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Condensins and the evolution of torsion-mediated genome organization
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Condensins and the evolution of torsion-mediated genome organization Tatsuya Hirano Chromosome Dynamics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
At first glance, bacteria and eukaryotes appear to use different strategies to pack and organize their genomes. At the basal level, bacterial genome compaction relies on unconstrained, negative supercoils, whereas eukaryotic genomes are packaged into nucleosomes via constrained, negative supercoils. Here, I integrate the action of condensins, chromosome-packaging complexes conserved from bacteria to humans, into this picture, and discuss how torsional stress on DNA might have dual impacts on genome organization and function. A common theme is that organisms have evolved flexible and reversible strategies to pack their genomes while keeping them readily accessible to many activities such as gene expression. ‘Nothing in biology makes sense except in the light of evolution’, T. Dobzhansky (1973)[1].
Pack the genomes, but not too tightly Genomes in all organisms face two big challenges [2,3]. Long DNA molecules comprising a genome must be packaged tightly to fit within the limited spaces of the cell. At the same time, they must be readily accessible to many activities such as gene expression. How do they deal with these apparently conflicting problems? One of the crucial strategies used by bacteria is to introduce negative ( ) supercoils through the action of a specialized type II topoisomerase known as DNA gyrase (see Glossary and Box 1 for the basics of DNA topology). Although a group of small proteins associate with genomic DNA, roughly half of the ( ) supercoils are present in an unconstrained, plectonemic form in bacterial cells [3], thereby allowing local unwinding of DNA that promotes replication and transcription (Figure 1A). In eukaryotes, genomic DNA is also negatively supercoiled, but most of the supercoils are present in a constrained, toroidal form within nucleosomes (Figure 1B). Loss or conformational changes of nucleosomes not only make transcription factors accessible, but also introduce local changes of DNA structure, including unwinding. It is also important to note that the two types of ( ) supercoils (unconstrained or constrained) contribute to Corresponding author: Hirano, T. ([email protected]). Keywords: DNA supercoiling; nucleosomes; condensins; chromosomes; genomes; evolution. 0962-8924/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2014.06.007
the compaction of DNA molecules (Figure 1A,B). Thus, in both bacteria and eukaryotes, the superhelical status of DNA plays a fundamental role in the structural and functional organization of genomes. On top of this basal level of organization, both bacteria and eukaryotes possess a higher-order genome-packaging system, which is mediated by a class of multisubunit protein complexes, known as condensins [4]. Although it has been suggested that condensins also modulate or cooperate with torsional stress for genome organization, the importance of DNA topology is often overlooked in modern cell biology, and comparisons between bacterial and eukaryotic systems are rarely discussed, with only a few exceptions [2,3,5,6]. Here, I focus on the role of condensins, proposing that superhelical torsion imposed on DNA might have dual impacts on higher-order genome organization and function. The purpose of this article is not to summarize well-established concepts: rather I attempt to offer bold speculations that might help stimulate further discussions in the field. Bacterial genome organization: beyond unconstrained negative supercoils The condensin complex was originally identified in Xenopus egg extracts as a major component of metaphase Glossary Condensins: a class of large protein complexes involved in chromosome condensation and segregation. DNA topology: a structural property of DNA derived from its intrinsic doublehelical nature. Depending on how many times the two strands cross each other, a circular DNA molecule is subject to topological strain and can have different configurations (see Figure I in Box 1). A linear DNA molecule is also subject to topological strains when it is very long and interacts with intracellular structures. Negative supercoil/(S) supercoil: a type of supercoils in which DNA has fewer helical turns than would be expected for the typical (B form) structure. An underwound form of DNA produces negative supercoils. Node conversion: an event converting the crossing statuses of entry/exit DNA of nucleosomes. Nucleosome array: a DNA segment on which nucleosomes are arrayed at regular intervals. Nucleosome fiber: a nucleoprotein fiber composed of DNA and histones. Plectonemic supercoil: a type of supercoils in which a double-helical DNA molecule is twisted over itself. This type can be present as an unconstrained form without protein binding. Positive supercoil/(+) supercoil: a type of supercoils in which DNA has more helical turns than would be expected for the typical (B form) structure. An overwound form of DNA produces positive supercoils. Superhelical torsion: an action of twisting that changes the superhelical state of DNA. Topoisomerases: a class of enzymes that changes the topological state of DNA by transiently cutting and re-ligating one or two of the DNA strands. Toroidal supercoil: a type of supercoils in which a double-helical DNA molecule is wound in a cylindrical manner. This type is stabilized when it is constrained by protein binding.
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Box 1. Basics of DNA topology Shown here are different topological forms of a closed circular DNA. A negatively supercoiled DNA can be present as different forms, namely, ( ) plectonemic, ( ) toroidal (not shown) and underwound forms. Likewise, a positively supercoiled DNA is present as (+) plectonemic, (+) toroidal (not shown) and overwound forms. These forms are interconvertible without cutting DNA strands. By contrast, topoisomerases change the DNA topology by transiently cutting and
Underwound
religating one or two of the DNA strands [39]. For instance, eukaryotic topoisomerase I (euk topo I) can relax both ( ) and (+) supercoils, whereas Escherichia coil topoisomerase I (E. coli topo I) relaxes ( ) supercoils only. Bacteria possess DNA gyrase that introduces ( ) supercoils in an ATP-dependent manner. Some thermophilic bacteria have a unique topoisomerase (reverse gyrase) that is able to introduce (+) supercoils in an ATP-dependent manner (Figure I).
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Figure I. Basics of DNA topology.
chromosomes that plays a crucial role in mitotic chromosome assembly [7]. Subsequent studies demonstrated that many bacteria possess similar complexes (henceforth referred to as bacterial condensins) whose mutations cause defects in
nucleoid organization and segregation [8,9]. Interestingly, defects in condensin mutants are suppressed when excess ( ) supercoils are introduced in a topoisomerase mutant background [10], implicating that ( ) supercoiling and Replicaon/ transcripon machinery
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Figure 1. The basal level of genome organization in bacteria and eukaryotes. (A) In bacteria, roughly half of ( ) supercoils are present in an unconstrained, plectonemic form, which allows local unwinding of DNA required for replication and transcription. A group of proteins that partially constrain ( ) supercoils are indicated by the ovals with different colors. (B) In eukaryotes, most ( ) supercoils are present in a constrained, toroidal form within nucleosomes (indicated in the yellow discs). Loss or remodeling of nucleosomes causes local changes in DNA structure, making it possible for the replication/transcription machinery to work.
