Hit the Brakes – a New Perspective on the Loop Extrusion Mechanism of Cohesin and Other SMC Complexes Avi Matityahu and Itay Onn*

HYPOTHESIS SUBJECT COLLECTION: EXPLORING THE NUCLEUS Hit the brakes – a new perspective on the loop extrusion mechanism of cohesin and other SMC complexes Avi Matityahu and Itay Onn*

ABSTRACT have shown that ATP binding and hydrolysis also induce major The three-dimensional structure of chromatin is determined by the conformational changes in the SMC proteins (Hassler et al., 2019; action of protein complexes of the structural maintenance of Kamada et al., 2017; Muir et al., 2020; Orgil et al., 2016; Rajasekar chromosome (SMC) family. Eukaryotic cells contain three SMC et al., 2019; Sedeno Cacciatore and Rowland, 2019). The SMC complexes, cohesin, condensin, and a complex of Smc5 and Smc6. heads are connected by a kleisin subunit that interacts Initially, cohesin was linked to sister chromatid cohesion, the process asymmetrically with the heads, docking at the emerging coiled that ensures the fidelity of chromosome segregation in mitosis. In coil on one of the SMC proteins and the head base on the other one. recent years, a second function in the organization of interphase The kleisin serves as a binding hub for regulatory factors, which are chromatin into topologically associated domains has been characterized by the presence of HEAT repeats, a structural motif – determined, and loop extrusion has emerged as the leading associated with protein protein interactions. In addition to the core mechanism of this process. Interestingly, fundamental mechanistic subunits, other factors regulate the SMC complex either by differences exist between mitotic tethering and loop extrusion. As interacting or post-translationally modifying its core subunits. The distinct molecular switches that aim to suppress loop extrusion in structure and regulatory factors of SMCs have been the subject of different biological contexts have been identified, we hypothesize many studies and extensively reviewed (see, for example, Aragon, here that loop extrusion is the default biochemical activity of cohesin 2018; Hassler et al., 2018; Makela and Sherratt, 2020; Onn et al., and that its suppression shifts cohesin into a tethering mode. With this 2008; Palecek, 2018; Yatskevich et al., 2019). model, we aim to provide an explanation for how loop extrusion and Eukaryotes encode six different SMC proteins (Smc1 to Smc6), tethering can coexist in a single cohesin complex and also apply it to which are organized into three distinct heterodimers, while most the other eukaryotic SMC complexes, describing both similarities and bacteria encode a sole SMC protein that homodimerizes. The three differences between them. Finally, we present model-derived eukaryotic SMC complexes are cohesin, which contains Smc1 and – molecular predictions that can be tested experimentally, thus Smc3, condensin, including the Smc2 Smc4 heterodimer, and a offering a new perspective on the mechanisms by which SMC complex of Smc5 and Smc6 (denoted hereafter as Smc5/6) (Fig. 1) complexes shape the higher-order structure of chromatin. (Aragon, 2018; Hassler et al., 2018; Onn et al., 2008; Palecek, 2018; Yatskevich et al., 2019). These complexes are involved in many and KEY WORDS: SMC complex, Cohesin, Condensin, Smc5/6, Loop various cellular functions. Cohesin is a multifunctional complex extrusion, Sister chromatid cohesion involved in sister chromatid cohesion, DNA repair and gene expression regulation (Carretero et al., 2010; Moronta-Gines et al., Introduction 2019; Nishiyama, 2019), while condensin is a critical factor in the Structural maintenance of chromosome (SMC) complexes are an compaction of interphase chromatin into mitotic chromosomes evolutionarily conserved family of protein complexes that control (Kalitsis et al., 2017; Paul et al., 2019). Lower eukaryotes have the the spatial organization of chromatin (Fig. 1). SMC complexes have canonical condensin, whereas vertebrates contain two variants of a common architecture in all domains of life (Aragon, 2018; Hassler condensin, condensin I and condensin II (Onn et al., 2007, 2004, et al., 2018; Makela and Sherratt, 2020; Onn et al., 2008; Palecek, 2003). These complexes share the core Smc2–Smc4 heterodimer, 2018; Yatskevich et al., 2019). The core of the complex is composed but have a different set of kleisin and HEAT-repeat subunits, and of a dimer of SMC proteins. These are elongated, rod-like proteins they play distinct roles in the condensation process. Recently, it has characterized by two globular domains, the head and hinge, which been suggested that condensins, in addition to their role in mitotic are connected by a long coiled-coil domain of about 50 nm. The chromosome organization, regulate transcription and senescence by head domain contains two halves of an ATP-binding cassette mediating the local compaction of chromatin during interphase (ABC)-type ATPase domain. The dimerization of the SMC proteins (Hassan et al., 2020; Iwasaki et al., 2019; Marshall et al., 2020). is mediated through the hinges and co-alignment of the coiled coils Smc5/6 plays an essential role in sculpting chromatin during DNA that result in the spatial proximity of the heads. The binding of two replication and repair (Aragon, 2018; Palecek, 2018). However, its ATP molecules to the Walker box motifs of one head and the ABC exact functions are not fully understood, and this complex has been signature motifs of the other head induces their engagement less characterized than the other SMCs. (Aragon, 2018; Hassler et al., 2018; Makela and Sherratt, 2020; Onn Several families of SMC complexes that participate in et al., 2008; Palecek, 2018; Yatskevich et al., 2019). Recent studies chromosome organization and partitioning have been found in bacteria (Fig. 1) (Badrinarayanan et al., 2015; Hirano, 2016; Makela 8 Henrietta Szold St., The Azrieli Faculty of Medicine, Bar-Ilan University, P.O. Box and Sherratt, 2020). The most common is the ScpAB family, which 1589 Safed, Israel. shares a high degree of similarity with the eukaryotic condensin and *Author for correspondence ([email protected]) is also found in archaea. The second SMC family, known as MukBEF, is found in enterobacteria and several other related orders

