The EMBO Journal (2010) 29, 3544–3557
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|&
2010 European Molecular Biology Organization | All Rights Reserved 0261-4189/10
THE
EMBO
JOURNAL
The hsSsu72 phosphatase is a cohesin-binding
protein that regulates the resolution of sister
chromatid arm cohesion
Hyun-Soo Kim1,2, Kwan-Hyuck Baek1,
Geun-Hyoung Ha1,2, Jae-Chul Lee1,
Yu-Na Kim3, Janet Lee1, Hye-Young Park1,
Noo Ri Lee1, Ho Lee4, Yunje Cho5 and
Chang-Woo Lee1,2,*
1
Department of Molecular Cell Biology, Sungkyunkwan University
School of Medicine, Suwon, Korea, 2Center for Molecular Medicine,
Samsung Biomedical Research Institute, Sungkyunkwan University
School of Medicine, Suwon, Korea, 3Department of Microbiology,
College of Medicine, Gyeonsang National University, Jinju, Korea,
4
Division of Cancer Biology, Research Institute, National Cancer Center,
Goyang, Korea and 5Department of Life Science, Pohang University of
Science and Technology, Pohang, Korea
Cohesin is a multiprotein complex that establishes sister
chromatid cohesion from S phase until mitosis or meiosis.
In vertebrates, sister chromatid cohesion is dissolved in a
stepwise manner: most cohesins are removed from the
chromosome arms via a process that requires polo-like
kinase 1 (Plk1), aurora B and Wapl, whereas a minor
amount of cohesin, found preferentially at the centromere,
is cleaved by separase following its activation by the
anaphase-promoting complex/cyclosome. Here, we report
that our budding yeast two-hybrid assay identified
hsSsu72 phosphatase as a Rad21-binding protein. Additional experiments revealed that Ssu72 directly interacts
with Rad21 and SA2 in vitro and in vivo, and associates
with sister chromatids in human cells. Interestingly,
depletion or mutational inactivation of Ssu72 phosphatase
activity caused the premature resolution of sister chromatid arm cohesion, whereas the overexpression of Ssu72
yielded high resistance to this resolution. Interestingly,
it appears that Ssu72 regulates the cohesion of chromosome arms but not centromeres, and acts by counteracting
the phosphorylation of SA2. Thus, our study provides
important new evidence, suggesting that Ssu72 is a novel
cohesin-binding protein capable of regulating cohesion
between sister chromatid arms.
The EMBO Journal (2010) 29, 3544–3557. doi:10.1038/
emboj.2010.217; Published online 3 September 2010
Subject Categories: proteins; cell cycle
Keywords: protein phosphatase; Rad21/Scc1; SA2/Scc3; sister chromatid cohesion; Ssu72
*Corresponding author. Department of Molecular Cell Biology,
Sungkyunkwan University School of Medicine, Suwon 440-746, Korea.
Tel.: þ 82 31 299 6121; Fax: þ 82 31 299 6109;
E-mail: cwlee@med.skku.ac.kr
Received: 27 February 2010; accepted: 12 August 2010; published
online: 3 September 2010
3544 The EMBO Journal VOL 29 | NO 20 | 2010
Introduction
Proper chromosome alignment and segregation during
mitosis rely on the cohesion between sister chromatids.
This is mediated by a protein complex called cohesin,
which includes heterodimeric ATPases made of Smc1 and
Smc3 proteins in association with the regulatory subunit,
Rad21 (a mammalian isoform of Scc1), which is in turn
associated with the SA1 or SA2 variants of the Scc3 protein
(Michaelis et al, 1997; Losada et al, 1998, 2000; Sumara et al,
2000; Haering et al, 2002). In higher eukaryotes, the removal
of cohesin from sister chromatids occurs in a stepwise
manner. During prophase, the bulk of cohesin is removed
from chromosome arms via a process that requires Wapl and
involves the polo-like kinase 1 (Plk1) and aurora B kinases,
but does not result in the proteolytic cleavage of Rad21
(Losada et al, 1998, 2002; Waizenegger et al, 2000;
Gimenez-Abian et al, 2004; Gandhi et al, 2006; Kueng et al,
2006). Recent studies have shown that protein phosphatase
2A (PP2A) and shugoshin (Sgo) together function to protect
centromeric cohesin during early mitosis (Kitajima et al,
2006; Tang et al, 2006). More specifically, PP2A localizes at
the centromere via Sgo, and prevents the phosphorylation of
cohesin by Plk1 and potentially by aurora B (McGuinness
et al, 2005; Tang et al, 2006). Therefore, PP2A counteracts the
Plk1-mediated phosphorylation of the SA1/2 cohesin subunits, thereby preventing their dissociation from centromeric
chromatin. After prophase, a minor amount of cohesin, found
preferentially at the centromeres, is cleaved by anaphasepromoting complex/cyclosome-activated separase, which
cleaves Rad21 at the metaphase-to-anaphase transition
(Hauf et al, 2001; Nasmyth, 2002). This process removes all
the remaining cohesin from the chromosomes and triggers
the separation of sister chromatids. Despite the appeal of
such a simple model, however, the related mechanisms are
not yet fully understood.
The yeast protein, Ssu72, was initially identified as a
transcription/RNA processing factor through its physical
interaction with the TFIIB transcription factor (Pappas and
Hampsey, 2000). Subsequent studies implicated Ssu72 in the
regulation of cell viability in yeast, and showed that it is
highly conserved among eukaryotic organisms (Pappas and
Hampsey, 2000). Two groups independently reported that
Ssu72 has phosphatase activity and its sequence contains
the CX5R signature motif of PTPases; based on this, Ssu72
was thought to be a member of a new phosphatase subfamily
in which the so-called aspartate loop is the phosphatase
active site (Ganem et al, 2003; Meinhart et al, 2003).
Recombinant yeast Ssu72 proteins were found to exhibit
phosphatase activity against the synthetic substrate, p-nitrophenyl phosphate, and both phosphatase domain and aspartate mutants of Ssu72 showed decreased phosphatase activity
(Ganem et al, 2003; Meinhart et al, 2003). Another study
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Regulation of cohesion by Ssu72
H-S Kim et al
found that the phosphatase activity of Ssu72 could dephosphorylate serine 5 at the C-terminal domain (CTD) of RNA
polymerase II (Krishnamurthy et al, 2004). However, the
exact functions and physiological substrates of Ssu72 remain
unclear even in yeast.
In this study, we report that our yeast two-hybrid assay
identified hsSsu72 as a Rad21-binding protein. Additional
experiments revealed that Ssu72 directly interacts with
Rad21 and SA2 in vitro and in vivo, and associates with
mitotic sister chromatids. Interestingly, Ssu72 depletion or
mutational inactivation of Ssu72 phosphatase activity causes
premature sister chromatid arm separation, whereas the
overexpression of Ssu72 yields high resistance to the resolution of arm cohesion. Further studies showed that Ssu72
counteracts the phosphorylation of SA2 but not that of
other cohesin subunits, and it dephosphorylates SA2.
Collectively, these results provide important new evidence,
suggesting that Ssu72 is a novel cohesin-binding phosphatase
capable of regulating the cohesion between sister chromatid
arms.
Results
Ssu72 associates and co-localizes with Rad21 and SA2
To gain insight into the mechanisms underlying sister
chromatid dissociation, we performed a conventional
yeast two-hybrid assay using a human fetal kidney cDNA
library, and identified Ssu72 as an hsRad21-binding protein
(Figure 1A; Supplementary Figure S1). To confirm this finding, we first raised both rabbit polyclonal and mouse
monoclonal antibodies against a peptide from hsSsu72
(LDRNKRIKPRPERFQNC). Experiments showed that the
affinity-purified antibodies specifically and efficiently recognized both endogenous and exogenous Ssu72 proteins
(Supplementary Figure S2). Specifically, the antibodies recognized a 28-kDa Ssu72 polypeptide in HeLa cell extracts,
and this immunoreactivity could be depleted by transfection
of HeLa cells with Ssu72-targeting shRNAs. Extracts from
asynchronously growing HeLa cells were immunoprecipitated with the polyclonal anti-Ssu72 antibody or normal IgG
(control), and immunoblotting was performed with antibodies against Rad21, SA2, Smc1, Smc3 and Mad2 (negative
control) (Figure 1B, upper panel). Similarly, HeLa cell
extracts were immunoprecipitated with an anti-Rad21 antibody or control IgG, and the resulting immunoprecipitates
were immunoblotted as indicated (Figure 1B, lower panel).
Notably, Ssu72 and Rad21 were present in the complexes
in vitro and in vivo, and Ssu72 also showed interactions
with the cohesin subunits, SA2, Smc1 and Smc3. Next, we
prepared cellular extracts from HeLa cells stably expressing a
Myc-tagged Rad21/Scc1 (Myc-Rad21) fusion protein under
the control of the inducible ‘Tet-on’ promoter (Figure 1C;
Hauf et al, 2001). Subsequent immunoprecipitation and immunoblotting analyses revealed that the Myc-Rad21 fusion
proteins formed a complex with endogenous Ssu72, as well
as with Smc1 and Smc3 (positive controls) (Figure 1C). To
further determine whether Ssu72 interacts directly with
Rad21, we generated full-length His- and Glutathione
S-transferase (GST)-tagged fusion proteins (His-Ssu72 and
GST-Rad21, respectively) (Figure 1D, left panel). Pull-down
assays revealed that His-Ssu72 bound to GST-Rad21. In
addition, we generated full-length GST-SA2 and incubated it
& 2010 European Molecular Biology Organization
with His-Ssu72 (Figure 1D, right panel). Unexpectedly, we
found that Ssu72 also interacted directly with SA2.
