SUPPLEMENTARY INFORMATION

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1 Supplementary table 1 S.pombe strains used in this study Fig. 1a to c PM158 h 90 leu1 ade6-m216 cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r PL240 h 90 leu1 ade6-m216 cdc13-m7<<hyg r cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r PL368 h - leu1 ade6-m216 cdc Z::P adh15 -mcherry-atb2 + <<nat r 1d PM374 h + leu1 cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r PM375 h + leu1 bir1-8a cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r PL240 h 90 leu1 ade6-m216 cdc13-m7<<hyg r cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r 1e, g JY902 h + leu1 nda3-km311 1f PM255 h 90 leu1 ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r prep1(mad2 + ) 1h SH211 h + leu1 ade6-m216 nda3-km311 plo1-gfp<<kan r PL286 h + leu1 ade6 nda3-km311 bir1-8a plo1-gfp<<kan r 1i SH211 h + leu1 ade6-m216 nda3-km311 plo1-gfp<<kan r PL286 h + leu1 ade6 nda3-km311 bir1-8a plo1-gfp<<kan r PL287 h - leu1 nda3-km311 cdc13-m7<<hyg r plo1-gfp<<kan r 1j, k PM158 h 90 leu1 ade6-m216 cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r PM375 h + leu1 bir1-8a cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r PL278 h + leu1 bir1-8d cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r PL240 h 90 leu1 ade6-m216 cdc13-m7<<hyg r cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r PL281 h 90 leu1 cdc13-m7<<hyg r bir1-8a cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r PL241 h 90 leu1 ade6 cdc13-m7<<hyg r bir1-8d cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r Fig. 2a PM243 h 90 leu1 ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<hyg r PM293 h 90 leu1 bir1-8a ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r PM369 h + leu1 bir1-8d ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r 2b PM371 h 90 leu1 bir1-8a ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r prep81(gar1 + -CFP) PL370 h 90 leu1 ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<hyg r prep81(gar1 + -CFP) 2c, d PM359 h + leu1 ade6-m216 bir1-8a lys1::p adh81 -CFP-2CD<<hyg r PM360 h + leu1 ade6-m216 bir1-8a lys1::p adh81 -bir1 + -CFP-2CD<<hyg r PM361 h + leu1 ade6-m216 bir1-8a lys1::p adh81 -bir1 + -3pk<<hyg r PM362 h + leu1 ade6-m216 bir1-8a lys1::p adh81 -bir1-8a-cfp-2cd<<hyg r PM363 h + leu1 ade6-m216 bir1-8a lys1::p adh81 -bir1-8a-3pk<<hyg r PM158 h 90 leu1 ade6-m216 cen2-gfp Z::P adh15 -mcherry-atb2 + -nat r PM375 h + leu1 bir1-8a cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r PL283 h + leu1 bir1-8a cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r lys1::p adh81 -CFP-2CD<<hyg r PL284 h + leu1 bir1-8a cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r lys1::p adh81 -bir1 + -CFP-2CD<<hyg r PL285 h + leu1 bir1-8a cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r lys1::p adh81 -bir1-8a-cfp-2cd<<hyg r 2i PM255 h 90 leu1 ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r prep1(mad2 + ) PL236 h 90 leu1 sgo2-13myc<<kan r ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r prep1(mad2 + ) PL237 h 90 leu1 bir1-8a sgo2-13myc<<kan r ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r prep1(mad2 + ) Fig. S1 PM158 h 90 leu1 ade6-m216 cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r 1

