Might any of the non-mitotic functions of con- field for stimulating discussions. The work from the author’s densins be linked to human diseases in the future? laboratory was supported by grants from the National Given their fundamental functions in chromosome Institutes of Health. segregation, it is not surprising to find that serendipi- References tous knocking-out of the gene encoding the CAP-G2 1. Hirano, T., and Mitchison, T.J. (1994). A heterodimeric coiled-coil subunit caused embryonic lethality in mice [80]. protein required for mitotic chromosome condensation in vitro. Cell Recent studies, however, show that haploinsufficiency 79, 449–458. 2. Hirano, T., Kobayashi, R., and Hirano, M. (1997). 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