InteractiveFly: GeneBrief

Topoisomerase 2: Biological Overview | References

Gene name - Topoisomerase 2

Synonyms -

Cytological map position - 37E1-37E1

Function - enzyme

Keywords - ATP-dependent homodimeric enzyme that transiently cleaves double stranded DNA, passes a second DNA double helix through the break, and then reseals the break - plays a role in homolog association in meiosis - modulates insulator function

Symbol - Top2

FlyBase ID: FBgn0284220

Genetic map position - chr2L:19,447,418-19,453,490

Classification - DNA topoisomerase 2-like protein

Cellular location - nuclear

NCBI link: EntrezGene
Top2 orthologs: Biolitmine
Recent literature
Apte, M.S. and Meller, V.H. (2015). Sex differences in Drosophila melanogaster heterochromatin are regulated by non-sex specific factors. PLoS One 10: e0128114. PubMed ID: 26053165
The eukaryotic genome is assembled into distinct types of chromatin. Gene-rich euchromatin has active chromatin marks, while heterochromatin is gene-poor and enriched for silencing marks. In spite of this, genes native to heterochromatic regions are dependent on their normal environment for full expression. Expression of genes in autosomal heterochromatin is reduced in male flies mutated for the noncoding roX RNAs, but not in females. roX mutations also disrupt silencing of reporter genes in male, but not female, heterochromatin, revealing a sex difference in heterochromatin. This study adopted a genetic approach to determine how this difference is regulated, and found no evidence that known X chromosome counting elements, or the sex determination pathway that these control, are involved. This suggested that the sex chromosome karyotype regulated autosomal heterochromatin by a different mechanism. To address this, candidate genes that regulate chromosome organization were examined. In XX flies mutation of Topoisomerase II (Top2), a gene involved in chromatin organization and homolog pairing, made heterochromatic silencing dependent on roX, and thus male-like. Interestingly, Top2 also bound to a large block of pericentromeric satellite repeats (359 bp repeats) that are unique to the X chromosome. Deletion of X heterochromatin also made autosomal heterochromatin in XX flies dependent on roX and enhanced the effect of Top2 mutations, suggesting a combinatorial action. The study postulates that Top2 and X heterochromatin in Drosophila comprise a novel karyotype-sensing pathway that determines the sensitivity of autosomal heterochromatin to loss of roX RNA.

Piskadlo, E., Tavares, A. and Oliveira, R. A. (2017). Metaphase chromosome structure is dynamically maintained by Condensin I-directed DNA (de)catenation. Elife 6. PubMed ID: 28477406
Mitotic chromosome assembly remains a big mystery in biology. Condensin complexes are pivotal for chromosome architecture yet how they shape mitotic chromatin remains unknown. Using acute inactivation approaches and live-cell imaging in Drosophila embryos, this study dissects the role of condensin I in the maintenance of mitotic chromosome structure with unprecedented temporal resolution. Removal of condensin I from pre-established chromosomes results in rapid disassembly of centromeric regions while most chromatin mass undergoes hyper-compaction. This is accompanied by drastic changes in the degree of sister chromatid intertwines. While wild-type metaphase chromosomes display residual levels of catenations, upon timely removal of condensin I, chromosomes present high levels of de novo Topoisomerase II (TopoII)-dependent re-entanglements, and complete failure in chromosome segregation. TopoII is thus capable of re-intertwining previously separated DNA molecules and condensin I continuously required to counteract this erroneous activity. It is proposed that maintenance of chromosome resolution is a highly dynamic bidirectional process.
Tang, X., Cao, J., Zhang, L., Huang, Y., Zhang, Q. and Rong, Y. S. (2017). Maternal Haploid, a metalloprotease enriched at the largest satellite repeat and essential for genome integrity in Drosophila embryos. Genetics [Epub ahead of print]. PubMed ID: 28615282
The incorporation of the paternal genome into the zygote during fertilization requires chromatin remodeling. The maternal haploid (mh) mutation in Drosophila affects this process and leads to the formation of haploid embryos without the paternal genome. mh encodes the Drosophila homolog of SPRTN, a conserved protease essential for resolving DNA-protein cross-linked products. This study characterized the role of MH in genome maintenance. It is not understood how MH protects the paternal genome during fertilization particularly in lights of the finding that MH is present in both parental pro-nuclei during zygote formation. Maternal chromosomes in mh-mutant embryos were shown to experience instabilities in the absence of the paternal genome, which suggests that MH is generally required for chromosome stability during embryogenesis. This is consistent with the finding that MH is abundantly present on chromatin throughout the cell cycle. Remarkably, MH is prominently enriched at the 359bp satellite repeats during interphase, which becomes unstable without MH. This dynamic localization and specific enrichment of MH at the 359 repeats resemble that of Topoisomerase 2 (Top2), suggesting that MH regulates Top2 possibly as a protease for the resolution of Top2-DNA intermediates. It is proposed that maternal MH removes proteins specifically enriched on sperm chromatin. In the absence of that function, paternal chromosomes are precipitously lost. This mode of paternal chromatin remodeling is likely conserved and the unique phenotype of the Drosophila mh mutants represents a rare opportunity to gain insights into the process that has been difficult to study.
Yang, D. W., Mok, J. W., Telerman, S. B., Amson, R., Telerman, A. and Choi, K. W. (2021). Topoisomerase II is regulated by translationally controlled tumor protein for cell survival during organ growth in Drosophila. Cell Death Dis 12(9): 811. PubMed ID: 34453033
Regulation 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, a human Topoisomerase II (TOP2) was identified as a TCTP partner, but its role in vivo has been unknown. To determine the significance of this interaction, their roles in developing Drosophila organs was examined. 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.
Vicars, H., Karg, T., Warecki, B., Bast, I. and Sullivan, W. (2021). Kinetochore-independent mechanisms of sister chromosome separation. PLoS Genet 17(1): e1009304. PubMed ID: 33513180
Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, this study discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, 117 RNAi gene knockdowns were screened for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on this analysis of EB1 localization and knockdown phenotypes, it is proposed that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.
Brand, C. L. and Levine, M. T. (2022). Cross-species incompatibility between a DNA satellite and the Drosophila Spartan homolog poisons germline genome integrity. Curr Biol. PubMed ID: 35643081
Satellite-rich genomic regions mediate strictly conserved, essential processes such as chromosome segregation and nuclear structure. A leading resolution to this paradox posits that satellite DNA and satellite-associated chromosomal proteins coevolve to preserve these essential functions. This study experimentally test this model of intragenomic coevolution by conducting the first evolution-guided manipulation of both chromosomal protein and DNA satellite. The 359bp satellite spans an 11 Mb array in Drosophila melanogaster that is absent from its sister species, Drosophila simulans. This species-specific DNA satellite colocalizes with the adaptively evolving, ovary-enriched protein, Maternal haploid (MH), the Drosophila homolog of Spartan. To determine if MH and 359bp coevolve, the D.simulans version of MH ("MH[sim]") was swapped into D.melanogaster. MH[sim] triggers ovarian cell death, reduced ovary size, and loss of mature eggs. Surprisingly, the D.melanogaster mh-null mutant has no such ovary phenotypes, suggesting that MH[sim] is toxic in a D.melanogaster background. Using both cell biology and genetics, it was discovered that MH[sim] poisons oogenesis through a DNA-damage pathway. Remarkably, deleting the D.melanogaster-specific 359bp satellite array completely restores mh[sim] germline genome integrity and fertility, consistent with a history of coevolution between these two fast-evolving loci. Germline genome integrity and fertility are also restored by overexpressing topoisomerase II (Top2), suggesting that MH[sim] interferes with Top2-mediated processing of 359bp. The observed 359bp-MH[sim] cross-species incompatibility supports a model under which seemingly inert repetitive DNA and essential chromosomal proteins must coevolve to preserve germline genome integrity.

DNA topoisomerases have been classified into two different categories: type I enzymes, which mediate the transient breakage of one DNA strand at a time; and type II enzymes, which generate nicks in both DNA strands. Among the first class, type IB DNA topoisomerases (like budding yeast Top1) are very efficient in relaxing positively supercoiled naked DNA molecules and could contribute to the removal of positive supercoils accumulating at unreplicated regions. Type II enzymes (like yeast Top2) can relax positively overwounded DNA molecules and could also reduce the torsional stress by resolving sister chromatid intertwining at precatenate nodes (Postow, 2001; Wang 2002). Type II enzymes are also involved in chromosome condensation and segregation during mitosis. Type IA enzymes (such as budding yeast Top3) seem to be preferentially implicated in recombinational DNA repair, although it has been proposed that they could also act in replication termination (Postow, 2001; Wang 2002; Bermejo, 2007 and references therein).

To investigate how DNA topoisomerases deal with the topological constrains of replicating chromosomes and their range of action within the topological domains generated by active replicons in Saccharomyces cerevisiae, this study analyzed the timing and the sites of recruitment of Top1 and Top2 on chromosomes. How Top1 and Top2 dysfunctions alter the dynamics of replication forks, and how the DNA damage checkpoint responds to the resulting topological abnormalities was also investigated. This study shows that while both Top1 and Top2 localize in close proximity to replication forks, Top2 exhibits additional clusters at specific intergenic loci in unreplicated DNA regions, mostly at promoters. TOP1 ablation does not affect fork progression and stability and does not lead to activation of the Rad53-dependent DNA damage checkpoint. Conversely, top2 mutants accumulate during S-phase cruciform DNA structures tethering sister chromatids, without affecting fork progression, and activate Rad53 upon completion of mitosis. The simultaneous attenuation of Top1 and Top2 causes the block of replication forks, their aberrant mass reduction, and the generation of checkpoint signals leading to Rad53 activation in S phase. Ablation of the Exo1 exonuclease counteracts the mass reduction of replication intermediates in top1 top2 double mutants and delays the accumulation of active Rad53 in top1 top2 cells (Bermejo, 2007).

Altogether, the results indicate that Top1 and Top2 act coordinately within a 600-base-pair (bp) region spanning the moving forks to assist their progression and prevent their pathological processing. The fork-confined action of Top1 and Top2 would counteract the diffusion of topological changes along large chromosomal regions, thus protecting chromatin integrity, and would prevent the generation of aberrant replication intermediates causing DNA damage checkpoint activation and, possibly, chromosome breakage during segregation (Bermejo, 2007).

The analysis of mutant alleles of different strength reveals multiple functions of topoisomerase 2 in regulation of Drosophila chromosome structure

Topoisomerase II is a major component of mitotic chromosomes but its role in the assembly and structural maintenance of chromosomes is rather controversial, as different chromosomal phenotypes have been observed in various organisms and in different studies on the same organism. In contrast to vertebrates that harbor two partially redundant Topo II isoforms, Drosophila and yeasts have a single Topo II enzyme. In addition, fly chromosomes, unlike those of yeast, are morphologically comparable to vertebrate chromosomes. Thus, Drosophila is a highly suitable system to address the role of Topo II in the assembly and structural maintenance of chromosomes. This study shows that modulation of Top2 function in living flies by means of mutant alleles of different strength and in vivo RNAi results in multiple cytological phenotypes. In weak Top2 mutants, meiotic chromosomes of males exhibit strong morphological abnormalities and dramatic segregation defects, while mitotic chromosomes of larval brain cells are not affected. In mutants of moderate strength, mitotic chromosome organization is normal, but anaphases display frequent chromatin bridges that result in chromosome breaks and rearrangements involving specific regions of the Y chromosome and 3L heterochromatin. Severe Top2 depletion resulted in many aneuploid and polyploid mitotic metaphases with poorly condensed heterochromatin and broken chromosomes. Finally, in the almost complete absence of Top2, mitosis in larval brains was virtually suppressed and in the rare mitotic figures observed chromosome morphology was disrupted. These results indicate that different residual levels of Top2 in mutant cells can result in different chromosomal phenotypes, and that the effect of a strong Top2 depletion can mask the effects of milder Top2 reductions. Thus, these results suggest that the previously observed discrepancies in the chromosomal phenotypes elicited by Topo II downregulation in vertebrates might depend on slight differences in Topo II concentration and/or activity (Mengoli, 2014).

