Yoshida MM et al. (2016), SUMOylation of DNA topoisomerase IIα regulates ...

XB-ART-52238

J Cell Biol 2016 Jun 20;2136:665-78. doi: 10.1083/jcb.201511079.

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SUMOylation of DNA topoisomerase IIα regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis.

Yoshida MM , Ting L , Gygi SP , Azuma Y .

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DNA topoisomerase II (TOP2) plays a pivotal role in faithful chromosome separation through its strand-passaging activity that resolves tangled genomic DNA during mitosis. Additionally, TOP2 controls progression of mitosis by activating cell cycle checkpoints. Recent work showed that the enzymatically inert C-terminal domain (CTD) of TOP2 and its posttranslational modification are critical to this checkpoint regulation. However, the molecular mechanism has not yet been determined. By using Xenopus laevis egg extract, we found that SUMOylation of DNA topoisomerase IIα (TOP2A) CTD regulates the localization of the histone H3 kinase Haspin and phosphorylation of histone H3 at threonine 3 at the centromere, two steps known to be involved in the recruitment of the chromosomal passenger complex (CPC) to kinetochores in mitosis. Robust centromeric Haspin localization requires SUMOylated TOP2A CTD binding activity through SUMO-interaction motifs and the phosphorylation of Haspin. We propose a novel mechanism through which the TOP2 CTD regulates the CPC via direct interaction with Haspin at mitotic centromeres.

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Species referenced: Xenopus laevis
Genes referenced: haspin top2a

