© 2014. Published by The Company of Biologists Ltd.
STAG2 promotes error correction in mitosis by regulating
kinetochore-microtubule attachments
Journal of Cell Science
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Marianna Kleyman, Lilian Kabeche and Duane A. Compton*
Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH
03755;
Norris Cotton Cancer Center, Lebanon, NH 03766
USA
* corresponding author
Department of Biochemistry
413 Remsen Bldg.
Geisel School of Medicine at Dartmouth
Hanover, N.H. 03755
Tel: (603) 650-1190
FAX: (603) 650-1128
email: [email protected]
Running Title: STAG2 influences kMT stability
JCS Advance Online Article. Posted on 29 July 2014
Summary
Mutations in the STAG2 gene are present in approximately 20% of tumors from different
tissues of origin. STAG2 encodes a subunit of the cohesin complex, and tumors with
loss of function mutations are usually aneuploid and display elevated frequencies of
lagging chromosomes during anaphase.
Lagging chromosomes are a hallmark of
chromosomal instability (CIN) arising from persistent errors in kinetochore-microtubule
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of kMT attachment errors or decreases the rate of their correction, we examined mitosis
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(kMT) attachment. To determine whether loss of STAG2 increases the rate of formation
overexpression of the microtubule destabilizing enzymes MCAK and Kif2B decreased
in STAG2-deficient cells. STAG2 depletion does not impair bipolar spindle formation or
delay mitotic progression. Instead, loss of STAG2 permits excessive centromere stretch
along with hyper-stabilization of kMT attachments. STAG2-deficient cells mislocalize
Bub1 kinase, Bub3 and the chromosome passenger complex. Importantly, strategically
destabilizing kMT attachments in tumor cells harboring STAG2 mutations by
the rate of lagging chromosomes and reduced the rate of chromosome missegregation.
These data demonstrate that STAG2 promotes the correction of kMT attachment errors
to ensure faithful chromosome segregation during mitosis.
2
Introduction
Chromosomes must be faithfully segregated during mitosis to allow for normal
cellular growth and development of organisms. Chromosome missegregation leads to
aneuploidy, a common state of cells in solid tumors (Fang and Zhang, 2011; Holland and
Cleveland, 2009).
Evidence suggests that an important distinction between normal
diploid cell populations and aneuploid cancer cells is the tolerance for aneuploid
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the cell cycle through a p53-dependent mechanism (Thompson and Compton, 2010) or
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genomes. Diploid cells are intolerant of chromosome missegregation and either arrest in
et al., 2008; Choi et al., 2009; Heilig et al., 2009; Lee et al., 2011; Bakhoum et al., 2011;
display growth retardation (Williams et al., 2008). In addition to a state of aneuploidy,
many solid tumors continuously missegregate chromosomes at high rates in a
phenomenon called chromosomal instability, or CIN (Lengauer et al., 1998; Holland and
Cleveland, 2009; Thompson et al., 2010). This high rate of missegregation has been
clinically correlated to metastasis, drug resistance, and poor patient prognosis (Walther
Bakhoum and Compton, 2012). Direct analysis of CIN cancer cells has shown that the
most common cause of whole chromosome missegregation is the persistence of errors
in the attachment of chromosomes to spindle microtubules (Thompson and Compton,
2008).
These errors involve the attachment of single kinetochores to microtubules
oriented toward both spindle poles (i.e. merotely) leading to lagging chromosomes in
anaphase and chromosome non-disjunction (Salmon et al., 2005; Cimini, 2007).
Dysfunction of multiple mitotic processes can give rise to persistent merotelic
kinetochore-microtubule (kMT) attachments (Thompson et al., 2010; Bakhoum and
Compton, 2012). For example, defects that increase the rate of formation of merotelic
kMT attachments cause CIN.
Extra centrosomes cause multipolar spindles prior to
bipolarization, and this transient defect in spindle geometry substantially elevates the
3
frequency of merotelic attachments (Loncarek et al., 2007; Ganem et al., 2009; Silkworth
and Cimini, 2012)). Alterations in centromere geometry also increase the propensity for
kMT attachment errors (Manning et al., 2010). On the other hand, defects that decrease
the rate of error correction also contribute to CIN. KMT attachment errors are corrected
by the release of microtubules from kinetochores making error correction dependent on
the dynamic association/dissociation of microtubules from kinetochores. Studies have
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shown that CIN cancer cells have hyper-stable kMT attachments, which erodes their
ability to correct attachment errors (Bakhoum et al., 2009a; Bakhoum et al., 2009b).
Importantly,
strategic
destabilization
of
kMT
attachments
can
restore
faithful
chromosome segregation to otherwise CIN cancer cells (Bakhoum et al., 2009a)
indicating a causal relationship between kMT attachment errors and CIN.
Genome sequencing of human cancers has revealed few recurrent mutations in
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genes responsible for mitosis with the exception of members of the cohesin complex
(Barbero, 2011; Kon et al., 2013; Mannini and Musio, 2011; Thol et al., 2013; Bailey et
al., 2013; The Cancer Genome Atlas Research Network, 2014). The vertebrate cohesin
core complex is comprised of two Structural Maintenance of Chromosome proteins
(Smc1 and Smc3), the kleisin subunit Scc1/RAD21, and a stromal antigen protein
(STAG). This complex is responsible for sister chromatid cohesion and participates in
important cellular functions including transcription regulation, DNA repair, and
chromosome segregation during mitosis (Peters et al., 2008; Barbero, 2009; Mehta et
al., 2013). Inherited mutations in genes encoding cohesin subunits or their regulators
cause cohesinopathies such as Cornelia de Lange Syndrome and Roberts Syndrome.
Somatically acquired mutations in CIN cancers have been identified in genes encoding
SMC1, SMC3, and other cohesin-related proteins (Barber et al., 2008; Barbero, 2011;
Yan et al., 2012). Recently, mutations causing loss of expression of the cohesin subunit
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STAG2, also referred to as SA2, have been identified in numerous cancers (Solomon et
al., 2011; Kim et al., 2012; Bernardes et al., 2013; Bailey et al., 2013). Microarray
analysis showed that STAG2 deficiency did not significantly alter gene expression
(Solomon et al., 2011). Instead, fixed cell imaging revealed that STAG2-deficient cells
showed an increase in the frequency of chromosome bridges and lagging chromosomes
at anaphase (Solomon et al., 2011). This suggests that loss of STAG2 generates CIN
attachment errors in mitosis. Here we use cell based assays to show that STAG2 plays
a role in regulating kMT attachment stability to promote efficient error correction thereby
ensuring faithful chromosome segregation.
