Chromosome condensation by a human condensin complex in Xenopus egg extracts.

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.

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 mitosisspecific 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
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 Cterminal 3/4 of XCAP-G. The following two polymerase chain reaction primers were designed to amplify a human cDNA fragment using a gt10 library as a template: 5hG1, 5Ј-CCCTCTAGAGCTATGCAGA-* 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. This 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 GenBank TM  AGCATCTTC-3Ј (XbaI tag sequence is underlined); and 3hG1, 5Ј-T-AGGATCCAGGGATATTGGGATTGTGGG-3Ј (BamHI tag sequence is underlined). A resulting ϳ530-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 . Immunization and affinity-purification of antibodies were performed as described previously (4).
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.

RESULTS AND DISCUSSION
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. cerevi-

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siae (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 ϳ1:1:1:1:1, 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 ϳ13 S (Fig. 2B, upper panel). A small fraction of hCAP-C and hCAP-E cosedimented at a second peak of ϳ8 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 (ϳ1/10 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 cdc2cyclin B (Fig. 3A, lane 2). This treatment phosphorylated the three non-SMC subunits, hCAP-D2, hCAP-G, and hCAP-H, as judged by [ 32 P] 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 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 cdc2cyclin B (lanes 2 and 4) in the presence of [␥-32 P]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 ϳ9:1 (lanes 2 and 6), ϳ18:1 (lanes 3 and 7), or ϳ36:1 (lanes 4 and 8). 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 4Ј,6diamidino-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 m.
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 cdc2cyclin 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.