2 PL240 h 90 leu1 ade6-m216 cdc13-m7<<hyg r cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r PM65 h - leu1 ade6 ark1-t7<<kan r Z::P adh15 -mcherry-atb2 + <<nat r Fig. S2a JY902 h + leu1 nda3-km311 S2b JY902 h + leu1 nda3-km311 PM367 h + ade6 leu1 nda3-km311 bir1-8a Fig. S3 PL324 h - cdc PL323 h - cdc bir1-8a PL325 h - cdc bir1-8d JY334 h + leu1 ade6-m216 PL319 h - cdc25-22 cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r PL320 h - cdc25-22 cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r bir1-8a PL321 h - cdc25-22 cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r bir1-8d Fig. S4 PL367 h 90 leu1 ade6-m216 ura4 ura4 + <<GFP-bir1 + Z::P adh15 -mcherry-atb2 + <<nat r prep81(gar1 + -CFP) PL366 h + leu1 ade6-m216 ura4 ura4 + <<GFP-bir1-8A Z::P adh15 -mcherry-atb2 + <<nat r prep81(gar1 + -CFP) Fig. S5 PL335 h + ade6 leu1 nda3-km311 ark1-gfp<<kan r PL336 h - ade6 leu1 nda3-km311 ark1-gfp<<kan r bir1-8a Fig. S6a SH211 h + leu1 ade6-m216 nda3-km311 plo1-gfp<<kan r PL287 h - leu1 nda3-km311 plo1-gfp<<kan r cdc13-m7<<hyg r PL373 h - leu1 nda3-km311 plo1-gfp<<kan r cdc13-m7<<hyg r bir1-8d S6b PM243 h 90 leu1 ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<hyg r PL231 h - ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r cdc13-m7<<hyg r PL232 h - ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r cdc13-m7<<hyg r bir1-8d S7 JY902 h + leu1 nda3-km311 PL335 h + ade6 leu1 nda3-km311 ark1-gfp<<kan r PL336 h - ade6 leu1 nda3-km311 ark1-gfp<<kan r bir1-8a Fig. S9 PM243 h 90 leu1 ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<hyg r PL374 h + leu1 sgo2-δcoiledcoil ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + -nat r PP336 h 90 ade6 leu1 ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<hyg r sgo2::kan r PM293 h 90 leu1 bir1-8a ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r Fig. S10a PM243 h 90 leu1 ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<hyg r PP198 h - leu1 ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<hyg r kan r <<bir1-5a PM293 h 90 leu1 bir1-8a ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r S10b PP117 h - sgo2-gfp<<kan r kan r <<bir1 + PP119 h - sgo2-gfp<<kan r kan r <<bir1-5a PP153 h - sgo2-gfp<<kan r kan r <<bir1-5d PM251 h - ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r PM252 h - kan r <<bir1-5a ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r PM284 h - bir1-8a ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r Fig. S13 JY902 h + leu1 nda3-km311 Fig. S17a PP125 h - sgo2-gfp<<kan r Z::P adh15 -CFP-atb2 + <<nat r PM392 h - bir1-8a sgo2-gfp<<kan r Z::P adh15 -CFP-atb2 + <<nat r PM393 h - bir1-8d sgo2-gfp<<kan r Z::P adh15 -CFP-atb2 + <<nat r 2

3 S17b PL304 h + leu1 nda3-km311 sgo2-gfp<<kan r PL305 h + leu1 nda3-km311 sgo2-gfp<<kan r bir1-8a Fig. S18a PM243 h 90 leu1 ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<hyg r PP337 h 90 ade6 leu1 ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<hyg r swi6::kan r PM293 h 90 leu1 bir1-8a ark1-gfp<<kan r Z::P adh15 -mcherry-atb2 + <<nat r S18b PY653 h - swi6::kan r PM357 h + leu1 ade6-m216 bir1-8a S18c PM158 h 90 leu1 ade6-m216 cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r PM160 h 90 leu1 ade6-m216 cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r sgo2::kan r PL220 h 90 leu1 cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r bir1-8a PY615 h 90 leu1 ade6-m210 ura4d-18 swi6::ura4 + PL221 h 90 leu1 cen2-gfp Z::P adh15 -mcherry-atb2 + <<nat r bir1-8a sgo2::kan r PZ917 h 90 leu1 ade6-m210 swi6::kan r sgo2::ura4 + 3

4 a 26 C 28 C cdc13-m7 ark1-t7 TBZ 6 µg/ml b Cell length at division (µm) cdc13 -M7 28 C ark1 -T7 c Chromosome segregation (%) 7 28 C cdc13 -M7 ark1 -T7 lagging mis Supplementary Figure 1. The cdc13-m7 mutant shows a very similar phenotype to that of the ark1-t7 temperature-sensitive mutant. a, Serial dilutions of the indicated strains were spotted onto YE plates containing 0 or 6 µg/ml TBZ and incubated at 26 C. YE plate incubation at 28 C is also shown. b, Cultures of the indicated strains were shifted from 25 C to 28 C for 6 hr and then fixed. Lengths of septated cells were measured by Image J software (n > 50 cells). c, Cells in b were stained for DNA. The spindles were visualized by expressing mcherry-atb2. Frequencies of lagging chromosomes and mis-segregation at anaphase cells (n > 100 cells) were examined. Error bars represent s.d. (n = 3 experiments). 4