Type II topoisomerases are large ATP-dependent homodimeric enzymes that transiently cleave double stranded DNA, pass a second DNA double helix through the break, and then reseal the break (Wang, 2002; Nitiss, 2009). In this way, Topo II enzymes solve a variety of topological problems that normally arise in double stranded DNA during processes such as replication, transcription, recombination and sister chromatid segregation. Topo II enzymes are structurally and functionally conserved, and the genomes of all eukaryotes harbor at least one Topo II enzyme. Vertebrates have two Topo II isoforms, alpha and beta; these enzymes have identical catalytic activities but distinct localization patterns during mitosis. The beta isoform is primarily cytoplasmic, while most of Topo II alpha is concentrated in mitotic chromosomes. In contrast, yeast and Drosophila have a single Topoisomerase II (Top2) gene. Notably, both the Drosophila Top2 and each of the human Topo II genes can rescue the phenotype of yeast Top2 mutants, highlighting the strong functional conservation of type II topoisomerases (Mengoli, 2014 and references therein).

Topo II alpha is a major component of vertebrate mitotic chromosomes. In vivo studies have shown that Topo II alpha has a dynamic behavior and that chromosome-associated Topo II alpha is rapidly exchanged with the cytoplasmic pool. In fixed mitotic chromosomes, Topo II alpha exhibits a discontinuous localization pattern with Topo II alpha alternating with cohesin along chromatid axes (Maeshima, 2003; Samejima, 2012). There is also evidence that in some systems Topo II alpha accumulates at centromeres in prometaphase and metaphase, suggesting a role of this enzyme in the regulation of centromere structure and/or cohesion (Mengoli, 2014).

Studies in yeast have shown that Top2 is not required for completion of DNA synthesis but plays essential roles in mitotic chromosome condensation and sister chromatid segregation. Failure to decatenate sister chromatids results in anaphase chromatin bridges that cause chromosome breakage during anaphase or cytokinesis. Loss of Topo II activity does not affect S phase progression and disrupts sister chromatid separation also in vertebrate cells. However, the role of Topo II in vertebrate chromosome structure is rather controversial, possibly due to species-specific differences in chromosome organization and/or the different methods used to inhibit Topo II function (chemical inhibitors, immunodepletion, mutations or RNAi). For example, treatment of Indian muntjac cells with the Topo II inhibitor ICRF-193 caused frequent failures in sister chromatid individualization, the process by which duplicated DNA is resolved into two distinct chromatids. In contrast, Topo II inhibition with ICRF-193 did not affect sister chromatid resolution in both Chinese hamster ovary cells (CHO) and baby hamster kidney (BHK) cells. Moreover, Topo II alpha inhibition in different vertebrate systems resulted in a variety of chromosome morphologies ranging from relatively mild effects on the axial compaction of chromosomes to severe defects in chromosome condensation (Mengoli, 2014 and references therein).

Although Topo II beta is not normally able to compensate for Topo II alpha loss, overexpression of Topo II beta in human cells can correct the defects caused by Topo II alpha depletion. In addition, it has been shown that DT40 avian cells and human cells depleted of both Topo II alpha and Topo II beta display chromosomal defects more severe than those observed in cells lacking Topo II alpha alone. These results suggest that the two Topo II isoforms are partially redundant. Thus, studies of cells depleted of both Topo II alpha and Topo II beta are particularly relevant to define the role of Topo II in the maintenance of proper chromosomal architecture. An analysis of DT40 avian cells conditionally depleted of both Topo II isoforms showed that they exhibit extensive anaphase chromatin bridges, defective cytokinesis and polyploid cells. Similar defects were observed in human cells lacking both Topo II alpha and Topo II beta. In addition, cytological analysis showed that the latter cells exhibit severe defects in chromosome structure ranging from chromosome entangling to disrupted chromosome morphology (Mengoli, 2014).

Another controversial issue is the existence of a decatenation checkpoint triggered by loss of Topo II activity. The existence of such a checkpoint was suggested by studies in human cells showing that catalytic inhibitors of Topo II such as ICRF-187 or ICFR-193 are able to induce a caffeine-sensitive G2 delay that is dependent on ATR and BRCA1, but apparently independent of the DNA damage checkpoint. However, a decatenation checkpoint is not present in both S. cerevisiae and S. pombe, in which Top 2 mutations cause minimal cell cycle delays. Recent RNAi-based studies have also shown that depletion of Topo II alpha alone or both Topo II alpha and II beta does not trigger a decatenation checkpoint in vertebrate cells. In addition, no G2 delay was observed in Topo II-depleted vertebrate cells that were also treated with ICRF-193 . In contrast, expression of certain mutant forms of Top2 resulted in a G2 arrest in budding yeast cells. Collectively, these results indicate that loss of Topo II, and thus DNA catenation per se, is not able to induce a cell cycle delay and that a G2 checkpoint is instead activated by specific DNA lesions caused by catalytically inactive forms of Topo II (Mengoli, 2014).

Several studies have addressed the role of Top2 in Drosophila. Early work showed that injection of anti-Top2 antibodies or Top2 inhibitors into live Drosophila embryos result in strong defects in chromosome condensation and sister chromatid segregation at anaphase (Buchenau, 1993). Two RNAi-based studies on S2 tissue culture cells showed that Top2-depleted fixed cells exhibit defects in longitudinal compaction of chromosomes, defective sister chromatid segregation and extensive anaphase bridges (Chang, 2003; Somma, 2008). Another study performed on live S2 cells, in which Top2 activity was reduced by either RNAi or chemical inhibition, did not detect defects in chromosome condensation and suggested that Top2 is required for centromere resolution and to prevent incorrect microtubule-kinetochore attachment (Coelho, 2008). Remarkably, downregulation of Top2 did not affect the mitotic index, indicating that Top2 deficiency does not activate cell cycle checkpoints in S2 cells (Chang, 2003; Coelho, 2008). Other studies suggested that Drosophila Top2 is involved in homolog pairing in cell cultures (Williams, 2007), modulation of insulator function (Ramos, 2011), and regulation of polytene chromosome structure (Hohl, 2012; Mengoli, 2014 and references therein).

Surprisingly, the role of Top2 in the maintenance of mitotic and meiotic chromosome structure in living flies has never been investigated. In a previous study viable mutants were identified in the solofuso (suo) gene. suo1 and suo2 mutant spermatocytes exhibit severely defective ana-telophases with extensive chromatin bridges; these telophases give rise to achromosomal secondary spermatocytes that are able to assemble bipolar spindles and divide in the complete absence of chromosomes (Bucciarelli, 2003). This study shows that suo1 and suo2 are weak mutant alleles of the Top2 gene and describes the isolation and characterization of a stronger Top2 mutation. Modulation of Top2 function by means of these mutant alleles and in vivo RNAi results are shown to result in multiple cytological phenotypes including site-specific chromosome aberrations, heterochromatin undercondensation, polyploidy, and complete disruption of chromosome morphology accompanied by a cell cycle arrest. These phenotypes recapitulate most of the phenotypes observed in vertebrate cells and indicate that Drosophila chromosomes are exquisitely sensitive to the residual level of Top2 in the cell (Mengoli, 2014).

This study shows that mutations in Top2 affect chromatin organization within the primary spermatocyte nuclei. During prophase I, wild type spermatocytes exhibit 3 main distinct chromatin clusters, which correspond to the major Drosophila bivalents (X-Y, 2-2 and 3-3); the small fourth chromosome bivalent is either separated from these chromatin masses or associated with the X-Y bivalent. At early growth stages (S1 and S2) the chromatin distribution within the spermatocyte nuclei of Top2suo1/Df males was not substantially different from wild type, suggesting that spermatogonial divisions are not severely affected. However, at stages S4 and S5 mutant spermatocytes displayed approximately twice as many chromatin masses as their wild type counterparts. In addition, in most mutant nuclei masses of similar size were closely apposed, suggesting a separation of the homologs within each chromatin territory. In the subsequent stages of spermatocyte growth, the number of chromatin masses in mutant nuclei progressively decreased, so that at prometaphase they displayed 3 compact chromatin clumps like their wild type counterparts (Mengoli, 2014).

Chromosome behavior during spermatocyte growth and male meiosis has been investigated in previous studies, which revealed a complex pairing mechanism. The X and the Y pair through their rDNA regions, while no specific euchromatic or heterochromatic pairing sites have been identified for the major autosomes, which are thus likely to exploit a homology-based pairing mechanism. Tagging of allelic chromosome sites using the GFP-Lac repressor/lacO system or fluorescent in situ hybridization (FISH) showed that the major autosomes are tightly paired during the S1 and S2 stages. However, pairing is suddenly lost at the S2/S3 transition; the chromosomes remain then unpaired throughout the rest of meiosis but are included in a common nuclear territory until they condense prior to meiotic division. One open problem is the mechanism underlying homolog co-mingling within the territories. Such co-mingling is unlikely to be the result of a canonical meiotic pairing, as the homologs remain uncondensed throughout prophase. It has been thus postulated that during early prophase the homologs might be held together in a single territory by chromatin entanglements. The results of this study are consistent with this idea and lead to a hypothesis that Topo II plays an active role in generating the entanglements that mediate homolog association. However, it is equally possible that Topo II is required for some kind of chromatin modifications that are important for homolog conjunction within the territories (Mengoli, 2014).

The chromatin organization defects within the prophase nuclei of Top2suo1/Df spermatocytes are very different from those previously observed in mutants of the condensins Cap-H2 and Cap-D3. In these mutants the chromatin remains diffuse within the spermatocyte nuclei from stage S4 through S6, indicating that the Cap-H2 and Cap-D3 condensin II subunits are required for the formation of the intranuclear territories that comprise the homologous chromosomes. The chromatin organization defect in Top2suo1/Df spermatocytes is also different from that caused by mutations in genes mediating achiasmate homolog pairing in Drosophila males (teflon, MNM and SNM). Spermatocytes of mutants in these genes display diffuse and slightly expanded chromatin territories during stages S4-S6; at prometaphase they show up to eight distinct chromatin clumps corresponding to unpaired univalents (Mengoli, 2014).

Collectively, the available results suggest that condensins (Cap-H2 and Cap-D3), the proteins required for homolog conjunction (teflon, MNM and SNM) and Top2 play distinct roles in chromatin organization during spermatocyte growth. As previously suggested, condensins are essential for territory formation and appear to function in opposition to homolog conjunction. Top2, Teflon, MNM and SNM are all required for proper territory formation and homolog pairing. Top2 is primarily required for homolog conjunction and correct territory organization during stages S4-S6 of spermatocyte growth, whereas Teflon, MNM and SNM are primarily required for meiotic chromosome pairing during prometaphase and metaphase. However, Top2 might have a redundant role in metaphase chromosome pairing that would be masked by the activity of Teflon, MNM and SNM, which would be able to mediate homolog pairing even when Top2 is reduced. The finding that Top2 mediates an aspect of homologous chromosome pairing in males is intriguing, as this enzyme ensures proper biorientation of achiasmatic homologs in females (Hughes and Hawley, 2014). Thus, despite the profound differences between Drosophila male and female meiosis, both types of meiotic divisions share a common Top2-depedent mechanism to facilitate achiasmate chromosome pairing (Mengoli, 2014).