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by either increasing the formation rate or decreasing the correction rate of kMT
5
Results
To determine how loss of function mutations in the STAG2 gene cause lagging
chromosomes in anaphase we used siRNA to knock down STAG2 expression in
immortalized, non-transformed and diploid RPE1 cells, transformed and near-diploid
HCT 116 cells, as well as aneuploid, chromosomally unstable and STAG2-deficient H4
cells (Fig. 1 and supplementary material Fig. S1A&B). Immunoblot analysis for STAG2
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shows that the efficiency of knockdown with this siRNA treatment varied from 40% to
70%, and that this effect was specific because no significant changes were observed in
the levels of other mitotic proteins such as Bub1 or Aurora B kinase (Supplementary
material Fig. S1A). The fractional diminution of STAG2 levels observed by immunoblot
under these conditions is reflected in the fraction of cells displaying loss of STAG2 as
judged by fluorescence microscopy (Supplementary material Fig. S1B) consistent with
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the effects of STAG2 siRNA previously published (Kong et al., 2014).
RPE1 and
HCT116 cells displayed a significant increase in the frequency of lagging chromosomes
in anaphase as judged by staining for chromosomes and centromeres, but H4 cells did
not (Fig. 1A and B). H4 cells harbor a frameshift mutation in the STAG2 gene and do
not express the STAG2 protein (Solomon et al., 2011). Thus, H4 cells are insensitive to
siRNA targeting STAG2 (Fig. 1B) and demonstrates that the lagging chromosomes
induced by STAG2 siRNA are likely to be due to a specific effect of the siRNA on
STAG2. Thus, depletion of STAG2 increases the frequency of lagging chromosomes
during mitosis as previously reported (Solomon et al., 2011). The frequency of lagging
chromosomes in anaphase under these conditions following STAG2 knockdown are
underestimates since we did not pre-select STAG2 negative cells prior to quantifying
lagging chromosomes.
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An increase in the frequency of lagging chromosomes in anaphase can result
from insults that either elevate the rate of formation of merotelic kMT attachments or
decrease the efficiency of correction of merotelic kMT attachments (Thompson et al.,
2010). To discriminate between these mechanisms, we examined different aspects of
mitosis in STAG2-depleted cells. Depletion of STAG2 from RPE1 or HCT116 cells did
not alter mitotic progression as measured by the fractions of cells in each phase of
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S1C). Moreover, STAG2-depleted cells are checkpoint proficient because disruption of
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mitosis (Fig. 1C) or the mitotic index of the population (supplementary material Fig.
STAG2 depleted cells also did not show a significant difference in the frequency of
spindles using nocodazole increases the mitotic index (supplementary material Fig.
S1C). We observed an increase in cell death in STAG2-depleted cells during prolonged
mitotic arrest which explains the difference in mitotic index between control cells and
STAG2-depleted cells after extended nocodazole treatment (e.g. 6 and 12 hours)
although we currently do not know if these cells die in mitosis or following mitotic exit.
multipolar spindles (supplementary material Fig. S1D). Thus, loss of STAG2 does not
elevate the frequency of lagging chromosomes in anaphase by disruption of mitotic
progression, the spindle assembly checkpoint, or spindle bipolarity.
Since STAG2 functions in centromere cohesion (Canudas and Smith, 2009), we
measured centromere stretch by staining with human centromere-specific antiserum or
antibodies against the outer kinetochore component Hec1. Both HCT116 (Fig. 1D) and
RPE1 (supplementary material Fig. S2A) cells depleted of STAG2 displayed a significant
increase in centromere and/or inter-kinetochore stretch relative to control cells in
metaphase. Centromere distance was unchanged in STAG2-deficient cells relative to
control cells when treated with nocodazole (supplementary material Fig. S2B) indicating
that excessive centromere stretch in cells lacking STAG2 requires force from MT
attachments.
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Next, we measured the stability of attachment of microtubules to kinetochores
using fluorescence dissipation after photoactivation (Zhai et al., 1995) (Fig. 2). The
turnover of fluorescently activated GFP-tubulin in spindle microtubules undergoes a
double exponential decay with the fast component representing the non-kMT and the
slow component representing the kMT (Fig. 2A & B). STAG2 depleted cells showed a
significant increase in the stability of kMT compared to control cells in both
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of non-kMT dynamics between control cells and STAG2 depleted cells (Fig. 2D). These
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prometaphase and metaphase (Fig. 2C). There was no significant change in the stability
microscopy we found that Aurora B kinase did not localize efficiently to the inner
data demonstrate that loss of STAG2 results in hyper-stable kMT attachments. This
leads to improper regulation of kMT attachments such that erroneous attachments
persist into anaphase and thereby cause lagging chromosomes and CIN.
To determine how STAG2 affected kMT dynamics we examined the distribution
of proteins responsible for regulating kMT attachments (Fig. 3 & 4). Using fluorescence
centromere in STAG2 depleted cells (Fig. 3A). STAG2 depletion did not alter the overall
quantity of Aurora B kinase in cells (supplementary material Fig. S1A), but there was a
40% decrease in Aurora B kinase localized to centromeres. Instead, Aurora B kinase
localized along the length of chromosomes arms similar to when Bub1, Sgo1, or Wapl
are disrupted (Ricke et al., 2012; Rivera et al., 2012; Haarhuis et al., 2013). This change
in localization of Aurora B kinase was mimicked by the distribution of INCENP (Fig. 3B),
demonstrating that the entire chromosome passenger complex (CPC) is improperly
localized in STAG2 depleted cells.
Moreover, the activity of Aurora B kinase was
reduced without STAG2 as judged by the quantity of phospho-histone H3 serine 10 and
phospho-Knl1 (Fig. 3C & D), two common Aurora B substrates (Crosio et al., 2002;
Hirota et al., 2005; Welburn et al., 2010). In STAG2 depleted cells, the quantity of
phospho-histone H3 declined by 20% in prometaphase cells and 37% in metaphase
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cells and phospho-Knl1 at kinetochores was decreased by approximately 30% in
prometaphase cells. These data support the role of STAG2 in recruitment and activity of
Aurora B kinase during mitosis.
Recently, it was demonstrated that Bub1 is required for efficient centromere
localization of the CPC (Ricke et al., 2012). One function of Bub1 is to localize the CPC
to the centromere by phosphorylating threonine 120 on histone H2A (pH2A T120)
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Therefore, we examined both Bub1
localization and phosphorylation activity in STAG2 depleted cells. Bub1 kinetochore
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(Kawashima et al., 2010, 1; Watanabe, 2010).
