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The second international meeting on “SMC proteins: Chromosomal Organizers from Bacteria to Human” (SMC2017) was held in Nanyo City, Yamagata, Japan, from 13 to 16 June 2017. The meeting was attended by 134 participants (among them, 76 from outside of Japan) who were interested in one of the highly conserved classes of chromosomal proteins regulating large-scale chromosome structure and function. A keynote lecture was followed by 41 oral presentations and 71 poster presentations in the four-day meeting. Diverse topics surrounding eukaryotic SMC protein complexes (cohesins, condensins and SMC5/6) and prokaryotic SMCs, and a wide range of cutting-edge approaches (from polymer physics through medical genetics) were presented. Dominant themes discussed in the meeting included mechanistically how the SMC protein complexes might form chromatin loops and domains. The participants enjoyed both exciting debate about chromosome organization and warm welcome offered by local people in a small city located in the northern part of Japan.

The mitotic chromosome is a macromolecular assembly that ensures error-free transmission of the genome during cell division. It has long been a big mystery how long stretches of DNA might be folded into rod-shaped chromosomes or how such an elaborate process might be accomplished at a mechanistic level. Cell-free extracts made from frog eggs offer a unique opportunity to address these questions by enabling mitotic chromosomes to be assembled in a test tube. Moreover, the core part of the chromosome assembly reaction can now be reconstituted with a limited number of purified factors. A combination of these in vitro assays makes it possible not only to prepare a complete list of proteins required for chromosome assembly but also to dissect functions of individual proteins and their cooperation with unparalleled clarity. Emerging lines of evidence underscore the paramount importance of condensins in building mitotic chromosomes and shed new light on the functional cross talk between nucleosomes and condensins in this process.

Molecular dynamics simulation is a powerful tool used in modern molecular modeling, which enables a deeper comprehension of the physical behavior of atoms and molecules at a micro level. In this study, we simulated mitotic chromosome assembly mediated by condensins, a class of large protein complexes containing a pair of structural maintenance of chromosomes (SMC) subunits that are central to this process. In this chapter, we present the construction of a coarse-grained physical model of chromosomal DNA fibers and condensin molecules, and monitoring of the function of condensins in mitotic chromosome assembly, using computer-based molecular dynamics simulation. We explain how our model of chromosomes and condensins may be simulated using a package of molecular dynamics simulation. Procedures involved in calculating the observables of dynamics are described, together with an example of the simulation results.

Condensins are large protein complexes that play a central role in chromosome organization and segregation in the three domains of life. They display highly characteristic, rod-shaped structures with SMC (structural maintenance of chromosomes) ATPases as their core subunits and organize large-scale chromosome structure through active mechanisms. Most eukaryotic species have two distinct condensin complexes whose balanced usage is adapted flexibly to different organisms and cell types. Studies of bacterial condensins provide deep insights into the fundamental mechanisms of chromosome segregation. This Review surveys both conserved features and rich variations of condensin-based chromosome organization and discusses their evolutionary implications.

Although condensins play essential roles in mitotic chromosome assembly, Ki-67, a protein localizing to the periphery of mitotic chromosomes, had also been shown to make a contribution to the process. To examine their respective roles, we generated a set of HCT116-based cell lines expressing Ki-67 and/or condensin subunits that were fused with an auxin-inducible degron for their conditional degradation. Both the localization and the dynamic behavior of Ki-67 on mitotic chromosomes were not largely affected upon depletion of condensin subunits, and vice versa. When both Ki-67 and SMC2 (a core subunit of condensins) were depleted, ball-like chromosome clusters with no sign of discernible thread-like structures were observed. This severe defective phenotype was distinct from that observed in cells depleted of either Ki-67 or SMC2 alone. Our results show that Ki-67 and condensins, which localize to the external surface and the central axis of mitotic chromosomes, respectively, have independent yet cooperative functions in supporting the structural integrity of mitotic chromosomes.List of Abbreviations usedAIDauxin-inducible degronDOXdoxycyclineFRAPfluorescence recovery after photobleachingIAAindol-3-acetic acidmAClmAID-mClovermAChmAID-mCherryNEBDnuclear envelope breakdownSMCstructural maintenance of chromosomesSTLCS-Trityl-L-cysteinetopo IIαtopoisomerase IIα

A new study uses a Hi-C technique to demonstrate that condensin has a major role in remodeling interphase chromatin into mitotic chromosomes. This study provides insight into the mechanism whereby a centimeters-long DNA molecule is folded into a micrometers-long rod-shaped chromosome.

Spatiotemporal dynamics of condensins I and II in the primitive red alga Cyanidioschyzon merolae is surprisingly similar to that observed in mammalian cells. Condensin II is not essential for mitosis under laboratory growth conditions but is required for sister centromere resolution in the presence of a microtubule drug., Condensins are multisubunit complexes that play central roles in chromosome organization and segregation in eukaryotes. Many eukaryotic species have two different condensin complexes (condensins I and II), although some species, such as fungi, have condensin I only. Here we use the red alga Cyanidioschyzon merolae as a model organism because it represents the smallest and simplest organism that is predicted to possess both condensins I and II. We demonstrate that, despite the great evolutionary distance, spatiotemporal dynamics of condensins in C. merolae is strikingly similar to that observed in mammalian cells: condensin II is nuclear throughout the cell cycle, whereas condensin I appears on chromosomes only after the nuclear envelope partially dissolves at prometaphase. Unlike in mammalian cells, however, condensin II is confined to centromeres in metaphase, whereas condensin I distributes more broadly along arms. We firmly establish a targeted gene disruption technique in this organism and find, to our surprise, that condensin II is not essential for mitosis under laboratory growth conditions, although it plays a crucial role in facilitating sister centromere resolution in the presence of a microtubule drug. The results provide fundamental insights into the evolution of condensin-based chromosome architecture and dynamics.

