Biochemical and genetic dissection of mitotic chromosome condensation

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T~BS 20 - SEPTEMBER1995 ~ H~GHER EU~YOT~C cells, dupD cation and expression of genetic inforo mation occur within the cell nucleus. At the onset of mitosis, the interphase nucleus disassembles and the du#icared chromatin is packaged into compact vehicles, the mitotic chromesomes, before being transported into two daughter cells. This packaging process, termed mitotic chromosome condensation, is believed to be essential for maintaining the integrity of genetic information during mitosis. Although the fascination with this phenomenon pre-dates the recognition that chromosomes are the carriers of genetic information, the molecular mechanisms underlying the dynamic changes of chromosome structure in the ceil cycle are poorly understood. What Rind of molecular machinery is involved in the condensation of mitotic chromosomes? Two decades ago, histone HI was found to be hyperphosphorylated during mitosis,and for some time this modification was thought to drive mitotic chromosome condensation (reviewed in Ref. 1). Recent studies, however, provide strong evidence against this notion ~-4 and the actual function of H1 phosphorylation remains to be determined. Histone H3 is also phosphorylated during mitosis at a single site in its amino terminus ] and this event is tightly coupled to chromosome condensation 4. Again, it is not known whether this modification actively participates in the regulation of nucleosomal and higher-order chromosome structure. One of the best-characterized nonhistone chromosomal proteins is topoisomerase II, an enzyme that catalyses strand passing of double-stranded DNA. It is also functionally implicated in mitotic chromosome condensation (for recent reviews, see Refs 5, 6). Recently, biochemical and genetic studies independently identified a new class of putative ATPases that play a fundamental role in this process TM. Sequence analyses revealed that they belong to the same protein family, now called the SMC family. In this review, I describe recent technical advances leading to the identification of SMC family members, and discuss their potential functions in comparison with topoisomerase il. While the original SMC acronym was derived from the yeast I". Hirano is at the Cold Spring Harbor Laboratory,PO Box 100, Cold SpringHarbor,

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ica and geaetk dioa of mitotic chromo me condensation Tatsuya Hirano The moSecu~ar mechanisms responsible for mitotic chromosome condensation are unknown. Two independent approaches, biochemicaJ studies in vertebrate cells and genetic analyses in yeasts, have converged recently, leading to the identification of a family of putative ATPases that play a fundamental rage in this process. Further characterization of these proteins promises to uncover a highly dynamic aspect of mitotic chromosome architecture. gone SMCI whose mutation affects 'stability of minichromosome q~-, redefinition of SMC as 'structural maintenance of chromosomes' has been proposed".

B[echem[camappreach~ to mitotic chromosomeorganization The first step towards the biochemical characterization of chromosomal components is to isolate 'pure' mitotic chromosomes from the cell. However, this is technically difficult, because mitotic chromosomes are fragile structures whose morphology is easily perturbed, even under near-physiological conditions; they are also sticky, which makes it difficult to separate them from cellular contaminants, particularly cytoskeletal proteins. Despite these difficulties, several procedures for chromosome isolation and subsequent biochemical dissection have been established. Perhaps one of the b.estcharacterized subchromosomal fractions is the chromosome scaffold. This fraction consists of a subset of nonhistone chromosomal proteins that remain insoluble after treatment of isolated metaphase chromosomes with nuclease and subsequent extraction under a variety of conditions ~3. A decade ago the first abundant polypepfide of this fraction, Scl, was identified as topoisomerase II (Refs 14, 15~°Th',~ qnding, combined with other structural and biochemical studies, led to the proposal that topoisomerase If acts as a chromatin-loop fastener by interacting directly with specific DNA sequences called scaffold-associated regions

