From the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724
Received for publication, December 11, 2000, and in revised form, December 28, 2000
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ABSTRACT |
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13S condensin is a five-subunit protein
complex that plays a central role in mitotic chromosome condensation.
The condensin complex was originally identified and purified from
Xenopus egg extracts and shown to have an
ATP-dependent positive supercoiling activity in
vitro. We report here the characterization of a human condensin
complex purified from HeLa cell nuclear extracts. The human 13S complex
has exactly the same composition as its Xenopus counterpart, being composed of two structural maintenance of
chromosomes (human chromosome-associated polypeptide (hCAP)-C
and hCAP-E) subunits and three non-structural maintenance of
chromosomes (hCAP-D2/CNAP1, hCAP-G, and hCAP-H/BRRN) subunits. Human
condensin purified from asynchronous HeLa cell cultures fails to
reconfigure DNA structure in vitro. When phosphorylated by
purified cdc2-cyclin B, however, it gains the ability to introduce
positive supercoils into DNA in the presence of ATP and topoisomerase
I. Strikingly, human condensin can induce chromosome condensation when
added back into a Xenopus egg extract that has been
immunodepleted of endogenous condensin. Thus, the structure and
function of the condensin complex are highly conserved between
Xenopus and humans, underscoring its fundamental importance
in mitotic chromosome dynamics in eukaryotic cells.
Chromosome condensation is an essential cellular process that
ensures the faithful segregation of chromosomes in both mitosis and
meiosis (1, 2). Recent studies in Xenopus egg cell-free extracts led to the identification of a five-subunit protein complex, termed 13S condensin, that plays a key role in this process (3, 4). The
Xenopus 13S condensin complex is composed of two
subcomplexes, an 8S core subcomplex
(8SC)1 consisting of two
structural maintenance of chromosomes (SMC) subunits (XCAP-C and -E)
and an 11S regulatory subcomplex (11SR) containing three non-SMC
subunits (XCAP-D2, -G, and -H) (3-6). Similar five-subunit complexes
have also been identified from Schizosaccharomyces pombe (7)
and Saccharomyces cerevisiae (8). Each of the condensin
subunits is essential for cell viability in yeasts, and their mutations
lead to defects in chromosome condensation and segregation in mitosis
(7-12). Subunit composition of the putative condensin complex in human
cells is not fully understood, although a complex containing hCAP-C,
hCAP-E, and CNAP1 (homologous to XCAP-C, XCAP-E, and XCAP-D2,
respectively) has been reported very recently (13).
13S condensin, when purified from Xenopus egg mitotic
extracts, displays a DNA-stimulated ATPase activity and changes DNA structure in an ATP-dependent manner in vitro.
It introduces positive supercoils into relaxed circular DNA in the
presence of type I topoisomerases (14) and converts nicked circular DNA
into positively knotted forms in the presence of a type II
topoisomerase (15). The interphase form of 13S condensin lacks these
activities, although its subunit composition is the same as that of the
mitotic form. It was found that mitosis-specific phosphorylation of the
non-SMC subunits by purified cdc2-cyclin B can activate the
ATP-dependent activities of 13S condensin in
vitro (5, 15). Moreover, the ability of 13S condensin to induce
DNA supercoiling in the purified system is tightly coupled with its
ability to promote chromosome condensation in the cell-free extracts
(6). These results suggest strongly that the supercoiling and knotting
activities are fundamental to condensin function and directly
contribute to mitotic chromosome condensation. However, these in
vitro activities have so far been detected only in the
Xenopus condensin complex purified from egg extracts. It is
therefore very important to determine whether the functional, as well
as structural, properties of the condensin complex are conserved in
different organisms and at different developmental stages.
In this paper, we report the purification of 13S condensin from HeLa
cell nuclear extracts and describe its complete subunit composition. We
show that the human complex displays ATP-dependent supercoiling and knotting activities that are regulated by
phosphorylation by cdc2-cyclin B in vitro. Finally, a
functional complementation assay demonstrates that the human condensin
complex can induce chromosome condensation in Xenopus egg extracts.
