LETTERS. Chfr is required for tumor suppression and Aurora A regulation

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1 25 Nture Pulishing Group Chfr is required for tumor suppression nd Auror A regultion Xiohun Yu 1, Ktherine Minter-Dykhouse 1, Liviu Mlurenu 2, Wei-Meng Zho 3, Dongwei Zhng 4, Crolin J Merkle 1, Irene M Wrd 1, Hideyuki Sy 4, Guowei Fng 3, Jn vn Deursen 2 & Junjie Chen 1 Tumorigenesis is onsequene of loss of tumor suppressors nd tivtion of onogenes. Expression of the mitoti hekpoint protein Chfr is lost in 2 5% of primry tumors nd tumor ell lines. To explore whether downregultion of Chfr ontriutes diretly to tumorigenesis, we generted Chfr knokout mie. Chfr-defiient mie re ner-prone, develop spontneous tumors nd hve inresed skin tumor inidene fter tretment with dimethylenz()nthrene. Chfr defiieny leds to hromosoml instility in emryoni firolsts nd regultes the mitoti kinse Auror A, whih is frequently upregulted in vriety of tumors. Chfr physilly interts with Auror A nd uiquitintes Auror A oth in vitro nd in vivo. Colletively, our dt suggest tht Chfr is tumor suppressor nd ensures hromosoml stility y ontrolling the expression levels of key mitoti proteins suh s Auror A. Figure 1 Trgeted disruption of Chfr in mie. () Strtegy for generting the trgeted Chfr llele. Prt of mouse Chfr (; top), the trgeting vetor (middle) nd the disrupted Chfr llele (KO; ottom) re shown. () Multiplex PCR genotype nlyses for neo (KO; upper nd) nd the trgeted exon (; lower nd) were done to onfirm the genotypes of wild-type nd Chfrdefiient mie. () Western lots of ell lystes prepred from MEFs with indited genotypes were done with immunopreipittes using ntiody to Chfr (upper pnel). Immunolotting with ntiody to tin ws used s loding ontrol (lower pnel). Preise regultion of DNA ondenstion nd hromosome segregtion in mitosis is importnt for norml ell yle progression. The progression of ells in mitosis is driven y severl key mitoti kinses inluding CDC2, PLK1 nd Auror kinses. Filure or improper regultion of these kinses leds to neuploidy nd my ontriute to tumorigenesis in humns. For exmple, Auror A kinse is overexpressed or mplified in numer of primry tumors nd tumor ell lines 1,2. Mouse NIH-3T3 ells trnsfeted with Auror A develop into tumors when implnted in nude mie, suggesting tht Auror A is onogeni 3 5. The most studied mitoti hekpoint is the spindle ssemly hekpoint. Proteins involved in the spindle hekpoint sense mirotuulekinetohore tthment nd llow the tivtion of the nphsepromoting omplex (APC) only when ll hromosomes re properly tthed to the spindles nd ligned on the metphse plte 6 8. Although the integrity of the spindle hekpoint is essentil for the mintenne of hromosoml stility, spindle hekpoint proteins nd APC omponents re rrely downregulted or mutted in tumors 9,1, suggesting tht the disruption of other mitoti hekpoint pthwys my hve greter role in tumor-ssoited hromosoml instility. Chfr (hekpoint protein with FHA nd Ring domin) ws reently identified s defining new erly mitoti hekpoint tht delys trnsition to metphse in response to mitoti stress 11,12. The physiologil funtion of Chfr is still not known. Chfr ontins n N-terminl FHA domin 13, whih is involved in phosphoprotein intertion, nd Ring domin, whih