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Cohesion and cohesin-dependent chromatin organization
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ScienceDirect Cohesion and cohesin-dependent chromatin organization Tomoko Nishiyama Cohesin, one of structural maintenance of chromosomes (SMC) complexes, forms a ring-shaped protein complex, and mediates sister chromatid cohesion for accurate chromosome segregation and precise genome inheritance. The cohesin ring entraps one or two DNA molecules to achieve cohesion, which is further regulated by cohesin-binding proteins and modification enzymes in a cell cycle-dependent manner. Recent significant advancements in Hi-C technologies have revealed numerous cohesin-dependent higher-order chromatin structures. Simultaneously, single-molecule imaging has also unveiled the detailed dynamics of cohesin on DNA and/or chromatin. Thus, those studies are providing novel visions for the authentic chromatin structure regulated by cohesin. Address Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan Corresponding author: Nishiyama, Tomoko ([email protected])
Current Opinion in Cell Biology 2019, 58:8–14 This review comes from a themed issue on Cell nucleus Edited by Daniel R Larson and Naoko Imamoto
https://doi.org/10.1016/j.ceb.2018.11.006 0955-0674/ã 2018 Elsevier Ltd. All rights reserved.
Introduction Cohesin is a ring-shaped protein complex comprising four core subunits, Smc1, Smc3, Scc1/Rad21, and stromal antigen (SA)/STAG, and is conserved among eukaryotes (Figure 1) [1,2]. Cohesin is essential but not sufficient for sister chromatid cohesion, for which several regulatory proteins are required. For instance, cohesin is loaded on chromatin before DNA replication through a cohesin loader Scc2/ Nipbl-Scc4/Mau2, and cohesion is established during DNA replication in S phase. For cohesion establishment, acetylation of Smc3 by acetyltransferases Eco1/Esco/Deco and, in metazoans, loading of a cohesin-binding protein Sororin/ Dalmatian are essential. Sororin is recruited on chromatinbound cohesin in a Smc3 acetylation-dependent manner. These Sororin and Smc3 acetylation antagonize the function of ‘anti-establishment factors’ Wapl and Pds5, both being cohesin-binding proteins, to establish cohesion [3]. Current Opinion in Cell Biology 2019, 58:8–14
In addition to sister chromatid cohesion, numerous recent studies have investigated another aspect of cohesin function in regulating higher order chromatin structure. This review summarizes recent progress in the understanding of how cohesion is achieved and further discusses cohesinmediated organization of chromatin structure.
Cohesin loading and establishment of cohesion One of the most remarkable features of cohesin is topological entrapment of DNA. It is clear at least that a cohesin loader Scc2-Scc4 and ATPase activity of cohesin are required for its loading [4–6]. However, it is still debatable as to how DNA enters the cohesin ring during loading. Currently, two possibilities have been suggested regarding the entry gate on the cohesin ring (Figure 2). One possibility is opening of the Smc1-Smc3 hinge interface to load DNA. Evidence supporting this possibility is forced tethering experiments using FKBP-FRB and rapamycin system in budding yeast [7]. Tethering of the Smc1-Smc3 hinge interface inhibits cohesin loading and increases sister chromatid separation. The same tethering experiments applied to human somatic cells also support the hinge-opening hypothesis [8]. Moreover, triple mutations on lysine and arginine residues in the hinge domain hinders cohesin loading in budding yeast [9], suggesting that the hinge structure and/or a basic charge is important for the cohesin loading step (Figure 2a). Another interface between Smc3 and Scc1 has also been proposed for the entry gate on the basis of biochemical studies on fission yeast cohesin [10]. The Smc3-Scc1 interface is known as an ‘exit gate’ from where DNA is liberated in a Wapl-dependent manner [8,11–13]. Murayama and Uhlmann showed an in vitro reconstitution system wherein Wapl-Pds5 not only removes cohesin but also loads it by dissociating the N-terminus of Scc1 from Smc3. Cohesin loader Scc2-Scc4 associates with both hinge and STAG [10,13] and ATPase activity on head domain of cohesin is required for cohesin loading [4– 6]. These lines of evidence suggest the possibility of cohesin loading through the Scc1-Smc3 gate (Figure 2b). Further in vivo analyses are required to clarify if either or both gates are used in vivo. The mechanism underlying cohesion establishment is also unclear, especially with respect to the relevance to the replication machinery. Genetic studies in budding yeast, have revealed several cohesion establishment factors among replication-related genes such as RFCCtf18, Tof1/Timeless complex, Ctf4/And1 and Chl1/DDX11. All events required for www.sciencedirect.com
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Figure 1
cells [20–22], although it was originally reported in Xenopus early embryo that pre-replicative complex formation is a prerequisite for cohesin loading [23–25]. These findings clearly indicate the association between cohesion and the replication system.
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One of the most confounding issues regarding cohesion establishment is how cohesin topologically loaded on unreplicated DNA allows DNA synthesis to proceed, such that cohesion can subsequently be established. This issue raises two questions: first, whether topological cohesin is dissociated from DNA when a huge replisome is passing through, and second, what mechanism underlies this phenomenon. The first question was addressed via visual inspections of single molecule observation and fluorescence recovery after photobleaching (FRAP) experiments, showing that cohesin can remain associated with chromatin during replication [26,27], although both studies do not rule out the possibility of a newly loaded cohesin fraction in the S-phase is present. Furthermore, in budding yeast, under replication stress, cohesin is transiently dissociated proximal to replication forks by Rsp5Bul2- and Cdc48-dependent cohesin ubiquitylation [28]. Therefore, both remaining and dissociating fractions are likely to be present. If cohesin is once dissociated and reloaded during replication, how does dissociated cohesin recognize, entrap, and establish cohesion specifically on replicated DNA? An intriguing possibility has
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Cohesin core complex entrapping one dsDNA molecule into the ring. Two Smc family members, Smc1 and Smc3, are connected to each other through their internal globular domains, forming hinge. Nterminus and C-terminus of Smc proteins form head domain, possessing ATPase activity. A kleisin subunit Scc1 bridges two head domains of Smc1 and Smc3, and it binds SA/STAG. All 4 subunits are required for chromatin binding of cohesin.