($ARs)~! The gone encoding a second major component of the chromosome scaffold, Scll, was cloned recently and found to encode a member of the SMC family9. While colocalizafion of topoisomerase II and ScH on hypotonically swollen chromosomes is intriguing, the functional role of Scll in chromosome organization remains to be determined. A celffree extract derived from Xenopus laevis eggs provides a powerful system for dissecting the biochemical processes of mitotic chromosome assembly in vitro. This system has been used to address the specific roles of known chromosomal proteins, such as topoisomerase 11~ and histone HI, in chromosome assembly by inhibitor treatment or immunodepletion 3'17-2°. More recently, the finding that a singlestep sedimentation of in vitro assembled chromosomes yields a 'clean' chromosomal fraction has provided a good st.arting point for identifying novel chromosomal components l°. Xenopus members of the SMC family, XCAP-Cand XCAPoE, are two of the major polypeptides recovered in this fraction. They were tightly associated with mitotic chromosomes assembled in vitro, consistent with the behavior of Scll in chromosome kactionation 9. lmmunofluorescent staining of tissue culture cells showed that XCAP-C and XCAP-E do indeed localize to mitotic chromosomes in vivo. The role of XCAP-C in mitotic chromosome assembly was tested in vitro by using sperm chromatin as a substrate. The addition of an antibody against XC,M~'Cto an extract

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mitotic arrest) 21. Recent development of fluorescent in situ hybridization (FISH) techniques has made it possible to examine the extent of chromosome condensation more precisely in both yeasts ~-2-24, thereby promising fruitful genetic approaches to this problem. Two SMC family members have been found from both S. cereoisiae (Smclp and Smc2p) and S. pombe (cut3 and cutl4) z,s.n. Most of the yeast members were originally identified as gene prod: ucts .that affect proper segregation of mitotic chromosomes ~2,2s,as.One exception is Smc2p, which was initially classified as a member of the SMC family on the basis of sequence homology, and was subsequently shown, using mutants, to be required for chromosome segregation u. To address the question of whether these mutants have any defects in chromosome condensation, two different FISH assays Genetic appmadms to mitotic chromosome were used (Fig. 2). In S. pombe, a whole dynamics arm of a particular chromosome was It ,~sed to be thought that mitotic 'painted' with mixed hybridization chromosome conde:,~ation does not probes, enabling mitosis-dependent occur in yeasts. Indeed, in the budding contraction to be visualized in wildyeast Saccharomyces cerevisiae, con- type cells (Fig. 2a)s. In cut3 and cut14 densation is not detectable by the mutants, the contraction was greatly conventional DNA-staining methods. In impaired, demonstrating a dramatic the fission yeast Schizosaccharomyces defect in mitotic chromosome condenpombe, a certain degree of compaction sation. A similar, but less severe, defect of the chromatln mass has been was observed in a topoisomerase II observed in mitotic cells. An early mutant (top2). in S. cereoisiae, the disgenetic study showed that topoisom- tance between two FiSH signals on a erase il is required for chromosome single chromosome was measured to condensation, but this was only judge the condensation state in a mitotirevealed under a restricted experimen- cally arrested condition (Fig. 21))u. tal condition On the background of a ShlRing the cells to a restrictive temperalS-tubulin mutation that leads to hyper- ture caused a significant increase in the condensation owing to prolonged separation of the two spots in temperablocked chromosome assembly at a late stage, causing condensation intermediates of entangled prophase-like fibers to accumulate (Fig. 1)m. When this antibody was added after chromosome assembly, rod-shaped chromosomes were disrupted and progressively converted into a mass of prophase-like fibers. Thus, XCAP-C function is essential for both assembly and structural maintenance of mitotic chromosomes in this system. This role of XCAP-C is distinct from that of topoisomerase lla in the same assay: previous studies showed that topoisomerase ila is required at an early stage of chromosome assembly, when entangled chromatin fibers need to be resolved (Fig. 1)2°. Once condensation was comple'~ed, unlike XCAP-C, topoisomerase Ha activity was not apparently required for maintaining the condensed state.