I.O., 0000-0002-7689-5520 of γ-proteobacteria (Badrinarayanan et al., 2015; Hirano, 2016). Journal of Cell Science

1 HYPOTHESIS Journal of Cell Science (2021) 134, jcs247577. doi:10.1242/jcs.247577

Eukaryotes Bacteria

Cohesin Condensin I Condensin II Smc5/6 ScpAB Hinge

Coiled coil

Head Kleisin

HEAT repeats

Fig. 1. SMC protein complexes. SMC complexes appear in all domains of life. Eukaryotic cells contain six SMC proteins (Smc1 to Smc6) organized in three heterodimers that serve as the core of the distinct SMC complexes. The canonical condensin (in vertebrates, known as condensin I), which is common to all eukaryotes, is accompanied in vertebrates by condensin II. Both share the same SMC proteins but contain a different set of regulatory proteins. Most bacteria and archaea contain the ScpAB complex with an SMC homodimer core. All SMC complexes feature this complex architecture. Elongated SMC proteins dimerize through their globular hinge domains. A long coiled-coil region emerges from the hinge and extends to ∼50 nm, until it ends in a second globular domain called the ‘head’. Heads contain two halves of ABC-type ATPase domains, which induce their ATP-dependent engagement. The heads are connected by a kleisin subunit that serves as a binding hub for HEAT repeat proteins. Smc5 is unique among SMCs as it contains a mid-protein binding site for the SUMO ligase NSMCE2. The similar architecture of all SMC complexes implies a common mechanism of action. Protein nomenclature shown is based on human proteins and yeast ortholog names appear in parentheses. The small red circles represent ATP molecules bound to the SMC head.

