Article

ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 4, pp. 601-625 © Pleiades Publishing, Ltd., 2024.

601

REVIEW

Cohesin-Dependent Loop Extrusion:

Molecular Mechanics and Role in Cell Physiology

Arkadiy K. Golov

1,2,a

* and Alexey A. Gavrilov

1,b

Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia

Technion – Israel Institute of Technology, 3525433 Haifa, Israel

e-mail: golovstein@gmail.com

e-mail: aleksey.a.gavrilov@gmail.com

Received October 21, 2023

Revised December 29, 2023

Accepted February 15, 2024

Abstract— The most prominent representatives of multisubunit SMC complexes, cohesin and condensin, are best

known as structural components of mitotic chromosomes. It turned out that these complexes, as well as their bac-

terial homologues, are molecular motors, the ATP-dependent movement of these complexes along DNA threads

leads to the formation of DNA loops. In recent years, we have witnessed an avalanche-like accumulation of data on

the process of SMC dependent DNA looping, also known as loop extrusion. This review briefly summarizes the cur-

rent understanding of the place and role of cohesin-dependent extrusion in cell physiology and presents a number

of models describing the potential molecular mechanism of extrusion in a most compelling way. We conclude the

review with a discussion of how the capacity of cohesin to extrude DNA loops may be mechanistically linked to its

involvement in sister chromatid cohesion.

DOI: 10.1134/S0006297924040023

Keywords: cohesin, SMC complexes, loop extrusion, cohesion, DNA gripping state

Abbreviations: CAR, cohesin associated region; E-P, en-

hancer-promoter (interactions); FRET, Förster resonance en-

ergy transfer; HAWK,HEAT protein associated with Kleisin;

SMC,structural maintenance of chromosomes.

* To whom correspondence should be addressed.

INTRODUCTION

Cohesin is a protein complex, which is absolute-

ly essential for reproduction of eukaryotic cells [1, 2].

First cohesin subunits were discovered more than

25years ago as factors participating in pairing of sis-

ter chromatids in mitosis [3-5]. It has been found out

later that this phenomenon termed ‘cohesion’ is based

on the fact that the pairs of sister chromosomes af-

ter replication end up being threaded through multi-

ple cohesion complexes (each of which having ring-

shaped structure with a relatively large intersubunit

pore), similar to two threads passing through a se-

ries of beads (Fig. 1a) [6-8]. Maintenance of cohesion

during the G2-phase of cell cycle and its controlled re-

lease in anaphase occurring due to proteolysis of the

RAD21 subunit of cohesin ensures correct attachment

of spindle microtubules to kinetochores and subse-

quent equal distribution of genetic material between

the two daughter cells [9].

No less important activity of cohesin besides co-

hesion is its ability to form DNA loops via the mecha-

nism called extrusion [2, 10, 11]. Extrusion begins with

cohesin binding to small DNA fragment followed by

the ATP-dependent movement of the complex along

the DNA resulting in processive pulling of the flank-

ing DNA inside the growing loop, the length of which

could eventually reach hundreds of thousands of base

pairs (kbp). Theoretically flanking DNA can be contin-

uously pulled inside from one side of the loop held by

the complex (in the case of unidirectional extrusion) or

from both sides (in the case of bidirectional extrusion).

Extrusion is typical not only for cohesin, but for an en-

tire group of protein complexes, known as SMC com-

plexes (structural maintenance of chromosomes pro-

teins), with cohesin being one of the representatives

of this group [12-14]. Synergistic activity of the typeII

DNA topoisomerases and SMC-dependent extrusion is

required for post-replicative individualization of sis-

ter genomes in all cells (prokaryotic and eukaryotic).

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BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

Fig. 1. Cohesin structure and its participation in sister chromatid cohesion. a)Topological loading of cohesin rings onto sis-

ter chromatids during cohesion. b)Schematic representation of core trimer forming the cohesin ring; recruitment of auxiliary

HAWK subunits to the core trimer. c)Dimerization of head domains of SMC subunits required for ATP hydrolysis.

