Volume 27, Issue 7, Pages 992-1004 (April 2017) The N-Terminal Non-Kinase-Domain-Mediated Binding of Haspin to Pds5B Protects Centromeric Cohesion in Mitosis  Linli Zhou, Cai Liang, Qinfu Chen, Zhenlei Zhang, Bo Zhang, Haiyan Yan, Feifei Qi, Miao Zhang, Qi Yi, Youchen Guan, Xingfeng Xiang, Xiaoqing Zhang, Sheng Ye, Fangwei Wang  Current Biology  Volume 27, Issue 7, Pages 992-1004 (April 2017) DOI: 10.1016/j.cub.2017.02.019 Copyright © 2017 Elsevier Ltd Terms and Conditions

Current Biology 2017 27, 992-1004DOI: (10.1016/j.cub.2017.02.019) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Defective Mitosis Progression in Haspin-KO Cells (A) HeLa and the indicated Haspin-KO clones were treated with nocodazole for 15 hr. Mitotic cells were immunoblotted. (B) HeLa and clone D2 cells were treated with S-Trityl-L-cysteine (STLC) for 5 hr. Mitotic chromosome spreads were immunostained. (C) The mitosis progression of HeLa and clone D2 cells stably expressing H2B-GFP were analyzed by time-lapse live imaging. The time from nuclear envelope breakdown (NEB) to metaphase chromosome alignment and from metaphase to anaphase onset was determined. (D) HeLa and clone D2 cells, either untreated or released from nocodazole treatment, were fixed and stained with anti-human centromere autoantibodies (ACAs) and DNA. The percentage of cells with lagging chromosomes was determined in 100 anaphase cells. (E and F) HeLa and clone D2 cells stably expressing H2B-GFP were released from 3 hr treatment with STLC, followed by live imaging of mitosis progression. Fate profiles of mitotic cells and the selected frames of the movies are shown. Time stated in hours: minutes. See Movies S1, S2, S3, and S4. (G) Clone D2 cells stably expressing H2B-GFP were treated as in (E) and then released from STLC into DMSO or the Mps1 inhibitor AZ3146. The time from STLC washout to mitotic exit was determined. (H) The indicated stable cell lines were released from 5 hr treatment with STLC, and then the mitosis progression was determined. Means and SDs are shown (C, E, and G). Scale bars, 10 μm. See also Figure S1. Current Biology 2017 27, 992-1004DOI: (10.1016/j.cub.2017.02.019) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Weakened Centromeric Cohesion in Haspin-KO Cells (A) HeLa and clone D2 cells were released from 3 hr treatment with STLC into MG132-containing medium and then fixed at the indicated time points for DNA staining. The percentage of mitotic cells with near standard metaphase plate (fewer than three misaligned chromosomes) was determined in around 300 cells (n = 3). (B) HeLa and Haspin-KO cells were treated with 3.3 μM nocodazole for 3 hr. Mitotic chromosome spreads were subjected to ACA and DNA staining. The number of separated chromatids per cell was determined in 50 cells. (C) HeLa and Haspin-KO cells were treated with STLC for 5 hr. The morphology of chromosome spreads in 20 cells was classified and quantified. Means and ranges are shown (n = 2). (D and E) HeLa and Haspin-KO clones were exposed to MG132 for 4 hr. Using mitotic chromosome spreads, the percentage of cells with at least 26 separated chromatids was determined in 100 cells (D). Example images are shown (E). (F) HeLa and Haspin-KO cells stably expressing H2B-GFP were exposed to MG132 and subjected to live imaging. The time from the achievement of metaphase chromosome alignment to chromosome scattering was determined. See Movies S5 and S6. (G) HeLa and clone D2 cells were exposed to MG132, and fixed at the indicated time points to stain DNA. The percentage of mitotic cells with near standard metaphase plate was determined in 300 cells (n = 3). (H) HeLa and clone D2 cells were treated as indicated. Mitotic chromosome spreads were stained with ACAs and DNA. The inter-KT distance was measured on over 500 chromosomes in 20 cells. (I and J) HeLa and clone D2 cells were exposed to 1.3 