Download Shaping the metaphase chromosome: coordination of cohesion and

Review articles
Shaping the metaphase chromosome:
coordination of cohesion and
condensation
Ana Losada and Tatsuya Hirano*
Summary
Recent progress in our understanding of mitotic chromosome dynamics has been accelerated by the identification of two essential protein complexes, cohesin and
condensin. Cohesin is required for holding sister chromatids (duplicated chromosomes) together from S phase
until the metaphase-to-anaphase transition. Condensin
is a central player in chromosome condensation, a
process that initiates at the onset of mitosis. The main
focus of this review is to discuss how the mitotic
metaphase chromosome is assembled and shaped by a
precise balance between the cohesion and condensation
machineries. We argue that, in different eukaryotic
organisms, the balance of cohesion and condensation
is adjusted in such a way that the size and shape of the
resulting chromosomes are best suited for their accurate
segregation. BioEssays 23:924±935, 2001.
ß 2001 John Wiley & Sons, Inc.
Introduction
Chromosomes undergo dramatic structural changes during
the cell cycle, which ensure faithful transmission of the genetic
information into daughter cells. This ``chromosome cycle'' is
summarized in Figure 1. After cell division, each chromosome
consists of a single chromatid with a rather extended configuration. During S phase, it is entirely duplicated, producing a
pair of sister chromatids. The physical linkage between the
sister chromatids (sister chromatid cohesion) is established at
this stage and must be maintained throughout G2 phase.
When cells enter mitosis, chromatids condense to form a
metaphase chromosome, in which the close juxtaposition of
the two chromatids becomes apparent cytologically. At the
metaphase-to-anaphase transition, cohesion is suddenly lost
along the entire length of the chromatids, allowing them to be
pulled apart by microtubules that emanate from opposite poles
of the spindle. When separation is completed, the chromatids
decondense and a new cell cycle starts. During the past
Cold Spring Harbor Laboratory, Cold Spring Harbor, USA. Funding
agencies: NIH and the Human Frontier Science Program. A. L. is
supported by a fellowship from the Robertson Research Fund.
*Correspondence to: Tatsuya Hirano, Cold Spring Harbor Laboratory,
PO Box 100, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA.
E-mail: [email protected]
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decade, we have witnessed great advances in our understanding of the biochemical basis of the mechanisms of cell
cycle progression that underlie these changes in chromosome
structure. Much less has been learnt, however, about how
built-in components of the chromosome contribute to its
morphological transformation. The recent identification of
chromosomal protein complexes directly involved in cohesion
and condensation provides us with a golden opportunity to
address this fundamental aspect of chromosome biology. It
also raises a new set of questions regarding how these
processes are coordinated with each other at a mechanistic
level. In this review, we will first describe the central players in
cohesion and condensation, and then discuss how a precise
balance between cohesion and condensation might help
determine the shape of the metaphase chromosome in
mitosis. Finally, we propose the occurrence of a regulatory
network that may coordinate the two processes.
Cohesion and condensation are mediated by
two SMC protein complexes
During the past several years, genetic studies in yeast and
biochemical analyses in Xenopus have led to the identification
of two multiprotein complexes, cohesin and condensin, that
play a central role in cohesion and condensation, respectively.(1±3) The two complexes are distinct, but both contain
structural maintenance of chromosomes (SMC) proteins as
their core subunits. Members of the SMC family of chromosomal ATPases are present in most, if not all, organisms from
bacteria to human, and are involved in diverse aspects of
chromosome dynamics.(4±6) Eukaryotic SMC proteins have
been classified into four subfamilies (SMC1±SMC4). The
cohesin and condensin complexes contain heterodimeric
pairs of SMC1/SMC3 and SMC2/SMC4, respectively, and
distinct sets of non-SMC subunits (see below). An electron
microscopy study showed that bacterial SMC proteins form
V-shaped homodimers with two long coiled-coil arms connected by a flexible hinge.(7) ATP- and DNA-binding activities
reside in two globular domains at the distal end of each arm. It
is most likely (although by no means proved) that eukaryotic
SMC heterodimers adopt an analogous two-armed configuration. On the basis of the structural similarity and functional
differentiation of cohesin and condensin, it has been proposed
BioEssays 23:924±935, ß 2001 John Wiley & Sons, Inc.
