Download Chromosome segregation: Samurai separation

Dispatch
R531
Chromosome segregation: Samurai separation of Siamese sisters
Michael Glotzer
How do cells ensure that sister chromatids are precisely
partitioned in mitosis? New studies on budding yeast
have revealed that sister chromatid separation at
anaphase requires endoproteolytic cleavage of a protein
that maintains the association between sister chromatids.
Address: Research Institute of Molecular Pathology, Dr. Bohr-Gasse 7,
A-1030 Vienna, Austria.
E-mail: [email protected]
Current Biology 1999, 9:R531–R534
http://biomednet.com/elecref/09609822009R0531
© Elsevier Science Ltd ISSN 0960-9822
Chromosomes are segregated with high fidelity; in yeast,
chromosome loss occurs only about once per 105–106 cell
divisions [1,2]. The machinery responsible for this critical
task is of course the mitotic spindle, a microtubule-based,
bipolar structure, in which microtubules attach to
chromosomes at a specialized site called the kinetochore.
It has long been appreciated that, during mitosis, sister
chromatids — the DNA molecules that arise by replication
of a single chromosome — align on the mitotic spindle by
the binding of sister kinetochores to microtubules growing
from opposite poles of the mitotic spindle. A combination
of forces contributed by microtubule dynamics and microtubule-based molecular motors balances the chromosomes
midway between the poles of the mitotic spindles. This
structural organization is well suited to the equal partitioning of the genome. Moreover, defects in various components are thought to contribute to the development of
certain cancers [3]. I shall focus here on the connection
between sister chromatids and how it is dissolved in an
orderly fashion at anaphase.
In eukaryotes there is a temporal delay between the time
of replication of the genome (S phase) and the time of its
segregation (M phase). To ensure that chromosomes are
segregated equally, sister chromatids are linked to each
other from the time of their synthesis to the time of their
segregation. Although mechanisms do exist that allow
similar chromosomes to find each other, as evidenced by
the pairing of homologous chromosomes during meiosis,
during mitotic cell cycles linkage is established at the time
of DNA replication and is maintained between sister
chromatids until anaphase [4]. In recent years, the nature
of this linkage has been under intense scrutiny, and a
recent study has uncovered a surprising twist to the story
of how the linkage is dissolved at anaphase.
Anaphase onset is not only the abrupt moment at which
sister chromatids separate from each other, but it is also the
moment at which the cell begins to transit from a mitotic
state to an interphase state. This transition is, in part, mediated by the destruction of cyclin, the regulatory subunit of
the central regulator of mitosis, a kinase complex containing cyclin B and cyclin-dependent kinase 1 (Cdk1) [5].
Early experiments indicated that sister separation and
cyclin destruction are independently regulated by multiubiquitination of specific substrates by the anaphase promoting complex (APC) and their subsequent destruction
by the 26S proteasome.
Specific inhibition of cyclin proteolysis or general inhibition of the APC both confer a mitotic arrest phenotype.
But while inhibition of the APC causes a metaphase
arrest, inhibition of cyclin proteolysis allows chromosomes
to separate from each other [6,7]. This suggests that a
protein other than cyclin must be degraded in an APCdependent fashion to allow anaphase to occur. One interpretation of these results is that this protein forms a
proteinaceous ‘glue’ that holds sister chromatids together.
An equally plausible explanation is that this protein is a
soluble inhibitor of anaphase, and that its APC-mediated
destruction indirectly permits dissolution of the bond that
holds sisters chromatids together.
Over the last several years the simpler interpretation, the
‘glue’ hypothesis, has suffered a significant setback, and
the alternative ‘anaphase inhibitor’ hypothesis has gained
considerable support. A mutation was identified in a
budding yeast gene called PDS1 that caused premature
dissociation of sister chromatids (hence the gene name)
[8,9]. The encoded Pds1 protein contains a destruction
box characteristic of APC substrates; the protein was
found to be degraded at the metaphase-to-anaphase transition, and expression of a non-degradable version blocked
cells in metaphase [10]. Moreover, Pds1 is the only protein
that must be targeted for degradation by the APC to allow
anaphase to occur [11]. However, PDS1 is not an essential
gene in budding yeast, nor is its product associated with
chromosomes, as one would expect if it were a protein that
physically holds sisters together.
What does Pds1 do to prevent sisters from separating? And
what holds them together, if it is not Pds1? A clue to the
first question came in the form of a protein, Esp1, that was
found to be associated with Pds1 in budding yeast cells
[11]. Esp1 is a 180 kDa protein with a conserved carboxyterminal domain [12]. ESP1 function is required for the
separation of sister chromatids and its activity is negatively
regulated by Pds1 [11]. Thus, for anaphase to occur, Pds1
is degraded by APC-mediated ubiquitination, allowing
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Current Biology, Vol 9 No 14
Esp1 to become active. Esp1, like most of the known
regulators of the cell cycle, is quite highly conserved; Esp1
homologues can be readily recognized across the animal
kingdom. The sequence conservation reflects an underlying functional conservation, as the fission yeast homolog
of Esp1, Cut1, is also required for sister separation and it
associates with a protein, Cut2, that is functionally analogous to Pds1 [13].
