Cell Press Reviews: Core Concepts in Cell Biology

USA

Preface

We are very pleased to present Cell Press Reviews: Core Concepts in Cell Biology, which brings together review articles from Cell Press journals in order to offer readers a comprehensive and accessible entry point into some of the most important topics in cell biology today. Articles were selected by the editorial staff at Cell Press with an eye toward providing readers an introduction to timely and cutting-edge research written by leaders in the field. While Cell Press Reviews: Core Concepts in Cell Biology is not an exhaustive overview of current cell biological advances, our aim is to give readers insight into some of the most exciting recent developments and the challenges that remain. A wide range of topics are covered within this publication, including the cell biology of genomes, mechanochemical patterning in cell polarity, mechanisms of membrane curvature, and insights into processes such as organelle growth, cell motility, and morphogenesis.

We are pleased to be able to include contributions from Tom Misteli, National Cancer Institute; Galit Lahav, Harvard Medical School; Scott D. Emr, Cornell University; David G. Drubin, University of California, Berkeley; Tom Rapoport, Harvard Medical School; Anthony A. Hyman, Max Planck Institute of Molecular and Cell Biology, Dresden; and many other prominent researchers in the field. Their insights will offer readers, both experts and those new to the field, a fascinating perspective into this critically important and evolving area of research.

Cell Press Reviews: Core Concepts in Cell Biology is one in a series of books being published as part of an exciting new collaboration between Cell Press and Elsevier Science and Technology Books. Each book in this series is focused on a highly timely topic in the biological sciences. Editors at Cell Press carefully select recently published review articles in order to provide a comprehensive overview of the topic. With the wide range of journals within the Cell Press family, including research journals such as Cell, Current Biology, and Developmental Cell as well as review journals like Trends in Cell Biology, these compilations provide a diverse and accessible assortment of articles appropriate for a wide variety of readers. You can find additional titles in this series at http://www.store.elsevier.com/CellPressReviews. We are happy to be able to offer this series to such a wide audience via the collaboration with Elsevier Science and Technology Books, and we welcome all feedback from readers on how we might continue to improve the series.

Chapter 1

The Cell Biology of Genomes

Bringing the Double Helix to Life

Tom Misteli¹,∗,    ¹National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA,    ∗Correspondence: mistelit@mail.nih.gov

The recent ability to routinely probe genome function at a global scale has revolutionized our view of genomes. One of the most important realizations from these approaches is that the functional output of genomes is affected by the nuclear environment in which they exist. Integration of sequence information with molecular and cellular features of the genome promises a fuller understanding of genome function.

Keywords

genome; stochastic; epigenetic; genome organization

Acknowledgments

Due to space limitations, mostly review articles were cited. Work in the author’s laboratory is supported by the Intramural Research Program of the National Institutes of Health (NIH), NCI, Center for Cancer Research.

Cell, Vol. 152, No. 6, March 14, 2013 © 2013 Elsevier Inc.

http://dx.doi.org/10.1016/j.cell.2013.02.048

Summary

The recent ability to routinely probe genome function at a global scale has revolutionized our view of genomes. One of the most important realizations from these approaches is that the functional output of genomes is affected by the nuclear environment in which they exist. Integration of sequence information with molecular and cellular features of the genome promises a fuller understanding of genome function.

Introduction

It was a moment of scientific amazement in 1953 when Watson and Crick revealed the structure of DNA. The magnificence of the double helix and its elegant simplicity were awe inspiring. But more than just being beautiful, the double helix immediately paved the way forward; its structure implied fundamental biological processes such as semiconservative replication and the notion that chemical changes in its composition may alter heritable traits. The linear structure of DNA laid the foundation for the concept that a string of chemical entities could encode the information that determines the very essence of every living organism. The beauty of the double helix was the promise that, if the sequence of bases in the genome could be mapped and decoded, the genetic information that underlies all living organisms would be revealed and the secret of biological systems would be unlocked.

The idea of linearly encoded genetic information has been spectacularly successful, culminating in the recent development of powerful high-throughput sequencing methods that now allow the routine reading of entire genomes. The conceptual elegance of the genome is that the information contained in the DNA sequence is absolute. The order of bases can be determined by sequencing, and the result is always unequivocal. The ability to decipher and accurately predict the behavior of genome sequences was appealing to the early molecular biologists, has given rise to the discipline of molecular genetics, and has catalyzed the reductionist thinking that has driven and dominated the field of molecular biology since its inception.