cohesion establishment depend on replication machinery [3]. For instance, in metazoans, recruitment of Sororin/Dalmatian depends on both DNA replication and Smc3 acetylation [14– 16]. The Smc3 acetylation itself also requires replicative helicase Mcm [17–19]. Furthermore, recent studies suggest that even cohesin recruitment on chromatin is facilitated by replication machineries both in budding yeast and human Figure 2
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Two possible entry gates for cohesin loading. Cohesin is loaded on DNA in a Scc2-Scc4- and ATPase-dependent manner. Though it is not clear how hinge is opened, ATPase activity on head domain may facilitate the hinge opening (a). If Smc3-Scc1 interface is the entry gate, it may be in two steps: first, Wapl-Pds5 dissociates Smc3-Scc1 interface, then second, ATP hydrolysis allows disengagement of head domains and let DNA enter into the ring (b). www.sciencedirect.com
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been proposed using an in vitro reconstitution system [29]. Purified fission yeast cohesin can capture only single-stranded DNA (ssDNA) but not double-stranded DNA (dsDNA) after entrapment of the 1st dsDNA. Hence, it is plausible that cohesin is reloaded during replication first on the replicated leading strand dsDNA, followed by entrapment of the unreplicated lagging strand ssDNA. Lagging strand replication then results in entrapment of two dsDNAs by one cohesin ring. Consistent with this idea, overexpression of dominantnegative RPA, a ssDNA-binding protein, in budding yeast restores Ctf18-dependent cohesion, suggesting that increased exposure of ssDNA can restore defects in cohesion [29]. This may explain how newly loaded cohesin during replication can hold two replicated DNAs together.
Cohesin and higher-order chromatin architecture Recent technical advances in chromosome conformation capture-sequencing methods including 3C, 4C, 5C, Hi-C, or ChIA-PET accelerated studies of higher-order chromatin structure. In particular, Hi-C has enabled the identification of higher-order genome-wide chromatin interactions [30–32]. Although the nomenclature of topologically associated domains (TADs), sub-TADs, or loops is not yet well defined [31], a high resolution Hi-C study of several kilobase (kb) resolution has identified 10,000 of small loops ranging in size from 40 kb to 3 Mb (median
size 185 kb) in the human genome. Among these loops, approximately 90% of the loop peaks are associated with CCCTC-binding factor (CTCF) motifs in a convergent orientation and with cohesin subunits Rad21 and Smc3 [33] (Figure 3). The idea that ring-like motor proteins (i.e. cohesin, condensin, or other SMC protein family complexes) may form a chromosomal axis and construct higher-order chromatin or chromosome structures by extruding DNA loops has been debated for decades [34–38]. Several studies have experimentally suggested that cohesin forms chromosome axes or replication foci, implying that cohesin can serve as a basement of chromatin loops [39–41]. Recent Hi-C-based highresolution studies render the hypothesis of cohesin- and condensin-dependent loop formation testable. Indeed, Rao et al. reported that auxin degron-driven degradation of Rad21 eliminates loop domains of hundreds kilobase in size, and the loop domains are formed again after the obliteration of auxins [42]. Similarly, TADs and the associated peaks are diminished globally on the mouse genome in Nipbl/Scc2-deficient cells [43], where cohesin is not loaded on chromatin. In contrast, in Wapl-deficient cells, where cohesin is highly stabilized on chromatin, extended loops are increased with abnormal combinations of CTCF-binding sites [44,45,46]. Therefore, it is conceivable that Wapl restricts loop size by rendering cohesin dynamic, such that cells maintain the chromatin loop structure with an appropriate combination/orientation of
Figure 3
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Current Opinion in Cell Biology
A model of cohesin-dependent DNA loop formation and Hi-C. Several lines of evidence suggest that cohesin and CTCF are frequently colocalized on the genome in ‘convergent’ CTCF sites, where two consensus CTCF motifs are pointing toward each other, and forms a DNA loop (a). To create such a DNA loop, if cohesin possesses motor-like activity or with help of other motor proteins, cohesin may extrude DNA loop until it encounters another CTCF motif of opposite direction (b). Illustrated example of Hi-C map is shown in (c). When DNA loop is formed as in (a) between 2 genomic loci 2 and 3, Hi-C map will give a ‘peak 1’. When cohesin is highly stabilized in the absence of Wapl, loop size restriction between loci 2 and 3 is relieved, then abnormally extended loop with locus 1 and 3 (peak 2) is additionally appeared. If loop extrusion is proceeding from one anchoring site (locus 3) as shown in (b), a ‘stripe’ structure should be seen on Hi-C matrix. These anchoring sites ‘stripe anchors’ are frequently colocalized with NIPBL and CTCF. Current Opinion in Cell Biology 2019, 58:8–14
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CTCF binding sites. Interestingly, different type of cohesin subunits (i.e. cohesin complex harboring different variant of SA subunit: either SA1 or SA2) seems to regulate different chromatin structure. Cohesin-SA1 preferentially contributes to TADs boundaries together with CTCF, while cohein-SA2 promotes cell-type-specific enhancerpromoter-contacts independently of CTCF [46]. Loss of cohesin not only eliminates local loop domain formation but also affects higher-order genome compartmentalization. Compartments are thought to be closely associated with open (type-A) and closed (type-B) chromatin conformations [32,33]. In the absence of cohesin, genome compartmentalization retains or becomes rather stronger [42,43]. Schwarzer et al. further revealed that deletion of Nipbl alters compartment structure and, interestingly, newly emerging compartments reflect transcriptional activity showing a strong correlation with active marks of H3K27ac, H3K4me3, DNase hypersensitivity, and transcription factor binding [43], suggesting that higher-order chromatin conformation simply depends on epigenetic marks in the absence of cohesin. In contrast, the compartmentalization is weakened in Wapl-deficient cells [44,45 ,47]. These findings suggest that cohesin plays an inhibitory role in genome compartmentalization presumably by promoting loop domain formation. Although these Hi-C studies have reported that cohesin is located on the bases of loop domains and is required for the formation of the domain structure throughout the genome, the underlying mechanisms are unclear. Loop extrusion is one possibility to form such domains. However, there is no direct evidence showing loop extrusion by cohesin so far. Recently, a Hi-C study using ultra-deep sequencing reported that cohesin-dependent formation of loop domains requires ATP but not transcription or replication process, implying that ATPase activity of cohesin is required for loop domain formation. Furthermore, by inspecting ultra-deep Hi-C maps, architectural ‘stripes’ can he detected as vertical and/or horizontal straight lines from a single locus along the edge of loop domain (Figure 3). Stripe anchors frequently colocalize with cohesin and CTCF. Stripes range from a few to hundreds of kilobases, and they often tether super-enhancers to cognate promoters [48]. Though this finding is the first evidence implying the cohesin-dependent loop extrusion on the genome, the direct proof is still missing. Currently, most of these Hi-C studies are based on population-averaged data, which may be different from what we observe in each single cell. Indeed, it was reported recently that, by using super-resolution chromatin tracking in single cells, TAD-like domain structures are preserved even after cohesin degradation, while their preferential positioning of boundaries at CTCF-cohesin sites become uniform distribution [49]. Interestingly, in this condition, TAD-like structure is apparently disappeared in the population-averaged contact map. Thus, more detailed analyses at single cell resolution would www.sciencedirect.com
reveal bona fide view of higher-order structure organized by cohesin.