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Opinion condensins synergistically contribute to proper compaction and segregation of bacterial nucleoids. When considering the structural organization of bacterial nucleoids, it is important to keep in mind that there are no discrete cell-cycle stages in rapidly dividing bacteria. Bacterial condensins must therefore organize and segregate their genomes at the same time that they are replicated and actively transcribed [8,9]. Thus, the primary target of bacterial condensins is most likely to be ( ) supercoiled DNA. Consistent with this prediction, it has been shown that the core subunit of bacterial condensins displays a binding preference to ( ) supercoils over (+) supercoils [11]. It is not fully understood, however, to what extent bacterial condensins might have the ability to actively introduce superhelical torsion into DNA [12]. Currently available lines of evidence suggest that bacterial condensins could support genome organization either as a protein clamp [13] or through large molecular assembly [14]. Eukaryotic genome organization: beyond constrained negative supercoils The nucleosome, the fundamental unit of eukaryotic chromatin, wraps DNA in a left-handed direction. Consequently, ( ) supercoils are constrained by histone octamers and present as a toroidal form within nucleosomes. During the past two decades, it has become increasingly clear that the nucleosome structure is highly dynamic: mobilization or disruption of nucleosomes occurs locally, providing an opportunity to regulate gene expression precisely in locus-specific manners during interphase [15]. In contrast to bacterial condensins, eukaryotic condensins function in a relatively short period of the cell cycle (i.e., mitosis) when duplicated chromosomes undergo large conformational changes to prepare for their segregation. Equally important, gene expression is largely suppressed during this stage of the eukaryotic cell cycle. Despite the long history of research, however, it remains unknown exactly how nucleosome fibers might be folded and organized into rod-shaped, mitotic chromosomes [16–18]. Could torsional stress be involved, at least in part, in the assembly of mitotic chromosomes? A vital hint to this question was provided immediately after the discovery of the eukaryotic condensin I complex: it was shown that condensin I purified from Xenopus egg extracts has the ability to introduce (+) superhelical torsion into DNA in an ATPdependent manner [19,20]. This activity was stimulated in a mitosis-specific manner through direct phosphorylation of condensin subunits by cyclin-dependent kinase (Cdk)1 [21], implicating that it represents one of the physiologically relevant activities that drive chromosome condensation during mitosis. Although condensin complexes purified from humans, Caenorhabditis elegans, and Saccharomyces cerevisiae also possess the same activity in vitro [22–24], attempts to search for the corresponding activity in bacterial condensins have so far been unsuccessful (e.g., [12]). It therefore seems likely that the ATPdependent introduction of (+) superhelical torsion into DNA is an activity that is unique to eukaryotic condensins. Somewhat puzzlingly, however, single-molecule analysis has failed to detect the supercoiling-dependence of ‘the
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interactions of condensin with naked DNA [25], leaving many mechanistic questions that remain to be resolved. Mechanical and functional responses of nucleosome fibers to torsional stress: a role for condensins? Here, I wish to set up two sets of questions regarding the action of condensins in eukaryotic genome packaging. First, if the ability of condensins to introduce supercoiling does indeed contribute to the assembly of mitotic chromosomes, why do they utilize (+) superhelical rather than ( ) superhelical torsion? By analogy to bacterial nucleoids, could additional ( ) supercoiling be sufficient to build eukaryotic chromosomes? Second, little is currently known about how condensins might work on nucleosome fibers. Could condensins have the capacity to impose torsional stress on nucleosome fibers as well as on DNA? If so, exactly how do the nucleosome fibers respond to such stress? I argue below that understanding the mechanical properties of nucleosome fibers is crucial in addressing and answering these sets of questions. How might eukaryotic condensins work on nucleosome arrays? A pioneering study by Strick et al. (1996) used magnetic tweezers to probe structural elasticity of single DNA molecules in response to torsional stress [26]. More recently, Bancaud et al. (2006) applied the same technique to single nucleosome arrays to study their mechanical response under torsion [27]. It was shown that nucleosome arrays are more resilient compared to naked DNA, accommodating a large amount of either ( ) or (+) torsional stress without much change in their length (Figure 2A). The authors reasoned that this is because torsional stress imposed on nucleosome arrays first emerges as changes in crossing statuses of the entry/exit DNA of nucleosomes. According to a three-state model, when a nucleosome array is subjected to ( ) torsional stress, ( ) crossings accumulate at the nucleosome nodes. Once all nodes are converted into ( ) crossings, additional rotations create a ( ) plectonemic supercoiled array of nucleosomes, resulting in a rapid decrease in length. The same is true for (+) torsional stress, although (+) plectonemic supercoiling may be initiated before all nodes are converted into (+) crossings. It is important to note that this series of structural changes of nucleosome arrays occurs under the condition where nucleosome core structures are not largely affected. By contrast, it has been hypothesized that higher levels of (+) torsion might induce a chiral transition of nucleosomes, resulting in the formation of ‘reversomes’ [28,29]. How could condensins have an impact on this mechanical response? I propose here that condensins might introduce (+) superhelical torsion into nucleosome arrays in an ATPdependent manner, as has been shown for naked DNA [19,20], thereby facilitating (+) node conversion of nucleosomes (Figure 2B). Without active introduction of torsional stress, nucleosome arrays would be relaxed, having a mixture of open and ( ) crossing conformations [27]. These conformations likely correspond to the default state of interphase chromatin (Figure 3A). Upon entry into mitosis, condensins could act on a region with open nucleosomes (or a nucleosome-free region) and initiate the reaction of (+) node 3
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Figure 2. A model for the action of eukaryotic condensins on nucleosome arrays. (A) An extension-versus-rotation curve at a fixed force in a single nucleosome-array experiment [27]. The nucleosome assay used was 7.5-kb long (36 nucleosomes on average), sandwiched between two naked DNA spacers (0.6 kb each). According to the authors’ three-state model, individual nucleosomes can accommodate different crossing statuses of the entry/exit DNA, namely, ( ) crossings, open and (+) crossings. The nucleosome arrays #1, #2 and #3 are dominated by nucleosomes with ( ), open and (+) conformations, respectively. When more (+) rotations are introduced into the array structure #3, (+) plectonemic loops are formed, resulting in a rapid decrease in length. Adapted from [27]. (B) In this model, condensins are hypothesized to cause the node conversion of nucleosomes by introducing (+) superhelical torsion into DNA. Topoisomerase II (topo II) is predicted to cooperate with condensin to modulate this reaction. In principle, the strand passage activity of topo II is able to support the reaction in both directions. When ‘the action of condensin is robust in early mitosis, topo II might help accumulate (+) crossings by removing compensatory ( ) crossings. When the action of condensin attenuates in late mitosis, topo II would help convert (+) crossings back into ( ) crossings, thereby contributing to decondensation of chromosomes. (C) Binding of histone H1 (red) to linker DNA could limit the rate of node conversion, whereas phosphorylation of H1 (shown in yellow) could reverse such a suppressive effect. The blue triangle indicates the nucleosome dyad.
