CENP-A and topoisomerase-II antagonistically affect chromosome length

The size of mitotic chromosomes is coordinated with cell size in a manner dependent on nuclear trafficking. In this study, we conducted an RNA interference screen of the Caenorhabditis elegans nucleome in a strain carrying an exceptionally long chromosome and identified the centromere-specific histone H3 variant CENP-A and the DNA decatenizing enzyme topoisomerase-II (topo-II) as candidate modulators of chromosome size. In the holocentric organism C. elegans, CENP-A is positioned periodically along the entire length of chromosomes, and in mitosis, these genomic regions come together linearly to form the base of kinetochores. We show that CENP-A protein levels decreased through development coinciding with chromosome-size scaling. Partial loss of CENP-A protein resulted in shorter mitotic chromosomes, consistent with a role in setting chromosome length. Conversely, topo-II levels were unchanged through early development, and partial topo-II depletion led to longer chromosomes. Topo-II localized to the perimeter of mitotic chromosomes, excluded from the centromere regions, and depletion of topo-II did not change CENP-A levels. We propose that self-assembly of centromeric chromatin into an extended linear array promotes elongation of the chromosome, whereas topo-II promotes chromosome-length shortening.

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Transfection of human topoisomerase IIα into etoposide-resistant cells: transient increase in sensitivity followed by down-regulation of the endogenous gene

Biochemical Journal ◽

10.1042/bj3190307 ◽

1996 ◽

Vol 319 (1) ◽

pp. 307-313 ◽

Cited By ~ 28

Author(s):

Takeshi ASANO ◽

Taeha AN ◽

Janice MAYES ◽

Leonard A. ZWELLING ◽

Eugenie S. KLEINERMAN

Keyword(s):

Topoisomerase Ii ◽

Dexamethasone Treatment ◽

Down Regulation ◽

Topoisomerase Iiα ◽

Endogenous Gene ◽

Protein Levels ◽

Mammalian Expression ◽

Protein Complex Formation ◽

Mmtv Promoter ◽

Topo Ii

We have investigated the possibility of overcoming the resistance of human brain tumour cells (HBT20) to etoposide by transferring the normal human topoisomerase IIα (H-topo II) gene into these cells. H-topo II in a mammalian expression vector containing a glucocorticoid-inducible mouse mammary tumour virus (MMTV) promoter was transfected into etoposide-resistant HBT20 cells (HBT20-hTOP2MAM). HBT20 cells transfected with pMAMneo vector alone served as control cells (HBT20-MAM). These were stable transfections. Following a 2 h dexamethasone treatment, H-topo II mRNA expression, protein production, etoposide-induced DNA-protein complex formation and sensitivity to etoposide were increased in HBT20-hTOP2MAM cells compared with control HBT20-MAM cells and with HBT20-hTOP2MAM cells not treated with dexamethasone. However, mRNA and protein levels and cell sensitivity returned to baseline when incubation with dexamethasone was continued for 24 h. This decrease from the 2 h values could not be explained by a loss of the MMTV promoter response to dexamethasone. (H-topo IIα promoter)-(chloramphenicol acetyltransferase) constructs containing regions -559–0 and -2400–0 were significantly down-regulated in HBT20-hTOP2MAM cells treated for 24 h with dexamethasone compared with dexamethasone-treated control cells. H-topo II mRNA stability after 24 h of dexamethasone treatment was not altered compared with that in control cells. Our data indicate that the exogenously produced H-topo II may have a negative-feedback effect on the endogenous topoisomerase II promoter, causing down-regulation of the endogenous gene.