Previous studies have shown that loss of Topo II function results in species-specific meiotic defects. In top2 mutant cells of S. cerevisiae, premeiotic DNA synthesis, recombination and chromosome condensation are not affected but cells arrest at metaphase I and do not undergo the first meiotic division. However, top2 rad50 double mutants, in which recombination and synaptonemal complex formation are suppressed, perform the first meiotic division but not the second. A similar meiotic phenotype has been observed in Top2 mutant cells of S. pombe, which exhibit only a mild defect in the final steps of meiotic chromosome condensation and arrest at metaphase. This arrest is relieved by mutations in rec7 that strongly reduce recombination. Thus, in both budding and fission yeast, Topo II has little or no role in chromosome condensation but its activity is required for segregation of recombinant chromosomes at meiosis I and, at least in S. cerevisiae, for sister chromatid separation at meiosis II (Mengoli, 2014 and references therein).

Studies on meiotic cells from mouse and Chinese hamster injected with Topo II inhibitors did not reveal gross defects in chromosome condensation at the doses used in the experiments. However, the inhibitors induced a substantial meiotic delay and resulted in anaphase bridges and lagging chromosomes at both the first and the second meiotic anaphase. It has been also suggested that the defect in homolog separation at meiosis I was due to a primary defect in chiasmata resolution. In contrast, studies on mouse pachytene spermatocyte cultured in vitro showed that treatments with the Topo II inhibitors ICRF-193 and teniposide cause drastic defects in chromosome condensation. In teniposide-treated spermatocytes, both chromatin condensation and sister chromatid individualization were strongly affected. The effects of ICRF-193 were milder and some chromosomes managed to condense reaching a diplotene-like configuration (Mengoli, 2014).

This study has shown that weak Drosophila Top2 mutants (Top2suo1/Top2suo1 and Top2suo2/Df) with virtually no defects in brain cell mitoses exhibit strong defects in chromosome segregation during both meiotic divisions of males (this report and Bucciarelli, 2003). In addition, this study has shown that in Top2suo1/Df and Top2suo1/Top2suo3 testes all meiotic divisions exhibit severe defects in chromosome structure and segregation. In most cells, the chromosomes formed amorphous metaphase I masses where the sister chromatids were no longer discernible. In addition, these chromatin masses often emanated protrusions that are likely to correspond to stretched pericentric regions. Despite the strong defect in chromosome structure, Drosophila spermatocytes did not arrest at metaphase like yeast cells or mouse spermatocytes treated with teniposide. This finding is consistent with the limited delay caused by the spindle checkpoint in Drosophila male meiosis and by the inability of this checkpoint to prevent spermatid formation and differentiation. Given that Drosophila male meiosis is achiasmatic, these observations strongly suggest that the aberrant meiotic chromosome segregation observed in Top2 mutant males is the consequence of a primary defect in chromatin folding within the chromosomes. This defect is particularly evident when the chromosomes are pulled away by the meiotic spindle. In the anaphase-like figures of Top2 mutants, the chromatids are not individualized and the spindle poles are connected by an irregular network of chromatin threads. This suggests that the chromatin fibers of both the homologous and the heterologous chromosomes as well as those of the sister chromatids remain trapped by multiple entanglements, which prevent correct chromosome segregation during both meiotic divisions (Mengoli, 2014).

The observation that Top2 is required for homolog conjunction during early meiotic prophase and then for correct chromosome segregation during anaphase is intriguing. The hypothesis is favored that Top2 has two independent activities, one required for catenation of the homologs within each chromosome territory and one required for proper chromatin folding within the metaphase chromosomes. However it cannot be excluded that Top 2 activity during early prophase results in aberrant chromatin configurations that interfere with the chromatin folding processes leading to proper chromosome assembly (Mengoli, 2014).

Frequent anaphase bridges have been also observed in spermatocytes from mutants in the Cap-H2 and Cap-D3 genes. However, the morphology of the meiotic chromosomes of these mutants is not severely disrupted as occurs in Top2 mutants. Judging from the published micrographs, the chromatids of Cap-H2 and Cap-D3 mutants are clearly individualized and their appearance is not very different from that of their wild type counterparts. These data do not provide an explanation of why the meiotic chromosomes of Drosophila males are much more sensitive to Top2 depletion than brain chromosomes. It can only be envisage that the two types of chromosomes contain different proteins and/or different numbers of high affinity Top2 binding sites. If these binding sites were more frequent in meiotic than in mitotic chromosomes, then a ~50% reduction of the Top2 protein would be sufficient to disrupt meiotic chromosome organization in males, but would have only a limited effect on mitotic chromosomes of larval brains (Mengoli, 2014).

A meiotic phenotype reminiscent of that seen in Top2 mutant males has been observed after RNAi-mediated depletion of Top2 in females (Hughes and Hawley, 2014). In wild type female meiosis, the heterochromatic regions of the homologous chromosomes remain connected during prometaphase I by chromatin threads that ensure proper biorientation of achiasmatic homologues; these homologous connections are then resolved at later stages of meiosis allowing chromosome segregation. In Top2- depleted oocytes, heterochromatic regions of chromosomes usually fail to separate during prometaphase and metaphase I, and are often stretched into long protrusions with centromeres at their tips (Hughes and Hawley and references therein). These findings indicate that Top2 is required for resolution of the DNA entanglements that normally connect homologous heterochromatic regions during female meiosis and suggest that the pulling forces exerted by the spindle generate chromatin protrusions. However, the meiotic phenotypes elicited by Top2 depletion in males and females are similar but not identical. While in female meiosis only the heterochromatic regions appear to be affected, in male meiosis both euchromatin and heterochromatin are affected. A higher sensitivity of heterochromatin to Top2 depletion is consistent with observations on Top2suo1/Df and Top2suo1/Top2suo3 mitotic cells, which exhibit chromosome breaks that preferentially involve the 3L and the Y heterochromatin (Mengoli, 2014).

This study has shown that relatively weak mutant combinations of Top2 alleles (Top2suo1/Df and Top2suo1/Top2suo3) only exhibit chromosome aberrations (CABs), most of which involve specific regions of the Y and third chromosome heterochromatin. Severe RNAi-mediated Top2 depletion results in extensive chromosome breakage involving all chromosome regions with a preference for heterochromatin. Previous studies with pharmacological inhibitors of Topo II have also shown that treatments with these drugs cause CABs, but it is currently unclear to which extent these drugs directly induce DNA lesions, cause DNA damage via Topo II inhibition, or affect DNA stability through other off target effects (Nitiss, 2009). However, CABs have been also observed in Top2 mutants of budding and fission yeasts and in vertebrate cells depleted of Topo II by RNAi. In both yeast and vertebrate systems, most CABs induced by Topo II deficiency are thought do be produced by breakage of the anaphase chromatin bridges generated by failure to decatenate sister chromatids (Mengoli, 2014).

This study has shown that a relatively modest reduction of the Top2 level results in many isochromatid breaks and chromosome exchanges (translocations and dicentric chromosomes) that primarily involve 4 regions of the entirely heterochromatic Y chromosome (regions h1-2; h4-6, h19-21 and h24-25) and a specific region of the 3L heterochromatin (region h47). Previous studies did not detect site-specific chromosome aberrations after inhibition of Topo II function. What is then the mechanism underlying the chromosome damage specificity in weak Top2 Drosophila mutants? Two observations help answering this question. First, in mutant brain cells not treated with colchicine, 37% of the anaphases displayed chromatin bridges or lagging chromosome fragments generated by severing of the bridges. Second, most CABs observed in colchicine-treated cells were 'incomplete' chromosome type aberrations (i.e involving both sister chromatids). Namely, they consisted in broken centric chromosomes not accompanied by a corresponding acentric fragment, in normal chromosome complements with an additional acentric fragment, in Y-3 translocations lacking the reciprocal element, or Y-3 dicentric chromosomes lacking the acentric fragment. These aberrations are likely to be the consequence of chromosome breaks generated during the anaphase of the previous cell cycle. It is proposed that these breaks preferentially occur in chromosomal sites whose stability is particularly dependent on Top2-mediated DNA decatenation. In Top2 deficient cells, these sites would not be properly untangled and would break when the sister chromatids are pulled apart by the mitotic spindle. It has been shown that a prominent Top2 cleavage target is the 359 bp Drosophila satellite DNA, which is mainly found in the centric heterochromatin of the X chromosome. The extant maps of satellite DNA and transposable element distribution along Drosophila heterochromatin were examined but no sequence was found that uniquely maps to the Top2-sensitive regions. Thus these regions might share an as yet unidentified DNA or might correspond to junctions between different DNA sequences (e. g. satellite-satellite; satellite transposon, or transposon-transposon) (Mengoli, 2014).

Top2 RNAi brain cells contain very small amounts of Top2 and exhibit only a few divisions, most of which are hyperploid or polyploid. The few scorable diploid figures almost invariably displayed incomplete aberrations involving the Y or third chromosome heterochromatin, often accompanied by breaks in other chromosomes. It was not possible to assess the presence of incomplete aberrations in polyploid metaphases, most of which displayed many apparent breaks of the centric heterochromatin of the major autosomes. These discontinuities in chromosome structure could be either due to drastic failures of heterochromatin condensation or to real breaks generated by the rupture of chromatin bridges during anaphase. The results do not permit discrimination between these possibilities, but the first is favored (Mengoli, 2014).

Observations on different Top2 mutant combinations and Top2 RNAi cells revealed different and apparently contradictory effects on cell cycle progression. In Top2suo1/Df brains that exhibit a ~60% reduction in the wild type Top2 level, the mitotic index (MI) was comparable to that of wild type controls, but mutant brains displayed an increase in the frequency of anaphases. These data are consistent with previous studies on Drosophila S2 cells showing that RNAi-mediated depletion of Top2 does not affect the MI and causes only a small increase in the anaphase frequency. The MI was not substantially affected also in DT40 and human cells depleted of both Topo II alpha and Topo II beta (Mengoli, 2014).

In contrast, in Top2 RNAi brains and Topsuo3/Df brains the MI was reduced by one and two orders of magnitude, respectively. Top2 RNAi brains also displayed many aneuploid and polyploid cells. Polyploidy has been also observed in chicken and human cells lacking Topo II activity, and was attributed either to defects in cytokinesis or to a reentry into interphase following a mitotic arrest (restitution). The low MI and the extensive chromosome damage in RNAi brains did not allow reliable pinpointing of the mechanism of polyploid cells formation. Polyploidy in Drosophila brains can be generated by either restitution or cytokinesis failure. It is thus possible that the polyploid cells of Top2 RNAi brains were generated through both mechanisms (Mengoli, 2014).

The observations on weak Top2 mutants (Top2suo1/Df and Top2suo1/Top2suo3; this study) and Top2 RNAi S2 cells (Chang, 2003; Coelho, 2003) strongly suggest that Drosophila does not have a decatenation checkpoint that arrests cell cycle in response to loss of Top2 function. This conclusion agrees with recent data indicating that Topo II depletion and the resulting excess of DNA catenation does not trigger a G2 arrest in vertebrate cells. However, a catenation-independent but Topo II-dependent checkpoint is activated by interruptions of the decatenation process caused by catalytically inactive forms of Topo II (Mengoli, 2014 and references therein).

This study found that in Top2suo3/Df and Top2 RNAi brains the MI is drastically reduced, indicating that cells are blocked in interphase. This study also showed that this block is not relieved by mutations in either mei-41 (ATR) or tefu (ATM). Because these kinases are involved in the signaling pathways that mediate most cell cycle checkpoints, and because ATR has been previously implicated in the decatenation checkpoint, it is believed that the interphase block observed in the nearly complete absence of Top2 is not due to the activation of a checkpoint. It is instead believed that this block could depend on the failure to remove supercoils during DNA replication, which would cause extensive DNA damage and make the cell unable to sustain cell cycle progression (Mengoli, 2014).