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	Figure 1. Inhibition of SUMOylation reduces Aurora B kinase on mitotic chromosomes. (A) Schematic method for the preparation of mitotic replicated chromosomes from XEEs. (B) Mitotic replicated chromosomes isolated as in A with (+dnUbc9) or without (control [Cont.]) dnUbc9 were subjected to immunoblotting. Histone H3 was used for the loading control for the mitotic chromosomes. (C) Quantification of Aurora B and Aurora B T248p levels on the mitotic chromosome, as seen in B, relative to levels of Cont. chromosomes from three independent experiments (n = 3) with levels normalized to histone H3 levels. Error bars represent SD. , P < 0.05 (Student’s t test). (D) Mitotic replicated chromosomes prepared as in A with or without dnUbc9 (Cont.) were subjected to immunofluorescence staining with antibodies as indicated with Hoechst 33342. Bar, 10 µm. (E) Quantification of the Aurora B signal intensity at mitotic centromeres, as seen in D, relative to signal intensities of Cont. centromeres from three independent experiments (n = 3, 50 centromeres per n) with levels normalized to CENP-A signal. Error bars represent SD. , P < 0.05 (Student’s t test).
	Figure 2. Haspin binding to TOP2A CTD is dependent on SUMOylation and SIMs. (A) Silver stain of the pulled-down proteins using TOP2A CTD. S-tagged non-SUMOylated (CTD) and SUMOylated CTD (CTD-SUMO) through in vitro SUMOylation assay were bound to S-agarose beads and incubated with CSF XEEs for pull-down assay. After incubation with SENP2 CD, proteins were eluted with urea and precipitated with trichloroacetic acid (TCA precip.). Lanes 1 and 2 represent 5% of the S-tagged CTD and CTD-SUMO bound onto S-agarose beads as bait. Proteins in each fraction were visualized with silver stain. After elution, samples were the proteins remaining on S-agarose beads. Trichloroacetic acid–precipitated fractions were subjected to protein identification by LC-MS/MS. (B) SENP2-digested pull-down samples were analyzed by immunoblotting for Haspin. SENP2-digested S-tagged CTD was used as a loading control for the bait used in the pull-down assay. CSF lane represents 0.75% of the volume of XEEs used for each pull-down sample. CSF Haspin indicates the endogenous band found in the CSF XEEs, and Bound Haspin indicates the Haspin band in the pull-down sample. (C) Schematic representation of the primary structure of X. laevis Haspin. SIMs are located at aa 343–346 (VICL) and 364–367 (VLCL). Point mutations in each SIM are indicated in red for the disrupted SIM mutant protein (2-SIM). (D) mRNAs of GFP-tagged WT or 2-SIM Haspin were supplemented in XEEs to express Haspin-GFP, and Haspin-GFP–expressing CSF XEEs were subjected to the pull-down assay with S-tagged CTD SUMOylated (CTD-SUMO) through in vitro SUMOylation assay and bound onto S-agarose beads (middle). After SENP2-CD incubation, CTD-SUMO–bound Haspin-GFP was analyzed by immunoblotting (right). SENP2-digested S-tagged CTD was used as a loading control for the bait used in the pull-down assay. CSF XEE lanes represent 0.5% of the volume of the Haspin-GFP–expressing CSF XEEs used for each pull-down sample. (E) Quantification of pulled-down Haspin-GFP levels by CTD-SUMO, as seen in D, relative to Haspin-GFP WT levels from three independent experiments (n = 3) with levels normalized by CTD levels. Error bar represents SD. ***, P < 0.001 (Student’s t test).
	Figure 3. SUMOylation on mitotic chromosomes regulates Haspin binding and H3T3 phosphorylation. (A) Mitotic replicated chromosomes prepared as in Fig. 1 A with (Cont.) or without (+dnUbc9) mitotic SUMOylation. Isolated chromosomes were analyzed by immunoblotting with indicated antibodies. Histone H3 was used as a loading control for the mitotic replicated chromosomes. (B) Quantification of Haspin and H3T3p levels on the mitotic replicated chromosomes, as seen in A, relative to levels of Cont. chromosomes from three independent experiments (n = 3) with levels normalized to histone H3 levels. Error bar represents SD. , P < 0.05 (Student’s t test). (C) Mitotic replicated chromosomes prepared from CSF XEEs with (Cont.) or without (+dnUbc9) mitotic SUMOylation were subjected to immunofluorescence staining with antibodies as indicated with Hoechst 33342. (D) Quantification of H3T3p signal intensity at the mitotic centromeres, as seen in C, relative to signal intensities of Cont. centromeres from three independent experiments (n = 3, 40 centromeres per n) with levels normalized to CENP-A. Error bar represents SD. **, P < 0.001 (Student’s t test).
	Figure 4. SUMOylation regulates centromeric Haspin localization during mitosis. (A) Haspin-GFP mRNA was supplemented in XEEs for protein expression (top), and mitotic replicated chromosomes prepared without or with dnUbc9 were subjected to immunofluorescence staining with indicated antibodies with Hoechst 33342. β-Tubulin was used as a loading control for Haspin-GFP expression levels in XEEs. Bar, 10 µm. (B) Quantification of centromeric Haspin-GFP signal intensity, as seen in A, relative to signal intensities of +Haspin-GFP centromeres from three independent experiments (n = 3, 50 centromeres per n) with levels normalized to CENP-A. Error bar represents SD. **, P < 0.01 (Student’s t test).