Prokaryotic SMC complexes are considered to be ancestors of the proteins regulate cohesin activity by either binding to the core eukaryotic condensins (Makela and Sherratt, 2020), although some complex or modifying it. Cohesin was initially found to have an evidence suggests that they are more related to the Smc5/6 complex essential role in sister chromatid cohesion (Guacci et al., 1997; (Palecek and Gruber, 2015). Their function has been linked to a Michaelis et al., 1997). Here, newly replicated DNA molecules, compaction of the bacterial chromosome around an axial core known as sister chromatids, are tethered once they are formed (Badrinarayanan et al., 2012a,b; Rajasekar et al., 2019; Wang et al., during S-phase, which ensures their bipolar attachment to the 2017, 2018). spindle and their accurate segregation to the two daughter cells The cellular functions of SMCs relate to their activity in during mitosis. organizing the three-dimensional structure of chromatin, mainly Cohesin is loaded onto chromatin in an Scc2–Scc4-dependent through the formation of chromatin loops (Hassler et al., 2018; manner during mitotic exit (vertebrate cells) or in G1 phase (yeast) Makela and Sherratt, 2020; Rowley and Corces, 2018; Sedeno (Ciosk et al., 2000; Hinshaw et al., 2015; Kuleszewicz et al., 2013; Cacciatore and Rowland, 2019; Yatskevich et al., 2019). The similar Shwartz et al., 2016; Watrin et al., 2006). Scc2 has also been overall structure of all SMC complexes implies a common identified as an activator of ATPase activity of cohesin, which is mechanism of action. However, the biological roles with regard to mediated by the SMC head domain (Petela et al., 2018). WAPL and their effects on chromosome structure, stability and dynamics Pds5 are loading antagonists that release cohesin from the chromatin significantly differ between the different SMC complexes and even (Bernard et al., 2008; Gandhi et al., 2006; Kueng et al., 2006; in different functions of the same complex. In this Hypothesis, we Kulemzina et al., 2012; Rowland et al., 2009; Sutani et al., 2009; examine the two main mechanisms that have been associated with Tanaka et al., 2001). During the S-phase of the cell cycle, in cohesin and propose a unified model for their co-existence in a coordination with DNA replication and the formation of the sister single complex. Finally, we discuss how this mechanism can be chromatid, Eco1 acetylates the Smc3 subunit of cohesin (Rolef Ben- extended and interpreted for both condensin and Smc5/6. As Shahar et al., 2008; Rowland et al., 2009; Unal et al., 2008). This, in bacterial SMCs are considered to be ancestors of eukaryotic turn, counteracts the effect of WAPL and Pds5 (Vaur et al., 2012) condensin, they will not be further scrutinized here. and blocks the ATPase activity (Camdere et al., 2015; Elbatsh et al., 2016), and the chromatids become entrapped by cohesin. The dual role of cohesin in sister chromatid cohesion and Interestingly, during the G2 phase of the cell cycle, Pds5 adopts a interphase chromatin organization new function and protects the cohesin from premature dissociation Apart from the Smc1–Smc3 heterodimer, cohesin contains the from the chromatin until the onset of prophase (Hartman et al., kleisin family protein Scc1 (RAD21 in humans) and Scc3 (Stag1/ 2000; Losada et al., 2005; Panizza et al., 2000), when cohesin is Sa1 and Stag2/Sa2 in humans) that together define the cohesin core removed from chromatin to allow the segregation of the sister (Fig. 1) (Michaelis et al., 1997; Nasmyth and Haering, 2009; chromatids. Yatskevich et al., 2019). Key regulatory proteins are the loading The molecular mechanism of tethering has been at the center of a dimer, Scc2–Scc4 (NIPBL–MAU2 in humans), the cohesin release long-standing dispute, and several models have been proposed factors WAPL and Pds5 (Pds5A and Pds5B in humans), a throughout the years. Three main models have been described: multitasking protein involved in both cohesin release during G1/S (1) the ring model, in which the sister chromatids are topologically phases of the cell cycle and cohesin maintenance during G2 entrapped in the central ring formed by the Smc1–Smc3–kleisin (Hartman et al., 2000; Losada et al., 2005; Panizza et al., 2000), and subunits (Fig. 2A) (Haering et al., 2008; Ivanov and Nasmyth, the acetyltransferase Eco1 (Esco1 and Esco2 in humans) (Nasmyth 2005, 2007); (2) the modified ring model, in which one chromatid is and Haering, 2009; Onn et al., 2008; Yatskevich et al., 2019). These topologically entrapped in the cohesin ring, while the second Journal of Cell Science