Single molecule dynamics of cohesin Accumulating evidence for cohesin localization on the genome and chromatin structures in 3C and Hi-C studies have suggested that cohesin is a motor protein that extrudes DNA loops. However, there is no evidence showing the motor activity of cohesin so far. Recent studies on cohesin single-molecule observations directly addressed this issue and unveiled the cohesin dynamics on DNA. One of the most important questions is whether cohesin has a motor activity. If indeed the case, cohesin must be able to translocate unidirectionally along DNA, as reported in budding yeast condensin [50,51]. Cohesin has been suggested to be translocated on the budding yeast genome from their sites of loading [52,53]. In single molecule studies using either DNA curtains [54] or a flowcell system [26,55], it was reported that eukaryotic cohesin (budding yeast, human, and frog) is translocated on DNA. However, translocation occurs via diffusion, which does not require ATP. Salt-resistant cohesin loaded by the loader Scc2-Scc4 is dissociated from DNA after artificial cleavage of Scc1, indicating that cohesin topologically entrapping DNA is translocated along DNA via one-dimensional diffusion. Interestingly, however, AMPPCP, a non-hydrolyzable analog of ATP, inhibits this translocation, implying that head-to-head engagement of cohesin bound to ATP or AMP-PCP is an inhibitory conformation deterring translocation [26]. These single molecule studies have also provided an insight into structural properties of the cohesin ring topologically bound to DNA. The inner diameter of the cohesin ring has been estimated from previous electron microscopic observations to be between 30 to 35 nm, which can theoretically hold two 10-nm nucleosome fibers [1,13,56]. However, the actual permissive size of the ring seems to be much smaller, because cohesin cannot bypass only 20 nm-sized molecules tethered to DNA. Consistently, though one nucleosome incorporated into DNA can be passed through by cohesin, a nucleosome array including 10–50 nucleosomes, which is predicted as 30 nm in size on assuming the 30 nm-fiber model, highly restricts cohesin translocation. Not only nucleosomes but also motor proteins such as FtsK translocase (12.6 nm) or T7 RNA polymerase (12 nm with tag) restrict cohesin diffusion or even push cohesin towards its direction of travel [54,55]. These properties of cohesin (i. e. diffusion and restricted size of the ring) suggest that cohesin basically diffuses along the genome after its loading, but the direction of translocation depends on surrounding genomic events or circumstance including transcriptional activity and/or nucleosome density. Thereafter, these relocated cohesin could be finally Current Opinion in Cell Biology 2019, 58:8–14
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accumulated at CTCF binding sites [57,58] and, consequently, it may form loop domains as observed in vivo [42]. Consistently, transcription-dependent relocation of cohesin was indeed observed in budding yeast and mouse cells [59,60].
Acknowledgements
In addition to transcription, replication is a critical event, which encounters cohesin ring throughout the genome. In eukaryotes, as initial loading of cohesin occurs before DNA replication [24,61,62], cohesin must either be displaced when replisomes pass through or permit replisomes progression, followed by establishment of cohesion. How do the cells execute this process? This has been a long-standing question, whose answer is yet unclear. Single-molecule observations have suggested an answer to this question via coupling the flow-cell system with replication-compatible Xenopus egg extracts [26,63]. In interphase egg extracts, DNA tethered onto the coverslip at the both ends can be replicated, and the expansion of the replication bubbles can be monitored in real-time under the microscope. Using this system, cohesin behavior during DNA replication was observed [26]. In this assay, most of DNA-bound cohesins were dissociated from DNA when encountered with replication bubbles or thrusted in the direction of its expansion, similar to that observed in other motor proteins. However, interestingly, one-third of total DNA-bound cohesin was retained at its location even after replication, indicating that cohesin permitted the replisome to pass through. This observation shows for the first time the possibility of cohesin to establish cohesion without dissociating from DNA. Indeed, cohesin can remain associated with chromatin during replication, as revealed via FRAP experiments in human living cells [27], supporting the single-molecule observation. Together, genomic distribution of topologically bound cohesin is affected by every chromosomal event such as replication, transcription, and CTCF bindings, and the distribution defines higher-order chromatin structure.