prtiiptes in protein uiquitintion. The puttive Chfr fission yest ortholog Dm1 lso ontins oth n FHA domin nd Ring domin nd prtiiptes in multiple mitoti events, inluding CDC2 tivtion, spindle ssemly nd ytokinesis 14,15. The Ring domin of Chfr hs E3 uiquitin ligse tivity in vitro One potentil Chfr sustrte is PLK1 (ref. 16), ut the expression of PLK1 does not lwys orrelte with Chfr downregultion nd Chfr funtion in rest ner ell lines, suggesting tht other Chfr sustrtes re involved in mitoti hekpoint ontrol 19. Although Chfr is uiquitously expressed in norml tissues, it is frequently downregulted in humn ners, mostly owing to hypermethyltion of its promoter region Chfr downregultion ATG (nt ) 5.7 k 4.2 k 1.7 k PGK-neo 4.1 k Arm1 Arm2 7.4 k PGK-neo Trgeting onstruts KO / / KO Chfr Atin 1 Deprtment of Onology, nd 2 Peditri nd Adolesent Mediine, Myo Clini nd Foundtion, Rohester, Minnesot 5595, USA. 3 Deprtment of Biologil Sienes, Stnford University, Stnford, Cliforni 9435, USA. 4 Deprtment of Tumor Genetis nd Biology, Kummoto University, Kummoto, , Jpn. Correspondene should e ddressed to J.C. (hen.junjie@myo.edu). Pulished online 27 Mrh 25; doi:1.138/ng1538 NATURE GENETICS VOLUME 37 [ NUMBER 4 [ APRIL 25 41

2 1 9/1 Thymus Liver Ovry Ded mie (%) /123 1/188 Spleen 25 Nture Pulishing Group / Liver Spleen hs een found in primry lung, olon, esophgus nd gstri rinoms nd tumor ell lines of lung, olon, esophgel, rin, one, rest, gstri nd hemtopoieti origin 11, Muttions in CHFR hve lso een identified in primry lung ners nd ell lines inluding osteosrom nd rest ner 11,19,24. These oservtions rise the possiility tht Chfr my ontriute to tumorigenesis in humns. To explore the physiologil funtion of Chfr nd its role in tumorigenesis, we generted Chfr-null mie y disrupting Chfr using stndrd gene-trgeting tehniques. We repled n exon tht enodes prt of the sequene of the Chfr FHA domin with neomyin Ovry Figure 2 Chfr defiieny inreses tumor inidene in mie. () Deth rtes of wild-type nd Chfrdefiient mie ( 4 weeks). () Tumors derived from Chfr / mie. We inluded exmples of spontneous lymphoms otined from Chfr / mie nd the hemtoxylin nd eosin stined setions. () DMBA tretment indued skin tumor formtion in Chfr / mie. A hemtoxylin nd eosin stined skin tumor oserved in Chfr / mie is shown. (d) Inidenes of DMBA-indued skin tumors in wildtype nd Chfr-defiient mie >5 Chromosome numer d Mie with skin tumor (%) Time (d) (12/26) / (6/34) (/24) seletion ssette (Fig. 1). We used two independent emryoni stem (ES) ell lones to generte Chfr-defiient mie. We rried out PCR nlyses to onfirm tht we otined mie of genotype Chfr / (Fig. 1). We lso onfirmed y western lotting using ntiodies to Chfr tht Chfr ws not expressed in Chfr / mie (Fig. 1). Chfr / mie were vile nd hd no ovious developmentl defets, suggesting tht Chfr is not required for norml ell yle progression during emryoni development. We proeeded to monitor tumor inidene in these mie over time. After 4 weeks, nine Chfr / mie (9%) developed lymphom, whih invded thymus, spleen, liver nd ovry (Fig. 2,), nd died. Ten Chfr / mie (5.3%) 2N 8 / 6 2N 4 >4N >4N 4N 4N 2 Kryotypes (%) G1: 69.3% S: 9.9% 4N: 19.9% >4N:.9% G1: 43.6% S: 4.9% 4N: 34.7% >4N: 16.8% f Anphse Cytokinesis Lgging hromosomes