But the apparent simplicity and deterministic nature of genomes can be deceptive. One of the most important lessons learned from our ability to exhaustively sequence DNA and to probe genome behavior at a global scale by mapping chromatin properties and expression profiling is that the sequence is only the first step in genome function. In intact living cells and organisms, the functional output of genomes is modulated, and the hard-wired information contained in the sequence is often amplified or suppressed. While mutations are an extreme case of genome modulation, most commonly occurring changes in genome function are more subtle and consist of fluctuations in gene expression, temporary silencing, or temporary activation of genes. Although not caused by mutations, these genome activity changes are functionally important.

Several mechanisms modulate genome function (Figure 1). At the transcription level, the limited availability of components of the transcription machinery at specific sites in the genome influences the short-term behavior of genes and may make their expression stochastic. Epigenetic modifications are capable of overriding genetically encoded information via chemical modification of chromatin. Similarly, changes in higher-order chromatin organization and gene positioning within the nucleus alter functional properties of genome regions.

FIGURE 1 From Primary Sequence to Genome Output

The hard-wired primary information contained in the genome sequence is modulated at short or long timescales by several molecular and cellular events. Modulation may lead to activation (green) or silencing (red) of genome regions.

The existence of mechanisms that modulate the output of genomes makes it clear that a true understanding of genome function requires integration of what we have learned about genome sequence with what we are still discovering about how genomes are modified and how they are organized in vivo in the cell nucleus.

The Stochastic Genome

The genome is what defines an organism and an individual cell. It is therefore tempting to assume that identical genomes behave identically in a population of cells. We now know that this is not the case. Individual, genetically identical cells can behave very differently even in the same physiological environment. It is rare to find a truly homogeneous population of cells even under controlled laboratory conditions, as anyone who has tried to make a cell line stably expressing a transgene knows. Much of the variability in biological behavior between individual cells comes from stochastic activity of genes (Raj and van Oudenaarden, 2008).

Genes are by definition low-copy-number entities, as each typically only exists in two copies in the cell. Similarly, many transcription factors are present in relatively low numbers in the cell nucleus. The low copy number of genes and transcription factors makes gene expression inherently prone to stochastic effects (Raj and van Oudenaarden, 2008). Numerous observations make it clear that gene expression is stochastic in vivo. For example, dose-dependent increases in gene expression after treatment of cell populations with stimulating ligands, such as hormones, are often brought about by high expression of target genes in a relatively small number of cells in the population rather than by a uniform increase in the activity in all cells. Stochastic gene behavior is most evident in single-cell imaging approaches, and mapping by fluorescence in situ hybridization of multiple genes, which according to population-based PCR analysis are active in a given cell population, shows that only a few cells transcribe all constitutively active genes at any given time. Most cells only express a subset of genes, and the combinations vary considerably between individual cells. These observations suggest that many genes blink on and off and are expressed in bursts rather than in a continuous fashion (Larson et al., 2009).

The molecular basis for stochastic gene expression is unknown. There are several candidate mechanisms, all of which are related to genome or nuclear organization. Most genes require some degree of chromatin remodeling for activity, which is thought to make regulatory regions accessible to the transcription machinery. Several observations suggest that chromatin remodeling contributes to the stochastic bursting of gene expression. Maybe most compelling is the finding that genes located near each other on the same chromosome show correlated blinking behavior, indicating that a local chromosome property, such as chromatin structure, drives stochastic behavior (Becskei et al., 2005). Furthermore, altering chromatin, for example by deletion of chromatin remodeling machinery, affects stochastic variability in yeast. It can be envisioned that the stochastic behavior of genes is caused by the requirement for cyclical opening of chromatin regions. Open chromatin has a limited persistence time, and maintaining chromatin in an open state requires the cyclical action of chromatin remodelers. Whether an active gene is transcribed at any given time may thus depend on the transient condensation status of its chromatin at a particular moment.

A second mechanism to impose nonuniform stochastic genome activity may be the local availability of the transcription machinery at a gene. Although transcription factors are able to relatively freely diffuse through the nuclear space, and in this way effectively scan the genome for binding sites, their availability and functionality at a given local site may undergo significant temporal fluctuations (Misteli, 2001). The local availability of transcription complexes may affect transcription frequency positive or negatively. On the one hand, it is possible that relatively stable preinitiation complexes persist on a given gene, where they may support multiple rounds of transcription and in this way boost initiation frequency. On the other hand, assembly of the full polymerase is a stochastic and relatively inefficient event itself. In order for a functional polymerase complex to assemble, individual transcription machinery components associate with chromatin in a step-wise fashion, and formation of the mature polymerase complex involves multiple partially assembled intermediates, many of which are unstable and disintegrate before a functionally competent complex is formed (Misteli, 2001). The inefficiency of polymerase assembly may create stochasticity at an individual locus.