References and recommended reading
Conclusion and perspective Accumulating evidence from yeast genetics, biochemistry, and cell biology have revealed the mechanism underlying sister chromatid cohesion, ever since the discovery of SMC proteins. Nevertheless, fundamental questions regarding the mechanism of cohesion establishment or cause of cohesinopathies are yet unsolved. Technical improvement of resolution in time and space in future studies would help further these understandings. Observation of cohesin-regulated higher-order chromatin structures at kilobase resolution constitutes outstanding progress in the field in most recent years, clearly depending on newly developing Hi-C methods. A comprehensive understanding of chromatin structure, which explains both sister chromatid cohesion and cohesin-dependent higher-order chromatin structure would be expected in the next decade. Current Opinion in Cell Biology 2019, 58:8–14
The author would like to thank numerous colleagues in the field, for their insightful discussions. The author apologizes to many colleagues whose studies could not be cited because of space limitation. Studies in the author’s laboratory were chiefly supported by JST-PRESTO (25-J-J4301) and JSPS Grant-in-Aid for Young Scientists (25711002).
Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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34. Alipour E, Marko JF: Self-organization of domain structures by DNA-loop-extruding enzymes. Nucleic Acids Res 2012, 40:11202-11212. 35. Fudenberg G, Imakaev M, Lu C, Goloborodko A, Abdennur N, Mirny LA: Formation of chromosomal domains by loop extrusion. Cell Rep 2016, 15:2038-2049. 36. Nasmyth K: Disseminating the genome: joining, resolving, and separating sister chromatids during mitosis and meiosis. Annu Rev Genet 2001, 35:673-745. 37. Riggs AD: DNA methylation and late replication probably aid cell memory, and type I DNA reeling could aid chromosome folding and enhancer function. Philos Trans R Soc Lond B Biol Sci 1990, 326:285-297. 38. Sanborn AL, Rao SS, Huang SC, Durand NC, Huntley MH, Jewett AI, Bochkov ID, Chinnappan D, Cutkosky A, Li J et al.: Chromatin extrusion explains key features of loop and domain formation in wild-type and engineered genomes. Proc Natl Acad Sci U S A 2015, 112:E6456-6465. 39. Guillou E, Ibarra A, Coulon V, Casado-Vela J, Rico D, Casal I, Schwob E, Losada A, Mendez J: Cohesin organizes chromatin loops at DNA replication factories. Genes Dev 2010, 24:2812-2822. 40. Novak I, Wang H, Revenkova E, Jessberger R, Scherthan H, Hoog C: Cohesin Smc1beta determines meiotic chromatin axis loop organization. J Cell Biol 2008, 180:83-90. 41. Tedeschi A, Wutz G, Huet S, Jaritz M, Wuensche A, Schirghuber E, Davidson IF, Tang W, Cisneros DA, Bhaskara V et al.: Wapl is an essential regulator of chromatin structure and chromosome segregation. Nature 2013, 501:564-568. 42. Rao SSP, Huang SC, Glenn St Hilaire B, Engreitz JM, Perez EM, Kieffer-Kwon KR, Sanborn AL, Johnstone SE, Bascom GD, Bochkov ID et al.: Cohesin loss eliminates all loop domains. Cell 2017, 171:305-320 e324. By means of auxin-inducible degradation system and Hi-C, this study shows loop domains are lost and compartments are strengthened after cohesin degradation. 43. Schwarzer W, Abdennur N, Goloborodko A, Pekowska A, Fudenberg G, Loe-Mie Y, Fonseca NA, Huber W, HH C, Mirny L et al.: Two independent modes of chromatin organization revealed by cohesin removal. Nature 2017, 551:51-56. This study, concurrently published with previous one, reaches the same conclusion in Hi-C, but here Nipbl, instead of cohesin, is deleted in conditional Nipbl knockout mice. 44. Haarhuis JHI, van der Weide RH, Blomen VA, Yanez-Cuna JO, Amendola M, van Ruiten MS, Krijger PHL, Teunissen H, Medema RH, van Steensel B et al.: The cohesin release factor WAPL restricts chromatin loop extension. Cell 2017, 169:693-707 e614. Hi-C in DWapl haploid cells shows that loop length is extended with abnormal combination of CTCF sites in the absence of Wapl. This extension is reduced by further depletion of Scc4, indicating the loop extension is caused by excessively stabilized cohesin in DWapl cells. 45. Wutz G, Varnai C, Nagasaka K, Cisneros DA, Stocsits RR, Tang W, Schoenfelder S, Jessberger G, Muhar M, Hossain MJ et al.: Topologically associating domains and chromatin loops depend on cohesin and are regulated by CTCF, WAPL, and PDS5 proteins. EMBO J 2017, 36:3573-3599. In human somatic cells, this study shows that Pds5 depletion causes reduction of chromatin loop number and violation of CTCF convergence rule, suggesting that Pds5 is required for boundary function of cohesinCTCF. 46. Gassler J, Brandao HB, Imakaev M, Flyamer IM, Ladstatter S, Bickmore WA, Peters JM, Mirny LA, Tachibana K: A mechanism of cohesin-dependent loop extrusion organizes zygotic genome architecture. EMBO J 2017, 36:3600-3618. 47. Kojic A, Cuadrado A, De Koninck M, Gimenez-Llorente D, Rodriguez-Corsino M, Gomez-Lopez G, Le Dily F, MartiRenom MA, Losada A: Distinct roles of cohesin-SA1 and cohesin-SA2 in 3D chromosome organization. Nat Struct Mol Biol 2018, 25:496-504. This study shows majority of cohesin-SA2 localizes to enhancers lacking CTCF, which cannot be compensated by cohesin-SA1. While cohesin-SA1 functions in CTCF-dependent TAD formation, cohesin-SA2 promotes celltype-specific enhancer-promoter contacts independently of CTCF. Current Opinion in Cell Biology 2019, 58:8–14
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48. Vian L, Pekowska A, Rao SSP, Kieffer-Kwon KR, Jung S, Baranello L, Huang SC, El Khattabi L, Dose M, Pruett N et al.: the energetics and physiological impact of cohesin extrusion. Cell 2018, 173:1165-1178 e1120. Using the same degron system as [42] but with ultra-deep Hi-C, this study identifies ‘stripe’ pattern on Hi-C matrix. As stripe anchors are highly colocalized with CTCF and Nipbl, it is implicated that the stripe could be a trace of loop extrusion. 49. Bintu B, Mateo LJ, Su JH, Sinnott-Armstrong NA, Parker M, Kinrot S, Yamaya K, Boettiger AN, Zhuang X: Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science 2018, 362. By means of super-resolution chromatin tracking in single cells, this study shows that the TAD-like domain structure is preserved even after cohesin degradation, while their preferential boundaries at CTCF-cohesin sites are lost and become uniform distribution. Nevertheless, importantly, population-averaging data shows disappearance of TAD-like structure after cohesin degradation as many studies had already reported. 50. Terakawa T, Bisht S, Eeftens JM, Dekker C, Haering CH, Greene EC: The condensin complex is a mechanochemical motor that translocates along DNA. Science 2017, 358:672-676. 51. Ganji M, Shaltiel IA, Bisht S, Kim E, Kalichava A, Haering CH, Dekker C: Real-time imaging of DNA loop extrusion by condensin. Science 2018, 360:102-105.