The SMC-ScpAB complex plays a crucial role in chromosome organization and segregation in many bacteria. It is composed of a V-shaped SMC dimer and an ScpAB subcomplex that bridges the two Structural Maintenance of Chromosomes (SMC) head domains. Despite its functional significance, the mechanistic details of SMC-ScpAB remain obscure. Here we provide evidence that ATP-dependent head-head engagement induces a lever movement of the SMC neck region, which might help to separate juxtaposed coiled-coil arms. Binding of the ScpA N-terminal domain (NTD) to the SMC neck region is negatively regulated by the ScpB C-terminal domain. Mutations in the ScpA NTD compromise this regulation and profoundly affect the overall shape of the complex. The SMC hinge domain is structurally relaxed when free from coiled-coil juxtaposition. Taken together, we propose that the structural parts of SMC-ScpAB are subjected to the balance between constraint and relaxation, cooperating to modulate dynamic conformational changes of the whole complex.

Cell-free extracts made from Xenopus laevis eggs enable us to recapitulate many chromosomal events associated with cell cycle progression in a test tube. When sperm chromatin is incubated with these extracts, it is first duplicated within an assembled nucleus, and is then transformed into mitotic chromosomes, in each of which sister chromatids are juxtaposed with each other in a cohesin-dependent manner. Here we describe our protocols for assembling duplicated chromosomes using egg extracts, along with cytological and biochemical assays for addressing the molecular mechanisms of sister chromatid cohesion. A powerful approach involving immunodepletion of cohesin and its regulators is also included.

Cellular proteins do not work in isolation. Instead, they often function as part of large macromolecular complexes, which are transported and concentrated into specific cellular compartments and function in a highly crowded environment. A central theme of modern cell biology is to understand how such macromolecular complexes are assembled efficiently and find their destinations faithfully. In this Opinion article, we will focus on HEAT repeats, flexible arrays of amphiphilic helices found in many eukaryotic proteins, such as karyopherins and condensins, and discuss how these uniquely designed helical repeats might underlie dynamic protein–protein interactions and support cellular functions in crowded environments. We will make bold speculations on functional similarities between the action of HEAT repeats and intrinsically disordered regions (IDRs) in macromolecular phase separation. Potential contributions of HEAT–HEAT interactions, as well as cooperation between HEATs and IDRs, to mesoscale organelle assembly will be discussed.

In mouse oocytes, condensin I localizes around centromeric regions, whereas condensin II is concentrated onto chromatid axes of Meta-I bivalent chromosomes. Both condensins are required for many aspects of meiotic chromosome dynamics, including individualization, resolution, and segregation, as well as monopolar attachment of sister kinetochores., In many eukaryotes, condensins I and II associate with chromosomes in an ordered fashion during mitosis and play nonoverlapping functions in their assembly and segregation. Here we report for the first time the spatiotemporal dynamics and functions of the two condensin complexes during meiotic divisions in mouse oocytes. At the germinal vesicle stage (prophase I), condensin I is present in the cytoplasm, whereas condensin II is localized within the nucleus. After germinal vesicle breakdown, condensin II starts to associate with chromosomes and becomes concentrated onto chromatid axes of bivalent chromosomes by metaphase I. REC8 “glues” chromosome arms along their lengths. In striking contrast to condensin II, condensin I localizes primarily around centromeric regions at metaphase I and starts to associate stably with chromosome arms only after anaphase I. Antibody injection experiments show that condensin functions are required for many aspects of meiotic chromosome dynamics, including chromosome individualization, resolution, and segregation. We propose that the two condensin complexes play distinctive roles in constructing bivalent chromosomes: condensin II might play a primary role in resolving sister chromatid axes, whereas condensin I might contribute to monopolar attachment of sister kinetochores, possibly by assembling a unique centromeric structure underneath.

To understand how chromosome shapes are determined by actions of condensins and cohesin, we devised a series of protocols in which their levels are precisely changed in Xenopus egg extracts. When the relative ratio of condensin I to II is forced to be smaller, embryonic chromosomes become shorter and thicker, being reminiscent of somatic chromosomes. Further depletion of condensin II unveils its contribution to axial shortening of chromosomes. Cohesin helps juxtapose sister chromatid arms by collaborating with condensin I and counteracting condensin II. Thus, chromosome shaping is achieved by an exquisite balance among condensin I and II and cohesin.

MCPH1, a protein linked to primary microcephaly, directly modulates condensin II to regulate chromosome condensation and shape., Mutations in human MCPH1 (hMCPH1) cause primary microcephaly, which is characterized by a marked reduction of brain size. Interestingly, hMCPH1 mutant patient cells display unique cellular phenotypes, including premature chromosome condensation (PCC), in G2 phase. To test whether hMCPH1 might directly participate in the regulation of chromosome condensation and, if so, how, we developed a cell-free assay using Xenopus laevis egg extracts. Our results demonstrate that an N-terminal domain of hMCPH1 specifically inhibits the action of condensin II by competing for its chromosomal binding sites in vitro. This simple and powerful assay allows us to dissect mutations causing primary microcephaly in vivo and evolutionary substitutions among different species. A complementation assay using patient cells revealed that, whereas the N-terminal domain of hMCPH1 is sufficient to rescue the PCC phenotype, its central domain plays an auxiliary role in shaping metaphase chromosomes by physically interacting with condensin II. Thus, hMCPH1 acts as a composite modulator of condensin II to regulate chromosome condensation and shaping.

