Volume 42, Issue 2, Pages 181-189.e3 (July 2017) Association of M18BP1/KNL2 with CENP-A Nucleosome Is Essential for Centromere Formation in Non-mammalian Vertebrates  Tetsuya Hori, Wei-Hao Shang, Masatoshi Hara, Mariko Ariyoshi, Yasuhiro Arimura, Risa Fujita, Hitoshi Kurumizaka, Tatsuo Fukagawa  Developmental Cell  Volume 42, Issue 2, Pages 181-189.e3 (July 2017) DOI: 10.1016/j.devcel.2017.06.019 Copyright © 2017 Elsevier Inc. Terms and Conditions

Developmental Cell 2017 42, 181-189. e3DOI: (10. 1016/j. devcel. 2017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Localization of the Mis18 Complex throughout the Cell Cycle in Chicken DT40 Cells (A) Immunostaining with anti-CENP-T antibody (red) in chicken DT40 cells expressing GFP-tagged chicken M18BP1/KNL2 (green). Chromosomes and nuclei were stained with DAPI (blue). Cells at various cell-cycle stages are shown. Scale bar, 10 μm. See also Figure S1. (B) Immunostaining with anti-Mis18α (green) and anti-FLAG (red) antibodies in chicken DT40 cells expressing CENP-C-FLAG. Chromosomes and nuclei were stained with DAPI (blue). Cells at various cell-cycle stages are shown. Scale bar, 10 μm. Developmental Cell 2017 42, 181-189.e3DOI: (10.1016/j.devcel.2017.06.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 New CENP-A Is Not Incorporated into Centromeres in KNL2-Deficient DT40 Cells, and M18BP1/KNL2 Localization Depends on CENP-A (A) Immunostaining (red) with anti-M18BP1/KNL2, Mis18α, CENP-H, and CENP-O antibodies in Aid-based KNL2 conditional knockout cells. In the absence of IAA, cells express M18BP1/KNL2 (KNL2 ON). In the presence of IAA, the M18BP1/KNL2 protein is degraded in 1 hr (KNL2 OFF). Scale bar, 10 μm. See also Figures S1 and S2. (B) Top: outline of quench-chase-pulse experiments in KNL2 ON or OFF cells stably expressing SNAP-CENP-A. Middle: representative images of G1 cells in which newly synthesized CENP-A was labeled with TMR-Star in KNL2 ON or OFF cells. CENP-T was used as a centromere marker. Punctate SNAP-CENP-A signals were not detected in KNL2 OFF cells, while SNAP-CENP-A signals co-localized with CENP-T in KNL2 ON cells. Scale bar, 10 μm. Bottom: quantification of intensities by TMR-Star at KNL2 ON or OFF cells. Signal intensities of TMR-Star in each centromere were quantified (n = 100). Error bars represent the SD. Relative intensities are shown. Statistical significance was evaluated by Student's t test. (C) Immunostaining (red) with anti-M18BP1/KNL2 antibody in Aid-based CENP-A conditional knockout cells. In the absence of IAA, cells express Aid-CENP-A-GFP (CENP-A ON, green). In the presence of IAA, the CENP-A protein was degraded within 2 hr (CENP-A OFF). Scale bar, 10 μm. See also Figures S2 and S3. Developmental Cell 2017 42, 181-189.e3DOI: (10.1016/j.devcel.2017.06.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 CENP-C-like Motif in Chicken M18BP1/KNL2 Is Essential for Its Centromere Localization and Function (A) Diagram of chicken M18BP1/KNL2 (top) and CENP-C (bottom). The position of the SANT domain, CENP-C (CC)-like motif, and SANTA domain is shown. Sequences of CENP-C-like motifs in various organisms are shown. Corresponding sequences for chicken and human CENP-C are also shown. Human CENP-C has two CENP-A binding regions and the second region (C terminus) corresponds to chicken CENP-A binding region. Red text indicates highly conserved residues between CENP-C (in CENP-A binding region) and the CENP-C-like motif in M18BP1/KNL2. (B) Growth curve of KNL2-deficient cell lines expressing either full-length KNL2 (KNL2 WT), KNL2 with the SANT domain deleted (KNL2 ΔSANT), KNL2 with the SANTA domain deleted (KNL2 ΔSANTA), or KNL2 with the CENP-C-like motif deleted (KNL2 ΔCC #01 or KNL2 ΔCC #02). (C) Localization of KNL2 ΔCC-GFP, KNL2 ΔSANTA-GFP, and KNL2 ΔSANT-GFP. Immunostaining with anti-CENP-T (a centromere marker: red) was also performed. Scale bar, 10 μm. Percentages for co-localization of KNL2 with CENP-T in each cell line are shown. (D) Immunostaining with anti-Mis18α antibody (red) in DT40 cells expressing KNL2 ΔCC-GFP. Scale bar, 10 μm. (E) Newly synthesized CENP-A (red) was detected by TMR staining for SNAP-CENP-A in DT40 cells expressing KNL2 ΔCC-GFP (green). Immunostaining with anti-CENP-T (blue) was also performed. Scale bar, 10 μm. (F) Immunostaining with anti-CENP-A (red) and -CENP-C (red) antibodies in DT40 cells expressing KNL2 ΔCC-GFP (green). Scale bar, 10 μm. Developmental Cell 2017 42, 181-189.e3DOI: (10.1016/j.devcel.2017.06.019) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 CENP-C-like Motif in Chicken M18BP1/KNL2 Directly Binds to CENP-A Nucleosome (A) Immunoprecipitation (IP) with anti-M18BP1/KNL2 antibody in DT40 whole-cell extract or chromatin fraction followed by western blot analysis with the indicated antibodies. Each blot was cut around expected band and presented. Molecular weight information is shown. (B) GST pull-down experiments of various GST-fused M18BP1/KNL2 recombinant proteins with in vitro reconstituted CENP-A- or histone H3.1-containing nucleosomes. Pull-down products were subjected to SDS-PAGE, followed by Coomassie brilliant blue (CBB) staining. M18BP1/KNL2-CC pulled down CENP-A nucleosomes, but not H3.1 nucleosomes. Asterisk indicates a degraded product from GST-CC-FLAG protein. See also Figure S4. (C) Current model of CENP-A deposition in chicken cells. The Mis18 complex directly associated with CENP-A nucleosome (old CENP-A) before CENP-A deposition. HJURP, bound with CENP-A-H4 (new CENP-A), recognizes the Mis18 complex, and new CENP-A is deposited near existing CENP-A nucleosome. Developmental Cell 2017 42, 181-189.e3DOI: (10.1016/j.devcel.2017.06.019) Copyright © 2017 Elsevier Inc. Terms and Conditions