ture sensitive smc2 mutants (smc2~s) but not in smcV s mutants, suggesting that Smc2p is required for maintenance of the condensed state. Taken together, these results suggested that SMC function is required for chromosome condensation in both yeasts and that condensation may be a prerequisitefor successful sister chromatid segregation during mitosis. Gene disruption experiments showed that each SMC family member is essential for mitotic growth, indicating that they execute nonredundant functions 7&ll. However, an overlapping function was suggested by the fact that overexpression of curl4 ~ partially suppressed the temperature-sensitivity of a cut3 alleles. These results are best explained if the two proteins interact physically with each other, which seems to be the case, at least in S. cerevisioe (see below) n. ¢onne©tions to interphase nuclear functions The biochemical and genetic evidence suggests strongly that SMC fam-

ily members are major structural components of mitotic chromosomes and play a fundamental role in mitotic chromosome dynamics. Do they play any roles in interphase chromatin organization? Chicken Scll cofractionated with interphase nuclei, but leaked out of the nuclei readily during subcellular fractionation, suggesting that chromatin association of Scll was very weak in lnterphase cells, if it occurred at alP.Consistent with thisobservation, chromosomal targeting o[ XCAP-C and XCAP-E was mitosis specific in the Xenopus in vi~,o system i°. Genetic

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Pathway of mitotic chromosome assembly from sI~rm chromatin in Xenopusegg extracts. Sperm chromatin exhibits a snake-like compact shape. Upon incubation with a mitotic extract, the compact template chromatin rapidly swells and undergoes local condensation. Further incubation induces the formation of entangled, prophase-like chromatin fibers that eventually resolve into highly condensed individual chromosomes~. Since no DNA replication occurs in this assay, the assembled chromosomes consist of single chromatids. Immunodepletion of topoisomerase Ila or addition of antibodies agai2noSttopoisomerase Ila (anti-tope Ii) blocks an early stage of chromosome assemblywhere entangled chromatin fibers need to be resolved , whereas anti-XCAP-Cblocks a late stage of assembly1°.

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TSBS 2 0 - SEPTEMBER 1995 studies also showed that SMC function is not required for DNA re#ication and only becomes essential for ceil viability during mitosisT,8,~L Two lines of evidence from S. pombe, however, suggested a possible requirement for SMC function in interphase nuclear organizationS: (1) cut3Misrupted cetts exhibited an aberrant, swollen morphology of interphase chromatin; (2) a cut~ mutation, when combined with a topoisomerase I mutation (topl), showed a phenotype indicative of defective organization of interphase chromatin (single cut3 and topi mutants did not show such a phenotype). Furthermore, a genetic study in Caenorhabditis e~egans revealed that DPY-27, another member of the SMC family, is involved in dosage compensation, a process that specifically downregulates transcript levels of X-linked genes to compensate for differences in their copy number between the sexes ~-7.Importantly, DPY27 is not required for mitotic chromosome segregation, and the mitotic function of SMC proteins is thought to be provided by other family members. These results indicate that novel nuclear functions, perhaps linked to higherorder chromosome structures, may be found in the existing and/or unidentified members of the SMC family. $M8 proteins: m0iecuiarstructure afl~ funoti0n Members of the SMC family (SMC proteins) share a common 'head-rodtail' organization 7-1z,z7 (Fig. 3): the amino-terminal 'head' contains a nucleotide-binding motif (the P-loop motif) and the carboxy-terminal 'tail' contains a unique conserved sequence termed the DA box [because it is rich in aspartate (D) and alanine (A)]. The central 'rod' consists of two long c~-helical coiled-coil regions, which are connected by a nonhelical 'hinge' sequence. Although the function of the DA box is unknown, recent sequence analysis has identified related sequences in other ATP-binding proteins, and suggested that the motif, together with the P-loop motif, might be involved in ATP hydrolysis9'28. On the basis of sequence identities, SMC family members can be further classified into at least two subfamilies: subfamily I comprises Smclp, cut3 and XCAP-C, whereas subfamily II includes Smc2p, cutl4, XCAP-E and Scll (Fig. 3). In Xenopus egg extracts, XCAP-C and ]{CAPE exist as a heterodimer (XCAP-C-E), as demonstrated by co-

