ACCELERATED PUBLICATION
Chromosome Condensation by a Human Condensin Complex in Xenopus Egg Extracts*

Keiji KimuraDagger, Olivier Cuvier, and Tatsuya Hirano§

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



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 lambda 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.

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 beta -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). [gamma -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.

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).


    RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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).



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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.

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).



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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.

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.



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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 [gamma -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.

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.



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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.

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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.

Dagger 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


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


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