d Multinuleted ells (%) e Men time of eh phse of ell mitosis (min) * * Prophseprometphse Prometphsemetphse Metphsenphse Anphsetelophse (ytokinesis) Filed nuler segregtion Filed ytokinesis Figure 3 Loss of Chfr leds to hromosome instility. () Metphse spreds with norml kryotype in wild-type MEFs (left) nd neuploidy in Chfr / MEFs (right). () Chfr / ells hve inresed neuploidy. We ounted the hromosome numers of 1 metphse spreds per smple from P4 MEFs. () Cell yle profile of unsynhronized MEFs (t P4) ws determined y fluoresene-tivted ell-sorting nlysis. Aneuploidy nd polyploidy were oserved in Chfr / MEFs. (d) Multinuleted ells oserved in Chfr / MEFs. We exmined 2,5 ells for eh genotype y DAPI stining nd fluoresene mirosopy. The perentges of inuleted nd multinuleted ells is indited (P o.1). (e,f) Chfr / MEFs hve errors in mitoti progression. MEFs were infeted with retrovirus enoding histone H2B EGFP. The mitoti progress of Chfr (n ¼ 4) nd Chfr / (n ¼ 51) MEFs ws followed y time-lpse mirosopy. (e) Men time of eh mitoti phse is shown. Prophse nd nphse were prolonged in Chfr / MEFs ompred with wild-type MEFs (*P o.5). (f) Mitoti errors were reorded. Differentil interferene ontrst imges re lso inluded. Cells with mitoti errors (%) Lgging hromosomes Filed nuler segregtion Filed ytokinesis 42 VOLUME 37 [ NUMBER 4 [ APRIL 25 NATURE GENETICS

3 25 Nture Pulishing Group / IP: nti-chfr Blot: nti-chfr Blot: nti-auror A Blot: nti-plk1 Blot: nti-auror B Blot: nti-auror A Blot: nti-auror B Blot: nti-auror A Blot: nti-auror B lso died during this period from lymphom (Fig. 2 nd dt not shown). Only two wild-type mie (1.6%) died during this period, nd we oserved no tumors in wild-type mie. From 4 to 8 weeks, 38 Chfr / mie died. At lest 29 of them hd ovious tumors in gross pthologil exmintion. Most tumors found during this period were epithelil tumors in mjor orgns, espeilly lung, liver nd gstrointestinl trt (Supplementry Fig. 1 nd Supplementry Tle 1 MG132 IP Anti-Auror B FHA Ring Cys-rih F R C 1 48 F R C Anti-Auror A Blot: nti-chfr IP: nti-chfr Blot: nti-chfr Blot: nti-auror A Blot: nti-auror B IP: nti-flag (Auror A) Blot: nti-my (Chfr) Blot: nti-my (Chfr) Blot: nti-flag (Auror A) P1 P1 4N >4N P4 4N: 25.2% >4N: 1.% 4N: 25.6% >4N: 2.1% d P4 4N: 19.1% >4N: 1.7% 4N: 33.9% >4N: 17.5% D1 D2 D3 D4 A-ox 1 61 D D1 D2 D3 D4 Ativtion loop Pull-down: GST-Chfr Figure 4 Chfr negtively regultes expression of Auror A. () Expression of Auror A ws upregulted in Chfr / MEFs. Lystes derived from wild-type or Chfr-defiient MEFs were immunopreipitted (IP) nd immunolotted with indited ntiodies. () Auror A levels were determined using P1 or P4 wild-type nd Chfr / MEFs. Cell lystes were lotted with the indited ntiodies. Cell yle distriutions were determined y fluoresenetivted ell-sorting nlysis. () Expression of Auror A ws higher in Chfr / tissues. Liver nd spleen were homogenized nd lysed. Lystes were immunolotted with indited ntiodies. online). In the sme time frme, eight Chfr mie died, three of whih hd tumors. Tumor inidene in Chfr / mie ws intermedite etween tht in Chfr nd Chfr / mie. The spetrum of tumors identified in Chfr / mie ws similr to tht in Chfr / mie. The higher deth rte nd tumor inidene in Chfr / mie suggest tht Chfr is importnt