The dramatic condensation of chromosomes that occurs during mitosis is widely thought to be largely controlled by a protein complex termed condensin. Here, we describe a conditional knockout of the condensin subunit ScII/SMC2 in chicken DT40 cells. In cells lacking this condensin subunit, chromosome condensation is delayed, but ultimately reaches near-normal levels. However, these chromosomes are structurally compromised. Kinetochores appear normal, but the localization of nonhistone proteins such as topoisomerase II and INCENP is aberrant. Both proteins also fail to partition into the chromosome scaffold fraction, which appears to be largely missing in the absence of condensin. Furthermore, the chromosomes lack structural integrity, as defined by an assay that tests the stability of the chromosomal higher-order structure. Thus, a major function of condensin is to promote the correct association of nonhistone proteins with mitotic chromosomes, and this is essential for establishment of a robust chromosome structure.

SummaryDespite many decades of study, mitotic chromosome structure and composition remain poorly characterized. Here, we have integrated quantitative proteomics with bioinformatic analysis to generate a series of independent classifiers that describe the ∼4,000 proteins identified in isolated mitotic chromosomes. Integrating these classifiers by machine learning uncovers functional relationships between protein complexes in the context of intact chromosomes and reveals which of the ∼560 uncharacterized proteins identified here merits further study. Indeed, of 34 GFP-tagged predicted chromosomal proteins, 30 were chromosomal, including 13 with centromere-association. Of 16 GFP-tagged predicted nonchromosomal proteins, 14 were confirmed to be nonchromosomal. An unbiased analysis of the whole chromosome proteome from genetic knockouts of kinetochore protein Ska3/Rama1 revealed that the APC/C and RanBP2/RanGAP1 complexes depend on the Ska complex for stable association with chromosomes. Our integrated analysis predicts that up to 97 new centromere-associated proteins remain to be discovered in our data set.

The reversible condensation of chromosomes during cell division remains a classic problem in cell biology. Condensation requires the condensin complex 1 in certain experimental systems 2-8, but not in many others 9-15. Anaphase chromosome segregation almost always fails in condensin-depleted cells, leading to the formation of prominent chromatin bridges and cytokinesis failure 4, 9-17. Here, live cell analysis of chicken DT40 cells bearing a conditional knockout of condensin subunit SMC2 reveals that condensin-depleted chromosomes abruptly lose their compact architecture during anaphase and form massive chromatin bridges. The compact chromosome structure can be preserved and anaphase chromosome segregation rescued by preventing the phosphatase targeting subunit RepoMan from recruiting PP1 to chromatin at anaphase onset. This study identifies an activity critical for mitotic chromosome structure that is inactivated by Repo-Man/PP1 during anaphase. This activity, RCA (regulator of chromosome architecture), cooperates with condensin to preserve the characteristic chromosome architecture during mitosis.Mitosis is normal in SMC2 conditional knockout (SMC2 ON/OFF ) chicken DT40 cells grown without doxycycline (SMC2 ON ) 12. By 30 hours after addition of doxycycline to the culture medium (SMC2 OFF ) SMC2 mRNA levels drop at least 160-fold (QRT-PCR, Supplementary Figure 1a) and the protein becomes undetectable in immunoblots. The cells begin to die within 24-48 hours as anaphase chromosome segregation fails and massive chromatin bridges block cytokinesis (Figure 1a-d). The loss of SMC2 is accompanied by loss of other condensin subunits (e.g. CAP-H) from mitotic chromosomes (Supplementary Figure 1b- d).While this anaphase failure is unlikely to be due to defects in cohesin dynamics (see 18 , our unpublished results), it could reflect a loss of DNA topoisomerase II (topo II) function, because topo II localisation is altered in condensin-depleted chromosomes 12,18 , and the activity of extracted Drosophila topo II against an exogenous substrate is decreased following condensin RNAi 18. We therefore examined topo II activity in vivo at a physiological site by quantitating in situ topo II cleavage within the highly characterized 2.1 Mb centromeric α-satellite DXZ1 array of the human X chromosome 19 in four independent SMC2 ON/OFF DT40 hybrid cell lines. No significant differences in topo II activity at this site were found in the presence or absence of condensin (Supplementary Figure 2). Therefore, Correspondence should be addressed to WCE. telephone -44-(0)131-650-7101, fax -44-(0)131-650-7100, Bill.Earnshaw@ed.ac.uk.