To define the domains responsible for the Ssu72–Rad21
and Ssu72–SA2 interactions, we incubated a series of GSTfused Ssu72 deletion mutants with HeLa cell extracts. As
shown in Figure 1E, the Ssu72 COOH-terminal domain
(amino acids 121–194) formed a complex with Rad21,
whereas both the central and COOH-terminal regions
(amino acids 61–194) of Ssu72 were required for the interaction with SA2. To exclude the possibility that the interactions
between Ssu72 and the cohesin subunits might be indirectly
mediated through chromatin, we treated nuclear extracts
from HeLa cells stably overexpressing HA-tagged Ssu72
(HeLa-HA-Ssu72) with DNase, and then subjected the
extracts to immunoprecipitation with an anti-HA antibody
or control IgG, followed by immunoblotting with the indicated antibodies (Figure 1F). Again, all of the tested cohesin
subunits (Smc1, Smc3, SA2 and Rad21) were found in
complexes with Ssu72, as shown by silver nitrate staining
(data not shown) and immunoblotting (Figure 1F), indicating
that Ssu72 forms a complex with the cohesin subunits.
As cohesins are loaded onto chromatin, most cohesin
complexes are detected in chromatin fractions (Losada
et al, 2002; Kueng et al, 2006). To test whether Ssu72 could
also be found as a chromatin-bound protein, we removed the
soluble proteins from HeLa cells by pre-extraction, and costained the remaining fraction with anti-Ssu72 and anti-Rad21
antibodies, and CREST antiserum (Figure 1G; Supplementary
Figure S3). Both Ssu72 and Rad21 were detected near
chromosomes during interphase (G2) and prophase (pro).
However, consistent with the behaviour of Rad21, Ssu72
was dissociated from metaphase (meta) chromosomes. We
further separated HeLa cell lysates into soluble cytoplasmic
supernatants, insoluble pellets and chromatin-bound fractions for additional experiments. Consistent with previous
findings (Losada et al, 2002; Kueng et al, 2006), we observed
the presence of the cohesin subunits, Smc3, SA2 and Rad21,
in the insoluble pellet and chromatin-bound fractions
(Figure 1H). Under our experimental conditions, Ssu72 was
also detected in the insoluble pellet and chromatin-bound
fractions of asynchronously grown cells. In contrast (and
consistent with our immunostaining results), the levels of
Ssu72 were significantly reduced in the chromatin-bound
fractions of mitotic cells (Figure 1H).
Aberrant expression of Ssu72 causes defects in the
dissociation of cohesin from chromatin
To examine whether aberrant expression of Ssu72 affects the
association or dissociation of chromatin cohesion in mitotic
cells, we generated doxycycline-inducible HeLa cells expressing Rad21-RFP or SA2-RFP fusion proteins (Figure 2A).
These HeLa-Rad21-RFP and HeLa-SA2-RFP cells were cotransfected with or without expression plasmids encoding
Ssu72 and GFP-tagged H2B (H2B-GFP) or CFP-tagged H2B
(H2B-CFP) and maltose-binding protein fused with GFP
(MBP-GFP). The RFP fusion proteins were induced by doxycycline, and the RFP emissions (reflecting the intensity of
Rad21 or SA2 protein expression) were digitally monitored by
time-lapse microscopy (Figure 2B). Nuclear envelope breakdown (NEBD), which occurs as a cell enters mitosis, was
determined by both the appearance of sister chromatid
disorganization, as indicated by H2B-GFP or H2B-CFP, and
The EMBO Journal
VOL 29 | NO 20 | 2010 3545
Regulation of cohesion by Ssu72
H-S Kim et al
A
PBB
PPase
MPSSPLRVAVVCSSNQNRSMEAHNILSKRGFSVRSFGTGTHVKLPGPRVAAPDKPNVYDFT
YDQMYNDLLRKDKELYTQNGILHMLDRNKRIKPRPERLNSREQETCQPVHVVNV DIQDNHE
aspartate loop
EFQNCKDLFDLILTCEERVYDQVVEDATLGAFLICELCQCIQHTEDMENEIDELLQEFEEKSG
LxCxE
RTFLHTVCFY
D-box
SA2
G-SA2
Rad21
+ His-Ssu72
Input (1/20)
Input IgG Ssu72
G
Input (1/40)
+ His-Ssu72
G
D
G-Rad21
B
Smc1
Ssu72
Ssu72
Smc3
Rad21
Mad2
G-Rad21
SA2
G-SA2
Ssu72
Ponceau
Ponceau
G
G
Input IgG Rad21
+
Myc (Rad21)
Rad21 (endo)
G-WT
G
D – box
SA2
Rad21
Input (1/20)
–
LxCxE
NE+DNase
+
194
NE
–
120
F
IP (Myc)
Input
Doxy
60
Maker
C
PBB PPase
WT
(1–60)
(61–194)
(121–194)
Input
Ssu72
Smc3
Rad21
G-(1–60)
E
Mad2
G-(121–194)
SA2
G-(61–194)
Ssu72
IP
IgG
HA
Smc1
Smc3
Ssu72
Smc1
Smc3
SA2
Rad21
Actin
HA (Ssu72)
Pro
Meta
Chro
Noco
Pellets
Media
Supt
G2
H
Chro
Ssu72 Rad21 CREST Merge
Pellets
DNA
Supt
G
SA2
Rad21
Smc1
Smc3
Ssu72
ERK2
Lamin B
Figure 1 The hsSsu72 phosphatase forms a complex with cohesin subunits and associates with sister chromatids. (A) The amino-acid
sequence of the hsSsu72 protein. The polo-box-binding motif (PBB, red), a conserved protein phosphatase domain (PPase, purple), the putative
LxCxE motif (blue), and the putative destruction box (D-box, orange) are indicated. (B) Asynchronized HeLa cell extracts were immunoprecipitated with normal immunoglobulin IgG or a polyclonal anti-Ssu72 antibody, and immunoprecipitates were immunoblotted with anti-Rad21,
anti-SA2, anti-Smc1, anti-Smc3 or anti-Mad2 (negative control) antibodies. Similarly, immunoprecipitation was performed with normal IgG or
an anti-Rad21 antibody, followed by immunoblotting with anti-Ssu72, anti-SA2 or anti-Mad2 (a negative control) antibodies. Note that the
generated anti-SA2 antibody specifically recognized endogenous SA1 and SA2 polypeptides in the HeLa cells. (C) HeLa cells expressing Myctagged Rad21/Scc1 (HeLa-Myc-Rad21) were cultured in the absence () or presence ( þ ) of 2 mg/ml doxycycline (Doxy) and further incubated
with 100 ng/ml nocodazole. Cell extracts were immunoprecipitated with an anti-Myc antibody, and subsequently immunoblotted with the
indicated antibodies. (D) Purified His-Ssu72 was incubated with beads bound to either GST (G)-Rad21 (left) or GST (G)-SA2 (right). The beads
were analysed by immunoblotting with anti-Ssu72, anti-Rad21 or anti-SA2 antibodies. (E) HeLa cell lysates were incubated with beads bound
to GST (G) alone or to a series of GST (G)-fused Ssu72 deletion mutants. Bound proteins were resolved and immunoblotted with anti-SA2
and anti-Rad21 antibodies. (F) Nuclear extracts (NE) from HeLa-HA-Ssu72 cells (overexpressing HA-tagged Ssu72) were treated with DNase
(0.5 U/mg) for 30 min, and then immunoprecipitated with an anti-HA antibody or control IgG. The immunoprecipitates were analysed by
immunoblotting with anti-Smc1, anti-Smc3, anti-SA2, anti-Rad21 and anti-HA antibodies (right panels). Chromosomal DNA from nuclear
extracts was analysed by ethidium bromide staining (left panels). (G) HeLa cells were extracted with 0.2% Triton X-100, and then fixed and
qco-stained with an anti-Ssu72 antibody (green), an anti-Rad21 antibody (red) and CREST (purple). DNA was visualized by DAPI staining.‘Pro’
and ‘Meta’ indicate prophase and metaphase, respectively. (H) Extracts from HeLa cells cultured in the absence or presence (Noco) of
nocodazole were separated into soluble cytoplasmic supernatant (Supt), insoluble pellet (Pellet) and chromatin-bound (Chro) fractions. The
fractions were analysed by immunoblotting using anti-Smc1, anti-Smc3, anti-SA2, anti-Rad21, anti-Ssu72 and anti-ERK1 (as a control for
cytoplasmic supernatant fractions) or anti-lamin B (as a control for chromatin-bound fractions) antibodies.
3546 The EMBO Journal VOL 29 | NO 20 | 2010
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Regulation of cohesion by Ssu72
H-S Kim et al
the nuclear envelope permeability of MBP-GFP after cytoplasmic photobleaching (Figure 2B; Supplementary Figure S5).
The Rad21-RFP signals on chromatin decreased within 4 min
after NEBD in control cells, whereas this signal was markedly
prolonged for up to 16 min post-NEBD in Ssu72-overexpressing cells (Figure 2C and D; Supplementary Figure S4A). The
SA2-RFP signals on chromatin gradually decreased in control
cells, becoming barely detectable after 3 min post-NEBD
(Figure 2E and F; Supplementary Figure S4B). In Ssu72overexpressing cells, however, the SA2-RFP signal was clearly
visible at 4 min post-NEBD and was maintained even in
metaphase chromosomes, indicating that Ssu72 overexpression appears to decrease the dissociation of cohesin from
mitotic chromatin. We then investigated the effect of Ssu72
expression on cell cycle progression, and found that cells
overexpressing Ssu72 showed a significant mitotic delay
compared with control cells (Supplementary Figure S6A–D);
this delay arose largely from the elongation of prometaphase
and metaphase, while the timings of interphase and mitotic
exit were unaffected (Supplementary Figure S6C–E).