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Chromosome disentanglement driven via optimal compaction of loop-extruded brush structures

10.1101/616102 ◽

2019 ◽

Cited By ~ 1

Author(s):

Sumitabha Brahmachari ◽

John F. Marko

Keyword(s):

Expression Patterns ◽

Polymer Brush ◽

Chromosome Size ◽

Mitotic Chromosomes ◽

Mechanical Stiffness ◽

Structural Reorganization ◽

Metaphase Chromosomes ◽

Topo Ii ◽

Strand Passage

AbstractEukaryote cell division features a chromosome compaction-decompaction cycle that is synchronized with their physical and topological segregation. It has been proposed that lengthwise compaction of chromatin into mitotic chromosomes via loop extrusion underlies the compaction-segregation/resolution process. We analyze this disentanglement scheme via considering the chromosome to be a succession of DNA/chromatin loops - a polymer “brush” - where active extrusion of loops controls the brush structure. Given topoisomerase (TopoII)-catalyzed topology fluctuations, we find that inter-chromosome entanglements are minimized for a certain “optimal” loop that scales with the chromosome size. The optimal loop organization is in accord with experimental data across species, suggesting an important structural role of genomic loops in maintaining a less entangled genome. Application of the model to the interphase genome indicates that active loop extrusion can maintain a level of chromosome compaction with suppressed entanglements; the transition to the metaphase state requires higher lengthwise compaction, and drives complete topological segregation. Optimized genomic loops may provide a means for evolutionary propagation of gene-expression patterns while simultaneously maintaining a disentangled genome. We also find that compact metaphase chromosomes have a densely packed core along their cylindrical axes that explains their observed mechanical stiffness. Our model connects chromosome structural reorganization to topological resolution through the cell cycle, and highlights a mechanism of directing Topo-II mediated strand passage via loop extrusion driven lengthwise compaction.

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Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts.

The Journal of Cell Biology ◽

10.1083/jcb.120.3.601 ◽

1993 ◽

Vol 120 (3) ◽

pp. 601-612 ◽

Cited By ~ 139

Author(s):

T Hirano ◽

T J Mitchison

Keyword(s):

Topoisomerase Ii ◽

Mitotic Chromosome ◽

Chromosome Morphology ◽

Sperm Chromatin ◽

Mitotic Chromosomes ◽

Egg Extracts ◽

Xenopus Egg ◽

Topo Ii ◽

Xenopus Egg Extracts

We have investigated the role of topoisomerase II (topo II) in mitotic chromosome assembly and organization in vitro using Xenopus egg extracts. When sperm chromatin was incubated with mitotic extracts, the highly compact chromatin rapidly swelled and concomitantly underwent local condensation. Further incubation induced the formation of entangled thin chromatin fibers that eventually resolved into highly condensed individual chromosomes. This in vitro system made it possible to manipulate mitotic chromosomes in their assembly condition without any isolation or stabilization steps. Two complementary approaches, immunodepletion and antibody blocking, demonstrated that topo II activity is required for chromosome assembly and condensation. Once condensation was completed, however, blocking of topo II activity had little effect on the chromosome morphology. Immunofluorescent studies showed that topo II was uniformly distributed throughout the condensed chromosomes and was not restricted to the chromosomal axis. Surprisingly, all detectable topo II molecules were easily extracted from the chromosomes under mild conditions where the shape of chromosomes was well preserved. Our results show that topo II is essential for mitotic chromosome assembly, but does not play a scaffolding role in the structural maintenance of chromosomes assembled in vitro. We also present evidence that changes of DNA topology affect the distribution of topo II in mitotic chromosomes in our system.

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Mitotic chromosomes are constrained by topoisomerase II–sensitive DNA entanglements

The Journal of Cell Biology ◽

10.1083/jcb.200910085 ◽

2010 ◽

Vol 188 (5) ◽

pp. 653-663 ◽

Cited By ~ 38

Author(s):

Ryo Kawamura ◽

Lisa H. Pope ◽

Morten O. Christensen ◽

Mingxuan Sun ◽

Ksenia Terekhova ◽

...