	Figure 5. SUMOylation of TOP2A CTD regulates Haspin binding and H3T3 phosphorylation on mitotic chromosomes. (A) Schematic representation of the primary structure of X. laevis TOP2A. Three lysines indicated in the CTD were mutated to arginine for a TOP2A mutant that could not be SUMOylated in the CTD (3KR). (B) Endogenous TOP2A in CSF XEEs was immunodepleted and replaced with either recombinant full-length T7-TOP2A WT or 3KR (left). β-Tubulin was used as a loading control of TOP2A levels in CSF XEEs. Mitotic chromosomes assembled in TOP2A-replaced CSF XEEs were analyzed by immunoblotting with indicated antibodies (right). Histone H4 was used as a loading control for mitotic chromosomes. (C) Quantification of Haspin and H3T3p levels on the mitotic chromosomes, as seen in B, relative to levels of TOP2A WT chromosomes from three independent experiments (n = 3) with levels normalized to histone H4 levels. Error bar represents SD. , P < 0.05; *, P < 0.01 (Student’s t test).
	Figure 6. Cell cycle–dependent Haspin T206 phosphorylation regulates SUMOylated TOP2A CTD–Haspin interaction. (A) Either CSF XEEs or interphase XEEs (Int.) expressing Haspin-GFP (left) were used in pull-down assays with S-tagged non-SUMOylated (CTD) and SUMOylated CTD (CTD-SUMO) bound to S-agarose beads (middle), and Haspin-GFP binding was analyzed by immunoblotting (right). β-Tubulin was used as a loading control for Haspin-GFP levels in XEEs (loading 0.5% of the volume of XEEs used in each pull-down sample). SENP2-digested S-tagged CTD was used as the loading control for the bait used in the pull-down assay. (B) Quantification of pulled-down Haspin-GFP levels with CTD and CTD-SUMO, as seen in A, relative to levels from the pull-down sample using CSF XEEs with CTD-SUMO from three independent experiments (n = 3) with levels normalized to TOP2A CTD levels. Error bar represents SD. *, P < 0.01 (Student’s t test). (C) Schematic representation of X. laevis Haspin mutants. Threonine 206 (T206) was mutated to alanine (T206A) to eliminate the mitotic phosphorylation site. T206A/2-SIM indicates the combined T206A and the double SIM mutations. (D) Expressed WT, T206A, 2-SIM, and T206A/2-SIM Haspin-GFP in CSF XEEs (top) were used in pull-down assays with S-tagged CTD and CTD-SUMO bound onto S-agarose beads with Haspin-GFP binding analyzed by immunoblotting (bottom). β-Tubulin was used as a loading control for Haspin-GFP levels in XEEs (loading 0.5% of the volume of XEEs used in each pull-down sample; top). SENP2-digested S-tagged CTD was used as the loading control for the bait used in the pull-down assay (bottom). (E) Quantification of pulled-down Haspin-GFP levels with CTD and CTD-SUMO, as seen in D, relative to Haspin-GFP WT levels of CTD-SUMO from three independent experiments (n = 3) with levels normalized to TOP2A CTD levels. Error bar represents SD. , P < 0.05; **, P < 0.01 (Student’s t test).
	Figure 7. Haspin SIMs and the phosphorylation of Haspin contribute to its localization at mitotic centromeres. (A) Mitotic replicated chromosomes prepared from Haspin-GFP–expressing XEEs with different mRNA concentrations of Haspin WT, T206A, 2-SIM, or T206A/2SIM mutant were subjected to immunofluorescence staining. Immunofluorescence staining of chromosomes from XEEs with similar levels of expressed Haspin-GFP is shown and compared (lane 2, WT; lane 5, T206A; lane 8, 2-SIM; and lane 11, T206A/2-SIM). β-Tubulin was used as a loading control for Haspin-GFP levels in CSF XEEs (top). Bars, 10 µm. (B) Chromosomes from XEEs with similar levels of expressed Haspin-GFP were quantified using centromeric Haspin-GFP signal intensity, as seen in A, relative to signal intensities of Haspin-GFP WT from three independent experiments (n = 3, 30 centromeres per n) with levels normalized to SUMO2/3. Error bar represents SD. , P < 0.05; *, P < 0.01 (Student’s t test).
	Figure 8. Model for centromeric Haspin recruitment by DNA topoisomerase IIα. (1) Centromeric DNA topoisomerase IIα is SUMOylated (S) at the C-terminal domain by SUMO E3 ligase PIASy, whereas Haspin is phosphorylated (P) by Cdk1 at T206 during the onset of mitosis. Plk1 binds to phosphorylated T206 to phosphorylate other sites on Haspin to create active Haspin kinase. (2) SUMOylated topoisomerase IIα recruits active Haspin to the centromere to allow for the phosphorylation of histone H3 (purple) at threonine 3 (T3). Phosphorylated T3 recruits CPC members to the centromere through direct interaction with Survivin. H2A (dark purple) T120 phosphorylation mediated by Bub1 additionally contributes to the recruitment of CPC members to the centromere through the binding of Shugoshin 1/2 (Sgo1/2) that interacts with Borealin.