The mechanistic details underlying the assembly of rod-shaped chromosomes during mitosis and how they segregate from each other to act as individually mobile units remain largely unknown. Here, we construct a coarse-grained physical model of chromosomal DNA and condensins, a class of large protein complexes that plays key roles in these processes. We assume that condensins have two molecular activities: consecutive loop formation in DNA and inter-condensin attractions. Our simulation demonstrates that both of these activities and their balancing acts are essential for the efficient shaping and segregation of mitotic chromosomes. Our results also demonstrate that the shaping and segregation processes are strongly correlated, implying their mechanistic coupling during mitotic chromosome assembly. Our results highlight the functional importance of inter-condensin attractions in chromosome shaping and segregation., Author summary Immediately before a cell divides, chromosomal DNA in a eukaryotic cell is packaged into a discrete set of rod-shaped chromosomes. This process, known as mitotic chromosome assembly or condensation, secures the faithful segregation of genetic information into daughter cells. Central to this mechanistically complex process is a class of protein complexes known as condensins. However, how condensins support the assembly and segregation of mitotic chromosomes at a mechanistic level remains elusive. Here we construct a coarse-grained physical model of chromosomal DNA fibers and condensin molecules, and study how condensins work in the mitotic chromosome assembly using computer simulations. Our results show that two activities of condensins, formation of consecutive loops in chromosomal DNA fibers and inter-condensin attractions, are necessary for both the shaping and segregation of mitotic chromosomes, and balancing acts of these activities help to coordinate the efficient progress of the processes. Importantly, chromosome shaping and segregation in our results are strongly correlated, implying that they are controlled by the same underlying mechanism mediated by condensins.

The mitotic chromosome, which is composed of a pair of sister chromatids, is a large macromolecular assembly that ensures faithful transmission of genetic information into daughter cells. Despite its fundamental importance, how a nucleosome fiber is folded and assembled into a large-scale chromatid structure remains poorly understood. To address this question, we have established a biochemically tractable system in which mitotic chromatids can be reconstituted in vitro by mixing a simple substrate (sperm nucleus) and a limited number of purified factors. The minimum set of required factors includes core histones, three histone chaperones, topoisomerase II, and condensin I. In this article, we describe a set of protocols for the preparation of key reagents and the setup of reconstitution reactions. We believe that this classical approach of biochemical reconstitution will be of great help to dissect the mechanisms of action of individual factors during mitotic chromatid assembly and to assess the contribution of nucleosome dynamics to this process from a fresh angle. © 2018 by John Wiley & Sons, Inc.

When chromosomes start to assemble in mitotic prophase, duplicated chromatids are not discernible within each chromosome. As condensation proceeds, they gradually show up, culminating in two rod-shaped structures apposed along their entire length within a metaphase chromosome. This process, known as sister chromatid resolution, is thought to be a prerequisite for rapid and synchronous separation of sister chromatids in anaphase. From a mechanistic point of view, the resolution process can be dissected into three distinct steps: (1) release of cohesin from chromosome arms; (2) formation of chromatid axes mediated by condensins; and (3) untanglement of inter-sister catenation catalyzed by topoisomerase II (topo II). In this review article, we summarize recent progress in our understanding the molecular mechanisms of sister chromatid resolution with a major focus on its first step, cohesin release. An emerging idea is that this seemingly simple step is regulated by an intricate network of positive and negative factors, including cohesin-binding proteins and mitotic kinases. Interestingly, some key factors responsible for cohesin release in early mitosis also play important roles in controlling cohesin functions during interphase. Finally, we discuss how the step of cohesin release might mechanistically be coordinated with the actions of condensins and topo II.

Mitotic chromosomal assembly in vertebrates is regulated by condensin I and condensin II, which work cooperatively but have different chromosomal localization profiles and make distinct mechanistic contributions to this process. We show here that protein phosphatase 2A (PP2A), which interacts with condensin II but not condensin I, plays an essential role in targeting condensin II to chromosomes. Unexpectedly, our data indicate that PP2A acts as a recruiter protein rather than a catalytic enzyme to target condensin II to chromosomes. This recruiting activity of PP2A was inhibited by okadaic acid, but not by fostriecin, even though both molecules strongly inhibited the catalytic activity of PP2A. Additionally, we found that the chromokinesin KIF4a is also targeted to chromosomes via the noncatalytic activity of PP2A. Thus, our studies reveal a previously unknown contribution of PP2A to chromosome assembly.

Chromosome condensation is a hallmark of mitosis in eukaryotes and is a prerequisite for faithful segregation of genetic material to daughter cells. Here we show that condensin, which is essential for assembling condensed chromosomes, helps to preclude the detrimental effects of gene transcription on mitotic condensation. ChIP-seq profiling reveals that the fission yeast condensin preferentially binds to active protein-coding genes in a transcription-dependent manner during mitosis. Pharmacological and genetic attenuation of transcription largely rescue bulk chromosome segregation defects observed in condensin mutants. We also demonstrate that condensin is associated with and reduces unwound DNA segments generated by transcription, providing a direct link between an in vitro activity of condensin and its in vivo function. The human condensin isoform condensin I also binds to unwound DNA regions at the transcription start sites of active genes, implying that our findings uncover a fundamental feature of condensin complexes., Chromosome condensation is a prerequisite for faithful segregation of chromosomes to daughter cells. Here, the authors show that the condensin complex binds to protein-coding genes in a transcription-dependent manner during condensation, and reduces unwound DNA segments generated by transcription.

In addition to inter-chromatid cohesion, mitotic and meiotic chromatids must have three physical properties: compaction into 'threads' roughly co-linear with their DNA sequence, intra-chromatid cohesion determining their rigidity, and a mechanism to promote sister chromatid disentanglement. A fundamental issue in chromosome biology is whether a single molecular process accounts for all three features. There is universal agreement that a pair of Smc-kleisin complexes called condensin I and II facilitate sister chromatid disentanglement, but whether they also confer thread formation or longitudinal rigidity is either controversial or has never been directly addressed respectively. We show here that condensin II (beta-kleisin) has an essential role in all three processes during meiosis I in mouse oocytes and that its function overlaps with that of condensin I (gamma-kleisin), which is otherwise redundant. Pre-assembled meiotic bivalents unravel when condensin is inactivated by TEV cleavage, proving that it actually holds chromatin fibres together.

Structural maintenance of chromosomes (SMC) proteins are ubiquitous in organisms from bacteria to humans, and function as core components of the condensin and cohesin complexes in eukaryotes. SMC proteins adopt a V-shaped structure with two long arms, each of which has an ATP-binding head domain at the distal end. It is important to understand how these uniquely designed protein machines interact with DNA strands and how such interactions are modulated by the ATP-binding and -hydrolysis cycle. An emerging idea is that SMC proteins use a diverse array of intramolecular and intermolecular protein-protein interactions to actively fold, tether and manipulate DNA strands.