A further contributor to stochastic gene expression may be the organization of transcription events in transcription factories. These hubs of transcription consist of accumulations of transcription factors to which multiple genes, often located on distinct chromosomes, are recruited (Edelman and Fraser, 2012). Typically only a few hundred such transcription factories are observed in a mammalian cell nucleus. It is possible that some genes need to physically relocate from nucleoplasmic locations to transcription factories. A nominally active gene locus that is not associated with a transcription factory may thus be stochastically silent. The relatively low number of transcription sites makes them a limiting factor in the transcription process and thus a potential mediator of stochastic gene expression.

Epigenetics—and When Epigenetics is not Epigenetics

Stochastic effects modulate genome output on short timescales. A mechanism to modulate the hardwired information of genomes on longer timescales is via epigenetics. The Greek-derived Epi means over or above, and epigenetic effects are defined as heritable changes in genome activity caused by mechanisms other than changes in DNA sequence. Epigenetic events are mediated by chemical modifications of DNA or core histones in complex patterns by methylation, acetylation, ubiquitination, phosphorylation, etc. These modifications alter gene expression by changing the chromatin surface and in this way affect the binding of regulatory factors. Well-established examples of such effects include binding of the DNA-methylation-dependent binding of the MeCP2 protein or the binding of PHD-domain-containing proteins to trimethylated histone H3 tails. Prominent biological effects based on epigenetic regulation are phenotypic differences between homozygous twins or imprinted genes that are expressed from only one allele in a diploid organism.

A central tenet in the definition of epigenetic regulation is that its effects are heritable, i.e., transmittable over generations. In fact, the concept of epigenetics was inspired by epidemiological findings that nutrient availability in preadolescents during the 19th century Swedish famine determined life expectance of their grandchildren. The epidemiological studies have recently been complemented by controlled laboratory studies in mice (Rando, 2012), and they have been extended to the molecular level by the findings that loss of the histone H3K4-trimethylation prolongs lifespan in C. elegans in a heritable fashion for several generations (Greer et al., 2011).

A complicating aspect of epigenetics is that the same modifications that mediate heritable epigenetic regulation may also bring about nonheritable transient modulations of the genome. In fact, the term epigenetic is nowadays often used in a very cavalier manner to refer to any biological effect, heritable or not, that is affected by histone modifications. Even if they are not heritable, histone modifications are biologically relevant modulators of genome function. The system of histone modifications is in many ways akin to the mechanisms by which signal transduction pathways work (Schreiber and Bernstein, 2002). Just as in signal transduction pathways, posttranslational modifications on histone tails create binding sites that are then recognized by adaptor or reader proteins, which in turn elicit downstream effects such as activation of kinases in the case of signaling cascades or recruitment of transcription factors in the case of histone modifications. In further analogy to the reversible events in signaling pathways, histone modifications can be altered or erased by modifying enzymes. Such transient and reversible modulatory effects of histone modifications have been implicated in every step of gene expression, starting from chromatin remodeling to recruitment of transcription machinery and even to downstream events that were thought to be chromatin independent, such as alternative pre-mRNA splicing (Luco et al., 2011). It is often difficult to determine heritability of these histone modification effects, and it therefore remains unclear how many of them are truly epigenetic. Regardless, DNA and histone modifications are an obvious source of modulation of the information contained in the genome sequence.

Genome Organization as a Modulator of Genome Function

Genomes of course do not exist as linear, naked DNA in the cell nucleus but are organized into higher-order chromatin fibers, chromatin domains, and chromosomes. Many correlations between genome organization and activity have been made—most prominently, the findings that transcriptionally active genes are generally located in decondensed chromatin and that transcriptionally repressed genome regions are often found at the nuclear periphery. These observations point to the possibility that the spatial organization of the genome modulates its functional output.

But in considering the relationship of genome structure with its function, we are faced with a perpetual chicken-and-egg problem. Does structure drive function, or is structure merely a reflection of function? Much of the thinking on this topic has been guided by observations on individual genes. How representative these were for the genome as a whole has been a confounding concern. Recent unbiased genome-wide analysis of structure/function relationships has validated the tight link between structure and function. Large-scale analysis of chromatin structure, histone modifications, and expression profiles shows that genomes are portioned into well-defined domains that closely correlate with their activity status and the presence of active or repressive histone marks (Sexton et al., 2012). The domains are separated by sharp boundaries marked by particular histone modification patterns and binding sites for chromatin insulator proteins such as CTCF. Even stronger evidence comes from the analysis of physical interactions between chromatin domains. At least in fruit flies, functionally equivalent domains tend to preferentially interact; that is, domains containing silent regions cluster in three-dimensional space, as do domains containing active regions (Sexton et al., 2012).