Review articles
Figure 1. The chromosome cycle. Chromosomes are highly dynamic structures subject to multiple morphological changes throughout
the cell cycle (see text for details).
that the two SMC protein complexes may act as different types
of ATP-modulated DNA cross-linkers.(4) Cohesin would hold
together two different DNA molecules (intermolecular DNA
cross-linker), whereas condensin would bind two segments
within a single DNA molecule to facilitate its folding (intramolecular DNA cross-linker). Biochemical properties of the two
complexes in vitro are largely consistent with this model.(8±10)
Cohesin and sister chromatid cohesion
Cohesion between the sister chromatids is achieved by two
types of physical linkages, one mediated by DNA catenation,
and the other by chromatid-linking proteins.(11) The DNA
catenation-based linkage arises as a result of the duplication
process per se, which leaves a certain number of intertwinings
between the duplicated DNA strands in the region where
adjacent replication forks meet.(12) Most of the tangles are
resolved during S and G2 phase by topoisomerase II (topo II),
but full decatenation only occurs at anaphase.(13) Accumulating lines of genetic and biochemical evidence, discussed
below, suggest that cohesin participates in the protein-mediated linkage between the sister chromatids. This complex,
highly conserved from yeast to humans, consists of a heterodimer of SMC1 and SMC3, and two additional non-SMC
subunits called Scc1/Mcd1/Rad21 and Scc3/SA.(14±20) Yeast
cohesin mutants lose chromosomes at high frequency and
show premature separation of the sister chromatids.(14±17)
Similarly, chromosomes assembled in Xenopus egg extracts
immunodepleted of cohesin show severe cohesion defects,
indicating that a cohesin-mediated linkage also exists in higher
eukaryotes.(18)
The cohesin complex purified from HeLa cells can bind to
double-stranded DNA and induce the formation of large
protein±DNA aggregates in vitro.(10) In the presence of topoisomerase II, cohesin stimulates intermolecular catenation of
circular DNA molecules. This activity is in striking contrast to
the intramolecular knotting directed by condensin.(9) Cohesin
also increases the probability of intermolecular ligation in the
presence of DNA ligase. These results fit a model in which the
complex acts as a physical bridge between the sister chromatids (i.e. an intermolecular DNA cross-linker). The role of
ATP-binding and hydrolysis in cohesin function remains to be
determined since none of the activities supported by the
complex requires ATP.
In Saccharomyces cerevisiae, cohesin binds to chromatin
in G1 phase and establishes cohesion during S phase concomitantly with DNA replication.(21) Cohesin remains bound to
chromosomes and maintains cohesion until the metaphaseto-anaphase transition.(14,15) This transition is triggered by the
activation of the anaphase-promoting complex (APC), which
targets a protein called securin (Pds1) for degradation by
ubiquitin-dependent proteolysis. Destruction of securin liberates its binding partner, known as separin or separase (Esp1),
whose cysteine protease activity specifically cleaves one of
the cohesin subunits, Scc1/Mcd1. This cleavage destabilizes
cohesin-mediated cohesion, and leads to a single-step
separation of the sister chromatids(22±24) (Fig. 2A). A similar,
although not identical, mechanism appears to operate in
Schizosaccharomyces pombe.(17)
In Xenopus egg extracts, cohesin binds to chromatin during
interphase but most of it ( 95%) dissociates upon entry into
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Figure 2. One-step versus two-step dissolution of cohesion. A: In yeast, the extent of condensation is limited and chromosome
structure changes very little at the onset of mitosis. Dissolution of cohesion occurs in a single step at the metaphase-to-anaphase
transition by an APC-dependent mechanism that involves cleavage of one of the cohesin subunits. B: In metazoans, two different
pathways regulate the sequential loss of cohesion at two different stages of mitosis. Most of cohesin is released from chromatin during
prophase (step 1) by a mechanism that might involve phosphorylation of the SA subunit. Dissociation of the remaining complexes at the
metaphase-to-anaphase transition is probably triggered by a cleavage-dependent pathway (step 2), analogous to the one in yeast.