Keyword(s):

Topoisomerase Ii ◽

Mitotic Chromosome ◽

Specific Inhibitor ◽

Elastic Stiffness ◽

Type I ◽

Mitotic Chromosomes ◽

Relaxation Effects ◽

Double Stranded Dna ◽

Topo Ii ◽

Mitotic Chromatin

We have analyzed the topological organization of chromatin inside mitotic chromosomes. We show that mitotic chromatin is heavily self-entangled through experiments in which topoisomerase (topo) II is observed to reduce mitotic chromosome elastic stiffness. Single chromosomes were relaxed by 35% by exogenously added topo II in a manner that depends on hydrolysable adenosine triphosphate (ATP), whereas an inactive topo II cleavage mutant did not change chromosome stiffness. Moreover, experiments using type I topos produced much smaller relaxation effects than topo II, indicating that chromosome relaxation by topo II is caused by decatenation and/or unknotting of double-stranded DNA. In further experiments in which chromosomes are first exposed to protease to partially release protein constraints on chromatin, ATP alone relaxes mitotic chromosomes. The topo II–specific inhibitor ICRF-187 blocks this effect, indicating that it is caused by endogenous topo II bound to the chromosome. Our experiments show that DNA entanglements act in concert with protein-mediated compaction to fold chromatin into mitotic chromosomes.

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Chromosome length and perinuclear attachment constrain resolution of DNA intertwines

The Journal of Cell Biology ◽

10.1083/jcb.201404039 ◽

2014 ◽

Vol 206 (6) ◽

pp. 719-733 ◽

Cited By ~ 21

Author(s):

Iris Titos ◽

Tsvetomira Ivanova ◽

Manuel Mendoza

Keyword(s):

Chromosome Segregation ◽

Topoisomerase Ii ◽

Chromosome Length ◽

Genomic Region ◽

Critical Feature ◽

Topological Constraints ◽

Rdna Array ◽

Topo Ii ◽

Mutant Cells ◽

Segregation Of Chromosomes

To allow chromosome segregation, topoisomerase II (topo II) must resolve sister chromatid intertwines (SCI) formed during deoxynucleic acid (DNA) replication. How this process extends to the full genome is not well understood. In budding yeast, the unique structure of the ribosomal DNA (rDNA) array is thought to cause late SCI resolution of this genomic region during anaphase. In this paper, we show that chromosome length, and not the presence of rDNA repeats, is the critical feature determining the time of topo II–dependent segregation. Segregation of chromosomes lacking rDNA also requires the function of topo II in anaphase, and increasing chromosome length aggravates missegregation in topo II mutant cells. Furthermore, anaphase Stu2-dependent microtubule dynamics are critical for separation of long chromosomes. Finally, defects caused by topo II or Stu2 impairment depend on attachment of telomeres to the nuclear envelope. We propose that topological constraints imposed by chromosome length and perinuclear attachment determine the amount of SCI that topo II and dynamic microtubules resolve during anaphase.

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Linker histone H1.8 inhibits chromatin-binding of condensins and DNA topoisomerase II to tune chromosome length and individualization

eLife ◽

10.7554/elife.68918 ◽

2021 ◽

Vol 10 ◽

Author(s):

Pavan Choppakatla ◽

Bastiaan Dekker ◽

Erin E Cutts ◽

Alessandro Vannini ◽

Job Dekker ◽

...

Keyword(s):

Topoisomerase Ii ◽

Mitotic Chromosome ◽

Chromosome Length ◽

Chromosome Morphology ◽

Linker Histone ◽

Dna Topoisomerase Ii ◽

Dna Amount ◽

Dna Topoisomerase ◽

Topo Ii

DNA loop extrusion by condensins and decatenation by DNA topoisomerase II (topo II) are thought to drive mitotic chromosome compaction and individualization. Here, we reveal that the linker histone H1.8 antagonizes condensins and topo II to shape mitotic chromosome organization. In vitro chromatin reconstitution experiments demonstrate that H1.8 inhibits binding of condensins and topo II to nucleosome arrays. Accordingly, H1.8 depletion in Xenopus egg extracts increased condensins and topo II levels on mitotic chromatin. Chromosome morphology and Hi-C analyses suggest that H1.8 depletion makes chromosomes thinner and longer through shortening the average loop size and reducing the DNA amount in each layer of mitotic loops. Furthermore, excess loading of condensins and topo II to chromosomes by H1.8 depletion causes hyper-chromosome individualization and dispersion. We propose that condensins and topo II are essential for chromosome individualization, but their functions are tuned by the linker histone to keep chromosomes together until anaphase.