conversion similar to a nucleation step (Figure 3B). Torsional stress propagates in cis, therefore, the (+) node conversion events would spread along a nucleosome fiber (Figure 3C). When a certain level of nucleosomes is converted into the (+) configuration (Figure 3D), the fiber would start forming (+) plectonemic, nucleosome loops (Figure 3E; [27]). These structural changes would naturally bring multiple condensin complexes together at the base of the loops, thereby promoting the assembly of a precursor of the chromatid axis. It is anticipated that topoisomerase II, the major relaxase of nucleosomal DNA [30], supports the series of events through its DNA strand passage activity (Figure 2B). In fact, numerous studies have observed functional cooperation between topoisomerase II and condensins; both of which are major components of mitotic chromosomes enriched along their axis [31–33]. A possible contribution of higher-order coiling of chromatin fibers to mitotic chromosome structure is not a new idea in the field (e.g., [34]). Indeed, previous studies of condensins led to two models of chromosome condensation that involve active manipulations of DNA topology [17], although neither of these models considered the mechanistic response of nucleosome fibers to torsional stress. Another prevailing model in the field suggests that condensins might function as crosslinkers that constitute the network of a folded chromatin fiber (e.g., [18,35]). However, this model does not explain how condensins might work as 4
intrachromosomal crosslinkers rather than interchromosomal crosslinkers. By contrast, propagation of superhelical torsion operates only in cis, and therefore is a prime candidate for the active mechanism that drives large structural changes of individual chromatin fibers. The idea of node conversion might further our understanding of chromosome architecture. First, (+) superhelical torsion prevents unwinding of double-stranded DNA segments in the context of both naked DNA molecules [26] and nucleosome fibers [27]. Moreover, due to the intrinsic chirality of DNA, double helices are packed more stably into (+) crossings than ( ) crossings [36]. Thus, (+) superhelical torsion, as opposed to ( ) superhelical torsion, would effectively help stabilize DNA as well as its folded structure. Second, the potential involvement of histone modifications in regulating nucleosome arrays could be viewed from a new angle. For instance, the exact function of mitotic phosphorylation of linker histones has long been debated [37]. The globular domain of histone H1 binds near the nucleosome dyad, while its C-terminal tail holds linker DNA [38]. One possibility could be that H1 binding limits the rate of node conversion, whereas its mitotic phosphorylation liberates this suppression (Figure 2C). Intriguingly, the N-terminal tail of histone H3, another major target of mitotic phosphorylation, is also located near the nucleosome dyad, implicating its involvement in the regulation of node conversion.
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Figure 3. (+) Superhelical torsion might spread along a chromatin fiber and contribute to its folding. (A) In an interphase chromatin fiber, the default status would be a mixture of nucleosomes with open and ( ) crossing configurations. (B,C) In early mitosis, condensins might act on an open region to initiate the node conversion reaction (nucleation). The (+) configuration might then spread along the chromatin fiber in both directions (spreading). When an advancing RNA polymerase encounters a wave of (+) crossings approaching from the opposite direction, the polymerase would stall, thereby preventing transcriptional elongation. (D,E) When (+) crossings accumulate to a threshold, the fiber starts undergoing large structural changes, forming (+) plectonemic loops [27], which in turn bring multiple condensin complexes together at the base of the loops, assembling the chromatid axis.