2 HYPOTHESIS Journal of Cell Science (2021) 134, jcs247577. doi:10.1242/jcs.247577

1 2 3

Fig. 2. Models for cohesin-mediated sister-chromatid tethering and loop extrusion. (A) Three models of sister chromatid tethering are shown: (1) Entrapment of the sister chromatids in the central lumen of cohesin; (2) entrapment of one chromatid in the central lumen and the second between the kleisin and the HEAT repeat subunits; and (3) a single chromatid entrapped in the lumen, with cohesion mediated by the dimerization of cohesins. Shown here is dimerization through the HEAT repeats subunits, but other possibilities exist. (B) A model of loop extrusion. Chromatin is locked between the kleisin and HEAT repeat subunits and threads through the central lumen. ATP (red circles) binding and hydrolysis induce a conformational change that pushes chromatinto form a loop. Cycles of ATP binding and hydrolysis result in an increase in loop size. Notably, several other models describing the mechanism of loop extrusion have been proposed (Fudenberg et al., 2017; Hassler et al., 2018; Rowley and Corces, 2018; Shi et al., 2020b; Yatskevich et al., 2019). However, while these models differ in the molecular details, they are conceptually similar and all include a conformational change providing the extruding force. chromatid is locked between the kleisin and the HEAT-repeats called topologically associating domains (TADs). These mega- subunits (Fig. 2A) (Chapard et al., 2019; Xu and Yanagida, base-pair regions are characterized by extensive interactions 2019); and (3) a model that is based on the entrapment of a single between chromatin entrapped inside the domain but only show chromatid in the central lumen of cohesin, while cohesion is rare inter-domain interactions. (Belton et al., 2012; Dekker et al., achieved through the dimerization of cohesins (Fig. 2A) (Huang 2013; Dixon et al., 2012; Rowley and Corces, 2018; Yu and Ren, et al., 2005; Zhang et al., 2008). More recent structural and 2017). TADs are a distinctive feature of eukaryotic chromatin during spectroscopic studies provide strong support that tethering involves interphase, and they are believed to play roles in the regulation of a single copy of the cohesin complex with two distinct chromatin- gene expression and, presumably, also DNA replication and repair binding sites (model 2) (Chapard et al., 2019; Liu et al., 2020; Xu (Luppino and Joyce, 2020; Mamberti and Cardoso, 2020; Matthews and Yanagida, 2019). However, evidence pointing to cohesin and Waxman, 2018; Noordermeer and Feil, 2020). TAD size varies dimerization (Eng et al., 2014, 2015) needs to be reconciled with in different organisms, and similar chromatin structures have been this model. found in yeast (Eser et al., 2017). However, as yeast does not have a Elucidating the molecular details of tethering and discussing CTCF homolog, the stabilization of their loops is different, as which one of these models is correct has been the source of discussed below. many disputes, as recently discussed elsewhere (Skibbens, 2019). Loop extrusion is the currently accepted mechanism to explain Nonetheless, despite the fundamental differences between these chromatin loop formation, while cohesin has been identified as the models, a common concept is that sister chromatid cohesion is main extruder of this process (Alipour and Marko, 2012; Nasmyth, mediated by physical and stable tethering of the chromatids by 2001; Wutz et al., 2020). Cohesin acts as a motor protein through cohesin in order to prevent their separation during the cell cycle by which the chromatin is progressively threaded, resulting in the diffusion or premature pulling by the spindle (Fig. 2A). formation of extended loops (Fig. 2B). This model shifted the role For many years, cohesin was not been considered to have a role in of cohesin from a passive tethering factor in mitosis and stabilizer of interphase chromatin organization. However, this dramatically TADs, as we describe above, into being the principal extruder changed with the identification of cohesin as a regulator of gene (Alipour and Marko, 2012; Nasmyth, 2001). Structural studies have expression (Dorsett et al., 2005; Rollins et al., 1999) and the shown that chromatin is anchored between the SMC heads and development of genomics techniques that show that cohesin is the kleisin, while the ATP-induced conformational change provides involved in the organization of interphase chromatin into chromatin the mechanical threading of chromatin. Notably, several models loops together with the CCCTC-binding factor (CTCF), a sequence- for the exact nature of the conformations have been proposed specific DNA-binding protein known to act as a chromatin insulator (Gligoris et al., 2014; Haering et al., 2008; Higashi et al., 2020; (Dixon et al., 2012; Hansen et al., 2017; Lieberman-Aiden et al., Petela et al., 2018; Srinivasan et al., 2018). 2009; Nora et al., 2012). The colocalization of cohesin and CTCF at The loop extrusion activity of cohesin has been confirmed chromatin loop boundaries is important for their stability. The experimentally in recent single-molecule spectroscopy in vitro

CTCF–cohesin loops represent a subset of chromatin domains studies using recombinant cohesin (Davidson et al., 2019; Kim Journal of Cell Science