mitosis.(18) The same behavior is also observed in vivo in
Xenopus, mouse and human cells.(19,20,25 ±27) Recent studies
have shown that a small population of cohesin remains bound
to metaphase chromosomes and is likely to contribute to
holding sister chromatids together until the onset of anaphase(19,28±30) (Fig. 3B). These observations have led to a
model in which two different pathways regulate the two-step
dissolution of cohesion in higher eukaryotes (Fig. 2B). The first
one, which takes place during prophase, is APC-independent
and may involve phosphorylation of the SA subunit of
cohesin.(19,20) The second pathway, which is activated at the
metaphase-to-anaphase transition, is APC-dependent and
involves the separase-mediated cleavage of the Scc1/Rad21
subunit, as has been shown in yeast.(28) It remains to be
determined how the two pathways distinguish between the
two populations of cohesin and differentially regulate their
dissociation from chromosomes. One possibility is that a
cohesin fraction enriched around the centromeric regions is
``protected'' against the prophase release from chromatin.
Centromere-specific factors, such as Drosophila MEIS322,(31) may contribute to such a selective stabilization of
cohesin.
Genetic studies in yeast and other fungi have shown that,
along with cohesin, a number of proteins are involved in the
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establishment and maintenance of sister chromatid cohesion.
First, binding of cohesin to chromatin is facilitated by a distinct
complex containing Scc2 /Mis4 and Scc4.(17,32) Second, a
protein called Eco1/Ctf7/Eso1 is essential for the establishment of cohesion during S phase, but neither for loading of
cohesin on chromatin nor for the maintenance of cohesion
during G2 phase and mitosis.(16,33,34) Ctf7 interacts genetically
with some DNA replication factors (e.g., PCNA), implicating a
functional link between cohesion and DNA replication.(33)
Further support for this idea comes from the study of Trf4, a
novel DNA polymerase (termed pol k) whose mutation leads to
partial defects in sister chromatid cohesion in S. cerevisiae.(35)
Although it remains to be determined how a DNA polymerase
participates in cohesion, one attractive hypothesis is that pol k
is required to allow passage of the replication forks through the
barrier created by cohesin bound to chromatin during G1
phase.(36) Finally, recent results have shown that a gene
product called BimD/Spo76/Pds5 may work in close association with cohesin in both mitosis and meiosis.(20,37±39) The
amino acid sequence of BimD/Spo76/Pds5 contains multiple
HEAT repeats that could serve as a flexible ``scaffolding'' for
the assembly of other proteins.(39,40) Interestingly, HEAT
repeats are also found in two of the non-SMC subunits of
condensin (CAP-D2 and CAP-G; see below), further empha-
Review articles
Figure 3. Condensin and cohesin in Xenopus mitotic chromosomes. Immunofluorescent staining of chromosomes assembled in vitro
in Xenopus egg extracts with anti-XCAP-E (condenrin; A) or anti-XSA1 (cohesin; B) [the latter reproduced from The Journal of Cell
Biology, 2000, vol.150, pp. 405±416, by copyright permission of the Rockefeller University Press]. Antibody and DNA staining are shown
in yellow and red, respectively.
sizing the structural and functional similarity between the
cohesion and condensation machineries.(40)
Condensin and chromosome condensation
Chromosome condensation is a highly ordered process in
which the two sister chromatids are sorted out and compacted
without losing the linkage between them.(1,41) How condensation is accomplished mechanistically is far from understood,
but one of the major insights in the field came from the
identification of condensin from Xenopus egg extracts.(42,43)
Condensin is one of the most abundant components of mitotic
chromosomes and distributes throughout the chromosome
arms (Fig. 3A). The complex is made up of two SMC subunits
(CAP-C [SMC4-type] and CAP-E [SMC2-type]) and three nonSMC subunits (CAP-D2, CAP-G and CAP-H).(43,44) Genetic
studies in yeast and Drosophila have showed that each of the
condensin subunits is essential and is required for proper
condensation and segregation of mitotic chromosomes.(45±52)
In Xenopus egg extracts, depletion of condensin completely
impairs chromosome assembly in vitro, and the defect can be
complemented by addition of either the Xenopus or human
complex.(43,53) Purified Xenopus and human condensin complexes show two ATP-dependent activities: positive supercoiling of a closed circular plasmid in the presence of
topoisomerase I, and knotting of a nicked circular plasmid in
the presence of topoisomerase II.(8,9,53) These energydependent activities are likely to reflect fundamental aspects
of the condensin action that drives chromosome condensation
in vivo.