Remarkably, despite their functional conservation Pds1
and Cut2 are only 13% identical in sequence, which is
not usually considered statistically significant. This lack
of sequence conservation greatly hampered the identification of vertebrate equivalents of Pds1. A lead has
come, however, from the recent identification of a
Xenopus protein, xPDS1, on the basis of its instability in
mitotic, but not interphase, extracts [14]. The xPDS1
protein is about 30% identical in sequence to a human
protein encoded by a gene called pituitary tumor transforming gene (PTTG). Each of these vertebrate proteins
is only about 15% identical in sequence to yeast Pds1,
but their genes appear to be functional homologs of
PDS1 and Cut2. PTTG associates with hESP1 and nondegradable mutant forms of xPDS1 cause dominant
mitotic arrest. Thus, in both vertebrates and yeast,
anaphase onset requires the destruction of a rapidly
evolving protein that acts as an inhibitor of Esp1. What
does active Esp1 actually do? The answer to this question requires a digression into the nature of the linkage
that holds sister chromatids together.
In budding yeast, sister chromatid linkage, or cohesion, is
mediated by a complex of proteins that contains, at the
least, two Smc proteins and two additional conserved
components, Scc3 and Scc1/Mcd1, which together form
the so-called cohesin complex [13–15]. Smc proteins,
named for their involvement in the structural maintenance
of chromosomes, are required for chromosome condensation, recombination and dosage compensation [16]. These
proteins are a structural curiosity. Their sequences reveal
three distinct regions: an amino-terminal region that
contains part of an NTP-binding motif; a central coiledcoil region; and a carboxy-terminal region that contains the
remaining parts of the NTP-binding motif and a domain
that binds DNA [17]. Electron microscopy showed that
Smc proteins form antiparallel dimers with a central hinge
and two identical terminal globular domains that bind
nucleotide and DNA [18]. Though still not fully established, the cohesin complex fits the bill for a proteinaceous link between sister chromatids. It is required from S
phase until M phase, and during this time it is bound to
chromosomes. The complex then dissociates at the onset
of anaphase in a reaction that depends on Esp1.
In order to examine how Esp1 causes cohesin dissociation,
an in vitro assay was developed in which cohesins bound
to chromatin could be mixed with soluble extracts from
cells overproducing Esp1. Using this assay, Esp1-containing extracts were found to cause Scc1 to dissociate from
chromatin [19]. The surprising result was that one cohesin
subunit, Scc1, underwent a dramatic mobility shift of
approximately 40 kDa in response to Esp1 activity. Such a
significant mobility shift suggested a proteolytic event.
Two controls suggested that this processing was specific:
firstly, the Scc1 associated with the chromatin was much
more sensitive to the processing event than soluble Scc1;
and secondly, the reaction was inhibited by Pds1. The
nature of the processing event was established by analysis
of a derivative of Scc1 carrying distinct epitope tags at
either end. When this substrate was subjected to Esp1containing extracts, the two epitopes migrated at distinct
mobilities, confirming that Esp1 indeed induced
proteolytic cleavage of Scc1. A cleaved product of the
appropriate molecular weight could also be observed
in vivo when a synchronous culture of cells exited mitosis.
These data suggest that the cleavage of Scc1 is stimulated
by Esp1, raising the possibility that this cleavage might
regulate separation of sister chromatids. To test this
hypothesis, a version of Scc1 that could not be cleaved was
generated and tested in vivo [19]. First, the site of cleavage was identified by sequencing the carboxy-terminal
fragment of Scc1. An arginine residue immediately
upstream of the first residue of the carboxy-terminal fragment was mutated, and the resulting mutant protein
inducibly expressed in cells. Yeast cells expressing this
mutant protein could still grow quite well; biochemical
analysis, however, revealed that this protein was still
subject to Esp1-induced cleavage, albeit at a site aminoterminal to the previously identified cleavage site.
Sequence analysis revealed a second sequence motif very
similar to the initial cleavage site.
A double mutant form of Scc1 was therefore designed to
abolish both cleavage sites. The double-mutant protein
was found to be resistant to Esp1-mediated cleavage and
toxic to yeast cells in which it was expressed. In cells
expressing the double-mutant protein, sister chromatids
failed to separate and the cohesin complex remained associated with chromosomes. The double-mutant protein
could, however, mediate cohesion between sister chromosomes, so it appears to be specifically defective in the dissociation step at anaphase. Esp1-induced cleavage of Scc1
is thus an essential step in the separation of sister chromatids at the metaphase-to-anaphase transition.
The glue hypothesis has thus undergone a dramatic
renaissance, only the mechanism and its regulation is far
more complex than previously thought (Figure 1). At the
metaphase-to-anaphase transition, ubiquitin-mediated proteolysis of a non-cyclin protein, Pds1, is required for sister
chromatid separation. Pds1 acts to inhibit Esp1, which in
Dispatch
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Figure 1
A model of the reactions required for the
separation of sister chromatids at the
metaphase-to-anaphase transition. The
anaphase promoting complex (APC)
ubiquitinates Pds1, thus targeting it for
degradation and relieving Esp1 from inhibition.