Condensins are multi-subunit protein complexes that play a central role in mitotic chromosome assembly and segregation. The complexes contain ‘structural maintenance of chromosomes’ (SMC) ATPase subunits, and induce DNA supercoiling and looping in an ATP-hydrolysis-dependent manner in vitro. Vertebrate cells have two different condensin complexes, condensins I and II, each containing a unique set of regulatory subunits. Condensin II participates in an early stage of chromosome condensation within the prophase nucleus. Condensin I gains access to chromosomes only after the nuclear envelope breaks down, and collaborates with condensin II to assemble metaphase chromosomes with fully resolved sister chromatids. The complexes also play critical roles in meiotic chromosome segregation and in interphase processes such as gene repression and checkpoint responses. In bacterial cells, ancestral forms of condensins control chromosome dynamics. Dissecting the diverse functions of condensins is likely to be central to our understanding of genome organization, stability and evolution.

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIα and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150–200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200–300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial “glue.”

Background: Condensin is thought to contribute to large-scale DNA compaction during mitotic chromosome assembly. It remains unknown, however, how the complex reconfigures DNA structure at a mechanistic level. Results: We have performed single-molecule DNA nanomanipulation experiments to directly measure in real-time DNA compaction by the Xenopus laevis condensin I complex. Condensin can bind to the nanomanipulated DNA in the absence of ATP, but it compacts the DNA only in the presence of hydrolyzable ATP. Linear compaction is evidenced by a reduction in the end-to-end extension of nanomanipulated DNA. The reaction results in total compaction of the DNA (i.e., zero end-to-end extension). Discrete and reversible DNA compaction events are observed in the presence of competitor DNA when the DNA is subjected to weak stretching forces ( F = 0.4 picoNewton [pN]). The distribution of step sizes is broad and displays a peak at ∼60 nm (∼180 bp) as well as a long tail. This distribution is essentially unaffected by the topological state of the DNA substrate. Increasing the force to F = 10 pN drives the system toward step-wise reversal of compaction. The distribution of step sizes observed upon disruption of condensin-DNA interactions displays a sharp peak at ∼30 nm (∼90 bp) as well as a long tail stretching out to hundreds of nanometers. Conclusions: The DNA nanomanipulation assay allows us to demonstrate for the first time that condensin physically compacts DNA in an ATP-hydrolysis-dependent manner. Our results suggest that the condensin complex may induce DNA compaction by dynamically and reversibly introducing loops along the DNA.

The canonical condensin complex (henceforth condensin I) plays an essential role in mitotic chromosome assembly and segregation from yeast to humans. We report here the identification of a second condensin complex (condensin II) from vertebrate cells. Condensins I and II share the same pair of structural maintenance of chromosomes (SMC) subunits but contain different sets of non-SMC subunits. siRNA-mediated depletion of condensin I- or condensin II-specific subunits in HeLa cells produces a distinct, highly characteristic defect in chromosome morphology. Simultaneous depletion of both complexes causes the severest defect. In Xenopus egg extracts, condensin I function is predominant, but lack of condensin II results in the formation of irregularly shaped chromosomes. Condensins I and II show different distributions along the axis of chromosomes assembled in vivo and in vitro. We propose that the two condensin complexes make distinct mechanistic contributions to mitotic chromosome architecture in vertebrate cells.

Condensin, a five-subunit protein complex essential for mitotic chromosome condensation from yeast to humans, introduces positive supercoils into DNA in an ATP-dependent manner in vitro. We report here the direct visualization of this supercoiling reaction by electron spectroscopic imaging. In the presence of ATP, a single condensin complex is capable of introducing two or more compensatory supercoils into the protein-free region of a closed circular DNA. Within the condensin-bound region, approximately 190 bp of DNA is organized into a compact structure with two distinct domains, indicative of the formation of two oriented gyres. The current results suggest that the action of condensin is more dynamic and more efficient than that postulated before, providing fundamental insight into the energy-dependent mechanism of higher order chromatin folding.

A-kinase (or PKA)-anchoring protein AKAP95 is a zinc-finger protein implicated in mitotic chromosome condensation by acting as a targeting molecule for the condensin complex. We have identified determinants of chromatin-binding, condensin-targeting and chromosome-condensation activities of AKAP95. Binding of AKAP95 to chromatin is conferred by residues 387–450 and requires zinc finger ZF1. Residues 525–569 are essential for condensation of AKAP95-free chromatin and condensin recruitment to chromosomes. Mutation of either zinc finger of AKAP95 abolishes condensation. However, ZF1 is dispensable for condensin targeting, whereas the C-terminal ZF2 is required. AKAP95 interacts with Xenopus XCAP-H condensin subunit in vitro and in vivo but not with the human hCAP-D2 subunit. The data illustrate the involvement of overlapping, but distinct, domains of AKAP95 for condensin recruitment and chromosome condensation and argue for a key role of ZF1 in chromosome condensation and ZF2 in condensin targeting. Moreover, condensin recruitment to chromatin is not sufficient to promote condensation.

Structural maintenance of chromosomes (SMC) proteins play central roles in higher-order chromosome dynamics from bacteria to humans. In eukaryotes, two different SMC protein complexes, condensin and cohesin, regulate chromosome condensation and sister chromatid cohesion, respectively. Each of the complexes consists of a heterodimeric pair of SMC subunits and two or three non-SMC subunits. Previous studies have shown that a bacterial SMC homodimer has a symmetrical structure in which two long coiled-coil arms are connected by a flexible hinge. A catalytic domain with DNA- and ATP-binding activities is located at the distal end of each arm. We report here the visualization of vertebrate condensin and cohesin by electron microscopy. Both complexes display the two-armed structure characteristic of SMC proteins, but their conformations are remarkably different. The hinge of condensin is closed and the coiled-coil arms are placed close together. In contrast, the hinge of cohesin is wide open and the coiled-coils are spread apart from each other. The non-SMC subunits of both condensin and cohesin form a globular complex bound to the catalytic domains of the SMC heterodimers. We propose that the “closed” conformation of condensin and the “open” conformation of cohesin are important structural properties that contribute to their specialized biochemical and physiological functions.