μM nocodazole for 3 hr. Mitotic cells were cytospun onto coverslips and fixed for immunostaining (I). Lysates of mitotic cells were immunoblotted (J). Means and SDs are shown (A and F–H). Scale bars, 10 μm. See also Figure S2. Current Biology 2017 27, 992-1004DOI: (10.1016/j.cub.2017.02.019) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Haspin Interacts with Pds5B through a Conserved YGA/R Motif and Impedes Wapl Binding to Pds5B (A) U2OS-LacO cells expressing EGFP-LacI-Pds5B-N or EGFP-LacI together with SFB-Haspin were immunostained with anti-Haspin antibodies. (B) Lysates of nocodazole-arrested mitotic HeLa cells transiently expressing Myc-Pds5B and SFB-Haspin were subjected to IP followed by immunoblotting. (C) MBP-Pds5B-N was subjected to pulldown by Haspin-N-GST followed by immunoblotting with anti-MBP antibodies and Coomassie brilliant blue (CBB) staining. (D) MBP-Pds5B-N was subjected to pulldown by Wapl-N28 and Haspin-N30 peptides immobilized on beads followed by immunoblotting. (E) Isothermal titration calorimetry (ITC) curves of the binding between MBP-Pds5B-N and Haspin-N30 peptide. Kd is shown (n = 3). (F) Alignment of Haspin N-terminal sequences surrounding the conserved YGA/R motif and the YSR motifs of human Wapl and Sororin. (G) MBP-Pds5B-N was subjected to pulldown by Haspin-N30 peptides immobilized on beads followed by immunoblotting. AE represents the Y16A/A18E mutation. (H) Lysates of nocodazole-arrested mitotic HeLa cells transiently expressing Myc-Pds5B and SFB-Haspin were subjected to IP followed by immunoblotting. (I) The model of Pds5B in complex with the Haspin peptide of RTYGA. Pds5B is shown in light-green ribbon, with the Haspin interacting residues in light-green stick representations. The bound Haspin peptide is shown in yellow stick representation. (J) U2OS-LacO cells transiently expressing SFB-Haspin-K511A and EGFP-LacI-Pds5B-N were immunostained as in (A). (K) U2OS-LacO cells transiently expressing EGFP-LacI-Pds5B-N and Myc-Wapl together with SFB-Haspin or a control vector were immunostained with anti-Myc and anti-Haspin antibodies (Figure S3G). The relative enrichment of Myc-Wapl was quantified. (L) U2OS-LacO cells transfected with the indicated plasmids were analyzed as described in (K) for the relative enrichment of SFB-Haspin-K511A. (M and N) Asynchronous HeLa cell lysates were subjected to pulldown with GST-Wapl-N in the absence or presence of the WT (M and N) or mutant (N) Haspin peptides followed by immunoblotting and CBB staining. (O) MBP-Pds5B-N pulldown by Haspin-N-GST as described in (C), with or without Wapl-N28 peptides. Means and SDs are shown (E, K, and L). Scale bar, 10 μm. See also Figure S3. Current Biology 2017 27, 992-1004DOI: (10.1016/j.cub.2017.02.019) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Loss of Haspin-Pds5B Interaction Weakens Mitotic Centromere Cohesion (A) HeLa and Haspin-Y16A/A18E mutant clones were exposed to nocodazole for 15 hr. Mitotic cells were immunoblotted. (B and C) HeLa and clone 4-16 cells were exposed to 0.3 μM nocodazole for 3 hr. Mitotic chromosome spreads were immunostained. Example images are shown (B). The relative intensity of centromeric H3pT3 was determined as the ratio of H3pT3/CENP-C on 200 chromosomes from 20 cells (C). (D) HeLa and Haspin mutant clones were treated with MG132 and analyzed for metaphase chromosome alignment as in Figure 2G (n = 3). (E) The indicated cell lines were exposed to MG132 for 4 hr or 8 hr, and then PCS was analyzed as in Figure 2D. (F and G) HeLa and clone 2A3 cells were exposed to 3.3 μM nocodazole for 3 hr. Mitotic chromosome spreads were immunostained (F). The inter-KT distance was determined as in Figure 2H (G). (H–J) HeLa and the indicated stable cell lines were subjected to immunoblotting (H) or treated with MG132 to analyze metaphase chromosome alignment (I) or the PCS (J). Means