Extrusion can also facilitate orderly compaction of DNA;

formation of mitotic chromatids in prophase/metaphase

in the vertebrate cells is the best example of such com-

paction. Chromatids are elongated structures consist-

ing of the densely packed chromatin loops anchored

on the proteinaceous axial core. The main structural

component of this axis is condensin– SMC complex re-

sponsible for mitotic loop extrusion, which eventually

results in formation of chromatids. The cohesin-depen-

dent loop extrusion contributes to individualization of

eukaryotic chromosomes, in many cases is responsible

for chromatin compaction and also has a number of

secondary functions.

Cohesion, unlike extrusion, is typical exclusively

for cohesin, and not for any other representatives of

SMC complexes[1,2]. Cohesive activity likely emerged

in the primordial cohesin during early eukaryogen-

esis [15], this acquisition did not result, however, in

the loss of extrusive activity of the complex [16-18].

This commonly accepted scenario raises a number of

interesting questions on mechanistic, functional, and

evolutionary interrelationships between the cohesion

phenomenon and the process of loop extrusion.

In this review we summarize current under-

standing of cohesin-dependent loop extrusion, its role

in cellular processes, and its molecular mechanisms.

We also briefly discuss disparate data indicating pos-

sible mechanistic relationship between extrusion and

cohesion. Detailed description of the cohesin complex

structure and principles of its interactions with chro-

matin can be found in the first part of the review pub-

lished in the same issue of the journal [19].

STRUCTURE OF COHESIN COMPLEX

Cohesin is a protein complex with a ring-shaped

structure, which is based on the trimer of core pro-

teins: SMC1 (Smc1)

, SMC3 (Smc3), and RAD21 (Scc1).

All three proteins have elongated shape and interact

with each other through terminal globular domains

(Fig. 1b). Such organization results in formation of

the extensive intersubunit pore, which is able to let

through globular particles with diameter up to around

10 nm [20, 21]. Presence of the intersubunit pore makes

possible topological entrapment of DNA within the

complex with DNA being threaded through the protein

ring [6-8].

SMC1 and SMC3 subunits are paralogs belong-

ing to the family of ATPases called SMC proteins [22].

SMC1 and SMC3 form a stable V-shaped heterodimer

via homotypic interaction between the hinge domains

of two subunits [20,23]. Head domains, which are lo-

cated at the opposite end of the rod-shaped molecule

from the hinge domains, are responsible for ATPase

activity. In the presence of ATP intermittent engage-

ment of the head domains of two SMC subunits occurs,

such dimerization is required for hydrolysis of bound

In the paper names of human proteins are presented in the main text; names of Saccharomyces cerevisiae homologs are

shown in parenthesis (at first mention).

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BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

ATP molecules (Fig. 1c). The hinge and head domains

of each of the SMC subunits are separated by the long

and relatively flexible coiled-coil arm domain. Flexibil-

ity of the arm domain is to a large degree associated

with the presence of evolutionary conserved defect in

the regular coiled-coil structure, the so-called elbow

region (Fig. 1b). Bending of ‘elbows’ has rather large

amplitude; and simultaneous bending of elbow sites

in both SMC subunits could facilitate direct physical

interaction of the hinge and head domains of the com-

plex. The RAD21 protein, also called kleisin subunit,

forms a constant bridge between the head domains of

the two SMC subunits, thus closing the ring structure.

Auxiliary subunits belonging to the family of

HAWK proteins (HEAT protein associated with Klei-

sin) including STAG1/2

 (Scc3), NIPBL (Scc2), and

PDS5A/B (Pds5) bind to the core trimer [16, 20, 23].

Theprimary site of interaction of the HAWK subunits

with the core trimer is the kleisin subunit, however,

only STAG1/2 forms a stable contact with RAD21 and,

hence, is a constitutive component of the complex.

NIPBL and PDS5A/B compete for the shared binding

site at the kleisin subunit, and both these proteins

interact transiently with the stable cohesin tetramer

(SMC3–SMC1–RAD21–STAG1/2) [24-26]. Therefore, at

each particular moment the complex could contain

one (STAG1/2) or two (STAG1/2 + NIPBL or STAG1/2 +

+ PDS5A/B) HAWK subunits(Fig.1b). Binding of NIPBL

to the core cohesin complex (and presence of DNA)

isrequired for effective hydrolysis of ATP, replacement

of NIPBL with PDS5A/B dramatically changes activity

of the complex [26].