STAG2 depleted cells relative to control cells (Fig 4B). Phosphorylation of histone H3 on
localization was decreased in both prometaphase (~50%) and metaphase (~90%)
relative to control cells (Fig. 4A), although Bub1 protein levels remained unchanged
(supplementary material Fig. S1A) and there must be sufficient Bub1 acitivity to mount a
checkpoint response (supplementary material Fig. S1C). Correspondingly, the levels of
phospho-histone H2A decreased by 15% in prometaphase and 8% in metaphase in
threonine 3 (pH3 T3) by Haspin is also involved in CPC localization (Dai et al., 2005),
but we found no change in pH3 T3 levels in either prometaphase or metaphase (Fig.
4C). Conversely, the quantity of Bub3 at kinetochores was increased by nearly 50%
during prometaphase in STAG2 depleted cells (Fig. 4D). Bub3 is a binding partner of
Bub1 so it is curious that these proteins would respond differently to the loss of STAG2.
Perhaps the change in abundance of Bub3 at kinetochores contributes to altering the
stability of kMT attachments since Bub3 overexpression has been shown to rescue
some cold-sensitive microtubule mutants in budding yeast (Guénette et al., 1995).
These data indicate that STAG2 promotes the correction of errors in kMT
attachment. This predicts that loss of function mutations in STAG2 could be rescued by
destabilizing kMT to enhance error correction. To test this prediction, we overexpressed
GFP alone, GFP-STAG2, and the microtubule destabilizing kinesins GFP-MCAK and
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GFP-Kif2b in H4 cells, which harbor a frameshift mutation in the STAG2 gene and do not
produce functional STAG2 protein.
Immunoblot confirms the expression of each
transgene in these cells (supplementary material Fig. 3A).
We then quantified the
frequency of lagging chromosomes in anaphase (Fig. 5A & B), and, as expected, the
expression of STAG2 significantly reduced the fraction of anaphase cells displaying
lagging chromosomes relative to cells expressing GFP alone (Fig. 5B). Importantly,
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anaphase cells displaying lagging chromosomes with an efficiency that matched the
Journal of Cell Science
cells expressing GFP-MCAK or GFP-Kif2b also significantly reduced the fraction of
chromosomes induced by loss of STAG2 expression (supplementary material Fig. S3B).
expression of STAG2 (Fig. 5A & B).
To ensure the specificity of this strategy, we
overexpressed GFP alone and GFP-Kif2B in HCT116 cells that were depleted of STAG2
by transfection with STAG2-specific siRNA. GFP expression did not alter the fraction of
anaphase cells with lagging chromosomes relative to control cells. However, GFP-Kif2b
expression significantly reduced the fraction of anaphase cells displaying lagging
Lagging chromosomes are symptomatic of chromosomal instability, but they do
not always result in nondisjunction causing aneuploidy (Thompson and Compton, 2011).
To directly test if altering kMT error correction efficiency alters CIN in STAG2-deficient
cells, we isolated clones of H4 cells expressing GFP alone, GFP-STAG2, or the
microtubule destabilizing kinesins GFP-MCAK or GFP-Kif2b. Each clone was grown for
approximately 25 generations and then processed for fluorescence in situ hybridization
(FISH) using centromere-specific probes. Using FISH probes specific for four different
chromosomes, we identified the modal number for each of these chromosomes and
quantified the fraction of cells in the population deviating from that mode (Table 1). As
expected, H4 cells expressing GFP alone are aneuploid and chromosomally unstable as
judged by the high percentage of cells in the population that have a chromosome
number that deviates from the modal number for each chromosome (ranging from 19%-
10
65% depending on the chromosome). Expression of GFP-STAG2 decreased karyotypic
deviance from the mode for all four chromosomes tested, with two of the chromosomes
showing significant reductions relative to cells expressing GFP alone.
Similarly,
expression of GFP-MCAK decreased karyotypic deviance from the mode for all four
chromosomes tested, again with two of the chromosomes showing significant reductions
relative to cells expressing GFP alone.
Finally, expression of GFP-Kif2b induced a
tested relative to cells expressing GFP alone. These data demonstrate that strategically
destabilizing kMT attachments to increase the efficiency of error correction in STAG2
deficient cells decreases not only the rate of lagging chromosomes in anaphase, but the
frequency of whole chromosome missegregation.
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significant decrease in karyotypic deviation from the mode for all four chromosomes
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Discussion
Loss of STAG2 has recently been detected in a variety of tumors and tumorderived cell lines (Solomon et al., 2011; Kim et al., 2012; Bailey et al., 2013; BalbásMartínez et al., 2013; Bernardes et al., 2013; Taylor et al., 2013; Lawrence et al., 2014;
The Cancer Genome Atlas Research Network, 2014). In most of these cases, the loss
of STAG2 correlates with aneuploidy and chromosomal instability. Moreover, STAG2 is
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in human tumors (Lawrence et al., 2014). These results suggest that the aneuploidy and
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one of only a handful of genes with a defined mitotic function that is frequently mutated
In this work, we provide evidence that STAG2 ensures genome integrity by
chromosomal instability caused by the loss of STAG2 expression can act as a driver for
cancer initiation.
Moreover, since tissue context determines whether aneuploidy
promotes or suppresses cancer (Weaver et al., 2007), these results indicate that the
tumors that most often harbor mutations in STAG2 may be derived from tissues that are
most sensitive to transformation caused by karyotypic change.
promoting efficient correction of kMT attachment errors. Loss of STAG2 induces the
hyper-stabilization of kMT attachments, which impairs the error correction process, and
leads to elevated rates of chromosome missegregation (Fig. 5C). Several molecular
models could explain how the loss of STAG2 stabilizes kMT attachments.
One
possibility is that STAG2 promotes the localization of Bub1 to kinetochores allowing it to
efficiently phosphorylate its substrates, like histone H2A, and thereby encouraging
proper CPC localization. Without STAG2, the CPC does not localize properly to the
centromere, and Aurora B kinase fails to efficiently phosphorylate substrates involved in
regulating kMT attachment stability (e.g. Knl1). This may precipitate a feedback network
that is detrimental to kMT regulation since Bub1 kinase has been shown to be a
substrate of Aurora B kinase, especially for its checkpoint function. In this context,
reductions in Aurora B activity may contribute to the mislocalization of Bub1 as was
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previously observed using small molecule inhibitors of Aurora B (Becker et al., 2010).
Consistently, cells may have a more complicated response to STAG2 loss because we
observed increases in Bub3 localization at kinetocores.
Regardless of the specific
molecular defects, we show that the strategic destabilization of kMT attachments in cells
lacking STAG2 function is sufficient to increase the efficiency of correction of kMT
attachment errors and restore faithful chromosome segregation in cells devoid of
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cause of chromosomal instability caused by STAG2 loss of function.