Could (+) torsional stress make dual contributions to chromosome compaction and gene repression? The conformation of interphase chromatin fibers dynamically changes in response to numerous chromosomal activities, including transcription and replication [39]. For instance, progression of RNA polymerase produces a (+) supercoiled domain ahead of it and a ( ) supercoiled domain behind it [40] (Figure 3A). Such superhelical torsion would first be absorbed into the nucleosome fiber and eventually be removed by the action of topoisomerases [41]. What would happen to transcription when condensins start introducing (+) torsional stress along a chromatin fiber? When an advancing RNA polymerase encounters the wave of (+) torsional stress approaching from the opposite direction, the movement of the polymerase would be blocked, thereby shutting down ongoing transcription [41,42]. Although multiple mechanisms are known to be responsible for transcriptional repression during mitosis (e.g., [43]), the torsion-mediated mechanism is unique because it can operate along the entire length of the chromosome (Figure 3B,C). If correct, then spreading of (+) torsional stress would make dual contributions to chromosome compaction and gene repression during mitosis. Torsion-mediated gene repression during mitosis may also provide mechanistic implications in our understanding of dosage compensation in C. elegans, which utilizes a
condensin-like complex (condensin IDC) to repress the transcript level from both X chromosomes by half [44]. It remains unknown exactly how such fine-tuning of gene repression might be achieved at a mechanistic level. Also unknown is how dosage compensation machinery might act at a distance to control gene expression along the X chromosome [45]. The formation and propagation of (+) nucleosome nodes proposed here could function as a highly flexible and elaborate scheme for establishing and maintaining chromosome-wide gene repression. Finally, it may be worth considering the naı¨ve question of why nucleosomes might be retained during mitosis. If the final goal of chromosome condensation were to simply pack genomic DNA as compact as possible, one extreme strategy would be to discard all histones before entry into mitosis. It is easy to imagine, however, that complete (or even substantial) loss of nucleosomes during mitosis would be problematic when cells exit mitosis, because they have to reassemble nucleosomes de novo and re-establish all epigenetic makers. It would therefore be reasonable to pack genomic DNA while keeping individual nucleosome structures intact during mitosis. Together with the mechanistic considerations discussed above, it is tempting to speculate that eukaryotic condensins have coevolved with histones and nucleosome structures. Moreover, the ability of eukaryotic condensins to introduce (+) superhelical torsion could contribute not only to the stable folding of 5
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nucleosome fibers, but also to decatenation of duplicated nucleosome fibers for their segregation in mitosis [46]. Concluding remarks To read genetic information stored within DNA, bacteria and eukaryotes have evolved different strategies to destabilize and unwind its double-helical structure (Figure 4). In bacteria, DNA gyrase maintains ( ) supercoils largely in an unconstrained form, and condensins are most likely to contribute to higher-order organization of the ( ) supercoiled loops (Figure 4A). It has been speculated that (+) supercoiling would potentially be harmful in mesophilic bacteria: in fact, reverse gyrase, a specialized type I topoisomerase capable of introducing (+) supercoils into DNA, is found only in thermophilic species ([47]). By contrast, eukaryotes store ( ) supercoils in a constrained form within individual nucleosomes, and additional superhelical stress [either ( ) or (+)] can be accommodated in nucleosome arrays. Like bacteria, eukaryotes also utilize ( ) superhelical torsion to support chromosomal activities such as transcription during interphase (Figure 4B). As opposed to bacterial condensins, eukaryotic condensins work when most transcriptional activities are repressed. For this reason, eukaryotic condensins might have devised the unique strategy of utilizing (+) superhelical torsion to build and stabilize mitotic chromosomes (Figure 4C). Although many of the ideas presented here are highly speculative at present, to test rigorously and further
extend these ideas, two lines of future research will be of particular interest. First, it is important to understand mechanistically how eukaryotic condensins interact with nucleosome fibers and potentially induce their conformational change. Single-molecule analyses using nucleosome arrays should provide vital information about their mechanical response to torsional stress imposed by condensins. Potential functional crosstalk between condensins and mitosis-specific histone modifications is also an important issue that needs to be clarified [48]. Second, genomewide approaches should be applied to probe structural differences between interphase chromatin and mitotic chromosomes. For instance, a recent Hi-C analysis in human cells has shown that megabase-sized local structures of interphase chromatin, known as the topologically associated domains (TADs), disappear during mitosis [49]. Combined with computer simulations, the authors propose that mitotic chromosomes are homogeneous structures in which an array of consecutive loops is linearly organized and compressed. This simple picture of mitotic chromosome organization is largely compatible with the mechanism proposed above. Equally exciting, a psoralenmediated method to probe the superhelical state of DNA has successfully been applied to the mammalian genome, and demonstrated that transcriptional activities are responsible for creating negatively supercoiled domains [50]. It will be highly informative to perform the same type of experiments using mitotically synchronized cell populations.
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Figure 4. A hypothetical view of torsion-mediated genome organization in bacteria and eukaryotes. (A) The roles of unconstrained ( ) supercoils and bacterial condensins in bacterial genome organization are depicted. (B) In eukaryotes, nucleosomes with ( ) crossings or an open configuration produce dynamic chromatin fibers during interphase. (C) During mitosis, eukaryotic condensins convert the nucleosome nodes into (+) crossings and form (+) plectonemic nucleosome loops. The series of structural changes contribute to not only mitotic chromosome condensation but also transcriptional repression.
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Opinion In the future, critical comparisons between bacteria and eukaryotes will become even more important in answering the fundamental question of how evolution has shaped the balancing acts between genome packaging and genome function. Acknowledgments I am grateful to H. Kurumizaka (Waseda University) and J. Marko (Northwestern University) for insightful discussions. I also thank H. Kurumizaka and members of the Hirano laboratory for critically reading the manuscript. The work in the author’s laboratory was supported by Grant-in-Aid for Specially Promoted Research and Grant-in-Aid for Scientific Research A.