Mutations in Top2 have been shown to cause a specific alteration of the X chromosome morphology in male polytene chromosomes. Hohl (2012) has shown that in polytene nuclei of Top2 mutants the X chromosome retains the ability to recruit the MSL dosage compensation complex. In agreement with this, it was found that the bloated X chromosomes of Top2 mutants are decorated by anti-Mle, anti-Msl3 and anti-Mof antibodies. However, it was not possible to assess whether the staining intensity is the same as that of a normally condensed wild type X. Thus, it is quite possible that a reduction in Top2 expression partially affects the association of the MSL complex with the male X chromosome as recently suggested (Cugusi, 2013). Regardless of the role of Top2 in recruitment and/or stabilization of the MSL complex, the observation that loss or inhibition of Top2 activity specifically disrupts the X chromosome morphology in males is fully consistent with the ChIP/Mass Spec experiments indicating that Top2 is the major MSL interactor (Wang, 2013) and with the co-IP assays showing that Top2 interacts with MSL through its Mle component (Cugusi; Mengoli, 2014 and references therein).

Previous studies have shown that the X chromosome of polytene nuclei from Top2 mutants is decorated by antibodies against histone H4 acetylated at lysine 16 (H4K16ac). This post-translational modification is mediated by the Mof histone acetyltransferase, whose association with the X chromosome depends on Mle; a mutation in mle or blocking H4k16 acetylation rescues the X chromosome condensation defects caused by mutations in ISWI. This study found that the X chromosome phenotype elicited by mutations in Top2 is rescued in mof; Top2 double mutants, and that both the bloated X of Top2 mutants and the reconstituted X of mof; Top2 double mutants normally recruit the ISWI protein. These results suggest that loss of Top2 does not affect condensation of the dosage compensated chromatin by inhibiting ISWI recruitment. However, in the absence of Mof-mediated H4k16 acetylation the chromatin compaction functions of Top2 and ISWI are both dispensable. The genetic interaction between Top2 and mof is consistent with the previously shown physical and functional interactions between topoisomerase II and histone deacetylases (HDACs) (Nitiss, 2009), and with the synergistic cytotoxic effects caused by simultaneous inhibition of HDAC and Topo II (Mengoli, 2014).

This study has shown that meiotic chromosomes are extremely sensitive to Top2 depletion and exhibit drastic defects in chromosome morphology even in weak Top2 mutants. Top2 downregulation in brain mitotic cells by either mutations or in vivo RNAi produced different cytological phenotypes. Moderate Top2 depletion (Top2suo1/Df) did not affect chromosome structure, and produced site-specific chromosome aberrations generated by the rupture of anaphase bridges. Severe Top2 depletion (Top2 RNAi) strongly reduces the MI and induces heterochromatin undercondensation, extensive chromosome breakage, aneuploidy and polyploidy. Finally, complete (or nearly complete) Top2 deficiency (Top2suo3/Df) caused an interphase block and disrupted chromatid individualization in the rare diving cells. These phenotypes indicate that Drosophila chromosomes are exquisitely sensitive to the residual level of Top2 in the cell. In addition, they recapitulate most, if not all, phenotypes previously observed in vertebrate cells exposed to Topo II inhibitors or RNAi against Topo II. Thus, the results suggest that previously observed discrepancies in vertebrate chromosome phenotypes elicited by Topo II downregulation might depend on the type of chromosomes examined (e.g. mitotic vs meiotic), slight differences in Topo II activity, or both (Mengoli, 2014).

Topoisomerase II is required for the proper separation of heterochromatic regions during Drosophila melanogaster female meiosis

Heterochromatic homology ensures the segregation of achiasmate chromosomes during meiosis I in Drosophila melanogaster females, perhaps as a consequence of the heterochromatic threads that connect achiasmate homologs during prometaphase I. This study asked how these threads, and other possible heterochromatic entanglements, are resolved prior to anaphase I. The knockdown of Topoisomerase II (Top2) by RNAi in the later stages of meiosis results in a specific defect in the separation of heterochromatic regions after spindle assembly. In Top2 RNAi-expressing oocytes, heterochromatic regions of both achiasmate and chiasmate chromosomes often failed to separate during prometaphase I and metaphase I. Heterochromatic regions were stretched into long, abnormal projections with centromeres localizing near the tips of the projections in some oocytes. Despite these anomalies, bipolar spindles were observed in most Top2 RNAi-expressing oocytes, although the obligately achiasmate 4th chromosomes exhibited a near complete failure to move toward the spindle poles during prometaphase I. Both achiasmate and chiasmate chromosomes displayed defects in biorientation. Given that euchromatic regions separate much earlier in prophase, no defects were expected or observed in the ability of euchromatic regions to separate during late prophase upon knockdown of Top2 at mid-prophase. Finally, embryos from Top2 RNAi-expressing females frequently failed to initiate mitotic divisions. These data suggest both that Topoisomerase II is involved in the resolution of heterochromatic DNA entanglements during meiosis I and that these entanglements must be resolved in order to complete meiosis (Hughes, 2014).

This study has shown that knockdown of Top2 during prophase of meiosis I in Drosophila oocytes results in defects in homolog segregation and sterility. Heterochromatic regions of all four chromosomes failed to properly separate, leading to a failure of chromosomes to properly biorient during meiosis I. Additionally, achiasmate chromosomes showed defects in their ability to move away from the autosomes towards the spindle poles in prometaphase I. Instead, abnormal chromosomal projections were present. These DNA projections displayed several differences compared to the heterochromatic threads observed in wild-type prometaphase I oocytes. Specifically, the projections did not appear to directly connect two chromosomes and, more importantly, contained centromeres, which are not present in wild-type DNA threads. These attributes suggest that chromosomes initiate centromere-led movement but are anchored by DNA entanglements at the center of the spindle, resulting in the stretching out of chromosomal regions (Hughes, 2014).

One might imagine that one component of these defects reflects a role of Top2 in resolving chiasmata. Indeed, in yeast, conditional mutants of top2 caused a meiotic cell cycle arrest that was partially alleviated by simultaneously eliminating recombination, indicating that in yeast, some of the targets of Top2 during meiosis are recombination dependent. Several lines of evidence suggest that this is not the case in Drosophila. First, the matαGAL driver used in the Drosophila oocytes does not appear to be strongly expressed until after recombination is thought to be finished. Second, recombination is suppressed in heterochromatic regions near the centromeres. Therefore, it seems more likely that the defects in heterochromatic separation during Top2 knockdown are due to the failure to resolve the heterochromatic threads observed during prometaphase I rather than a failure of Top2 to resolve DNA entanglements during the repair of double-strand breaks initiated in early prophase. Third, the AATAT heterochromatic repeat of the 4th chromosomes also shows defects in separation. Since the 4th chromosomes, which never undergo crossing over and are thus obligately achiasmate, also fail to properly separate heterochromatic regions, the defects in 4th chromosome heterochromatin separation cannot be due to homologous connections formed as a result of recombination. Finally, the heterochromatic threads observed during prometaphase I in Drosophila oocytes are not dependent on recombination, since they are observed in mutants of the Drosophila spo11 homolog, mei-W68. While Top2 appears to affect the heterochromatic regions by resolving DNA entanglements that are recombination independent, it is unknown whether or not Top2 plays a role in the resolution of chiasmata at anaphase I. Since knockdown of Top2 results in metaphase I arrest, a role at anaphase I in chiasmata resolution cannot be assessed under these conditions (Hughes, 2014).

The observation that 4th chromosome sequences are present within the DNA projections in Drosophila oocytes is not surprising given that the 4th chromosomes often move precociously towards the spindle poles during prometaphase I. However, it is more difficult to explain the stretching of 3rd chromosome heterochromatic sequences into some projections and the multiple CID foci within the projections. These results may indicate that the centromeres of the chiasmate chromosomes are also attempting to move towards the poles in Top2 RNAi-expressing oocytes. One possibility is that these projections are the consequence of a failed attempt by the oocyte to separate the heterochromatic regions. The four heterochromatic regions examined varied in the extent that they failed to separate. These differences may be due to a difference in the number of DNA entanglements between homologous heterochromatic regions in the oocytes (Hughes, 2014).

The 359-bp repeat region displayed the highest failure to separate upon Top2 knockdown. Several lines of evidence have suggested that the 359-bp heterochromatic region of the X may be handled differently by the cell than other heterochromatic regions. First, Ferree (2009) demonstrated that the hybrid lethality between D. simulans females and D. melanogaster males is due to the formation of anaphase bridges containing the 359-bp repeat region during mitosis in hybrid embryos. The authors speculate that D. simulans may lack factors for proper condensation of the 359-bp region. More recently, Ferree (2014) demonstrated that the lethality caused by some circularized X-Y ring chromosomes is also due to anaphase bridging of the 359-bp repeat in embryos. In both instances, Top2 localized to the anaphase bridges. Additionally, during the mitotic divisions of the Drosophila germarium, the 359-bp region is highly paired while the AACAC and Dodeca regions of the 2nd and 3rd chromosomes are mostly unpaired (Christophorou, 2013). These results, as well as the complete failure of the 359-bp region of the X to separate in over 90% of oocytes when Top2 is knocked down, suggest that the 359-bp region may be especially prone to form DNA entanglements during replication (both between homologs and sisters) or that these entanglements may be processed differently than those in other heterochromatic regions. Additionally, in vivo studies of Top2 cleavage sites during mitosis in Drosophila showed a major cleavage site in the 359-bp repeat (Kas, 1992). A failure to cleave this site when Top2 levels are decreased in meiosis I may contribute to the high failure of the 359-bp repeat to separate in Top2 RNAi oocytes (Hughes, 2014).

In a parallel study examining decreased Top2 levels during Drosophila male meiosis, in which recombination does not occur (Mengoli, 2014) observed phenotypes similar to those seen in this study in oocytes. Homologs, as well as sister chromatids, frequently failed to separate during meiosis I and meiosis II in Drosophila males, despite the formation of bipolar spindles similar to Drosophila oocytes. Homologs were stretched out into anaphase bridges at meiosis I, a phenotype which has similarities to the stretched out chromosomal projections in oocytes. These results indicate that Topoisomerase II may resolve similar DNA connections in both sperm and oocytes. Thus, the cells are responding in a similar fashion to deal with the failure of the resolution of these homologous connections. It is worth noting, however, that Mengoli observed defects in euchromatic regions as well as heterochromatic regions of the chromosomes during male meiosis, while in oocytes, only heterochromatic regions were affected. This difference likely reflects the fact that Mengoli examined mutations affecting Top2 levels at the start of meiosis while in Drosophila females the Top2 RNAi construct is not expressed until mid-late prophase (Hughes, 2014).

Decreasing the level of Top2 in mitotic cell types causes phenotypes that are both similar to and divergent from those phenotypes observed during Drosophila female and male meiosis (Mengoli, 2014). Top2 RNAi in mitotic Drosophila S2 cells led to the formation of DNA projections (Chang, 2003). Although these chromosomal projections appeared similar to the ones in oocytes, CID foci were not observed within the DNA projections in S2 cells, while one or more CID foci were present in the projections in oocytes. This suggests that while the projections in oocytes are at least partially centromere led, the S2 projections are composed of the arms of the chromosomes. Top2 RNAi expression in S2 cells results in extensive chromosome lagging, chromosome bridging during anaphase, and chromosome missegregation (Coelho, 2008). Oocytes expressing Top2 RNAi seem to arrest in metaphase I before chromosome bridging can manifest, but chromosome missegregation is evident in oocytes as well (Hughes, 2014).