55. Davidson IF, Goetz D, Zaczek MP, Molodtsov MI, Huis In’ t Veld PJ, Weissmann F, Litos G, Cisneros DA, Ocampo-Hafalla M, Ladurner R et al.: Rapid movement and transcriptional relocalization of human cohesin on DNA. EMBO J 2016, 35:26712685. 56. Anderson DE, Losada A, Erickson HP, Hirano T: Condensin and cohesin display different arm conformations with characteristic hinge angles. J Cell Biol 2002, 156:419-424. 57. Parelho V, Hadjur S, Spivakov M, Leleu M, Sauer S, Gregson HC, Jarmuz A, Canzonetta C, Webster Z, Nesterova T et al.: Cohesins functionally associate with CTCF on mammalian chromosome arms. Cell 2008, 132:422-433. 58. Wendt KS, Yoshida K, Itoh T, Bando M, Koch B, Schirghuber E, Tsutsumi S, Nagae G, Ishihara K, Mishiro T et al.: Cohesin mediates transcriptional insulation by CCCTC-binding factor. Nature 2008, 451:796-801. 59. Busslinger GA, Stocsits RR, van der Lelij P, Axelsson E, Tedeschi A, Galjart N, Peters JM: Cohesin is positioned in mammalian genomes by transcription, CTCF and Wapl. Nature 2017, 544:503-507. 60. Ocampo-Hafalla M, Munoz S, Samora CP, Uhlmann F: Evidence for cohesin sliding along budding yeast chromosomes. Open Biol 2016, 6.
52. Hu B, Itoh T, Mishra A, Katoh Y, Chan KL, Upcher W, Godlee C, Roig MB, Shirahige K, Nasmyth K: ATP hydrolysis is required for relocating cohesin from sites occupied by its Scc2/4 loading complex. Curr Biol 2011, 21:12-24.
61. Hinshaw SM, Makrantoni V, Harrison SC, Marston AL: The kinetochore receptor for the cohesin loading complex. Cell 2017, 171:72-84 e13.
53. Lengronne A, Katou Y, Mori S, Yokobayashi S, Kelly GP, Itoh T, Watanabe Y, Shirahige K, Uhlmann F: Cohesin relocation from sites of chromosomal loading to places of convergent transcription. Nature 2004, 430:573-578.
62. Lopez-Serra L, Lengronne A, Borges V, Kelly G, Uhlmann F: Budding yeast Wapl controls sister chromatid cohesion maintenance and chromosome condensation. Curr Biol 2013, 23:64-69.
54. Stigler J, Camdere GO, Koshland DE, Greene EC: Singlemolecule imaging reveals a collapsed conformational state for DNA-bound cohesin. Cell Rep 2016, 15:988-998.
63. Yardimci H, Loveland AB, Habuchi S, van Oijen AM, Walter JC: Uncoupling of sister replisomes during eukaryotic DNA replication. Mol Cell 2010, 40:834-840.
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ScienceDirect Cohesion and cohesin-dependent chromatin organization Tomoko Nishiyama Cohesin, one of structural maintenance of chromosomes (SMC) complexes, forms a ring-shaped protein complex, and mediates sister chromatid cohesion for accurate chromosome segregation and precise genome inheritance. The cohesin ring entraps one or two DNA molecules to achieve cohesion, which is further regulated by cohesin-binding proteins and modification enzymes in a cell cycle-dependent manner. Recent significant advancements in Hi-C technologies have revealed numerous cohesin-dependent higher-order chromatin structures. Simultaneously, single-molecule imaging has also unveiled the detailed dynamics of cohesin on DNA and/or chromatin. Thus, those studies are providing novel visions for the authentic chromatin structure regulated by cohesin. Address Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, Japan Corresponding author: Nishiyama, Tomoko ([email protected])
Current Opinion in Cell Biology 2019, 58:8–14 This review comes from a themed issue on Cell nucleus Edited by Daniel R Larson and Naoko Imamoto
https://doi.org/10.1016/j.ceb.2018.11.006 0955-0674/ã 2018 Elsevier Ltd. All rights reserved.
Introduction Cohesin is a ring-shaped protein complex comprising four core subunits, Smc1, Smc3, Scc1/Rad21, and stromal antigen (SA)/STAG, and is conserved among eukaryotes (Figure 1) [1,2]. Cohesin is essential but not sufficient for sister chromatid cohesion, for which several regulatory proteins are required. For instance, cohesin is loaded on chromatin before DNA replication through a cohesin loader Scc2/ Nipbl-Scc4/Mau2, and cohesion is established during DNA replication in S phase. For cohesion establishment, acetylation of Smc3 by acetyltransferases Eco1/Esco/Deco and, in metazoans, loading of a cohesin-binding protein Sororin/ Dalmatian are essential. Sororin is recruited on chromatinbound cohesin in a Smc3 acetylation-dependent manner. These Sororin and Smc3 acetylation antagonize the function of ‘anti-establishment factors’ Wapl and Pds5, both being cohesin-binding proteins, to establish cohesion [3]. Current Opinion in Cell Biology 2019, 58:8–14
In addition to sister chromatid cohesion, numerous recent studies have investigated another aspect of cohesin function in regulating higher order chromatin structure. This review summarizes recent progress in the understanding of how cohesion is achieved and further discusses cohesinmediated organization of chromatin structure.