CELLULAR CONTEXT

OF COHESIN-DEPENDENT LOOP EXTRUSION

Cohesin-dependent loop extrusion throughout

the cell cycle. Cohesin-dependent extrusion is realized

in eukaryotic cells throughout interphase and mitosis

(Fig.2a). Various estimates indicate that in the G1-phase

vertebrate cells there are around 100,000 cohesin rings,

and this number doubles in the G2-phase [27, 28].

Nucleoplasm of the cells in G1-phase contains two co-

existing subpopulations of cohesin complexes of ap-

proximately the same size, which are in dynamic equi-

librium: (1) freely diffusing cohesin and (2) cohesin

involved in extrusion (with chromatin residence time of

about 10-30min in vertebrate cells and around 1min

in yeast cells) [28-31]. When cohesion is established

in the S-phase a third subpopulation emerges: stably

bound cohesive rings excluded from extrusion initia-

tion/termination cycle [28, 29, 32]. Complete arrest of

cohesin-dependent extrusion occurs during mitosis. In

vertebrates phosphorylation of the HAWK subunits in

prophase induces termination of cohesion in chromo-

some arms; most likely this phosphorylation also leads

to dissociation of extruding cohesin from DNA [29,33].

Resumption of cohesin-dependent extrusion occurs at

the end of telophase [34,35]. In the budding yeasts the

cohesin-dependent extrusion is realized in mitotic cells

up to the start of anaphase [36], when the separase-de-

pendent proteolysis of the kleisin subunits results in

degradation of the whole cellular pool of cohesin com-

plexes and is resumed only at the end of G1-phase with

restoration of the population of intact cohesin rings

[4,36].

Genomic distribution of initiation sites. Initi-

ation of the cohesin-dependent loop extrusion is not

strictly localized to particular genomic sites, howev-

er, there are some preferences in extrusion complex

binding to DNA. Centromeres and regions adjacent to

them are examples of such sites [8, 26]. Initiation of ex-

trusion, however, also occurs constantly outside of the

centromere regions. It has been assumed up until now

that initiation of the cohesin-dependent extrusion in

the chromosome arms is mainly associated with open

chromatin [37-39]. However, this model is questioned

now. The recently reported experimental data on the

genomic distribution of cohesin binding, as well as the

results of computer modeling indicate that probability

of extrusion initiation is distributed evenly along the

genome outside of the centromeres [40].

WAPL and PDS5A/B are negative regulators of

processivity, NIPBL – positive. The final size of the

loops formed in the process of extrusion is determined

by processivity of the cohesin complexes and genom-

ic distribution of the extrusion pause sites [36, 41-45],

itvaries from several dozens of kb in yeasts [44, 46] to

hundreds of kb in vertebrates[41, 42].

Processivity of the cohesin-dependent extrusion is

suppressed by the HAWK subunit PDS5A/B, as well as

by the WAPL protein recruited by this subunit. Deple-

tion of PDS5A/B and WAPL (together or individually)

significantly increases chromatin residence time of

the extruding cohesin, as well as length of the form-

ing DNA loops [36, 41, 42, 44,45]. Interestingly enough,

both these proteins also participate in non-proteolyt-

ic termination of cohesion as a part of the so-called

prophase cascade: recruitment of the WAPL protein

to cohesin complexes containing PDS5A/B subunit re-

sults in non-proteolytic opening of the protein rings

in the prophase cells of vertebrates and their removal

from the chromosomal arms [47,48]. This dual activity

Vertebrate genomes generally encode a pair of somatically expressed paralogs for both Scc3 and Pds5 HAWK proteins:

STAG1/STAG2 and PDS5A/PDS5B. These paralogs in the majority of cases are structurally and functionally equivalent, hence,

here and further in the text the designations STAG1/2 and PDS5A/B are used.