HeLa-Rad21-RFP cells were transfected with CFP-tagged
H2B (H2B-CFP) and GFP-tagged MBP (MBP-GFP) expression
plasmids, along with shRNA specifically targeting Ssu72
(which significantly depleted Ssu72 expression (data not
shown)) or luciferase (as a control), and cultured in the
presence of doxycycline for 48 h (Figure 2G and H;
Supplementary Figure S4B). In control cells, the RFP signal
appeared on mitotic chromosomes, remained present until
prophase, and then decreased beginning at 180 s post-NEBD.
In Ssu72-depleted cells, however, the RFP signal sharply
decreased at 120 s post-NEBD. To further investigate the
effects of Ssu72 depletion on cell cycle progression, we
analysed mitotic progression by flow cytometry. However,
Ssu72-depleted cells did not show any meaningful change in
the proportion of cells at interphase or mitotic cell cycle
progression (Supplementary Figure S7).
Ssu72 regulates sister chromatid cohesion between
chromosome arms
To determine the effect of Ssu72 expression on sister chromatid cohesion, we transfected HeLa cells stably expressing
Myc-tagged versions of Rad21/Scc1 (Myc-Rad21) (Hauf et al,
2001) or SA2 (Riedel et al, 2006) with either control luciferase
shRNA or Ssu72 shRNA, and analysed Rad21 and SA2 staining by confocal microscopy. As shown in Figure 3A, both
Myc-Rad21 and Myc-SA2 clearly appeared as chromatinbound proteins in prophase/prometaphase control cells,
whereas Ssu72-depleted cells showed significant displacement of these cohesin proteins from prophase/prometaphase
chromatin. About 87% of the prophase/prometaphase control cells showed chromatin binding of Myc-Rad21, whereas
only 40–50% of Ssu72-depleted prophase/prometaphase cells
were Myc-Rad21 positive (Figure 3B), indicating that depletion of Ssu72 leads to the premature dissociation of cohesin
from mitotic chromosomes. Next, we generated expression plasmids encoding Ssu72 WT, Ssu72 C12S (in
which cysteine 12, located in the CX5RS signature motif of
the protein phosphatase (PPase), was mutated to a serine)
(Supplementary Figure S8), or the Ssu72 shRNA. HeLa cells
transfected with these plasmids were cultured and treated
with the microtubule inhibitor, colcemid, and sister chromatid cohesion was analysed by chromosome spreading and
& 2010 European Molecular Biology Organization
Giemsa staining. Three types of mitotic chromosome patterns
were observed: (1) ‘closed’ (cohesed chromosome arms), (2)
‘partial open’ (partial loss of arm cohesion) and (3) ‘open’
(complete loss of arm cohesion) (Figure 3C). Treatment of
HeLa cells with a microtubule inhibitor normally abrogates
cohesion between chromosome arms but not at the centromeres, forming the X-shaped ‘open’ arm pattern. However,
asynchronous HeLa cells treated with a microtubule inhibitor
usually show mixed-type mitotic chromosome spreads comprising about 45% open, 45% partial open and B10% closed
conformations (Figure 3D). Interestingly, cells overexpressing
Ssu72 showed a significant increase in the percentage of
closed arms, from 10% (in mock-transfected cells) to 50%
in Ssu72-overexpressing cells, and a sharp reduction in the
open type, from 45 to 15%, respectively (Figure 3D). In
contrast, cells overexpressing the phosphatase-dead mutant
of Ssu72 (Ssu72 C12S) showed a very similar pattern to
control cells, with only a slight increase in the open phenotype. These data indicate that overexpression of WT but not
phosphatase-dead mutant Ssu72 appears to prevent the dissociation of chromosome arm cohesion.
We next transfected HeLa cells with control luciferase
shRNA or Ssu72 shRNA and treated the cells with colcemid.
In contrast to the behaviour of the Ssu72-overexpressing cells
described above, the chromosomes from these Ssu72-depleted cells showed a significant increase in the open phenotype (about 75%) compared with control cells (about 35%),
and a marked decrease in the partial open (from 47% in
controls to 15% in Ssu72-depleted cells) and closed (from 18
to 10%, respectively) phenotypes (Figure 3E), indicating that
the depletion of Ssu72 causes premature dissociation of arm
cohesion. Notably, we did not observe any changes in the
dissociation of cohesion at the centromeres of Ssu72-overexpressing or -depleted cells.
To confirm that sister chromatid arm cohesion is maintained by Ssu72 expression, we generated shRNA-insensitive
versions of Myc-tagged Ssu72 (shi Myc-Ssu72 WT and -Ssu72
C12S), and examined the abilities of these constructs to
rescue the phenotypes of Ssu72-depleted cells (Figure 3F).
As shown in Figure 3G, cells transfected with shSsu72
alone clearly showed endogenous Ssu72 knockdown,
whereas cells co-transfected with shSsu72 and shi MycSsu72 WT or -Ssu72 C12S MT expressed Ssu72 WT and
C12S MT, respectively. Chromosome spreading assays
revealed that overexpression of shi Myc-Ssu72 WT in
Ssu72-depleted HeLa cells significantly recovered the resolution of sister chromatid arm cohesion (the open phenotype
increased from 15 to 50%) (Figure 3G). In contrast, cells
overexpressing the phosphatase-dead mutant of Ssu72 (Ssu72
C12S) showed a pattern similar to that of Ssu72-depleted cells.
Collectively, our results suggest that Ssu72 regulates the
maintenance and resolution of sister chromatid cohesion at
the chromosome arms.
Depletion of Ssu72 causes the premature dissociation
of cohesin
In vertebrate cells, cohesin complexes are removed from
sister chromatids in a stepwise manner. During prophase/
prometaphase, cohesin is first dissociated from the chromosome arms by phosphorylation of the cohesin subunits,
which is mediated by phosphorylation of SA2 (Hauf et al,
2005). Therefore, we tested the effect of Ssu72 depletion on
The EMBO Journal
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Regulation of cohesion by Ssu72
H-S Kim et al
Ssu72, anti-SA2, anti-Rad21 and anti-phospho SA2 S1224
(which recognizes SA2 phosphorylated at serine 1224) antibodies (Figure 4A). Consistent with a previous report (Kueng
et al, 2006), the hyperphosphorylated form of SA2 was detected in the non-chromatin fractions of mitotic cellular extracts.
Interestingly, however, the levels of hyperphosphorylated
SA2 were significantly increased in Ssu72-depleted cells
(Figure 4A), indicating that Ssu72 depletion augments the
the expression and phosphorylation of cohesin subunit proteins during the various stages of the cell cycle. HeLa cells
were transfected with shLuciferase (shLuc) or shSsu72 and
then synchronized by double-thymidine block (G1/S phases),
doxorubicin treatment (G2), or nocodazole treatment
(mitosis). Extracts from these synchronized HeLa cells were
separated into chromatin and non-chromatin soluble fractions, and these fractions were immunoblotted with anti-
A
B
Doxy
+
–
H2B-GFP
MBP-GFP H2B-CFP
G1/S
Rad21-RFP
Rad21 (endo)
Actin
G2
Bleaching
NEBD
NEBD
SA2-RFP
SA2 (endo)
Actin
H2B
-GFP
HeLa-Rad21-RFP + pMyc
0m
2m
6m
4m
pMyc
D
8m
10 m 12 m 14 m 16 m
Fluorescence
(Rad21-RFP)
C
Rad21
-RFP
HeLa-Rad21-RFP + pMyc-Ssu72
0
H2B
-GFP
pMyc-Ssu72
1.2
1
0.8
0.6
0.4
0.2
0
2
4
6
8
10 12 14 16 18
Time (min)
2m
0m
6m
4m
8m
10 m 12 m 14 m 16 m 18 m 20 m
Rad21
-RFP
HeLa-SA2-RFP + pHA
E
H2B
-GFP
0m
1m
2m
3m
4m
5m
6m
HeLa-SA2-RFP + pHA-Ssu72
1m
2m
3m
4m
0m
5m
14 m 16 m
SA2
-RFP
6
8
10 12 14 16 18
Time (min)
0
4
20
2
shSsu72
80
10
0
12
0
14
0
16
0
18
0
0
shLuc
1.2
1
0.8
0.6
0.4
0.2
0
60
Fluorescenec
(Rad21-RFP)
Fluorescence
(SA2-RFP)
H
1.2
1
0.8
0.6
0.4
0.2
0
0
20
40
pHA-Ssu72
pHA
F
Time (sec)
G
Bleaching
MBP-GFP
NEBD
0S
20 S
40 S
60 S
80 S 100 S 120 S 140 S 160S 180 S 200 S
HeLa-Rad21-RFP
+ shLuc
Rad21-RFP
H2B-CFP
MBP-GFP
0S
20 S
40 S
60 S
Rad21-RFP
80 S 100 S 120 S 140 S
HeLa-Rad21-RFP
+ shSsu72
H2B-CFP
3548 The EMBO Journal VOL 29 | NO 20 | 2010
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Regulation of cohesion by Ssu72
H-S Kim et al
hyperphosphorylation of SA2. In addition, we observed a
slower migrating band of SA2 polypeptides in mitotic
non-chromatin fractions, which becomes faster migrating
after lphosphatase treatment (Figure 4B). To further analyse
the effect of Ssu72 depletion on cohesin subunit expression
and phosphorylation levels in mitotic chromosomes, HeLa
cells were transfected with shLuc or shSsu72, synchronized
by double-thymidine block followed by a 6-h release, and
then treated with nocodazole (as indicated) to enrich cells at
the early phase of mitosis, prior to metaphase (Figure 4C).
Interestingly, the relative level of hyperphosphorylated SA2
(Figure 4C, arrowhead) in the soluble non-chromatin supernatant was significantly increased in Ssu72-depleted cells,
whereas that of hypophosphorylated SA2 in the chromatin
fraction was sharply decreased in Ssu72-depleted cells after
only 2 h of nocodazole treatment. In contrast, the control
cells still retained hypophosphorylated SA2 in the chromatin
fraction following 6 h of nocodazole treatment. Similarly, the
amount of Rad21 detected in the chromatin fraction was also
decreased in Ssu72-depleted cells subjected to only 2 h of
nocodazole treatment. These results suggest that Ssu72
depletion leads to the premature dissociation of cohesin
from chromosome arms.