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STAG2. This demonstrates that alterations of kMT attachment stability are the root
STAG1 or that the role played by STAG2 in cohesion is regulatory and not mechanical.
STAG2 is not essential to the mechanical cohesion of sister chromatids during
mitosis since its loss does not induce premature sister separation as observed when
other components of the cohesin ring are lost (Michaelis et al., 1997; Hoque and
Ishikawa, 2002; Toyoda and Yanagida, 2006; Canudas and Smith, 2009; Solomon et al.,
2011). This suggests that there may be partial compensation by other factors such as
Consistent with a regulatory role, the loss of STAG2 disrupts the localization and activity
of multiple proteins and protein complexes at centromeres including Aurora B kinase,
Bub1 kinase, Bub3, and may also disrupt the localization or activity of other centromere
components to which it has been linked including Plk1 and Sgo1 (Kettenbach et al.,
2011; Liu et al., 2013; Shintomi and Hirano, 2009).
However, these changes are
selective because not all kinases are affected by the loss of STAG2 as demonstrated by
the quantity of pH3 T3, which is a surrogate for Haspin kinase activity.
How
STAG2
influences
the
localization
and
activity
of
some
centromeric/kinetochore proteins remains an open question. One possibility is that it
influences the activity or accessibility of the kinetochore protein Knl1, which, when
phosphorylated by Mps1, has been shown to be a docking site for Bub3/Bub1 kinase
and a factor in Aurora B kinase localization and activity (Shepperd et al., 2012;
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Yamagishi et al., 2012; Caldas et al., 2013). Another possibility is that STAG2 stabilizes
the positioning of cohesin rings on centromeric chromatin to create platforms for the
appropriate phosphorylation of histone subunits and the recruitment of centromeric
binding proteins.
These views are consistent with a recent report showing that the
cohesin-interacting protein Pds5b also promotes Aurora B localization at centromeres
(Carretero et al., 2013), though it affected Haspin kinase activity, and not Bub1. This
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multiple factors to ensure faithful chromosome segregation.
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indicates that cohesin structure and function at centromeres is subject to regulation by
tumor recurrence (Choi et al., 2009; Heilig et al., 2009; Bakhoum et al., 2011; Bakhoum
In conclusion, these data show that loss of function mutations in STAG2 directly
undermine the fidelity of chromosome segregation leading to whole chromosome
instability. This provides a straight-forward explanation for why tumors that have loss of
function mutations in STAG2 are often aneuploid and CIN. CIN generates intra-tumor
genetic heterogeneity and correlates with tumor aggressiveness, drug-resistance, and
and Compton, 2012). Our data provide insight into strategies to suppress CIN caused
by loss of STAG2 and, perhaps, alter the aggressiveness of tumors.
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Materials and Methods
Cell culture
RPE1, PA-RPE1 and H4 cells were grown in Dulbecco's Modification of Eagle's
Medium (DMEM; Corning) and HCT 116 cells were grown in McCoy's 5A (Iwakata and
Grace Modification; Corning) at 37°C in a humidified atmosphere with 5% CO2. All
media was supplemented with 10% Bovine Growth Serum (HyClone) and penicillin-
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streptomycin at 10,000 U penicillin/10,000 mg streptomycin per mL (Lonza). Media for
cells stably expressing GFP constructs was supplemented with 1 mg/mL G418 Sulfate
(InvivoGen) for clonal selection, and 0.5 mg/mL for outgrowth.
Nocodazole (EMD
Millipore) for mitotic arrest was used at 300 ng/mL.
Cell transfection
Plasmids encoding GFP-tagged STAG2 (Addgene), MCAK, Kif2b, and GFP
vector control (Manning et al., 2007) were transfected using FuGENE 6 (Roche
Diagnostics) and cells analyzed at 24 hours post-transfection. For stable transgene
expression, cells were placed under G418 selection. Clones were isolated after 14-18
days and screened for GFP expression. siRNA transfections were conducted using
Oligofectamine (Invitrogen) and cells were analyzed 48 hours later. RNA duplex for
STAG2 (5’-CCGAAUGAAUGGUCAUCAC-3’) was purchased from Ambion. For STAG2
rescue experiments, cells were transfected using STAG2 siRNA with Oligofectamine
(Invitrogen), and 24 hours later transfected with GFP vector or GFP-Ki2B plasmids using
FuGENE 6 (Roche Diagnostics); cells were analyzed 24 hours after GFP transfection
(48 hours after RNAi).
Antibodies
Antibodies used for this study were: ACA (CREST; Geisel School of Medicine,
Dartmouth College, Hanover NH), Hec1 (Novus Biologicals), Actin (Seven Hills
15
Bioreagents), tubulin (DM1α; Sigma-Aldrich), GFP (Invitrogen), STAG2 (Santa Cruz),
Aurora B (Novus Biologicals), Bub1 (Abcam), Bub3 (Abcam), pH3 S10 (Cell Signaling
Technologies), pKnl1 (gift from Iain Cheeseman), pH3 T3 (gift from Jonathan
Higgins), and pH2A T120 (Active Motif). Secondary antibodies were conjugated to
fluorescein isothyocyante (FITC) (Jackson ImmunoResearch), Texas Red (Jackson
ImmunoResearch), Cy5 (Invitrogen), and HRP (Jackson ImmunoResearch). DNA was
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stained with HOECHST (VWR). Immunoblots were detected using Lumiglow (KPL).
Fluorescence in situ hybridization (FISH)
FISH analyses were performed as previously described (Thompson and
Compton, 2008). Briefly, clones were isolated using glass cloning rings, grown in 100
mm dishes to >90% confluency, and collected by trypsinization. Cells were washed with
PBS and treated with 75 mM KCl for 15 min. Cells were then fixed in and washed twice
with 75% methanol and 25% acetic acid fixation solution. FISH probes used in this
study were Aquarius satellite enumeration probes (Cytocell, Rainbow Scientific) for
chromosomes 3 (red, 3r), 4 (red, 4r), 7 (green, 7g), and 10 (green, 10g).
Photoactivation
Photoactivation was performed as previously described (Zhai et al., 1995;
Kabeche and Compton, 2013).
Fluorescent and differential interference microscopy
images were acquired using Quorum WaveFX-X1 spinning disk confocal system
(Quorum Technologies Inc., Guelph, Canada) equipped with Hamamatsu ImageEM
camera (Bridgewater, New Jersey). Microtubules were locally activated on one half of
the spindle using a Mosaic digital mirror (Andor Technology, South Windsor,
Connecticut). Fluorescence images were captured every 15 s for 4 min with a 100X oilimmersion 1.4 numerical aperture objective. DIC microscopy was used to select mitotic
16
cells and to verify that a bipolar spindle was maintained throughout image acquisition
and that cells had not entered anaphase.