for tumor suppression. To onfirm tht Chfr defiieny leds to inresed tumor inidene, we treted wild-type nd Chfr-defiient mie with single dose of dimethylenz()nthrene (DMBA), hemil rinogen, on the dorsl skin 3 5 d fter irth. After 4 months, nerly 5% of Chfr / mie, ut no wild-type mie, developed skin tumors (Fig. 2,d). Chfr / mie lso developed skin tumors, leit t lower frequeny thn Chfr / mie (Fig. 2d). Tken together, these results support the ide tht Chfr is required for tumor suppression in mie. IP: nti-flag (Auror A) IP: nti-my (Chfr) Blot: nti-flag (Auror A) IP: nti-flag (Auror A) Blot: nti-flag (Auror A) IP: nti-my (Chfr) Blot: nti-chfr D-ox e f My-Chfr: FLAG-Auror A: HA-U: U-Auror A (U)n-Auror A Auror A Chfr Chfr Time (min) _ C IP: nti-flag (Auror A) Blot: nti-ha (U) Blot: nti-flag (Auror A) Blot: nti-my (Chfr) Blot: nti-tin U-Auror A FLAG-Auror A: My-Chfr: HA-U: _ D1 Figure 5 Chfr interts with Auror A nd uiquitintes Auror A oth in vitro nd in vivo. () Chfr interts with endogenous Auror A, ut not Auror B. 293T ells were treted with MG132 (15 mm) or left untreted. Cell lystes were immunopreipitted (IP) nd immunolotted with indited ntiodies. () The C-terminl ysteine-rih region of Chfr is required for its intertion with Auror A. 293T ells were otrnsfeted with plsmids enoding My-tgged Chfr nd FLAG-tgged Auror A. Cell lystes were immunopreipitted (IP) nd immunolotted with indited ntiodies., wild-type; DF, FHA domin deletion; DR, Ring domin deletion; DC, ysteine-rih domin deletion. () The N-terminl region of Auror A medites its ssoition with Chfr. In vitro trnslted wild-type or deletion mutnts (D1 D4) of Auror A were pulled-down y GST-Chfr nd exmined y utordiogrphy. IP, immunopreipittion. (d) 293T ells were trnsfeted with plsmids enoding My-tgged Chfr nd FLAG-tgged wild-type or D1 mutnt Auror A. Cell lystes were immunopreipitted (IP) nd immunolotted with indited ntiodies. (e) In vitro trnslted Auror A ws uiquitinted (U) in the presene or sene of reominnt GST-Chfr. The uiquitintion of Auror A ws nlyzed y SDS-PAGE. (f) Chfr uiquitintes (U) Auror A in vivo. HCT116 ells were trnsfeted with plsmids enoding FLAG-tgged wild-type or D1 mutnt Auror A, hemgglutinin-tgged uiquitin (HA-U) nd My-tgged wild-type or DC mutnt Chfr. Cells were treted with MG132 (15 mm) for 3 h nd then olleted for nlyses. NATURE GENETICS VOLUME 37 [ NUMBER 4 [ APRIL 25 43

4 25 Nture Pulishing Group shrna Auror A PLK1 Auror A PLK1 Blot: nti-auror A Blot: nti-plk1 Blot: nti-auror B Beuse Chfr is involved in mitoti hekpoint ontrol, we next exmined whether hromosoml stility ws omprised in Chfrdefiient ells. We prepred wild-type nd Chfr-defiient mouse emryoni firolsts (MEFs) from emryos t emryoni dy 13.5 for in vitro ulture. After four pssges (P4), we prepred metphse spreds of Chfr nd Chfr / ells nd determined hromosome numers. More thn 3% of metphse Chfr / MEFs showed sustntil neuploidy or polyploidy, wheres Chfr MEFs hd norml or nerly norml kryotypes (Fig. 3,). In ddition, Chfr / MEFs formed spontneous olonies in in vitro ulture (Supplementry Fig. 2 online), suggesting tht Chfr / ells hve trnsformtion potentil. Flow ytometry nlysis onfirmed tht nerly 17% of Chfr / MEFs ontined more thn 4N genomi DNA (Fig. 3). Compred with wild-type