To further validate that Ssu72 is involved in sister chromatid arm cohesion, we performed chromatin immunoprecipitation (ChIP) assays against two cohesin-associated regions
on the chromosome arms: an arm region on chromosome 7
(116357745–116357949: primer 88) and the H19 imprinting
control region (ICR) on chromosome 11 (1977613–1977821:
primer 96). These regions reportedly include binding sites for
cohesins and the zinc finger insulator protein, CTCF, which is
required for the positioning of cohesin on DNA (Wendt et al,
2008). We used ChIP-qPCR to measure the relative amounts
of cohesin (Rad21) at these cohesin-binding sites in Ssu72depleted cells at G2 and early mitosis (Figure 4D and E). As a
control, we analysed Scc2-depleted cells, in which the
chromosomal loading of cohesin is sharply reduced. The
abundance of bound cohesin sites was reduced in mitotic
cells versus those in G2; this was as expected, because the
arm cohesins at these sites are dissociated from the chromosome through the so-called ‘prophase removal’. In addition,
and consistent with a previous report (Wendt et al, 2008),
Scc2-depleted cells showed a remarkable reduction in the
cohesin levels at these cohesin-binding sites. With regard to
Ssu72, the cohesin signals were reduced at the cohesinbinding sites of Ssu72-depleted cells synchronized at both
G2 and mitosis. Thus, our results suggest that Ssu72 is
involved in sister chromatid arm cohesion as early as the
G2 phase of the cell cycle.
Ssu72 regulates cohesion at the chromosome arms
in a Wapl-independent manner
Previous important studies have shown that depletion of
Wapl prevents arm cohesins from dissociating from sister
chromatids during mitosis, indicating that Wapl is essential
for the resolution of chromosome arm cohesin and cell
cycle progression (Gandhi et al, 2006; Kueng et al, 2006).
To examine the potential interplay between Ssu72 and Wapl,
we used shSsu72-encoding plasmids to generate HeLa cells in
which Ssu72 was stably knocked down (hereafter called
‘Ssu72 KD cells’) (Figure 5A). Control HeLa and Ssu72 KD
cells transfected with siLuc or siWapl were synchronized by
double-thymidine block followed by a 6-h release, and further
synchronized by nocodazole treatment for 4 h to enrich for
cells at early mitosis. As shown in Figure 5B, the depletion of
Wapl from control cells led to a significant increase of the
cohesin protein (SA2 and Rad21) levels in the chromatin
fractions; this is consistent with a previous report that Wapl
is essential for the prophase removal of sister chromatid arm
cohesins (Kueng et al, 2006). In Ssu72 KD cells, however,
Wapl depletion failed to rescue the premature dissociation of
arm cohesion caused by Ssu72 depletion, raising the possibility that Ssu72 regulates cohesion at the chromosome arms
in a Wapl-independent manner. Next, we analysed the relative timing kinetics of cohesin dissociation from sister chromatids in Ssu72- and Wapl-depleted cells (Figure 5C and D).
Quantitative live-cell imaging analysis revealed that after
NEBD, the majority of the SA2-RFP proteins diffused from
the nucleus; o5% of SA2-RFP was detected on the sister
chromatids of shLuc- or shSsu72-transfected cells after NEBD
(Figure 5D; t ¼ 5 min). In Wapl-depleted cells, however, SA2RFP largely remained on the sister chromatids, with only
about 20% of the SA2-RFP signal diffused to the non-chromatin cell regions (Figure 5D; t ¼ 5 min).
To further examine the links between Ssu72 and Wapl in
regulating sister chromatid arm cohesion, we tested whether
Ssu72 could antagonize the functional interaction of Wapl
with sister chromatid cohesin. Inducible HeLa-SA2-RFP cells
were co-transfected with shLuc, siWapl and shSsu72 singly or
in combination, along with the H2B-GFP expression plasmid
(Figure 5E–G). Our results revealed that Wapl depletion
sharply inhibited the dissociation of SA2-RFP from sister
Figure 2 Aberrant expression of Ssu72 causes defects in the dissociation of cohesin from chromatin. (A–H) Inducible HeLa cells expressing
Rad21-RFP (HeLa-Rad21-RFP) or SA2-RFP (HeLa-SA2-RFP) were co-transfected with expression plasmids encoding GFP-tagged H2B (H2B-GFP)
and Ssu72 (pMyc-Ssu72 or pHA-Ssu72) or empty backbone vector (pMyc or pHA; controls). At 12 h post-transfection, cells were induced with
2 mg/ml doxycycline (Doxy) and further cultured for 48 h. (A) Cell extracts were analysed by immunoblotting as indicated. ‘Endo’ designates
the endogenous Rad21 or SA2 proteins. (B) Representative time-lapse microscopic images of chromosomes in the G1/S, G2 and nuclear
envelope breakdown (NEBD) phases of the cell cycle (left panels). Representative time-lapse images of cells expressing MBP-GFP and H2B-CFP
before and after photobleaching, and NEBD-phase cells. NEBD was defined by the appearance of sister chromatid disorganization (indicated by
arrowheads) and the dispersal of MBP-GFP throughout the cell after photobleaching. (C, D) Time-lapse images taken at 2-min intervals show
the false-coloured GFP (H2B-GFP) and RFP (Rad21-RFP) emissions. The time of NEBD is shown as t0. The relative amounts of Rad21-RFP on
the chromatin were quantified in Ssu72-overexpressing (n ¼ 11) and control (n ¼ 6) cells (mean±s.d.). (E, F) Time-lapse images taken at 1-min
intervals show the false-coloured GFP (H2B-GFP) and RFP (SA2-RFP) emissions. The relative amounts of SA2-RFP on the chromatin were
quantified in Ssu72-expressing (n ¼ 7) and control (n ¼ 4) cells (mean±s.d.). (G, H) Inducible HeLa-Rad21-RFP cells were co-transfected with
an expression plasmid encoding H2B-CFP (to visualize the chromosomes), MBP-GFP (as a marker for NEBD) and shRNAs against Ssu72
(shSsu72) or luciferase (shLuc, control). At 12 h post-transfection, cells were induced with doxycycline. Time-lapse images taken at 20-s
intervals show the false-coloured CFP (H2B-CFP), GFP (MBP-GFP) and RFP (Rad21-RFP) emissions from control luciferase shRNA- or Ssu72
shRNA-transfected cells. The relative amounts of Rad21-RFP on the chromatin were quantified in Ssu72-depleted (n ¼ 15) and control (n ¼ 11)
cells (mean±s.d.).
& 2010 European Molecular Biology Organization
The EMBO Journal
VOL 29 | NO 20 | 2010 3549
Regulation of cohesion by Ssu72
H-S Kim et al
shSsu72
Myc-SA2
shLuc
shSsu72
Closed
Partial open
Myc-Rad21 staining
Weak or no staining
100%
80%
60%
40%
20%
0%
#2
shLuc
C
B
Merge
#1
Ssu72 Crest
shLuc
Myc
Myc-Rad21
DNA
Prophase/
prometaphase cells
A
shSsu72
E
Open
100%
80%
60%
40%
20%
D
0%
Open
Partial open
Closed
100%
80%
60%
G
40%
shLuc
shSsu72
Ssu72
1
2
3
4
Myc-Ssu72
Ssu72
20%
0%
Myc
100%
Myc-WT Myc-C12S
80%
Myc
60%
F
40%
0%
shRNA insensitive Ssu72 ctc ttc gac ctc att
L F
D
L
I
+
–
–
+
–
+
–
+
shi MycSsu72 C12S
20%
shi MycSsu72 WT
Ssu72 WT ctg ttt gat ctg atc
shLuc
shSsu72
Figure 3 Ssu72 regulates the maintenance and resolution of cohesion between sister chromatid arms. (A) HeLa cells stably expressing Myctagged Scc1/Rad21 (HeLa-Myc-Rad21) or Myc-tagged SA2 (HeLa-Myc-SA2) were transfected with control luciferase shRNA or Ssu72 shRNA
and treated with 200 ng/ml nocodazole for 4 h, and the shaking-off method was used to collect a population enriched in cells at the early stage
of mitosis. Mitotic cells (2 105/ml) were incubated in a hypotonic buffer, spun onto coverslips using Cytospin, and stained with CREST serum
(purple), and anti-Myc (green) and anti-Ssu72 (red) antibodies. DNA was stained with DAPI. (B) HeLa-Myc-Rad21 cells were transfected with
luciferase shRNA, Ssu72 shRNA #1 or Ssu72 shRNA #2 (see Materials and methods), and stained as above. Prophase/prometaphase cells
displaying chromatin-bound Myc-Rad21 fusion protein staining were scored, and the results are expressed as percentages out of more than 100
prophase/prometaphase cells. (C–E) HeLa cells were transfected with expression plasmids encoding Myc-tagged Ssu72 WT, Ssu72 C12S
(a phosphatase-inactive mutant), or a control Myc vector (pMyc), and then cultured and treated with colcemid (0.04 mg/ml). Chromosomes
were spread and visualized by Giemsa staining. The percentage of mitotic chromosome spreads was calculated from at least 600 cells per
transfectant. HeLa cells were transfected with shRNA expression plasmids targeting either Ssu72 (shSsu72) or luciferase (shLuc, negative control),
and then cultured and treated with colcemid, and chromosomes were analysed as described above. Extracts from HeLa cells transfected with
expression plasmids encoding the Myc epitope, Myc-Ssu72 WT, Myc-Ssu72 C12S, shLuc or shSsu72 were immunoblotted with anti-Myc or antiSsu72 antibodies. (F, G) To generate an shRNA-insensitive Ssu72-encoding construct, five silent mutations were introduced into the Ssu72 gene
sequence, which was then cloned into a Myc-tagged vector (F). Extracts from HeLa cells transfected with the indicated plasmids were
immunoblotted with an anti-Ssu72 antibody (G). Chromosome spreads prepared from HeLa cells transfected with the indicated plasmids were
visualized by Giemsa staining. The percentage of mitotic chromosome spreads was calculated from at least 300 cells per transfectant.
chromatids (Figure 5F, middle panels, and 5G). However,
Ssu72 depletion clearly counteracted the association of SA2RFP with sister chromatids in Wapl-depleted cells (Figure 5F,
lower panels, and 5G), suggesting that the depletion of Ssu72
results in the premature dissociation of cohesin subunits from
sister chromatids. We believe that this is likely to occur via
the augmentation of SA2 hyperphosphorylation at the stage
before Wapl regulates the dissociation of arm cohesion during
3550 The EMBO Journal VOL 29 | NO 20 | 2010
early mitosis. Taken together, these results further support
our contention that Ssu72 selectively regulates sister chromatid arm cohesion.