5 a Bir1-pS244 Bir1 wt His-Bir1 8A ATP IP b wt bir1-8a Bir1- ps244 G2 M M Bir1 Supplementary Figure 2. Bir1-S244 is phosphorylated in vivo in M-phase. a, Preparation of phosopho-specific antibodies against Bir1-pS244. Recombinant His-Bir1-WT and -8A proteins were incubated with Suc1-bound Cdc2 in the absence or presence of ATP, and analyzed by immunoblot using anti-bir1-ps244 or anti-bir1 antibodies. b, Immunoprecipitated Bir1 proteins from extracts of wild-type cells (asynchronous and prometaphase) and bir1-8a cells (prometaphase) were similarly analyzed. 5

6 cdc cdc bir1-8a cdc bir1-8d TBZ 6µg/ml TBZ 8µg/ml cdc25-22 cdc25-22 bir1-8a cdc25-22 bir1-8d Supplementary Figure 3. The bir1-8d mutation does not suppress the sensitivities of cdc or cdc25-22 to TBZ. Serial dilutions of the indicated strains were spotted onto YE plate or YE plates containing TBZ and incubated at 30 C. 6

7 Interphase Metaphase Anaphase bir1-8a wt Tubulin Bir1Nucleolus Supplementary Figure 4. Bir1-8A fails to localize at centromeres. GFP-Bir1 was detected at interphase, metaphase and anaphase in wild-type and bir1-8a cells expressing mcherry-atb2 and Gar1-CFP (nucleolus marker). Scale bar, 2µm. 7

8 ChIP (%) 0.08 Ark1-GFP ChIP (%) 0.6 Bir mes1 56F2 mes1 56F2 mes1 56F2 mes1 56F2 cen arm cen arm bir1-8a cen arm cen arm bir1-8a ChIP (%) 16 H3 4 H3S10P/H mes1 56F2 mes1 56F2 mes1 56F2 mes1 56F2 cen arm cen arm bir1-8a cen arm cen arm bir1-8a Supplementary Figure 5. Chromatin Immunoprecipitation (ChIP) assay shows the reduced centromeric localization of Ark1, Bir1 and histone H3-S10 phosphorylation in bir1-8a cells. Wild type and bir1-8a cells expressing Ark1-GFP were arrested at prometaphase by nda3-km311 inactivation and examined by ChIP assay using anti-gfp, anti-bir1, anti-h3 and anti-h3-ps10 antibodies. Error bars represent s.d. (n = 2 PCR experiments). 8

9 a 0.16 Bir1 ChIP (%) b 0.12 Metaphase Ark1 Tubulin cen arm cen arm cdc13-m7 H3 ChIP (%) cen arm cdc13-m7 bir1-8d cdc13-m7 bir1 + cdc13-m7 bir1-8d Signal intensity (metaphase) cen arm cen arm cdc13-m7 cen arm cdc13-m7 bir1-8d Supplementary Figure 6. Centromeric localization of Bir1 in cdc13-m7 cells. a, The indicated cells were arrested at prometaphase by nda3-km311 inactivation and examined by ChIP assay using anti-bir1 and anti- H3 antibodies. Error bars represent s.d. (n = 2 PCR experiments). Note that centromeric localization of Bir1 is impaired, while that of Bir1-8D is intact, in cdc13-m7 cells. b, Ark1-GFP was detected at metaphase in the indicated cells expressing mcherry-atb2 and measured for signal intensities. Error bars indicate s.e.m. (n > 25 cells). Scale bar, 2µm. 9