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chromatid segregation during mitosis and meiosis. Genes Dev. 16, 729–742 St-Pierre, J. et al. (2009) Polo kinase regulates mitotic chromosome condensation by hyperactivation of condensin DNA supercoiling activity. Mol. Cell 34, 416–426 Kimura, K. et al. (2001) Chromosome condensation by a human condensin complex in Xenopus egg extracts. J. Biol. Chem. 276, 5417–5420 Strick, T.R. et al. (2004) Real-time detection of single-molecule DNA compaction by condensin I. Curr. Biol. 14, 874–880 Strick, T.R. et al. (1996) The elasticity of a single supercoiled DNA molecule. Science 271, 1835–1837 Bancaud, A. et al. (2006) Structural plasticity of single chromatin fibers revealed by torsional manipulation. Nat. Struct. Mol. Biol. 13, 444–450 Bancaud, A. et al. (2007) Nucleosome chiral transition under positive torsional stress in single chromatin fibers. Mol. Cell 27, 135–147 Recouvreux, P. et al. (2011) Linker histones incorporation maintains chromatin fiber plasticity. Biophys. J. 100, 2726–2735 Salceda, J. et al. (2006) Topoisomerase II, not topoisomerase I, is the proficient relaxase of nucleosomal DNA. EMBO J. 25, 2575–2583 Hirano, T. and Mitchison, T.J. (1994) A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro. Cell 79, 449–458 Saitoh, N. et al. (1994) ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure. J. Cell Biol. 127, 303–318 Ohta, S. et al. (2010) The protein composition of mitotic chromosomes determined using multiclassifier combinatorial proteomics. Cell 142, 810–821 Rattner, J.B. (1992) Integrating chromosome structure with function. Chromosoma 101, 259–264 Cuylen, S. and Haering, C.H. (2011) Deciphering condensin action during chromosome segregation. Trends Cell Biol. 21, 552–559 Timsit, Y. and Va´rnai, P. (2010) Helical chirality: a link between local interactions and global topology in DNA. PLoS ONE 5, e9326 Wang, F. and Higgins, J.M.G. (2013) Histone modifications and mitosis: countermarks, landmarks, and bookmarks. Trends Cell Biol. 23, 175–184 Zhou, B-R. et al. (2013) Structural insights into the histone H1nucleosome complex. Proc. Natl. Acad. Sci. U.S.A. 110, 19390–19395 Wang, J.C. (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat. Rev. Mol. Cell Biol. 3, 430–440 Liu, L.F. and Wang, J.C. (1987) Supercoiling of the DNA template during transcription. Proc. Natl. Acad. Sci. U.S.A. 84, 7024–7027 Joshi, R.S. et al. (2012) Topoisomerase II is required for the production of long Pol II gene transcripts in yeast. Nucleic Acids Res. 40, 7907– 7915 Schultz, M.C. et al. (1992) Topoisomerases and yeast rRNA transcription: negative supercoiling stimulates initiation and topoisomerase activity is required for elongation. Genes Dev. 6, 1332–1341 Gottesfeld, J.M. and Forbes, D.J. (1997) Mitotic repression of the transcriptional machinery. Trends Biochem. Sci. 22, 197–202 Meyer, B.J. (2010) Targeting X chromosomes for repression. Curr. Opin. Genet. Dev. 20, 179–189 Jans, J. et al. (2009) A condensin-like dosage compensation complex acts at a distance to control expression throughout the genome. Genes Dev. 23, 602–618 Baxter, J. et al. (2011) Positive supercoiling of mitotic DNA drives decatenation by topoisomerase II in eukaryotes. Science 331, 1328– 1332 Valenti, A. et al. (2011) Positive supercoiling in thermophiles and mesophiles: of the good and evil. Biochem. Soc. Trans. 39, 58–63 Wilkins, B.J. et al. (2014) A cascade of histone modifications induces chromatin condensation in mitosis. Science 343, 77–80 Naumova, N. et al. (2013) Organization of the mitotic chromosome. Science 342, 948–953 Naughton, C. et al. (2013) Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures. Nat. Struct. Mol. Biol. 20, 387–395
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Condensins and the evolution of torsion-mediated genome organization Tatsuya Hirano Chromosome Dynamics Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
At first glance, bacteria and eukaryotes appear to use different strategies to pack and organize their genomes. At the basal level, bacterial genome compaction relies on unconstrained, negative supercoils, whereas eukaryotic genomes are packaged into nucleosomes via constrained, negative supercoils. Here, I integrate the action of condensins, chromosome-packaging complexes conserved from bacteria to humans, into this picture, and discuss how torsional stress on DNA might have dual impacts on genome organization and function. A common theme is that organisms have evolved flexible and reversible strategies to pack their genomes while keeping them readily accessible to many activities such as gene expression. ‘Nothing in biology makes sense except in the light of evolution’, T. Dobzhansky (1973)[1].
Pack the genomes, but not too tightly Genomes in all organisms face two big challenges [2,3]. Long DNA molecules comprising a genome must be packaged tightly to fit within the limited spaces of the cell. At the same time, they must be readily accessible to many activities such as gene expression. How do they deal with these apparently conflicting problems? One of the crucial strategies used by bacteria is to introduce negative ( ) supercoils through the action of a specialized type II topoisomerase known as DNA gyrase (see Glossary and Box 1 for the basics of DNA topology). Although a group of small proteins associate with genomic DNA, roughly half of the ( ) supercoils are present in an unconstrained, plectonemic form in bacterial cells [3], thereby allowing local unwinding of DNA that promotes replication and transcription (Figure 1A). In eukaryotes, genomic DNA is also negatively supercoiled, but most of the supercoils are present in a constrained, toroidal form within nucleosomes (Figure 1B). Loss or conformational changes of nucleosomes not only make transcription factors accessible, but also introduce local changes of DNA structure, including unwinding. It is also important to note that the two types of ( ) supercoils (unconstrained or constrained) contribute to Corresponding author: Hirano, T. ([email protected]). Keywords: DNA supercoiling; nucleosomes; condensins; chromosomes; genomes; evolution. 0962-8924/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2014.06.007
the compaction of DNA molecules (Figure 1A,B). Thus, in both bacteria and eukaryotes, the superhelical status of DNA plays a fundamental role in the structural and functional organization of genomes. On top of this basal level of organization, both bacteria and eukaryotes possess a higher-order genome-packaging system, which is mediated by a class of multisubunit protein complexes, known as condensins [4]. Although it has been suggested that condensins also modulate or cooperate with torsional stress for genome organization, the importance of DNA topology is often overlooked in modern cell biology, and comparisons between bacterial and eukaryotic systems are rarely discussed, with only a few exceptions [2,3,5,6]. Here, I focus on the role of condensins, proposing that superhelical torsion imposed on DNA might have dual impacts on higher-order genome organization and function. The purpose of this article is not to summarize well-established concepts: rather I attempt to offer bold speculations that might help stimulate further discussions in the field. Bacterial genome organization: beyond unconstrained negative supercoils The condensin complex was originally identified in Xenopus egg extracts as a major component of metaphase Glossary Condensins: a class of large protein complexes involved in chromosome condensation and segregation. DNA topology: a structural property of DNA derived from its intrinsic doublehelical nature. Depending on how many times the two strands cross each other, a circular DNA molecule is subject to topological strain and can have different configurations (see Figure I in Box 1). A linear DNA molecule is also subject to topological strains when it is very long and interacts with intracellular structures. Negative supercoil/(S) supercoil: a type of supercoils in which DNA has fewer helical turns than would be expected for the typical (B form) structure. An underwound form of DNA produces negative supercoils. Node conversion: an event converting the crossing statuses of entry/exit DNA of nucleosomes. Nucleosome array: a DNA segment on which nucleosomes are arrayed at regular intervals. Nucleosome fiber: a nucleoprotein fiber composed of DNA and histones. Plectonemic supercoil: a type of supercoils in which a double-helical DNA molecule is twisted over itself. This type can be present as an unconstrained form without protein binding. Positive supercoil/(+) supercoil: a type of supercoils in which DNA has more helical turns than would be expected for the typical (B form) structure. An overwound form of DNA produces positive supercoils. Superhelical torsion: an action of twisting that changes the superhelical state of DNA. Topoisomerases: a class of enzymes that changes the topological state of DNA by transiently cutting and re-ligating one or two of the DNA strands. Toroidal supercoil: a type of supercoils in which a double-helical DNA molecule is wound in a cylindrical manner. This type is stabilized when it is constrained by protein binding.