Cloning of cDNA for hCAP-G--
By searching the human
expressed sequence tag (EST) data base, we identified a set of partial
cDNA sequences that potentially encode the human ortholog of XCAP-G
(AW503468, AW194979, AW401913, BE278549, AI628901, and AI761782). A
nucleotide sequence assembled from these clones encoded a 768-amino
acid polypeptide that is homologous to the C-terminal 3/4 of XCAP-G. The following two polymerase chain reaction primers were designed to
amplify a human cDNA fragment using a Antibody Production--
Rabbit polyclonal antisera were raised
against synthetic peptides corresponding to the C-terminal sequences of
hCAP-C (VAVNPKEIASKGLC; see Ref. 16), hCAP-E (KSKAKPPKGAHVEV; see Ref.
16), hCAP-D2/CNAP1 (TTPILRASARRHRS; see Refs. 5 and 13), hCAP-G
(EKSKLNLAQFLNEDLS; this study), and hCAP-H/BRRN (GTEDLSDVLVRQGD; see
Refs. 4 and 17). Immunization and affinity-purification of antibodies
were performed as described previously (4).
Affinity Purification of Human Condensin--
HeLa cell nuclear
extracts were prepared as described previously (18) in a buffer
containing 20 mM K-Hepes (pH 8.0), 100 mM KCl,
2 mM MgCl2, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM
Immunodepletion and Rescue--
Immunodepletion of condensin
from Xenopus egg extracts was performed as described
previously (4, 6). For the rescue experiment, an amount of
Xenopus or human condensin equivalent to the endogenous
level was added back into the depleted extract.
Other Assays--
Sucrose gradient centrifugation, supercoiling,
and knotting assays were performed as described previously (4, 14,
15).
Cloning of hCAP-G--
A search of the human EST data base
identified a set of partial cDNA sequences that potentially encode
the human ortholog of XCAP-G, a 130-kDa subunit of the
Xenopus 13S condensin complex (4). On the basis of this
information, we designed polymerase chain reaction primers, amplified a
human cDNA fragment, and used it as a hybridization probe to screen
a HeLa cell cDNA library. The longest open reading frame deduced
from multiple clones encoded a 1,015-amino acid polypeptide with a
calculated molecular mass of 114.1 kDa, which was highly
homologous to XCAP-G along its entire length (62% identical; 74%
conserved). We named this polypeptide human chromosome-associated
polypeptide-G (hCAP-G). Members of this class of condensin subunits had
been reported from S. pombe (Cnd3; see Ref. 7) and S. cerevisiae (Ycg1/Ycs5; see Refs. 8 and 12). An alignment of these
sequences is shown in Fig. 1. We found
that hCAP-G contains HEAT repeats, a highly degenerate repeating motif
found in a number of proteins with diverse functions (19) (Fig. 1,
red rectangles). This is in agreement with our recent
sequence analysis showing that each of the CAP-G family members has at
least nine copies of this motif (20). During the preparation of this
manuscript, the same human cDNA was cloned by serological screening
in an attempt to identify melanoma antigens (21).
Subunit Composition of the Human Condensin Complex--
Very
recently, Schmiesing et al. (13) reported a human protein
complex that contains hCAP-C, hCAP-E, and CNAP1 (homologous to XCAP-D2;
see Ref. 5). It remains unknown, however, whether the complex also
contains hCAP-G (this study) and BRNN (17) (homologous to XCAP-H; see
Ref. 4). In this manuscript, we refer to CNAP1 and BRNN as hCAP-D2 and
hCAP-H, respectively, in accordance with the nomenclature of their
Xenopus orthologs. We raised a set of peptide antibodies
against the putative subunits of human condensin (see "Experimental
Procedures"). It was found that an hCAP-G antibody
coimmunoprecipitates five discrete bands from a HeLa cell nuclear
extract as judged by silver stain (Fig. 2A, lane 1).
Immunoblotting analysis clearly showed that the five bands correspond
to hCAP-C (170 kDa), -D2 (155 kDa), -E (135 kDa), -G (120 kDa), and -H
(100-105 kDa) (Fig. 2A, lanes 2-6). The
stoichiometry of the five subunits was apparently ~1:1:1:1:1,
although hCAP-H always appeared as a fuzzy band (presumably because of
multiple phosphorylation) as we had also observed for XCAP-H in
Xenopus egg extracts (4).