10 WCE α-gfp IP bir1 : 8A 8A ark1-gfp : Ark1-GFP Bir1 Cdc2 Supplementary Figure 7. Bir1-8A forms a intact complex with Ark1 in vivo. Co-immunoprecipitation of Bir1 and Ark1-GFP. Whole cell extracts (WCE) were prepared from mitotic S. pombe cells expressing Ark1-GFP. Ark1-GFP was immunoprecipitated (IP) with anti-gfp antibodies to examine the co-precipitation of Bir1. 10

11 a 1 BIR Bir Bir1-N 997 CC b Bait (Left)p53 Nbl1 Bir1 Pic1 Ark1 Ark1 Pic1 Prey Supplementary Figure 8. Control experiment for Fig. 2e. a, Schematic diagrams of fission yeast Bir1 and Bir1-N. Tandemly positioned BIR domains (blue), C terminus coiled coil region (CC; gray) and eight CDK consensus residues (arrowhead) are highlighted. b, The bait used for two-hybrid assays in Fig. 2e gives an interaction with the positive control, verifying the significance of the assays. 11

12 a wt sgo2-δcc sgo2 TBZ 10 µg/ml b sgo2- cc wt metaphase Ark1 signal intensity (metaphase) bir1-8a sgo2 Ark1-GFP Tubulin 2 wt sgo2 - cc sgo2 Supplementary Figure 9. Cells expressing Sgo2- cc, which lacks coiled-coil region (10-63 a.a.), show TBZ sensitivity and defects in CPC targeting to centromeres. a, Serial dilutions of the indicated strains were spotted onto YE plates containing 10 µg/ml TBZ and incubated at 28 C. b, Ark1-GFP was detected at metaphase in the indicated cells expressing mcherry- Atb2 and measured for signal intensities. Error bars indicate s.e.m. (n > 25 cells). Scale bar, 2µm. 12

13 a metaphase wt Ark1-GFPTubulin Ark1-GFP b wt TBZ 12.5 µg/ml bir1-5a bir1-5a bir1-5d 120 Signal intensity (metaphase) TBZ 10 µg/ml wt bir1-5a bir1-8a 20 0 bir1-5a bir1-8a Supplementary Figure 10. As bir1-8a cells, bir1-5a cells shows defects in CPC targeting to centromeres and TBZ sensitivity. a, Ark1-GFP was detected at metaphase in the indicated cells expressing mcherry-atb2 and measured for signal intensities. Error bars indicate s.e.m. (n > 25 cells). Scale bar, 2µm. b, Serial dilutions of the indicated strains were spotted onto YE plates containing 10 or 12.5 µg/ml TBZ and incubated at 28 C. 13

14 a haurora B ACA DNA haurora B hsgo1 c Hec1 ACADNA Hec1 ACA hsgo1/2 hsgo1 RNAi Control haurora B ACA DNA haurora B hsgo2 Control hsgo1/2 RNAi Hec1/ACA Control hsgo2 RNAi Control haurora B ACA DNA haurora B hbub1 Control hsgo1/2 RNAi CENP-A ACADNA CENP-A ACA hsgo1/2 CENP-A/ACA hbub1 RNAi b hsgo1 hsgo2 hborealin control WCE hsgo1/2 RNAi IgG IP control hborealin hsgo1/2 RNAi hborealin Control hsgo1/2 RNAi pcenp-a ACADNA pcenp-a ACA haurora B pcenp-a/aca haurora B/ACA hincenp haurora B hsurvivin GAPDH Supplementary Figure 11. Analysis of sirna treated HeLa cells. a, haurora B signals at prometaphase were examined by immunostaining control and hsgo1, hsgo2 and hbub1 RNAi cells as in Fig. 3a. The ratio of centromere and arm haurora B signals was quantified and is shown in Fig. 3a. b, Whole cell extracts (WCE) of nocodazole-arrested HeLa cells treated with control or Sgo1/2 sirna were immunoprecipitated (IP) with anti-hborealin antibodies and analyzed by immunoblot. c, Centromeric signals of Hec1, CENP-A, Ser7 phosphorylation of CENP-A (pcenp-a), haurora B and ACA at prometaphase were examined by immunostaining control and hsgo1/2 RNAi cells. Relative fluorescent intensities of centromeric signals toward ACA were quantified at 10 centromeres in each cell. The average value over 5 cells is shown. Error bars represent s.e.m. (n = 5 cells). Scale bars, 5 µm. Note that hsgo1/hsgo2 knockdown preserves the intact localization of CENP-A as well as Hec1/ Ndc80, an outer kinetochore protein responsible for kinetochore-microtubule attachment, implying that delocalization of CPC in hsgo1/hsgo2 knockdown is not an indirect consequence of kinetochore/centromere disruption and that the phosphorylation of a centromeric substrate of haurora B is impaired under these conditions such that the CPC is insufficiently targeted to centromeres. 14