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A role of topoisomerase II in linking DNA replication to chromosome condensation

The Journal of Cell Biology ◽

10.1083/jcb.200209023 ◽

2003 ◽

Vol 160 (5) ◽

pp. 645-655 ◽

Cited By ~ 67

Author(s):

Olivier Cuvier ◽

Tatsuya Hirano

Keyword(s):

Dna Replication ◽

Topoisomerase Ii ◽

Atp Hydrolysis ◽

Early Stage ◽

Chromosome Condensation ◽

Binding Property ◽

Dependent Manner ◽

Mitotic Chromosomes ◽

Topo Ii

The condensin complex and topoisomerase II (topo II) have different biochemical activities in vitro, and both are required for mitotic chromosome condensation. We have used Xenopus egg extracts to investigate the functional interplay between condensin and topo II in chromosome condensation. When unreplicated chromatin is directly converted into chromosomes with single chromatids, the two proteins must function together, although they are independently targeted to chromosomes. In contrast, the requirement for topo II is temporarily separable from that of condensin when chromosome assembly is induced after DNA replication. This experimental setting allows us to find that, in the absence of condensin, topo II becomes enriched in an axial structure within uncondensed chromatin. Subsequent addition of condensin converts this structure into mitotic chromosomes in an ATP hydrolysis–dependent manner. Strikingly, preventing DNA replication by the addition of geminin or aphidicolin disturbs the formation of topo II–containing axes and alters the binding property of topo II with chromatin. Our results suggest that topo II plays an important role in an early stage of chromosome condensation, and that this function of topo II is tightly coupled with prior DNA replication.

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Anticancer Activity and Topoisomerase II Inhibition of Naphthalimides with ω-Hydroxylalkylamine Side-Chains of Different Lengths

Medicinal Chemistry ◽

10.2174/1573406414666180912105851 ◽

2019 ◽

Vol 15 (5) ◽

pp. 550-560

Author(s):

Mateusz D. Tomczyk ◽

Anna Byczek-Wyrostek ◽

Klaudia Strama ◽

Martyna Wawszków ◽

Przemysław Kasprzycki ◽

...

Keyword(s):

Anticancer Activity ◽

Cancer Cells ◽

Cell Lines ◽

Inhibitory Activity ◽

Topoisomerase Ii ◽

Significant Activity ◽

Anticancer Activities ◽

Human Colon Cancer ◽

Substitution Pattern ◽

Topo Ii

Background: The substituted 1,8-Naphthalimides (1H-benzo[de]isoquinoline-1,3(2H)- diones) are known as DNA intercalators stabilizing DNA-Topoisomerase II complexes. This interaction disrupts the cleavage-relegation equilibrium of Topo II, resulting in formation of broken strands of DNA. Objective: To investigate the influence of type of substituents and substitution positions in 1,8- naphthalimde skeleton on the inhibition of Topoisomerase II activity. Methods: The starting 1,8-naphthalimide were prepared from acenaphthene by introduction of appropriate substituents followed by condensation with ω-hydroxylakylamines of different chain length. The substituents were introduced to 1,8-naphthalimide molecule by nucleophilic substitution of leaving groups like nitro or bromo present in 4 or 4,5- positions using the ω- hydroxylalkylamines. The bioactivity of obtained compounds was examined in model cell lines. Results: Antiproliferative activity of selected compounds against HCT 116 human colon cancer cells, human non-small cell lung cells A549 and non-tumorigenic BEAS-2B human bronchial epithelium cells was examined. Several of investigated compounds exhibit a significant activity (IC50 µM to 7 µM) against model cancer cell lines. It was demonstrated that upon treatment with concentration of 200 µM, all derivatives display Topo II inhibitory activity, which may be compared with activity of Amonafide. Conclusion: The replacement of the nitro groups in the chromophore slightly reduces its anticancer activities, whereas the presence of both nitro group and ω-hydroxylalkylamine chain resulted in seriously increased anticancer activity. Obtained compounds showed Topo II inhibitory activity, moreover, influence of the substitution pattern on the ability to inhibit Topo II activity and cancer cells proliferation was observed.