To quantify fluorescence dissipation after photoactivation, pixel intensities were
measured within a 1 µm rectangular area surrounding the region of highest fluorescence
intensity and background subtracted using an equal area from the non-activated half
spindle. The values were corrected for photobleaching by treating cells with 10 µM
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acquisition after photoactivation. Fluorescence values were normalized to the first time-
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Taxol and determining the percentage of fluorescence loss during 4 min of image
population of microtubules.
point after photoactivation for each cell and the average intensity at each time point was
fit to a double exponential curve using MatLab (Mathworks): A1 × exp(–k1t)+A2 × exp(–
k2t). Here, A1 represents the less stable non-kinetochore-microtubule population and
A2 the more stable kinetochore-microtubule population with decay rates of k1 and k2,
respectively. The turnover half-life for each process was calculated as ln2/k for each
Immunofluorescence Microscopy
Cells were fixed with 3.5% paraformaldehyde for 15 min, quenched twice with
500 mM ammonium chloride in PBS for 10 min each, washed with Tris-buffered saline
with 5% bovine serum albumin (TBS-BSA) plus 0.5% Triton X-100 for 5 min, and
washed with TBS-BSA for 5 min. For Bub3, pKnl1, and pH3 T3, cells were pre-treated
with microtubule-stabilizing buffer (MTSB [pH 6.8]: 4 M glycerol, 100 mM PIPES, 1 mM
EGTA, and 5 mM MgCl2), MTSB plus 0.05% Triton-X, washed with MTSB again for 2
min each before fixing with paraformaldehyde. For Aurora B, pH2A T120, Bub3, pKnl1,
and pH3 T3 staining, cells were permeabilized with -20oC methanol for 5 min after
paraformaldehyde fixation. Antibodies were diluted in TBS-BSA plus 0.1% Triton X-100
and coverslips incubated for 1 hour at room temperature. After incubation, coverslips
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were washed with TBS-BSA for 5 minutes with shaking. Secondary antibodies were
diluted in TBS-BSA plus 0.1% Triton X-100 with HOECHST and coverslips were
incubated for 1 hour at room temperature.
Immunofluorescence staining for STAG2 was used to directly identify specific
STAG2-deficient cells when scoring for Bub1 and phospho-histone H3 S10 localization.
In other assays, STAG2 levels were not directly assessed indicating that the reported
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tagged plasmids pre-selected individual cells based on GFP expression and localization
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values underestimate the effect of loss of STAG2. All experiments done with GFP-
Bub1/Bub3pH3 S10/pKnl1/pH3 T3 staining images were taken with 0.2 µm optical
of the transgene, if applicable. P-values for immunofluorescence quantification were
calculated with the 2-way ANOVA test using Graph Pad Prism 5 software.
Images for FISH, Bub1, pH3 S10, Bub3, pH3 T3, pKnl1, mitotic analyses, and
lagging chromosomes were acquired with Orca-ER Hamamatsu cooled CCD camera
mounted on an Eclipse TE 2000-E Nikon microscope.
Sister separation and
sections in the z-axis were collected with a plan Apo 60X or 100X 1.4 NA oil immersion
objectives at room temperature. Iterative restoration was performed using Phylum Live
software (Improvision) for DNA and centromere images; all but pKnl1 panels are raw
images to show pixel intensities. Images for Aurora B, INCENP, and pH2A T120 were
acquired using Quorum WaveFX-X1 spinning disk confocal system (Quorum
Technologies Inc., Guelph, Canada); 0.1 µm optical sections were taken in the z-axis at
room temperature.
Anaphase chromatids were counted as lagging if they contained both HOECHST
and centromere staining (using CREST antibody) and persisted in the spindle midzone,
separate from centromeres segregating to the poles. For quantitative assessments,
cells were fixed and stained for Aurora B/Bub1/pH3 S10/pH2A T120/Bub3/pKnl1/pH3
T3, CREST, STAG2, and DNA.
Raw pixel intensities for Aurora B/Bub1/pH2A
18
T120/Bub3/pKnl1 staining were measured in 10-15 regions over the entire cell. Raw
pixel intensities for pH3 S10 and pH3 T3 were measured at the chromosome region for
each cell. Background fluorescence was subtracted and the ratio of intensities was
calculated and averaged over multiple kinetochores from multiple mitotic cells in three
independent experiments (n ≥ 10 cells).
Measurements of inter-centromere and inter-kinetochore distances were made
Measurements were performed on ≥10
centromeres pairs per cell in n ≥ 10 cells for three independent experiments. Error bars
represent standard errors (SEM).
The Student’s t-test was used to calculate the
significance of the differences between samples.
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with Phylum Live software (Improvision).
19
Abbreviations
CIN, chromosomal instability
kMT, kinetochore-microtubule
CPC, chromosome passenger complex
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STAG2 (also referred to as SA2), stromal antigen 2
20
Acknowledgements
This work was supported by National Institutes of Health grants GM51542 (D.A.C) and
Journal of Cell Science
Accepted manuscript
GM008704 (L.K.).
21
References
Bailey, M. L., O’Neil, N. J., van Pel, D. M., Solomon, D. A., Waldman, T. and
Hieter, P. (2013). Glioblastoma cells containing mutations in the cohesin
component, STAG2, are sensitive to PARP inhibition. Mol. Cancer Ther.
Bakhoum, S. F. and Compton, D. A. (2012). Chromosomal instability and cancer: a
complex relationship with therapeutic potential. J. Clin. Invest. 122, 1138–1143.
Journal of Cell Science
Accepted manuscript
Bakhoum, S. F., Thompson, S. L., Manning, A. L. and Compton, D. A. (2009a).
Genome stability is ensured by temporal control of kinetochore-microtubule
dynamics. Nat. Cell Biol. 11, 27–35.
Bakhoum, S. F., Genovese, G. and Compton, D. A. (2009b). Deviant kinetochore
microtubule dynamics underlie chromosomal instability. Curr. Biol. CB 19,
1937–1942.
Bakhoum, S. F., Danilova, O. V., Kaur, P., Levy, N. B. and Compton, D. A. (2011).
Chromosomal Instability Substantiates Poor Prognosis in Patients with Diffuse
Large B-cell Lymphoma. Clin. Cancer Res. 17, 7704 –7711.