The function of condensin is tightly regulated during the cell
cycle by multiple mechanisms that appear to vary among
different organisms. The first mechanism concerns nuclear
import. In S. pombe, Cdc2-dependent phosphorylation of a
condensin subunit mediates the mitosis-specific accumulation
of the whole complex in the nucleus.(54) Such import-mediated
regulation is, however, not observed in S. cerevisiae.(49) In
metazoan cells, a tight association of condensin with mitotic
chromosomes is well documented but whether condensin
subunits are nuclear or cytoplasmic during interphase is still
unresolved.(42,52,55 ±57) Second, condensin function is regulated at the level of chromosomal binding. In Xenopus egg
extracts, the non-SMC subunits of condensin are hyperphosphorylated in mitosis. The complex associates with mitotic
chromosomes, but not with interphase chromatin even when
no nuclear envelope is assembled.(43) Mitosis-specific phosphorylation of histone H3 is suspected to be part of this
recruiting mechanism(58) and, in fact, phosphorylated H3
colocalizes with condensin during the G2±prophase transition
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in human cells.(57) However, neither phosphorylation of
condensin nor phosphorylation of histone H3 is sufficient to
account for the mitosis-specific targeting of condensin in
vitro.(44) Thus, a non-histone ``receptor'' may participate in the
recruitment of condensin to chromosomes. One candidate for
such a receptor, AKAP95, has been reported recently.(56) The
third mechanism of regulation involves phoshorylation-dependent activation of the supercoiling and knotting activities of
Xenopus and human condensin. Several lines of evidence
suggest that Cdc2 is likely to be one of the physiological
kinases that mediate this activation.(9,53,59)
Given the ability of condensin to modulate DNA topology in
vitro, it is of great interest to determine how the complex
interacts with topo II, a protein also required for chromosome
condensation and segregation. An early study showed that
Drosophila Barren protein (homologous to CAP-H) physically
interacts with topo II and directly modulates its activity in
vitro.(47) Recent analysis of barren mutants of S. cerevisiae,
however, provides little support for this idea: the mutant cells
neither accumulate catenated DNA molecules nor show the
pattern of chromosome breakage typically observed in topo II
mutants.(50) Furthermore, topo II and condensin associate
independently with chromatin in Xenopus egg extracts.(43) It is
most likely that the two proteins work in concert (but not
through direct physical interactions) to coordinate chromosome condensation and sister chromatid resolution.(1)
The structure of the metaphase chromosome:
finding the balance between cohesion and
condensation
In the previous section, we have emphasized that the
molecular machineries involved in cohesion and condensation
and their mechanisms of action are likely to be conserved from
yeast to humans. From a cytological and evolutionary point of
view, however, the size and morphology of metaphase
chromosomes are highly divergent among different eukaryotic
species. What is the molecular basis of these differences? In
this section, we discuss how the differential contribution of the
condensation and cohesion machineries could produce
seemingly different chromosome structures among different
organisms.
Compaction ratio and the density of cohesin
and condensin
First, it is important to compare the basic parameters that
characterize an ``average-sized'' metaphase chromosome
from S. cerevisiae and X. laevis (Table 1). Based on the
genome size and the number of chromosomes in each
species, the length of the DNA molecule within a chromosome
can be calculated. The result of dividing this value by the mean
chromosome length (obtained empirically) is the linear compaction ratio. Our estimation yields a linear compaction ratio of
3,000 for mitotic chromosomes assembled in Xenopus egg
extracts. Although individual chromosomes cannot be visualized in the mitotic cell cycle of S. cerevisiae, their compaction
ratio has been estimated to be 140 by fluorescence in situ
hybridization (FISH).(60) This gives 20-fold difference in
compaction ratios between Xenopus and S. cerevisiae. The
compaction ratio for human somatic chromosomes is even
greater ( 10,000). In contrast, the compaction ratio in
interphase chromatin ( 80±100) is similar between yeast
and vertebrate cells.(60,61) Thus, as generally believed, the
extent of mitotic chromosome condensation is modest in yeast
but very high in vertebrate cells.
