Current Biology 22, R1012–R1021, December 4, 2012 ª2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.10.

023

Condensin, Chromatin Crossbarring Review

and Chromosome Condensation

Rahul Thadani1, Frank Uhlmann1,*, and Sebastian Heeger1 Accumulating lines of evidence over two decades indicate
that the chromosomal condensin complex is the principal
effector of condensation [10,11]. Condensin is a large, evolu-
The processes underlying the large-scale reorganisation tionarily conserved multisubunit protein assembly that is
of chromatin in mitosis that form compact mitotic found, with a broadly similar architecture, throughout the
chromosomes and ensure the fidelity of chromosome domains of life, including bacteria, archaea and eukarya
segregation during cell division still remain obscure. The (Figure 1). Along with cohesin and Smc5/6, it is one of three
chromosomal condensin complex is a major molecular complexes built from dimers of SMC proteins, members
effector of chromosome condensation and segregation of the structural maintenance of chromosomes family of
in diverse organisms ranging from bacteria to humans. ATPases, that are intimately involved in diverse aspects of
Condensin is a large, evolutionarily conserved, multisubu- higher order chromosome organisation. Indeed, condensin
nit protein assembly composed of dimers of the structural has been ascribed roles in several cellular processes apart
maintenance of chromosomes (SMC) family of ATPases, from chromosome condensation; these have been exten-
clasped into topologically closed rings by accessory sively described elsewhere [12,13]. Conversely, the related
subunits. Condensin binds to DNA dynamically, in a poorly SMC complexes might also contribute to chromosome
understood cycle of ATP-modulated conformational condensation [14]. In this review, we focus on the role of
changes, and exhibits the ability to positively supercoil condensin in mitotic chromosome condensation.
DNA. During mitosis, condensin is phosphorylated by the
cyclin-dependent kinase (CDK), Polo and Aurora B kinases Molecular Architecture of Condensin
in a manner that correlates with changes in its localisation, Eukaryotic condensin is a large pentameric complex that
dynamics and supercoiling activity. Here we review the re- comprises a core catalytic Smc2–Smc4 heterodimer. As is
ported architecture, biochemical activities and regulators characteristic of SMC proteins, Smc2 and Smc4 contain
of condensin. We compare models of bacterial and eukary- three globular parts — two terminal and one central — linked
otic condensins in order to uncover conserved mecha- by long coiled coils. Each SMC protein folds back on itself
nistic principles of condensin action and to propose through antiparallel coiled-coil arm interactions. This forms
a model for mitotic chromosome condensation. an SMC dimerisation hinge domain from the central part at
one end, and an ATPase head domain from association of
Introduction the terminal globular parts at the other (Figure 1). The cata-
The propagation of the blueprint of life, at a molecular level, lytic head domain features canonical ATP-binding cassette
can be described as the accurate transmission of replicated motifs: the amino-terminal ‘Walker A’ motif, and carboxy-
genetic material to daughter cells. To enable this, cells must terminal ‘Walker B’ and ‘C/signature’ motifs. The amino-
compact centimetre-long DNA molecules, within the con- terminal globular part of one SMC subunit engages in trans
fines of micrometre-sized nuclei, into stable chromosomes with a carboxy-terminal part from the other to form a bipartite
that can withstand the forces generated during segregation. ATP-binding pocket. Three accessory subunits bind to the
This condensation of chromatin — into the thread-like SMC heterodimer and regulate its activity. Kleisin I/Brn1,
chromosomes that give mitosis its name (from the Greek a member of the kleisin family [15], interacts at its amino
mitos, i.e., thread) — is one of the most striking morpholog- terminus with Smc2, and at its carboxyl terminus with
ical events of the cell cycle. Yet more than a century after Smc4 to form a topologically closed ring [10,16,17]. HEAT
Walther Flemming first observed mitotic chromosomes [1], IA/Ycs4 [18,19] and HEAT IB/Ycg1 [20] contain HEAT
and Theodor Boveri proposed they maintained their identity repeats, and interact with the amino- and carboxy-terminal
through interphase [2], mechanistic explanations of chromo- halves of Kleisin I/Brn1, respectively, and weakly with each
some condensation remain elusive. other [21]. All three accessory subunits of condensin are
Early indications of a mitosis-specific condensation factor required for its association with chromatin and function in
in cells began to emerge in the 1970s: classical cell fusion chromosome condensation [22]. It is noteworthy that while
experiments showed that premature chromosome conden- the integrity of the ring-like structure of condensin is neces-
sation could be induced in interphase HeLa cells that were sary for its function [17], details of its mechanistic signifi-
fused to mitotic ones [3]. In a cell-free system derived from cance remain to be determined. This is in contrast to the
Xenopus eggs, metaphase chromosomes were assembled case of cohesin, where it has been shown that the complex
in interphase nuclei incubated with mitotic extracts [4,5]. topologically encircles sister chromatids until separase-
Subsequent studies in budding and fission yeasts [6,7], driven cleavage of the kleisin Scc1 at anaphase onset
Xenopus egg extracts [8] and chicken cells [9] led to the enables them to segregate [23,24].
identification of Smc2 and Smc4, core components of the Most eukaryotes possess two isoforms of condensin,
condensin complex, as proteins essential for chromosome termed condensin I and II. These are built from identical
condensation and segregation. core heterodimers of Smc2 and Smc4 but differing acces-
sory subunits (Table 1), which may modulate the differential
localisation patterns, dynamics, and functions of the two
1Chromosome Segregation Laboratory, Cancer Research UK London condensins. Condensin I is termed the canonical condensin
Research Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK. due to its phylogenetic ubiquity (Figure 2) and relative
*E-mail: [email protected] cellular abundance, although the ratio of condensin I and II
Review
R1013

Table 1. Condensin subunits involved in mitotic chromosome

condensation.

