CELL CYCLE 2016, VOL. 15, NO. 23, 3151–3152 http://dx.doi.org/10.1080/15384101.2016.1216928
EDITORIALS: CELL CYCLE FEATURES
Mechanisms behind Topoisomerase II SUMOylation in chromosome segregation Makoto M. Yoshida and Yoshiaki Azuma Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA ARTICLE HISTORY Received 8 July 2016; Accepted 22 July 2016 KEYWORDS Aurora B; centromere; chromosome segregation; CPC; decatenation; Haspin; SUMO; TOP2
One of the major substrates discovered to be SUMOylated during mitosis is DNA topoisomerase IIa (TOP2A), an enzyme that regulates the topology of genomic DNA. Interestingly, while TOP2A binds throughout the chromosome the majority of the SUMOylated TOP2A is likely clustered at the mitotic centromeres during early mitosis when SUMOylation occurs.1 Genetic studies in budding yeast on the SUMOylation sites of TOP2 have revealed the importance of TOP2A SUMOylation during mitosis. TOP2 CTD has been found to be SUMOylated at multiple lysines, and mutations to prevent SUMOylation at the CTD led to defects in the cohesion of the centromeric DNA in budding yeast, and led to a defect in the spindle checkpoint that senses tension at the microtubules-kinetochore attachment sites.2,3 This suggests that TOP2A CTD SUMOylation may regulate Aurora B kinase, the key regulator of the spindle checkpoint and metaphase-to-anaphase progression. An additional site discovered to be SUMOylated during mitosis is on Xenopus laevis TOP2A lysine 660 (K660) that is within the catalytic core of the enzyme.1 SUMOylation of K660 greatly inhibits TOP2A decatenation activity. The function of this inhibition is likely to maintain the cohesion of the sister chromatids by keeping the entangled centromeric DNA of the sister chromatids from being resolved. Interestingly, biochemical analysis of TOP2A SUMOylation has determined that SUMOylating the CTD lysine sites does not affect TOP2A decatenation activity.4 Therefore, the mechanism behind the regulation of the spindle checkpoint through TOP2A CTD SUMOylation has remained unknown. Not until recently was the pathway of TOP2A CTD SUMOylation in the regulation of Aurora B discovered. Recent work from our laboratory determined that the atypical histone H3 kinase Haspin interacts with TOP2A as a SUMO-binding protein.5 Haspin phosphorylates centromeric histone H3 at threonine 3 (H3T3), which provides a binding site for the chromosomal passenger complex (CPC) including Aurora B at the mitotic centromeres. Our results determined that TOP2A CTD SUMOylation can regulate Haspin, whose centromeric localization mechanism was previously unclear, by recruiting it onto the mitotic chromosomes, and that inhibition of SUMOylation mis-localizes Haspin off of the centromeres in the Xenopus egg extract model system. Additionally, a concurrent study in
budding yeast showed that both TOP2 mutant strain lacking the CTD and non-SUMOylatable TOP2 CTD mutant strain yielded failure in the proper localization of Aurora B at the centromeres, which suggests a conserved role of the TOP2 CTD between yeast and vertebrates through SUMO conjugation.6 It is interesting to think about how the SUMO modification on TOP2A can regulate different functions during the same stage in mitosis. The SUMOylation of the K660 within the catalytic core inhibits TOP2A decatenation activity necessary to resolve the entangled centromeric DNA, and therefore, maintains the cohesion of the sister chromatids.4 On the other hand, SUMOylation of the CTD recruits CPC including Aurora B to the centromere via the Haspin-H3T3p pathway for a successful metaphase-to-anaphase transition.5,6 At first glance, these 2 functions of SUMO modification on TOP2A act in opposite manners: one that prevents the sister chromatids from being separated, and one that promotes the transition to anaphase for chromosome segregation. How can these 2 functions of TOP2A SUMO modifications that occur during early mitosis coordinate to lead to the proper segregation of chromosomes into the daughter cells? While they act through different mechanisms, the functions of TOP2A through the SUMOylation at the catalytic core and through the SUMOylation on the CTD work together for the same overall purpose of maintaining the characteristics