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Box 1. Basics of DNA topology Shown here are different topological forms of a closed circular DNA. A negatively supercoiled DNA can be present as different forms, namely, ( ) plectonemic, ( ) toroidal (not shown) and underwound forms. Likewise, a positively supercoiled DNA is present as (+) plectonemic, (+) toroidal (not shown) and overwound forms. These forms are interconvertible without cutting DNA strands. By contrast, topoisomerases change the DNA topology by transiently cutting and
Underwound
religating one or two of the DNA strands [39]. For instance, eukaryotic topoisomerase I (euk topo I) can relax both ( ) and (+) supercoils, whereas Escherichia coil topoisomerase I (E. coli topo I) relaxes ( ) supercoils only. Bacteria possess DNA gyrase that introduces ( ) supercoils in an ATP-dependent manner. Some thermophilic bacteria have a unique topoisomerase (reverse gyrase) that is able to introduce (+) supercoils in an ATP-dependent manner (Figure I).
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Figure I. Basics of DNA topology.
chromosomes that plays a crucial role in mitotic chromosome assembly [7]. Subsequent studies demonstrated that many bacteria possess similar complexes (henceforth referred to as bacterial condensins) whose mutations cause defects in
nucleoid organization and segregation [8,9]. Interestingly, defects in condensin mutants are suppressed when excess ( ) supercoils are introduced in a topoisomerase mutant background [10], implicating that ( ) supercoiling and Replicaon/ transcripon machinery
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Figure 1. The basal level of genome organization in bacteria and eukaryotes. (A) In bacteria, roughly half of ( ) supercoils are present in an unconstrained, plectonemic form, which allows local unwinding of DNA required for replication and transcription. A group of proteins that partially constrain ( ) supercoils are indicated by the ovals with different colors. (B) In eukaryotes, most ( ) supercoils are present in a constrained, toroidal form within nucleosomes (indicated in the yellow discs). Loss or remodeling of nucleosomes causes local changes in DNA structure, making it possible for the replication/transcription machinery to work.
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Opinion condensins synergistically contribute to proper compaction and segregation of bacterial nucleoids. When considering the structural organization of bacterial nucleoids, it is important to keep in mind that there are no discrete cell-cycle stages in rapidly dividing bacteria. Bacterial condensins must therefore organize and segregate their genomes at the same time that they are replicated and actively transcribed [8,9]. Thus, the primary target of bacterial condensins is most likely to be ( ) supercoiled DNA. Consistent with this prediction, it has been shown that the core subunit of bacterial condensins displays a binding preference to ( ) supercoils over (+) supercoils [11]. It is not fully understood, however, to what extent bacterial condensins might have the ability to actively introduce superhelical torsion into DNA [12]. Currently available lines of evidence suggest that bacterial condensins could support genome organization either as a protein clamp [13] or through large molecular assembly [14]. Eukaryotic genome organization: beyond constrained negative supercoils The nucleosome, the fundamental unit of eukaryotic chromatin, wraps DNA in a left-handed direction. Consequently, ( ) supercoils are constrained by histone octamers and present as a toroidal form within nucleosomes. During the past two decades, it has become increasingly clear that the nucleosome structure is highly dynamic: mobilization or disruption of nucleosomes occurs locally, providing an opportunity to regulate gene expression precisely in locus-specific manners during interphase [15]. In contrast to bacterial condensins, eukaryotic condensins function in a relatively short period of the cell cycle (i.e., mitosis) when duplicated chromosomes undergo large conformational changes to prepare for their segregation. Equally important, gene expression is largely suppressed during this stage of the eukaryotic cell cycle. Despite the long history of research, however, it remains unknown exactly how nucleosome fibers might be folded and organized into rod-shaped, mitotic chromosomes [16–18]. Could torsional stress be involved, at least in part, in the assembly of mitotic chromosomes? A vital hint to this question was provided immediately after the discovery of the eukaryotic condensin I complex: it was shown that condensin I purified from Xenopus egg extracts has the ability to introduce (+) superhelical torsion into DNA in an ATPdependent manner [19,20]. This activity was stimulated in a mitosis-specific manner through direct phosphorylation of condensin subunits by cyclin-dependent kinase (Cdk)1 [21], implicating that it represents one of the physiologically relevant activities that drive chromosome condensation during mitosis. Although condensin complexes purified from humans, Caenorhabditis elegans, and Saccharomyces cerevisiae also possess the same activity in vitro [22–24], attempts to search for the corresponding activity in bacterial condensins have so far been unsuccessful (e.g., [12]). It therefore seems likely that the ATPdependent introduction of (+) superhelical torsion into DNA is an activity that is unique to eukaryotic condensins. Somewhat puzzlingly, however, single-molecule analysis has failed to detect the supercoiling-dependence of ‘the
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interactions of condensin with naked DNA [25], leaving many mechanistic questions that remain to be resolved. Mechanical and functional responses of nucleosome fibers to torsional stress: a role for condensins? Here, I wish to set up two sets of questions regarding the action of condensins in eukaryotic genome packaging. First, if the ability of condensins to introduce supercoiling does indeed contribute to the assembly of mitotic chromosomes, why do they utilize (+) superhelical rather than ( ) superhelical torsion? By analogy to bacterial nucleoids, could additional ( ) supercoiling be sufficient to build eukaryotic chromosomes? Second, little is currently known about how condensins might work on nucleosome fibers. Could condensins have the capacity to impose torsional stress on nucleosome fibers as well as on DNA? If so, exactly how do the nucleosome fibers respond to such stress? I argue below that understanding the mechanical properties of nucleosome fibers is crucial in addressing and answering these sets of questions. How might eukaryotic condensins work on nucleosome arrays? A pioneering study by Strick et al. (1996) used magnetic tweezers to probe structural elasticity of single DNA molecules in response to torsional stress [26]. More recently, Bancaud et al. (2006) applied the same technique to single nucleosome arrays to study their mechanical response under torsion [27]. It was shown that nucleosome arrays are more resilient compared to naked DNA, accommodating a large amount of either ( ) or (+) torsional stress without much change in their length (Figure 2A). The authors reasoned that this