15 a CC Bait SGO hborealin Prey T CC Bait SGO hborealin Prey hsgo1 hsgo2 T p53 p53 b hsurvivin Prey (Left) T hborealin hborealin hincenp haurora B hsurvivin haurora B hincenp Bait Supplementary Figure 12. Two-hybrid assays. a, Two-hybrid assay indicates that the coiled-coil domains (CC) of both hsgo1 and hsgo2 interact with hborealin. b, The prey used for two-hybrid assays in Fig. 3b gives an interaction with the positive control, verifying the significance of the assays. 15

16 a hborealin b H1 GST-hBorealin T106 CC CBB 32 P Supplementary Figure 13. In vitro Phosphorylation of hborealin by Cdc2. a, Putative CDK phosphorylation sites in human Borealin. Full consensus CDK site T106 (black arrowheads) and its related sites (S/TP, grey arrowheads) are shown. b, Wild-type GST-hBorealin or its mutant versions were phosphorylated in vitro using Suc1-bound Cdc2 purified from S. pombe extracts, indicating that not only T106, but also one or more of six other related sites, are phosphorylated by Cdc2. 16

17 a (Chromatin) hborealin haurora B hsgo1 hsgo2 Actin WCE IgG IP hborealin : Roscovitine (Cytoplasm +Chromatin) Securin Cyclin B haurora B b ACA DNA haurora B Cells with defective (%) haurora B localization abolished diffused Roscovitine : (min) - + normal diffused abolished Supplementary Figure 14. Roscovitine treatment of prometaphase cells disrupts the complex formation between hborealin and shugoshin as well as centromeric haurora B localization. HeLa cells were synchronized by single thymidine block and release, and then arrested at prometaphase by adding nocodazole. Prometaphase cells were further treated with nocodazole and MG-132 in the presence or absence of 100 µm Roscovitine. After 40 min treatment, the cells were analyzed by immunoprecipitation (a), while cells treated for 0, 40, 80 min were analyzed for immunostaining (b). a. After Roscovitine treatment, the cells were cross-linked with dithiobis(succinimidylpropionate) (DSP). Immunoprecipitates (IPs) were obtained form chromatin extracts using control and anti-hborealin antibodies and analyzed by immunoblot using the indicated antibodies. Securin and cyclin B were also analyzed to demonstrate that the cell cycle remained intact prior to anaphase onset. Note that hsgo1 and hsgo2 co-precipitated with hborealin only in the absence of Roscovitine, while haurora B co-precipitated regardless of the presence or absence of Roscovitine. b, After Roscovitine treatment, the cells were stained with anti-cyclin B, anti-haurora B and anti-aca antibodies. the number of cells (%) showing diffused or abolished haurora B signals were counted. Error bars represent s.d. (n = 3 experiments) In this time frame, almost all cells retained full cyclin B signals (not shown), while haurora B signals were largely diffused or abolished. Scale bar, 5 µm. 17