Balbás-Martínez, C., Sagrera, A., Carrillo-de-Santa-Pau, E., Earl, J., Márquez, M.,
Vazquez, M., Lapi, E., Castro-Giner, F., Beltran, S., Bayés, M., et al. (2013).
Recurrent inactivation of STAG2 in bladder cancer is not associated with
aneuploidy. Nat. Genet. advance online publication,.
Barbero, J. L. (2009). Cohesins: chromatin architects in chromosome segregation,
control of gene expression and much more. Cell. Mol. Life Sci. CMLS 66, 2025–
2035.
Barbero, J. L. (2011). Sister chromatid cohesion control and aneuploidy. Cytogenet.
Genome Res. 133, 223–233.
Becker, M., Stolz, A., Ertych, N. and Bastians, H. (2010). Centromere localization of
INCENP-Aurora B is sufficient to support spindle checkpoint function. Cell Cycle
9, 1360–1372.
Bernardes, V. F., Correa, G. T. B., Loyola, A. M., Cardoso, S. V., de Paula, A. M. B.,
Cabral, M. M. D. Á., Gomez, R. S. and Gomes, C. C. (2013). STAG2
expression in oral cancer and potentially malignant lesions. Tumour Biol. J. Int.
Soc. Oncodevelopmental Biol. Med.
Caldas, G. V., DeLuca, K. F. and DeLuca, J. G. (2013). KNL1 facilitates
phosphorylation of outer kinetochore proteins by promoting Aurora B kinase
activity. J. Cell Biol. 203, 957–969.
22
Canudas, S. and Smith, S. (2009). Differential regulation of telomere and centromere
cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells. J.
Cell Biol. 187, 165–173.
Carretero, M., Ruiz-Torres, M., Rodríguez-Corsino, M., Barthelemy, I. and Losada,
A. (2013). Pds5B is required for cohesion establishment and Aurora B
accumulation at centromeres. EMBO J. advance online publication,.
Journal of Cell Science
Accepted manuscript
Choi, C.-M., Seo, K. W., Jang, S. J., Oh, Y.-M., Shim, T.-S., Kim, W. S., Lee, D.-S.
and Lee, S.-D. (2009). Chromosomal instability is a risk factor for poor prognosis
of adenocarcinoma of the lung: Fluorescence in situ hybridization analysis of
paraffin-embedded tissue from Korean patients. Lung Cancer Amst. Neth. 64, 66–
70.
Cimini, D. (2007). Detection and Correction of Merotelic Kinetochore Orientation by
Aurora B and its Partners. Cell Cycle 6, 1558–1564.
Crosio, C., Fimia, G. M., Loury, R., Kimura, M., Okano, Y., Zhou, H., Sen, S., Allis,
C. D. and Sassone-Corsi, P. (2002). Mitotic Phosphorylation of Histone H3:
Spatio-Temporal Regulation by Mammalian Aurora Kinases. Mol. Cell. Biol. 22,
874–885.
Dai, J., Sultan, S., Taylor, S. S. and Higgins, J. M. G. (2005). The kinase haspin is
required for mitotic histone H3 Thr 3 phosphorylation and normal metaphase
chromosome alignment. Genes Dev. 19, 472–488.
Fang, X. and Zhang, P. (2011). Aneuploidy and Tumorigenesis. Semin. Cell Dev. Biol.
22, 595–601.
Ganem, N. J., Godinho, S. A. and Pellman, D. (2009). A Mechanism Linking Extra
Centrosomes to Chromosomal Instability. Nature 460, 278–282.
Guénette, S., Magendantz, M. and Solomon, F. (1995). Suppression of a conditional
mutation in alpha-tubulin by overexpression of two checkpoint genes. J. Cell Sci.
108 ( Pt 3), 1195–1204.
Haarhuis, J. H. I., Elbatsh, A. M. O., van den Broek, B., Camps, D., Erkan, H.,
Jalink, K., Medema, R. H. and Rowland, B. D. (2013). WAPL-Mediated
Removal of Cohesin Protects against Segregation Errors and Aneuploidy. Curr.
Biol.
Heilig, C. E., Löffler, H., Mahlknecht, U., Janssen, J. W. G., Ho, A. D., Jauch, A.
and Krämer, A. (2009). Chromosomal instability correlates with poor outcome
in patients with myelodysplastic syndromes irrespectively of the cytogenetic risk
group. J. Cell. Mol. Med.
23
Hirota, T., Lipp, J. J., Toh, B.-H. and Peters, J.-M. (2005). Histone H3 serine10
phosphorylation by Aurora B causes HP1 dissociation from heterochromatin.
Nature 438, 1176–1180.
Holland, A. J. and Cleveland, D. W. (2009). Boveri revisited: chromosomal instability,
aneuploidy and tumorigenesis. Nat Rev Mol Cell Biol 10, 478–487.
Journal of Cell Science
Accepted manuscript
Hoque, M. T. and Ishikawa, F. (2002). Cohesin Defects Lead to Premature Sister
Chromatid Separation, Kinetochore Dysfunction, and Spindle-assembly
Checkpoint Activation. J. Biol. Chem. 277, 42306–42314.
Kabeche, L. and Compton, D. A. (2013). Cyclin A regulates kinetochore microtubules
to promote faithful chromosome segregation. Nature advance online
publication,.
Kawashima, S. A., Yamagishi, Y., Honda, T., Ishiguro, K. and Watanabe, Y. (2010).
Phosphorylation of H2A by Bub1 prevents chromosomal instability through
localizing shugoshin. Science 327, 172–177.
Kettenbach, A. N., Schweppe, D. K., Faherty, B. K., Pechenick, D., Pletnev, A. A.
and Gerber, S. A. (2011). Quantitative Phosphoproteomics Identifies Substrates
and Functional Modules of Aurora and Polo-Like Kinase Activities in Mitotic
Cells. Sci Signal 4, rs5.
Kim, M. S., Kim, S. S., Je, E. M., Yoo, N. J. and Lee, S. H. (2012). Mutational and
expressional analyses of STAG2 gene in solid cancers. Neoplasma 59, 524–529.
Kon, A., Shih, L.-Y., Minamino, M., Sanada, M., Shiraishi, Y., Nagata, Y., Yoshida,
K., Okuno, Y., Bando, M., Nakato, R., et al. (2013). Recurrent mutations in
multiple components of the cohesin complex in myeloid neoplasms. Nat. Genet.
advance online publication,.
Lawrence, M. S., Stojanov, P., Mermel, C. H., Robinson, J. T., Garraway, L. A.,
Golub, T. R., Meyerson, M., Gabriel, S. B., Lander, E. S. and Getz, G. (2014).
