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Borealin-nucleosome interaction secures chromosome association of the chromosomal passenger complex.
Abad MA
,
Ruppert JG
,
Buzuk L
,
Wear M
,
Zou J
,
Webb KM
,
Kelly DA
,
Voigt P
,
Rappsilber J
,
Earnshaw WC
,
Jeyaprakash AA
.
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Chromosome association of the chromosomal passenger complex (CPC; consisting of Borealin, Survivin, INCENP, and the Aurora B kinase) is essential to achieve error-free chromosome segregation during cell division. Hence, understanding the mechanisms driving the chromosome association of the CPC is of paramount importance. Here using a multifaceted approach, we show that the CPC binds nucleosomes through a multivalent interaction predominantly involving Borealin. Strikingly, Survivin, previously suggested to target the CPC to centromeres, failed to bind nucleosomes on its own and requires Borealin and INCENP for its binding. Disrupting Borealin-nucleosome interactions excluded the CPC from chromosomes and caused chromosome congression defects. We also show that Borealin-mediated chromosome association of the CPC is critical for Haspin- and Bub1-mediated centromere enrichment of the CPC and works upstream of the latter. Our work thus establishes Borealin as a master regulator determining the chromosome association and function of the CPC.
Figure 1. Borealin nucleosome binding is essential for chromosome association of the CPC. (A) Domain architecture of the subunits of the CPC. (B) Representative fluorescence images of a siRNA rescue assay for Borealin10–109 fragment. Immunofluorescent staining of Myc and Survivin in HeLa cells cotransfected with siRNA duplexes targeting the 3′ UTR region of Borealin and Myc-Borealin constructs. Hoechst was used for DNA staining. Scale bar, 10 µm. All cells transfected with the siRNA and Myc-Borealin10–109 fragment showed exclusion of the CPC complex from the chromatin. (C and E) Native PAGE analysis of EMSA assays performed with increasing concentrations of recombinant CPC_LM containing different Borealin fragments and either 20 nM phosphorylated (H3T3ph; C) or unmodified IR700-labeled (E) NCPs. (D) EMSA assays performed with increasing concentrations of Survivin with 20 nM IR700-labeled H3T3ph NCPs. (F) Representative SPR sensorgrams of the interaction between different CPC_LM complexes (CPC_LM, CPC_LMSUR MUT) or Survivin and unmodified (top) or H3T3ph (middle) NPCs or DNA (bottom) immobilized on the surface of a neutravidin sensor chip. Mean values (n ≥ 3, ±SEM) determined for the equilibrium Kd are shown in boxes underneath the sensorgrams. For a detailed description of the CPC domain architecture, refer to Fig. S1 C.
Figure 2. N-terminal nine amino acids and C-terminal half of Borealin are required for CPC–chromatin interaction. (A) Native PAGE analysis of EMSA assays performed with recombinant CPC_LM containing different Borealin truncations binding to IR700-labeled H3T3ph NCPs (left) and unmodified NCPs (middle) and quantification of binding (right). Concentrations of the NCP and the CPC used in the assay were 20 and 160 nM, respectively. Mean of percentage of binding ± SD; n = 5; **, P ≤ 0.01; ***, P ≤ 0.001, unpaired t test. (B) Representative SPR sensorgrams of the interaction between different CPC_LM complexes (CPC_LMBOR 10–end, CPC_LMBOR Δloop, and CPC_LMBOR 10–221) and unmodified (top) or H3T3ph (middle) NCPs or DNA (bottom). Mean values (n ≥ 3, ±SEM) determined for the equilibrium Kd are shown in boxes underneath the sensorgrams. For a detailed description of the CPC domain architecture, refer to Fig. S1 C. (C) Representative fluorescence images of a rescue assay for Borealin, Borealin10–end, BorealinΔloop, and Borealin10–221 constructs. Immunofluorescent staining of Myc, Survivin, and ACA in HeLa cells cotransfected with siRNA duplexes targeting the 3′ UTR region of Borealin and Myc-Borealin constructs. Hoechst was used for DNA staining. Scale bar, 10 µm. (D) Quantification of Survivin levels at the centromeres for the siRNA-rescue assays with Myc-Borealin (n = 111 cells), Myc-Borealin10–end (n = 101 cells), Myc-BorealinΔloop (n = 85 cells), and Borealin10–221 (n = 72 cells) shown in C (three independent experiments, mean ± SEM, Mann–Whitney U test; ****, P < 0.0001). (E) Quantification of uncongressed chromosomes observed for the siRNA-rescue assay of Myc-Borealin, Myc-Borealin10–end, Myc-BorealinΔloop, and Myc-Borealin10–221 fragments shown in C. A minimum of 25 cells were counted for each construct.