18 a hborealin b ACA DNA GFP-hBorealin GFP-hBorealin c WCE GFP-hBorealin : - 7A IP GFP - 7A hsgo1 GFP-hBorealin hsurvivin Tubulin 7A haurora B GAPDH d hborealin-wt pcenp-a DNAACA pcenp-a ACA GFP-hBorealin 0.8 pcenp-a/ ACA 1.2 GFPhBorealin/ ACA hborealin-7a A 7A Supplementary Figure 15. Characterization of hborealin-7a cells. a, Immunoblot of hborealin in RNAi cells confirming the RNAi efficiency. b, hborealin RNAi cells expressing GFP-hBorealin-WT and -7A were synchronized by single thymidine block and release, and the subsequent addition of MG-132. Centromeric localization of hborealin was examined after treatment with the pre-extraction buffer (1 mm CaCl 2, 80 mm PIPES [ph6.1], 1 mm EGTA, 1 mm MgCl 2, 0.5% Triton X-100) for 15 min. Scale bars, 5 µm. c, Chromatin extracts were prepared from nocodazole-arrested control and hborealin RNAi cells, which express no GFP-hBorealin-WT or-7a, immunoprecipitated with anti-gfp antibody and analyzed by immunoblot. Note that CPC complex formation is intact in the hborealin -7A mutant protein. d, the phosphorylation of CENP-A was examined in hborealin RNAi cells expressing GFP-hBorealin-WT and -7A by immunostaining. Relative fluorescent intensities of centromeric signals of pcenp-a and GFP-hBorealin toward ACA were quantified. Error bars represent s.e.m. (n = 5 cells). Scale bars, 5 µm. These data indicate that the hborealin-7a protein hampers the CPC targeting to centromeres as well as phosphorylating centromeric substrate of haurora B without affecting complex formation of the CPC. 18

19 Fungi Metazoan Neurospora crassa Schizosaccharomyces pombe human Survivin Borealin Aspergillus nidulans Saccharomyces cerevisiae Drosophila Survivin Borealin (CDK consensus motif) strict consensus (S/T-P-X-K/R) consensus (S/T-P) BIR domain of Survivin C-terminal coiled-coil of Survivin N-terminal coiled-coil of Borealin Supplementary Figure 16. Distribution of putative CDK phosphorylation sites in Survivin and Borealin differs among organisms. Metazoans carry the putative phosphorylation cluster only in Borealin. In contrast, a majority of fungi have the cluster in both Survivin and Borealin, while fission yeast and budding yeast might have lost it from Borealin. Notably, this truncation of Borealin in these yeasts occurred independently during evolution, raising the possibility that the precise function of these subunits is not always conserved between two yeasts in the context of their different centromere structures 5. So far the dependency of CPC targeting on shugoshin is under debate in budding yeast 6,7, although the functional link between the CPC and shugoshin seems conserved 1, 8. 19

20 b a (Metaphase) ChIP (%) 0.05 Sgo2-GFP wt bir1-8a bir1-8d wt bir1-8a bir1-8d TubulinSgo2 Sgo2 (Anaphase) Signal intensity (A.U.) at centromeres wt 8A 8D Signal intensity (A.U.)at spindle midzone wt 8A 8D mes1 H3 mes1 cen arm cen arm bir1-8a Supplementary Figure 17. Sgo2 localization is influenced by Bir1-8A or Bir1-8D. a, Sgo2-GFP was detected at metaphase and anaphase in wild-type, bir1-8a and bir1-8d cells expressing CFP-Atb2 (tubulin). The Sgo2-GFP signals were measured at metaphase and anaphase. Error bars indicate s.e.m. (n > 25 cells). Scale bar, 2µm. b, Wild-type and bir1-8a cells expressing Sgo2-GFP were arrested at prometaphase by nda3-km311 inactivation and examined for ChIP assay using anti-gfp and anti-h3 antibodies. Error bars represent s.d. (n = 2 PCR experiments). Because Bir1 is important for the centromeric localization of Sgo2 (ref1), we examined the localization of Sgo2 in bir1-8a or -8D mutants. In wild-type cells, Sgo2-GFP locates as a dot on the spindle in metaphase, and relocates to the nucleus at anaphase. In bir1-8a cells, Sgo2 localization is defective in metaphase but intact in anaphase. In bir1-8d cells, however, Sgo2 abnormally localizes to the spindle midzone in anaphase, while its metaphase localization is intact. This may be because of the ectopically retained interaction between Sgo2 and Bir1-8D in anaphase. These results further suggest that the dephosphorylation of Bir1/Survivin is not always required for the release from centromeres or targeting to the spindle midzone at anaphase. In this regard, it is noteworthy that in budding yeast dephosphorylation of INCENP is sufficient for this anaphase relocalization 2. Thus, phosphorylation acting on CPC targeting at prophase and relocalization at anaphase may differ in character, although both phosphorylation may be relevant to Cdk1-cyclin B (also see ref 3, 4). 20