of metaphase. While the CTD SUMOylation allows for the recruitment of Aurora B to the centromeres for proper kinetochore-microtubule attachment, K660 SUMOylation helps maintain the cohesion of the entangled centromeric DNA until TOP2A is signaled to resolve the entangled chromatids for their separation (Fig. 1). That signal occurs at the onset of anaphase when the SUMO conjugations are removed by the SUMO proteases through de-SUMOylation. The removal of the SUMO modifications at the CTD is also important during anaphase since Aurora B is no longer needed at the mitotic centromeres, but rather, is needed at the spindle midzone. The discovery of the TOP2A-Haspin interaction through SUMOylation not only clarifies how Haspin localizes at the mitotic centromeres to phosphorylate centromeric H3T3, but it also introduces another protein involved in the already complicated recruitment pathways of Aurora B kinase to the mitotic
CONTACT Yoshiaki Azuma [email protected] 1200 Sunnyside Ave., Haworth Hall 3037, Lawrence, KS, 66045 USA. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kccy. Feature to: Yoshida MM, et al. SUMOylation of DNA topoisomerase IIa regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis. J Cell Biol 2016; 213(6):66578; PMID: 27325792; http://dx.doi.org/10.1083/jcb.201511079. © 2016 Taylor & Francis
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Figure 1. Model for the dual role of SUMOylation (green) of centromeric TOP2A (orange) during mitosis. K660 SUMOylation inhibits TOP2A activity to maintain centromeric cohesion of sister chromatids (gray). CTD SUMOylation recruits CPC to centromeres to regulate metaphase-to-anaphase transition via Haspin-H3T3p. TOP2A is de-SUMOylated at all lysine sites at the onset of anaphase and resolves centromeric DNA for chromosome segregation by microtubules (brown).
centromeres. A previous report in budding yeast suggested that a mitotic checkpoint could be activated by affecting the DNA strand passaging reaction activity of TOP2A and transmit the signal for checkpoint activation through the TOP2 CTD.7 These findings support the importance of TOP2A SUMOylation at both the catalytic core and the CTD in proper mitotic progression. Further studies will determine how TOP2 inhibitors that disrupt the normal TOP2A activities affect these functions regulated by SUMOylation.
Disclosure of potential conflict of interests
[3]
[4]
[5]
No potential conflicts of interest were disclosed. [6]
References [1] Ryu H, Furuta M, Kirkpatrick D, Gygi SP, Azuma Y. PIASy-dependent SUMOylation regulates DNA topoisomerase IIa activity. J Cell Biol 2010; 191:783-94; PMID:21079245; http://dx.doi.org/10.1083/ jcb.201004033 [2] Bachant J, Alcasabas A, Blat Y, Kleckner N, Elledge SJ. The SUMO-1 isopeptidase Smt4 is linked to centromeric cohesion through SUMO-
[7]
1 modification of DNA topoisomerase II. Mol Cell 2002; 9:1169-82; PMID:12086615; http://dx.doi.org/10.1016/S1097-2765(02)00543-9 Warsi TH, Navarro MS, Bachant J. DNA topoisomerase II is a determinant of the tensile properties of yeast centromeric chromatin and the tension checkpoint. Mol Biol Cell 2008; 19:4421-33; PMID:18701701; http://dx.doi.org/10.1091/mbc. E08-05-0547 Ryu H, Yoshida MM, Sridharan V, Kumagai A, Dunphy WG, Dasso M, Azuma Y. SUMOylation of the C-terminal domain of DNA topoisomerase IIa regulates the centromeric localization of Claspin. Cell Cycle 2015; 14:2777-84; PMID: 26131587; http://dx.doi.org/10.1080/ 15384101.2015.1066537 Yoshida MM, Ting L, Gygi SP, Azuma Y. SUMOylation of DNA topoisomerase IIa regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis. J Cell Biol 2016; 213:665-78; PMID:27325792; http:// dx.doi.org/10.1083/jcb.201511079 Edgerton H, Johansson M, Keifenheim D, Mukherjee S, Chac on JM, Bachant J, Gardner MK, Clarke DJ. A noncatalytic function of the topoisomerase II CTD in Aurora B recruitment to inner centromeres during mitosis. J Cell Biol 2016; 213:651-64; PMID:27325791; http:// dx.doi.org/10.1083/jcb.201511080 Furniss KL, Tsai H-J, Byl JAW, Lane AB, Vas AC, Hsu W-S, Osheroff N, Clarke DJ. Direct monitoring of the strand passage reaction of DNA topoisomerase II triggers checkpoint activation. PLoS Genet 2013; 9:e1003832; PMID:24098144; http://dx.doi.org/10.1371/journal. pgen.1003832