But can genome structure drive its function? The best example for structure-mediated gene expression effects is the silencing of genes when they become juxtaposed to heterochromatin domains, be it in the nuclear interior or at the nuclear periphery (Beisel and Paro, 2011). Gene activity has also frequently been linked to the position of a gene within the cell nucleus. The strongest evidence for such a relationship is experiments in which genes are transplanted from the nuclear interior to the lamina, leading to their repression or making them refractory to activation (Geyer et al., 2011). Based on these and similar experiments, it is often quite categorically stated that active genome regions are found in the interior of the nucleus and inactive ones at the periphery. This is a somewhat misleading oversimplification. Although lamina-associated genome regions are generally gene poor and are not transcribed, transcription labeling experiments reveal numerous active transcription sites at the periphery, and genes that are near the periphery, but not physically associated with it, are often active. On the other hand, inactive genes are frequently found in the interior. As far as we can tell, nuclear position per se does not determine activity, but association with repressive regions of the nucleus, be it at the periphery or the interior, does.

So, how then should we think about the chicken-and-egg problem of nuclear structure and function? How can it be that clear evidence exists for both function-driving-structure as well for structure-driving-function? The likely answer is that both effects are at play and are part of an overarching principle in which the mutual interplay of structure and function at multiple levels influences gene expression. The fact that there are very few known heterochromatic active genes suggests that a structural change in the form of chromatin decondensation is a crucial early step in gene activation. However, because chromatin states are generally unstable, mechanisms that reinforce a decondensed chromatin state must be in force for a gene to remain active. Such reinforcing mechanisms are dependent on gene activity and represent the activity-drives-function aspect of gene expression. Reinforcement mechanisms might be mediated by what we consider active histone modifications, some of which are known to be deposited during transcription as the polymerases traverse genes. On the flipside, a chromatin domain may also impose its effect on neighboring regions, either in cis on the same chromosome by spreading or in trans on distinct chromosomes. This effect represents the structure-drives-function aspect of genome function. Such a bidirectional, self-enforcing function-structure-function model accounts for most experimental observations on structure-function relationships in gene expression.

Facing the Complexity

Since the discovery of the double helix, we have come to realize that understanding genomes requires more than reading their sequence and that the information contained in the sequence is modulated by the cellular environment. How then do we gain full knowledge of the functional information encoded in genomes?

To get a comprehensive picture of the functional output of genomes, the sequence information needs to be integrated with other information parameters such as epigenetic patterns, higher-order chromatin landscapes, and noncoding RNA profiles. The technology to do this is now available, and intense efforts are currently underway to comprehensively gather these data sets in various biological systems. The first examples of such multilevel mapping analyses are emerging, such as the recent flurry of reports from the ENCODE consortium, which has systematically mapped genome properties ranging from histone modification profiles to regulatory elements and chromatin structure (Ecker et al., 2012). Given the scale and complexity of the generated data, not to mention the technical difficulties in gathering it, this is a challenging undertaking that will require a series of progressively larger studies. Ideally, future studies should be designed to systematically map multiple genome properties for focused biological systems such as specific human diseases.

Large-scale mapping of genome-related parameters and their comparison is a logical and necessary next step in the exploration of genomes and their function. These efforts will create invaluable catalogs of genome properties, and the hope is that, by cross-comparing data sets, insight into the rules that govern genome regulation will be gleaned. One can go one step further and advocate for an even more comprehensive approach in which genome expression data are then compared to other cellular characteristics such as proteomic, metabolomic, morphological, and physiological data to systematically link genome activity to biological behavior. The ultimate version of such an approach was recently described in a report by the US National Academies of Sciences entitled Toward Precision Medicine, which envisioned a fully minable biomedical data repository that would include information ranging from genomic and epigenetic parameters to physiological features and clinical symptoms.

The elegant simplicity of the DNA structure revealed by Watson and Crick is still stunning. True to its promise when it was first discovered, it opened up the floodgates to understanding heredity. But one of the most profound lessons from the ensuing decades of genome exploration must be that the linear arrangement of bases in the DNA is not an absolute set of instructions but is malleable by the cellular environment. We are just beginning to uncover some of the mechanisms that are responsible for these effects. As is the rule in biology, wherein the whole is often greater than the sum of its parts, we are realizing that the genome is far more complex than the sequence of its DNA.