21 a wt Ark1-GFP Tubulin Ark1 signal intensity (metaphase) b sgo2 bir1-8a swi6 6 4 swi6 bir1-8a sgo2 bir1-8a 2 sgo2 swi6 bir1-8a swi6 Synthetic lethal c Tetrad dissection (swi6 x bir1-8a) d Swi6 CPC Bir1 P Sgo2 CDK +/+ s/+ +/b +/+ +/+ +/b s/+ s/b s/b s/b s/+ +/b s/+ s/b s/b s/b +/b +/+ +/+ +/+ Centromere targeting (Bi-orientation) Supplementary Figure 18. Redundancy in centromere CPC targeting. a, Ark1-GFP was detected at metaphase in the indicated cells expressing mcherry-atb2 and measured for signal intensities. Error bars indicate s.e.m. (n > 25 cells). Scale bar, 2µm. b, Serial dilutions of the indicated strains were spotted onto a YE plate and incubated at 30 C. c, Spores from bir1-8a (b) and swi6δ (s) cross were tetrad dissected on a YE plate and incubated at at 30 C. The genotype of each segregant as judged by PCR is shown at the bottom. d, A schematic model illustrating the redundancy of the mechanism of CPC targeting to centromeres in fission yeast. Although centromeric CPC plays an essential role in chromosome bi-orientation 9, sgo2δ or bir1-8a cells are viable, suggesting that a redundant pathway for CPC centromere targeting exists. Indeed, we previously demonstrated that the heterochromatin protein Swi6 (S. pombe HP1 homolog) contributes somewhat to targeting the CPC to centromeres 1 (also see Sup. Fig. 18a), in a redundant capacity with Sgo2; sgo2δ and swi6δ produce synergetic defects in bi-orientation as well as mitotic growth 1 (also see Sup. Fig. 18b). If the phosphorylation of Bir1 acts in the Sgo2 pathway, then bir1-8a and swi6δ should show synergetic growth defects. Indeed, swi6δ and bir1-8a show synthetic lethality (Sup. Fig. 18c), while the sgo2δ bir1-8a double mutant is viable (Sup. Fig. 18b). Thus, heterochromatin assembly contributes to the viability of bir1-8a cells, presumably by supporting residual CPC localization at centromeres (Sup Fig. 18d). 21

22 Supplementary references: 1. Kawashima, S. A. et al., Shugoshin enables tension-generating attachment of kinetochores by loading Aurora to centromeres. Genes Dev 21 (4), 420 (2007). 2. Pereira, G. and Schiebel, E., Separase regulates INCENP-Aurora B anaphase spindle function through Cdc14. Science 302 (5653), 2120 (2003). 3. Parry, D. H., Hickson, G. R., and O Farrell, P. H., Cyclin B destruction triggers changes in kinetochore behavior essential for successful anaphase. Curr Biol 13 (8), 647 (2003). 4. Oliveira, R. A. et al., Cohesin cleavage and Cdk inhibition trigger formation of daughter nuclei. Nat Cell Biol 12 (2), 185 (2010). 5. Nakajima, Y. et al., Nbl1p: a Borealin/Dasra/CSC-1-like protein essential for Aurora/ Ipl1 complex function and integrity in Saccharomyces cerevisiae. Mol Biol Cell 20 (6), 1772 (2009). 6. Yu, H. G. and Koshland, D., The Aurora kinase Ipl1 maintains the centromeric localization of PP2A to protect cohesin during meiosis. J Cell Biol 176 (7), 911 (2007). 7. Kiburz, B. M., Amon, A., and Marston, A. L., Shugoshin promotes sister kinetochore biorientation in Saccharomyces cerevisiae. Mol Biol Cell 19 (3), 1199 (2008). 8. Indjeian, V. B., Stern, B. M., and Murray, A. W., The centromeric protein Sgo1 is required to sense lack of tension on mitotic chromosomes. Science 307 (5706), 130 (2005). 9. Hauf, S. et al., Aurora controls sister kinetochore mono-orientation and homolog biorientation in meiosis-i. EMBO J 26 (21), 4475 (2007). 22