When a HeLa nuclear extract was subject to sucrose gradient
centrifugation, the five polypeptides cofractionated with a
sedimentation coefficient of ~13 S (Fig. 2B, upper
panel). A small fraction of hCAP-C and hCAP-E cosedimented at a
second peak of ~8 S, suggesting the presence of an SMC core
subcomplex (8SC). These sedimentation properties of the human condensin
subunits were very similar to those found in Xenopus egg
extracts (4). In this experiment, we did not detect the presence of an
11SR consisting of the non-SMC subunits only (6). This was not
surprising, however, because even in the Xenopus egg
extracts this subcomplex is present at a very low level (~1/10 of the
13S complex) and not detectable in the same assay (4). We then purified
the human 13S complex by immunoaffinity column chromatography using the
hCAP-G peptide antibody and fractionated it by sucrose gradient
centrifugation (Fig. 2B, lower panel). Again, all
the five subunits cofractionated at a single peak of 13S, confirming
that they tightly associate with each other and form a complex. Taking
these results together, we conclude that the 13S holocomplex of human
condensin has exactly the same size and subunit composition as its
Xenopus counterpart.
Phosphorylation-dependent Supercoiling and Knotting
Activities--
The Xenopus 13S condensin complex
introduces positive supercoils into relaxed circular DNA in the
presence of ATP and topoisomerase I in vitro (supercoiling
assay; see Ref. 14). It also converts nicked circular DNA into
positively knotted forms in the presence of ATP and topoisomerase II
(knotting assay; see Ref. 15). The two activities are regulated by
mitosis-specific phosphorylation of the non-SMC subunits (5, 15). We
wished to test whether human condensin displays a similar set of
activities. When the complex was affinity-purified from a nuclear
extract of an asynchronously grown HeLa cell culture (Fig.
3A, lane 1), it
exhibited little activity in the supercoiling and knotting assays (Fig.
3B, lanes 1-4). We reasoned that most of the
purified complexes were in the interphase (unphosphorylated) form,
thereby producing the negative result. To test this possibility, the
purified condensin fraction was treated with cdc2-cyclin B (Fig.
3A, lane 2). This treatment phosphorylated the
three non-SMC subunits, hCAP-D2, hCAP-G, and hCAP-H, as judged by
[32P] labeling (Fig. 3A, lane 4).
Remarkably, we found that the phosphorylated form of condensin was
active in both of supercoiling and knotting assays (Fig. 3B,
lanes 5-8). Neither of these activities was found in the
purified cdc2-cyclin B fraction alone. The apparently less effective
stimulation of the knotting activity compared with the supercoiling
activity (Fig. 3B, lanes 4 and 8) is
probably because of the less quantitative nature of the former assay; a
similar observation was made with Xenopus condensin (15). As
expected, two-dimensional gel electrophoresis demonstrated that the
final products of the supercoiling assay were positively supercoiled (data not shown). These results show that the human condensin complex
displays the same set of biochemical activities as its Xenopus counterpart. The cdc2-mediated stimulation of these
activities also suggests that they contribute directly to
mitosis-specific condensation of chromosomes in human somatic
cells.
Human Condensin Induces Chromosome Condensation in Xenopus Egg
Extracts--
To further test for the functional similarity between
the human and Xenopus condensin complexes, we set up a
complementation assay in Xenopus egg extracts. When sperm
chromatin was incubated in a control extract containing endogenous
condensin (Fig. 4A, lane
1), it was converted into a mass of mitotic chromosomes (Fig. 4B, panel a). In a condensin-depleted extract
(Fig. 4A, lane 2), however, no chromosome
assembly occurred (Fig. 4B, panel b). When purified Xenopus condensin (Fig. 4A, lane
3) was added back into the extract, it restored the ability of the
extract to condense chromosomes (Fig. 4B, panel
c) as we reported previously (4, 6). Strikingly, we found that
human condensin purified from a HeLa nuclear extract (Fig.
4A, lane 4) could also functionally complement
the extract, inducing chromosome condensation very effectively (Fig.
4B, panel d). No pre-treatment with cdc2-cyclin B
was required in this assay, suggesting that the human complex was
phosphorylated by a protein kinase(s) present in the Xenopus egg extract and converted into an active complex.