Ssu72 phosphatase dephosphorylates SA2
As specific phosphorylations of the Rad21 and SA1/2 subunits are required for the prophase/prometaphase removal of
arm cohesion (Losada et al, 2000; Hauf et al, 2005), we tested
& 2010 European Molecular Biology Organization
Regulation of cohesion by Ssu72
H-S Kim et al
G1
M
– –
+ + – –
SA2
Rad21
shLuc
shSsu72
λ PP – –
M
+ +
shLuc
shSsu72
B
M
shLuc
shSsu72
G2
shLuc
shSsu72
G1
shLuc
shSsu72
M
shSsu72
shLuc
shSsu72
G2
shLuc
shSsu72
shLuc
shSsu72
shLuc
shSsu72
G1
shLuc
shSsu72
shLuc
A
SA2
SA2
(S1224)
Erk2
Tubulin
b li
H3
H3
Ssu72
(Non-chromatin
supt)
(Chromatin)
(Chromatin)
(Non-chromatin
supt)
C
Noco (6 h)
Chro
SA2
siScc2
shLuc
shSsu72
D
shSsu72
shLuc
shSsu72
shLuc
Supt
shSsu72
shLuc
shSsu72
Noco (4 h)
Supt
Chro
shLuc
shSsu72
shLuc
shSsu72
shLuc
Noco (2 h)
Supt
Chro
Scc2
Rad21
Ssu72
Ssu72
Actin
Erk2
H3
E
Primer 88
G2 Phase
Mitosis
Fold change
Fold change
Primer 96 (H19 ICR)
1.0
0.8
0.6
0.4
0.2
0.0
siLuc
shSsu72
siScc2
G2 Phase
1.0
0.8
0.6
0.4
0.2
0.0
siLuc
shSsu72
Mitosis
siScc2
Figure 4 Depletion of Ssu72 causes the premature dissociation of cohesion. (A, B) HeLa cells were transfected with shRNAs against Ssu72
(shSsu72) or luciferase (shLuc; control). At 12 h post-transfection, cells were synchronized at the G1-S boundary by double-thymidine block, at
the G2 phase by doxorubicin treatment, or at mitosis by nocodazole treatment. (A) Synchronized cells were separated into chromatin and nonchromatin supernatant fractions, resolved by SDS–PAGE (10%), and immunoblotted with anti-SA2, anti-Rad21, anti-phospho SA2 serine 1224
( SA2-S1224), anti-tubulin (as a non-chromatin-fraction marker), anti-histone H3 (as a chromatin-fraction marker) and anti-Ssu72 antibodies.
(B) Synchronized cells were separated into chromatin and non-chromatin supernatant fractions, and cellular extracts were incubated with or
without l phosphatase (l PP), resolved by SDS–PAGE (6%), and immunoblotted with anti-SA2, anti-Erk2 (as a non-chromatin-fraction marker)
and anti-histone H3 antibodies. (C) HeLa cells were transfected as indicated, synchronized by a double-thymidine block followed by a 6-h
release, and then treated with nocodazole (as indicated) to enrich for cells in the early phase of mitosis, prior to metaphase. The cells were then
separated into soluble non-chromatin supernatant (Supt) and insoluble chromatin (Chro) fractions, resolved by SDS–PAGE (10%), and
immunoblotted with anti-SA2, anti-Rad21, anti-Ssu72, anti-Erk2 and anti-histone H3 antibodies. (D, E) HeLa cells were transfected with siLuc,
shSsu72 and siScc2 (a positive control), respectively, and synchronized at G2 and mitosis. Cellular extracts were immunoblotted with antiSsu72, anti-Scc2 and anti-actin antibodies. ChIP was performed with Rad21 or control IgG antibodies on cells synchronized at G2 or mitosis,
and qPCR analyses were performed using primer pairs specific for the cohesin and CTCF-binding sites. The relative transcript levels were
normalized with respect to that of the control siLuc in G2 phase (mean of n ¼ 3; error bars, ±s.d.).
the phosphorylations of SA2 and/or Rad21 by Cdk1 and Plk1
(Figure 6A, data not shown), and then examined whether
these phosphorylations could be counteracted by the phosphatase activity of Ssu72 (Figure 6B–D). We generated a Histagged CTD of the SA2 cohesin subunit (residues 895–1232,
His C-SA2) (Figure 6B); this fragment contained the mitosisspecific phosphorylation sites, which comprise a cluster of
12 serine and threonine residues (Hauf et al, 2005). Although
full-length SA2 was efficiently phosphorylated by Plk1
& 2010 European Molecular Biology Organization
in vivo (data not shown), the purified SA2, GST-SA2 and
His C-SA2 proteins appeared to be more efficiently phosphorylated by Cdk1 than by Plk1 in vitro (Figure 6A). We
then reacted His C-SA2 with recombinant Cdk1/cyclin B
kinase in the presence of [g32P]ATP, and reacted the resulting
in vitro-phosphorylated His C-SA2 with GST (negative control), GST-Ssu72 WT, the GST-Ssu72 C12S mutant or l PPase
(positive control) (Supplementary Figure S8E; Figure 6B). In
agreement with the above results, the levels of phosphorylated
The EMBO Journal
VOL 29 | NO 20 | 2010 3551
Regulation of cohesion by Ssu72
H-S Kim et al
C
Ssu72 KD #2
Con
Ssu72 KD #1
A
shLuc
shSsu72
siWapl
H2B-GFP
0m
Ssu72
5m
0m
5m
0m
5m
siLuc
shSsu72 siWapl
SA2-RFP
Actin
P-SA2
SA2
Rad21
SA2 (S1224)
Relative ratio of nonchromatin binding
SA2-RFP
D
siWapl
siLuc
siWapl
siLuc
siLuc
siWapl
Ssu72 KD Con Ssu72 KD
siWapl
Con
siLuc
B
1.2
0.9
0.6
0.3
0
Erk2
shLuc
Ssu72
(Non-chromatin
supt)
F
siWapl
+ shLuc
E
Wapl
(Chromatin)
siWapl
+ shSsu72
Histone H3
RFP (SA2)
Wapl
Ssu72
Actin
shLuc
H2B-GFP
0m
1m
2m
3m
4m
5m
6m
7m
SA2-RFP
G
shLuc
siWapl+ shLuc
siWapl + shSsu72
H2B-GFP
0m
1m
2m
3m
4m
5m
6m
7m 8m
SA2-RFP
siWapl + shSsu72
H2B-GFP
0m
1m
2m
3m
4m
5m
6m
7m
SA2-RFP
Fluorescence
(SA2-RFP)
siWapl + shLuc
1.2
1
0.8
0.6
0.4
0.2
0
0
2
4
6 8 10 12 14 16
Time (min)
Figure 5 Ssu72 regulates chromosome arm cohesion in a Wapl-independent manner. (A) Extracts from control HeLa cells and two different
Ssu72 KD cells (Ssu72 KD #1 and #2) were immunoblotted with anti-Ssu72 and anti-actin antibodies. (B) Control HeLa and Ssu72 KD cells were
transfected with siRNAs against Wapl (siWapl) or luciferase (siLuc). At 12 h post-transfection, cells were synchronized by double-thymidine
block followed by a 6-h release, and then treated with nocodazole for 4 h to enrich for cells at the early phase of mitosis. The synchronized cells
were separated into insoluble chromatin and soluble non-chromatin fractions, and immunoblotted with anti-SA2, anti-Rad21, anti-phospho
SA2 S1224 ( SA2 (S1224)), anti-histone H3, anti-Erk2, anti-Wapl and anti-Ssu72 antibodies. (C–G) Inducible HeLa-SA2-RFP cells were cotransfected with the H2B-GFP expression plasmid and shLuc, siWapl and shSsu72 singly or in combination. (C) The SA2-RFP signals on both
chromatin and non-chromatin regions were digitally monitored by time-lapse microscopy. (D) The relative ratio of non-chromatin-binding
SA2-RFP fluorescence to the total SA2-RFP signal was determined by densitometric analysis (mean±s.d.); shLuc-transfected cells (n ¼ 5),
shSsu72-transfected cells (n ¼ 12) and siWapl-transfected cells (n ¼ 11). (E) Extracts from cells transfected as indicated were immunoblotted
with anti-RFP (SA2), anti-Ssu72, anti-Wapl and anti-actin antibodies. (F) The SA2-RFP signal was digitally monitored by time-lapse
microscopy. (G) The relative amounts of SA2-RFP on the chromatin were quantified in Wapl-depleted (n ¼ 8), Ssu72-depleted (n ¼ 11) and
shLuc-transfected control (n ¼ 4) cells (mean±s.d.).
SA2 were significantly reduced by incubation with GST-Ssu72
WT, but not with GST or the GST-Ssu72 C12S mutant.
