Activating the abscission checkpoint: Top2α senses chromatin bridges in cytokinesis

How chromatin bridges are detected by the abscission checkpoint during mammalian cell division is unknown. Here, we discuss recent findings from our lab showing that the DNA topoisomerase IIα (Top2α) enzyme binds to catenated (“knotted”) DNA next to the midbody and forms abortive Top2‐DNA cleavage complexes (Top2ccs) on chromatin bridges. Top2ccs are then processed by the proteasome to promote localization of the DNA damage sensor protein Rad17 to Top2‐generated double‐strand DNA ends on DNA knots. In turn, Rad17 promotes local recruitment of the MRN protein complex and downstream ATM‐Chk2‐INCENP signaling to delay abscission and prevent chromatin bridge breakage in cytokinesis.


INTRODUCTION
During mitotic cell division, chromosome segregation is followed by the separation of the cytoplasm to two daughter cells through cytokinesis.In animal cells, cytokinesis starts with ingression of the actomyosin contractile ring to generate a cleavage furrow between the two spindle poles and ends with abscission, in which the narrow intercellular canal is cut to release the two daughter cells.As a result, abscission is required for cell proliferation and its mis-regulation can cause DNA damage and cancer formation. [1]r abscission to occur, remodeling of the plasma membrane at the constriction sites and reorganization of the cytoskeleton inside the intercellular canal are required.The Endosomal Sorting Complex Required for Transport (ESCRT) machinery that constricts and cuts membranes delivers the membrane scission step during abscis-This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.© 2024 The Authors.BioEssays published by Wiley Periodicals LLC.sion.More specifically, in late cytokinesis in mammalian cells, the microtubule bundling protein Cep55 localizes to the midbody where it promotes recruitment of ESCRT-III subunits, such as Chmp4b, through several adaptor proteins. [1,2]Just before abscission, ESCRT-III polymers form spiral structures with progressively smaller diameters that extend from the midbody to the abscission site to perform membrane scission; furthermore, this reorganization requires ESCRT binding to the ATPase Vps4 which promotes remodeling of the ESCRT-III filaments by subunit turnover. [1,2]In parallel, ESCRT proteins recruit microtubule severing enzymes to the future abscission site to coordinate membrane cutting with local disassembly of midbody microtubules. [2]romatin bridges are long DNA fibers stretching between the two segregating masses of chromosomes and have been linked to tumorigenesis. [3]They can arise from segregation of catenated chromosomes after incomplete DNA replication or imperfect resolution of double-strand DNA catenates, or from chromosome end-to-end fusions resulting from telomere crisis. [3]In the presence of chromatin bridges in cytokinesis, wild-type eukaryotic cells delay abscission to prevent chromatin breakage or tetraploidization by regression of the cleavage furrow, which are associated with genomic instability and cancer predisposition. [2]The abscission delay in response to chromosome segregation defects was first identified in budding yeast and termed "NoCut." [4]In higher eukaryotic cells, the abscission delay in response to chromatin bridges is called the "abscission checkpoint" and is dependent on the catalytic activity of Aurora B kinase at the midbody. [2]rora B is the catalytic component of the Chromosomal Passenger Complex (CPC), a tetrameric protein complex that also includes the scaffolding protein INCENP and the nonenzymatic subunits Survivin and Borealin.The CPC localizes to specific sites at different stages of mitosis to coordinate proper chromosome segregation with cytokinesis.In late cytokinesis in human cells, the CPC localizes to the central area of the midbody where it imposes the abscission checkpoint. [2,5]r this purpose, Aurora B phosphorylates the ESCRT-III subunit Chmp4c at several serine residues and this phosphorylation sustains a ternary complex of Chmp4c with Vps4 and the Abscission/NoCut checkpoint regulator (ANCHR) protein. [2]It is proposed that formation of the above complex delays relocalization of Vps4 from the midbody to the secondary ingression site where it is required for membrane scission by Chmp4b filaments, thus delaying abscission; however, inhibition of additional abscission executor proteins is likely required to fully implement the abscission checkpoint delay. [2]e MRN (Mre11-Rad50-Nbs1) double-strand break sensor complex that processes DNA ends regulates Aurora B localization to the midbody in cytokinesis with chromatin bridges. [5]More specifically, the MRN complex recruits the DNA damage ataxia-telangiectasia mutated (ATM) kinase at the midbody.ATM phosphorylates and activates the effector kinase Chk2; in turn, Chk2 phosphorylates INCENP-serine 91 to promote stable binding of INCENP to Mklp2 kinesin, to induce localization of the CPC-Mklp2 complex to the midbody through Mklp2-interaction with the central midbody protein Cep55 and to delay abscission. [5]However, how chromatin bridges are sensed and promote localization of the MRN complex to the midbody has remained elusive.In a recent paper, Petsalaki et al.