In summary, the current work identifies, for the first time, all the
five subunits of the 13S condensin complex purified from HeLa cells.
Like Xenopus condensin, the human complex displays ATP- and
phosphorylation-dependent supercoiling and knotting
activities in vitro, providing strong lines of evidence that
they are fundamental to condensin function (and thereby mitotic
chromosome condensation), not only in early embryonic cells but
also in somatic cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
gt10 library as a
template: 5hG1, 5'-CCCTCTAGAGCTATGCAGAAGCATCTTC-3'
(XbaI tag sequence is underlined); and 3hG1,
5'-TAGGATCCAGGGATATTGGGATTGTGGG-3' (BamHI tag sequence is underlined). A resulting ~530-base pair
fragment was used as a hybridization probe to screen a HeLa cell
cDNA library (Stratagene). Eight positive clones were analyzed, and
seven of them were found to contain the full coding sequence. One of
the full-length clones (pHG104) was fully sequenced.
-mercaptoethanol, and 10% glycerol. After immunoaffinity purification of cohesin (18), 2 ml of the flowthrough fraction (equivalent to 9 × 108 cells) were incubated with 200 µg of anti-hCAP-G coupled to 200 µl of protein A-agarose beads
(Life Technologies, Inc.) at 4 °C for 1 h. The mixture was
poured into a 2-ml column, washed consecutively with 80 column volumes
of XBE2-gly (10 mM K-Hepes (pH 7.7), 100 mM
KCl, 2 mM MgCl2, 0.1 mM
CaCl2, 5 mM EGTA, and 10% glycerol), 10 volumes of XBE2-gly containing 400 mM KCl, and 10 volumes
of XBE2-gly. To elute condensin from the column, the hCAP-G tail peptide was added at a final concentration of 0.4 mg/ml in XBE2-gly. The peak fractions were pooled (~150 µl), supplemented with 0.1 mg/ml ovalbumin and 1 mM DTT, and concentrated 3-fold with
Microcon-30 tubes (Amicon). This procedure yielded ~1.5-2.0 µg of
human condensin of >95% purity as judged by SDS polyacrylamide gel
electrophoresis (PAGE). For phosphorylation, purified condensin (1 µg) was incubated at 22 °C for 30 min in 40 µl of
phosphorylation buffer (XBE2-gly containing 1 mg/ml ovalbumin, 1 mM DTT, and 1 mM MgATP) in the presence or
absence of cdc2-cyclin B (1 ng) purified from Xenopus egg
extracts (5). [
-32P]ATP (specific radioactivity of
>5000 Ci/mmol) was added at a final concentration of 0.05 mCi/ml in
the labeling reaction. For the rescue experiment (see Fig. 4),
condensin was eluted in XBE6 (10 mM K-Hepes (pH 7.7), 100 mM KCl, 6 mM MgCl2, 0.1 mM CaCl2, 5 mM EGTA, and 50 mM sucrose) instead of XBE2-gly and concentrated in the
same way. The amounts of purified complexes were determined by SDS-PAGE
followed by Coomassie Blue stain using bovine serum albumin as a standard.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Sequence alignment of the CAP-G family.
hCAP-G was aligned with its orthologs in Xenopus
laevis (XCAP-G; AF111423),
S. pombe (Cnd3; AB030214), and
S. cerevisiae (Ycg1/Ycs5; YDR325W)
using ClustalW. HEAT repeats are shown by rectangles.
Amino acid residues that match the consensus of HEAT repeats (20) are
shown by red, and other conserved residues are shown by
blue.
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Fig. 2.
Characterization of human condensin purified
from HeLa cell nuclear extracts. A, human condensin was
immunoprecipitated from a HeLa cell nuclear extract with an antibody
against the C-terminal peptide sequence of hCAP-G. The
immunoprecipitate was recovered on protein A-agarose beads, resolved by
7.5% SDS-PAGE, and stained with silver (lane 1). The same
fraction was analyzed by immunoblotting with antibodies specific for
hCAP-C (lane 2; 2 µg/ml), hCAP-D2 (lane 3; 1 µg/ml), hCAP-E (lane 4; 2 µg/ml), hCAP-G (lane
5; 1 µg/ml), or hCAP-H (lane 6; 1 µg/ml).