MEFs, Chfr / MEFs hd smller S-phse popultion nd greter 4N popultion. To exmine whether the 4N ells were in G2-M phse or were the polyploid ells, we rried out immunofluoresene mirosopy using mitoti mrker, phosphorylted histone H3. We did not oserve ny sustntil inrese in G2 (indited y puntuted stining with ntiody to histone H3 phosphorylted t Ser1) or M phse ells (indited y stining with DAPI nd ntiody to histone H3 phosphorylted t Ser1) in Chfr / MEFs. Insted, we found mny multinuleted ells in Chfr / MEFs, whih my represent the inresed popultions of 4N nd 44N ells (Fig. 3d). To exmine mitosis in Chfr / MEFs, we followed individul mitoti ells using time-lpse mirosopy. We oserved prolonged prophse nd nphse in Chfr / MEFs (Fig. 3e). We lso oserved severl types of mitoti errors, inluding lgging hromosomes, filed nuler segregtion nd ytokinesis filure (Fig. 3f), suggesting tht Chfr is required for proper mitoti trnsitions. We oserved inresed tumor inidene, neuploidy nd defetive hromosoml segregtion nd ytokinesis in Chfr-defiient mie nd ells. These phenotypes re very similr to those oserved in ells with overexpression of Auror A, puttive onoprotein involved in mitosis 26. Amplifition nd overexpression of Auror A hve een oserved in mny primry tumors inluding rest, oloretl nd 4N: 21.7% 4N: 36.8% >4N:.8% >4N: 16.4% 4N >4N Auror A shrna 4N: 27.2% >4N: 5.1% PLK1 shrna 4N: 37.7% >4N: 15.9% Kryotypes (%) >5 Chromosome numer Auror APLK1 shrna 4N: 22.8% >4N: 1.% Auror A shrna PLK1 shrna Auror APLK1 shrna Figure 6 Downregultion of Auror A dereses the neuploidy oserved in Chfr / MEFs. P1 MEFs were treted with Auror A or PLK1 shrna until P4. () Cell lystes were immunolotted with indited ntiodies. () Chromosome numers of 1 metphse spreds per smple. () Cell yle distriutions of these smples. gstri ner 3,4,27,28. Like Chfr, overexpression of Auror A in wildtype MEFs indues hromosome segregtion defets nd ytokinesis filure, whih results in neuploidy nd polyploidy 29,3.Themrked similrities etween Chfr defiieny nd Auror A overexpression prompted us to explore potentil mehnisti link etween these two proteins. Beuse Chfr is n E3 uiquitin ligse tht my e involved in regulting protein degrdtion, we first exmined the expression levels of Auror A in wild-type nd Chfr-defiient ells. The expression levels of Auror A inversely orrelted with those of Chfr in MEFs (Fig. 4), suggesting tht Chfr my negtively regulte the expression of Auror A. PLK1, known sustrte of Chfr 16, ws lso upregulted in Chfr / ells. As ontrol, we showed tht the level of nother mitoti kinse, Auror B, ws unhnged in Chfr / MEFs. In vitro ulture led to inresed 4N nd 44N popultions in Chfr / MEFs (Fig. 3). To exlude the possiility tht the inresed Auror A expression is due to the hnge in ell yle profile, we determined the levels of Auror A in first-pssge (P1) wild-type nd Chfr / MEFs, whih hve omprle ell yle distriutions. We still oserved inresed Auror A levels in Chfr / MEFs (Fig. 4). We lso oserved inresed Auror A expression in tissues from Chfr / mie (Fig. 4). As n E3 uiquitin ligse, Chfr my diretly trget Auror A for uiquitintion nd degrdtion. Beuse mny E3 ligses intert with their sustrtes, we exmined whether Chfr ound to Auror A. Endogenous Chfr oimmunopreipitted