shed new light on this important mechanism that safeguards genome integrity. [6] analyzing human cell lines in cytokinesis with high resolution confocal microscopy, Petsalaki et al. showed that spontaneous or replication-stress induced chromatin bridges exhibit DNA "knots" containing catenated and supercoiled DNA next to the ring-shaped midbody (Figure 1). [6]The authors found that the DNA topoisomerase IIα (Top2α), an enzyme that can relax DNA supercoils and untangle catenated DNA molecules, localizes to DNA knots where it forms irreversible Top2 cleavage complexes (Top2ccs) exhibiting covalent Top2α-DNA adducts and Top2α-associated double-strand DNA ends. [6]The MRN, ATM and Chk2 proteins also localize to DNA knots, in agreement with the reported role of these proteins in abscission F I G U R E 1 Chromatin bridge sensing by the abscission checkpoint in human cells.Cartoon of a cell undergoing cytokinesis in the presence of a chromatin bridge derived from catenated DNA connecting the two daughter nuclei.An enlarged area of the intercellular canal around the midbody (MB) ring through which the chromatin bridge passes is shown.In a recent study, [6]  checkpoint signaling. [5]Furthermore, Chk2 also localizes to the midbody ring, which is consistent with Chk2 phosphorylating the CPC subunit INCENP at the midbody. [5]By inhibiting Top2α or by reconstituting human cells with mutant Top2α proteins, the authors showed that the DNA cleavage, but not decatenation, activity of Top2α is required for proper localization of MRN, ATM, Chk2, and CPC proteins to DNA knots/the midbody, and for stable chromatin bridges in cytokinesis. [6]These results show that Top2α recognizes knotted DNA on chromatin bridges and activates the abscission checkpoint by generating Top2α-linked double-strand DNA ends on the bridge DNA next to the midbody.
The authors then went on to further dissect the molecular mechanism involved.Top2-linked DNA ends are usually processed by the proteasome to activate downstream responses.Consistently, the authors proposed that Top2α molecules that are covalently bound to DNA knots are ubiquitinated.Furthermore, inhibition of the proteasome or ubiquitination enzymes impaired localization of the MRN complex to DNA knots and promoted chromatin bridge breakage in cytokinesis, indicating that ubiquitination and degradation of Top2α which is bound to DNA ends on DNA knots is required to expose the Top2αlinked double-strand DNA ends to downstream proteins, to activate the abscission checkpoint. [6]e authors also identified the DNA damage sensor protein Rad17 as an adaptor that tethers the MRN complex to DNA knots.Rad17 localizes to DNA knots on a Top2α-dependent manner; furthermore, Rad17 is required for MRN binding to DNA knots and stable chromatin bridges in cytokinesis. [6]To verify a role for Rad17 in recruiting the MRN complex on chromatin bridges, the authors engineered an siRNAresistant Top2α protein that targets Rad17 to DNA knots without cleaving the DNA. [6]Expression of this fusion protein rescued localization of MRN proteins to DNA knots and restored abscission checkpoint signaling in Top2α-deficient cells, showing that Rad17 implements the Top2α-dependent abscission checkpoint by promoting binding of the MRN complex to DNA knots.
By using an inducible cell line that produces uncapped chromosome ends, the authors also investigated whether dicentric chromatin bridges activate the abscission checkpoint in human cells. [6]They found that dicentric chromosomes generated by telomere end-fusion lack DNA knots, fail to recruit Top2α and downstream abscission checkpoint proteins to the midbody, and break in cytokinesis.Significantly, introducing DNA knots on dicentric chromosome bridges by DNA replication stress rescued localization of Top2α and abscission checkpoint proteins to the bridge DNA/midbody, and prevented chromatin bridge breakage in cytokinesis. [6]Collectively, the above findings show that in chromatin bridges exhibiting catenated DNA, but not in dicentrics, Top2α localizes to DNA knots and forms irreversible Top2ccs next to the midbody.These complexes are then processed by the proteasome to recruit Rad17 on Top2α-linked double-strand DNA ends.In turn, Rad17 recruits the MRN complex to DNA knots and promotes ATM-Chk2-CPC signaling, resulting in inhibition of abscission executer proteins at the midbody ring by the CPC to delay abscission and prevent chromatin bridge breakage in cytokinesis (Figure 1).