Similarly, we purified GST-Rad21, which is also known to be
phosphorylated by Plk1 (Sumara et al, 2002; Hornig and
Uhlmann, 2004), incubated it with Plk1 in the presence of;
[g32P]ATP (Figure 6C), and then further reacted the in vitrophosphorylated GST-Rad21 proteins with GST, GST-Ssu72 WT,
the GST-Ssu72 C12S mutant or l PPase. In contrast to our
findings with SA2, the phosphorylation of Rad21 was not
counteracted by Ssu72 WT, but the level of phosphorylated
Rad21 was markedly reduced by control l PPase treatment
3552 The EMBO Journal VOL 29 | NO 20 | 2010
(Figure 6C). To confirm these findings, we isolated the SA2–
cohesin complexes from synchronized mitotic cell extracts,
and then incubated the complexes with purified GST-Ssu72
WT, GST-Ssu72 C12S or l PPase (positive control) (Supplementary Figure S8E; Figure 6D). Phosphorylation of SA2,
which was clearly recognized by both the anti-phospho-serine
and anti-phospho-threonine antibodies, was markedly counteracted by the addition of purified Ssu72 WT but not the
phosphatase-inactive mutant Ssu72 (Ssu72 C12S).
To further examine the hypophosphorylation or dephosphorylation of SA2 in Ssu72-overexpressing cells, HeLa-Con
& 2010 European Molecular Biology Organization
+ λ PPase
Plk1
+ Ssu72 WT (4)7
+ Ssu7
+ GST (4)
Cdk1
+ Ssu72 C12S
Thr
Ser
SA2
C
+ λ PPase
GST
– + – + Plk1
– – + + Cdk1
+ Ssu72 WT
B
GST-SA2
+ GST
A
+ Ssu72 WT (2)
Regulation of cohesion by Ssu72
H-S Kim et al
GST-Rad21
His C-SA2
+
+
Arbitrary
intensity
E
SA2
SA2
αSA2
Ser
Thr
HeLa-Con
Con
n
150
SA2
HeLa-Ssu72
150
(kDa)
αSA2
IgG
Ssu
u72 KD
Input
Con
G
Ssu
u72 KD
8
Con
n
pI
*
u72 KD
Ssu
Smc1
4
IgG
(IP)
SA2
SA2
Ser
Thr
F
0
He
eLa-Ssu72
+
0
0.5
HeLa-Con
λ PPase
+
0.5
1
He
eLa-Ssu72
Ssu72 C12S (2)
SA2 (IP)
Ssu72 C12S (1)
D
Ssu72 WT (2)
Thr
Rad21
1
HeLa-Con
– + – + Plk1
– – + + Cdk1
Arbitrary
intensity
GST
GST-Rad21
SA2
Ser
SA2
Thr
SA2
Figure 6 Ssu72 dephosphorylates SA2. (A) Purified GST-SA2 or GST-Rad21 was reacted with recombinant Cdk1/cyclin B1 or Plk1 in the
presence of radio-unlabelled ATP. In vitro-phosphorylated GST-SA2 or GST-Rad21 was incubated with purified His-Ssu72. The eluted beads
were immunoblotted with anti-phospho-threonine ( Thr), anti-phospho-serine ( Ser), anti-SA2 and anti-Ssu72 antibodies. (B) His-tagged
C-terminal SA2 peptides (His C-SA2) were incubated with recombinant Cdk1/cyclin B1 in the presence of [g32P]ATP, washed and then reacted
with purified GST, GST-Ssu72 WT, GST-Ssu72 C12S or l phosphatase (l PPase). The graph shows the relative signal intensities of the radiolabelled His C-SA2 peptides. (C) Purified GST-Rad21 proteins were incubated with Plk1 proteins in the presence of [g32P]ATP, washed and then
reacted with purified GST, GST-Ssu72 WT, GST-Ssu72 C12S (2 or 4 mg/ml) or l PPase. (D) Dephosphorylation of SA2 by Ssu72. Nocodazoletreated (100 ng/ml, 12 h) HeLa cells were lysed and immunoprecipitated with an anti-SA2 antibody, and the resulting SA2–cohesin complexes
were incubated with purified GST-Ssu72 WT, GST-Ssu72 C12S MT (1 or 2 mg/ml) or l PPase (control). The bound SA2 proteins were resolved by
SDS–PAGE and detected using antibodies against phospho-serine ( Ser), phospho-threonine ( Thr), SA2 or Smc1. (E) HeLa-Con and HeLaSsu72 cells were cultured and treated with nocodazole for 4 h. Endogenous SA2 was immunoprecipitated from cell extracts using an anti-SA2
antibody or normal IgG (negative control), and the eluted SA2 complexes were immunoblotted with anti-SA2, anti-phospho-serine and
anti-phospho-threonine antibodies. (F) SA2–cohesin complexes were isolated from HeLa-Con or HeLa-Ssu72 cell extracts using a polyclonal
anti-SA2 antibody, and the SA2 immunocomplexes were analysed by two-dimensional SDS–PAGE followed by immunoblotting with a
polyclonal anti-SA2 antibody. (G) Nocodazole-treated (100 ng/ml, 12 h) control and Ssu72-knockdown cells were lysed and immunoprecipitated with an anti-SA2 antibody, and the resulting SA2 complexes were immunoblotted with anti-SA2, anti-phospho-serine ( Ser) and antiphospho-threonine ( Thr) antibodies.
(control HeLa cells) and HeLa-Ssu72 cells were treated with
nocodazole, and endogenous SA2 complexes were immunoprecipitated from cellular extracts (Figure 6E). Interestingly,
significantly less phosphorylated SA2 was recognized by the
anti-phospho-threonine antibody (Figure 6E, asterisk) in
Ssu72-overexpressing cells. In these cells, small amounts of
SA2 were detected only in the faster electrophoretic migration
range (indicative of dephosphorylated SA2), suggesting that
SA2 phosphorylation may be counteracted by Ssu72 overexpression. In contrast, Rad21 phosphorylation was not
affected by Ssu72 overexpression (data not shown). We
then analysed the SA2 immunocomplexes by two-dimensional SDS–PAGE and subsequent immunoblotting with an
& 2010 European Molecular Biology Organization
anti-SA2 antibody (Figure 6F). As expected, the higher molecular weight and higher isoelectric point (pI) polypeptides
of SA2 were clearly reduced in cells overexpressing Ssu72,
implying that Ssu72 may be involved in the dephosphorylation or hypophosphorylation of SA2. Similarly, we examined
whether the depletion of Ssu72 affected the hyperphosphorylation of SA2 in vivo. Control HeLa and Ssu72 KD cells were
treated with nocodazole and the endogenous SA2 complexes
were immunoprecipitated from cellular extracts (Figure 6G).
As expected, more highly phosphorylated SA2 was recognized by the anti-phospho-threonine antibody in Ssu72depleted cells compared with control cells, but a little change
by anti-phospho-serine antibody in consistent with Figure 6E.
The EMBO Journal
VOL 29 | NO 20 | 2010 3553
Regulation of cohesion by Ssu72
H-S Kim et al
Furthermore, we tested whether the expression of a nonphosphorylatable SA2 mutant (SA2 4A; Hauf et al, 2005)
could prevent the dissociation of arm cohesins in Ssu72depleted cells. We first generated plasmids encoding
SA2 WT or SA2 4A, in which four putative phosphorylation sites were mutated, and transfected into HeLa cells
together with shSsu72 (Supplementary Figure S9A and B).
Interestingly, the expression of SA2 WT in Ssu72-depleted
cells clearly rescued the resolution of sister chromatid arm
cohesion induced by Ssu72 depletion (Supplementary Figure
S9C). Moreover, the expression of SA2 4A significantly
augmented sister chromatid cohesion compared with that in
Ssu72-depleted cells expressing SA2 WT (the partial open
phenotype was 40% in SA2 4A-expressing cells versus 25% in
SA2 WT-expressing cells) (Supplementary Figure S9C), indicating that the non-phosphorylatable SA2 mutant rescued
the premature dissociation of chromatid cohesin caused by
Ssu72 depletion. Collectively, these findings indicate that the
function of Ssu72 in the hypophosphorylation/dephosphorylation of SA2 depends on its phosphatase activity.
Discussion
It is vital for cells to shield chromosome arm cohesion prior
to mitosis. The dissolution of chromosome arm cohesion is
triggered by the mitotic kinase-mediated phosphorylation of
cohesin subunits. Recent studies have shown that phosphorylation of SA2 by Plk1 is important for the dissociation of the
cohesin complex from chromosome arms during prophase/
prometaphase (Sumara et al, 2002; Hauf et al, 2005). The
expression of a mutated, non-phosphorylatable SA2 was
found to prevent the loss of cohesion between sister chromatid arms (Hauf et al, 2005), suggesting that protection of
cohesin subunits from phosphorylation may be required to
stabilize sister chromatid arm cohesion. Recent studies have
shown that PP2A and Sgo collaborate to function as a
‘protector’ that prevents phosphorylation of cohesin by Plk1
and aurora B, thereby shielding centromeric cohesins
(Kitajima et al, 2006; Riedel et al, 2006; Tang et al, 2006).
In terms of arm cohesion, we herein propose a model in
which the Ssu72 phosphatase appears to shield chromosome
arm cohesion by interacting with Rad21 and SA2, and potentially by protecting SA2 from hyperphosphorylation by a yetunknown mechanism. Notably, the amino-acid sequence of
Ssu72 includes a polo-box-binding (PBB) motif, which is
recognized by Plk1, as well as potential phosphorylation
sites for aurora B kinase (HS Kim and CW Lee, unpublished
data). This raises the possibility that the Plk1 and/or aurora B
kinase could phosphorylate Ssu72, thereby contributing to its
stability or activity. Future studies will be required to determine whether this model involves the phosphorylation of
Ssu72 by Plk1 or one or more of the aurora kinases.