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Figure 2 Two fluorescent in situ hybridization (RSH) assays for chromosome condensation in yeasts. (a) A collectioa of hybridization probes (shown by the small black boxes) are used to 'paint' a whole arm of a particular chromosome. A rod-shaped RSH signal (red) in the interphase nucleus is converted into a small sphere during mitosis, demonstrating an aspect of mitotic chromosome condensation. In cut3 and cut14 mutants, this conversion is greatly impaired. (b) Two hybridization probes (also shown by the small black boxes) are used to mark two spots in the nucleus. In a wild-typecell, the distance between the two spots is significantly shorter i~ mitosis than in interphase. When a temperature-sensitive smc2 mutant is first arrested during mitosis at the permissive temperature and then transferred to the restrictive one (to inactivate Smc2p), the mitotic distance significantly increases, suggesting a requirement of Smc2p function for the maintenance of the condensed state.

immunoprecipitation and biochemical cofractionation ~°. In S. cerevisiae, Smc2p associates with other Smc2p molecules as well as Smclp, indicating the formation of homo- and heterodimers or a larger oligomer u. This result may provide the first evidence in support of the self-assembly model, in which SMC proteins might self-assemble to form a filamentous structure that determines the rod shape of mitotic chromosomes ~°. The assembly of a higher-order structure from heteromeric coiled-coil proteins is reminiscent of intermediate filaments or yeast bud-neck filaments. An alternative model for SMC function, the so-called chromatin motor model, was proposed on the basis of the overall structural similarity between SMC proteins and known mechanochemical proteins 7. Such motor activity could drive condensation by reeling in or twisting a chromatin fiber. Whatever the mechanism may be, it is most likely that a putative ATp-binding/hydrolysis cycle

of SMC proteins plays a key role in the dynamic organization of mitotic chromosomes. SMC homologs are also conserved in non-eukaryotic species 7, suggesting that the primary target of these proteins might be DNA itself, rather than eukaryote-specific chromatin components. Recent purification of XCAPC-E from Xenopus eggs has shown that this protein interacts directly with DNA in a cooperative fashion (3". Hirano, unpublished).

Possible interaction with topoisomerases What kinds of proteins interact funco

tionaily with SMC proteins? Genetic interactions have been reported between cut3 and topoisomerases in S. pombeS: (1) overexpression of the gone encoding topoisomerase 1 (top19 partially suppressed temperature-sensitivity of a cut3 mutation; (2) the combination of cut3ts and cold-sensitive top2 (top2CO was lethal at any temperature;

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Rgure 3 Overall structures of SMC proteins. Members of the two subfamilies share a common 'head=rod=taiV structura6 organization. The centrag 'rod' domain, comprising the coiled-coil motif (blue), is interrupted by a nonhelical 'h,nge' sequence. The three homoaogous regions between subfamily I and subfamily II are shown in red. The positions of the nucleotide-binding (P-loop) motif and the DA box are shown by the black boxes. Some members of subfamily I (cut3 and XCAP-C)have an additional sequence in their amino termini.

(3) a top:-cut3 double mutant showed disorganized chromatin morphology, which was reminiscent of topl-top2 double mutants. On the basis of these results, it was proposed that cut3 might be involved in the regulation of chromosomal DNA topology. In addition, two lines of biochemical evidence suggested that Scli and topoisomerase II might interact physically: (1) Scll and topoisomerase il (SoD are major components of the chromosome scaffold fraction 13 and colocalize in spread mitotic chromosomesg; (2) the two polypeptides copuri[y in a sequence-specific DNA-blnding protein complex called UB2 (Re[. 29). However, in Xenopus extracts, no evidence has been found for physical interaction between topoisomerase i| and XCAP-C-E before they are targeted to chromosomes: the two proteins do not coimmunoprecipltate ~° and their chromosomal targeting appears to occur independently (T. Hirano, unpublished). Furthermore, the two proteins appear to have distinct functions in chromosome assembly, as revealed by the in vitro functional assay 1°,2°.Clearly, more analyses must be done both with purified proteins and in the context of assembled chromosomes.

k~dtdlon andc o m ~ : two d~tln~ in mb~© ©hmm~e ~ b l y In principle, mitotic chromosome assembly can be dissected into two distinct processes, as suggested previously~. Rrst is the resolution process, in which entangled chromatin fibers are resolved into individual chromosome units. This is followed by the compaction process, in which resolved fibers condense further cad