with Auror A ut not with Auror B (Fig. 5). Auror A expression nd ssoition with Chfr lso inresed fter tretment with the proteosome inhiitor MG132. We further determined tht the C-terminl ysteine-rih domin of Chfr medites this intertion with the N terminus of Auror A (Fig. 5 d). Next, we exmined whether Auror A ws sustrte of Chfr. Chfr promoted polyuiquitintion of Auror A in vitro (Fig. 5e). To test whether Chfr ould uiquitinte Auror A in vivo, we otrnsfeted HCT116 ells with plsmids enoding uiquitin, Auror A nd Chfr. Auror A (Fig. 5f), ut not Auror B (Supplementry Fig. 3 online), ws uiquitinted only when it ws otrnsfeted with wild-type Chfr, suggesting tht Chfr ould promote Auror A uiquitintion in vivo (Supplementry Fig. 4 online). Neither the Chfr deletion mutnt missing the C-terminl ysteinerih domin nor the Auror A N terminus deletion mutnt ould promote the Chfr-dependent uiquitintion of Auror A (Fig. 5f), suggesting tht speifi intertion etween Chfr nd Auror A is required for the Chfr-dependent uiquitintion of Auror A in vivo. Tken together, these dt suggest tht Auror A is physiologil sustrte of Chfr in vivo. To explore whether overexpression of Auror A ounts for the inresed neuploidy oserved in Chfr / ells, we used short hirpin RNA (shrna) to downregulte expression of Auror A (Fig. 6). Deresing expression of Auror A, ut not tht of PLK1, redued neuploidy nd the numer of multinuleted ells in Chfr / MEFs (Fig. 6,), suggesting tht Auror A is the key omponent downstrem of Chfr in mitoti ontrol. Notly, downregultion of oth Auror A nd PLK1 ws more effetive in preventing hromosoml instility in Chfr / MEFs (Fig. 6,), implying tht Auror A nd PLK1 my work together to ensure norml mitoti progression. In summry, we show tht Chfr is tumor suppressor. Chfr funtions y regulting the expression level of Auror A nd mintining genomi stility. Our results imply tht disruption of the Chfr Auror A pthwy promotes tumorigenesis nd support the hypothesis tht hromosoml instility ould ontriute to tumorigenesis in humns. 44 VOLUME 37 [ NUMBER 4 [ APRIL 25 NATURE GENETICS

5 25 Nture Pulishing Group METHODS Genertion of knokout mie. We designed the Chfr gene-trgeting vetor to reple n exon spnning nt of the mouse gene Chfr with the PGK-neo ssette. We loned genomi DNA frgments flnking this exon into the pko Srmler NTKV-191 vetor (Strtgene). We eletroported 129/SvE ES ells with the trgeting vetor nd sreened B3 G418-resistnt ES lones y Southern-lot nlysis using proe tht hyridizes to 5.7-k restrition frgment in wild-type ells nd 7.4-k frgment in homologous reominnts. We injeted two independent ES lones with homologous integrtion t the trgeting site into C57BL/6 lstoysts to generte himeri mie. We then rossed these himers with C57BL/6 femles nd used heterozygous mie with suessful germline trnsmission of the trgeted llele to generte Chfr / mie. We used PCR to distinguish mouse genotypes using ommon ntisense primer outside of the trget exon, sense primer in the trget exon nd sense primer in the neo gene (primer sequenes ville on request). Plsmids nd ntiodies. DNA enoding FLAG Auror A inserted into psg5 ws gift from D.