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BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

Fig. 2. Cohesin activity throughout the cell cycle and chromatin compaction due to SMC-dependent loop extrusion. a)Intact

cohesin complex quantity and their activity throughout mitotic cycle in vertebrate(1) and S. cerevisiae(2) cells. b)Metaphase

chromosomes of vertebrates are formed due to extrusion activity of condensin complexes eventually accumulated in axial struc-

tures. Typical X-shaped structure is maintained due to residual cohesion in centromeres of two metacentric sister chromosomes.

c)Compact chromatid-like “vermicelli” structures formed due to cohesin-dependent loop extrusion in interphase vertebrate

cells with suppressed WAPL activity. Cohesin is the primary structural component of the axial structures in such chromosomes.

ofPDS5A/B and WAPL implies existence of mechanis-

tic relatedness between the process of loop extrusion

and the phenomenon of cohesion.

Another HAWK subunit, NIPBL, on the contrary,

is a positive regulator of the extrusion processivity.

NIPBL is commonly considered as a cohesin loader;

however, this point of view probably requires recon-

sideration as the new data have been reported demon-

strating that NIPBL could be dispensable for the pri-

mary loading of cohesin onto chromatin [49]. It has

been firmly established that recruitment of NIPBL to

the complex is necessary for active extrusion: func-

tional depletion of NIPBL results in suppression of co-

hesin-dependent loop interactions in vertebrates and

inhibition of the cohesin translocation from the pri-

mary loading sites in yeasts [26, 42, 50]. During each

round of extrusion initiation/termination cycle NIPBL

is repeatedly recruited to and released from the com-

plex: chromatin residence time of NIPBL subunit in

the vertebrate G1-cells is around 1 min, which is an

order of magnitude less than average duration of each

round of extrusion [25, 31]. During NIPBL absence its

binding site can be occupied by the PDS5A/B subunit

recruiting WAPL, which, likely, leads to extrusion ter-

mination by not yet elucidated mechanism [2].

Cohesin-dependent interphase extrusion, unlike

mitotic condensin-dependent extrusion, usually does

not result in formation of condensed chromatid-like

structures with proteinaceous axial cores to which

DNA loops are anchored. This can be a consequence

of low processivity of the cohesin-dependent extru-

sion. Suppression of activity of PDS5A/B and WAPL in

the vertebrate cells results in interphase condensation

of chromatin accompanied by the formation of micro-

scopically visible elongated structures with axial cores

containing cohesin [41, 42, 51]. Such compact structures

with characteristic shape resembling metaphase chro-

matids (Fig.2b) have been called ‘vermicelli’(Fig.2c).

Interphase ‘vermicelli’ in a structural sense is simi-

lar to chromatids formed in the meiosisI prophase[52].

Compaction of meiotic chromosomes is achieved by the

extruding activity of cohesin that eventually accumu-

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BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

lates in the axial structures, which later become an im-

portant component of synaptonemal complex. Discov-

ery of cohesin-mediated formation of the condensed

structures similar to metaphase chromatids in the

meiosis I prophase became one of the early indica-

tions that extrusion of DNA loops might be a universal

activity of all SMC complexes [51,53].

Site-specific arrest of loop extrusion. Cohesin-

dependent extrusion differs from extrusion mediated

by other SMC complexes by the ability for a regulat-

ed arrest at the specific genome loci. It is not known

exactly whether such arrest is a temporary pause or

final termination of extrusion. In any case, stability of

the formed DNA loop is maintained by the extrusion

complex for a certain time after arrest until dissoci-

ation of the complex from DNA [31, 54]. At least two

mechanisms of site-specific arrest of extrusion cohes-

in complexes have been described: CAR-dependent

(Cohesin Associated Region) and CTCF-dependent.

Thefirst one, which is evolutionary more ancient and

typical for the cells of lower eukaryotes, is associated

with extended genome regions of cohesin accumula-

tion, CAR-regions [44, 46, 55]. CAR-regions are prefer-

ably immunoprecipitated by the antibodies against

cohesin subunits and, as a rule, are located at the

3′-ends of the convergently transcribed genes [56, 57].

The second mechanism of extrusion arrest associated

with activity of the insulator protein CTCF is realized

in the vertebrate cells [58, 59]. Blocking of extrusion

complexes at the CTCF-sites results in colocalization of

the vast majority of strong cohesin binding sites with

the CTCF binding sites in the vertebrate cells.