3 HYPOTHESIS Journal of Cell Science (2021) 134, jcs247577. doi:10.1242/jcs.247577 et al., 2019). Notably, these studies show that under these A unified mechanism for the different functions of cohesin experimental conditions, extrusion is highly processive and SMC complexes are evolutionary related to ABC transporters, a depends on both the ATP-hydrolysis activity of cohesin and its large superfamily of transmembrane protein complexes (Hopfner interaction with the Scc2 subunit. As described above, the Scc2– and Tainer, 2003; Nasmyth and Haering, 2005). ABC transporters Scc4 dimer is required for cohesin loading onto the chromatin in are located in the cell membrane; they are activated by the presence cells, but not in vitro (Murayama and Uhlmann, 2017; Onn and of their substrate and use ATP to induce a conformational change Koshland, 2011). The structural basis for the interaction between that is associated with the active passage of molecules from the Scc2 and cohesin has only recently been determined (Shi et al., cytoplasm through the transporter cavity into the cell exterior. In 2020b), and this interaction has been shown to activate the ATPase many mechanistic aspects, loop extrusion of chromatin by cohesin activity of cohesin (Petela et al., 2018). is similar to the shuttling of molecules through ABC transporters. In Comparing the mechanisms of cohesin function during mitosis both cases, the molecular mechanism depends on cycles of ATP- and loop extrusion reveals fundamental differences between them. induced conformational changes that, in turn, push the substrate Sister chromatids are tethered by cohesin from the time of their molecule through the complex (Fig. 2B). With this similarity in formation until cohesin removal in anaphase initiation (Nasmyth mind, we suggest that loop extrusion, rather than passive tethering, and Haering, 2009; Onn et al., 2008). Therefore, this cohesion is the fundamental biochemical activity of cohesin. Indeed, natural function requires physical and stable entrapment of the chromatids. shifting between conformational changes has been described in By contrast, loop extrusion is a dynamic process that requires cycles purified SMC dimers in vitro (Eeftens et al., 2016). The interactions of attachment and dissociation of cohesin from the chromatin, until between SMC proteins, kleisin and the regulatory subunits restrain the loop reaches its final size of tens of kilobases at a rate of ∼0.5 and coordinate these spontaneous conformational changes. kilobases per second (Davidson et al., 2019; Kim et al., 2019). Notably, the nature of this conformational change is not fully Thereafter, cohesin engages in passive tethering that ensures loop characterized. Different models that include compartmentalization stability by preventing the diffusion of the chromatin fibers (Collier et al., 2020; Elbatsh et al., 2016; Srinivasan et al., 2018) or (Haarhuis et al., 2017) (Fig. 2). Hence, a full understanding of the head–hinge engagement (Rowland et al., 2009; Shi et al., 2020b) molecular basis of cohesin function requires elucidating how the have been suggested to describe this conformational change. dynamic loop extrusion activity is turned on and off to mediate Extrusion is therefore initiated when cohesin interacts with Scc2 physical passive tethering, as during TAD stabilization and in and chromatin, which stimulates its ATPase activity (Fig. 3) mitosis. (Hassler et al., 2018; Hassler et al., 2019; Petela et al., 2018; Shi

TAD formationtion

Cohesin Esco1 acetylation

Scc1 ATP Scc2Scc2 Scc2 Loop stabilizationzation CTCF-WAPL interactions

WAPL Pds5 Stag

CTCF Sister chromatid cohesion Loader complex dissociation

Esco2 acetylation

Cohesion establishment

Cohesin complex dissociation

Fig. 3. A unified model for tethering and loop extrusion by cohesin. Cohesin, for simplicity represented as a ring shape, is attached to chromatin. Both cohesin loading and activation of its loop extrusion activity depend upon interaction with SCC2. The loop extrusion activity of cohesin terminates when it encounters CTCF, which defines the boundaries of the TAD. Acetylation of cohesin by Esco1 (indicated by a yellow star) stabilizes cohesin in its tethering mode by inhibiting its ATPase activity, and serves as a safety mechanism that maintains the loop structure and thus the TAD (upper mechanism). In order to mediate mitotic cohesion, Scc2 dissociates from cohesin post loading. This results in suppression of loop extrusion, and cohesin is moved by the transcription machinery along the chromatin. However, cohesin frequently dissociates from the chromatin. Sister chromatid cohesion is induced after DNA replication and the formation of the sister chromatids. After replication, Esco2-mediated acetylation of cohesin stabilizes the cohesin–chromatin complex and inhibits its reactivation of loop extrusion activity by the reassociation of Scc2 with cohesin (lower mechanism). Journal of Cell Science