is because torsional stress imposed on nucleosome arrays first emerges as changes in crossing statuses of the entry/exit DNA of nucleosomes. According to a three-state model, when a nucleosome array is subjected to ( ) torsional stress, ( ) crossings accumulate at the nucleosome nodes. Once all nodes are converted into ( ) crossings, additional rotations create a ( ) plectonemic supercoiled array of nucleosomes, resulting in a rapid decrease in length. The same is true for (+) torsional stress, although (+) plectonemic supercoiling may be initiated before all nodes are converted into (+) crossings. It is important to note that this series of structural changes of nucleosome arrays occurs under the condition where nucleosome core structures are not largely affected. By contrast, it has been hypothesized that higher levels of (+) torsion might induce a chiral transition of nucleosomes, resulting in the formation of ‘reversomes’ [28,29]. How could condensins have an impact on this mechanical response? I propose here that condensins might introduce (+) superhelical torsion into nucleosome arrays in an ATPdependent manner, as has been shown for naked DNA [19,20], thereby facilitating (+) node conversion of nucleosomes (Figure 2B). Without active introduction of torsional stress, nucleosome arrays would be relaxed, having a mixture of open and ( ) crossing conformations [27]. These conformations likely correspond to the default state of interphase chromatin (Figure 3A). Upon entry into mitosis, condensins could act on a region with open nucleosomes (or a nucleosome-free region) and initiate the reaction of (+) node 3
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Figure 2. A model for the action of eukaryotic condensins on nucleosome arrays. (A) An extension-versus-rotation curve at a fixed force in a single nucleosome-array experiment [27]. The nucleosome assay used was 7.5-kb long (36 nucleosomes on average), sandwiched between two naked DNA spacers (0.6 kb each). According to the authors’ three-state model, individual nucleosomes can accommodate different crossing statuses of the entry/exit DNA, namely, ( ) crossings, open and (+) crossings. The nucleosome arrays #1, #2 and #3 are dominated by nucleosomes with ( ), open and (+) conformations, respectively. When more (+) rotations are introduced into the array structure #3, (+) plectonemic loops are formed, resulting in a rapid decrease in length. Adapted from [27]. (B) In this model, condensins are hypothesized to cause the node conversion of nucleosomes by introducing (+) superhelical torsion into DNA. Topoisomerase II (topo II) is predicted to cooperate with condensin to modulate this reaction. In principle, the strand passage activity of topo II is able to support the reaction in both directions. When ‘the action of condensin is robust in early mitosis, topo II might help accumulate (+) crossings by removing compensatory ( ) crossings. When the action of condensin attenuates in late mitosis, topo II would help convert (+) crossings back into ( ) crossings, thereby contributing to decondensation of chromosomes. (C) Binding of histone H1 (red) to linker DNA could limit the rate of node conversion, whereas phosphorylation of H1 (shown in yellow) could reverse such a suppressive effect. The blue triangle indicates the nucleosome dyad.
conversion similar to a nucleation step (Figure 3B). Torsional stress propagates in cis, therefore, the (+) node conversion events would spread along a nucleosome fiber (Figure 3C). When a certain level of nucleosomes is converted into the (+) configuration (Figure 3D), the fiber would start forming (+) plectonemic, nucleosome loops (Figure 3E; [27]). These structural changes would naturally bring multiple condensin complexes together at the base of the loops, thereby promoting the assembly of a precursor of the chromatid axis. It is anticipated that topoisomerase II, the major relaxase of nucleosomal DNA [30], supports the series of events through its DNA strand passage activity (Figure 2B). In fact, numerous studies have observed functional cooperation between topoisomerase II and condensins; both of which are major components of mitotic chromosomes enriched along their axis [31–33]. A possible contribution of higher-order coiling of chromatin fibers to mitotic chromosome structure is not a new idea in the field (e.g., [34]). Indeed, previous studies of condensins led to two models of chromosome condensation that involve active manipulations of DNA topology [17], although neither of these models considered the mechanistic response of nucleosome fibers to torsional stress. Another prevailing model in the field suggests that condensins might function as crosslinkers that constitute the network of a folded chromatin fiber (e.g., [18,35]). However, this model does not explain how condensins might work as 4
intrachromosomal crosslinkers rather than interchromosomal crosslinkers. By contrast, propagation of superhelical torsion operates only in cis, and therefore is a prime candidate for the active mechanism that drives large structural changes of individual chromatin fibers. The idea of node conversion might further our understanding of chromosome architecture. First, (+) superhelical torsion prevents unwinding of double-stranded DNA segments in the context of both naked DNA molecules [26] and nucleosome fibers [27]. Moreover, due to the intrinsic chirality of DNA, double helices are packed more stably into (+) crossings than ( ) crossings [36]. Thus, (+) superhelical torsion, as opposed to ( ) superhelical torsion, would effectively help stabilize DNA as well as its folded structure. Second, the potential involvement of histone modifications in regulating nucleosome arrays could be viewed from a new angle. For instance, the exact function of mitotic phosphorylation of linker histones has long been debated [37]. The globular domain of histone H1 binds near the nucleosome dyad, while its C-terminal tail holds linker DNA [38]. One possibility could be that H1 binding limits the rate of node conversion, whereas its mitotic phosphorylation liberates this suppression (Figure 2C). Intriguingly, the N-terminal tail of histone H3, another major target of mitotic phosphorylation, is also located near the nucleosome dyad, implicating its involvement in the regulation of node conversion.
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Figure 3. (+) Superhelical torsion might spread along a chromatin fiber and contribute to its folding. (A) In an interphase chromatin fiber, the default status would be a mixture of nucleosomes with open and ( ) crossing configurations. (B,C) In early mitosis, condensins might act on an open region to initiate the node conversion reaction (nucleation). The (+) configuration might then spread along the chromatin fiber in both directions (spreading). When an advancing RNA polymerase encounters a wave of (+) crossings approaching from the opposite direction, the polymerase would stall, thereby preventing transcriptional elongation. (D,E) When (+) crossings accumulate to a threshold, the fiber starts undergoing large structural changes, forming (+) plectonemic loops [27], which in turn bring multiple condensin complexes together at the base of the loops, assembling the chromatid axis.