B, upper panel, a 0.1-ml HeLa cell nuclear
extract (equivalent to 4.5 × 107 cells) was
fractionated in a 5-ml sucrose gradient (5-20%) and analyzed by
immunoblotting. The positions of the ~8S and ~13S peaks are
indicated. Lower panel, human condensin was
affinity-purified using anti-hCAP-G antibody, fractionated in a 5-20%
sucrose gradient, and analyzed by immunoblotting.
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Fig. 3.
Phosphorylation-dependent
supercoiling and knotting activities of human condensin.
A, human condensin was purified from a HeLa cell nuclear
extract and treated with phosphorylation buffer alone (lanes
1 and 3) or with the same buffer containing cdc2-cyclin
B (lanes 2 and 4) in the presence of
[ -32P]ATP. The complexes were fractionated by 7.5%
SDS-PAGE and visualized by silver stain (lanes 1 and
2) or autoradiography (lanes 3 and 4).
B, supercoiling (upper panel) and knotting
(lower panel) assays were performed using human condensin
that had been treated with phosphorylation buffer alone (lanes
1-4) or the same buffer containing cdc2-cyclin B (lanes
5-8). 6 ng of calf thymus topoisomerase I (upper
panel) or 6.5 ng of T2 topoisomerase II (lower panel)
were used per reaction. The molar ratio of condensin to DNA were ~9:1
(lanes 2 and 6), ~18:1 (lanes 3 and
7), or ~36:1 (lanes 4 and 8). No
protein was added in lanes 1 and 5. The positions
of relaxed circular (RC), supercoiled (SC),
nicked circular (NC), and knotted (Kn) DNAs are
indicated.
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Fig. 4.
Reconstitution of chromosome condensation in
Xenopus egg extracts. A,
Xenopus egg mitotic extracts were immunodepleted with
control IgG (mock depletion (M), lane 1) or with
a mixture of anti-XCAP-C, -D2, -E, and -G (condensin depletion
(C), lane 2). Aliquots of each extract were
analyzed by immunoblotting using a mixture of condensin antibodies. The
13S condensin complex was purified from a Xenopus egg
extract (Xe, lane 3) or a HeLa cell extract
(He, lane 4), resolved by 7.5% SDS-PAGE, and
stained with Coomassie Blue. B, sperm chromatin was
incubated with mock-depleted (a) or condensin-depleted
(b) extracts at 22 °C for 2 h, fixed, and stained
with 4',6-diamidino-2-phenylindole. For rescue, condensin purified from
a Xenopus egg extract (c) or a HeLa nuclear
extract (d) was added back into the depleted extracts before
incubation with sperm chromatin. In each case, an amount equivalent to
the endogenous level of 13S condensin was added back. Bar,
10 µ m.
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ACKNOWLEDGEMENTS |
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We thank Ana Losada for donation of HeLa cell nuclear extracts and David MacCallum and Michiko Hirano for technical assistance and instructions. We are also grateful to the members of the Hirano laboratory for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported in part by a grant from the National Institutes of Health (R01-GM53926), by the Pew Scholars Program in the Biomedical Sciences (to T. H.), by fellowships from the Leukemia and Lymphoma Society and the Robertson Research Fund (to K. K.), and by the Cold Spring Harbor Laboratory Association (to O. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF331796.
Present address: Cellular Physiology Laboratory, The Institute of
Physical and Chemical Research (Riken), 2-1 Hirosawa, Wako, Saitama
351-01, Japan.
§ To whom correspondence should be addressed: Cold Spring Harbor Laboratory, One Bungtown Rd., P. O. Box 100, Cold Spring Harbor, NY 11724. Tel.: 516-367-8370; Fax: 516-367-8815; E-mail: hirano@cshl.org.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.C000873200
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ABBREVIATIONS |
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The abbreviations used are: 8SC, 8S core subcomplex; SMC, structural maintenance of chromosomes; CAP, chromosome-associated polypeptides; 11SR, 11S regulatory subcomplex; EST, expressed sequence tag; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; h, human; X, Xenopus.
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