Discovery and saturation analysis of cancer genes across 21 tumour types. Nature
505, 495–501.
Lee, A. J. X., Endesfelder, D., Rowan, A. J., Walther, A., Birkbak, N. J., Futreal, P.
A., Downward, J., Szallasi, Z., Tomlinson, I. P. M., Howell, M., et al. (2011).
Chromosomal Instability Confers Intrinsic Multidrug Resistance. Cancer Res. 71,
1858–1870.
Lengauer, C., Kinzler, K. W. and Vogelstein, B. (1998). Genetic instabilities in human
cancers. Nature 396, 643–649.
Liu, H., Jia, L. and Yu, H. (2013). Phospho-H2A and Cohesin Specify Distinct
Tension-Regulated Sgo1 Pools at Kinetochores and Inner Centromeres. Curr.
Biol.
24
Loncarek, J., Kisurina-Evgenieva, O., Vinogradova, T., Hergert, P., La Terra, S.,
Kapoor, T. M. and Khodjakov, A. (2007). The centromere geometry essential
for error-free mitosis is controlled by spindle forces. Nature 450, 745–749.
Manning, A. L., Ganem, N. J., Bakhoum, S. F., Wagenbach, M., Wordeman, L. and
Compton, D. A. (2007). The Kinesin-13 Proteins Kif2a, Kif2b, and Kif2c/MCAK
Have Distinct Roles during Mitosis in Human Cells. Mol. Biol. Cell 18, 2970–
2979.
Journal of Cell Science
Accepted manuscript
Manning, A. L., Longworth, M. S. and Dyson, N. J. (2010). Loss of pRB causes
centromere dysfunction and chromosomal instability. Genes Dev 24, 1364–1376.
Mannini, L. and Musio, A. (2011). The dark side of cohesin: the carcinogenic point of
view. Mutat. Res. 728, 81–87.
Mehta, G. D., Kumar, R., Srivastava, S. and Ghosh, S. K. (2013). Cohesin: Functions
beyond sister chromatid cohesion. FEBS Lett. 587, 2299–2312.
Michaelis, C., Ciosk, R. and Nasmyth, K. (1997). Cohesins: Chromosomal Proteins
that Prevent Premature Separation of Sister Chromatids. Cell 91, 35–45.
Peters, J.-M., Tedeschi, A. and Schmitz, J. (2008). The cohesin complex and its roles
in chromosome biology. Genes Dev. 22, 3089 –3114.
Ricke, R. M., Jeganathan, K. B., Malureanu, L., Harrison, A. M. and Deursen, J. M.
van (2012). Bub1 kinase activity drives error correction and mitotic checkpoint
control but not tumor suppression. J. Cell Biol. 199, 931–949.
Rivera, T., Ghenoiu, C., Rodriguez-Corsino, M., Mochida, S., Funabiki, H. and
Losada, A. (2012). Xenopus Shugoshin 2 regulates the spindle assembly pathway
mediated by the chromosomal passenger complex. EMBO J. 31, 1467–1479.
Salmon, E. D., Cimini, D., Cameron, L. A. and DeLuca, J. G. (2005). Merotelic
kinetochores in mammalian tissue cells.
Shepperd, L. A., Meadows, J. C., Sochaj, A. M., Lancaster, T. C., Zou, J., Buttrick,
G. J., Rappsilber, J., Hardwick, K. G. and Millar, J. B. A. (2012).
Phosphodependent Recruitment of Bub1 and Bub3 to Spc7/KNL1 by Mph1
Kinase Maintains the Spindle Checkpoint. Curr. Biol. 22, 891–899.
Shintomi, K. and Hirano, T. (2009). Releasing cohesin from chromosome arms in early
mitosis: opposing actions of Wapl–Pds5 and Sgo1. Genes Dev. 23, 2224–2236.
Silkworth, W. T. and Cimini, D. (2012). Transient defects of mitotic spindle geometry
and chromosome segregation errors. Cell Div. 7, 19.
Solomon, D. A., Kim, T., Diaz-Martinez, L. A., Fair, J., Elkahloun, A. G., Harris, B.
T., Toretsky, J. A., Rosenberg, S. A., Shukla, N., Ladanyi, M., et al. (2011).
25
Mutational Inactivation of STAG2 Causes Aneuploidy in Human Cancer. Science
333, 1039 –1043.
Taylor, C. F., Platt, F. M., Hurst, C. D., Thygesen, H. H. and Knowles, M. A. (2013).
Frequent inactivating mutations of STAG2 in bladder cancer are associated with
low tumor grade and stage and inversely related to chromosomal copy number
changes. Hum. Mol. Genet. ddt589.
Journal of Cell Science
Accepted manuscript
The Cancer Genome Atlas Research Network (2014). Comprehensive molecular
characterization of urothelial bladder carcinoma. Nature advance online
publication,.
Thol, F., Bollin, R., Gehlhaar, M., Walter, C., Dugas, M., Suchanek, K. J., Kirchner,
A., Huang, L., Chaturvedi, A., Wichmann, M., et al. (2013). Mutations in the
cohesin complex in acute myeloid leukemia: clinical and prognostic implications.
Blood.
Thompson, S. L. and Compton, D. A. (2008). Examining the link between
chromosomal instability and aneuploidy in human cells. J. Cell Biol. 180, 665–
672.
Thompson, S. L. and Compton, D. A. (2010). Proliferation of aneuploid human cells is
limited by a p53-dependent mechanism. J Cell Biol 188, 369–381.
Thompson, S. L. and Compton, D. A. (2011). Chromosome missegregation in human
cells arises through specific types of kinetochore–microtubule attachment errors.
Proc. Natl. Acad. Sci. 108, 17974 –17978.
Thompson, S. L., Bakhoum, S. F. and Compton, D. A. (2010). Mechanisms of
Chromosomal Instability. Curr. Biol. CB 20, R285–R295.
Toyoda, Y. and Yanagida, M. (2006). Coordinated Requirements of Human Topo II
and Cohesin for Metaphase Centromere Alignment under Mad2-dependent
Spindle Checkpoint Surveillance. Mol. Biol. Cell 17, 2287–2302.
Walther, A., Houlston, R. and Tomlinson, I. (2008). Association between
chromosomal instability and prognosis in colorectal cancer: a meta-analysis. Gut
57, 941–950.
Watanabe, Y. (2010). Temporal and spatial regulation of targeting aurora B to the inner
centromere. Cold Spring Harb. Symp. Quant. Biol. 75, 419–423.
Weaver, B. A. and Cleveland, D. W. (2006). Does aneuploidy cause cancer? Curr.