References

1. Becskei A, Kaufmann BB, van Oudenaarden A. Contributions of low molecule number and chromosomal positioning to stochastic gene expression. Nat Genet. 2005;37:937–944.

2. Beisel C, Paro R. Silencing chromatin: comparing modes and mechanisms. Nat Rev Genet. 2011;12:123–135.

3. Ecker JR, Bickmore WA, Barroso I, Pritchard JK, Gilad Y, Segal E. Genomics: ENCODE explained. Nature. 2012;489:52–55.

4. Edelman LB, Fraser P. Transcription factories: genetic programming in three dimensions. Curr Opin Genet Dev. 2012;22:110–114.

5. Geyer PK, Vitalini MW, Wallrath LL. Nuclear organization: taking a position on gene expression. Curr Opin Cell Biol. 2011;23:354–359.

6. Greer EL, Maures TJ, Ucar D, et al. Transgenerational epigenetic inheritance of longevity in Caenorhabditis elegans. Nature. 2011;479:365–371.

7. Larson DR, Singer RH, Zenklusen D. A single molecule view of gene expression. Trends Cell Biol. 2009;19:630–637.

8. Luco RF, Allo M, Schor IE, Kornblihtt AR, Misteli T. Epigenetics in alternative pre-mRNA splicing. Cell. 2011;144:16–26.

9. Misteli T. Protein dynamics: implications for nuclear architecture and gene expression. Science. 2001;291:843–847.

10. Raj A, van Oudenaarden A. Nature, nurture, or chance: stochastic gene expression and its consequences. Cell. 2008;135:216–226.

11. Rando OJ. Daddy issues: paternal effects on phenotype. Cell. 2012;151:702–708.

12. Schreiber SL, Bernstein BE. Signaling network model of chromatin. Cell. 2002;111:771–778.

13. Sexton T, Yaffe E, Kenigsberg E, et al. Three-dimensional folding and functional organization principles of the Drosophila genome. Cell. 2012;148:458–472.

Chapter 2

Condensin, Chromatin Crossbarring and Chromosome Condensation

Rahul Thadani¹, Frank Uhlmann¹,∗ and Sebastian Heeger¹,    ¹Chromosome Segregation Laboratory, Cancer Research UK London Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK,    ∗Correspondence: frank.uhlmann@cancer.org.uk

The processes underlying the large-scale reorganisation of chromatin in mitosis that form compact mitotic chromosomes and ensure the fidelity of chromosome segregation during cell division still remain obscure. The chromosomal condensin complex is a major molecular effector of chromosome condensation and segregation in diverse organisms ranging from bacteria to humans. Condensin is a large, evolutionarily conserved, multisubunit protein assembly composed of dimers of the structural maintenance of chromosomes (SMC) family of ATPases, clasped into topologically closed rings by accessory subunits. Condensin binds to DNA dynamically, in a poorly understood cycle of ATP-modulated conformational changes, and exhibits the ability to positively supercoil DNA. During mitosis, condensin is phosphorylated by the cyclin-dependent kinase (CDK), Polo and Aurora B kinases in a manner that correlates with changes in its localisation, dynamics and supercoiling activity. Here we review the reported architecture, biochemical activities and regulators of condensin. We compare models of bacterial and eukaryotic condensins in order to uncover conserved mechanistic principles of condensin action and to propose a model for mitotic chromosome condensation.

Keywords

condensing; chromatin; chromosome; chromosome condensation; chromosome structure; holocomplex; supercoiling; multimerisation

Acknowledgements

We thank D. Agard for the chromosome reconstruction shown in Figure 4C. We are grateful to members of the Chromosome Segregation Laboratory for discussions and critical reading of the manuscript. This work was supported by Cancer Research UK and the European Research Council. R. Thadani and S. Heeger acknowledge support through a Boehringer Ingelheim Fonds PhD fellowship, and an EMBO long term fellowship, respectively.

Current Biology, Vol. 22, No. 23, R1012–R1021, December 4, 2012 © 2012 Elsevier Inc.

http://dx.doi.org/10.1016/j.cub.2012.10.023

Summary

The processes underlying the large-scale reorganisation of chromatin in mitosis that form compact mitotic chromosomes and ensure the fidelity of chromosome segregation during cell division still remain obscure. The chromosomal condensin complex is a major molecular effector of chromosome condensation and segregation in diverse organisms ranging from bacteria to humans. Condensin is a large, evolutionarily conserved, multisubunit protein assembly composed of dimers of the structural maintenance of chromosomes (SMC) family of ATPases, clasped into topologically closed rings by accessory subunits. Condensin binds to DNA dynamically, in a poorly understood cycle of ATP-modulated conformational changes, and exhibits the ability to positively supercoil DNA. During mitosis, condensin is phosphorylated by the cyclin-dependent kinase (CDK), Polo and Aurora B kinases in a manner that correlates with changes in its localisation, dynamics and supercoiling activity. Here we review the reported architecture, biochemical activities and regulators of condensin. We compare models of bacterial and eukaryotic condensins in order to uncover conserved mechanistic principles of condensin action and to propose a model for mitotic chromosome condensation.