References [+] :

Adams, INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow. 2000, Pubmed, Xenbase

	Figure 1. Inhibition of SUMOylation reduces Aurora B kinase on mitotic chromosomes. (A) Schematic method for the preparation of mitotic replicated chromosomes from XEEs. (B) Mitotic replicated chromosomes isolated as in A with (+dnUbc9) or without (control [Cont.]) dnUbc9 were subjected to immunoblotting. Histone H3 was used for the loading control for the mitotic chromosomes. (C) Quantification of Aurora B and Aurora B T248p levels on the mitotic chromosome, as seen in B, relative to levels of Cont. chromosomes from three independent experiments (n = 3) with levels normalized to histone H3 levels. Error bars represent SD. , P < 0.05 (Student’s t test). (D) Mitotic replicated chromosomes prepared as in A with or without dnUbc9 (Cont.) were subjected to immunofluorescence staining with antibodies as indicated with Hoechst 33342. Bar, 10 µm. (E) Quantification of the Aurora B signal intensity at mitotic centromeres, as seen in D, relative to signal intensities of Cont. centromeres from three independent experiments (n = 3, 50 centromeres per n) with levels normalized to CENP-A signal. Error bars represent SD. , P < 0.05 (Student’s t test).
	Figure 2. Haspin binding to TOP2A CTD is dependent on SUMOylation and SIMs. (A) Silver stain of the pulled-down proteins using TOP2A CTD. S-tagged non-SUMOylated (CTD) and SUMOylated CTD (CTD-SUMO) through in vitro SUMOylation assay were bound to S-agarose beads and incubated with CSF XEEs for pull-down assay. After incubation with SENP2 CD, proteins were eluted with urea and precipitated with trichloroacetic acid (TCA precip.). Lanes 1 and 2 represent 5% of the S-tagged CTD and CTD-SUMO bound onto S-agarose beads as bait. Proteins in each fraction were visualized with silver stain. After elution, samples were the proteins remaining on S-agarose beads. Trichloroacetic acid–precipitated fractions were subjected to protein identification by LC-MS/MS. (B) SENP2-digested pull-down samples were analyzed by immunoblotting for Haspin. SENP2-digested S-tagged CTD was used as a loading control for the bait used in the pull-down assay. CSF lane represents 0.75% of the volume of XEEs used for each pull-down sample. CSF Haspin indicates the endogenous band found in the CSF XEEs, and Bound Haspin indicates the Haspin band in the pull-down sample. (C) Schematic representation of the primary structure of X. laevis Haspin. SIMs are located at aa 343–346 (VICL) and 364–367 (VLCL). Point mutations in each SIM are indicated in red for the disrupted SIM mutant protein (2-SIM). (D) mRNAs of GFP-tagged WT or 2-SIM Haspin were supplemented in XEEs to express Haspin-GFP, and Haspin-GFP–expressing CSF XEEs were subjected to the pull-down assay with S-tagged CTD SUMOylated (CTD-SUMO) through in vitro SUMOylation assay and bound onto S-agarose beads (middle). After SENP2-CD incubation, CTD-SUMO–bound Haspin-GFP was analyzed by immunoblotting (right). SENP2-digested S-tagged CTD was used as a loading control for the bait used in the pull-down assay. CSF XEE lanes represent 0.5% of the volume of the Haspin-GFP–expressing CSF XEEs used for each pull-down sample. (E) Quantification of pulled-down Haspin-GFP levels by CTD-SUMO, as seen in D, relative to Haspin-GFP WT levels from three independent experiments (n = 3) with levels normalized by CTD levels. Error bar represents SD. ***, P < 0.001 (Student’s t test).
	Figure 3. SUMOylation on mitotic chromosomes regulates Haspin binding and H3T3 phosphorylation. (A) Mitotic replicated chromosomes prepared as in Fig. 1 A with (Cont.) or without (+dnUbc9) mitotic SUMOylation. Isolated chromosomes were analyzed by immunoblotting with indicated antibodies. Histone H3 was used as a loading control for the mitotic replicated chromosomes. (B) Quantification of Haspin and H3T3p levels on the mitotic replicated chromosomes, as seen in A, relative to levels of Cont. chromosomes from three independent experiments (n = 3) with levels normalized to histone H3 levels. Error bar represents SD. , P < 0.05 (Student’s t test). (C) Mitotic replicated chromosomes prepared from CSF XEEs with (Cont.) or without (+dnUbc9) mitotic SUMOylation were subjected to immunofluorescence staining with antibodies as indicated with Hoechst 33342. (D) Quantification of H3T3p signal intensity at the mitotic centromeres, as seen in C, relative to signal intensities of Cont. centromeres from three independent experiments (n = 3, 40 centromeres per n) with levels normalized to CENP-A. Error bar represents SD. **, P < 0.001 (Student’s t test).
	Figure 4. SUMOylation regulates centromeric Haspin localization during mitosis. (A) Haspin-GFP mRNA was supplemented in XEEs for protein expression (top), and mitotic replicated chromosomes prepared without or with dnUbc9 were subjected to immunofluorescence staining with indicated antibodies with Hoechst 33342. β-Tubulin was used as a loading control for Haspin-GFP expression levels in XEEs. Bar, 10 µm. (B) Quantification of centromeric Haspin-GFP signal intensity, as seen in A, relative to signal intensities of +Haspin-GFP centromeres from three independent experiments (n = 3, 50 centromeres per n) with levels normalized to CENP-A. Error bar represents SD. **, P < 0.01 (Student’s t test).