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Topoisomerase II is regulated by translationally controlled tumor protein for cell survival during organ growth in Drosophila

Cell Death and Disease ◽

10.1038/s41419-021-04091-y ◽

2021 ◽

Vol 12 (9) ◽

Author(s):

Dae-Wook Yang ◽

Jung-Wan Mok ◽

Stephanie B. Telerman ◽

Robert Amson ◽

Adam Telerman ◽

...

Keyword(s):

Cell Survival ◽

Topoisomerase Ii ◽

Wing Disc ◽

Organ Development ◽

Translationally Controlled Tumor Protein ◽

Protein Levels ◽

Eye Disc ◽

Mutual Regulation

AbstractRegulation of cell survival is critical for organ development. Translationally controlled tumor protein (TCTP) is a conserved protein family implicated in the control of cell survival during normal development and tumorigenesis. Previously, we have identified a human Topoisomerase II (TOP2) as a TCTP partner, but its role in vivo has been unknown. To determine the significance of this interaction, we examined their roles in developing Drosophila organs. Top2 RNAi in the wing disc leads to tissue reduction and caspase activation, indicating the essential role of Top2 for cell survival. Top2 RNAi in the eye disc also causes loss of eye and head tissues. Tctp RNAi enhances the phenotypes of Top2 RNAi. The depletion of Tctp reduces Top2 levels in the wing disc and vice versa. Wing size is reduced by Top2 overexpression, implying that proper regulation of Top2 level is important for normal organ development. The wing phenotype of Tctp RNAi is partially suppressed by Top2 overexpression. This study suggests that mutual regulation of Tctp and Top2 protein levels is critical for cell survival during organ development.

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Synthesis and anticancer evaluation of sulfur containing 9-anilinoacridines

Recent Patents on Anti-Cancer Drug Discovery ◽

10.2174/1574892816666210728122910 ◽

2021 ◽

Vol 16 ◽

Author(s):

Pranav Gupta ◽

Radhika V. Kumar ◽

Chul-Hoon Kwon ◽

Zhe-Sheng Chen

Keyword(s):

Cell Cycle ◽

Anticancer Activity ◽

Topoisomerase Ii ◽

Critical Role ◽

K562 Cells ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

In Vivo Models ◽

Topo Ii ◽

Sulfur Containing

Background: DNA topoisomerases are a class of enzymes that play a critical role in fundamental biological processes of replication, transcription, recombination, repair and chromatin remodeling. Amsacrine (m-AMSA), the best-known compound of 9-anilinoacridines series was one of the first DNA-intercalating agents to be considered as a Topoisomerase II inhibitor. Objective: A series of sulfur containing 9-anilinoacridines related to amsacrine were synthesized and evaluated for their anticancer activity. Methods: Cell viability was assessed by the MTT assay. The topoisomerase II inhibitory assay was performed using the Human topoisomerase II Assay kit and flow cytometry was used to evaluate the effects on cell cycle of K562 cells. Molecular docking was performed using Schrödinger Maestro program. Results: Compound 36 was found to be the most cytotoxic of the sulfide series against SW620, K562, and MCF-7. The limited SAR suggested the importance of the methansulfonamidoacetamide side chain functionality, the lipophilicity and relative metabolic stability of 36 in contributing to the cytotoxicity. Topoisomerase II α inhibitory activity appeared to be involved in the cytotoxicity of 36 through inhibition of decatenation of kinetoplast DNA (kDNA) in a concentration dependent manner. Cell cycle analysis further showed the Topo II inhibition through accumulation of K562 cells in G2/M phase of cell cycle. Docking of 36 into the Topo II α-DNA complex suggested that it may be an allosteric inhibitor of Topo II α. Conclusion: Compound 36 exhibits anticancer activity by inhibiting topoisomerase II and it could further be evaluated in in vivo models.

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