The findings by Petsalaki et al. are the first to describe a mechanism by which human cells detect chromatin bridges to activate the abscission checkpoint.Interestingly, they also highlight essential differences in the respective mechanisms between budding yeast and animal cells: In budding yeast, chromatin bridges induced by DNA replication stress, chromosome condensation or decatenation defects, but not dicentrics, lead to mitotic spindle stabilization during ring contraction and subsequent binding of Ipl1/Aurora to the chromosomal DNA at the midzone to impose the abscission delay. [4,7]In human cells on the other hand, Top2α forms stable Top2ccs on knotted DNA next to the midbody to activate abscission checkpoint signaling. [6]These distinct mechanisms may be due to fundamental differences in the mechanics of cell division between budding yeast and higher eukaryotes: Because yeast cells do not disassemble their nuclear envelope during the cell cycle, binding of Aurora B to chromosomal DNA may represent an efficient way to coordinate chromatin-sensing at the midzone with the implementation of the abscission delay while the integrity of the nucleus is also preserved.Animal cells on the other hand, perform an open mitosis and disassemble their nuclear membrane at the beginning of mitosis.In these cells, reformation of the nucleus after chromosome segregation requires Aurora B removal from chromatin giving rise to chromosome decondensation, and mitotic spindle disassembly by coordinated actions of ESCRT-III, Vps4 and spastin leading to local resealing of the nuclear envelope. [8,9]Therefore, one possibility is that, by uncoupling chromatin bridge sensing from Aurora B binding to the DNA and from mitotic spindle stabilization, animal cells can quickly reform their nuclear envelope while also maintaining an abscission delay in the presence of chromatin bridges.This could be important for preserving genome integrity because prolonged exposure of the chromosomal DNA to the cytoplasmic environment can promote DNA damage and mutagenesis. [10]tivation of the abscission checkpoint by detection of catenated DNA by Top2α can also explain why, contrary to budding yeast, [4] lagging chromosomes trapped inside the intercellular canal do not delay abscission in human cells.In higher eukaryotes, lagging chromosomes are often generated by merotelic kinetochore attachments in which a single kinetochore binds to microtubules coming from both spindle poles.As a result, lagging chromosomes are not expected to exhibit DNA knots and are unable to recruit Top2α to elicit an abscission checkpoint response.Thus, human cells favor segregation of merotelic chromosomes to the correct spindle pole by employing mechanisms that increase the probability of a high force differential between the correct and erroneous side of the lagging chromosome, in the absence of an abscission delay.Similarly, dicentric chromosome bridges generated by telomere end-fusion are likely properly condensed and decatenated; as a result, they do not induce an abscission delay in human cells.However, why dicentrics fail to activate NoCut in yeast is unclear and might be due to inability of this type of bridges to prevent spindle disassembly in cytokinesis. [7]e findings of Petsalaki et al. also raise several questions: For example, why are DNA knots typically found next to the midbody?It will be interesting to examine whether this is because DNA knots slide along the DNA molecule and become jammed at the narrow midbody ring, and also whether the close proximity of DNA knots with the midbody is important for an efficient abscission delay.How is the abscission delay signal transmitted from the chromatin bridge DNA, in the presence of a nuclear membrane, [5,6] to the midbody ring in the cytoplasm?Because Chk2 was detected both at DNA knots and at the midbody ring, it will be interesting to investigate whether a fraction of Chk2 dissociates from the DNA knot and exits to the cytoplasm to phosphorylate INCENP at the midbody.Can chromatin bridges that recruit Top2α be resolved before abscission and, if so, what is the DNA repair mechanism that could facilitate chromatin bridge resolution?How does the cell coordinate activation of the abscission checkpoint with generation of actin rich structures at the base of the intercellular canal ("actin patches"), which stabilize chromatin bridges? [2]Also, can the abscission checkpoint be employed for cancer therapy?For this purpose, it will be important to investigate whether inhibition of Topoisomerase II catalytic activity by anticancer drugs can sensitize cancer cells to replication stress-induced DNA bridges by relatively low doses of DNA polymerase inhibitors.This strategy may selectively target chromosomally unstable cancer cells while being potentially less toxic for normal tissues. [2]The discovered mechanism of chromatin bridge sensing by Top2α will be essential in addressing these significant questions in the future.
Petsalaki et al. show that Top2α covalently binds to "knots" of catenated DNA next to the midbody and generates Top2α-linked double-strand (ds) DNA ends.Proteasomal degradation of Top2α leads to the recruitment of Rad17 on DNA knots.Then, Rad17 recruits the Mre11-Rad50-Nbs1 (MRN) complex on DNA knots to activate local MRN-ATM-Chk2 signaling.A fraction of Chk2 also localizes to the midbody ring (not shown) where it phosphorylates the Chromosomal Passenger Complex subunit INCENP to promote stable recruitment of INCENP-Aurora B to the midbody ring.As a result, the INCENP-Aurora B complex inhibits abscission executer proteins at the midbody (red line) to delay abscission and prevent chromatin bridge breakage in cytokinesis.PM, plasma membrane; MB, midbody; NE, nuclear envelope.