Subsequently, Esp1 induces the proteolytic
cleavage of Scc1 and allows the mitotic
spindle to separate the sister chromatids.
Pds1
Esp1
Smc1+
Smc3
Scc1
Scc3
APC-mediated
Pds1 degradation
Esp1
Current Biology
turn either directly or indirectly causes one member of the
cohesin complex, Scc1, to be chopped into two parts,
allowing the sisters to separate. The remaining components of the cohesin complex may subsequently dissociate
after anaphase.
These results raise several important questions. Firstly,
how does Esp1 induce Scc1 proteolysis — is Esp1 itself
the Scc1 protease? Analysis of the Esp1 sequence has
failed to reveal any hallmarks of proteases; it is, however,
possible that Esp1 is a novel protease, or that the degree
of conservation is insufficient to allow recognition of a
known class of proteases. The answer to this question will
likely be uncovered by biochemical purification of the
Scc1 cleavage activity.
Secondly, have these studies in budding yeast revealed a
conserved mechanism for regulation of anaphase? This is a
very interesting question and several pieces of information
would suggest it is a universal mechanism, though other
data suggest otherwise. The Scc1 cleavage site is
conserved in a Scc1-like protein, Rec8, required for sisterchromatid cohesion during meiosis in budding yeast, and
in two related proteins of the fission yeast Schizosaccharomyces pombe, Rad21 and Rec8. The cleavage site is not,
however, conserved in vertebrate Scc1.
It is possible that the general mechanism — Esp1mediated cleavage of Scc1 — is conserved in vertebrate
cells, but that the recognition site for cleavage has
diverged beyond recognition. However, vertebrate Scc1
has been characterized in some detail, and it has been
found to dissociate from chromosomes upon entry into
mitosis, rather than at anaphase onset as in budding yeast
[20]. In view of this difference, why should Esp1 be conserved, and why does a non-degradable Pds1 block
anaphase onset in vertebrate cells? The explanation may
be that a small fraction of Scc1 remains on chromosomes,
or that there is a second Scc1-like protein that has not yet
been identified that remains on chromosomes until Esp1
induces its cleavage.
These questions are sure to be answered shortly, but the
answers to a host of other interesting questions may
require a longer wait. These include questions such as
why are the anaphase inhibitors evolving so rapidly? Is
Pds1 the only regulator of Esp1 activity? Are cohesins the
sole cellular substrates of Esp1-mediated cleavage? While
these regulatory issues are of great interest, the structural
aspects of chromosome cohesion and separation are
perhaps even more provocative and challenging. As the
recent discovery of a large duplex loop at eukaryotic
telomeres [21] has so vividly demonstrated, there is often
no substitute for direct visualization. How are chromosome cohesion sites arranged? Are they at the base of
chromatin loops? How many cohesin complexes are
present at each site? Does catenation of sister DNA molecules play any role in chromosome cohesion? Do all
cohesin complexes need to be destroyed by cleavage of
the Scc1 to allow sisters to separate? It will be satisfying
to scrutinize the system that enables Siamese sisters to
separate successfully.
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Current Biology, Vol 9 No 14
Acknowledgements
I thank Jan-Michael Peters and Jola Glotzer for helpful comments on
this dispatch.
If you found this dispatch interesting, you might also want
to read the April 1999 issue of
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Current Opinion in
Genetics & Development
which included the following reviews, edited
by James T Kadonaga and Michael
Grunstein, on Chromosomes and
expression mechanisms:
RNA polymerase II as a control panel for multiple
coactivator complexes
Michael Hampsey and Danny Reinberg
Coactivator and corepressor complexes in nuclear
receptor function
Lan Xu, Christopher K Glass and Michael G Rosenfeld
Chromatin-modifying and -remodeling complexes
Roger D Kornberg and Yahli Lorch
Activation by locus control regions?
Frank Grosveld
DNA methylation and chromatin modification
Huck-Hui Ng and Adrian Bird
Mechanisms of genomic imprinting
Camilynn I Brannan and Marisa S Bartolomei
Histone acetylation and cancer
Sonia Y Archer and Richard A Hodin
Modifying chromatin and concepts of cancer
Sandra Jacobson and Lorraine Pillus
Chromatin assembly: biochemical identities and
genetic redundancy
Christopher R Adams and Rohinton T Kamakaka
Stopped at the border: boundaries and insulators
Adam C Bell and Gary Felsenfeld
Nuclear compartments and gene regulation
Moira Cockell and Susan M Gasser
Centromere proteins and chromosome inheritance:
a complex affair
Kenneth W Dobie, Kumar L Hari, Keith A Maggert
and Gary H Karpen
Telomeres and telomerase: broad effects on
cell growth
Carolyn M Price
In vivo methods to analyze chromatin structure
Robert T Simpson
The full text of Current Opinion in Genetics &
Development is in the BioMedNet library at
http://BioMedNet.com/cbiology/gen