Cohesin loading and establishment of cohesion One of the most remarkable features of cohesin is topological entrapment of DNA. It is clear at least that a cohesin loader Scc2-Scc4 and ATPase activity of cohesin are required for its loading [4–6]. However, it is still debatable as to how DNA enters the cohesin ring during loading. Currently, two possibilities have been suggested regarding the entry gate on the cohesin ring (Figure 2). One possibility is opening of the Smc1-Smc3 hinge interface to load DNA. Evidence supporting this possibility is forced tethering experiments using FKBP-FRB and rapamycin system in budding yeast [7]. Tethering of the Smc1-Smc3 hinge interface inhibits cohesin loading and increases sister chromatid separation. The same tethering experiments applied to human somatic cells also support the hinge-opening hypothesis [8]. Moreover, triple mutations on lysine and arginine residues in the hinge domain hinders cohesin loading in budding yeast [9], suggesting that the hinge structure and/or a basic charge is important for the cohesin loading step (Figure 2a). Another interface between Smc3 and Scc1 has also been proposed for the entry gate on the basis of biochemical studies on fission yeast cohesin [10]. The Smc3-Scc1 interface is known as an ‘exit gate’ from where DNA is liberated in a Wapl-dependent manner [8,11–13]. Murayama and Uhlmann showed an in vitro reconstitution system wherein Wapl-Pds5 not only removes cohesin but also loads it by dissociating the N-terminus of Scc1 from Smc3. Cohesin loader Scc2-Scc4 associates with both hinge and STAG [10,13] and ATPase activity on head domain of cohesin is required for cohesin loading [4– 6]. These lines of evidence suggest the possibility of cohesin loading through the Scc1-Smc3 gate (Figure 2b). Further in vivo analyses are required to clarify if either or both gates are used in vivo. The mechanism underlying cohesion establishment is also unclear, especially with respect to the relevance to the replication machinery. Genetic studies in budding yeast, have revealed several cohesion establishment factors among replication-related genes such as RFCCtf18, Tof1/Timeless complex, Ctf4/And1 and Chl1/DDX11. All events required for www.sciencedirect.com
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Figure 1
cells [20–22], although it was originally reported in Xenopus early embryo that pre-replicative complex formation is a prerequisite for cohesin loading [23–25]. These findings clearly indicate the association between cohesion and the replication system.
hinge
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One of the most confounding issues regarding cohesion establishment is how cohesin topologically loaded on unreplicated DNA allows DNA synthesis to proceed, such that cohesion can subsequently be established. This issue raises two questions: first, whether topological cohesin is dissociated from DNA when a huge replisome is passing through, and second, what mechanism underlies this phenomenon. The first question was addressed via visual inspections of single molecule observation and fluorescence recovery after photobleaching (FRAP) experiments, showing that cohesin can remain associated with chromatin during replication [26,27], although both studies do not rule out the possibility of a newly loaded cohesin fraction in the S-phase is present. Furthermore, in budding yeast, under replication stress, cohesin is transiently dissociated proximal to replication forks by Rsp5Bul2- and Cdc48-dependent cohesin ubiquitylation [28]. Therefore, both remaining and dissociating fractions are likely to be present. If cohesin is once dissociated and reloaded during replication, how does dissociated cohesin recognize, entrap, and establish cohesion specifically on replicated DNA? An intriguing possibility has
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Cohesin core complex entrapping one dsDNA molecule into the ring. Two Smc family members, Smc1 and Smc3, are connected to each other through their internal globular domains, forming hinge. Nterminus and C-terminus of Smc proteins form head domain, possessing ATPase activity. A kleisin subunit Scc1 bridges two head domains of Smc1 and Smc3, and it binds SA/STAG. All 4 subunits are required for chromatin binding of cohesin.
cohesion establishment depend on replication machinery [3]. For instance, in metazoans, recruitment of Sororin/Dalmatian depends on both DNA replication and Smc3 acetylation [14– 16]. The Smc3 acetylation itself also requires replicative helicase Mcm [17–19]. Furthermore, recent studies suggest that even cohesin recruitment on chromatin is facilitated by replication machineries both in budding yeast and human Figure 2
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Two possible entry gates for cohesin loading. Cohesin is loaded on DNA in a Scc2-Scc4- and ATPase-dependent manner. Though it is not clear how hinge is opened, ATPase activity on head domain may facilitate the hinge opening (a). If Smc3-Scc1 interface is the entry gate, it may be in two steps: first, Wapl-Pds5 dissociates Smc3-Scc1 interface, then second, ATP hydrolysis allows disengagement of head domains and let DNA enter into the ring (b). www.sciencedirect.com
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been proposed using an in vitro reconstitution system [29]. Purified fission yeast cohesin can capture only single-stranded DNA (ssDNA) but not double-stranded DNA (dsDNA) after entrapment of the 1st dsDNA. Hence, it is plausible that cohesin is reloaded during replication first on the replicated leading strand dsDNA, followed by entrapment of the unreplicated lagging strand ssDNA. Lagging strand replication then results in entrapment of two dsDNAs by one cohesin ring. Consistent with this idea, overexpression of dominantnegative RPA, a ssDNA-binding protein, in budding yeast restores Ctf18-dependent cohesion, suggesting that increased exposure of ssDNA can restore defects in cohesion [29]. This may explain how newly loaded cohesin during replication can hold two replicated DNAs together.