It is interesting to point out that, despite the big difference in
compaction ratios, the density of condensin per unit length of
DNA is comparable between yeast and Xenopus mitotic
chromosomes (one complex per 8±10 kb of DNA).(9,48)
Table 1. Density of cohesin and condensin on mitotic chromosomes
X. laevis
Genome size
Chromosome number
Average DNA content/chromosome
Average DNA length/chromosome
Average chromosome length
Linear compaction ratio
Density of condensin on DNA
Density of condensin on chromosome
Density of cohesin on DNA(c)
Density of cohesin on chromosome(c)
Relative ratio cohesin/condensin
(a)
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(250)(a)
(230)
(230)
(10)
(20)
(20)
(0.5)
12.1 Mb
16
0.75 Mb
0.26 mm
2 mm
140
every ~8 kb(b)
40 per mm
every 9 kb
40 per mm
1
The values in parentheses indicate the fold-difference between Xenopus and S. cerevisiae for each parameter.
No quantitative data are available for the density of condensin in S. cerevisiae and we have used data from S. pombe instead.
These values have been estimated on the assumption that cohesin distributes uniformly along the entire length of the chromosomes.
(b)
(c)
3000 Mb
18
170 Mb
58 mm
20 mm
3,000
every 10 kb
800 per mm
every 400 kb
20 per mm
1/40
S. cerevisiae
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On the contrary, there is a huge difference in the density of
cohesin on metaphase chromosomes: one complex per 400
kb in Xenopus versus one per 9 kb in yeast (for simplicity, we
assume here that cohesin distributes uniformly along the
entire length of chromosomes).(18,62) This difference is created
largely by the dissociation from chromatin of 95% of cohesin
that occurs during prophase in Xenopus. In fact, during
interphase, the density of cohesin on chromatin is similar in
both organisms (one complex per 20 kb in Xenopus and
9 kb in S. cerevisiae).(18,62) Thus, cohesin is as abundant as
condensin in the yeast mitotic chromosomes, whereas much
less cohesin is present in the Xenopus chromosomes (Fig. 3).
It is tempting to speculate that the density of cohesin along the
chromosomes (or the relative ratio of cohesin and condensin)
may be a key determinant of the size and shape of the
metaphase chromosome. A high density of cohesin would
result in the formation of poorly condensed, elongated
chromosomes (Fig. 4A). Conversely, a low density of cohesin
would create chromatin loops of large size and thereby allow a
high degree of linear compaction (Fig. 4B,C). It is easy to
imagine that efficient compaction is beneficial to species with
larger genomes. Nevertheless, cohesion can not be infinitely
decreased in favor of compaction because it has to be strong
enough to resist the poleward pulling forces of spindle
microtubules from prometaphase to metaphase. To fulfill
these requisites, different mechanisms have arisen during
evolution that ensure the correct balance between cohesion
and condensation for each organism. We have already
described one of them, the massive release of cohesin that
takes place at the onset of mitosis in higher eukaryotes, but not
Figure 4. The balance between cohesion and condensation shapes the metaphase chromosome. Portions of three increasingly
condensed chromosomes are depicted from left to right. The chromatin fiber of each sister chromatid is folded in loops to which
condensin binds. Cohesin is likely to be present in the region where the two sister chromatids make a contact. We assume here that
the density of condensin is invariable along the chromatin fiber (see Table 1). A: A high density of cohesin on the chromosome limits
the size of the chromatin loops. In turn, the small size of the loops constrains a potential action of condensin, thereby leading to the
formation of a chromosome with a low compaction ratio. B, C: As the density of cohesin decreases and the loop size increases,
condensin-mediated folding of the chromatin loops makes a greater contribution to the linear compaction of a chromosome. For
simplicity, structural changes of chromatin supported by condensin in each loop are omitted.
