TOPBP1 recruits TOP2A to ultra-fine anaphase bridges to aid in their resolution

Abstract

During mitosis, sister chromatids must be faithfully segregated to ensure that daughter cells receive one copy of each chromosome. However, following replication they often remain entangled. Topoisomerase IIα (TOP2A) has been proposed to resolve such entanglements, but the mechanisms governing TOP2A recruitment to these structures remain poorly understood. Here, we identify TOPBP1 as a novel interactor of TOP2A, and reveal that it is required for TOP2A recruitment to ultra-fine anaphase bridges (UFBs) in mitosis. The C-terminal region of TOPBP1 interacts with TOP2A, and TOPBP1 recruitment to UFBs requires its BRCT domain 5. Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci. Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion. These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process.

Fig. 1. TOPBP1 is a novel TOP2A interacting protein that localises to UFBs

(a) Proteins identified in IP/MS analysis of GFP-Trap purification from HEK 293FT cells expressing GFP-TOP2A. (b) Endogenous TOP2A co-immunoprecipitates with GFP-tagged TOPBP1. U2OS control cells or U2OS cells stably expressing GFP-TOPBP1 were untreated or treated with nocodazole (0.1 μg ml⁻¹ for 16h) as indicated and subjected to GFP-nanotrap immunoprecipitation. Input = 0.4% of total IP (c) Endogenous TOPBP1 co-immunoprecipitates with GFP-tagged TOP2A. U2OS control cells or U2OS cells stably expressing GFP-TOP2A were treated with nocodazole (0.1 μg ml⁻¹ for 16h) and subjected to GFP-nanotrap immunoprecipitation. Input = 0.4% of total IP (d and e) Immunofluorescence microscopy in U2OS cells with antibodies against TOPBP1; BLM serves as a positive control. DAPI serves as a DNA stain. (f) Immunofluorescence microscopy of HeLa cells with antibodies against TOPBP1. DAPI serves as a DNA stain. (g) Immunofluorescence in human primary cells (human dermal fibroblasts) using antibody against TOPBP1. DAPI serves as a DNA stain. Scale bar = 6.4 μm.

Fig. 2. BRCT 5 of TOPBP1 mediates its recruitment to UFBs

(a) Schematic representation of GFP-TOPBP1 mutant constructs used in this study (WT TOPBP1, BRCT1 - K154A; BRCT5 - K704A). (b, c and d) Quantification of GFP-TOPBP1 WT, K154A and K704A mutant localisation to UFBs in pools of U2OS cells stably expressing GFP-TOPBP1 WT or mutant proteins (endogenous TOPBP1 was knocked down using siRNA directed against the 3′ UTR (si TOPBP1), scrambled pool siRNA was used as a control (si Control)). Mean from three independent experiments is depicted with bars representing +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (e, f and g) Analysis of localisation of GFP-TOPBP1 WT, K704A and K704E mutant localisation to UFBs in single U2OS clones expressing GFP-TOPBP1 WT or mutant proteins (endogenous TOPBP1 was depleted using siRNA directed against the 3′ UTR (si TOPBP1), scrambled pool siRNA was used as a control (si Control)). Mean from three independent experiments is depicted with bars representing +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance.

Fig. 3. TOP2A inhibition or depletion results in increased TOPBP1 localisation to UFBs

(a and b) Quantification of the percentage of ana/telophase cells staining positive for TOPBP1 localisation to UFBs in U2OS cells treated for 24h with DMSO as a control, or with 0.4 μM aphidicolin (APH) or 10 μM razoxane (RAZ). Bars represent mean values from three independent experiments +/− s.e.m. At least 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (c) Quantification of the percentage of U2OS ana/telophase cells staining positive for BLM-UFBs in cells treated for 24h with DMSO as a control or with 0.4 μM aphidicolin (APH). Bars represent mean values from three independent experiments +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance. (d) Quantification of the percentage of U2OS cells positive for TOPBP1-UFBs in cells treated with siRNA targeting TOP2A or in control siRNA treated cells. Mean from three independent experiments with bars indicating +/− s.e.m. At least 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance. (e and f) Quantification of the percentage of U2OS cells positive for BLM-UFBs and DAPI-positive anaphase bridges, respectively cells treated with siRNA directed against TOP2A or control siRNA treated cells. Mean from three independent experiments with bars indicating +/− s.e.m. At least 50 ana/telophase cells were scored per experiment, the Chi-square test was used to determine statistical significance.