Cohesin is subject to complicated temporal and spatial
regulation, thereby ensuring the proper establishment, maintenance and dissociation of cohesion. Cohesin becomes
stably cohesive once it is formed. In particular, cohesin
bound to chromatin during the G2 phase become cohesive
when a cell suffers a double-strand break in one of its
chromosomes (Ström et al, 2007; Unal et al, 2007). Recent
studies have revealed that Eco1 is critical for generating
cohesion through the acetylation of Smc3, and acts after
chromatin binding to help cohesin become cohesive
3554 The EMBO Journal VOL 29 | NO 20 | 2010
(Ben-Shahar et al, 2008; Unal et al, 2008). However, the
molecular mechanisms through which cohesin maintains
sister chromatid cohesion prior to the onset of mitosis are
not yet known. Our present results indicate that mutational
inactivation or depletion of Ssu72 phosphatase decreases the
cellular ability to maintain sister chromatid arm cohesion,
leading to premature chromosome resolution. In addition, we
found that the Ssu72 phosphatase is capable of dephosphorylating (or even hypophosphorylating) SA2. These findings
significantly advance our understanding of the way in which
cohesin maintains chromosome arm cohesion and protects
against the premature removal of cohesion at prophase/
prometaphase.
Given this, we examined some potential mechanisms
through which the loss or inactivation of Ssu72 might induce
the dissociation of chromosome arm cohesion. We tested
whether depletion of Ssu72 could alter the formation of
cohesin subunit-containing complexes, thereby decreasing
arm cohesion. Immunoprecipitation experiments on Ssu72depleted cell extracts showed that the depletion of Ssu72 did
not affect the interactions of Rad21 with SA2 with the two
structural subunits, Smc1 and Smc3 (data not shown).
However, the prevention of cohesin dissociation caused by
Wapl depletion was sharply antagonized in Ssu72-depleted
cells (Figure 5B and E–G). This prompted us to ask: When
does Ssu72 maintain the stability of chromosome arm cohesion? One possibility is that the phosphatase activity of Ssu72
promotes and sustains the premature dissociation of arm
cohesins by inhibiting SA2 hyperphosphorylation until the
onset of Wapl-mediated cohesin dissociation, which follows
mitotic entry. This model might be attractive because Ssu72
appears to be involved in maintaining sister chromatid cohesion in the G2 phase. Furthermore, cohesion between sister
chromatid arms in Ssu72-depleted cells was reduced in both
G2-phase and mitotic cells, indicating that the premature
dissociation of cohesin in Ssu72-depleted cells may begin
during the G2 phase of the cell cycle. In addition, preliminary
studies have indicated that the interaction between Rad21
and Wapl appears to be significantly increased in Ssu72depleted cells (HS Kim and CW Lee, unpublished data).
It is therefore possible that the recruitment of Ssu72 to the
cohesin complex may antagonize the interaction of cohesin
subunits with Wapl. We are currently working to corroborate
this model.
Ssu72 was originally identified based on its physical interaction with the yeast transcription factor, TFIIB (Pappas and
Hampsey, 2000). A subsequent study showed that Ssu72 was
essential in yeast and known to act as a CTD phosphatase to
dephosphorylate serine 5 of RNA pol II (Krishnamurthy et al,
2004). The phosphorylation of CTD regulates transcription
and facilitates the recruitment of RNA processing factors
during transcription (Orphanides and Reinberg, 2002).
However, although we cannot exclude the possibility that
Ssu72 functions to control the transcription of cohesin subunits, we found that Ssu72 overexpression did not significantly affect the mRNA levels of the genes encoding RAD21,
SA2, SMC1 and SMC3 (Supplementary Figure S10). These
findings are consistent with a previous report, suggesting that
Ssu72 is not involved in the regulation of basal transcriptional activity (St-Pierre et al, 2005).
Most of the prior studies on Ssu72 have focused on the
regulation of transcription and proliferation in yeast. Here,
& 2010 European Molecular Biology Organization
Regulation of cohesion by Ssu72
H-S Kim et al
we propose that human Ssu72 has an essential function in
maintaining sister chromatid arm cohesion by directly and
functionally interacting with Rad21 and SA2 during the
G2 and mitotic phases. This model is supported by our
observation that mutating the cysteine in the highly conserved CX5RS signature motif of Ssu72 not only abolished the
phosphatase activity of Ssu72, it also dysregulated sister
chromatin cohesion. Our structural analysis revealed that
this mutation would logically perturb the conformation of
the catalytic loop, potentially explaining the loss of phosphatase activity (Supplementary Figure S8).
In summary, we herein provide evidence that collectively
identifies Ssu72 as a novel cohesin-binding protein essential
for chromosome cohesion. These findings significantly
advance our understanding of the mechanisms responsible for maintaining chromosome arm cohesion and protecting against premature cohesion removal at prophase/
prometaphase.
Materials and methods
Generations of plasmids, shRNAs and siRNAs
The full-length cDNA sequences of the human Ssu72, Rad21 and
SA2 genes were PCR amplified using oligo-dT primers. The Ssu72
C12S allele was generated by site-directed mutagenesis. cDNAs for
Ssu72 WT, Ssu72 C12S and Ssu72 D1–12 were subcloned into the
Myc epitope- or HA epitope-encoding pcDNA3.1 vector to generate
pMyc-Ssu72 WT, C12S and D1–12 and pHA-Ssu72, respectively.
MBP-GFP plasmid was provided by EUROSCARF (Lénárt and
Ellenberg, 2006).
For shRNA synthesis, the following gene-specific sequences were
generated using pSuper vector (Oligoengine): Ssu72 shRNA #1, 50 -A
ACAGGGACTCACGTGAAGCT-30 ; Ssu72 shRNA #2, 50 -AAGACCTGTT
TGATCTGATCC-30 ; Luciferase shRNA, 50 -CTACGCGGAATACTTCGA-30 ,
and gene-specific sequences for siRNA synthesis were Luciferase
shRNA, 50 -CUACGCGGAAUACUUCGA-30 Wapl siRNA, 50 -CGGACU
ACCCUUAGCACAA-30 and Scc2 siRNA, 50 -GCAUCGGUAUCAAGU
CCCA-30 .
Constructions of inducible and stably transfected cell lines
To generate HeLa cells for inducible expression of Rad21-RFP or
SA2-RFP fusion proteins, HeLa Tet-on cells were transfected with
the pTRE2-hydro vector (BD Biosciences Clontech) containing the
respective cDNAs and the RFP tag fused in-frame. Hygromycinresistant clones were selected in culture media containing 200 mg/ml
hygromycin and induced with 2 mg/ml doxycycline for 48 h, and
expression of RFP-fused proteins was examined by immunoblotting
analysis. HeLa-HA-Ssu72 and Ssu72-knockdown cell lines were
generated by transfection of HeLa cells with the pIRES puro3 vector
(BD Biosciences Clontech) containing the full-length Ssu72 cDNA
sequence and with the pSuper puro vector (Oligoengine) containing the shRNA sequence against Ssu72 shRNA #2, respectively.
Puromycin-resistant clones were selected by growth in medium
containing 5 mg/ml puromycin, and were tested in immunoblotting
and immunofluorescence assays.
Cell culture, cellular fractionation and cell synchronization
HeLa cell lines were grown in DMEM containing 10% fetal bovine
serum (FBS; Hyclone). For fractionation of cell extracts, soluble
cytosolic supernatants were prepared using PA buffer (150 mM
Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM
DTT and a mixture of protease inhibitors). Pellet fractions were
collected by the dissolution of nuclei in XBE2 buffer (10 mM HEPES
(pH 7.5), 300 mM NaCl, 1 mM EDTA, 2 mM MgCl2, 1 mM PMSF,
1 mM DTT and a mixture of protease inhibitors). Chromatin
fractions were subsequently prepared by sonication of the insoluble
pellet fractions in XBE2 buffer. For synchronization at G1/S
boundary, cells were grown in the presence of 1 mM thymidine
(Sigma) for 14 h, washed with PBS, grown in fresh medium for 12 h
and then treated with thymidine. After an additional 14 h, the cells
were again washed in PBS and added with fresh medium. For
& 2010 European Molecular Biology Organization
synchronization G2, the cells were grown in the presence of 50 nM
doxorubicin. For synchronization at mitosis, the cells were grown in
the presence of 200 ng/ml nocodazole and collected by shake-off.
The cells were harvested at the indicated time points after release.
Antibodies
Rabbit polyclonal and mouse monoclonal antibodies to a
KLH-conjugated peptide corresponding to residues 85–101 of
human Ssu72 were generated and affinity purified (Supplementary
Figure S2). We also prepared peptide antibodies against
human SA2 (SSRGSTVRSKKSKPSTGKRKVV) and human SA1/SA2
(DLPPSKNRRERTELKPDFFD) peptides. Other antibodies used in
this study were obtained as follows: anti-Rad21 (Bethyl Laboratories
and upstate), anti-Smc1 (Bethyl Laboratories), anti-Smc3 (Bethyl
Laboratories), anti-Scc2 (Bethyl Laboratories), anti-Smc2 (Bethyl
Laboratories), anti-Topoisomerase II alpha (Bethyl Laboratories),
anti-Erk2 (Santa Cruz Biotechnology), anti-Lamin B1 (Abcam),
anti-Tubulin (Ab frontier), Histone H3 (upstate), anti-CREST
(Immunovision), anti-Plk1 (Santa Cruz Biotechnology), anti-actin
(Sigma), anti-Mad2 (BD Biosciences Clontech), anti-Cyclin B (Santa
Cruz Biotechnology), anti-Securin (Zymed), anti-Wapl (Bethyl
Laboratories), anti-Myc (Roche), anti-HA (Roche), anti-phosphoserine (Sigma), anti-phosphothreonine (Cell signaling) and
anti-phospho-SA2 Serine 1224 antibody was kindly provided by
Dr Jan-Michael Peters.