It should also be noted that knockdown of Top2 in Kc cells leads to a decrease in euchromatic pairing without affecting the pairing of heterochromatic regions (Williams, 2007; Joyce, 2012). However, evidence suggests Top2 is acting in different ways in each system. In meiosis, heterochromatic regions remain associated at higher levels than euchromatic regions during mid to late prophase (Dernburg, 1996), while euchromatic pairings persist longer than heterochromatic pairings during mitosis in cultured cells. The current data suggest that the lack of heterochromatin dissociation is due to the failure of Top2 to resolve DNA entanglements within the heterochromatin, while there is no evidence for the persistence of similar entanglements between euchromatic regions upon knockdown of Top2 in cell culture (Hughes, 2014).

The study by Mengoli (2014) suggests that different phenotypes manifest in Drosophila larval neuroblasts depending on the residual level of Top2. Additionally, different cell types, for example sperm, were more sensitive to decreases in Top2 levels. This study, as well as others in Drosophila, illustrates the complexity of understanding the function of Top2 in resolving various types of DNA entanglements. For example, expression of the Top2 RNAi construct using the nanos-Gal4:VP16 driver, which is expressed beginning in germline stem cells, led to minimal ovarian development, suggesting that high levels of Top2 expression are likely necessary to resolve DNA entanglements caused by replication in the germline stem cell divisions and/or the cystoblast divisions. This hinders the examination of the role of Top2 in such processes as replication, recombination, and chromosome condensation early in oogenesis (Hughes, 2014).

Knocking down topoisomerase II enzymes using RNAi or chemically inhibiting its two isoforms in mitotic human cell lines leads to a number of defects, including entangled chromosomes, chromosome segregation defects, cell cycle delays, and in some cases cell cycle arrest. Most interesting is that chemically inhibiting Topoisomerase IIα in HeLa cells has been reported to increase the number and duration of PICH (Plk1-interacting checkpoint helicase)-positive ultrafine DNA bridges that connect centromeres during anaphase of mitosis, including to non-centromeric regions of the chromosomes. These results indicate that Topoisomerase II enzymes resolve DNA entanglements prior to anaphase in addition to those observed at the centromeres in mitotic cells. These PICH-positive ultrafine bridges have several similarities to the DNA threads observed during prometaphase I of Drosophila oocytes, in that they are composed of heterochromatin and connect segregating chromosomes. While mitotic DNA entanglements are between sister chromatids and some of the meiotic entanglements are likely between homologs, the results suggest that Topoisomerase II enzymes may play a conserved role in resolving chromosomal entanglements in mitosis and meiosis (Hughes, 2014).

Determining the mechanism by which Topoisomerase II functions to resolve mitotic entanglements may provide insight into its potential role in resolving the meiotic. In HeLa cells, centromeric cohesion appears to protect centromeric DNA threads from resolution until anaphase I when this cohesion is lost, and a similar mechanism is believed to protect centromeric concatenations at centromeres until anaphase II during the mouse male meiotic divisions (Hughes, 2014).

Based on these studies, it is at least possible that in Drosophila oocytes entanglements may form during replication in both heterochromatic and euchromatic regions, but euchromatic entanglements may be more accessible to resolution by Top2 immediately after replication. These entanglements would be resolved before the Top2 RNAi construct is induced. Heterochromatic entanglements may be protected from early resolution due their conformation after replication or the presence of heterochromatin binding proteins. Top2 would be unable to resolve these entanglements until the karyosome reorganizes for prometaphase I or until tension is provided on the DNA by microtubules, as has been proposed for the resolution of entanglements by Top2 in yeast. At this stage Top2 levels would be reduced in Top2 RNAi-expressing oocytes, leading to a failure to resolve these entanglements. Alternatively, heterochromatic regions may be particularly prone to forming entanglements due to the repetitive nature of heterochromatic DNA, and thus more sensitive to decreased Top2 levels (Hughes, 2014).

Topoisomerase II enzymes have also been implicated in regulating chromosome condensation in a number of cell types and organisms. For example, Mengoli (2014) reported that the centric heterochromatin appeared undercondensed in some neuroblasts from Top2 RNAi-expressing larvae. Additionally, strong knockdown of Top2 levels in Drosophila S2 cells led to a large and quantitative change in chromosome condensation (Chang, 2003; Hughes, 2014 and references therein).

These observations led a question whether some of the phenotypes observed in this study might be the consequence of the effect of Top2 depletion on chromosome condensation, especially in the pericentric heterochromatin. Global chromosome condensation in Top2 RNAi-expressing oocytes looked similar to wild-type oocytes, but small changes in chromosome condensation would be obscured by the close proximity of the chromosomes at prometaphase I and the high level of condensation of the chromosomes. It is thus possible that chromosomes are undergoing mild decreases in condensation, particularly in heterochromatic regions, when Top2 levels are decreased. Decreased condensation could contribute to the stretched out phenotype observed with the 3rd and 4th chromosome FISH probes and to the centromere-led chromosomal projections. However, the effects on condensation that were observed appear to be too weak to account for the entanglements and stretching that were observed (Hughes, 2014).

These observations lead to a speculative model for the cause of the defects in Top2 RNAi-expressing oocytes. In wild-type oocytes, DNA entanglements form between the highly repetitive DNA sequences of heterochromatic regions. These entanglements could form during replication by stalled replications forks that can occur in the repetitive heterochromatic regions or by intertwinings that could form as chromosomes are replicated in close proximity. Entanglements within the heterochromatic regions would not be immediately resolved. Therefore, these entanglements could help hold heterochromatic regions of homologous chromosomes tightly together during prophase, including those chromosomes that, like the 4th chromosomes, fail to undergo recombination. As germinal vesicle breakdown approaches, many of these entanglements would have to be resolved by Top2 in order for chromosomes to separate and biorient properly during prometaphase I and metaphase I, possibly assisted by karyosome reorganization and/or microtubule attachments to the chromosomes. The chromatin threads observed during prometaphase I could be the entanglements that failed to be resolved during late prophase or those protected from resolution to facilitate the biorientation of achiasmate chromosomes. Top2 and/or other enzymes would then resolve these final DNA threads by anaphase I. In oocytes with decreased levels of Top2, many of the DNA entanglements would not be resolved during meiosis I, leading to a failure of homologous heterochromatic regions to separate. In some cases, heterochromatic regions appear to attempt separation, but the DNA entanglements hold the chromosomes together at one or more places and the rest of the heterochromatic regions of the chromosomes become highly stretched out. The centromere-led DNA projections apparently occur when chromosomes attempt separation despite the existence of heterochromatic entanglements. Since the heterochromatic regions would still be locked together at egg activation when meiosis resumes, chromosomes would be unable to segregate to opposite spindle poles at anaphase I and ultimately, the oocytes would fail to exit meiosis I. The results indicate that Top2 plays an important role in resolving homologous DNA entanglements in Drosophila oocytes. These results also suggest that the formation of such entanglements (by whatever mechanism) may be a characteristic of the meiotic process (Hughes, 2014).

Topoisomerase II plays a role in dosage compensation in Drosophila

In Drosophila, dosage compensation is mediated by the MSL complex, which binds numerous sites on the X chromosome in males and enhances the transcriptional rate of a substantial number of X-linked genes. This study has determined that topoisomerase II (Topo II) is enriched on dosage compensated genes, to which it is recruited by association with the MSL complex, in excess of the amount that is present on autosomal genes with similar transcription levels. Using a plasmid model, this study has showm that Topo II is required for proper dosage compensation and that compensated chromatin is topologically different from non-compensated chromatin. This difference, which is not the result of the enhanced transcription level due of X-linked genes and which represents a structural modification intrinsic to the DNA of compensated chromatin, requires the function of Topo II. The results suggest that Topo II is an integral part of the mechanistic basis of dosage compensation (Cugusi, 2013).

Restoration of topoisomerase 2 function by complementation of defective monomers in Drosophila

Type II topoisomerases are essential ATP-dependent homodimeric enzymes required for transcription, replication, and chromosome segregation. These proteins alter DNA topology by generating transient enzyme-linked double-strand breaks for passage of one DNA strand through another. The central role of type II topoisomerases in DNA metabolism has made these enzymes targets for anticancer drugs. This study describes a genetic screen that generated novel alleles of Drosophila Topoisomerase 2 (Top2). Fifteen alleles were obtained, resulting from nonsense and missense mutations. Among these, 14 demonstrated recessive lethality, with one displaying temperature-sensitive lethality. Several newly generated missense alleles carry amino acid substitutions in conserved residues within the ATPase, Topoisomerase/Primase, and Winged helix domains, including four that encode proteins with alterations in residues associated with resistance to cancer chemotherapeutics. Animals lacking zygotic Top2 function can survive to pupation and display reduced cell division and altered polytene chromosome structure. Inter se crosses between six strains carrying Top2 missense alleles generated morphologically normal trans-heterozygous adults, which showed delayed development and were female sterile. Complementation occurred between alleles encoding Top2 proteins with amino acid substitutions in the same functional domain and between alleles encoding proteins with substitutions in different functional domains. Two complementing alleles encode proteins with amino acid substitutions associated with drug resistance. These observations suggest that dimerization of mutant Top2 monomers can restore enzymatic function. These studies establish the first series of Top2 alleles in a multicellular organism. Future analyses of these alleles will enhance knowledge about the contributions made by type II topoisomerases to development (Hohl, 2012).

Mitotic chromosomes are compacted laterally by KIF4 and condensin and axially by topoisomerase IIalpha

Mitotic chromosome formation involves a relatively minor condensation of the chromatin volume coupled with a dramatic reorganization into the characteristic 'X' shape. This paper reports results of a detailed morphological analysis, which revealed that the mammalian chromokinesin KIF4 cooperates in a parallel pathway with condensin complexes to promote the lateral compaction of chromatid arms. In this analysis, KIF4 and condensin were mutually dependent for their dynamic localization on the chromatid axes. Depletion of either caused sister chromatids to expand and compromised the 'intrinsic structure' of the chromosomes (defined in an in vitro assay), with loss of condensin showing stronger effects. Simultaneous depletion of KIF4 and condensin caused complete loss of chromosome morphology. In these experiments, topoisomerase IIalpha contributed to shaping mitotic chromosomes by promoting the shortening of the chromatid axes and apparently acting in opposition to the actions of KIF4 and condensins. These three proteins are major determinants in shaping the characteristic mitotic chromosome morphology (Samejima, 2012).

DNA topoisomerase II modulates insulator function in Drosophila

Insulators are DNA sequences thought to be important for the establishment and maintenance of cell-type specific nuclear architecture. In Drosophila there are several classes of insulators that appear to have unique roles in gene expression. The mechanisms involved in determining and regulating the specific roles of these insulator classes are not understood. This study reports that DNA Topoisomerase II modulates the activity of the Su(Hw) insulator. Downregulation of Topo II by RNAi or mutations in the Top2 gene result in disruption of Su(Hw) insulator function. This effect is mediated by the Mod(mdg4)2.2 protein, which is a unique component of the Su(Hw) insulator complex. Co-immunoprecipitation and yeast two-hybrid experiments show that Topo II and Mod(mdg4)2.2 proteins directly interact. In addition, mutations in Top2 cause a slight decrease of Mod(mdg4)2.2 transcript but have a dramatic effect on Mod(mdg4)2.2 protein levels. In the presence of proteasome inhibitors, normal levels of Mod(mdg4)2.2 protein and its binding to polytene chromosomes are restored. Thus, Topo II is required to prevent Mod(mdg4)2.2 degradation and, consequently, to stabilize Su(Hw) insulator-mediated chromatin organization (Ramos, 2011).