Subunits S. cerevisiae S. pombe C. elegans others

Core (condensin I and II)
Smc2 Smc4 SMC2 Smc2 Cut14 MIX-1 CAP-E
MukB MukB SMC4 Smc4 Cut3 SMC-4 CAP-C
Condensin I-specific
Kleisin I (g) Brn1 Cnd2 DPY-26 CAP-H
HEAT IA Ycs4 Cnd1 DPY-28 CAP-D2
HEAT IB Ycg1 Cnd3 CAPG-1 CAP-G

C HEAT IB Condensin II-specific

Kleisin I N C (Ycg1) N C Kleisin II (b) – – KLE-2 CAP-H2
(Brn1) C N C N HEAT IIA – – HCP-6 CAP-D3
MukF HEAT IIB – – CAPG-2 CAP-G2
N MukF
MukE
HEAT IA MukE
(Ycs4) = ATP eukaryotic condensin, but note that the two prokaryotic
Current Biology
complexes are not strictly phylogenetically closer to eukary-
otic condensin than to other eukaryotic SMC complexes.
Figure 1. Molecular architecture of condensin.
Subunit composition of eukaryotic (left) and bacterial (right) conden-
sins. Condensins are composed of a core dimer of SMC or SMC-like
Differential Contributions of Condensin I and II
ATPases with a dimerisation hinge at one end and catalytic head to Chromosome Structure
domain at the other. The core dimers are closed into rings by kleisins, Condensin I and II exhibit distinct spatial staining patterns on
which are monomeric in eukaryotes and dimeric in prokaryotes. One or chromosome axes, as well as differing temporal localisation
more additional regulatory subunits interact with the kleisin and/or patterns through the cell cycle [25,36], suggesting that they
SMC core. may have non-redundant roles in chromosome organisation.
For instance, in HeLa cells, condensin I is excluded from the
nucleus in interphase and binds to chromatin only on nuclear
varies substantially in different organisms, ranging from 1:1 envelope breakdown (NEBD) in prometaphase. In contrast,
in HeLa cells and 5:1 in Xenopus egg extracts [25] to 10:1 condensin II is essentially nuclear in interphase, is stabilised
in chicken DT40 cells [26,27]. The presence of condensin I on chromatin in early prophase, and remains associated
and II in diverse eukaryotic taxa suggests that their last with chromosomes throughout mitosis [36–38].
common ancestor possessed both condensin isoforms The differential contributions of the two condensin com-
[13]. This implies that condensin II was independently lost plexes to chromosome condensation are as yet poorly
from the genomes of organisms such as fungi and ciliates understood. In Xenopus egg extracts, the phenotypes
that possess only a single known isoform of condensin. following immunodepletion of condensin I- or II-specific
Prokaryotes were believed to possess one of two SMC- subunits indicate that condensin I plays the major role in
related complexes: the SMC-ScpAB complex [28-30] wide- condensation [25]. By contrast, in HeLa cells, the siRNA-
spread in bacteria and archaea, or the MukBEF complex mediated knockdown of either condensin by RNAi leads
[31,32] present in g-proteobacteria such as Escherichia to only minor abnormalities in chromosome morphology
coli. These two complexes are divergent at the sequence [25,37], with condensin I depletion producing swollen
level but share a common architecture. Inactivation of the chromosomes, and condensin II depletion making them
two complexes produces defects reminiscent of condensin somewhat longer and curled. The varying severity of
mutants, including decondensed nucleoids, chromosome condensation phenotypes subsequent to condensin deple-
segregation failure, anucleate cell formation and tempera- tion in these two systems may be ascribed either to dose-
ture-sensitive growth [28–32]. SMC-ScpAB is composed of dependence, given the differing ratios of the two condensin
a catalytic SMC homodimer, the kleisin ScpA and accessory isoforms, or to incomplete silencing by RNAi. Consistent
protein ScpB, both of which are likely binary in the complex. with the observed aberrant chromosome morphologies in
Similarly, MukBEF comprises an SMC-like core MukB homo- HeLa cells, further studies in chicken DT40 cells [27] and
dimer, while the kleisin MukF and accessory protein MukE Xenopus egg extracts [39] implicate condensin II in the early
again form a dimeric frame that interacts with the MukB mitotic axial shortening of chromosome arms, and conden-
heads [33,34] (Figure 1). A third family of MukBEF-like SMC sin I in their later lateral compaction. More work is needed
protein, termed MksBEF, has recently been identified in to determine how two very similar complexes are able to
diverse bacterial genomes [35]. MksBEF is often present bind to distinct chromosomal regions, and whether it is their
alongside SMC-ScpAB, MukBEF, or even other MksBEFs, differential localisation or intrinsic activity that is responsible
suggesting that prokaryotic genome organisation may be for their separable contributions to condensation.
more complex than previously appreciated. This also raises
the possibility of as yet undiscovered molecular drivers of Cell-Cycle Regulation of Chromosome Condensation
chromosome condensation in the larger and incompletely Numerous aspects of condensin biology are, reportedly,
annotated genomes of eukaryotes. We refer to the bacterial subject to control by the cell cycle machinery, making
SMC-ScpAB and MukBEF complexes as prokaryotic con- possible a multi-layered regulation of its function. Condensin
densins, due to their condensin-like null phenotypes. In activity may be regulated at the level of holocomplex
the following sections, we draw mechanistic parallels with formation, subcellular localisation, chromosomal loading,
Current Biology Vol 22 No 23
R1014