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in yeast. A second mechanism would involve the spatial
compartmentalization of cohesion, as discussed next.
Differential distribution of cohesin in centromeric and
arm regions of chromosomes
Chromatin immunoprecipitation (ChIP) experiments indicate
that cohesin distributes along the entire length of chromosome
arms in S. cerevisiae, and is enriched in centromere-proximal
sequences.(62±65) Despite this enrichment, microtubulemediated pulling forces can overcome cohesion and, as a
result, centromeric regions separate and re-associate transiently prior to anaphase (Fig. 5A).(66±68) Under these
circumstances, arm cohesion seems to be essential to prevent
precocious sister chromatid separation, and this may explain
the high density of cohesin along the yeast metaphase
chromosome. In metazoans, immunofluorescent staining of
mitotic chromosomes shows that cohesin is more abundant
around the primary constriction than along the arms.(19,28±30)
Although transient splitting of centromere-proximal sequences before anaphase is sometimes observed in mammalian cells as well (Fig. 5B),(69) cytological studies reveal a much
closer apposition of the sister chromatids in the pericentromeric regions than along the chromosome arms.(70) Furthermore, when these cells are arrested at metaphase with drugs
that induce microtubule depolymerization (e.g., nocodazole,
colcemid), the two chromatids of each chromosome become
fully separated except around the centromeric regions
(Fig. 5B, ‡ drug). If the drug is removed from the culture
medium, cells exit the metaphase arrest and segregate their
chromosomes in an apparently normal fashion. This suggests
Figure 5. Spatial differences in cohesion along the chromosome. A: In yeast chromosomes, transient splitting of centromeric
sequences before anaphase occurs despite the enrichment of cohesin (represented in green). Arm cohesion is likely to be essential to
prevent premature separation of the sister chromatids. B: In metazoans, cohesion around the centromeric region of the metaphase
chromosome appears to be much tighter than along the arms. This spatial differentiation of cohesion is most clearly observed when cells
are arrested in metaphase with drugs that disrupt spindle assembly ( ‡ drug). In these cells, chromosome arms become fully separated
and the sister chromatids are joined only at their pericentromeric regions. When the drug is removed ( ÿ drug), poleward pulling forces of
the spindle microtubules may induce transient separation of the region underneath the kinetochore, but they are counterbalanced with
the strong cohesive forces supported by the pericentromeric heterochromatin even in the absence of arm cohesion.
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that pericentromeric cohesion is sufficient to prevent premature separation of the sister chromatids under this condition, and that arm cohesion may be less important in
metazoans than in S. cerevisiae.
What is the molecular basis for the stronger cohesion seen
in the pericentromeric regions of higher eukaryotes? These
regions consist mostly of heterochromatin that contains long
tracts of satellite DNAs and other repeated sequences, as well
as specific protein components.(71) Some observations suggest that tight cohesion is dictated by the intrinsic ``stickiness''
of heterochromatin. For example, the arms of the heterochromatic Y chromosome in Drosophila do not separate during a
prolonged metaphase arrest. Also, chromosomes with larger
amounts of heterochromatin take longer to complete segregation presumably because they need more time to dissolve
cohesion.(72) It is conceivable that the peculiar structure and
composition of heterochromatin could direct the recruitment of
more cohesin and/or make cohesin bound to this region
resistant to the release during prophase. Alternatively, heterochromatin-mediated cohesion may be independent of
cohesin. Detailed studies of the behavior of cohesin in heterochromatic regions, as well as analysis of heterochromatin
cohesiveness in cohesin mutants, will certainly help to distinguish between these possibilities. Whatever the mechanism might be, heterochromatin is likely to have evolved to
increase cohesion around centromeric regions and to alleviate
the need for strong arm cohesion in organisms that require
extensive condensation.