-S. Lim (Kore Advned Institute of Siene nd Tehnology). We inserted n expressed-sequene tg lone ontining the full-length Chfr open reding frme into the pa3m vetor to generte mmmlin expression onstrut for three My-epitope tgged Chfr. We otined the hemgglutinin-uiquitin onstrut from D. Hines (Temple University) nd the wild-type nd mutnt His-uiquitin onstruts from R. Ber (Columi University). We purhsed rit ntiody to humn Auror A from Cell Signling Tehnology. We rised rit ntiody to mouse Auror A ginst Auror A N terminus peptide (SRPVKTTVPFGPKRVLVTEQIPC) or C terminus peptide (CTGHTSKEPTSKSS). We purhsed rit ntiody to PLK1 from Upstte. We rised rit ntiody to Auror B ginst Auror B peptides (MAQKENSYPWPYGRQTAC nd AHPWVRANSRRVLPPSAKKC) nd rit ntiody to Chfr ginst the Ring domin of Chfr (residues ). We purhsed mouse ntiody to FLAG (M2) from Sigm nd mouse ntiody to His6 onjugted with peroxidse from Rohe. Genertion of Chfr nd Auror A mutnts, ell trnsfetion, immunopreipittion nd immunolotting. We generted ll Chfr nd Auror A mutnts using the Quikhnge site-direted mutgenesis kit (Strtgene). We rried out ell trnsfetion, preprtion of ell lystes, immunopreipittion nd immunolotting in ordne with stndrd protools. For oimmunopreipittion, we treted ells with MG132 (15 mm) for 1 h efore ell lysis. For the in vivo uiquitintion ssy, we treted trnsfeted ells with MG132 (15 mm) for 3 h nd used iotin-onjugted ntiody to hemgglutinin to detet uiquitinted Auror A. For demethyltion of the Chfr promoter in HCT116 ells, we inuted ells with 5 mm 5-z-2 -deoxyytidine for 8 h efore ell lysis. Tretment of neworn mie with DMBA. We pplied 5 ml of.5%dmba (Sigm) in etone to the dorsl skin on postntl dy 3 nd killed mie 4 months lter. Skin polyps (45 mm) were ounted, exised nd ssessed y stining with hemtoxylin nd eosin. Genertion nd ulture of MEFs, kryotype nlysis, flow ytometry nlysis, immunofluoresene stining nd time-lpse mirosopi nlysis. We isolted MEFs from emryos t emryoni dy 13.5 nd mintined them in Duleo s modified Egle medium with 1% fetl ovine serum. To prepre metphse spreds, we treted MEFs (t P4) with 5 ng ml 1 olemid (GIBCO BRL) for 6 h t 37 1C. We olleted ells, wshed them with phosphte-uffered sline nd suspended them in 75 mm KCl t room temperture for 15 min. We fixed ells in Crnoy s solution (75% methnol, 25% eti id), nd dropped 15-ml liquots onto slides nd stined them with 5% Giems solution. We oserved metphse spreds under light mirosopy nd determined hromosome numers. For fluoresene-tivted ell-sorting nlysis, we olleted MEFs nd stined them with propidium iodide. We determined ell yle profiles using Cell-Quest. To detet multinuleted ells, we fixed MEFs with 3% prformldehyde nd stined them with fluoresein isothioynte onjugted rit ntiody to -tuulin (SIGMA) nd DAPI. For time-lpse mirosopi nlysis, we infeted MEFs with retrovirus enoding histone H2B- EGFP twie. We exmined mitoti ells every 2.5 min. shrna infetion. Lentivirl shrna expression vetor pll3.7 ws gift from L. Vn Prijs (Msshusetts Institute of Tehnology). The trgeting sequenes of Auror A re GACCACTGTTCCCTTCGGT, GCAGGCATCCTTGCAGAAG nd GTTTGGAAATGTCTACTTG; the trgeting sequene of PLK1 is GAAAT TCTGGAGGTCCTAG. We inserted shrna sequenes into pll3.7 etween HpInd XhoI. We rried out lentivirus pkging nd infetion in ordne with instrutions from Invitrogen (Lentivirl support kit K497-). Beuse pll3.7 