Figure 2. Phylogenetic analysis of condensin

kleisin subunits.
γ) Phylogram representing the evolutionary
sin

M. musculus
relationships among eukaryotic kleisin sub-
ei

s
l

dyte
ns
(k units. A maximum likelihood unrooted tree

C. m

H. sapie
was constructed in PHYLIP [118] from a Clus-
I

D. rer
C.

oglo
n
si

ris
rus
talW multiple alignment [119] and rendered
rei
T.

ero
en

er

ilia
P. tr
n
th

st radially using iTOL [120]. The excavate Nae-

tau
io
nd

ha

lae
er

am
P. a
og gleria gruberi was used as an outgroup (top
m

rdt
Co

tri

B.

l. f
op

co i an left), from which taxa fan out clockwise in

el
i
rn
hi

C.
ut lis
la

N. um .m ti na order of increasing branch lengths. Note that

D tes kleisins from species with a single known
gru in
be
ri C. condensin isoform, such as fungi and the
be
om ciliate Tetrahymena thermophila, cluster with
S. p
the condensin I sequences. The scale bar
assa
D. rerio N. cr represents one unit of evolutionary distance
along branches, as computed by the Jones-
M. oryzae Taylor-Thornton method [121].
C. elegans
A. gossypii
stinalis
C. inte S. ce
is revis
am iliar K.
iae
C . l. f lac fluorescence recovery after photo-
u rus O.
tis
bleaching (FRAP) assays, would prove
B . ta s s.
ulu A. jap instructive in further elucidating regula-
c on
us th ica tion at the level of condensin holo-
s

al
en

.m ia
C.

complex formation.
pi

tes

M na
sa

C.

ele
tii
dy

D. m
H.

liana
ard

P. tricornu

ga
me
O. s. japonica
glo

ns

Subcellular Localisation
einh

rol
tro

elan
A. tha

ae

In bacteria and archaea that lack

P.

C. r

ogas

1 a nuclear envelope, condensin is free

tum

to interact with chromatin at any time,

ter

Co
nde Species containing limited only by the possible regulation
nsin condensin I of complex formation and DNA loading
II (kleisin β)
condensin II
reactions. In eukaryotes, however, the
Current Biology nuclear envelope offers a potential
barrier to chromatin access and conse-
quently a possible mode of regulation.
or chromatin binding dynamics, one or more of which are In organisms with a closed mitosis like the budding and
likely altered by post-translational modifications (Figure 3). fission yeasts, condensin has to be imported into the nucleus
at or before chromatin compaction in mitosis. Intriguingly, in
Holocomplex Formation the budding yeast Saccharomyces cerevisiae, condensin
In the test tube, condensin is often found in two major forms localises to the nucleus throughout the cell cycle, a behaviour
corresponding to the Smc2–Smc4 heterodimer and the hol- reminiscent of condensin II of higher eukaryotes, despite the
ocomplex, as seen in early immunoaffinity purifications complex being a homologue of condensin I at the sequence
from Xenopus egg extracts [10], as well as reconstitutions level (Figure 2). On the other hand, condensin in the fission
of recombinant human condensin [21]. In addition, yeast yeast Schizosaccharomyces pombe is predominantly cyto-
Smc2 and Smc4 can form a stable heterodimer in cell plasmic in interphase and nuclear during mitosis, an enrich-
extracts [40]. In vitro studies have ascribed differing activi- ment that requires the CDK-dependent phosphorylation of
ties to the SMC/MukB dimer and the holocomplex [10,41]. the T19 site in Smc4/Cut3 [45]. In higher eukaryotes with an
However, it should be noted that the uncomplexed SMC open mitosis, nuclear envelope breakdown (NEBD) at mitotic
dimer has not been directly observed in vivo. Interestingly, onset ensures that the chromatin association of condensin is
the reported role of condensin in disassembly of the not hindered. In Drosophila melanogaster [46], Caenorhabdi-
Drosophila nurse cell polytene chromosomes depends on tis elegans [47], zebrafish [48] and HeLa [36–38] cells, con-
a timely upregulation of only Kleisin II/CAP-H2 [42], indi- densin I is cytoplasmic in interphase and nuclear in
cating there may be a role for a limiting subunit in the regula- mitosis, while condensin II is nuclear throughout the cell
tion of complex assembly. Similarly, HEAT IA/CAP-D2 is cycle, at least in HeLa cells. An interesting but as yet unre-
a rate-limiting factor for the assembly of functional conden- solved question is how this differential subcellular localisa-
sin I complexes in Xenopus oocytes [43]. This is reminiscent tion of condensin I and II is achieved, either by
of cohesin, where levels of the kleisin Scc1 vary through the modifications to their regulatory subunits or recognition by
cell cycle, and determine the chromosomal association of additional factors. Intriguingly, in Drosophila embryos, Klei-
the complex [44]. This principle can be applied more broadly, sin I/Barren associates with chromatin several minutes
raising the possibility that varying expression levels of con- before NEBD [46]. In addition, HeLa cells depleted of
densin I- and II-specific subunits determine the changing condensin II still initiate chromosome compaction just
shapes of mitotic chromosomes in a developmental context before NEBD [38]. These observations suggest that chromo-
[39]. Investigations of complex assembly and DNA binding some compaction in these organisms may be functionally
dynamics of individual condensin subunits, for instance by coupled to disassembly of nuclear pore complexes (NPCs)
Review
R1015

Figure 3. Regulation of condensin activity.

Condensin can be regulated at several Cytoplasm Nucleus
different levels: complex formation, nuclear
import, chromosomal localisation, binding
dynamics, and ATPase activity. This regula-
tion is likely performed by posttranslational
modifications, which can modulate the bio- DNA loading Dynamics/
chemical activities of the complex. Scc2/4 Stabilisation
ParB
Aurora B

Complex
rather than the nuclear membrane formation
[38,49]. This is probably also the case
in C. elegans, where NEBD is com-
pleted only in anaphase but chromo-
some condensation is initiated in
prophase, accompanied by NPC
breakdown [47].

Dynamic Binding to DNA Nuclear

Chromosome condensation is a localisation Biochemical activity
Smc4/Cut3 T19-P Supercoiling ↑CDK, PLK1; ↓CKII
dynamic process, and condensin is
NEBD ATPase
required not only to assemble chromo- NPC breakdown
somes, but also to maintain them in
a condensed state throughout mitosis.
Immunodepletion of condensin from Current Biology
mitotic chromosomes assembled in
Xenopus egg extracts results in their
rapid decondensation [8]. In mitotic yeast cells, the inactiva- with chromosomes, and may in turn be recruited at least in
tion of condensin leads to dramatic defects in ribosomal part by the transcription factor TFIIIC [50]. In the same model
DNA (rDNA) compaction [22], and decondenses chromo- organism, replication fork barrier (RFB) sites at the 3’ end of
some arms to G1-like levels [50]. rDNA genes serve as cis elements for condensin recruitment
Interestingly, while condensin exchanges dynamically in a manner dependent on the RFB-binding protein Fob1, the
from chromosomes, condensin I and II exhibit different topoisomerase-I-interacting protein Tof2 and the monopolin
dynamic behaviour through the cell cycle. FRAP experi- subunits Csm1 and Lrs4 [54]. In addition, the fission yeast
ments on GFP-tagged kleisin subunits in HeLa cells have homologues of these monopolin subunits, Pcs1 and Mde4,
shown that condensin I, which is excluded from the nucleus act as recruitment factors for condensin enrichment at the
in interphase and binds to chromatin on nuclear envelope kinetochore [55]. In the case of condensin II, it has been re-
breakdown, dynamically exchanges from chromosomes ported that protein phosphatase 2/PP2A acts in a noncata-
throughout mitosis. In contrast, condensin II, which is lytic fashion to recruit the complex to frog and human
nuclear throughout the cell cycle, is stabilised on chromatin mitotic chromatin [56].
at the onset of condensation in prophase [38]. What Budding and fission yeast remain the only eukaryotes in
brings about this change in kinetic turnover, whether it is which condensin binding sites have been comprehensively
related to cell-cycle-dependent posttranslational modi- characterised [50,51,57]. These studies show condensin to
fications, and what the consequences are on the chromo- be enriched at centromeres, an enrichment that becomes
some condensation status are important questions to particularly striking in mitosis compared to interphase. The
explore. Curiously, the budding yeast condensin is bound molecular events governing this centromeric enrichment of
to chromatin even in interphase, and its levels and distribu- condensin remain unclear. Condensin underlies, in part,
tion along chromosome arms remain largely unaltered the stiff elastic properties of centromeres during spindle
through the cell cycle [50,51]; the dynamic behaviour of attachment [36,38,58,59]. Along chromosome arms, con-
this complex as a function of the cell cycle has not yet densin is found at TFIIIC-bound, RNA polymerase III-tran-
been investigated. scribed genes, notably tRNA genes, as well as a subset of
other strongly transcribed genes, including ribosomal
Condensin Loading protein genes. A TFIIIC binding element is sufficient to recruit
A critical but poorly understood aspect of condensin function condensin to previously unoccupied sites in S. cerevisiae
is its loading onto chromosomes. The two — not mutually [50], suggesting that condensin binding along chromosomes
exclusive — possibilities are that condensin directly recog- is determined by certain cis-acting elements. What exactly is
nises DNA sequence elements or chromatin features, or recognised by condensin, as well as the Scc2–Scc4 loading
that additional recruiting factors are responsible for loading complex that is found at the same sites, remains to be
condensin onto DNA. There is some evidence for the latter. elucidated. A chromosomal binding map of the Bacillus
In bacteria, the DNA-binding protein ParB recruits SMC to subtilis SMC complex has also been obtained, and shows
centromere-like parS sequences that cluster near the origin striking similarities to yeast condensin, with an enrichment
of replication [52,53]. In budding yeast, the cohesin loader at the bacterial centromere-like partitioning locus as well
Scc2–Scc4 promotes functional association of condensin as tRNA, ribosomal protein and other strongly expressed
Current Biology Vol 22 No 23
R1016

genes [60]. Little is as yet known about chromosomal con- in fission yeast Kleisin I/Cnd2 — S5, S41 and S52 — of which
densin binding patterns in higher eukaryotes, an area of S52 phosphorylation was shown to depend on Aurora B
great interest for forthcoming studies. kinase in vivo; substitutions of the three serine residues to
nonphosphorylatable alanines lead to chromosome misse-
Post-Translational Modifications gregation [80]. These observations have been confirmed
Mitotic CDK-dependent phosphorylation has been shown to and extended to mammals. A series of in vitro and in vivo
stimulate the ATPase and supercoiling activities of Xenopus experiments showed that mitotic phosphorylation of the
condensin I [61,62]. Subsequent studies using budding condensin I kleisin subunit triggers its interaction with the
yeast condensin have established that CDK-mediated phos- basic amino-terminal tail of histones H2A and H2A.Z, which
phorylation of Smc4 primes the complex for hyperphos- is required for chromatin association of the complex [55].
phorylation of its three regulatory subunits by the Polo The conservation of phosphorylation-dependent condensin
kinase PLK1/Cdc5. This hyperphosphorylation then further interactions with histone H2A variants between fission yeast
activates the DNA supercoiling activity of condensin [63]. and mammals, and the general requirement of Aurora B
Similarly, CDK has been shown to phosphorylate the T1415 kinase for chromosome condensation in various species,
residue of HEAT IIA/hCAPD3 in HeLa cells, priming the suggests this may be a fundamental mechanism common
complex for hyperphosphorylation by PLK1, and ensuring to all eukaryotes.
the fidelity of chromosome assembly [64]. Taken together, In addition to the kinases CDK, Polo and Aurora B, the
these observations establish CDK and PLK1 as major molec- phosphatase Cdc14 also plays a role in chromosome
ular cell cycle regulators of condensin function. Large-scale condensation in budding yeast. Cdc14 is sequestered in
phosphoproteomic studies [65–67] have described numer- the nucleolus until it is activated in anaphase by the Cdc-
ous additional cell-cycle-regulated condensin phosphory- Fourteen Early Anaphase Release (FEAR) pathway and the
lation events (some of which are summarised in [68]) the mitotic exit network (MEN) [81]. In early anaphase, Cdc14
role of which remains to be explored. A casein kinase is essential for anaphase-specific condensation and segre-
II-mediated, interphase-enriched phosphorylation of con- gation of the rDNA locus, in a manner dependent on con-
densin I in human cell cultures is the only known phosphor- densin and Aurora B [82]. Cdc14 promotes condensin
ylation that negatively regulates the supercoiling activity of association with rDNA, which correlates with the sumoyla-
condensin [69]. tion of HEAT IA/Ycs4 and phosphorylation of HEAT IB/
The chromosomal passenger complex (CPC) Aurora Ycg1 [83]. The Cdc14 target(s) that promote rDNA conden-
B kinase has been implicated in chromosome condensation sation and segregation are not known. One possibility is
in a number of species. In the fission yeast S. pombe, the that the Cdc14-dependent dephosphorylation of the CPC
CPC member Cut17/Bir1 is essential for proper localisation component Sli15 and the resultant anaphase-specific reloc-
of the Aurora B-like kinase Ark1, condensin recruitment alisation of Aurora B to the spindle midzone play a role [84].
and chromosome condensation [70]. In this system, Aurora Aurora B at the spindle midzone has been proposed to
B kinase activity is required for the mitotic chromatin associ- promote hyper-condensation of trailing chromosome arms
ation of condensin but not its nuclear localisation [71,72]. [55,85]. Potential roles of sumoylation or direct dephosphor-
In Drosophila Schneider cells, RNAi against Aurora B is ylation of condensin subunits by Cdc14 remain to be
associated with a loss of chromatin-bound Kleisin I/Barren, explored, as is the role of Cdc14 in anaphase condensation
leading to incomplete chromosome condensation, abnormal in other species. In contrast to its early anaphase role in
segregation, a failure of cytokinesis and polyploidy [73]. In compaction, Cdc14 promotes chromosome decondensation
budding yeast, the anaphase condensation of rDNA arrays subsequently in late anaphase. At this stage, CDK inhibition
requires Aurora B/Ipl1 [74]. Similarly, C. elegans Smc2/ and Cdc14 activity impair the association of Brn1 with chro-
MIX1 fails to be recruited to chromatin after Aurora B RNAi matin [86]. These results are consistent with the possibility
[75], and chromosome condensation is consequently de- that condensin dephosphorylation by Cdc14 promotes
layed [76]. In HeLa cells, RNAi, as well as treatment with chromosome decondensation at mitotic exit. Since phos-
the Aurora B inhibitor hesparadin, leads to a loss of chro- phorylation generally appears to stimulate the biochemical
matin association of condensin I but not condensin II [77]. activity of the condensin complex, such as DNA binding
Maximal anaphase chromosome compaction in rat kidney and supercoiling, its dephosphorylation may reverse these
cells also depends on Aurora B [78], and condensin I associ- effects to permit chromosome decondensation as cells re-
ation with chromatin is reduced after depletion of Aurora B turn to interphase. Consistent with this idea, the protein
from Xenopus egg extracts [79]. phosphatase PP2A, which plays a role in mitotic exit in
Two common themes emerge from these studies on the higher eukaryotes, has been implicated in the dephosphory-
role of Aurora B in chromosome condensation. First, the lation of HEAT IIA/CAP-D3 [87].
depletion of Aurora B impairs the association of condensin
I with chromatin, an observation consistent in S. pombe Towards a Mechanistic Understanding of Chromosome
[71], Drosophila [73] and HeLa [77] cells, as well as Xenopus Condensation
egg extracts [79]. Second, maximal chromosome compac- Biochemical characterisation of condensin has uncovered
tion occurs in anaphase in a manner requiring Aurora B a number of activities of the complex, including the ability
[74,78] and presumably its kinase activity [71,72]. However, to topologically encircle DNA, supercoil DNA and hydrolyse
a direct link between Aurora B kinase and condensin re- ATP. It is likely that models of mitosis-specific chromosome
mained elusive until Aurora B/Ipl1 was shown to promote condensation by condensin will incorporate some or all of
the mitosis-specific phosphorylation of the budding yeast these activities, each of which could be modulated by
condensin accessory subunits Kleisin I/Brn1, HEAT IA/ post-translational modifications. Notably, postulating the
Ycs4 and HEAT IB/Ycg1 in mitosis [63]. Mass spectrometry topological entrapment of two DNA strands within a single
studies have since identified three Aurora B consensus sites circular condensin complex provides a succinct explanation
Review
R1017

Figure 4. Models of condensin action. A B C

Proposed modes of condensin binding to Interphase
DNA. (A) Top: A ring-shaped condensin
complex topologically captures more than
one strand of DNA in a sequential manner to
bring about condensation. Bottom: A ring-
shaped condensin complex binds to a single
strand of DNA. The condensation reaction
then involves dimerisation of more than
one condensin complex in a handcuff-like
assembly, or even their multimerisation.
These models are not mutually exclusive,
and it is easy to imagine the multimerisation Metaphase
of condensin rings that entrap more than
one strand of DNA. (B) A meshwork of DNA
interactions bridged by condensin. In this
model, condensin constrains the expansion 100 nm
of a ‘nucleosome melt’ by bridging distant
DNA segments. The condensation reaction
involves a change in the dynamic binding of
condensin complexes to DNA or each other. Condensin
(C) Reconstruction of the DNA path and
DNA
condensin in reconstituted Xenopus chro-
Current Biology
mosomes by EM tomography, showing
condensin enriched at sites of chromatin
intersections. (Reproduced with permission
from König et al. [117] and Springer.)

of how condensin might mediate long-range chromosomal Future experiments using superresolution microscopy and
interactions [17]. Alternatively, a handcuff-like assembly of fluorescence resonance energy transfer (FRET) approaches
two tethered condensin rings, as is sometimes proposed will help to investigate this possibility.
in the case of cohesin [88], might bridge distant DNA
regions (Figure 4A). DNA Supercoiling and Topological Selectivity
Eukaryotic and bacterial condensins have both been shown
Multimerisation to possess the intrinsic ability to directly supercoil DNA
Evidence for the formation of multimeric condensin assem- in vitro, albeit with differing directionalities. Condensin puri-
blies stems largely from in vitro studies of bacterial conden- fied from Xenopus egg extracts [10,61] and yeast cells [63] is
sins. In electron micrographs, purified E. coli MukBEF has able to introduce positive supercoils in circular plasmid DNA
been seen as an oligomer, forming extended fibres and in the presence of topoisomerase I and ATP. The Xenopus
rosette-like configurations. In contrast, the MukB homo- condensin preparation also produces chiral knots in DNA
dimer is rarely multimeric, suggesting that intermolecular in the presence of topoisomerase II, leading to the idea
MukE or MukF interactions mediate oligomerisation [89]. that condensin reconfigures DNA by the introduction of a
Protein volume measurements via atomic force microscopy global positive writhe [62]. Like its eukaryotic counterparts,
show that B. subtilis SMC complexes form higher-order the E. coli MukB introduces right-handed knots into DNA in
structures in the presence of ScpA and ScpB, further indi- the presence of phage topoisomerase II; the net supercoiling
cating that the accessory subunits may have a role in the stabilised by MukB, however, is negative [96]. An interaction
organisation of SMC oligomers [90]. There are several indica- of condensin with type II topoisomerases has been demon-
tions that DNA compaction in vitro can proceed by the strated in Drosophila and E. coli [16,97,98]. However, this
concerted action of several condensin complexes. Direct interaction has been implicated in the role of condensin in
force measurements in single molecule experiments demon- the decatenation of sister chromatids to facilitate chromo-
strate that MukBEF compacts DNA into stable, repetitive some segregation [16,99]. Any presumed role for topoisom-
structures in a highly cooperative manner [91,92]. Similar erase II in chromosome condensation remains controversial.
cooperative behaviour of condensin I was observed during While supercoiling and the introduction of a writhe thus
the ATP-dependent compaction of single nanomanipulated remain striking in vitro effects of condensin on DNA, their
DNA fibres [93]. In contrast to these in vitro compaction relevance to chromosome condensation in vivo in the face
reactions in the presence of excess amounts of condensin, of abundant DNA topoisomerases that are adapted to relieve
the number of condensin complexes in vivo is relatively topological stress remains uncertain.
small. Thus, although MukB is found in clusters in living cells Condensin from Xenopus egg extracts was observed to
[94], the applicability of these results to the DNA condensa- preferentially bind cruciform DNA molecules over unstruc-
tion reaction at physiological concentrations of condensin tured linear duplexes, and longer DNA molecules over
remains undetermined. In both yeast and humans, chromo- shorter ones, in electromobility shift assays [100]. While
some condensation is achieved by one condensin complex such binding preferences to complex DNA structures
per 5–10 kb of DNA [51,95]. Whether and how interactions in vitro remain difficult to interpret, they may in this case
between more than one condensin complex contribute to point to an interesting feature of condensin. In the case of
chromosome condensation in vivo is as yet unknown. Our E. coli MukB, single molecule recordings have shown the
initial attempts to detect signs of condensin interactions protein to stabilise interactions between two strands of
in vivo have been unsuccessful (S. Heeger, unpublished). DNA, with a marked preference for right-handed DNA
Current Biology Vol 22 No 23
R1018

crossings [92]. These observations suggest that condensin two different ways. One scenario involves the sequential
does not just capture randomly colliding DNA molecules entrapment of two DNA strands by a single condensin ring.
but may recognise their topology. Such a topology distinc- A second is the dimerisation — or even multimerisation —
tion could contribute to a possible mechanism for dis- of condensin complexes that have captured one strand of
tinguishing interactions within a chromatid from those DNA each (Figure 4A). These two possibilities need not, of
between chromatids. course, be mutually exclusive. In order to understand chro-
mosome condensation, it is necessary to not only address
ATPase Activity the mechanism by which condensin associates with DNA,
SMC proteins lack sequence or structural similarity to but also determine which pairs of DNA sequences along
conventional motor proteins [101,102] and are thus unlikely a chromosome condensin brings together, and how this
to use the energy of ATP hydrolysis to move along chromo- pairing pattern changes as a function of cell cycle progres-
somes or physically reel in DNA. Instead the SMC ATPase sion. Techniques such as chromosome conformation
cycle drives a series of conformational changes at the capture and its variants [111] should be instructive in deter-
molecular level that likely influence the chromosomal associ- mining how condensin modulates intrachromosomal DNA
ation of the condensin complex. ATP binding to the SMC interactions to drive mitotic chromosome condensation.
head domains leads to their engagement, while ATP hydro- A model in which condensin promotes chromosome
lysis allows the heads to move apart [102]. It remains unclear condensation by providing a meshwork of interactions
how precisely the catalytic cycle of ATP-dependent head between distant DNA sequences on the same chromosome
engagement and disengagement is coupled to condensin’s is attractive for a number of reasons. Firstly, the biophysical
interaction with DNA, and whether this contributes to chro- properties of a mitotic vertebrate chromosome, as measured
mosome condensation. The SMC–kleisin interaction is by mechanical micromanipulation studies, suggest that
a candidate for regulation by the ATPase cycle. A striking chromosomes are a composite network of DNA, crosslinked
ATP-dependent conformational change has been observed by protein interactions [112]. In contrast to popular models,
in structural studies of the E. coli MukBEF complex, where no evidence for a contiguous protein scaffold has been
ATP-binding-mediated engagement of the two MukB heads found in native chromosomes. It should be emphasised
leads to the detachment of the dimeric MukF kleisin frame that while a localised axis-like enrichment of condensin
from one of the MukB heads [34]. This conformational has been observed in fixed chromosome preparations
change requires a transient loss of kleisin interaction with [113,114], the imaging of fluorescent-tagged condensin in
one of the MukB heads and thus might be coupled to topo- live cells does not support the notion of such a scaffold
logical DNA entry or exit from the MukB ring. Little is yet [37,38]. A scaffold would not be required if a broad network
known about the consequences of ATP hydrolysis for con- of condensin-mediated interactions between its binding
densin function in vivo, though in the case of cohesin it has sites compacts the chromosome. Recent structural studies
been established that ATP hydrolysis is required for chromo- of human mitotic chromosomes are also consistent with
some association of the complex [103,104]. A recent model this mode of condensin action. Cryo-electron microscopy
for cohesin suggests two distinct entry and exit gates for (cryo-EM) and X-ray scattering on close-to-native frozen
DNA in the complex. DNA in this model enters the ring chromosomes failed to find evidence for a hierarchical
through the Smc1–Smc3 hinge dimerisation interface, and chromosome folding pattern of the kind portrayed in most
exits via opening of the Smc3–kleisin interaction [105]. How molecular biology textbooks [115]. Instead of ordered struc-
conformational changes at the ATPase heads could be tures, chromosomes appear to consist of a ‘nucleosome
translated to the hinge domain some 50 nm away to allow melt’, and we would like to argue that a network of DNA
DNA entry remains to be investigated. Such a long range interactions between condensin binding sites would be
interaction has been demonstrated between the two parts ideally suited to constrain the expansion of such a melt.
of the cohesin complex [106]. In the case of condensin, Evidence from three-dimensional imaging of lac operator
a head–hinge interaction is apparent in atomic force micro- arrays on metaphase chromosomes, in addition, shows
scopic images of the fission yeast complex [107]. The that their folding pattern is irregular between cells and even
archaeal SMC hinge has also been implicated in its binding between sister chromatids, as would be expected from
to DNA. Notably, an enzymatic crosstalk between DNA a largely self-organising network of condensin binding site
binding close to the hinge and ATP hydrolysis by the interactions [116]. The modulation, by mitotic regulators, of
ATPase head domains has been observed [108,109]. At the on- or off-rates of condensin at its binding sites, or of
the same time, the hinges of the prokaryotic SMC and interactions between condensin complexes, could shift the
MukB proteins show substantial structural differences compaction equilibrium from a loose packing in interphase
[108,110]. The impact of ATP binding and hydrolysis on towards a more condensed state in mitosis (Figure 4B). A
SMC complexes therefore remains an important area of prediction from this model is that interphase chromosome
study that should shed light on their ability to dynamically architecture might similarly be governed by condensin.
associate with and condense chromosomes. Pictures of condensin in striking agreement with this model
have been obtained by EM tomography of reconstituted
A Model for Mitotic Chromosome Condensation Xenopus chromosomes [117] (Figure 4C).
Although the precise mechanism by which condensin More work is required to determine the details of conden-
promotes chromosome condensation still remains to be sin action, and the exact nature of the interplay between its
worked out, a few facts about its action are evident. Conden- DNA binding dynamics on the one hand, and its various
sin binds to specific sites along chromosomes, which likely biochemical activities and posttranslational modifications
involves the topological entrapment of DNA. Such conden- on the other. Future investigations towards uncovering
sin–DNA interactions could be translated into long-range mechanisms of chromosome condensation will, no doubt,
chromosomal interactions that bring about condensation in lead to a better understanding of the fascinating problem
Review
R1019

of how cells store, retrieve and transmit information using 23. Uhlmann, F., Lottspeich, F., and Nasmyth, K. (1999). Sister-chromatid
separation at anaphase onset is promoted by cleavage of the cohesin
DNA molecules several orders of magnitude longer than subunit Scc1. Nature 400, 37–42.
the spatial confines of a nucleus. 24. Gruber, S., Haering, C.H., and Nasmyth, K. (2003). Chromosomal cohesin
forms a ring. Cell 112, 765–777.
Acknowledgements 25. Ono, T., Losada, A., Hirano, M., Myers, M.P., Neuwald, A.F., and Hirano, T.
(2003). Differential contributions of condensin I and condensin II to mitotic
We thank D. Agard for the chromosome reconstruction shown in
chromosome architecture in vertebrate cells. Cell 115, 109–121.
Figure 4C. We are grateful to members of the Chromosome Segregation
26. Ohta, S., Bukowski-Wills, J.C., Sanchez-Pulido, L., Alves Fde, L., Wood, L.,
Laboratory for discussions and critical reading of the manuscript. Chen, Z.A., Platani, M., Fischer, L., Hudson, D.F., Ponting, C.P., et al. (2010).
This work was supported by Cancer Research UK and the European The protein composition of mitotic chromosomes determined using multi-
classifier combinatorial proteomics. Cell 142, 810–821.
Research Council. R. Thadani and S. Heeger acknowledge support
27. Green, L.C., Kalitsis, P., Chang, T.M., Cipetic, M., Kim, J.H., Marshall, O.,
through a Boehringer Ingelheim Fonds PhD fellowship, and an EMBO Turnbull, L., Whitchurch, C.B., Vagnarelli, P., Samejima, K., et al. (2012).
long term fellowship, respectively. Contrasting roles of condensin I and condensin II in mitotic chromosome
formation. J. Cell Sci. 125, 1591–1604.
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