Introduction

The propagation of the blueprint of life, at a molecular level, can be described as the accurate transmission of replicated genetic material to daughter cells. To enable this, cells must compact centimetre-long DNA molecules, within the confines of micrometre-sized nuclei, into stable chromosomes that can withstand the forces generated during segregation. This condensation of chromatin — into the thread-like chromosomes that give mitosis its name (from the Greek mitos, i.e., thread) — is one of the most striking morphological events of the cell cycle. Yet more than a century after Walther Flemming first observed mitotic chromosomes [1], and Theodor Boveri proposed they maintained their identity through interphase [2], mechanistic explanations of chromosome condensation remain elusive.

Early indications of a mitosis-specific condensation factor in cells began to emerge in the 1970s: classical cell fusion experiments showed that premature chromosome condensation could be induced in interphase HeLa cells that were fused to mitotic ones [3]. In a cell-free system derived from Xenopus eggs, metaphase chromosomes were assembled in interphase nuclei incubated with mitotic extracts [4,5]. Subsequent studies in budding and fission yeasts [6,7], Xenopus egg extracts [8] and chicken cells [9] led to the identification of Smc2 and Smc4, core components of the condensin complex, as proteins essential for chromosome condensation and segregation.

Accumulating lines of evidence over two decades indicate that the chromosomal condensin complex is the principal effector of condensation [10,11]. Condensin is a large, evolutionarily conserved multisubunit protein assembly that is found, with a broadly similar architecture, throughout the domains of life, including bacteria, archaea and eukarya (Figure 1). Along with cohesin and Smc5/6, it is one of three complexes built from dimers of SMC proteins, members of the structural maintenance of chromosomes family of ATPases, that are intimately involved in diverse aspects of higher order chromosome organisation. Indeed, condensin has been ascribed roles in several cellular processes apart from chromosome condensation; these have been extensively described elsewhere [12,13]. Conversely, the related SMC complexes might also contribute to chromosome condensation [14]. In this review, we focus on the role of condensin in mitotic chromosome condensation.

FIGURE 1 Molecular architecture of condensin.

Subunit composition of eukaryotic (left) and bacterial (right) condensins. Condensins are composed of a core dimer of SMC or SMC-like ATPases with a dimerisation hinge at one end and catalytic head domain at the other. The core dimers are closed into rings by kleisins, which are monomeric in eukaryotes and dimeric in prokaryotes. One or more additional regulatory subunits interact with the kleisin and/or SMC core.

Molecular Architecture of Condensin

Eukaryotic condensin is a large pentameric complex that comprises a core catalytic Smc2–Smc4 heterodimer. As is characteristic of SMC proteins, Smc2 and Smc4 contain three globular parts — two terminal and one central — linked by long coiled coils. Each SMC protein folds back on itself through antiparallel coiled-coil arm interactions. This forms an SMC dimerisation hinge domain from the central part at one end, and an ATPase head domain from association of the terminal globular parts at the other (Figure 1). The catalytic head domain features canonical ATP-binding cassette motifs: the amino-terminal ‘Walker A’ motif, and carboxy-terminal ‘Walker B’ and ‘C/signature’ motifs. The amino-terminal globular part of one SMC subunit engages in trans with a carboxy-terminal part from the other to form a bipartite ATP-binding pocket. Three accessory subunits bind to the SMC heterodimer and regulate its activity. Kleisin I/Brn1, a member of the kleisin family [15], interacts at its amino terminus with Smc2, and at its carboxyl terminus with Smc4 to form a topologically closed ring [10,16,17]. HEAT IA/Ycs4 [18,19] and HEAT IB/Ycg1 [20] contain HEAT repeats, and interact with the amino- and carboxy-terminal halves of Kleisin I/Brn1, respectively, and weakly with each other [21]. All three accessory subunits of condensin are required for its association with chromatin and function in chromosome condensation [22]. It is noteworthy that while the integrity of the ring-like structure of condensin is necessary for its function [17], details of its mechanistic significance remain to be determined. This is in contrast to the case of cohesin, where it has been shown that the complex topologically encircles sister chromatids until separase-driven cleavage of the kleisin Scc1 at anaphase onset enables them to segregate [23,24].