CAR-dependent and CTCF-dependent mechanisms

of extrusion arrest have much in common both

with each other and with the processes of stabiliza-

tion of cohesive ring binding to chromatin (Fig. 3a).

In both cases arrest is realized due to the fact that

the blocking sites favor replacement of NIPBL with

PDS5A/B[44, 58]. This replacement is facilitated by the

ESCO1(Eco1)-dependent acetylation of the SMC3 pro-

tein occurring at the arrest sites [43, 45]. Acetylation

of the SMC3 subunit at the conserved lysine residues

K105/K106 (K112 andK113– in yeasts) suppresses ex-

trusion activity of cohesin likely due to decrease in

affinity of the complex to NIPBL[60,61]. Additionally,

acetylation inhibits activity of the WAPL protein [62],

which, as has been mentioned above, removes from

chromatin not only the topologically engaged cohes-

in complexes, but also complexes participating in ex-

trusion [41, 42, 51]. Interestingly enough, stabilization

of the cohesive rings on chromatin in the G2-phase

occurs due to acetylation of SMC3 at the same amino

acid residues (in many species an auxiliary protein so-

rorin also participates in stabilization). Acetylation fa-

cilitating stabilization of cohesion is established in the

S-phase co-replicatively due to activity of the ESCO1

paralog, acetyltransferase ESCO2 (Eco1), and it also

suppresses activity of WAPL towards the acetylated

complexes.

Existence of the genomic elements blocking extru-

sion results in accumulation of cohesin complexes in-

teracting with CAR-elements and CTCF-sites, as well as

in formation of interphase chromatin typical folding

patterns: structural chromatic loops anchored in the

mentioned genomic elements and topologically-associ-

ated domains (TADs) (Fig.3,b andc) [44, 46, 58, 59,63].

In vertebrates the N-terminal fragment of CTCF is re-

sponsible for the arrest of extruding cohesin complex-

es, as well as for inhibition of the activity of WAPL sub-

unit [58, 59,64]. Steric characteristics of CTCF molecule

bound to the binding site allow effective interaction

of its N-terminal fragment with cohesin only if the

extrusion complex approaches the CTCF-motif from

the side of the 3′-end (N-terminal zinc-fingers of CTCF

bind there to DNA); this interaction blocks extrusion

and stabilizes cohesin at the CTCF-bound site (Fig.3a).

At the same time when extrusion complex approach-

es CTCF-motif from the side of the 5′-end it does not

cause prolonged stalling of cohesin. These features of

protein–protein interactions result in the peculiar reg-

ularity in orientation of the CTCF-motifs located at the

bases of structural loops and at the boundaries of to-

pological domains in vertebrates: pairs of CTCF-motifs

located at the bases of structural loops are general-

ly oriented in such a way that their 3′-ends face each

other (convergent orientation); at the same time sev-

eral CTCF-motifs (at least a pair) that are typically

present within each TAD boundary face interior of the

closest topological domain with their 3′-ends (divergent

orientation) (Fig.3,b andc)[63,65-67].

Interplay with transcriptional apparatus of

the cell. Why CAR-sites that stop extrusion in the

yeast cells are located at 3′-ends of the convergently

transcribed genes? Theoretically this could be a con-

sequence of the fact that collision of the transcribing

polymerase with extruding cohesin complexes moving

in the opposite direction results in translocation of

the latter to the end of the transcriptional units. This

model is in agreement with the fact that turning off

transcription leads to the removal of cohesin from the

CAR-sites[56].

The mechanisms underlying transcription-depen-

dent accumulation of cohesin rings at the 3′-end of the

genes are operating not only in yeast cells, they are

universal. For example, despite the fact that the verte-

brate cells do not have classic CAR-sites, simultaneous

depletion of the CTCF and WAPL results in relocation

of cohesin complexes from the CTCF-sites to the 3′-ends

of actively transcribing genes [38, 40]. These new co-

hesin-bound sites differ significantly from the original

genomic peaks colocalized with CTCF; they comprise

extended regions several kbp in length appropriately