Dataset Information

Topoisomerase II? is essential for maintenance of mitotic chromosome structure.

ABSTRACT: Topoisomerase II? (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in mitosis have been difficult to decipher due to its multiple functions across the cell cycle. To more precisely understand the role of TOP2A in mitosis, we used the auxin-inducible degron (AID) system to rapidly degrade the protein at different stages of the human cell cycle. Removal of TOP2A prior to mitosis does not affect prophase timing or the initiation of chromosome condensation. Instead, it prevents chromatin condensation in prometaphase, extends the length of prometaphase, and ultimately causes cells to exit mitosis without chromosome segregation occurring. Surprisingly, we find that removal of TOP2A from cells arrested in prometaphase or metaphase cause dramatic loss of compacted mitotic chromosome structure and conclude that TOP2A is crucial for maintenance of mitotic chromosomes. Treatments with drugs used to poison/inhibit TOP2A function, such as etoposide and ICRF-193, do not phenocopy the effects on chromosome structure of TOP2A degradation by AID. Our data point to a role for TOP2A as a structural chromosome maintenance enzyme locking in condensation states once sufficient compaction is achieved.

SUBMITTER: Nielsen CF

PROVIDER: S-EPMC7275761 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Topoisomerase IIα is essential for maintenance of mitotic chromosome structure.

Nielsen Christian F CF Zhang Tao T Barisic Marin M Kalitsis Paul P Hudson Damien F DF

Proceedings of the National Academy of Sciences of the United States of America 20200515 22

Topoisomerase IIα (TOP2A) is a core component of mitotic chromosomes and important for establishing mitotic chromosome condensation. The primary roles of TOP2A in mitosis have been difficult to decipher due to its multiple functions across the cell cycle. To more precisely understand the role of TOP2A in mitosis, we used the auxin-inducible degron (AID) system to rapidly degrade the protein at different stages of the human cell cycle. Removal of TOP2A prior to mitosis does not affect prophase ti ...[more]

PMID: 32414923

Similar Datasets

Project description:IntroductionThe nuclear enzyme topoisomerase IIα (TopoIIα) is able to cleave DNA in a reversible manner, making it a valuable target for agents such as etoposide that trap the enzyme in a covalent bond with the 5' DNA end to which it cleaves. This prevents DNA religation and triggers cell death in cancer cells. However, development of resistance to these agents limits their therapeutic use. In this study, we examined the therapeutic targeting of geminin for improving the therapeutic potential of TopoIIα agents.MethodsHuman mammary epithelial (HME) cells and several breast cancer cell lines were used in this study. Geminin, TopoIIα and cell division cycle 7 (Cdc7) silencing were done using specific small interfering RNA. Transit or stable inducible overexpression of these proteins and casein kinase Iε (CKIε) were also used, as well as several pharmacological inhibitors that target TopoIIα, Cdc7 or CKIε. We manipulated HME cells that expressed H2B-GFP, or did not, to detect chromosome bridges. Immunoprecipitation and direct Western blot analysis were used to detect interactions between these proteins and their total expression, respectively, whereas interactions on chromosomal arms were detected using a trapped in agarose DNA immunostaining assay. TopoIIα phosphorylation by Cdc7 or CKIε was done using an in vitro kinase assay. The TopoGen decatenation kit was used to measure TopoIIα decatenation activity. Finally, a comet assay and metaphase chromosome spread were used to detect chromosome breakage and changes in chromosome condensation or numbers, respectively.ResultsWe found that geminin and TopoIIα interact primarily in G2/M/early G1 cells on chromosomes, that geminin recruits TopoIIα to chromosomal decatenation sites or vice versa and that geminin silencing in HME cells triggers the formation of chromosome bridges by suppressing TopoIIα access to chromosomal arms. CKIε kinase phosphorylates and positively regulates TopoIIα chromosome localization and function. CKIε kinase overexpression or Cdc7 kinase silencing, which we show phosphorylates TopoIIα in vitro, restored DNA decatenation and chromosome segregation in geminin-silenced cells before triggering cell death. In vivo, at normal concentration, geminin recruits the deSUMOylating sentrin-specific proteases SENP1 and SENP2 enzymes to deSUMOylate chromosome-bound TopoIIα and promote its release from chromosomes following completion of DNA decatenation. In cells overexpressing geminin, premature departure of TopoIIα from chromosomes is thought to be due to the fact that geminin recruits more of these deSUMOylating enzymes, or recruits them earlier, to bound TopoIIα. This triggers premature release of TopoIIα from chromosomes, which we propose induces aneuploidy in HME cells, since chromosome breakage generated through this mechanism were not sensed and/or repaired and the cell cycle was not arrested. Expression of mitosis-inducing proteins such as cyclin A and cell division kinase 1 was also increased in these cells because of the overexpression of geminin.ConclusionsTopoIIα recruitment and its chromosome decatenation function require a normal level of geminin. Geminin silencing induces a cytokinetic checkpoint in which Cdc7 phosphorylates TopoIIα and inhibits its chromosomal recruitment and decatenation and/or segregation function. Geminin overexpression prematurely deSUMOylates TopoIIα, triggering its premature departure from chromosomes and leading to chromosomal abnormalities and the formation of aneuploid, drug-resistant cancer cells. On the basis of our findings, we propose that therapeutic targeting of geminin is essential for improving the therapeutic potential of TopoIIα agents.

Dataset Information

Topoisomerase II? is essential for maintenance of mitotic chromosome structure.

Publications

Topoisomerase IIα is essential for maintenance of mitotic chromosome structure.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets