RanBP2 is a nucleoporin with SUMO E3 ligase activity that functions in both nucleocytoplasmic transport and mitosis. However, the biological relevance of RanBP2 and the in vivo targets of its E3 ligase activity are unknown. Here we show that animals with low amounts of RanBP2 develop severe aneuploidy in the absence of overt transport defects. The main chromosome segregation defect in cells from these mice is anaphase-bridge formation. Topoisomerase IIalpha (Topo IIalpha), which decatenates sister centromeres prior to anaphase onset to prevent bridges, fails to accumulate at inner centromeres when RanBP2 levels are low. We find that RanBP2 sumoylates Topo IIalpha in mitosis and that this modification is required for its proper localization to inner centromeres. Furthermore, mice with low amounts of RanBP2 are highly sensitive to tumor formation. Together, these data identify RanBP2 as a chromosomal instability gene that regulates Topo IIalpha by sumoylation and suppresses tumorigenesis.

Proper control of entry into and progression through mitosis is essential for normal cell proliferation and the maintenance of genome stability1 -4. The mammalian mitotic kinase Pololike kinase 1 (Plk1) is involved in multiple stages of mitosis5. Here we report that Forkhead Box M1 (FoxM1), a substrate of Plk1 (refs 6 -8), controls a transcriptional programme that mediates Plk1-dependent regulation of cell-cycle progression. The carboxy-terminal domain of FoxM1 binds Plk1, and phosphorylation of two key residues in this domain by Cdk1 is essential for Plk1-FoxM1 interaction. Formation of the Plk1-FoxM1 complex allows for direct phosphorylation of FoxM1 by Plk1 at G2/M and the subsequent activation of FoxM1 activity, which is required for expression of key mitotic regulators, including Plk1 itself. Thus, Plk1-dependent regulation of FoxM1 activity provides a positivefeedback loop ensuring tight regulation of transcriptional networks essential for orderly mitotic progression.Transitions through cell-cycle phases require the coordination of multiple events and are tightly regulated by protein kinases 1,2,5 . In addition, transcriptional control of key cell-cycle regulators are important for cellcycle progression 9,10 . Thus, defining the links between protein kinases and transcriptional networks is essential for an understanding of normal cell-cycle progression and how specific factors may contribute to the misregulation of this process in diseases.In Saccharomyces cerevisiae, the polo kinase Cdc5p coordinates cellcycle-dependent transcription at G2/M by directly phosphorylating and regulating the activity of a co-activator © 2008 Macmillan Publishers Limited. All rights reserved. 6 Correspondence should be addressed to J.C. or D.J.T (e-mail: junjie.chen@yale.edu; tindall.donald@mayo.edu). AUTHOR CONTRIBUTIONS Z.F. performed most of experiments, analysed the data and wrote the paper; L.M. and J.M.V. analysed the time-lapse imaging data; J.H. performed the experiments shown in Fig. S2a and b; W. W. and H. L. synthesized ON01910; Z.F. and J.C. designed the experiments; J.C. and D.J. T. supervised the study and revised the paper. COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests. The mammalian transcription factor Forkhead Box M1 (FoxM1) is important in regulating mitotic entry and subsequent execution of the mitotic programme by controlling the expression of a cluster of G2/M target genes 6, 8 , 18. However, the mechanisms regulating the activation of FoxM1 at specific cell-cycle phases, and how this contributes to G2/M progression are largely unknown.Here, using yeast two-hybrid screening, we identified FoxM1 as a direct binding partner of Plk1. Six of the twenty-four positive clones encoded various lengths of the FoxM1 C terminus, with the smallest clone encoding residues 463-748 of FoxM1. These initial results suggest that there is a link between Plk1 and a transcription factor controlling a G2/M transcriptional programme.In vitro translated FoxM1 associated with GST-fused P...

The physiological role of the mitotic checkpoint protein Bub1 is unknown. To study this role, we generated a series of mutant mice with a gradient of reduced Bub1 expression using wild-type, hypomorphic, and knockout alleles. Bub1 hypomorphic mice are viable, fertile, and overtly normal despite weakened mitotic checkpoint activity and high percentages of aneuploid cells. Bub1 haploinsufficient mice, which have a milder reduction in Bub1 protein than Bub1 hypomorphic mice, also exhibit reduced checkpoint activity and increased aneuploidy, but to a lesser extent. Although cells from Bub1 hypomorphic and haploinsufficient mice have similar rates of chromosome missegregation, cell death after an aberrant separation decreases dramatically with declining Bub1 levels. Importantly, Bub1 hypomorphic mice are highly susceptible to spontaneous tumors, whereas Bub1 haploinsufficient mice are not. These findings demonstrate that loss of Bub1 below a critical threshold drives spontaneous tumorigenesis and suggest that in addition to ensuring proper chromosome segregation, Bub1 is important for mediating cell death when chromosomes missegregate.