Cohesin and higher-order chromatin architecture Recent technical advances in chromosome conformation capture-sequencing methods including 3C, 4C, 5C, Hi-C, or ChIA-PET accelerated studies of higher-order chromatin structure. In particular, Hi-C has enabled the identification of higher-order genome-wide chromatin interactions [30–32]. Although the nomenclature of topologically associated domains (TADs), sub-TADs, or loops is not yet well defined [31], a high resolution Hi-C study of several kilobase (kb) resolution has identified 10,000 of small loops ranging in size from 40 kb to 3 Mb (median
size 185 kb) in the human genome. Among these loops, approximately 90% of the loop peaks are associated with CCCTC-binding factor (CTCF) motifs in a convergent orientation and with cohesin subunits Rad21 and Smc3 [33] (Figure 3). The idea that ring-like motor proteins (i.e. cohesin, condensin, or other SMC protein family complexes) may form a chromosomal axis and construct higher-order chromatin or chromosome structures by extruding DNA loops has been debated for decades [34–38]. Several studies have experimentally suggested that cohesin forms chromosome axes or replication foci, implying that cohesin can serve as a basement of chromatin loops [39–41]. Recent Hi-C-based highresolution studies render the hypothesis of cohesin- and condensin-dependent loop formation testable. Indeed, Rao et al. reported that auxin degron-driven degradation of Rad21 eliminates loop domains of hundreds kilobase in size, and the loop domains are formed again after the obliteration of auxins [42]. Similarly, TADs and the associated peaks are diminished globally on the mouse genome in Nipbl/Scc2-deficient cells [43], where cohesin is not loaded on chromatin. In contrast, in Wapl-deficient cells, where cohesin is highly stabilized on chromatin, extended loops are increased with abnormal combinations of CTCF-binding sites [44,45,46]. Therefore, it is conceivable that Wapl restricts loop size by rendering cohesin dynamic, such that cells maintain the chromatin loop structure with an appropriate combination/orientation of
Figure 3
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Current Opinion in Cell Biology
A model of cohesin-dependent DNA loop formation and Hi-C. Several lines of evidence suggest that cohesin and CTCF are frequently colocalized on the genome in ‘convergent’ CTCF sites, where two consensus CTCF motifs are pointing toward each other, and forms a DNA loop (a). To create such a DNA loop, if cohesin possesses motor-like activity or with help of other motor proteins, cohesin may extrude DNA loop until it encounters another CTCF motif of opposite direction (b). Illustrated example of Hi-C map is shown in (c). When DNA loop is formed as in (a) between 2 genomic loci 2 and 3, Hi-C map will give a ‘peak 1’. When cohesin is highly stabilized in the absence of Wapl, loop size restriction between loci 2 and 3 is relieved, then abnormally extended loop with locus 1 and 3 (peak 2) is additionally appeared. If loop extrusion is proceeding from one anchoring site (locus 3) as shown in (b), a ‘stripe’ structure should be seen on Hi-C matrix. These anchoring sites ‘stripe anchors’ are frequently colocalized with NIPBL and CTCF. Current Opinion in Cell Biology 2019, 58:8–14
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Cohesin and chromatin organization Nishiyama 11
CTCF binding sites. Interestingly, different type of cohesin subunits (i.e. cohesin complex harboring different variant of SA subunit: either SA1 or SA2) seems to regulate different chromatin structure. Cohesin-SA1 preferentially contributes to TADs boundaries together with CTCF, while cohein-SA2 promotes cell-type-specific enhancerpromoter-contacts independently of CTCF [46]. Loss of cohesin not only eliminates local loop domain formation but also affects higher-order genome compartmentalization. Compartments are thought to be closely associated with open (type-A) and closed (type-B) chromatin conformations [32,33]. In the absence of cohesin, genome compartmentalization retains or becomes rather stronger [42,43]. Schwarzer et al. further revealed that deletion of Nipbl alters compartment structure and, interestingly, newly emerging compartments reflect transcriptional activity showing a strong correlation with active marks of H3K27ac, H3K4me3, DNase hypersensitivity, and transcription factor binding [43], suggesting that higher-order chromatin conformation simply depends on epigenetic marks in the absence of cohesin. In contrast, the compartmentalization is weakened in Wapl-deficient cells [44,45 ,47]. These findings suggest that cohesin plays an inhibitory role in genome compartmentalization presumably by promoting loop domain formation. Although these Hi-C studies have reported that cohesin is located on the bases of loop domains and is required for the formation of the domain structure throughout the genome, the underlying mechanisms are unclear. Loop extrusion is one possibility to form such domains. However, there is no direct evidence showing loop extrusion by cohesin so far. Recently, a Hi-C study using ultra-deep sequencing reported that cohesin-dependent formation of loop domains requires ATP but not transcription or replication process, implying that ATPase activity of cohesin is required for loop domain formation. Furthermore, by inspecting ultra-deep Hi-C maps, architectural ‘stripes’ can he detected as vertical and/or horizontal straight lines from a single locus along the edge of loop domain (Figure 3). Stripe anchors frequently colocalize with cohesin and CTCF. Stripes range from a few to hundreds of kilobases, and they often tether super-enhancers to cognate promoters [48]. Though this finding is the first evidence implying the cohesin-dependent loop extrusion on the genome, the direct proof is still missing. Currently, most of these Hi-C studies are based on population-averaged data, which may be different from what we observe in each single cell. Indeed, it was reported recently that, by using super-resolution chromatin tracking in single cells, TAD-like domain structures are preserved even after cohesin degradation, while their preferential positioning of boundaries at CTCF-cohesin sites become uniform distribution [49]. Interestingly, in this condition, TAD-like structure is apparently disappeared in the population-averaged contact map. Thus, more detailed analyses at single cell resolution would www.sciencedirect.com
reveal bona fide view of higher-order structure organized by cohesin.