Most eukaryotes possess two isoforms of condensin, termed condensin I and II. These are built from identical core heterodimers of Smc2 and Smc4 but differing accessory subunits (Table 1), which may modulate the differential localisation patterns, dynamics, and functions of the two condensins. Condensin I is termed the canonical condensin due to its phylogenetic ubiquity (Figure 2) and relative cellular abundance, although the ratio of condensin I and II varies substantially in different organisms, ranging from 1:1 in HeLa cells and 5:1 in Xenopus egg extracts [25] to 10:1 in chicken DT40 cells [26,27]. The presence of condensin I and II in diverse eukaryotic taxa suggests that their last common ancestor possessed both condensin isoforms [13]. This implies that condensin II was independently lost from the genomes of organisms such as fungi and ciliates that possess only a single known isoform of condensin.

Table 1

Condensin subunits involved in mitotic chromosome condensation

FIGURE 2 Phylogenetic analysis of condensin kleisin subunits.

Phylogram representing the evolutionary relationships among eukaryotic kleisin subunits. A maximum likelihood unrooted tree was constructed in PHYLIP [118] from a ClustalW multiple alignment [119] and rendered radially using iTOL [120]. The excavate Naegleria gruberi was used as an outgroup (top left), from which taxa fan out clockwise in order of increasing branch lengths. Note that kleisins from species with a single known condensin isoform, such as fungi and the ciliate Tetrahymena thermophila, cluster with the condensin I sequences. The scale bar represents one unit of evolutionary distance along branches, as computed by the Jones-Taylor-Thornton method [121].

Prokaryotes were believed to possess one of two SMC-related complexes: the SMC-ScpAB complex [28-30] widespread in bacteria and archaea, or the MukBEF complex [31,32] present in γ-proteobacteria such as Escherichia coli. These two complexes are divergent at the sequence level but share a common architecture. Inactivation of the two complexes produces defects reminiscent of condensin mutants, including decondensed nucleoids, chromosome segregation failure, anucleate cell formation and temperature-sensitive growth [28–32]. SMC-ScpAB is composed of a catalytic SMC homodimer, the kleisin ScpA and accessory protein ScpB, both of which are likely binary in the complex. Similarly, MukBEF comprises an SMC-like core MukB homodimer, while the kleisin MukF and accessory protein MukE again form a dimeric frame that interacts with the MukB heads [33,34] (Figure 1). A third family of MukBEF-like SMC protein, termed MksBEF, has recently been identified in diverse bacterial genomes [35]. MksBEF is often present alongside SMC-ScpAB, MukBEF, or even other MksBEFs, suggesting that prokaryotic genome organisation may be more complex than previously appreciated. This also raises the possibility of as yet undiscovered molecular drivers of chromosome condensation in the larger and incompletely annotated genomes of eukaryotes. We refer to the bacterial SMC-ScpAB and MukBEF complexes as prokaryotic condensins, due to their condensin-like null phenotypes. In the following sections, we draw mechanistic parallels with eukaryotic condensin, but note that the two prokaryotic complexes are not strictly phylogenetically closer to eukaryotic condensin than to other eukaryotic SMC complexes.

Differential Contributions of Condensin I and II to Chromosome Structure

Condensin I and II exhibit distinct spatial staining patterns on chromosome axes, as well as differing temporal localisation patterns through the cell cycle [25,36], suggesting that they may have non-redundant roles in chromosome organisation. For instance, in HeLa cells, condensin I is excluded from the nucleus in interphase and binds to chromatin only on nuclear envelope breakdown (NEBD) in prometaphase. In contrast, condensin II is essentially nuclear in interphase, is stabilised on chromatin in early prophase, and remains associated with chromosomes throughout mitosis [36–38].

The differential contributions of the two condensin complexes to chromosome condensation are as yet poorly understood. In Xenopus egg extracts, the phenotypes following immunodepletion of condensin I- or II-specific subunits indicate that condensin I plays the major role in condensation [25]. By contrast, in HeLa cells, the siRNA-mediated knockdown of either condensin by RNAi leads to only minor abnormalities in chromosome morphology [25,37], with condensin I depletion producing swollen chromosomes, and condensin II depletion making them somewhat longer and curled. The varying severity of condensation phenotypes subsequent to condensin depletion in these two systems may be ascribed either to dose-dependence, given the differing ratios of the two condensin isoforms, or to incomplete silencing by RNAi. Consistent with the observed aberrant chromosome morphologies in HeLa cells, further studies in chicken DT40 cells [27] and Xenopus egg extracts [39] implicate condensin II in the early mitotic axial shortening of chromosome arms, and condensin I in their later lateral compaction. More work is needed to determine how two very similar complexes are able to bind to distinct chromosomal regions, and whether it is their differential localisation or intrinsic activity that is responsible for their separable contributions to condensation.