We previously characterized major components of mitotic chromosomes assembled in Xenopus laevis egg extracts and collectively referred to them as Xenopuschromosome–associated polypeptides (XCAPs). They included five subunits of the condensin complex essential for chromosome condensation. In an effort to identify novel proteins involved in this process, we have isolated XCAP-F and found it to be theXenopus ortholog of ISWI, a chromatin remodeling ATPase. ISWI exists in two major complexes in Xenopus egg extracts. The first complex contains ACF1 and two low-molecular-weight subunits, most likely corresponding to Xenopus CHRAC. The second complex is a novel one that contains theXenopus ortholog of the human Williams syndrome transcription factor (WSTF). In the absence of the ISWI complexes, the deposition of histones onto DNA is apparently normal, but the spacing of nucleosomes is greatly disturbed. Despite the poor spacing of nucleosomes, ISWI depletion has little effect on DNA replication, chromosome condensation or sister chromatid cohesion in the cell-free extracts. The association of ISWI with chromatin is cell cycle regulated and is under the control of the INCENP-aurora B kinase complex that phosphorylates histone H3 during mitosis. Apparently contradictory to the generally accepted model, we find that neither chromosome condensation nor chromosomal targeting of condensin is compromised when H3 phosphorylation is drastically reduced by depletion of INCENP-aurora B.

During development of the cerebral cortex, neural stem cells (NSCs) divide symmetrically to proliferate and asymmetrically to generate neurons. Although faithful segregation of mitotic chromosomes is critical for NSC divisions, its fundamental mechanism remains unclear. A class of evolutionarily conserved protein complexes, known as condensins, is thought to be central to chromosome assembly and segregation among eukaryotes. Here we report the first comprehensive genetic study of mammalian condensins, demonstrating that two different types of condensin complexes (condensins I and II) are both essential for NSC divisions and survival in mice. Simultaneous depletion of both condensins leads to severe defects in chromosome assembly and segregation, which in turn cause DNA damage and trigger p53-induced apoptosis. Individual depletions of condensins I and II lead to slower loss of NSCs compared to simultaneous depletion, but they display distinct mitotic defects: chromosome missegregation was observed more prominently in NSCs depleted of condensin II, whereas mitotic delays were detectable only in condensin I-depleted NSCs. Remarkably, NSCs depleted of condensin II display hyperclustering of pericentric heterochromatin and nucleoli, indicating that condensin II, but not condensin I, plays a critical role in establishing interphase nuclear architecture. Intriguingly, these defects are taken over to postmitotic neurons. Our results demonstrate that condensins I and II have overlapping and non-overlapping functions in NSCs, and also provide evolutionary insight into intricate balancing acts of the two condensin complexes., Author Summary The cerebral cortex is built up of numerous neurons and cells supporting them, most of which originate from neural stem cells (NSCs). NSCs divide symmetrically to produce themselves and asymmetrically to generate neurons, and both types of divisions reply on faithful segregation of chromosomes into daughter cells. In the current study, we study the functions of evolutionarily conserved chromosome regulators, known as condensin I and condensin II, during development of the cerebral cortex in mice. We find that condensins I and II have both overlapping and non-overlapping functions in NSC divisions and survival: loss of either one of condensins causes distinct abnormalities in the process of chromosome segregation. Remarkably, loss of condensin II, but not of condensin I, also alters chromosome architecture during non-dividing stages. Our results demonstrate convincingly that an intricate balance between condensins I and II plays a crucial role in NSC divisions. It will be of great interest to test in the future whether such balancing acts of the two condensin complexes might be misregulated in tumorigenic NSCs that undergo uncontrolled cell divisions.

The establishment of sister chromatid cohesion during S phase and its dissolution at the metaphase-anaphase transition are essential for the faithful segregation of chromosomes in mitosis [1-4]. Recent studies in yeast genetics and Xenopus biochemistry have identified a large protein complex, cohesin, that plays a key role in sister chromatid cohesion [5-10]. The cohesin complex consists of a heterodimeric pair of SMC (structural maintenance of chromosomes) subunits and at least two non-SMC subunits. This structural organization is reminiscent of that of condensin, another major SMC protein complex that drives chromosome condensation in eukaryotic cells [11]. Condensin has been shown to reconfigure and compact DNA in vitro by utilizing the energy of ATP hydrolysis [12]. Very little is known, however, about how cohesin works at a mechanistic level. Here we report the first set of biochemical activities associated with an intact cohesin complex purified from HeLa cell extracts. The cohesin complex binds directly to double-stranded DNA and induces the formation of large protein-DNA aggregates. In the presence of topoisomerase II, cohesin stimulates intermolecular catenation of circular DNA molecules. This activity is in striking contrast to intramolecular knotting directed by condensin [13]. Cohesin also increases the probability of intermolecular ligation of linear DNA molecules in the presence of DNA ligase. Our results are consistent with a model in which cohesin functions as an intermolecular DNA crosslinker and is part of the molecular "glue" that holds sister chromatids together [14].

HEAT repeats correspond to tandemly arranged curlicue-like structures that appear to serve as flexible scaffolding on which other components can assemble. Using sensitive sequence analysis techniques we detected HEAT repeats in various chromosome-associated proteins, including four families of proteins associated with condensins and cohesins, which are nuclear complexes that contain structural maintenance of chromosome (SMC) proteins. Among the proteins detected were the XCAP-D2 and XCAP-G subunits of the Xenopus laevis 13S condensin complex, the Aspergillus BimD and Sordaria macrospora Spo76p proteins, the budding yeast Scc2p protein, and the related Drosophila transcriptional activator Nipped-B. Clathrin adaptor and COP-I coatomer subunits, which function in vesicle coat assembly and were previously noted to share weak sequence similarity to condensin subunits, also contain HEAT repeats. HEAT repeats were also found in the TBP-associated TIP120 protein, a global enhancer of transcription, and in the budding yeast Mot1p protein, which is a member of the SWI2/SNF2 family. SWI2/SNF2 proteins, some of which are helicases, perform diverse roles in transcription control, DNA repair, and chromosome segregation and form chromatin-remodeling complexes. HEAT repeats also were found in dis1-TOG family and cofactor D family microtubule-associated proteins, which, owing to their roles in microtubule dynamics, perform functions related to mitotic progression and chromosome segregation. Hence, our analysis predicts structural features of these proteins and suggests that HEAT repeats may play important roles in chromosome dynamics.