and ranges are shown (n = 2). (K and L) The indicated stable cell lines were treated and immunostained as in Figure 2I (K) and then analyzed for inter-KT distance as in Figure 2H (L). Means and SDs are shown (C, D, G, and L). Scale bars, 10 μm. See also Figure S4. Current Biology 2017 27, 992-1004DOI: (10.1016/j.cub.2017.02.019) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Centromere Targeting of Haspin N terminus Rescues Centromeric Cohesion Defects in Haspin-KO Cells (A) Lysates of Haspin-KO cells stably expressing the indicated CENP-B fusion proteins were immunoblotted. (B and C) The indicated cell lines were exposed to MG132 and analyzed for chromosome alignment (B) or the PCS (C). Means and ranges are shown (n = 2). (D and E) The indicated mitotic cells were immunostained (D) and analyzed for inter-KT distance as in Figures 4K and 4L (E). Means and SDs are shown. Scale bars, 10 μm. (F) The indicated cell lines released from 3 hr treatment with STLC were analyzed by live imaging as in Figure 1E. Fate profiles of cells are shown. (G–I) The indicated stable cell lines were exposed to MG132 and analyzed for chromosome alignment (G). Restoration of H3pT3 was confirmed by immunoblotting (H) and immunostaining (I) of mitotic cells. Means and ranges are shown (n = 2). See also Figure S5. Current Biology 2017 27, 992-1004DOI: (10.1016/j.cub.2017.02.019) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 6 Prevention of Wapl-mediated Cohesin Release Rescues Centromeric Cohesion Defects in Haspin Mutant Cells (A–C) Mitotic HeLa and Haspin-KO cells transfected with the indicated small interfering RNAs (siRNAs) were treated and immunostained as in Figure 2I (A), and inter-KT distance was measured as in Figure 2H (B). Cell lysates were immunoblotted (C). (D–F) Chromosome spreads prepared from mitotic clone D2 cells transfected with Myc-SA2 were immunostained (D), and inter-KT distance was measured as in Figures 4F and 4G (E). Cell lysates were immunoblotted (F). (G and H) Chromosome spreads prepared from the indicated mitotic cells transfected with control or Wapl siRNA were immunostained (G). Cell lysates were immunoblotted (H). Means and SDs are shown (B and E). Scale bars, 10 μm. See also Figure S6. Current Biology 2017 27, 992-1004DOI: (10.1016/j.cub.2017.02.019) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 7 Haspin Overexpression Suppresses Wapl Activity and Largely Bypasses Sgo1 for Cohesion Protection (A) HeLa and the indicated stable cell lines were treated with nocodazole, and then mitotic chromosome spreads were immunostained. (B) The indicated stable cell lines transfected with Myc-SA2 were treated with nocodazole, and then mitotic chromosome spreads were immunostained with anti-Myc and CENP-C antibodies. (C and D) The indicated cell lines were transfected with control or Sgo1 siRNA. After 26 hr, cells were either exposed to nocodazole for 14 hr to collect mitotic cells for immunoblotting (C) or fixed for DNA staining to quantify the mitotic index (D). (E and F) The indicated cell lines were transfected with control, Sgo1, or Sororin siRNA. After 26 hr, cells were analyzed by immunoblotting (E) and DNA staining (F) as in (C) and (D), respectively. (G and H) Nocodazole-arrested mitotic chromosome spreads prepared from cells treated as in (E) and (F) were immunostained. The percentage of cells with PCS was determined (G). Example images are shown (H). (D, F, and G) Means and ranges are shown (n = 2). Scale bars, 10 μm. (I) A schematic of Haspin-containing centromeric cohesin complex, in which Haspin binds Pds5B through a YGA/R motif to antagonize YSR-motif-dependent Wapl-Pds5B interaction. In the absence of Haspin, Sororin may bind to Pds5B (double-headed arrow). See also Figure S7. Current Biology 2017 27, 992-1004DOI: (10.1016/j.cub.2017.02.019) Copyright © 2017 Elsevier Ltd Terms and Conditions