EDITORIALS: CELL CYCLE FEATURES
Mechanisms behind Topoisomerase II SUMOylation in chromosome segregation Makoto M. Yoshida and Yoshiaki Azuma Department of Molecular Biosciences, University of Kansas, Lawrence, KS, USA ARTICLE HISTORY Received 8 July 2016; Accepted 22 July 2016 KEYWORDS Aurora B; centromere; chromosome segregation; CPC; decatenation; Haspin; SUMO; TOP2
One of the major substrates discovered to be SUMOylated during mitosis is DNA topoisomerase IIa (TOP2A), an enzyme that regulates the topology of genomic DNA. Interestingly, while TOP2A binds throughout the chromosome the majority of the SUMOylated TOP2A is likely clustered at the mitotic centromeres during early mitosis when SUMOylation occurs.1 Genetic studies in budding yeast on the SUMOylation sites of TOP2 have revealed the importance of TOP2A SUMOylation during mitosis. TOP2 CTD has been found to be SUMOylated at multiple lysines, and mutations to prevent SUMOylation at the CTD led to defects in the cohesion of the centromeric DNA in budding yeast, and led to a defect in the spindle checkpoint that senses tension at the microtubules-kinetochore attachment sites.2,3 This suggests that TOP2A CTD SUMOylation may regulate Aurora B kinase, the key regulator of the spindle checkpoint and metaphase-to-anaphase progression. An additional site discovered to be SUMOylated during mitosis is on Xenopus laevis TOP2A lysine 660 (K660) that is within the catalytic core of the enzyme.1 SUMOylation of K660 greatly inhibits TOP2A decatenation activity. The function of this inhibition is likely to maintain the cohesion of the sister chromatids by keeping the entangled centromeric DNA of the sister chromatids from being resolved. Interestingly, biochemical analysis of TOP2A SUMOylation has determined that SUMOylating the CTD lysine sites does not affect TOP2A decatenation activity.4 Therefore, the mechanism behind the regulation of the spindle checkpoint through TOP2A CTD SUMOylation has remained unknown. Not until recently was the pathway of TOP2A CTD SUMOylation in the regulation of Aurora B discovered. Recent work from our laboratory determined that the atypical histone H3 kinase Haspin interacts with TOP2A as a SUMO-binding protein.5 Haspin phosphorylates centromeric histone H3 at threonine 3 (H3T3), which provides a binding site for the chromosomal passenger complex (CPC) including Aurora B at the mitotic centromeres. Our results determined that TOP2A CTD SUMOylation can regulate Haspin, whose centromeric localization mechanism was previously unclear, by recruiting it onto the mitotic chromosomes, and that inhibition of SUMOylation mis-localizes Haspin off of the centromeres in the Xenopus egg extract model system. Additionally, a concurrent study in
budding yeast showed that both TOP2 mutant strain lacking the CTD and non-SUMOylatable TOP2 CTD mutant strain yielded failure in the proper localization of Aurora B at the centromeres, which suggests a conserved role of the TOP2 CTD between yeast and vertebrates through SUMO conjugation.6 It is interesting to think about how the SUMO modification on TOP2A can regulate different functions during the same stage in mitosis. The SUMOylation of the K660 within the catalytic core inhibits TOP2A decatenation activity necessary to resolve the entangled centromeric DNA, and therefore, maintains the cohesion of the sister chromatids.4 On the other hand, SUMOylation of the CTD recruits CPC including Aurora B to the centromere via the Haspin-H3T3p pathway for a successful metaphase-to-anaphase transition.5,6 At first glance, these 2 functions of SUMO modification on TOP2A act in opposite manners: one that prevents the sister chromatids from being separated, and one that promotes the transition to anaphase for chromosome segregation. How can these 2 functions of TOP2A SUMO modifications that occur during early mitosis coordinate to lead to the proper segregation of chromosomes into the daughter cells? While they act through different mechanisms, the functions of TOP2A through the SUMOylation at the catalytic core and through the SUMOylation on the CTD work together for the same overall purpose of maintaining the characteristics of metaphase. While the CTD SUMOylation