Summary BubR1 is an essential mitotic checkpoint protein with multiple functional domains. It has been implicated in mitotic checkpoint control -as an active kinase at unattached kinetochores and as a cytosolic inhibitor of APC/CCdc20 activity- as well as in mitotic timing and stable chromosome-spindle attachment. Using BubR1-conditional knockout cells and BubR1 domain mutants, we demonstrate that the N-terminal Cdc20 binding domain of BubR1 is essential for all of these functions, whereas its C-terminal Cdc20-binding domain, Bub3-binding domain, and kinase domain are not. We find that the BubR1 N terminus binds to Cdc20 in a KEN box-dependent manner to inhibit APC/C activity in interphase, thereby allowing accumulation of cyclin B in G2 phase prior to mitosis onset. Together, our results suggest that kinetochore-bound BubR1 is non-essential and that soluble BubR1 functions as a pseudosubstrate inhibitor of APC/CCdc20 during interphase to prevent unscheduled degradation of specific APC/C substrates.

The BubR1 gene encodes for a mitotic regulator that ensures accurate segregation of chromosomes through its role in the mitotic checkpoint and the establishment of proper microtubule-kinetochore attachments. Germline mutations that reduce BubR1 abundance cause aneuploidy, shorten lifespan, and induce premature aging phenotypes and cancer in both humans and mice. Reduced BubR1 expression is also a feature of chronological aging, but whether this age-related decline has biological consequences is unknown. Using a transgenic approach in mice, we show that sustained high expression of BubR1 preserves genomic integrity and reduces tumorigenesis, even in the presence of genetic alterations that strongly promote aneuplodization and cancer, such as oncogenic Ras. We find that BubR1 overabundance exerts its protective effect by correcting mitotic checkpoint impairment and microtubule-kinetochore attachment defects. Furthermore, sustained high expression of BubR1 extends lifespan and delays age-related deterioration and aneuploidy in several tissues. Collectively, these data uncover a generalized function for BubR1 in counteracting defects that cause whole chromosome instability and suggest that modulating BubR1 provides a unique opportunity to extend healthy lifespan.

Tumorigenesis is a consequence of loss of tumor suppressors and activation of oncogenes. Expression of the mitotic checkpoint protein Chfr is lost in 20-50% of primary tumors and tumor cell lines. To explore whether downregulation of Chfr contributes directly to tumorigenesis, we generated Chfr knockout mice. Chfr-deficient mice are cancer-prone, develop spontaneous tumors and have increased skin tumor incidence after treatment with dimethylbenz(a)anthracene. Chfr deficiency leads to chromosomal instability in embryonic fibroblasts and regulates the mitotic kinase Aurora A, which is frequently upregulated in a variety of tumors. Chfr physically interacts with Aurora A and ubiquitinates Aurora A both in vitro and in vivo. Collectively, our data suggest that Chfr is a tumor suppressor and ensures chromosomal stability by controlling the expression levels of key mitotic proteins such as Aurora A.

Cdc20 and Cdh1 are the activating subunits of the anaphase-promoting complex (APC), an E3 ubiquitin ligase that drives cells into anaphase by inducing degradation of cyclin B and the anaphase inhibitor securin. To prevent chromosome missegregation, APC activity directed against these mitotic regulators must be inhibited until all chromosomes are properly attached to the mitotic spindle. Here we show that in mitosis timely destruction of securin by APC is regulated by the nucleocytoplasmic transport factors Rae1 and Nup98. We show that combined Rae1 and Nup98 haploinsufficiency in mice results in premature separation of sister chromatids, severe aneuploidy and untimely degradation of securin. We find that Rae1 and Nup98 form a complex with Cdh1-activated APC (APC(Cdh1)) in early mitosis and specifically inhibit APC(Cdh1)-mediated ubiquitination of securin. Dissociation of Rae1 and Nup98 from APC(Cdh1) coincides with the release of the mitotic checkpoint protein BubR1 from Cdc20-activated APC (APC(Cdc20)) at the metaphase to anaphase transition. Together, our results suggest that Rae1 and Nup98 are temporal regulators of APC(Cdh1) that maintain euploidy by preventing unscheduled degradation of securin.

Aging is a highly complex biological process that is believed to involve multiple mechanisms. Mice that have small amounts of the mitotic checkpoint protein BubR1 age much faster than normal mice, but whether other mitotic checkpoint genes function to prevent the early onset of aging is unknown. In this study, we show that several aging-associated phenotypes appear early in mice that are double haploinsufficient for the mitotic checkpoint genes Bub3 and Rae1 but not in mice that are single haploinsufficient for these genes. Mouse embryonic fibroblasts (MEFs) from Bub3/Rae1 haploinsufficient mice undergo premature senescence and accumulate high levels of p19, p53, p21, and p16, whereas MEFs from single haploinsufficient mice do not. Furthermore, although BubR1 hypomorphic mice have less aneuploidy than Bub3/Rae1 haploinsufficient mice, they age much faster. Our findings suggest that early onset of aging-associated phenotypes in mice with mitotic checkpoint gene defects is linked to cellular senescence and activation of the p53 and p16 pathways rather than to aneuploidy.