	Figure 5. SUMOylation of TOP2A CTD regulates Haspin binding and H3T3 phosphorylation on mitotic chromosomes. (A) Schematic representation of the primary structure of X. laevis TOP2A. Three lysines indicated in the CTD were mutated to arginine for a TOP2A mutant that could not be SUMOylated in the CTD (3KR). (B) Endogenous TOP2A in CSF XEEs was immunodepleted and replaced with either recombinant full-length T7-TOP2A WT or 3KR (left). β-Tubulin was used as a loading control of TOP2A levels in CSF XEEs. Mitotic chromosomes assembled in TOP2A-replaced CSF XEEs were analyzed by immunoblotting with indicated antibodies (right). Histone H4 was used as a loading control for mitotic chromosomes. (C) Quantification of Haspin and H3T3p levels on the mitotic chromosomes, as seen in B, relative to levels of TOP2A WT chromosomes from three independent experiments (n = 3) with levels normalized to histone H4 levels. Error bar represents SD. , P < 0.05; *, P < 0.01 (Student’s t test).
	Figure 6. Cell cycle–dependent Haspin T206 phosphorylation regulates SUMOylated TOP2A CTD–Haspin interaction. (A) Either CSF XEEs or interphase XEEs (Int.) expressing Haspin-GFP (left) were used in pull-down assays with S-tagged non-SUMOylated (CTD) and SUMOylated CTD (CTD-SUMO) bound to S-agarose beads (middle), and Haspin-GFP binding was analyzed by immunoblotting (right). β-Tubulin was used as a loading control for Haspin-GFP levels in XEEs (loading 0.5% of the volume of XEEs used in each pull-down sample). SENP2-digested S-tagged CTD was used as the loading control for the bait used in the pull-down assay. (B) Quantification of pulled-down Haspin-GFP levels with CTD and CTD-SUMO, as seen in A, relative to levels from the pull-down sample using CSF XEEs with CTD-SUMO from three independent experiments (n = 3) with levels normalized to TOP2A CTD levels. Error bar represents SD. *, P < 0.01 (Student’s t test). (C) Schematic representation of X. laevis Haspin mutants. Threonine 206 (T206) was mutated to alanine (T206A) to eliminate the mitotic phosphorylation site. T206A/2-SIM indicates the combined T206A and the double SIM mutations. (D) Expressed WT, T206A, 2-SIM, and T206A/2-SIM Haspin-GFP in CSF XEEs (top) were used in pull-down assays with S-tagged CTD and CTD-SUMO bound onto S-agarose beads with Haspin-GFP binding analyzed by immunoblotting (bottom). β-Tubulin was used as a loading control for Haspin-GFP levels in XEEs (loading 0.5% of the volume of XEEs used in each pull-down sample; top). SENP2-digested S-tagged CTD was used as the loading control for the bait used in the pull-down assay (bottom). (E) Quantification of pulled-down Haspin-GFP levels with CTD and CTD-SUMO, as seen in D, relative to Haspin-GFP WT levels of CTD-SUMO from three independent experiments (n = 3) with levels normalized to TOP2A CTD levels. Error bar represents SD. , P < 0.05; **, P < 0.01 (Student’s t test).
	Figure 7. Haspin SIMs and the phosphorylation of Haspin contribute to its localization at mitotic centromeres. (A) Mitotic replicated chromosomes prepared from Haspin-GFP–expressing XEEs with different mRNA concentrations of Haspin WT, T206A, 2-SIM, or T206A/2SIM mutant were subjected to immunofluorescence staining. Immunofluorescence staining of chromosomes from XEEs with similar levels of expressed Haspin-GFP is shown and compared (lane 2, WT; lane 5, T206A; lane 8, 2-SIM; and lane 11, T206A/2-SIM). β-Tubulin was used as a loading control for Haspin-GFP levels in CSF XEEs (top). Bars, 10 µm. (B) Chromosomes from XEEs with similar levels of expressed Haspin-GFP were quantified using centromeric Haspin-GFP signal intensity, as seen in A, relative to signal intensities of Haspin-GFP WT from three independent experiments (n = 3, 30 centromeres per n) with levels normalized to SUMO2/3. Error bar represents SD. , P < 0.05; *, P < 0.01 (Student’s t test).
	Figure 8. Model for centromeric Haspin recruitment by DNA topoisomerase IIα. (1) Centromeric DNA topoisomerase IIα is SUMOylated (S) at the C-terminal domain by SUMO E3 ligase PIASy, whereas Haspin is phosphorylated (P) by Cdk1 at T206 during the onset of mitosis. Plk1 binds to phosphorylated T206 to phosphorylate other sites on Haspin to create active Haspin kinase. (2) SUMOylated topoisomerase IIα recruits active Haspin to the centromere to allow for the phosphorylation of histone H3 (purple) at threonine 3 (T3). Phosphorylated T3 recruits CPC members to the centromere through direct interaction with Survivin. H2A (dark purple) T120 phosphorylation mediated by Bub1 additionally contributes to the recruitment of CPC members to the centromere through the binding of Shugoshin 1/2 (Sgo1/2) that interacts with Borealin.