Live-cell imaging and fluorescence photobleaching assay
To estimate the signals of RFP and GFP emissions, HeLa-Rad21-RFP
and HeLa-SA2-RFP cells were transfected with an expression
plasmid encoding H2B-GFP, induced by doxycycline treatment,
and then imaged in DT 0.15-mm dishes in DMEM medium
containing 10% FBS. The confocal pinhole was adjusted to an
optical slice thickness larger than z-sampling rate and most timelapse recordings were performed in parallel at multiple stage
positions. Over the course of 24 h, 0.3-s exposures were taken every
20–60 s using an LSM500 META confocal microscope fitted with a 20 NA0.75 objective lens (Carl Zeiss). Photobleaching assay was
performed with a photobleaching program of the Carl Zeiss
confocal software, according to the manufacturer’s instructions.
Briefly, HeLa-Rad21-RFP and HeLa-SA2-RFP cells were transfected
with the expression plasmids encoding H2B-CFP and MBP-GFP, and
then bleached using 488 nm laser beam at 80–100% intensity. We
selected the cytoplasm (ROI) of whole cell for bleaching. The
bleaching time ranged from 20 to 30 s depending on the size and
localization of bleach ROIs.
Recombinant protein purification, GST-pull down and in vitro
binding assays
GST or His6-tagged fusion constructs for expression in Escherichia
coli cells were generated by in-frame insertion of PCR fragments
encoding Ssu72 WT, Rad21 and SA2 into the pGEX-KG or pVFT1S
vectors (Pharmacia). Recombinant protein purification method was
previously described (Kim et al, 2009). For the GST-pull-down
assay, the fusion proteins were adsorbed onto glutathione-Sepharose bead (Amersham Biosciences) and incubated with whole cell
extracts (2 mg) from asynchronized HeLa cells for 4 h at 41C. The
bound proteins were separated by SDS–PAGE and then analysed by
immunoblotting with the appropriate antibodies. For the in vitro
binding assay, purified His-Ssu72 and GST-Rad21 or SA2 were
incubated and pulled down with GST-Rad21 or SA2-containing
glutathione-Sepharose. The bound proteins were separated by
SDS–PAGE and then analysed by immunoblotting with Ssu72,
Rad21 and SA2 antibodies.
Immunoprecipitation, immunoblot and flow cytometer assay
For immunoprecipitation from total cell extracts, asynchronized or
nocodazole-treated cells were resuspended in buffer A (100 mM
Tris–HCl (pH 7.5), 20 mM EDTA, 1% NP40, 1 mM PMSF, 1 mM DTT
and a protease inhibitor cocktail). The supernatants (soluble
cytoplasmic fractions) were obtained and the cell pellets were
resuspended in buffer B (100 mM Tris–HCl (pH 7.5), 20 mM EDTA,
300 mM NaCl, 1% NP40, 1 mM PMSF, 1 mM DTT and a protease
inhibitor cocktail), centrifuged and then obtained the supernatants
(soluble pellet fractions) of pellet. The mixed extracts (soluble
cytoplasmic and pellet supernatants) were diluted with a salt-free
buffer to reduce the salt concentration to 150 mM, and the samples
were centrifuged and then analysed by immunoprecipitation. For
The EMBO Journal
VOL 29 | NO 20 | 2010 3555
Regulation of cohesion by Ssu72
H-S Kim et al
immunoblot assays, the cells were synchronized as described above
or left asynchronized, harvested by scraping, washed twice in cold
PBS, and then lysed in TNN buffer (50 mM Tris–HCl (pH 7.5),
150 mM NaCl, 1% NP40, 1 mM PMSF, 1 mM DTT and a protease
inhibitor cocktail). For flow cytometric analyses, cells were fixed
and stained with propidium iodide for 5 min and then the DNA
contents of 10 000 cells per sample were analysed on a Becton
Dickinson FACScan cytometer using the CellQuest and WinMD12.8
software packages.
Immunostaining and chromosome spreading assays
For immunostaining, cells were cultured directly on glass coverslips, washed with PBS (in the case of pre-extraction immunostaining, cells were pre-extracted with 0.2% Triton X-100 in PBS for
10 min and then washed with PBS), fixed in 4% paraformaldehyde,
and then incubated with the indicated primary and secondary
antibodies. For chromosome spreading assays, cells were treated
with 100 ng/ml colcemid or 200 ng/ml nocodazole for 4 h, and
mitotic cells were collected by the shaking-off method. Mitotic cells
(2 105/ml) were incubated in a hypotonic buffer (50 mM Tris
(pH 7.4) and 55 mM KCl), fixed with freshly made Carnoy’s solution
(75% methanol and 25% acetic acid), dropped onto glass slides,
and dried at 801C. Slides were stained with 5% Giemsa (Merck) or
DAPI, washed with PBS, air-dried, mounted and processed for
fluorescence microscopy.
ChIP and Chip–qPCR
For ChIP assays, cells were fixed in culture medium with 1%
formaldehyde for 15 min. The cells washed twice in PBS and
collected by centrifugation at 3000 r.p.m. at 41C. Cells were
resuspended in ChIP lysis buffer (50 mM Tris–HCl (pH 7.5),
150 mM NaCl, 5 mM EDTA, 1% NP40, 1 mM PMSF, 1 mM DTT
and a protease inhibitor cocktail), incubated on ice for 10 min, and
sonicated until chromatin DNA was sheared into 500–700 bp
fragments. Immunoprecipitations were performed in the cell
extracts using either chip grade anti-Rad21 (Abcam) or normal
IgG in combination with Protein-A Sepharose. Precipitates were
washed sequentially for 5 min each using TSE I (0.1% SDS, 1%
Triton X-100, 2 mM EDTA, 20 mM Tris–HCl, pH 8.1, 150 mM NaCl),
TSE II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl,
pH 8.1, 500 mM NaCl) and buffer III (0.25 M LiCl, 1% NP40, 1%
sodium deoxycholate, 1 mM EDTA, 10 mM Tris–HCl, pH 8.1),
respectively. Precipitates were then washed three times with 1 ml
of TE buffer, and extracted in the solution containing 1% SDS and
0.1 M NaHCO3. Elutes were pooled and heated at 651C for 6 h to
overnight to reverse the formaldehyde crosslinking. DNA fragments
were purified with a QIAquick spin kit (Qiagen). qPCR reactions
were performed with the SYBR Green PCR Master Mix in a
MicroAmp optical 96-well reaction plate (Applied Biosystems)
using the AMI PRISM 7000 SDS v1.1 instrument. The following
specific primers were used: Primer 96 (H19 ICR): Forward 50 -TG
TGGATAATGCCCGACCTGAAGATCTG-30 Reverse 50 -ACGGAATTGGT
TGTAGTTGTGGAATCGGAAGT-30 , and Primer 88: Forward 50 -AGATG
TTATCATTATGTGTCTCGC-30 , Reverse 50 -GGCATCTACCTATACTGCG-30 .
Real-time RT–PCR
Total RNAs were extracted by RNeasy Mini Kit (Qiagen) and
quantified by UV spectrometry. To prepare RNA for PCR analyses,
1 mg of RNAs were converted to cDNA using ImProm-II Reverse
Transcription System (Promega). qPCR reactions were performed
with the SYBR Green PCR Master Mix in a MicroAmp optical
96-well reaction plate (Applied Biosystems). Quantification of
mRNA expression for the genes was performed by real-time
quantitative PCR using the AMI PRISM 7000 SDS v1.1 instrument.
Two-dimensional SDS–PAGE
To test the dephosphorylation of SA2 in HeLa Con and HeLa HASsu72 cells by two-dimensional SDS–PAGE, SA2 immunocomplexes
were prepared from whole cell lysates and rehydrated in sample
buffer supplemented with 50 mM DTT and the appropriate
ampholytes. Isoelectric focusing was performed overnight using
the Protean IEF Cell System (BioRad) with pH 3–11 NL immobiline
DryStrip isoelectric focusing strips (Amersham PLC). The protein
complexes were separated in the second dimension by SDS–PAGE,
followed by transfer to a nitrocellulose membrane.
Purification of endogenous cohesin, in vitro kinase assay
and cohesin dephosphorylation assay
For the in vitro kinase assay, bead-bound cohesin or the various
purified GST-Ssu72 proteins were washed twice with kinase buffer
(100 mM Tris–HCl (pH 7.5), 2 mM EDTA (pH 8), 20 mM MgCl2,
10 mM MnCl2, 1 mM DTT and 1 mM PMSF) and reacted with 0.4 mg
of recombinant Cdk1/Cyclin B (Invitrogen), Plk1 (Invitrogen) or
aurora B (Sigma) proteins in the presence of radio-unlabelled ATP
or [g32P]ATP (10 mCi) at 301C for 1 h. The reaction was stopped by
the addition of SDS sample buffer, and resolved by SDS–PAGE and
visualized by autoradiography. For the cohesin dephosphorylation
assay, phosphorylated cohesin complexes were purified from
nocodazole-arrested cell extracts by immunoprecipitation using
anti-SA2 antibody. Bead-bound phosphorylated cohesin complex
was washed twice with phosphatase buffer and reacted
with purified GST-Ssu72 WT, GST-Ssu72 C12S or l phosphatase
(NEB) at 301C.
Supplementary data
Supplementary data are available at The EMBO Journal Online
(http://www.embojournal.org).
Acknowledgements
We thank Drs Jan-Michael Peters and Frank Uhlmann for materials,
and Drs Toru Hirota and Nori Shindo for helpful discussions and
comments. This work was supported by research grants from the
21C Frontier Functional Human Genome Project from the Ministry
of Science and Technology in Korea (FG07-21-01), and by a Research
Program for New Drug Target Discovery (M10748000198-08N480019810) grant from the Ministry of Science and Technology in Korea.
Conflict of interest
The authors declare that they have no conflict of interest.
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