Dual role of topoisomerase II in centromere resolution and aurora B activity

Chromosome segregation requires sister chromatid resolution. Condensins are essential for this process since they organize an axial structure where topoisomerase II can work. How sister chromatid separation is coordinated with chromosome condensation and decatenation activity remains unknown. This study combined four-dimensional (4D) microscopy, RNA interference (RNAi), and biochemical analyses to show that topoisomerase II plays an essential role in this process. Either depletion of topoisomerase II or exposure to specific anti-topoisomerase II inhibitors causes centromere nondisjunction, associated with syntelic chromosome attachments. However, cells degrade cohesins and timely exit mitosis after satisfying the spindle assembly checkpoint. Moreover, in topoisomerase II-depleted cells, Aurora B and INCENP fail to transfer to the central spindle in late mitosis and remain tightly associated with centromeres of nondisjoined sister chromatids. Also, in topoisomerase II-depleted cells, Aurora B shows significantly reduced kinase activity both in S2 and HeLa cells. Codepletion of BubR1 in S2 cells restored Aurora B kinase activity, and consequently, most syntelic attachments were released. Taken together, these results support that topoisomerase II ensures proper sister chromatid separation through a direct role in centromere resolution and prevents incorrect microtubule-kinetochore attachments by allowing proper activation of Aurora B kinase (Coelho, 2008).

Disruption of topoisomerase II perturbs pairing in Drosophila cell culture

Homolog pairing refers to the alignment and physical apposition of homologous chromosomal segments. Although commonly observed during meiosis, homolog pairing also occurs in nonmeiotic cells of several organisms, including humans and Drosophila. The mechanism underlying nonmeiotic pairing, however, remains largely unknown. This study explored the use of established Drosophila cell lines for the analysis of pairing in somatic cells. Using fluorescent in situ hybridization (FISH), pairing was assayed at nine regions scattered throughout the genome of Kc167 cells, observing high levels of homolog pairing at all six euchromatic regions assayed and variably lower levels in regions in or near centromeric heterochromatin. Extensive pairing was also observed in six additional cell lines representing different tissues of origin, different ploidies, and two different species, demonstrating homolog pairing in cell culture to be impervious to cell type or culture history. Furthermore, by sorting Kc167 cells into G1, S, and G2 subpopulations, it was shown that even progression through these stages of the cell cycle does not significantly change pairing levels. Finally, the data indicate that disrupting Drosophila topoisomerase II (Top2) gene function with RNAi and chemical inhibitors perturbs homolog pairing, suggesting Top2 to be a gene important for pairing (Williams, 2007).

Condensin-dependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis

Assembly of compact mitotic chromosomes and resolution of sister chromatids are two essential processes for the correct segregation of the genome during mitosis. Condensin, a five-subunit protein complex, is thought to be required for chromosome condensation. However, recent genetic analysis suggests that condensin is only essential to resolve sister chromatids. To study further the function of condensin DmSMC4, a subunit of the complex, was depleted from Drosophila S2 cells by dsRNA-mediated interference. Cells lacking DmSMC4 assemble short mitotic chromosomes with unresolved sister chromatids where Barren, a non-SMC subunit of the complex is unable to localise. Topoisomerase II, however, binds mitotic chromatin after depletion of DmSMC4 but it is no longer confined to a central axial structure and becomes diffusely distributed all over the chromatin. Furthermore, cell extracts from DmSMC4 dsRNA-treated cells show significantly reduced topoisomerase II-dependent DNA decatenation activity in vitro. Nevertheless, DmSMC4-depleted chromosomes have centromeres and kinetochores that are able to segregate, although sister chromatid arms form extensive chromatin bridges during anaphase. These chromatin bridges do not result from inappropriate maintenance of sister chromatid cohesion by DRAD21, a subunit of the cohesin complex. Moreover, depletion of DmSMC4 prevents premature sister chromatid separation, caused by removal of DRAD21, allowing cells to exit mitosis with chromatin bridges. These results suggest that condensin is required so that an axial chromatid structure can be organised where topoisomerase II can effectively promote sister chromatid resolution (Coelho, 2003).

This study has shown that dsRNAi can be used to severely deplete DmSMC4 in tissue culture cells resulting in mitotic phenotypes that are very similar to those previously described for dmSmc4 mutant Drosophila cells. Already 24 hours after RNAi treatment some mitotic cells show abnormal resolution of sister chromatids and later, cells in anaphase or telophase begin to show chromatin bridges indicating that the frequency with which these phenotypes are observed depends on the level of depletion of DmSMC4. Loss of DmSMC4 causes the formation of short mitotic chromosomes with poorly defined sister chromatids. These chromosomes are unlikely to contain other proteins of the condensin complex since immunofluorescence and mitotic chromatin immunoprecipitation shows that binding of Barren to condensing chromosomes is dependent on DmSMC4. These observations are in agreement with previous findings indicating that non-SMC condensins can only bind DNA in the presence of the entire condensin complex. Also, it was shown in S. pombe and S. cerevisiae that all members of the regulatory subcomplex are essential for chromatin association of yeast condensin in vivo. Together, these results strongly suggest that, in Drosophila, the assembly of the condensin complex to mitotic chromatin requires all protein subunits. Moreover, the results demonstrate that certain aspects of chromosome condensation, namely shortening of the longitudinal axis of sister chromatids, can occur in the absence of condensins (Coelho, 2003).

Although, chromosomes do not condense normally in DmSMC4-depleted cells, genetic studies in Drosophila showed that loss of DmSMC4 or Barren does not prevent cells from entering anaphase and attempting sister chromatid segregation. However, recently it has been suggested that the condensin complex might contribute to ensure proper function of the centromere. In S. cerevisiae, BRN1 the homologue of Barren, has been implicated in the formation of functional mitotic kinetochores and in C. elegans condensin activity is required for the normal orientation of the centromere towards the mitotic spindle. This study has shown that depletion of DmSMC4 does not affect the localisation of centromere or kinetochore proteins and that microtubules associate with kinetochores. Furthermore, it was observed that at early stages of mitosis, kinetochores associated with spindle microtubules appear to stretch poleward, sometimes well beyond the chromatin. However, when microtubules are depolymerised by colchicine, kinetochores localise only over mitotic chromatin, suggesting that stretching of kinetochores is microtubule dependent. Similar observations were reported after expressing GFP-tagged centromeres in a BRN1 mutant background. These results suggest that condensin is not required for the formation of functional kinetochores and that at metaphase-anaphase transition sister centromeres disjoin normally and segregate but sister chromatid arms remain attached causing stretching of the centromeres (Coelho, 2003).

Immunofluorescence and biochemical studies have suggested that condensed chromatids contain a central axial structure. Topoisomerase II and condensin have been identified at this elusive structure. Previously it was shown that in Drosophila S2 cells condensins associate with chromatin at prophase localising to the axis of sister chromatids throughout mitosis (Steffensen, 2001). This study shows that Topo II also localises to the axis of sister chromatids throughout mitosis. However, in prometaphase chromosomes it is clear that condensin and Topo II show only partial co-localisation. DmSMC4 and Topo II localise to discrete sites that alternate along the chromatid axis. Although similar patterns of localisation has been recently described for hBarren and Topo IIα in HeLa cells, hBarren appears to bind chromatin only during prometaphase while Topo IIα is present from prophase. This discrepancy in the kinetics of condensin accumulation at early stages of mitosis probably represents cell type-specific differences since it is unlikely that SMC and non-SMC subunits bind chromatin independently. Furthermore, this study shows that depletion of DmSMC4 abolishes the localisation of Topo II to a well-defined axial structure even though there is no significant reduction in the level of chromatin-associated Topo II. Similarly, in yeast, localisation of Topo II to mitotic chromatin has been shown to depend upon condensin function (Bhalla, 2002). However, more recent data has suggested that in chromatin assembled in Xenopus extracts, Topo II localisation to an axial structure of chromatids occurs independently of condensin. These apparently contradictory results could be explained if condensin was not completely depleted in the Xenopus extracts, allowing partial accumulation of Topo II to an axial chromatin structure. Since RNAi depletion studies no DmSMC4 was found either by immunoflourescence or western blotting, the results suggest that condensin plays an essential role in the organisation of the chromatin so that Topo II can localise to the chromatid axis. This structure is likely to be highly dynamic since recent live imaging and FRAP analysis in mammalian cells shows that Topo IIα exchanges rapidly between a cytoplasmic pool and that bound to chromosomes and centromeres. In vivo analysis of condensin accumulation to the axis of sister chromatids should provide valuable insights on the dynamics of this 'ill-defined' structure (Coelho, 2003).

The abnormal distribution of Topo II to mitotic chromatin resulting from depletion of DmSMC4 prompted a determination of whether its DNA decatenation activity was also compromised. This study showed, using an in vitro assay, that DNA decatenation activity of the endogenous Topo II is significantly reduced when DmSMC4 is depleted. Although these results are compatible with a direct interaction between DmSMC4 and Topo II, no co-immunoprecipitation was detected indicating that the interaction might be indirect. Nevertheless, the results suggest that proper activity of the enzyme requires condensin. A more direct interaction was reported previously since it was shown that Barren interacts in a yeast two-hybrid assay with Topo II and promotes its decatenating activity. However, it has been shown that BRN1, the yeast homologue, is not required for Topo II activity in vivo. Furthermore, it is unlikely that depletion of DmSMC4 completely abolishes Topo II activity since mutation or inhibition of its activity has been shown to cause arrest at the metaphase-anaphase transition, a phenotype not produced by DmSMC4 depletion. Accordingly, it is believed that the chromatin bridges observed after depletion of DmSMC4 are due to inappropriate activity of Topo II resulting in the maintenance of catenated DNA between sister chromatids (Coelho, 2003).

Previous reports have suggested a possible mechanistic interaction between cohesins and condensins. However, this study shows that depletion of DmSMC4 does not alter the localisation or removal of cohesins from mitotic chromatin in Drosophila S2 cells. Similarly, in S. cerevisiae it has been shown that although sister chromatid separation does not occur normally in Ycs4 mutants, MCD1/SCC1 is released from chromosomes at the metaphase-anaphase transition. Conversely, depletion of cohesins in higher eukaryotes does not appear to affect chromosome condensation and in Xenopus extracts the release of cohesin during prophase is not required for chromatin compaction mediated by condensin. Taken together, these results indicate that the removal of cohesins during mitosis is independent of condensin activity (Coelho, 2003).

Depletion of cohesins causes premature sister chromatid separation and a significant prometaphase arrest. This prometaphase arrest could be due to the activity of the spindle checkpoint, which prevents exit from mitosis if proper chromosome orientation and organisation of a metaphase plate is not achieved. Strikingly, it was observed that simultaneous depletion of DmSMC4 and DRAD21 does not lead to premature sister chromatid separation or arrest during prometaphase but cells progress into anaphase and telophase showing extensive chromatin bridges. It is proposed that sister chromatids do not separate prematurely in the absence of cohesins because depletion of DmSMC4 prevents sister chromatid resolution by compromising Topo II activity. These cells then proceed into prometaphase, kinetochores can now bind spindle microtubules and chromosomes congress to the metaphase plate, allowing cells to satisfy the spindle checkpoint and initiate mitotic exit. Thus, in the absence of DmSMC4, abnormal decatenation of sister chromatids appears to provide an alternate mechanism to hold sisters together during early stages of mitosis (Coelho, 2003).

From these results it is proposed that condensin is essential to organise a clearly defined axial structure of sister chromatids where Topo II can localise. In the absence of this specific localisation, Topo II can still bind chromatin but its decatenation activity is not specifically directed and sister chromatids cannot resolve properly (Coelho, 2003).