Crosstalk between cohesion, condensation
and resolution
As discussed above, the density of cohesin and its spatial
distribution could specify the different structures of yeast and
metazoan chromosomes. These intrinsic differences could
account, at least in part, for the conflicting observations
regarding the potential contribution of cohesin to chromosome
condensation. In Xenopus egg extracts, assembly of chromosomes in the absence of cohesin results in substantial defects
in sister chromatid cohesion whereas condensation is not
apparently compromised.(18) In S. cerevisiae, however, mutations in the cohesin subunit Mcd1(14) and another cohesion
factor, Pds5,(38) appear to affect both cohesion and condensation. The results in yeast have led to a model in which the
compaction of the mitotic chromosomes is achieved by two
mechanistically distinct actions. First, cohesion factors (like
cohesin and Pds5) drive a shortening of the chromosome axis
by inducing the association of adjacent cohesion sites.
Second, condensin drives the packaging of the DNA looped
out between these sites. If this is to be a general model, the
drastic difference in the density of cohesin between yeast and
Xenopus chromosomes must be accounted for (Table 1).
Given the high density of condensin (one complex per 8±10 kb)
and the low density of cohesin (one per 400 kb) in Xenopus
(and probably other metazoans), it is reasonable to speculate
that the action of condensin in chromosome compaction is
dominant whereas the contribution of cohesin to the axial
shortening, if any, is minor. In S. cerevisiae, it has been proposed that cohesin is required not only for the initiation of
condensation but also for the maintenance of a condensed
state.(14) It should be noted, however, that this conclusion was
drawn from the analysis of the rDNA locus, a 500 kb block of
highly repetitive genes. Since this locus has a peculiar
chromatin organization,(49) the general validity of these observations is unclear. In metazoan cells arrested in metaphase,
loss of cohesion along the chromatid arms is not accompanied
by a decrease in chromosome condensation. Rather the
metaphase arrest induces hypercondensation (Fig. 5B,
lower). This classical observation argues against a requirement for cohesin in the maintenance of compaction of the
chromosome arms.
Next it would be worth asking the opposite question: is
condensin function required for sister chromatid cohesion?
Premature separation of sister chromatids is not observed in
condensin mutants. Instead, they often display anaphase
segregation defects, indicative of poor resolution of DNA
catenation between sister chromatids.(45±52) This is consistent
with the idea that condensation is not a simple compaction
process; rather it helps topo II-mediated decatenation of sister
DNAs.(1,12) The importance of this aspect of chromosome
condensation can explain why condensin subunits are
essential in organisms like S. cerevisiae in which the linear
compaction of mitotic chromosomes is minimal.
A regulatory network that coordinates cohesion
and condensation in mitosis?
How is the balance between cohesion and condensation
precisely regulated during the cell cycle? It is attractive to
postulate that a regulatory network coordinates cohesin
dissociation from chromatin and condensin association
with chromosomes during mitotic prophase. Cdc2, the
master mitotic kinase, may be part of such a network. Although
Cdc2-cyclin B phosphorylates and activates condensin
in vitro,(9,53,59) it is unknown whether this phosphorylation
has a direct role in recruiting condensin to chromosomes. The
same kinase also phosphorylates the SA subunit of Xenopus
cohesin in vitro and reduces its affinity to chromatin and
DNA.(19) This phosphorylation may prevent a re-association of
released cohesin during mitosis, rather than directly induce
cohesin dissociation from chromatin.