Single molecule dynamics of cohesin Accumulating evidence for cohesin localization on the genome and chromatin structures in 3C and Hi-C studies have suggested that cohesin is a motor protein that extrudes DNA loops. However, there is no evidence showing the motor activity of cohesin so far. Recent studies on cohesin single-molecule observations directly addressed this issue and unveiled the cohesin dynamics on DNA. One of the most important questions is whether cohesin has a motor activity. If indeed the case, cohesin must be able to translocate unidirectionally along DNA, as reported in budding yeast condensin [50,51]. Cohesin has been suggested to be translocated on the budding yeast genome from their sites of loading [52,53]. In single molecule studies using either DNA curtains [54] or a flowcell system [26,55], it was reported that eukaryotic cohesin (budding yeast, human, and frog) is translocated on DNA. However, translocation occurs via diffusion, which does not require ATP. Salt-resistant cohesin loaded by the loader Scc2-Scc4 is dissociated from DNA after artificial cleavage of Scc1, indicating that cohesin topologically entrapping DNA is translocated along DNA via one-dimensional diffusion. Interestingly, however, AMPPCP, a non-hydrolyzable analog of ATP, inhibits this translocation, implying that head-to-head engagement of cohesin bound to ATP or AMP-PCP is an inhibitory conformation deterring translocation [26]. These single molecule studies have also provided an insight into structural properties of the cohesin ring topologically bound to DNA. The inner diameter of the cohesin ring has been estimated from previous electron microscopic observations to be between 30 to 35 nm, which can theoretically hold two 10-nm nucleosome fibers [1,13,56]. However, the actual permissive size of the ring seems to be much smaller, because cohesin cannot bypass only 20 nm-sized molecules tethered to DNA. Consistently, though one nucleosome incorporated into DNA can be passed through by cohesin, a nucleosome array including 10–50 nucleosomes, which is predicted as 30 nm in size on assuming the 30 nm-fiber model, highly restricts cohesin translocation. Not only nucleosomes but also motor proteins such as FtsK translocase (12.6 nm) or T7 RNA polymerase (12 nm with tag) restrict cohesin diffusion or even push cohesin towards its direction of travel [54,55]. These properties of cohesin (i. e. diffusion and restricted size of the ring) suggest that cohesin basically diffuses along the genome after its loading, but the direction of translocation depends on surrounding genomic events or circumstance including transcriptional activity and/or nucleosome density. Thereafter, these relocated cohesin could be finally Current Opinion in Cell Biology 2019, 58:8–14
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accumulated at CTCF binding sites [57,58] and, consequently, it may form loop domains as observed in vivo [42]. Consistently, transcription-dependent relocation of cohesin was indeed observed in budding yeast and mouse cells [59,60].
Acknowledgements
In addition to transcription, replication is a critical event, which encounters cohesin ring throughout the genome. In eukaryotes, as initial loading of cohesin occurs before DNA replication [24,61,62], cohesin must either be displaced when replisomes pass through or permit replisomes progression, followed by establishment of cohesion. How do the cells execute this process? This has been a long-standing question, whose answer is yet unclear. Single-molecule observations have suggested an answer to this question via coupling the flow-cell system with replication-compatible Xenopus egg extracts [26,63]. In interphase egg extracts, DNA tethered onto the coverslip at the both ends can be replicated, and the expansion of the replication bubbles can be monitored in real-time under the microscope. Using this system, cohesin behavior during DNA replication was observed [26]. In this assay, most of DNA-bound cohesins were dissociated from DNA when encountered with replication bubbles or thrusted in the direction of its expansion, similar to that observed in other motor proteins. However, interestingly, one-third of total DNA-bound cohesin was retained at its location even after replication, indicating that cohesin permitted the replisome to pass through. This observation shows for the first time the possibility of cohesin to establish cohesion without dissociating from DNA. Indeed, cohesin can remain associated with chromatin during replication, as revealed via FRAP experiments in human living cells [27], supporting the single-molecule observation. Together, genomic distribution of topologically bound cohesin is affected by every chromosomal event such as replication, transcription, and CTCF bindings, and the distribution defines higher-order chromatin structure.
References and recommended reading
Conclusion and perspective Accumulating evidence from yeast genetics, biochemistry, and cell biology have revealed the mechanism underlying sister chromatid cohesion, ever since the discovery of SMC proteins. Nevertheless, fundamental questions regarding the mechanism of cohesion establishment or cause of cohesinopathies are yet unsolved. Technical improvement of resolution in time and space in future studies would help further these understandings. Observation of cohesin-regulated higher-order chromatin structures at kilobase resolution constitutes outstanding progress in the field in most recent years, clearly depending on newly developing Hi-C methods. A comprehensive understanding of chromatin structure, which explains both sister chromatid cohesion and cohesin-dependent higher-order chromatin structure would be expected in the next decade. Current Opinion in Cell Biology 2019, 58:8–14
The author would like to thank numerous colleagues in the field, for their insightful discussions. The author apologizes to many colleagues whose studies could not be cited because of space limitation. Studies in the author’s laboratory were chiefly supported by JST-PRESTO (25-J-J4301) and JSPS Grant-in-Aid for Young Scientists (25711002).
Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
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14 Cell nucleus
48. Vian L, Pekowska A, Rao SSP, Kieffer-Kwon KR, Jung S, Baranello L, Huang SC, El Khattabi L, Dose M, Pruett N et al.: the energetics and physiological impact of cohesin extrusion. Cell 2018, 173:1165-1178 e1120. Using the same degron system as [42] but with ultra-deep Hi-C, this study identifies ‘stripe’ pattern on Hi-C matrix. As stripe anchors are highly colocalized with CTCF and Nipbl, it is implicated that the stripe could be a trace of loop extrusion. 49. Bintu B, Mateo LJ, Su JH, Sinnott-Armstrong NA, Parker M, Kinrot S, Yamaya K, Boettiger AN, Zhuang X: Super-resolution chromatin tracing reveals domains and cooperative interactions in single cells. Science 2018, 362. By means of super-resolution chromatin tracking in single cells, this study shows that the TAD-like domain structure is preserved even after cohesin degradation, while their preferential boundaries at CTCF-cohesin sites are lost and become uniform distribution. Nevertheless, importantly, population-averaging data shows disappearance of TAD-like structure after cohesin degradation as many studies had already reported. 50. Terakawa T, Bisht S, Eeftens JM, Dekker C, Haering CH, Greene EC: The condensin complex is a mechanochemical motor that translocates along DNA. Science 2017, 358:672-676. 51. Ganji M, Shaltiel IA, Bisht S, Kim E, Kalichava A, Haering CH, Dekker C: Real-time imaging of DNA loop extrusion by condensin. Science 2018, 360:102-105.
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