RNAi analysis reveals an unexpected role for topoisomerase II in chromosome arm congression to a metaphase plate

DNA topoisomerase II (Topo II) is a major component of mitotic chromosomes and an important drug target in cancer chemotherapy, however, its role in chromosome structure and dynamics remains controversial. This study used RNAi to deplete Topo II in Drosophila S2 cells in order to carry out a detailed functional analysis of the role of the protein during mitosis. Topo II was found not to be required for the assembly of a functional kinetochore or the targeting of chromosomal passenger proteins, nonetheless, it is essential for anaphase sister chromatid separation. In response to a long-running controversy, it was shown that Topo II does have some role in mitotic chromatin condensation. Chromosomes formed in its absence have a 2.5-fold decrease in the level of chromatin compaction, and are morphologically abnormal. However, it is clear that the overall programme of mitotic chromosome condensation can proceed without Topo II. Surprisingly, in metaphase cells depleted of Topo II, one or more chromosome arms frequently stretch out from the metaphase plate to the vicinity of the spindle pole. This is not kinetochore-based movement, as the centromere of the affected chromosome is located on the plate. This observation raises the possibility that further unexpected functions for Topo II may remain to be discovered (Chang, 2003).

Spindle assembly and cytokinesis in the absence of chromosomes during Drosophila male meiosis

A large body of work indicates that chromosomes play a key role in the assembly of both a centrosomal and centrosome-containing spindles. In animal systems, the absence of chromosomes either prevents spindle formation or allows the assembly of a metaphase-like spindle that fails to evolve into an ana-telophase spindle. This study shows that Drosophila secondary spermatocytes can assemble morphologically normal spindles in the absence of chromosomes. The Drosophila mutants fusolo (currently unmapped) and solofuso (later shown to be an allele of top2) are severely defective in chromosome segregation and produce secondary spermatocytes that are devoid of chromosomes. The centrosomes of these anucleated cells form robust asters that give rise to bipolar spindles that undergo the same ana-telophase morphological transformations that characterize normal spindles. The cells containing chromosome-free spindles are also able to assemble regular cytokinetic structures and cleave normally. In addition, chromosome-free spindles normally accumulate the Aurora B kinase at their midzones. This suggests that the association of Aurora B with chromosomes is not a prerequisite for its accumulation at the central spindle, or for its function during cytokinesis (Bucciarelli, 2003).

A two-step scaffolding model for mitotic chromosome assembly

Topoisomerase IIalpha (topoIIalpha) and 13S condensin are both required for mitotic chromosome assembly. This study shows that they constitute the two main components of the chromosomal scaffold on histone-depleted chromosomes. The structural stability and chromosomal shape of the scaffolding toward harsh extraction procedures are shown to be mediated by ATP or its nonhydrolyzable analogs, but not ADP. TopoIIalpha and 13S condensin components immunolocalize to a radially restricted, longitudinal scaffolding in native-like chromosomes. Double staining for topoIIalpha and condensin generates a barber pole appearance of the scaffolding, where topoIIalpha- and condensin-enriched 'beads' alternate; this structure appears to be generated by two juxtaposed, or coiled, chains. Cell cycle studies establish that 13S condensin appears not to be involved in the assembly of prophase chromatids; they lack this complex but contain a topoIIalpha-defined (-mediated?) scaffolding. Condensin associates only during the pro- to metaphase transition. This two-step assembly process is proposed to generate the barber pole appearance of the native-like scaffolding (Maeshima, 2003).

Drosophila chromosome condensation proteins Topoisomerase II and Barren colocalize with Polycomb and maintain Fab-7 PRE silencing

Mechanisms of cellular memory control the maintenance of cellular identity at the level of chromatin structure. An investigation was carried out to see whether the converse is true; namely, if functions responsible for maintenance of chromosome structure play a role in epigenetic control of gene expression. Topoisomerase II (TopoII) and Barren (Barr) are shown to interact in vivo with Polycomb group (PcG) target sequences in the bithorax complex of Drosophila, including Polycomb response elements. In addition, the PcG protein Polyhomeotic (Ph) interacts physically with TopoII and Barr and Barr is required for Fab-7-regulated homeotic gene expression. Conversely, defects in chromosome segregation have been found associated with ph mutations. It is proposed that chromatin condensation proteins are involved in mechanisms acting in interphase that regulate chromosome domain topology and are essential for the maintenance of gene expression (Lupo, 2001).

PcG genes have been proposed to act as chromosomal components maintaining transcriptional repression by 'heterochromatinizing' their target sites. However, the molecular mechanisms underlying chromosomal silencing by the PcG, heterochromatin formation, and the transmission of the silenced state through mitosis are not known. It was reasoned that chromosome condensation machineries could provide an important functional link between the regulation of chromosome domain structure, gene silencing, and mitotic inheritance. Thus, the interaction of the PcG with the machinery involved in orchestrating chromosome dynamics has been investigated and in particular with those machines enabling mitotic chromosome condensation. The in vivo formaldehyde-fixed chromatin immunoprecipitation (X-ChIP) method was used to analyze the distribution in the BX-C locus of two proteins: TopoII, an enzyme involved in the regulation of DNA supercoiling, chromosome condensation, and segregation, and Barr. Barr is the homolog of the Xenopus XCAP-H and C. elegans DPY26 proteins, a TopoII-interacting protein associated with the SMC2/4 condensins complexes, known to be involved in mitotic chromosome condensation (Lupo, 2001).

A striking colocalization of TopoII and Barr with previously mapped PC binding sites was found, suggesting that the two groups of functions are at least acting on the same DNA regions. A clear colocalization was found at major PREs (Fab-7, Mcp, iab-3, bxd, and bx). In particular, the Fab-7 element appears to be a major TopoII/Barr binding site. Strong association of PC to Fab-7 is found. No Barr/TopoII binding site was found at the Fab-8 PRE, which might define the border between the repressed and active BX-C domains in SL-2 cells (Lupo, 2001).

In iab-2 and iab-3, large fragments (11.0 and 11.5 kb, respectively) have PRE activity. Here specific Barr and TopoII sites are also found. These sites do not match the PC/GAGA peaks previously described. Yet, since these regions show considerable levels of PC, it is suggested that minor PC binding sites adjacent to the reported 'peaks' may also be functionally relevant. Another important aspect of PcG function is the interaction with promoters; major PC binding sites include core promoters, and it is known that PREs perform better when combined with their natural target promoters. Interestingly, a striking colocalization of TopoII and Barr is also found at promoters (AbdB gamma, abdA II, and Ubx) (Lupo, 2001).

Based on the mitotic phenotype and previous immunolocalization data, a direct association of TopoII and Barr with chromosomes mostly at mitosis is expected. In this context, the colocalization of TopoII and Barr in regulatory regions of the BX-C is striking. Although asyncronous tissue culture cells were used, it is believed that the association of Barr and TopoII with the regulatory regions of the BX-C occurs not only at mitosis but also in interphase. In particular, in X-ChIP experiments, the number of mitotic cells at the time of formaldehyde fixation is around 5%, thus, if only mitotic cells contributed to the overall precipitated DNA, this approach would have been below the detectable limit. Hence, it is proposed that TopoII and Barr are associated with their target sites throughout the cell cycle (Lupo, 2001).

The short proximal isoform of Ph (Ph 140p) can be copurified from nuclear extracts with TopoII and Barr. This isoform is not found coimmunoprecipitated with Pc and Psc, and neither Barr nor TopoII copurified with Pc and Psc. The three PcG members Pc, Psc, and the long proximal product Ph 170p have been shown to coimmunopurify from nuclear extracts with antibodies against one of the three. Due to the absence of Ph 140p signals in the Pc/Psc immunoprecipitations, these results might be taken to indicate that there is no functional connection between the presumptive TopoII/Barr/Ph 140p complex and the Pc/Psc/Ph 170p complex. For three reasons this is thought to be unlikely. (1) Both the 170p and the 140p isoforms of Ph are derived from the same transcript by posttranscriptional regulation and differ by a 244 N-terminal stretch of amino acids present only in the 170p isoform. Functional domains of Ph (zinc finger, coiled-coil region, GTP binding site, serine/threonine-rich region, and SAM/SPM domain) are all contained in both isoforms, suggesting that both proteins can fulfill related functions. (2) X-ChIP data, obtained with the same Ph antibodies used in this study, show an extended overlap of Pc and Ph binding regions in the BX-C. Together with the finding of a colocalization of TopoII and Barr with PcG binding sites in regulative regions of the BX-C, this suggests that these proteins act on the same DNA regions. (3) The data show that a reduction of the amount of Barren protein in barren heterozygotes parallels PcG-negative effects on the silencing function of the Fab-7 PRE (Lupo, 2001).

An additional finding supports the conclusion that Ph protein(s) are involved both in PcG function and mitotic chromosome condensation. ph null embryos show defects in chromosome segregation, the same phenotype observed for barren mutant embryos. Conversely, the results of Barren haplo-insufficiency on Fab-7 silencing are suggestive of a role for Barr in early embryogenesis. Since in early embryogenesis Ph 140p is the only Ph product made, these defects are diagnostic of a specific role of Ph 140p in mitosis. These results with regard to Barren protein and Fab-7 silencing are reminiscent of another previously documented role for SMCs in gene regulation. In C. elegans, the DPY27 protein, a homolog of the Xenopus XCAP-C (SMC4), has been shown to bind the X chromosome in females, whereas its absence results in lethality due to abnormally high gene expression levels from the X chromosome. Thus, it is concluded that Ph 140p shares an important role with the Barr/TopoII condensin complexes in mitosis and cell memory processes (Lupo, 2001).

In order to further study interactions between barren and the PcG the null alleles ph502 and ph602 were used for genetic analysis, and strains heterozygous for ph and barren mutations were crossed. Surprisingly, no effect was found. PcG genes, in contrast, show dosage effects, suggesting that the interaction between PcG and Barr/TopoII may imply a different, more dosage-insensitive regulation. However, it has been shown that barren mutations affect PRE silencing in the same way as mutations in PcG genes do. Taken together, these results may indicate a nonstoichiometric relationship between PcG and Barr/TopoII protein complexes. It is proposed that major PcG and condensin proteins belong to distinct protein complexes, but that they nevertheless cooperate at PREs and promoters to maintain the silenced state of homeotic genes. From the SMC standpoint, these results are intriguing because they show that proteins involved in chromosome condensation and segregation processes bind to regulatory elements in chromosomal domains responsible for the inheritance of transcription states. This would suggest that the 'structural maintenance of chromosome' function could also affect epigenetic control of gene expression (Lupo, 2001).

These data reveal novel molecular aspects of BX-C regulation. The distribution of PC and TopoII/Barr sites in the BX-C appears as a reiterated array suggestive of heterochromatic hallmarks, perhaps providing in cis information for higher-order organization of the BX-C chromosomal domain. In particular, TopoII oligomerizes in a DNA-dependent manner. Similar interactions in trans are proposed to occur between PcG proteins in vivo. According to this ability, spaced molecules at distant sites on the DNA could come into contact, giving rise to more condensed domains. A model has been proposed to explain how condensin proteins and Topoisomerases may act together in condensation. In this model, the size of the condensin complex (perhaps 1000 Å) could introduce (+) supercoils by affecting the global writhe of DNA, thus creating a more condensed state. In this study, Barr is found only at discrete sites, whereas PC and other PcG proteins are associated also with large chromosomal regions. Possibly, one aspect of PcG protein function and binding to chromatin in interphase is to stabilize and expand the condensed state by topological effects (Lupo, 2001 and references therein).