A large number of recent publications suggest that a
chromosome-bound kinase known as Aurora-B plays an
important role in mitotic chromosome dynamics. Aurora-B is
a member of the aurora kinase family. S. cerevisiae has only
one member (Ipl1) whereas at least three members (classified
as A, B, and C) have been found in higher eukaryotes.(73) In
C. elegans, Drosophila and human cells, Aurora-B is targeted
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to chromosomes along their length during prophase, becomes
concentrated around the centromeric regions at metaphase,
and is transferred to the spindle midzone when sister
chromatids separate in anaphase.(74±76) This localization is
characteristic of a class of proteins known as ``chromosomal
passengers'' with important (yet not fully understood) roles in
chromosome segregation and cytokinesis.(77) Interestingly,
the inner centromere protein (INCENP), the first chromosomal
passenger to be described,(78) forms a complex with AuroraB(75,79,80) and is required for proper targeting of Aurora-B to
chromosomes.(75) Strong lines of evidence suggest that
Aurora-B is the physiological kinase that phosphorylates
histone H3 during mitosis in S. cerevisiae,(81) C. elegans(82)
and Drosophila.(76) Drosophila cells depleted of Aurora-B by
RNA-mediated interference (RNAi) show decreased chromo-
somal levels of a condensin subunit (Barren) and defects in
chromosome condensation and segregation.(76) It remains to
be demonstrated whether Aurora-B recruits condensin to
chromosomes directly, by phosphorylating its subunits, or
indirectly, through phosphorylation of histone H3 or some
other ``chromatin receptor''. We would like to propose here an
additional role for Aurora-B in inducing the dissociation of
cohesin from chromatin during prophase. Although no
evidence is currently available for this idea, it could explain
some of the phenotypes observed in the absence of the
enzyme.(76,80,82) Aurora-B is present at the right place (the
chromosome arms) at the right time (prophase), which makes
it an ideal candidate for the job. The subsequent accumulation
of the enzyme around the centromeric regions could be
instrumental for its transfer to the spindle at anaphase, for
Figure 6. A speculative model for the regulation of cohesin and condensin in prophase. In this model, we assume that Cdc2 and
Aurora-B primarily act in the soluble compartment and on chromosomes, respectively. At the onset of mitosis, Cdc2 is activated and
phosphorylates the soluble pool of cohesin and condensin. INCENP(84) and Aurora-B(85) are phosphoproteins and may also be
substrates of Cdc2. An activated fraction of Aurora B-INCENP is targeted to the chromatin where it phosphorylates cohesin and induces
its release from chromatin. Once released, Cdc2 helps to maintain the phosphorylated state of cohesin to prevent its re-association with
chromatin. Phosphorylation of soluble condensin by Cdc2 stimulates its activities and may also promote its chromatin targeting directly
or indirectly. Aurora-B could contribute to keeping chromosome-bound condensin in its active form. Phosphorylation of histone H3 by
Aurora-B may be directly involved in condensin recruitment, cohesin release or both (black triangle). Putative dephosphorylation
reactions catalyzed by protein phosphatases are indicated by white arrows.
932
BioEssays 23.10
Review articles
mediating kinetochore function(83) or even for regulating the
centromere-enriched population of cohesin.
Taken all together, we propose a model for a regulatory
network that may contribute to finding the precise balance
between cohesion and condensation in metaphase chromosomes (Fig. 6). In this highly speculative model, Aurora-B and
Cdc2 function as key protein kinases in two different
compartments, on chromosomes and around chromosomes,
respectively. The two kinases may not only phosphorylate
several chromosomal components de novo but also maintain
their phosphorylation states in the corresponding compartments. One important question in this model is the role of
histone H3 phosphorylation in the coordination of a number of
chromosomal events that occur during mitosis. It will be
important to determine more precisely how cohesin and
condensin behave in the absence of Aurora B-INCENP in
different model systems.
Conclusions
We propose that the balance between cohesion and condensation is an essential determinant in shaping the mitotic
metaphase chromosome in eukaryotic cells. In organisms with
small chromosomes like S. cerevisiae, a modest compaction is
enough to make the chromosomes ``manageable'' during the
segregation process whereas cohesion along the entire length
of chromatids is essential to withstand the pulling forces of the
spindle microtubules. Consequently, there is no major structural change for yeast chromosomes at the G2 ±M transition.
In higher eukaryotes with much larger chromosomes, the size
of these chromosomes becomes a more serious problem for
segregation, and cohesion along the chromatid arms has to be
sacrificed to allow better compaction. Loosening of arm
cohesion must be counterbalanced in turn by an increased
cohesion around the centromeric regions. For these mechanistic requirements, metazoan chromosomes must go through
a major structural rearrangement at the onset of mitosis.
These important differences in chromosome architecture
emphasize the necessity of using different model organisms
to understand fully how chromosome cohesion and condensation work. Given the tremendous progress that we have
witnessed since the discovery of cohesin and condensin, it
would not be too optimistic to anticipate that the molecular
mechanisms underlying the mysterious behavior of chromosomes will be completely unveiled in the near future.
Acknowledgments
We would like to thank Alfredo Villasante for helpful discussions, and Juan MeÂndez and members of the Hirano Lab for
critically reading the manuscript.
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