Developmental Cell 11, 741–750, November, 2006 ª2006 Elsevier Inc. DOI 10.1016/j.devcel.2006.09.018 Short Article Regulation of Mitotic Chromosome Cohesion by Haspin and Aurora B Jun Dai, 1 Beth A. Sullivan, 2 and Jonathan M.G. Higgins 1, * 1 Division of Rheumatology, Immunology, and Allergy Department of Medicine Brigham & Women’s Hospital Harvard Medical School Boston, Massachusetts 02115 2 Institute for Genome Sciences & Policy Duke University Medical Center Durham, North Carolina 27708 Summary In vertebrate mitosis, cohesion between sister chro- matids is lost in two stages. In prophase and pro- metaphase, cohesin release from chromosome arms occurs under the control of Polo-like kinase 1 and Aurora B, while Shugoshin is thought to prevent removal of centromeric cohesin until anaphase. The regulatory enzymes that act to sustain centromeric co- hesion are incompletely described, however. Haspin/ Gsg2 is a histone H3 threonine-3 kinase required for normal mitosis. We report here that both H3 threo- nine-3 phosphorylation and cohesin are located at in- ner centromeres. Haspin depletion disrupts cohesin binding and sister chromatid association in mitosis, preventing normal chromosome alignment and acti- vating the spindle assembly checkpoint, leading to arrest in a prometaphase-like state. Overexpression of Haspin hinders cohesin release and stabilizes arm cohesion. We conclude that Haspin is required to maintain centromeric cohesion during mitosis. We also suggest that Aurora B regulates cohesin removal through its effect on the localization of Shugoshin. Introduction To ensure accurate chromosome segregation, cohesion between sister chromatids must be maintained until its release in a concerted fashion at anaphase. During mito- sis in vertebrates, cohesin removal occurs in two steps (Losada et al., 1998; Waizenegger et al., 2000). In pro- phase and prometaphase, cleavage-independent re- lease of cohesin from chromosome arms takes place under the control of Polo-like kinase 1 (Plk1) and Aurora B, while cohesion at centromeres and a few loci on the arms is maintained (Gimenez-Abian et al., 2004; Losada et al., 2002; Sumara et al., 2002). The protease Separase can cleave the Rad21/Scc1 cohesin subunit (Hauf et al., 2001), but it is kept inactive in early mitosis by two inhib- itory factors: association with Securin and phosphoryla- tion by Cyclin B/Cdk1. A ubiquitin ligase, the anaphase promoting complex/cyclosome (APC/C), promotes deg- radation of Securin and Cyclin B at the metaphase- anaphase transition, and Separase is activated. The resulting release of cohesin triggers the separation of sister chromatids (Nasmyth, 2002). The mechanisms that act to sustain centromeric co- hesion in the face of the prophase removal pathway remain poorly defined. Recently, Shugoshin (Sgo1) has been proposed to be a protector of centromeric cohe- sin. Repression of Sgo1 by RNA interference (RNAi) leads to premature loss of cohesion between chroma- tids and arrest at prometaphase with misaligned chro- mosomes (Kitajima et al., 2005; McGuinness et al., 2005; Salic et al., 2004; Tang et al., 2004). The centro- meric localization of Sgo1 relies on Bub1 (Kitajima et al., 2004, 2005; Riedel et al., 2006; Tang et al., 2004). After Bub1 repression, Sgo1 is relocated along the length of the chromosomes and appears then to prevent the dissociation of cohesin from the arms (Kitajima et al., 2005). Sgo1 may act in part by promoting the activity of the phosphatase PP2A at centromeres, counteracting the effects of mitotic kinases by preventing phosphory- lation of the cohesin subunit Scc3/SA2 (Kitajima et al., 2006; Riedel et al., 2006; Tang et al., 2006). We previously showed that the kinase Haspin associ- ates with chromosomes during mitosis. Phosphorylation of Haspin itself and its substrate histone H3 threonine-3 (H3T3ph) occurs specifically during mitosis. Haspin RNAi causes an accumulation of cells in which partial metaphase plates are present, but many chromosomes appear stranded near the spindle poles (Dai et al., 2005). In this study, we reveal that an important defect underlying this phenotype is a failure of mitotic chromo- some cohesion. Our results indicate that Haspin is a pos- itive regulator of centromeric cohesion. We also suggest that the complex influence of Aurora B on cohesion may be explained by its effect on the localization of Sgo1. Results Haspin Depletion Arrests Cells in Mitosis To better understand the basis for the mitotic defects caused by Haspin depletion, we optimized the RNAi pro- cedure (see Figure S1 in the Supplemental Data avail- able with this article online) and determined the DNA content and mitotic index of transfected HeLa cells by using flow cytometry. After 48 hr, 14% of control siRNA-transfected cells had a 4N or G2/M DNA content, while Haspin depletion resulted in an accumulation of more than 30% of cells in this state (Figure 1A). In contrast, Aurora B RNAi increased the number of poly- nuclear cells with greater than 4N DNA content (Fig- ure 1A and data not shown), suggesting endoreduplica- tion consistent with the known requirement for Aurora B in cytokinesis (Ditchfield et al., 2003; Hauf et al., 2003; Terada et al., 1998). Staining for the mitotic marker MPM-2 confirmed that Haspin RNAi caused an accumu- lation of cells in mitosis (reaching 12% of the population versus 2% for control RNAi; Figure 1A). This effect was less pronounced for Aurora B RNAi (Figure 1A), in keep- ing with the role of this kinase in establishing checkpoint signaling (Ditchfield et al., 2003; Hauf et al., 2003). These *Correspondence: jhiggins@rics.bwh.harvard.edu