4 HYPOTHESIS Journal of Cell Science (2021) 134, jcs247577. doi:10.1242/jcs.247577 et al., 2020b). Therefore, according to this scenario, we expect that whose specific activities were reported recently (Alomer et al., the dissociation of Scc2 from cohesin after loading of the DNA will 2017). Similar to canonical yeast Eco1, Esco2 plays a key role in the halt the loop extrusion activity. establishment of mitotic cohesion, whereas Esco1 acetylates As mentioned above, in vitro, Scc2 is not essential for the cohesin throughout interphase, but has little effect on mitotic association of cohesin with chromatin or its tethering activity cohesion and is non-essential for mitosis (Alomer et al., 2017). It is (Murayama and Uhlmann, 2017; Onn et al., 2008). However, important to note that yeast cells are significantly different from without Scc2, cohesin is unable to extrude DNA (Davidson et al., vertebrate cells in several aspects. First, yeast lacks the CTCF 2019; Kim et al., 2019). In cells, Scc2 is essential for the association of homolog. Second, while cohesin is essential for mitotic cohesion, it cohesinwithchromatinandforsisterchromatidcohesion(Michaelis plays a minor role in the regulation of gene expression in yeast et al., 1997), presumably by targeting cohesin to chromatin (Hinshaw (Bose et al., 2012; Skibbens et al., 2010). Cohesin is important for et al., 2015). In yeast, Scc2 dissociates from cohesin post-loading, and, yeast chromatin organization into TAD-like structures in interphase. in turn, cohesin is pushed away from the loading site and travels along However, cohesin is dispensable for maintaining their stability, the chromosome, moved along by the transcription machinery (Fig. 3) which instead depends on the forkhead family proteins Fkh1 and (Busslinger et al., 2017; Davidson et al., 2016; Hu et al., 2011; Fhd2 (Alipour and Marko, 2012). Therefore, we suggest that the Lengronne et al., 2004; Stigler et al., 2016; Wang et al., 2017). This acetylation of cohesin by Esco1 evolved as a safety mechanism in passive movement of cohesin can be explained by the suppression of vertebrates to maintain CTCF-dependent extrusion suppression of the loop extrusion activity of cohesin after dissociation of Scc2 and the cohesin in TADs. This activity is dispensable in yeast cells as we shift from binding to its passive-topological mode (Fig. 3). However, assume that the evolutionary divergence of Esco1 and Esco2 is Scc2 dissociation also reduces the stability of the cohesin–chromatin associated with the acquired function of cohesin in TAD complex; this results in frequent cohesin dissociations that can be stabilization. observed in cells by microscopy (Austin et al., 2009; Bernard et al., In higher eukaryotes, the composition of cohesin complexes can 2008; Gause et al., 2010; Gerlich et al., 2006). differ depending on the identity of the HEAT repeat subunit, either Interestingly, it has been suggested that in human cells, Scc2 Stag1 or Stag2 (homologs of yeast Scc3), and the presence of the exists in substoichiometric amounts relative to cohesin and ‘hops’ regulators Pds5A or Pds5B (homologs of yeast Pds5) (Losada et al., between different chromatin-bound cohesin complexes (Rhodes 2005, 2000; Sumara et al., 2000). Each one of these protein pairs is et al., 2017). This could fine-tune the extrusion activity by turning mutually exclusive, meaning that cells contain subpopulations of loop extrusion on and off at certain locations, while allowing cohesin complexes that differ in their STAG and Pds5 composition. cohesin to reside on the chromatin without inducing extrusion at The functional importance of these alternative complexes has been specific loci, such as centromeres, where it is enriched. Therefore, associated with differential localization and functions (Canudas and we suggest that dissociation of Scc2 is a key molecular event that Smith, 2009; Carretero et al., 2013; Casa et al., 2020; Kojic et al., suppresses the loop extrusion activity of cohesin but also reduces its 2018; Morales et al., 2020; Viny et al., 2019; Zhang et al., 2013). binding affinity to chromatin. The remaining issue is, therefore, how We suggest that an overlooked property of these alternative subunits cohesin is stabilized on chromatin in a tethering mode for either may affect cohesin loop extrusion processivity, which defines the TAD stabilization and or mitotic sister chromatid cohesion. residency time on chromatin. This property will eventually determine the final size of the extruded loop by allowing Molecular switches for turning off loop extrusion differential association times of cohesin with chromatin. To this Acetylation of the cohesin core subunit Smc3 by Eco1 on two end, the effect of alternative cohesin subunits on cohesin adjacent lysine residues located at the head domain in proximity to processivity has not been addressed in detail; it stands as a future the ATP binding site has been identified as a key event in the challenge to provide biochemical systems that can be used to test cohesin activity cycle (Rolef Ben-Shahar et al., 2008; Unal et al., loop extrusion activity for complexes with alternative subunit 2008). During the cell cycle, Smc3 is acetylated shortly after the composition to either confirm or reject this idea (Davidson et al., passage of the replication fork and the capturing of the newly 2019; Kim et al., 2019). Until further experimental data is collected, formed second chromatid by cohesin (Dauban et al., 2020; Rolef we hypothesize that high processivity enables the continuous Ben-Shahar et al., 2008; Unal et al., 2008). Mechanistically, the extrusion that is required for TAD formation in interphase until its acetylation of Smc3 inhibits both the ATPase activity of cohesin and suppression by CTCF, whereas low processivity results in frequent conformational changes (Camdere et al., 2018). Recently, it has dissociation of cohesin from the chromatin, as has been observed in been suggested that acetylation also induces cohesin dimerization mitotic cells (Austin et al., 2009; Bernard et al., 2008; Gause et al., (Shi et al., 2020a). Either way, we suggest that, as a result of the 2010; Gerlich et al., 2006). In support of this model, a recent study acetylation, cohesin loop extrusion is fully turned off, likely by revealed differences in the binding of cohesin–Stag1 and cohesin– modifying the ATPase activity at the SMC head. As a result, the Stag2 to DNA, with the former lasting hours versus only minutes in cohesin–DNA complex is stabilized in a tether-promoting case of the latter (Wutz et al., 2020). Furthermore, a distinct role has conformation (Fig. 3), such as the recently described conformations been reported for Stag1 in the formation of the TAD stem, whereas that enable the passive capture of the chromatids (Collier et al., 2020; Stag2 is involved in inter-TAD interactions (Cuadrado et al., 2019). Higashi et al., 2020; Sedeno Cacciatore and Rowland, 2019; Further studies are needed to unveil the differential functions of the Skibbens, 2015; Srinivasan et al., 2018). We imagine that even cohesin alternative subunits in tethering and TAD formation. after the dissociation of Scc2, conformational changes in cohesin occur at a slow rate as they may be essential to capturing the newly Loop extrusion suppression in condensin and Smc5/6 formed sister chromatid. As soon as this goal is achieved, the Loop extrusion is also considered a component of the mitotic acetylation of Smc3 ensures that loop extrusion is fully halted and chromosome assembly mechanism by the cohesin-related SMC cannot be reactivated by the reassociation of Scc2 with cohesin. complex condensin (Banigan et al., 2020; Ganji et al., 2018; Kim In contrast to yeast, which contain a sole Eco1, vertebrates have et al., 2020; Terakawa et al., 2017). Several computational two Eco1 orthologs, called Esco1 and Esco2 (Hou and Zou, 2005), simulations have suggested that condensation is initiated by Journal of Cell Science