Could (+) torsional stress make dual contributions to chromosome compaction and gene repression? The conformation of interphase chromatin fibers dynamically changes in response to numerous chromosomal activities, including transcription and replication [39]. For instance, progression of RNA polymerase produces a (+) supercoiled domain ahead of it and a ( ) supercoiled domain behind it [40] (Figure 3A). Such superhelical torsion would first be absorbed into the nucleosome fiber and eventually be removed by the action of topoisomerases [41]. What would happen to transcription when condensins start introducing (+) torsional stress along a chromatin fiber? When an advancing RNA polymerase encounters the wave of (+) torsional stress approaching from the opposite direction, the movement of the polymerase would be blocked, thereby shutting down ongoing transcription [41,42]. Although multiple mechanisms are known to be responsible for transcriptional repression during mitosis (e.g., [43]), the torsion-mediated mechanism is unique because it can operate along the entire length of the chromosome (Figure 3B,C). If correct, then spreading of (+) torsional stress would make dual contributions to chromosome compaction and gene repression during mitosis. Torsion-mediated gene repression during mitosis may also provide mechanistic implications in our understanding of dosage compensation in C. elegans, which utilizes a
condensin-like complex (condensin IDC) to repress the transcript level from both X chromosomes by half [44]. It remains unknown exactly how such fine-tuning of gene repression might be achieved at a mechanistic level. Also unknown is how dosage compensation machinery might act at a distance to control gene expression along the X chromosome [45]. The formation and propagation of (+) nucleosome nodes proposed here could function as a highly flexible and elaborate scheme for establishing and maintaining chromosome-wide gene repression. Finally, it may be worth considering the naı¨ve question of why nucleosomes might be retained during mitosis. If the final goal of chromosome condensation were to simply pack genomic DNA as compact as possible, one extreme strategy would be to discard all histones before entry into mitosis. It is easy to imagine, however, that complete (or even substantial) loss of nucleosomes during mitosis would be problematic when cells exit mitosis, because they have to reassemble nucleosomes de novo and re-establish all epigenetic makers. It would therefore be reasonable to pack genomic DNA while keeping individual nucleosome structures intact during mitosis. Together with the mechanistic considerations discussed above, it is tempting to speculate that eukaryotic condensins have coevolved with histones and nucleosome structures. Moreover, the ability of eukaryotic condensins to introduce (+) superhelical torsion could contribute not only to the stable folding of 5
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nucleosome fibers, but also to decatenation of duplicated nucleosome fibers for their segregation in mitosis [46]. Concluding remarks To read genetic information stored within DNA, bacteria and eukaryotes have evolved different strategies to destabilize and unwind its double-helical structure (Figure 4). In bacteria, DNA gyrase maintains ( ) supercoils largely in an unconstrained form, and condensins are most likely to contribute to higher-order organization of the ( ) supercoiled loops (Figure 4A). It has been speculated that (+) supercoiling would potentially be harmful in mesophilic bacteria: in fact, reverse gyrase, a specialized type I topoisomerase capable of introducing (+) supercoils into DNA, is found only in thermophilic species ([47]). By contrast, eukaryotes store ( ) supercoils in a constrained form within individual nucleosomes, and additional superhelical stress [either ( ) or (+)] can be accommodated in nucleosome arrays. Like bacteria, eukaryotes also utilize ( ) superhelical torsion to support chromosomal activities such as transcription during interphase (Figure 4B). As opposed to bacterial condensins, eukaryotic condensins work when most transcriptional activities are repressed. For this reason, eukaryotic condensins might have devised the unique strategy of utilizing (+) superhelical torsion to build and stabilize mitotic chromosomes (Figure 4C). Although many of the ideas presented here are highly speculative at present, to test rigorously and further
extend these ideas, two lines of future research will be of particular interest. First, it is important to understand mechanistically how eukaryotic condensins interact with nucleosome fibers and potentially induce their conformational change. Single-molecule analyses using nucleosome arrays should provide vital information about their mechanical response to torsional stress imposed by condensins. Potential functional crosstalk between condensins and mitosis-specific histone modifications is also an important issue that needs to be clarified [48]. Second, genomewide approaches should be applied to probe structural differences between interphase chromatin and mitotic chromosomes. For instance, a recent Hi-C analysis in human cells has shown that megabase-sized local structures of interphase chromatin, known as the topologically associated domains (TADs), disappear during mitosis [49]. Combined with computer simulations, the authors propose that mitotic chromosomes are homogeneous structures in which an array of consecutive loops is linearly organized and compressed. This simple picture of mitotic chromosome organization is largely compatible with the mechanism proposed above. Equally exciting, a psoralenmediated method to probe the superhelical state of DNA has successfully been applied to the mammalian genome, and demonstrated that transcriptional activities are responsible for creating negatively supercoiled domains [50]. It will be highly informative to perform the same type of experiments using mitotically synchronized cell populations.
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Figure 4. A hypothetical view of torsion-mediated genome organization in bacteria and eukaryotes. (A) The roles of unconstrained ( ) supercoils and bacterial condensins in bacterial genome organization are depicted. (B) In eukaryotes, nucleosomes with ( ) crossings or an open configuration produce dynamic chromatin fibers during interphase. (C) During mitosis, eukaryotic condensins convert the nucleosome nodes into (+) crossings and form (+) plectonemic nucleosome loops. The series of structural changes contribute to not only mitotic chromosome condensation but also transcriptional repression.
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Opinion In the future, critical comparisons between bacteria and eukaryotes will become even more important in answering the fundamental question of how evolution has shaped the balancing acts between genome packaging and genome function. Acknowledgments I am grateful to H. Kurumizaka (Waseda University) and J. Marko (Northwestern University) for insightful discussions. I also thank H. Kurumizaka and members of the Hirano laboratory for critically reading the manuscript. The work in the author’s laboratory was supported by Grant-in-Aid for Specially Promoted Research and Grant-in-Aid for Scientific Research A.
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