The positioning of TopoII at complex regulatory regions (e.g., abx/bx and iab-3-iab-8) may indicate the existence of minidomains providing tight control on the chromatin structure of intervening regulatory DNA sequences by localized changes of DNA superhelicity. The activity of TopoII could be locally regulated by the association with other proteins like Barr and perhaps some PcG and trxG members [e.g., Ph 140p, CCF, E(z), and Gaga]. Interestingly, Barr has been found to stimulate TopoII activity. It has to be pointed out that these data show, in a direct way, where in vivo TopoII binds to single-copy genes but they cannot tell if these sites correspond to TopoII cutting sites. However, it is likely that a tight association with DNA corresponds to enzymatic activity. Thus, it is proposed that in vivo TopoII activity may be enhanced at specific sites, whereas at others it could be reduced, resulting in local differences in chromatin condensation states controlled by DNA topology (Lupo, 2001).

The presence of multiple Barr and TopoII sites within the BX-C could thus provide a powerful way to fine-tune the structure of each of the parasegment-specific chromosomal subdomains. As a direct consequence of controlled condensation of specific parts of the BX-C, determined states could be fixed by enabling or not enabling specific interactions between cis elements. The mechanism by which Fab-7 regulates the AbdB promoters is, in fact, not known. It has been proposed that a combination of 'chromatin effects' and insulating activity may regulate enhancer-promoter interactions. It is proposed that the homeotic loss-of-function phenotypes observed in Fab-7 or Mcp deletions could be due to a change in local DNA topology altering the communication of segment-specific enhancers with the AbdB promoters. In this way, local differences in chromosome domain topology may contribute to stabilize or interfere with correct phasing between regulatory elements and promoters. If topological effects are at least part of Fab-7 function, this may also help to explain distance-dependent effects on enhancer-promoter interactions. Interestingly, in Drosophila, mutations in the Nipped-B gene facilitate enhancer-promoter interactions by overcoming the action of ectopic insulator elements in the Ubx domain. Nipped-B is the homolog of the yeast SMC-associated protein Scc2 (sister chromatid cohesion 2), suggesting that adherins may have a broader role in chromosomal domain organization and gene regulation. It is proposed that chromatin condensation proteins may be involved in a pathway acting also in interphase that regulates chromosome domain structure by DNA topology and is essential for maintenance of gene expression (Lupo, 2001).

Developmental regulation of DNA-topoisomerases during Drosophila embryogenesis

Type I and type II DNA-topoisomerases are essential enzymes that mediate replication, transcription, recombination, and mitosis in multicellular eukaryotes but the extent of their interchange for specific reactions in vivo is controversial. Expression patterns for topoisomerase I and topoisomerase II during the embryogenesis of Drosophila melanogaster were compared with patterns of DNA replication and expression of the histone genes. In late oogenesis the maternally supplied top2 mRNA was evenly distributed throughout the egg with elevated levels at the posterior tip, a pattern that is maintained in syncytial blastoderm embryos. During gastrulation, top2 mRNA became differentially localized only to regions of DNA replication, including new expression in the gonads preceding mitosis/meiosis. Significantly higher levels of top2 mRNA were found in mitotic compared to endoreplicating tissues. The total histone mRNA was exclusively associated with DNA replication but, in contrast to top2 mRNA, mitotic and endoreplicating cells contained similar expression levels with no expression in the gonads. Striking differences exist between the distribution of the top2 mRNA and topoisomerase II protein. The protein localizes to all evolving nuclei where it persists throughout embryogenesis. A high level of top1 mRNA transcript was present without differential tissue distribution throughout embryogenesis (Gemkow, 2001).

A nuclear matrix/scaffold attachment region co-localizes with the gypsy retrotransposon insulator sequence

The 5'-untranslated region of the Drosophila gypsy retrotransposon contains an "insulator," which disrupts the interactions between distally located enhancers and proximal promoter elements. The insulator effect is dependent on the suppressor of Hairy-wing (su[Hw]) protein, which binds to reiterated sites within the 350 base pairs of the gypsy insulator, and additionally acts as a transcriptional activator of gypsy. This study shows that the 350-base pair su(Hw) binding site-containing gypsy insulator behaves as a matrix/scaffold attachment region (MAR/SAR), involved in interactions with the nuclear matrix. In vitro experiments using nuclear matrices from Drosophila, murine, and human cells demonstrate specific binding of the gypsy insulator, not observed with any other sequence within the retrotransposon. Moreover, it is shown that the gypsy insulator, like previously characterized MAR/SARs, specifically interacts with topoisomerase II and histone H1, i.e. with two essential components of the nuclear matrix. Experiments within cells in culture demonstrate differential effects of the gypsy MAR sequence on reporter genes, namely no effect under conditions of transient transfection and a repressing effect in stable transformants, as expected for a sequence involved in chromatin structure and organization (Nabirochkin, 1998).

The presence of a MAR/SAR within gypsy is not totally unexpected, since 'boundary' elements are in general regions which contain not only enhancer and insulating elements, but also matrix attachment domains. The rather original feature of the gypsy sequence is that all three domains, which in general are sufficiently 'dispersed' so as to allow isolation of 'pure' enhancers, MAR/SAR, or insulators, are in the present case 'gathered' within a single and relatively short (350 bp) sequence. This rather uncommon situation might in fact be relevant to the pressure for compactness within retroviral sequences, as it is known that retroviruses can only package a limited amount of genetic information. A consequence of compaction is that the gypsy insulator and its associated components are most probably interacting, in vivo, with elements of the nuclear matrix. Accordingly, proteins of the nuclear matrix might play a role in the insulation process, and conversely the su(Hw) protein (which is essential for insulation) might interact with proteins of the matrix. Such interactions could actually account for the data on gypsy insulation and fit with previously proposed models for the gypsy effects (Nabirochkin, 1998).

A first series of data strongly suggested that the gypsy insulator, like all previously characterized insulators, essentially prevents interactions between distal enhancer and promoter, without any direct repressing effect on the enhancer itself. This directional effect can most easily be accounted for by the "looping model" involving generation of structural domains isolated one from the other by attachment of boundary sequences (MAR/SAR) to the nuclear matrix. Alternatively, a series of data on gypsy insulation (essentially in mod(mdg4) mutants) discloses bidirectional repressing effects, which can be accounted for by a model involving heterochromatinization. The present data (showing that the gypsy insulator behaves as a MAR/SAR) are clearly in agreement with the structural looping model, but also support the heterochromatinization model. Indeed, the gypsy MAR/SAR DNA per se, in the absence of su(Hw) protein, is involved in histone H1 nucleation (as shown in this paper), and it has been demonstrated that histone H1 nucleation is associated with both DNA compaction and transcriptional silencing. Additionally, Laemmli and co-workers have found that histone H1 can be removed from MAR/SAR domains by distamycin and distamycin-like proteins (D-like proteins, such as the high mobility group proteins); this has led to the proposal that MAR/SARs can activate or repress transcription of adjacent genes depending on the nucleation/depletion of histone H1. The gypsy MAR/SAR could then be responsible for the repressing effect observed in the mod(mdg4) mutants, as well as in the present assay within heterologous cells (assuming further that appropriate D-like proteins are absent in those cells). Taking into account, in addition, that mutations in the mod(mdg4) or the su(Hw) genes modify position-effect variegation, it could be further hypothesized that the su(Hw)/mod(Mdg4) complex acts as the D-like proteins and modifies the nucleation processes to allow the switch from a repressing to an active state. Accordingly, a model in which the su(Hw) binding sites and the associated su(Hw)/mod(Mdg4) complex modulate the effects of the MAR/SAR DNA sequence could rather simply account for the biological effects of the gypsy insulator in both the wild type and su(Hw)/mod(mdg4) mutants. The proposed model would then reconcile the two previous models for gypsy insulation, i.e. the heterochromatinization and the looping models (Nabirochkin, 1998 and references).

Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with Topoisomerase II

A Drosophila gene, barren (barr), is required for sister-chromatid segregation in mitosis. barr encodes a novel protein that is present in proliferating cells and has homologs in yeast and human. Mitotic defects in barr embryos become apparent during cycle 16, resulting in a loss of PNS and CNS neurons. Centromeres move apart at the metaphase-anaphase transition and Cyclin B is degraded, but sister chromatids remain connected, resulting in chromatin bridging. This phenotype is similar to that described in TOP2 mutants in yeast. Barren protein localizes to chromatin throughout mitosis. Colocalization and biochemical experiments indicate that Barren associates with Topoisomerase II throughout mitosis and alters the activity of Topoisomerase II. It is proposed that this association is required for proper chromosomal segregation by facilitating the decatenation of chromatids at anaphase (Bhat, 1996).

Consequences of topoisomerase II inhibition in early embryogenesis of Drosophila revealed by in vivo confocal laser scanning microscopy

The regulation of DNA topology by topoisomerase II from Drosophila melanogaster has been studied extensively by biochemical methods but little is known about its roles in vivo. This study has performed experiments on the inhibition of topoisomerase II in living Drosophila blastoderm embryos. The enzymatic activity can be specifically disrupted by microinjection of antitopoisomerase II antibodies as well as the epipodophyllotoxin VM26, a known inhibitor of topoisomerase II in vitro. By labeling the chromatin of live embryos with tetramethylrhodamine-coupled histones, the effects of inhibition on nuclear morphology and behaviour was followed in vivo using confocal laser scanning microscopy. Both the antibodies and the drug prevented or hindered the segregation of chromatin daughter sets at the anaphase stage of mitosis. In addition, high concentrations of inhibitor interfered with the condensation of chromatin and its proper arrangement into the metaphase plate. The observed effects yielded non-functional nuclei, which were drawn into the inner yolk mass of the embryo. Concurrently, undamaged nuclei surrounding the affected region underwent compensatory division, leading to the restoration of the nuclear population, and thereby demonstrating the regulative capacity of Drosophila blastoderm embryos (Buchenau, 1993).


Search PubMed for articles about Drosophila Topoisomerase2

Bermejo, R., Doksani, Y., Capra, T., Katou, Y. M., Tanaka, H., Shirahige, K. and Foiani, M. (2007). Top1- and Top2-mediated topological transitions at replication forks ensure fork progression and stability and prevent DNA damage checkpoint activation. Genes Dev 21: 1921-1936. PubMed ID: 17671091

Bhat, M. A., Philp, A. V., Glover, D. M. and Bellen, H. J. (1996). Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with Topoisomerase II. Cell 87: 1103-1114. 8978614

Bucciarelli, E., Giansanti, M. G., Bonaccorsi, S. and Gatti, M. (2003). Spindle assembly and cytokinesis in the absence of chromosomes during Drosophila male meiosis. J Cell Biol 160: 993-999. PubMed ID: 12654903

Buchenau, P., Saumweber, H. and Arndt-Jovin, D. J. (1993). Consequences of topoisomerase II inhibition in early embryogenesis of Drosophila revealed by in vivo confocal laser scanning microscopy. J Cell Sci 104 ( Pt 4): 1175-1185. PubMed ID: 8391015

Chang, C. J., Goulding, S., Earnshaw, W. C. and Carmena, M. (2003). RNAi analysis reveals an unexpected role for topoisomerase II in chromosome arm congression to a metaphase plate. J Cell Sci 116: 4715-4726. PubMed ID: 14600258

Coelho, P. A., Queiroz-Machado, J. and Sunkel, C. E. (2003). Condensin-dependent localisation of topoisomerase II to an axial chromosomal structure is required for sister chromatid resolution during mitosis. J. Cell Sci. 116(Pt 23): 4763-76. PubMed ID: 14600262

Coelho, P. A., Queiroz-Machado, J., Carmo, A. M., Moutinho-Pereira, S., Maiato, H. and Sunkel, C. E. (2008). Dual role of topoisomerase II in centromere resolution and aurora B activity. PLoS Biol 6: e207. PubMed ID: 18752348

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Biological Overview

date revised: 12 September 2022

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