5 HYPOTHESIS Journal of Cell Science (2021) 134, jcs247577. doi:10.1242/jcs.247577 individual condensins that are distributed along the chromatin. As these fundamentally different mechanisms could coexist within a chromatin condensation continues, the individual condensins single cohesin complex. aggregate to form the chromosome scaffold (Fig. 4) (Goloborodko Previous work in the field identified the need to develop a model et al., 2016). Notably, similar accumulation of bacterial SMC, which that will explain the co-existence of chromatid cohesion and loop is considered to be evolutionary ancestor of condensin, has been extrusion in a single complex (Yatskevich et al., 2019). We suggest reported (Rajasekar et al., 2019; Wang et al., 2017, 2018). We that loop extrusion activity represents a fundamental biochemical propose that inter-condensin contacts at the chromosome scaffold activity of SMC complexes that originated in their evolutionary suppress loop extrusion. This suppression can be mediated by ancestors. Various off-switches dependent on the intra- or inter- interactions between the HEAT repeat subunits, which is similar to molecular interactions evolved, with the capability of controlling the suppression of loop extrusion by cohesin that is mediated by the SMC complexes under different biological conditions, namely, CTCF–WAPL interaction (Haarhuis et al., 2017; Makrantoni and cohesin–CTCF interactions in TAD boundaries and acetylation of Marston, 2018). However, we cannot exclude the possibility that Smc3 by Eco1. Extrapolating this mechanism onto other eukaryotic other contacts involving the hinge or the coiled coil domains either SMC complexes, condensin and Smc5/6, we have advanced two mediate or contribute to the suppression. Alternatively, condensin hypotheses. First, we argue that condensing-mediated regulation of aggregation may contribute to the dynamic crowding of the nuclear loop extrusion is similar to that of cohesin, but here, the off-switch is nanoenvironment and chromatin viscoelasticity that hinders loop condensin crowding at the scaffold of the mitotic chromosome. extrusion activity by slowing down dynamic conformational changes Second, we suspect that Smc5/6 might have either lost or repressed (Kschonsak et al., 2017; Shim et al., 2020; Vivante et al., 2020). its extrusion activity and has thus become a passive tethering Notably, condensin activity depends on the phosphorylation of its member of the SMC family. HEAT repeat subunits by Cdc2 (known as Cdk1 in mammals) (Abe A unified mechanistic model for cohesion and loop extrusion et al., 2011; Kimura et al., 1998; Takemoto et al., 2004), and their should also be considered in light of the different cohesin tethering dephosphorylation may, therefore, serve as an off switch, models we present in Fig. 2. Although loop extrusion is mediated by corresponding to acetylation of Smc3 in cohesin. a monomer, in which the chromatin is thread through two binding The third eukaryotic SMC complex is Smc5/6, which is sites, a simple topological entrapment of the chromatin in the SMC involved in DNA replication and repair (Aragon, 2018; Palecek, ring is unlikely, as validated in recent studies that show 2018). However, its molecular activity is not fully understood, and compartmentalization and complex interactions of cohesin with loop extrusion activity has not yet been shown in this complex. DNA (Collier et al., 2020; Higashi et al., 2020; Liu et al., 2020; Another fundamental difference between Smc5/6 and the other Srinivasan et al., 2018). However, this model does not exclude SMC proteins is the presence of a docking site for the SUMO dimerization as a mechanism to suppress extrusion activity. In doing ligase Nse2 in the middle of the Smc5 coiled-coil region (Fig. 1). so, the interaction between monomers is expected to be weak. Thus, Nse2 sumoylates Smc5 and the kleisin subunit Nse4, which may identification of dimers by methods that do not involve crosslinking affect the structure of the complex (Varejao et al., 2018). Finally, will be difficult and might explain why the genetic evidence of we recently suggested that the organization of Smc5 is different dimerization has yet to be validated with molecular data. from that of other SMC proteins as it contains non-canonical Fluorescence resonance energy transfer (FRET) and similar breaks in the coiled coil domain (Matityahu and Onn, 2018). in vivo spectroscopic approaches will help to identify cohesin Taken together, the sequence and anticipated sumoylation- dimerization in vivo. Furthermore, FRET could be used to explore induced rigidity of Smc5/6 indicate that this SMC complex the conformation dynamics of SMC complexes in live cells. This might be less flexible than cohesin and condensin. Accordingly, could be accomplished through dual fluorescent labeling of subunits Smc5/6 may not have the structural flexibility required for the in the same complex, thereby allowing dynamic extruders to be conformational changes that underlie loop extrusion (Higashi distinguished from silent tethers. Under this experimental setup, an et al., 2020; Sedeno Cacciatore and Rowland, 2019). Thus, Smc5/ oscillating on–off fluorescent emission is expected from extruding 6 is unlikely to have the ability to mediate loop extrusion, and only complexes, while a constant emission would suggest stable passively tethers chromatin. conformation complexes involved in passive tethering. While such experiments may well offer exciting results, their success will Conclusions and perspectives depend upon the technical ability to differentiate between extruding Knowledge about the mechanism of cohesin activity has been and tethering complexes within a noisy cellular environment. accumulating rapidly, but along two separate paths – organization of Parts of the model we present here can be challenged interphase chromatin by loop extrusion and mitotic tethering of sister experimentally using in vitro loop extrusion assays for condensin chromatids. Herein, we have provided a rational explanation of how and cohesin (Banigan et al., 2020; Davidson et al., 2019; Eeftens

