COHESIN-DEPENDENT EXTRUSION 605
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.
Thefirst 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 andK113– 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 andc) [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 andc)[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