Figure 3. CPC–nucleosome binding is mediated by multivalent interactions predominantly involving Borealin. (A) Circle view of the cross-links observed between the subunits of the CPC (Borealin, purple; Survivin, green; INCENP, yellow) and histones from unmodified NCPs. Intermolecular contacts involving Borealin, Survivin, and INCENP and histones are shown as purple, green, or yellow lines, respectively, using XiNET (Kolbowski et al., 2018). (B) Cartoon representation of the crystal/NMR structures of the NCP (PDB: 1KX5; Davey et al., 2002) and CPC (CPC core PDB: 2QFA [Jeyaprakash et al., 2007] and Borealin dimerization domain PDB: 2KDD [Bourhis et al., 2009]; top). Surface representation of the NCP and the CPC colored based on the electrostatic surface potential calculated using APBS in Pymol v2.0.6 (bottom).
Figure 4. Borealin N-terminal region and the central loop contribute to NCP binding by interacting with the NCP acidic patch and DNA. (A) Representative SPR sensorgrams of the interaction between the cross-linking–based CPC_LM mutants (CPC_LMBOR K12/R17/K20E, CPC_LMBOR K135/K158/K183/K198/T199E, and CPC_LMBOR K198/T199E) and unmodified (top) or H3T3ph (middle) NCPs or DNA (bottom). Mean values (n ≥ 3, ±SEM) determined for the equilibrium Kd are shown in boxes underneath the sensorgrams. (B) Representative fluorescence images of rescue assays for Myc-Borealin, Myc-Borealin K12/R17/K20E, and Myc-Borealin K135/K158/K183/K198/T199E constructs in HeLa cells depleted of Borealin by siRNA. Immunofluorescent staining of Myc, Survivin, and ACA. Hoechst was used for DNA staining. Scale bar, 10 µm. (C) Quantification of Survivin levels at the centromeres for the siRNA-rescue assays with Myc-Borealin (n = 41 cells), Myc-Borealin K12/R17/K20E (n = 42 cells), and Myc-Borealin K135/K158/K183/K198/T199E (n = 43 cells) shown in B (three independent experiments, mean ± SEM, Mann–Whitney U test; ****, P < 0.0001). (D) Quantification of DNA binding by CPC_LM and CPC_LMΔloop. Binding was normalized to the steady-state response for 1 µM CPC_LM (n = 4). (E) Quantification of DNA binding by Sumo-Borealin110–206, Sumo-Borealin110–188, and Sumo-Borealin189–206 (n ≥ 2). Binding was normalized to the steady-state response for 1 µM Sumo-Borealin110–206. Representative sensorgrams are shown in inset. The apparent Kd values for each sample are indicated and were estimated from molecular weight–corrected responses in comparison to CPC_LM binding to unmodified NCPs. (F) Representative SPR sensorgrams for the interaction between Sumo-Borealin222–280 and NCPs (n = 1).
Figure 5. Borealin-mediated chromosome association of the CPC is an upstream requirement for its Haspin- and Bub1-mediated centromeric enrichment. (A and B) Immunofluorescence analysis of ACA and H3T3ph (A) or H2AT120ph (B) levels upon Borealin depletion using siRNA duplexes and rescue with different Borealin constructs. Hoechst was used for DNA staining. Scale bar, 10 µm. (C) Model for Borealin-mediated chromatin association of the CPC and subsequent centromere enrichment. Considering that Borealin can dimerize and is required for efficient NCP binding, we speculate that two copies of CPC likely bind symmetrically equivalent CPC-interaction sites on NCP. For clarity, just one CPC binding is shown in the cartoon.
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