Fig. 4. Organization of mitotic chromosome by condensin. In interphase, chromatin is compacted by condensing- mediated loop extrusion, which includes the clustering of individual condensins into a chromosome scaffold. We suggest that either inter-condensin interactions or molecular crowding is the molecular trigger that suppresses any loop extrusion activity of condensin. Journal of Cell Science

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The Escherichia coli SMC complex, MukBEF, shapes nucleoid organization 2008)] will determine the effects of these factors on loop extrusion independently of DNA replication. J. Bacteriol. 194, 4669-4676. doi:10.1128/JB. activity. These experiments will also reveal whether there are 00957-12 indeed any differences in processivity that depend upon subunit Badrinarayanan, A., Reyes-Lamothe, R., Uphoff, S., Leake, M. C. and Sherratt, composition, as we suggest here. The mechanism by which loop D. J. (2012b). In vivo architecture and action of bacterial structural maintenance of chromosome proteins. Science 338, 528-531. doi:10.1126/science.1227126 extrusion is suppressed can be challenged by isolating mutants that Banigan, E. J., van den Berg, A. A., Brandao, H. B., Marko, J. F. and Mirny, L. A. induce hyper-condensed mitotic chromosomes, such as those (2020). Chromosome organization by one-sided and two-sided loop extrusion. reported in two recent studies. 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