Opin. Cell Biol. 18, 658–667.
Welburn, J. P. I., Vleugel, M., Liu, D., Yates, J. R., Lampson, M. A., Fukagawa, T.
and Cheeseman, I. M. (2010). Aurora B phosphorylates spatially distinct targets
26
to differentially regulate the kinetochore-microtubule interface. Mol. Cell 38,
383–392.
Williams, B. R., Prabhu, V. R., Hunter, K. E., Glazier, C. M., Whittaker, C. A.,
Housman, D. E. and Amon, A. (2008). Aneuploidy Affects Proliferation and
Spontaneous Immortalization in Mammalian Cells. Science 322, 703 –709.
Yan, M., Xu, H., Waddell, N., Shield, K., Haviv, I., Authors, kConFab, McKay, M.
J. and Fox, S. B. (2012). Enhanced RAD21 cohesin expression confers poor
prognosis in BRCA2 and BRCAX, but not BRCA1 familial breast cancers. Breast
Cancer Res. 14, R69.
Zhai, Y., Kronebusch, P. J. and Borisy, G. G. (1995). Kinetochore microtubule
dynamics and the metaphase-anaphase transition. J. Cell Biol. 131, 721–734.
Journal of Cell Science
Accepted manuscript
Yamagishi, Y., Yang, C.-H., Tanno, Y. and Watanabe, Y. (2012). MPS1/Mph1
phosphorylates the kinetochore protein KNL1/Spc7 to recruit SAC components.
Nat. Cell Biol. 14, 746–752.
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Figure Legends
Figure 1. STAG2 deficiency causes lagging chromosomes and increases centromere
stretch. (A) Anaphase in HCT116 cells that were untreated (Control) or transfected with
siRNA specific to STAG2 (STAG2 KD) and stained for STAG2 (yellow), centromeres
(red), tubulin (green), and DNA (blue) as indicated.
The arrow indicates a lagging
chromosome in a STAG2 deficient cell. (B) Percentage of anaphase cells displaying at
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cells, and aneuploid H4 cells either untreated (Control) or transfected with siRNA
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least one lagging chromosome in anaphase in diploid RPE1 cells, near-diploid HCT116
100 centromeres, *p ≤ 0.001.
specific for STAG2 (STAG2 KD). N = 300 cells, *p ≤ 0.001. (C) Percentage of HCT116
cells that were untreated (Control) or transfected with siRNA specific to STAG2 (STAG2
KD) in different phases of mitosis. N = 1000 cells. (D) Centromere length in HCT116
cells that were untreated (Control) or transfected with siRNA specific to STAG2 (STAG2
KD) measured between centroids of staining intensity for anti-centromere antibody; N =
Figure 2. Measurement of kinetochore-microtubule dynamics in cells depleted of
STAG2. (A) DIC and time-lapse fluorescent images of mitotic RPE1 cells that were
untreated (control) or transfected with siRNA specific to STAG2 (STAG2 KD) before
(Pre-PA) and at indicated times after photoactivation. (B) Normalized fluorescence
intensity over time after photoactivation of spindles in untreated (white circles) and
STAG2-deficient (filled circles) prometaphase and metaphase cells. (C) kMT half-life in
untreated (Control) and STAG2-deficient (STAG2 KD) RPE1 cells in prometaphase and
metaphase. (D) Non-kMT microtubule half-life in RPE1 photoactivatable cells with or
without STAG2 KD. Data represents mean ± s. e. m., n ≥ 10 cells, *p ≤ 0.001.
28
Figure 3. STAG2 influences the centromeric localization and activity of Aurora B kinase
in HCT116 cells. Fluorescence intensities of (A) Aurora B kinase, (B) INCENP, (C)
phospho-Histone 3 serine 10 (pH3 S10), and (D) phospho-Knl1 (pKnl1) in prometaphase
and metaphase HCT116 cells that were untreated (Control) or transfected with siRNA
specific to STAG2 (STAG2 KD). Scale bar = 5 µm. Insets in panel A show a single
focal plane image of an enlarged (2.5X) chromosome arm with Aurora B localization.
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Panel D shows de-convolved pKnl1 staining for clarity, insets show an enlarged (3X)
centromere pair (red) with pKnl1 localization.
Graphs (A), (B), and (C) n ≥ 200
centromeres, *p ≤ 0.001. Graph (C) n = 10 cells, *p ≤ 0.01.
Figure 4. STAG2 influences the centromeric localization and activity of Bub1 kinase in
HCT116 cells. Fluorescence intensities of (A) Bub1,(B) phospho-Histone 2A threonine
120 (pH2A T120), (C) phospho-histone 3 threonine 3, and (D) Bub3 in prometaphase
and metaphase in HCT116 cells that were untreated (Control) or transfected with siRNA
specific to STAG2 (STAG2 KD). Scale bar = 5 µm. Graph (A) n = 25 centromeres, *p ≤
0.01. Graph (B) n = 200 centromeres, *p ≤ 0.001. Graph (C) n ≥ 25 prometaphase and
n ≥ 10 metaphase cells, p > 0.05. Graph (D) n > 200 centromeres, *p ≤ 0.001.
Figure 5. STAG2 promotes faithful chromosome segregation. (A) Anaphase in STAG2deficient H4 cells expressing GFP only (GFP) or GFP-tagged Kif2b (GFP-Kif2b) showing
GFP fluorescence (green), centromere staining (red), DNA (blue). (B) Quantification of
the percentage of anaphase cells with lagging chromosomes in H4 cells overexpressing
GFP, GFP-Kif2b, GFP-MCAK or GFP-STAG2. N ≥ 300 cells, *p ≤ 0.01. (C) Model of
STAG2 activity during mitosis. STAG2 promotes the localization and activity of Bub1
and Aurora B kinase to promote the destabilization of kMT attachments and enhance
error correction. In the absence of STAG2, Bub1 and Aurora B kinase do not localize
29
properly causing hyper-stabilization of kMT attachments and reduced error correction
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efficiency.
30
Cell line
3
% deviation
Chromosome
4
7
% deviation
% deviation
10
% deviation
H4 GFP
44 (3)
65 (6)
36 (5)
19 (2)
H4 GFP-STAG2
28* (3)
47* (4)
29 (5)
14 (2)
H4 GFP-Kif2b
27* (3)
28* (6)
16* (5)
9* (2)
H4 GFP-MCAK
29* (3)
59 (6)
27* (5)
16 (2)
Modal chromosome numbers are shown in brackets
* Chi-square test, p < 0.05.
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Table 1. Percent Deviance from Chromosomal Mode in H4 Cell Clones
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