allows for the recruitment of Aurora B to the centromeres for proper kinetochore-microtubule attachment, K660 SUMOylation helps maintain the cohesion of the entangled centromeric DNA until TOP2A is signaled to resolve the entangled chromatids for their separation (Fig. 1). That signal occurs at the onset of anaphase when the SUMO conjugations are removed by the SUMO proteases through de-SUMOylation. The removal of the SUMO modifications at the CTD is also important during anaphase since Aurora B is no longer needed at the mitotic centromeres, but rather, is needed at the spindle midzone. The discovery of the TOP2A-Haspin interaction through SUMOylation not only clarifies how Haspin localizes at the mitotic centromeres to phosphorylate centromeric H3T3, but it also introduces another protein involved in the already complicated recruitment pathways of Aurora B kinase to the mitotic
CONTACT Yoshiaki Azuma [email protected] 1200 Sunnyside Ave., Haworth Hall 3037, Lawrence, KS, 66045 USA. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kccy. Feature to: Yoshida MM, et al. SUMOylation of DNA topoisomerase IIa regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis. J Cell Biol 2016; 213(6):66578; PMID: 27325792; http://dx.doi.org/10.1083/jcb.201511079. © 2016 Taylor & Francis
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Figure 1. Model for the dual role of SUMOylation (green) of centromeric TOP2A (orange) during mitosis. K660 SUMOylation inhibits TOP2A activity to maintain centromeric cohesion of sister chromatids (gray). CTD SUMOylation recruits CPC to centromeres to regulate metaphase-to-anaphase transition via Haspin-H3T3p. TOP2A is de-SUMOylated at all lysine sites at the onset of anaphase and resolves centromeric DNA for chromosome segregation by microtubules (brown).
centromeres. A previous report in budding yeast suggested that a mitotic checkpoint could be activated by affecting the DNA strand passaging reaction activity of TOP2A and transmit the signal for checkpoint activation through the TOP2 CTD.7 These findings support the importance of TOP2A SUMOylation at both the catalytic core and the CTD in proper mitotic progression. Further studies will determine how TOP2 inhibitors that disrupt the normal TOP2A activities affect these functions regulated by SUMOylation.
Disclosure of potential conflict of interests
[3]
[4]
[5]
No potential conflicts of interest were disclosed. [6]
References [1] Ryu H, Furuta M, Kirkpatrick D, Gygi SP, Azuma Y. PIASy-dependent SUMOylation regulates DNA topoisomerase IIa activity. J Cell Biol 2010; 191:783-94; PMID:21079245; http://dx.doi.org/10.1083/ jcb.201004033 [2] Bachant J, Alcasabas A, Blat Y, Kleckner N, Elledge SJ. The SUMO-1 isopeptidase Smt4 is linked to centromeric cohesion through SUMO-
[7]
1 modification of DNA topoisomerase II. Mol Cell 2002; 9:1169-82; PMID:12086615; http://dx.doi.org/10.1016/S1097-2765(02)00543-9 Warsi TH, Navarro MS, Bachant J. DNA topoisomerase II is a determinant of the tensile properties of yeast centromeric chromatin and the tension checkpoint. Mol Biol Cell 2008; 19:4421-33; PMID:18701701; http://dx.doi.org/10.1091/mbc. E08-05-0547 Ryu H, Yoshida MM, Sridharan V, Kumagai A, Dunphy WG, Dasso M, Azuma Y. SUMOylation of the C-terminal domain of DNA topoisomerase IIa regulates the centromeric localization of Claspin. Cell Cycle 2015; 14:2777-84; PMID: 26131587; http://dx.doi.org/10.1080/ 15384101.2015.1066537 Yoshida MM, Ting L, Gygi SP, Azuma Y. SUMOylation of DNA topoisomerase IIa regulates histone H3 kinase Haspin and H3 phosphorylation in mitosis. J Cell Biol 2016; 213:665-78; PMID:27325792; http:// dx.doi.org/10.1083/jcb.201511079 Edgerton H, Johansson M, Keifenheim D, Mukherjee S, Chac on JM, Bachant J, Gardner MK, Clarke DJ. A noncatalytic function of the topoisomerase II CTD in Aurora B recruitment to inner centromeres during mitosis. J Cell Biol 2016; 213:651-64; PMID:27325791; http:// dx.doi.org/10.1083/jcb.201511080 Furniss KL, Tsai H-J, Byl JAW, Lane AB, Vas AC, Hsu W-S, Osheroff N, Clarke DJ. Direct monitoring of the strand passage reaction of DNA topoisomerase II triggers checkpoint activation. PLoS Genet 2013; 9:e1003832; PMID:24098144; http://dx.doi.org/10.1371/journal. pgen.1003832