Fig. 4. TOPBP1 depletion impedes C-UFBs resolution

(a) Quantification of BLM-positive UFBs in control U2OS cells or cells stably expressing GFP-TOPBP1 WT or GFP-TOPBP1 K704E, respectively in the presence or absence of endogenous TOPBP1. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment and the Chi-square test was used to determine statistical significance. (b) Quantification of UFBs emanating from centromeric loci as assayed by CENPB staining in TOPBP1-depleted cells. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment (c) Representative image of an anaphase cell with a BLM-positive UFB (Red) which emanates from a CENPB focus (Green). DAPI (Blue) serves as a DNA stain. Scale bar = 6.4 μm.

Fig. 5. TOPBP1-TOP2A interaction requires TOPBP1’s C-terminus

(a) Schematic representation of GFP-TOPBP1 truncation mutants used in panel b. (b) TOPBP1-TOP2A interaction requires TOPBP1’s C-terminus. HEK 293FT control cells, or cells transiently expressing WT GFP-TOPBP1 and indicated truncation mutants were nocodazole treated (0.1 μg ml⁻¹ for 16h) and subjected to GFP-nanotrap immunoprecipitation. (c) TOPBP1-TOP2A interaction requires BRCT 7/8. HEK 293FT control cells, or cells transiently expressing GFP-TOPBP1 WT or the indicated truncation mutant were nocodazole treated (0.1 μg ml⁻¹ for 16h) and subjected to GFP-nanotrap immunoprecipitation.

Fig. 6. TOP2A localises to UFBs

(a) Schematic describing the proximity ligation assay (PLA) employed to visualise TOPBP1-TOP2A co-localisation on UFBs. (b) Representative image of PLA assay carried out using antibodies raised against endogenous TOPBP1 and TOP2A in U2OS cells stably expressing GFP-TOPBP1 WT. GFP acts as a marker for UFBs, PLA signal indicates TOPBP1-TOP2A co-localisation. PLA signal co-localisation with a UFB is highlighted in the zoomed section. Scale bar = 6.4 μm. (c) Quantification of the frequency of PLA co-localisation with GFP-TOPBP1 UFBs. “Control” represents signal obtained for PLA assay when using only one of the antibodies (TOP2A antibody). Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 20 UFBs were scored per analysis. (d) Representative image of PLA assay carried out using antibodies raised against GFP and TOP2A in U2OS cells transiently expressing GFP-TOPBP1 WT or BRCT7/8 truncation mutant. GFP acts as a marker for UFBs, PLA signal indicates TOPBP1-TOP2A co-localisation. Scale bar = 6.4 μm. (e) Quantification of the frequency of PLA co-localisation with GFP-TOPBP1 WT and BRCT7/8 truncation mutant. Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 20 UFBs were scored per analysis and the Chi-square test was used to determine statistical significance.

Fig. 7. TOPBP1-TOP2A interaction promotes UFB resolution

(a) Western blot depicting the levels of expression of GFP-TOPBP1 WT and GFP-TOPBP1 ΔBRCT7/8 mutant transiently expressed in U2OS cells (GFP). α-Tubulin serves as a loading control. (b and c) Quantification of the frequency of BLM-UFBs or DAPI-positive anaphase bridges in mock-transfected U2OS cells (Mock) or cells transiently expressing GFP-TOPBP1 WT or ΔBRCT7/8 mutant (WT or ΔBRCT7/8). Mean from three independent experiments with bars indicating +/− s.e.m. A minimum of 50 ana/telophase cells were scored per experiment. Chi-square test was used to evaluate statistical significance. (d) Schematic model of TOPBP1-mediated UFB resolution: TOPBP1 binds to C-UFBs via its BRCT5 domain and recruits TOP2A via its C-terminus to aid their resolution.