In this issue of Molecular Cell, St-Pierre et al. (2009) present evidence that, in budding yeast, the polo kinase Cdc5 directly phosphorylates and activates condensin to promote anaphase-specific rDNA condensation.

Xenopus 13S condensin converts interphase chromatin into mitotic-like chromosomes, and, in the presence of ATP and a type I topoisomerase, introduces (+) supercoils into DNA. The specific production of (+) trefoil knots in the presence of condensin and a type II topoisomerase shows that condensin reconfigures DNA by introducing an ordered, global, (+) writhe. Knotting required ATP hydrolysis and cell cycle-specific phosphorylation of condensin. Condensin bound preferentially to (+) supercoiled DNA in the presence of ATP but not in its absence. Our results suggest a mechanism for the compaction of chromatin by condensin during mitosis.

The structural maintenance of chromosomes (SMC) family is a growing family of chromosomal ATPases. The founding class of SMC protein complexes, condensins, plays a central role in mitotic chromosome condensation. We report here a new class of SMC protein complexes containing XSMC1 and XSMC3, Xenopus homologs of yeast Smc1p and Smc3p, respectively. The protein complexes (termed cohesins) exist as two major forms with sedimentation coefficients of 9S and 14S. 9S cohesin is a heterodimer of XSMC1 and XSMC3, whereas 14S cohesin contains three additional subunits. One of them has been identified as a Xenopus homolog of the Schizosaccharomyces pombeRad21p implicated in DNA repair and the Saccharomyces cerevisiae Scc1p/Mcd1p implicated in sister chromatid cohesion. 14S cohesin binds to interphase chromatin independently of DNA replication and dissociates from it at the onset of mitosis. Immunodepletion of cohesins during interphase causes defects in sister chromatid cohesion in subsequent mitosis, whereas condensation is unaffected. These results suggest that proper assembly of mitotic chromosomes is regulated by two distinct classes of SMC protein complexes, cohesins and condensins.

13S condensin is a five-subunit protein complex that plays a central role in mitotic chromosome condensation in Xenopus egg extracts. Two core subunits of this complex, XCAP-C and XCAP-E, belong to an emerging family of putative ATPases, the SMC family. We report here that 13S condensin has a DNA-stimulated ATPase activity and exhibits a high affinity for structured DNAs such as cruciform DNA. 13S condensin is able to introduce positive supercoils into a closed circular DNA in the presence of bacterial or eukaryotic topoisomerase I. The supercoiling reaction is ATP-dependent. We propose that 13S condensin wraps DNA in a right-handed direction by utilizing the energy of ATP hydrolysis. This reaction may represent a key mechanism underlying the compaction of chromatin fibers during mitosis.

We report here purification and characterization of chro- mosome condensation protein complexes (termed condensins) containing XCAP-C and XCAP-E, two Xenopus members of the SMC family. Sucrose density gradient centrifugation reveals two major forms of condensins. The 8S form is a heterodimer of XCAP-C and XCAP-E, whereas the 13S form contains three additional subunits. One of them is identified as a homolog of the Drosophila Barren protein whose mutation shows a defect in chromosome segregation. Chromosomal targeting of condensins is mitosis-specific and is independent of topoisomerase IIα. 13S condensin is required for condensation, as demonstrated by immunodepletion and rescue experiments. Our results suggest that the condensin complexes represent the most abundant structural components of mitotic chromosomes and play a central role in driving chromosome condensation.

Condensin II initiates structural reorganization of duplicated chromosomes during S phase to prepare for their proper condensation and segregation in mitosis., Condensins I and II are multisubunit complexes that play essential yet distinct functions in chromosome condensation and segregation in mitosis. Unlike condensin I, condensin II localizes to the nucleus during interphase, but it remains poorly understood what functions condensin II might have before mitotic entry. Here, we report that condensin II changes its chromatin-binding property during S phase. Remarkably, advanced premature chromosome condensation (PCC) assays enabled us to visualize condensin II forming “sister axes” in replicated regions of chromosomes in S phase cells. Depletion of condensin II compromised PCC-driven sister chromatid resolution during S phase. Moreover, fluorescence in situ hybridization assays revealed that condensin II, but not condensin I, promotes disjoining duplicated chromosomal loci during S phase. Application of mild replicative stress partially impaired this process and further exacerbated phenotypes arising from condensin II depletion. Our results suggest that condensin II initiates structural reorganization of duplicated chromosomes during S phase to prepare for their proper condensation and segregation in mitosis.

Condensins are multisubunit protein complexes that play a fundamental role in the structural and functional organization of chromosomes in the three domains of life. Most eukaryotic species have two different types of condensin complexes, known as condensins I and II, that fulfill nonoverlapping functions and are subjected to differential regulation during mitosis and meiosis. Recent studies revealed that the two complexes contribute to a wide variety of interphase chromosome functions, such as gene regulation, recombination, and repair. Also emerging are their cell type- and tissue-specific functions and relevance to human disease. Biochemical and structural analyses of eukaryotic and bacterial condensins steadily uncover the mechanisms of action of this class of highly sophisticated molecular machines. Future studies on condensins will not only enhance our understanding of chromosome architecture and dynamics, but also help address a previously underappreciated yet profound set of questions in chromosome biology.