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eventually produce rod-shaped chromosomes. In normal chromosome assembly, the two processes are temporally and spatially coordinated, and cannot be separated (Fig. 4). It is very important, however, to distinguish between them when considering mechanistic aspects of chromosome assembly. Perhaps a tangle of chromatin fibers sterically blocks compaction, and there must be a certain amount of resolution before compaction is initiated. Conversely, as compaction proceeds, then resolution is likely to become more efficient. This notion might, at least in part, explain why topotsomerase II activity is required for apparently different 'stages' of condensation when assayed with different methods or systems~9"2L3LFor example, somatic and sperm nuclei produce seemingly distinct condensation intermediates in Xenopus egg extracts depleted of topoisomerase II: in the absence of this enzyme, erythrocyte nuclei form partially resolved, swollen chromosomes 19 whereas sperm nuclei show no indication of chromosome formation 2°. Maybe the chromatin fibers in the sperm nuclei are more tangled than those in the erythrocyte nuclei, and therefore fail to undergo compaction without any resolution reaction (Fig. 4). If this is true, then the nature of the chromatin substrate is crucial to interpret the results of the condensation assays. There is a growing consensus that topolsomerase II activity is required for the resolution process, but whether this enzyme is also involved in compaction remains an open question s,e.~o. Topoisomerase II activity has also been

speculated to stabilize a condensed state by facilitating local catenation o[ chromatin fibers 32. On the basis of the antibody-blocldng experiment in the Xenopus in vi~'o system TM,SMC function is most likely to be required for the compaction process. The mechanism by which SMC proteins drive or regulate this process has not yet been determined. Future biochemical analyses should elucidate their molecular activity and test the validity of the two models proposed for SMC functions 7'~°'28.

Condudlngremarks The unification of biochemical and genetic approaches has led to the identification of SMC proteins that play an essential function in mitotic chromosome condensation, and sequence analyses of SMC proteins have provided some insights into their potential mechanisms o[ action. SMC proteins might act as 'chromosome compaction proteins' whose activity is continuously required for the structural maintenance of chromosomes - a role distinct from that o[ topoisomerase I|, whose primary function is to decatenate (or catenate) chromatin fibers. This is an oversimplified view since compaction is unlikely to proceed by a single mechanism. However, having these two major chromosomal components in our hands, we are now in a good position to explore the generation and maintenance of higher-order chromosome structure at a molecular level. Reconstitution of chromosomes from purified proteins in vilro, combined with high-resolution structural analysis in si~, should help us to re-evaluate the existing models for mitotic chromosome architecture.

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F~gure 4 Coordination of resolution and compaction in mitotic chromosome assembly. Mitotic chromosome assembly can be dissected into two distinct processes, resolution [downward; steps (a) and {©)] and compaction [rightward; steps (b) and (d}]. In a simplified model topoisomerase H and SMC proteins are primarily respondbie for resoOution and compaction, respectively. On normal chromosome assembly, the two processes are temporally and spatially coordinated [from (a) to (d), or (b) to (d)]: resolution of chromatin fibers facilitates the compaction process whereas the progression of compaction increases the efficiency of resolution. The initial tangled state of chromatin substrates also affects the coordination. For example, if chromatin fibers are heavily tangled (such as in sperm nuclei), the initiation of compaction is completely blocked without topoisomerase il activity 2°. if the fibers are Hghtly tangled (such as in somatic nuclei), partial compaction may proceed (b) even without prior resolution 19. The partially compacted, but not fully resolved, structures are also observed in yeast21 or tissue culture cells 3~ that lack topoisomerase H activity. For simplicity, two chromosomes, each consisting of a single chromatid, are shown. The ellipse represents the nuclear envelope.

A©kn0wledgemenL~ I thank all the colleagues who contributed to the identification of the 5MC family, particularly A. Strunnikov, D. Koshland, P-T. Chuang, B. Meyer, W. Earnshaw and M. Yanagida for sharing their unpublished information. I am also especially grateful to T. J. Mitchison for his long-standing generous support and stimulating discussions. J. Swedlow made valuable comments on the manuscript. I am a Leukemia Society of America Special Fellow.

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