Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Cohesin release is required for sister chromatid resolution, but not for condensin-mediated compaction, at the onset of mitosis Ana Losada, Michiko Hirano, and Tatsuya Hirano1 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA The establishment of metaphase chromosomes is an essential prerequisite of sister chromatid separation in anaphase. It involves the coordinated action of cohesin and condensin, protein complexes that mediate cohesion and condensation, respectively. In metazoans, most cohesin dissociates from chromatin at prophase, coincident with association of condensin. Whether loosening of cohesion at the onset of mitosis facilitates the compaction process, resolution of the sister chromatids, or both, remains unknown. We have found that the prophase release of cohesin is completely blocked when two mitotic kinases, aurora B and polo-like kinase (Plx1), are simultaneously depleted from Xenopus egg extracts. Condensin loading onto chromatin is not affected under this condition, and rod-shaped chromosomes are produced that show an apparently normal level of compaction. However, the resolution of sister chromatids within these chromosomes is severely compromised. This is not because of inhibition of topoisomerase II activity that is also required for the resolution process. We propose that aurora B and Plx1 cooperate to destabilize the sister chromatid linkage through distinct mechanisms that may involve phosphorylation of histone H3 and cohesin, respectively. More importantly, our results strongly suggest that cohesin release at the onset of mitosis is essential for sister chromatid resolution but not for condensin-mediated compaction. [Keywords: Cohesin; condensin; sister chromatid cohesion; chromosome condensation; histone H3 phosphorylation] Received September 4, 2002; revised version accepted October 11, 2002. In eukaryotic cells, the duplication of chromosomal cycle-dependent transitions of chromosome structure DNA during S phase produces two sister DNA mol- can largely be explained by the dynamic behavior of two eculesthat are topologically intertwined. In addition, a distinct structural maintenance of chromosomes (SMC) proteinaceous linkage between the sister chromatids is protein complexes, cohesin and condensin. Cohesin established during this stage and is maintained through- plays a central role in sister chromatid cohesion, possi- out G2 phase. At the onset of mitosis, loosening of co- bly asthe main constituentof the proteinaceouslinkage hesion accompanies the compaction of each chromatid that holds sister chromatids together (for review, see Lee along itslongitudinal axis.The final product of this and Orr-Weaver 2001; Nasmyth 2001). In metazoan mechanistically complex process is the metaphase chro- cells, most cohesin complexes bound to interphase chro- mosome, in which two rod-shaped sister chromatids are matin dissociate during mitotic prophase (Losada et al. juxtaposed along their entire length. The establishment 1998, 2000; Waizenegger et al. 2000; Warren et al. 2000). of metaphase chromosome structure is an essential pre- Although it hasbeen proposedthat thisbulk releaseof requisite of the faithful segregation of sister chromatids cohesin may be required for proper chromosome conden- in anaphase. Despite its fundamental importance, very sation (e.g., Losada and Hirano 2001), no direct test for little isknown about the regulatory network that con- thishypothesishasbeenreported to date. The remaining tributesto the conversionof amorphousinterphasechro- cohesin complexes are removed from the chromosomes matin into highly organized metaphase chromosomes. via separase-mediated cleavage at the onset of anaphase, Accumulating lines of evidence suggest that the cell- leading to complete separation of the sister chromatids (Waizenegger et al. 2000; Hauf et al. 2001). On the other hand, condensin is loaded on chromosomes in prophase, 1Corresponding author. at the time when condensation is initiated and cohesin- E-MAIL [email protected]; FAX (516) 367-8815. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ mediated linkage is destabilized (for review, see Hirano gad.249202. 2000). Condensin is essential for the maintenance of 3004 GENES & DEVELOPMENT 16:3004–3016 © 2002 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/02 $5.00; www.genesdev.org Downloaded from genesdev.