In human diploid cells, 23 pairs of chromosomes become recognizable as a discrete set of rod-shaped entities during the mitotic stage of the cell cycle. Upon exit from mitosis, they decondense and are packed together into the nucleus, in which individual chromosomes are no longer discernible. Nonetheless, evidence accumulated during past decades suggests that the decondensed chromosomes are not randomly located within the nucleus, but instead occupy distinct subnuclear domains known as chromosome territories (CTs) [1]. This raises a number of obvious questions. What protein factor(s) is(are) involved in the formation of CTs? Is there any mechanistic link between mitotic chromosome structures and interphase chromosome territories? In this issue of PLOS Genetics, Bauer et al. provide a remarkable answer to these questions by demonstrating that condensin II, one of the major chromosome condensation factors in mitosis [2], also makes an important contribution to the formation of chromosome territories during interphase [3]. To address the question of how interphase chromosomes might be organized, the authors focused on nurse cells in the ovary of the fruit fly Drosophila melanogaster, which display a highly characteristic mode of chromosome organization and dynamics. The chromosomes in these cells are amplified by repeated rounds of DNA replication without intervening mitoses (i.e., endoduplication), giving rise to polytene chromosomes in which many copies of homologous chromosomes and chromatids are paired along their entire lengths. After the fifth cycle of endoduplication, however, the pairing of homologous chromatids is disrupted, allowing each set of chromosome arms to occupy a discrete globular territory that is reminiscent of a CT observed in mammalian cells. A previous study from the same group demonstrated that condensin II function is required for the disassembly of polytene chromosomes occurring at this stage [4]. In the current study, Bauer et al. followed the fate of these chromosomes further and found that CTs are not properly formed in the condensin II mutants [3]. The authors also noticed that chromosomes in these mutants maintain their initial orientation (known as the Rabl configuration) in which the centromeric regions of all chromosomes cluster near the nuclear periphery and the telomeres cluster near the opposite pole of the nucleus (Figure 1A). Figure 1 A model for how condensin II might contribute to the formation of chromosome territories in diploid cells. Why, then, do chromosomes fail to form CTs when condensin II function is impaired? An extensive set of fluorescence in situ hybridization (FISH) data suggested that condensin II promotes not only axial compaction of chromosomes, but also dispersal of heterologous centromeres. Importantly, the dispersed pericentromeric heterochromatin nonetheless stayed near the nuclear periphery. Conversely, when one of the condensin II–specific subunits (Cap-H2) was overexpressed in salivary glands, their polytene chromosomes were disassembled, leading to axial shortening of chromosomes and CT formation. Based on these data, the authors propose a simple and appealing model in which the combination of two events, condensin II–mediated axial compaction and sustained interactions of heterochromatic regions with the nuclear periphery, drives each chromosome into a globular CT (Figure 1B). They also suggest that intrachromosomal folding promoted by condensin II disfavors interchromosomal interactions, thereby contributing to the disassembly of polytene chromosomes. Although this is an interesting and plausible model, one could ask to what extent the insights obtained from the highly specialized polytene nuclei might be applicable to diploid nuclei. In Drosophila, unlike in other metazoans, homologous chromosomes are always paired in virtually all types of cells throughout development. It has long been known that the pairing of homologous chromosomes affects gene expression at a number of loci through a process known as transvection [5]. Notably, previous studies showed that condensin II antagonizes transvection in the imaginal wing disc [4], and also contributes to CT formation in diploid spermatocytes during meiosis [6]. Moreover, a more recent genome-wide screen identified condensin II subunits as factors that antagonize the pairing of homologous chromosomes in Drosophila tissue culture cells [7]. It will be of great importance to test whether condensin II–mediated axial shortening of chromosomes indeed underlies all these processes, although measurements of chromosome lengths are technically more challenging in diploid cells than in polyploid cells. The outcome of these emerging studies strongly implies that chromosome–chromosome interactions in cis (i.e., intrachromosomal folding) and those in trans (interchromosomal pairing) compete with each other. Interestingly, a similar principle has already been put forward to delineate the dynamic interaction between sister chromatids within a duplicated chromosome. Upon entry into mitosis, bulk cohesin is lost from chromatid arms, and condensin II coincidentally initiates intrachromatid folding. As a consequence, sister–sister interactions are loosened, leading to the formation of metaphase chromosomes in which rod-shaped sister chromatids are juxtaposed along their lengths, a process known as sister chromatid resolution [8]. A recent study using Xenopus laevis egg extracts has demonstrated that this process is indeed controlled by an intricate balance between the cohesin-mediated cohesive forces and condensin II–mediated resolving forces [9]. Moreover, condensin II has been shown to contribute to axial compaction of chromosomes during mitosis in Xenopus egg extracts [9] and in chicken DT40 cells [10], implying that condensin II can perform essentially the same job during interphase and mitosis. In summary, a unique and powerful experimental system, combined with a labor-intensive assay, enabled Bauer et al. to propose a model of how CTs might be formed in Drosophila interphase nuclei [3]. To what extent the current model might be applicable to organisms with more complex genomes remains to be determined. It is unlikely that condensin II is the sole factor contributing to CT formation. Searches for additional factors in different model systems would help advance our understanding of the organization of interphase chromosomes, and computational simulations could complement such efforts (e.g., [11]). The current work also reinforces the role of condensin II as one of the central organizers of chromosomes from interphase through mitosis. Future studies should be aimed at a mechanistic understanding of the fundamental principles of chromosome architecture and dynamics throughout the cell cycle as well as during the development of an organism.

Vertebrate cells possess two different condensin complexes, known as condensin I and condensin II, that play a fundamental role in chromosome assembly and segregation during mitosis. Each complex contains a pair of structural maintenance of chromosomes (SMC) ATPases, a kleisin subunit and two HEAT-repeat subunits. Here we use recombinant human condensin subunits to determine their geometry within each complex. We show that both condensin I and condensin II have a pseudo-symmetrical structure, in which the N-terminal half of kleisin links the first HEAT subunit to SMC2, whereas its C-terminal half links the second HEAT subunit to SMC4. No direct interactions are detectable between the SMC dimer and the HEAT subunits, indicating that the kleisin subunit acts as the linchpin in holocomplex assembly. ATP has little, if any, effects on the assembly and integrity of condensin. Cleavage pattern of SMC2 by limited proteolysis is changed upon its binding to ATP or DNA. Our results shed new light on the architecture and dynamics of this highly elaborate machinery designed for chromosome assembly.