Cell-Cycle Regulation of Chromosome Condensation

Numerous aspects of condensin biology are, reportedly, subject to control by the cell cycle machinery, making possible a multi-layered regulation of its function. Condensin activity may be regulated at the level of holocomplex formation, subcellular localisation, chromosomal loading, or chromatin binding dynamics, one or more of which are likely altered by post-translational modifications (Figure 3).

FIGURE 3 Regulation of condensin activity.

Condensin can be regulated at several different levels: complex formation, nuclear import, chromosomal localisation, binding dynamics, and ATPase activity. This regulation is likely performed by posttranslational modifications, which can modulate the biochemical activities of the complex.

Holocomplex Formation

In the test tube, condensin is often found in two major forms corresponding to the Smc2–Smc4 heterodimer and the holocomplex, as seen in early immunoaffinity purifications from Xenopus egg extracts [10], as well as reconstitutions of recombinant human condensin [21]. In addition, yeast Smc2 and Smc4 can form a stable heterodimer in cell extracts [40]. In vitro studies have ascribed differing activities to the SMC/MukB dimer and the holocomplex [10,41]. However, it should be noted that the uncomplexed SMC dimer has not been directly observed in vivo. Interestingly, the reported role of condensin in disassembly of the Drosophila nurse cell polytene chromosomes depends on a timely upregulation of only Kleisin II/CAP-H2 [42], indicating there may be a role for a limiting subunit in the regulation of complex assembly. Similarly, HEAT IA/CAP-D2 is a rate-limiting factor for the assembly of functional condensin I complexes in Xenopus oocytes [43]. This is reminiscent of cohesin, where levels of the kleisin Scc1 vary through the cell cycle, and determine the chromosomal association of the complex [44]. This principle can be applied more broadly, raising the possibility that varying expression levels of condensin I- and II-specific subunits determine the changing shapes of mitotic chromosomes in a developmental context [39]. Investigations of complex assembly and DNA binding dynamics of individual condensin subunits, for instance by fluorescence recovery after photobleaching (FRAP) assays, would prove instructive in further elucidating regulation at the level of condensin holocomplex formation.

Subcellular Localisation

In bacteria and archaea that lack a nuclear envelope, condensin is free to interact with chromatin at any time, limited only by the possible regulation of complex formation and DNA loading reactions. In eukaryotes, however, the nuclear envelope offers a potential barrier to chromatin access and consequently a possible mode of regulation. In organisms with a closed mitosis like the budding and fission yeasts, condensin has to be imported into the nucleus at or before chromatin compaction in mitosis. Intriguingly, in the budding yeast Saccharomyces cerevisiae, condensin localises to the nucleus throughout the cell cycle, a behaviour reminiscent of condensin II of higher eukaryotes, despite the complex being a homologue of condensin I at the sequence level (Figure 2). On the other hand, condensin in the fission yeast Schizosaccharomyces pombe is predominantly cytoplasmic in interphase and nuclear during mitosis, an enrichment that requires the CDK-dependent phosphorylation of the T19 site in Smc4/Cut3 [45]. In higher eukaryotes with an open mitosis, nuclear envelope breakdown (NEBD) at mitotic onset ensures that the chromatin association of condensin is not hindered. In Drosophila melanogaster [46], Caenorhabditis elegans [47], zebrafish [48] and HeLa [36–38] cells, condensin I is cytoplasmic in interphase and nuclear in mitosis, while condensin II is nuclear throughout the cell cycle, at least in HeLa cells. An interesting but as yet unresolved question is how this differential subcellular localisation of condensin I and II is achieved, either by modifications to their regulatory subunits or recognition by additional factors. Intriguingly, in Drosophila embryos, Kleisin I/Barren associates with chromatin several minutes before NEBD [46]. In addition, HeLa cells depleted of condensin II still initiate chromosome compaction just before NEBD [38]. These observations suggest that chromosome compaction in these organisms may be functionally coupled to disassembly of nuclear pore complexes (NPCs) rather than the nuclear membrane [38,49]. This is probably also the case in C. elegans, where NEBD is completed only in anaphase but chromosome condensation is initiated in prophase, accompanied by NPC breakdown