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Aurora B, polo and sister chromatid resolution condensation from metaphase to anaphase and dissoci- This in vitro system allows the assembly of chromo- ates from chromosomes in telophase when decondensa- somesindependentlyof other mitotic events(e.g., tion becomesapparent in the daughter cell nuclei (Hi- spindle assembly), enabling us to investigate the specific rano and Mitchison 1994; Schmiesing et al. 2000). contribution of different kinasestothisparticular pro- One important question is how multiple protein ki- cess. We have found that aurora B and polo-like kinase nases might regulate these changes of chromosome ar- (Plx1) act in concert to dissociate cohesin from chroma- chitecture. Three mitotic kinases, cdc2, polo, and aurora tin at the onset of mitosis. Condensin is still able to bind B, are of particular interest. We have shown previously to chromatin when cohesin release is prevented by si- that phosphorylation of condensin by cdc2–cyclin B ac- multaneousdepletion of both kinasesandleadsto the tivatesitspositivesupercoilingand knotting activities formation of rod-shaped chromosomes of apparently in vitro (Kimura et al. 1998, 1999, 2001). Although this normal length. However, the assembled chromosomes observation partially explains how chromosome conden- have a profound defect in the resolution of sister chro- sation is initiated at the onset of mitosis, additional matids. mechanisms are likely to dictate the mitosis-specific as- sociation of condensin with chromatin (Kimura and Hi- rano 2000; Steen et al. 2000). Cdc2 can also phosphory- Results late one of the regulatory subunits of vertebrate cohesin Characterization of Plx1 and aurora B in Xenopus (SA/Scc3) in vitro, and thisphosphorylationreducesthe egg extracts affinity of the complex for chromatin (Losada et al. 2000). However, cdc2 activity isnot required for cohesin We have shown previously that release of cohesin from release when a pseudomitotic state is induced by addi- chromatin at the onset of mitosis is not accompanied by tion of a phosphatase inhibitor to interphase extracts cleavage of the Rad21/Scc1 subunit but instead corre- (Sumara et al. 2000). In Saccharomyces cerevisiae, Cdc5/ lates with phosphorylation of the SA/Scc3 subunits in polo kinase phosphorylates the Scc1 subunit of cohesin Xenopus egg extracts(Losadaet al. 2000). In thisstudy, and thereby enhancesitscleavage by separaseat the on- we have focused on the role of two mitotic kinases, Plx1 set of anaphase (Alexandru et al. 2001). The specific role and aurora B, in cohesin behavior. If phosphorylation of of this kinase in sister chromatid separation has been cohesinindeed promotesitsrelease,one would expect revealed only recently, probably because polo plays mul- the responsible protein kinase(s) to act on chromatin. To tiple rolesthroughout mitosisanditsmutation causes test whether Plx1 and aurora B met this criterion, we pleiotropic phenotypes(Glover et al. 1998). investigated their chromatin-binding properties in the A similar level of complexity has been encountered in cell-free extracts. Sperm chromatin was first incubated the functional analysisofaurora B. Aurora B and itsbind- with an interphase extract and then a half volume of ing partner, INCENP, are referred to aschromosomal cytostatic factor (CSF)-arrested extract was added to in- passengers on the basis of their dynamic localization duce the transition to a mitotic state. Aliquots were during mitosis (for review, see Adams et al. 2001a). taken from the reaction mixture at different time points, These proteins associate with chromosomes early in mi- and chromatin fractionswere isolatedand analyzed by tosisandare detected at their centromeric regionsby immunoblotting. Consistent with our previous work metaphase. In anaphase, they redistribute to the spindle (Losada et al. 1998), cohesin was progressively loaded midzone and equatorial cortex and accumulate in the onto chromatin during interphase, and most complexes midbody during cytokinesis. Consistent with this dy- dissociated upon entry into mitosis (Fig. 1A, panel 1). namic behavior, mutation or depletion of aurora B causes Dissociation of cohesin coincided with association of defectsin multiple mitotic eventsincluding chromo- condensin with chromatin (Fig. 1A, panel 2). Although some segregation and cytokinesis. Recent studies have Plx1 and aurora B were detectable in both interphase and shown that aurora B phosphorylates the N-terminal tail mitotic chromatin (Fig. 1A, panels3,4), their kinaseac- of histone H3, implicating a direct contribution of this tivitiesappear to be regulated in a cell-cycle-dependent kinase to mitotic chromosome dynamics (Hsu et al. manner. Plx1 displayed a retarded migration during mi- 2000; Spelioteset al. 2000; Adamset al. 2001b; Giet and tosis(Fig.1A, panel 3, lanes9–13), which probably re- Glover 2001; Murnion et al. 2001). It has long been sus- flects its mitosis-specific activation by phosphorylation pected that thishistoneH3