The primary goal of mitosis is to partition duplicated chromosomes into daughter cells. Eukaryotic chromosomes are equipped with two distinct classes of intrinsic machineries, cohesin and condensins, that ensure their faithful segregation during mitosis. Cohesin holds sister chromatids together immediately after their synthesis during S phase until the establishment of bipolar attachments to the mitotic spindle in metaphase. Condensins, on the other hand, attempt to “resolve” sister chromatids by counteracting cohesin. The products of the balancing acts of cohesin and condensins are metaphase chromosomes, in which two rod-shaped chromatids are connected primarily at the centromere. In anaphase, this connection is released by the action of separase that proteolytically cleaves the remaining population of cohesin. Recent studies uncover how this series of events might be mechanistically coupled with each other and intricately regulated by a number of regulatory factors.

Autosomal recessive primary microcephaly (MCPH) is a neurodevelopmental disorder characterized by marked reduction in brain size and mental retardation. Mutations in the gene MCPH1, encoding microcephalin, cause MCPH and a unique cellular phenotype with premature chromosome condensation in early G2 phase and delayed decondensation post mitosis. Here, we show that in MCPH1 patient cells, siRNA-mediated depletions of condensin II subunits lead to a pronounced reduction of cells with the condensation defects in both G 1 and G2 phases of the cell cycle. Similar results are obtained when microcephalin and condensin II are simultaneously depleted in HeLa cells. In contrast, depletions of condensin I subunits do not reverse the cellular phenotype. Consistently, condensin I stays in the cytoplasm in the prophase-like cells of MCPH1 patients. Our results offer a molecular explanation for the aberrant chromosome condensation in MCPH1-deficiency and provide additional evidence that condensin I and II are regulated by distinct pathways. ©2006 Landes Bioscience.

Structural maintenance of chromosomes (SMC) proteins are chromosomal ATPases, highly conserved from bacteria to humans, that play fundamental roles in many aspects of higher-order chromosome organization and dynamics. In eukaryotes, SMC1 and SMC3 act as the core of the cohesin complexes that mediate sister chromatid cohesion, whereas SMC2 and SMC4 function as the core of the condensin complexes that are essential for chromosome assembly and segregation. Another complex containing SMC5 and SMC6 is implicated in DNA repair and checkpoint responses. The SMC complexes form unique ring- or V-shaped structures with long coiled-coil arms, and function as ATP-modulated, dynamic molecular linkers of the genome. Recent studies shed new light on the mechanistic action of these SMC machines and also expanded the repertoire of their diverse cellular functions. Dissecting this class of chromosomal ATPases is likely to be central to our understanding of the structural basis of genome organization, stability, and evolution.

Chromosome cohesion and condensation are essential prerequisites of proper segregation of genomes during mitosis and meiosis, and are supported by two structurally related protein complexes, cohesin and condensin, respectively. At the core of the two complexes lie members of the structural maintenance of chromosomes (SMC) family of ATPases. SMC proteins are also found in most bacterial and archaeal species, implicating the existence of an evolutionarily conserved theme of higher-order chromosome organization and dynamics. SMC dimers adopt a two-armed structure with an ATP-binding cassette (ABC)-like domain at the distal end of each arm. This article reviews recent work on the bacterial and eukaryotic SMC protein complexes, and discusses current understanding of how these uniquely designed protein machines may work at a mechanistic level. It seems most likely that the action of SMC proteins is highly dynamic and plastic, possibly involving a diverse array of intramolecular and intermolecular protein–protein interactions.

Structural maintenance of chromosomes (SMC) proteins are central regulators of higher-order chromosome dynamics from bacteria to humans. The Bacillus subtilis SMC (BsSMC) homodimer adopts a V-shaped structure with an ATP-binding catalytic domain at each end. We report here that two small proteins, ScpA and ScpB, associate with the catalytic domains of BsSMC in an ordered fashion and suppress its ATPase activity. When combined with a 'transition state' mutant of BsSMC that poorly hydrolyzes ATP, ScpA promotes stable engagement of two catalytic domains in an ATP-dependent manner. In solution, this occurs intramolecularly and closes the DNA-entry gate of an SMC dimer. ScpB further stabilizes this conformation and prevents BsSMC from binding to double-stranded DNA (dsDNA). In contrast, when the mutant BsSMC is first allowed to interact with dsDNA, subsequent addition of ScpA leads to assembly of large nucleoprotein complexes, possibly by stabilizing intermolecular engagement of the catalytic domains from different SMC dimers. We propose that the ATP-modulated engagement/disengagement cycle of SMC proteins plays both positive and negative roles in their dynamic interactions with dsDNA.

Two different condensin complexes make distinct contributions to metaphase chromosome architecture in vertebrate cells. We show here that the spatial and temporal distributions of condensins I and II are differentially regulated during the cell cycle in HeLa cells. Condensin II is predominantly nuclear during interphase and contributes to early stages of chromosome assembly in prophase. In contrast, condensin I is sequestered in the cytoplasm from interphase through prophase and gains access to chromosomes only after the nuclear envelope breaks down in prometaphase. The two complexes alternate along the axis of metaphase chromatids, but they are arranged into a unique geometry at the centromere/kinetochore region, with condensin II enriched near the inner kinetochore plate. This region-specific distribution of condensins I and II is severely disrupted upon depletion of Aurora B, although their association with the chromosome arm is not. Depletion of condensin subunits causes defects in kinetochore structure and function, leading to aberrant chromosome alignment and segregation. Our results suggest that the two condensin complexes act sequentially to initiate the assembly of mitotic chromosomes and that their specialized distribution at the centromere/kinetochore region may play a crucial role in placing sister kinetochores into the back-to-back orientation.

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.