lso enodes EGFP, positively infeted ells n e oserved under fluoresene mirosopy. Infetion effiieny is normlly more thn 8 9%. Preprtion of tissue lystes. We olleted liver (1 mg) nd spleen (1 mg) from 12-week-old wild-type nd Chfr / mie. We mined tissues etween two mirosope slides. We olleted ells nd lysed them with NETN uffer (.5% Nonidet-P4, 2 mm EDTA, 5 mm Tris-HCl (ph 8.) nd 1 mm NCl). In vitro trnsltion nd in vitro uiquitintion ssy. Full-length Auror A ws trnsried nd trnslted using the TNT quik oupled trnsription/ trnsltion system (Promeg). We rried out the in vitro uiquitintion ssy s previously desried 16. Note: Supplementry informtion is ville on the Nture Genetis wesite. ACKNOWLEDGMENTS We thnk D. Lim, D. Hines, E. Nigg, S. Sen, T. Tkhshi, J. Cheng, R. Ber, L. Vn Prijs, J. Chien nd K. Minn for regents nd help; J. Woods for proofreding the mnusript; nd S. Kufmnn nd W. Ernshw for suggestions. This work is supported in prt y the Myo Clini Cner Center nd the Brest Cner Reserh Foundtion. J.C. is reipient of the Deprtment of Defense rest ner reer development wrd. COMPETING INTERESTS STATEMENT The uthors delre tht they hve no ompeting finnil interests. Reeived 9 Novemer 24; epted 16 Ferury 25 Pulished online t 1. Crmen, M. & Ernshw, W.C. The ellulr geogrphy of uror kinses. Nt. Rev. Mol. Cell. Biol. 4, (23). 2. Ktym, H., Brinkley, W.R. & Sen, S. 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6 25 Nture Pulishing Group Bothos, J., Summers, M.K., Venere, M., Solnik, D.M. & Hlzonetis, T.D. The Chfr mitoti hekpoint protein funtions with U13-Mms2 to form Lys63-linked polyuiquitin hins. Onogene 22, (23). 19. Erson, A.E. & Petty, E.M. CHFR-ssoited erly G2/M hekpoint defets in rest ner ells. Mol. Crinog. 39, (24). 2. Mizuno, K. et l. Aerrnt hypermethyltion of the CHFR prophse hekpoint gene in humn lung ners. Onogene 21, (22). 21. Shit, Y. et l. Chfr expression is downregulted y CpG islnd hypermethyltion in esophgel ner. Crinogenesis 23, (22). 22. Corn, P.G. et l. Frequent hypermethyltion of the 5 CpG islnd of the mitoti stress hekpoint gene Chfr in oloretl nd non-smll ell lung ner. Crinogenesis 24, (23). 23. Toyot, M. et l. Epigeneti intivtion of CHFR in humn tumors. Pro. Ntl. Ad. Si. USA 1, (23). 24. Mritos, G. et l. Intivting muttions trgeting the hfr mitoti hekpoint gene inhumnlungner.cner Res. 63, (23). 25. Stoh, A. et l. Epigeneti intivtion of CHFR nd sensitivity to mirotuule inhiitors in gstri ner. Cner Res. 63, (23). 26. Ewrt-Tolnd, A. et l. Identifition of Stk6/STK15 s ndidte lowpenetrne tumor-suseptiility gene in mouse nd humn. Nt. Genet. 34, (23). 27. Sen, S., Zhou, H. & White, R.A. A puttive serine/threonine kinse enoding gene BTAK on hromosome 2q13 is mplified nd overexpressed in humn rest ner ell lines. Onogene 14, (1997). 28. Tnner, M.M. et l. Frequent mplifition of hromosoml region 2q12-q13 in ovrin ner. Clin. Cner Res. 6, (2). 29. Merldi, P., Hond, R. & Nigg, E.A. Auror A overexpression revels tetrploidiztion s mjor route to entrosome mplifition in p53 / ells. EMBO J. 21, (22). 3. Annd, S., Penrhyn-Lowe, S. & Venkitrmn, A.R. AURORA A mplifition overrides the mitoti spindle ssemly hekpoint, induing resistne to Txol. Cner Cell 3, (23). 46 VOLUME 37 [ NUMBER 4 [ APRIL 25 NATURE GENETICS