In vivo dissection of the chromosome condensation machinery: reversibility of condensation distinguishes contributions of condensin and cohesin

The machinery mediating chromosome condensation is poorly understood. To begin to dissect the in vivo function(s) of individual components, we monitored mitotic chromosome structure in mutants of condensin, cohesin, histone H3, and topoisomerase II (topo II). In budding yeast, both condensation establishment and maintenance require all of the condensin subunits, but not topo II activity or phospho-histone H3. Structural maintenance of chromosome (SMC) protein 2, as well as each of the three non-SMC proteins (Ycg1p, Ycs4p, and Brn1p), was required for chromatin binding of the condensin complex in vivo. Using reversible condensin alleles, we show that chromosome condensation does not involve an irreversible modification of condensin or chromosomes. Finally, we provide the first evidence of a mechanistic link between condensin and cohesin function. A model discussing the functional interplay between cohesin and condensin is presented.     

Pat1: a topoisomerase II-associated protein required for faithful chromosome transmission in Saccharomyces cerevisiae.

Saccharomyces cerevisiae top2 mutants deficient in topoisomerase II activity are defective in chromosome segregation during both mitotic and meiotic cell divisions. To identify proteins that act in concert with topoisomerase II during chromosome segregation in S.cerevisiae, we have used a two-hybrid cloning approach. We report the isolation of the PAT1 gene (for protein associated with topoisomerase II), which encodes a novel 90 kDa proline- and glutamine-rich protein that interacts with a highly conserved, leucine-rich region of topoisomerase II in vivo. Strains lacking Pat1p exhibit a slow growth rate and a phenotype reminiscent of conditional top2 mutants grown at the semi-permissive temperature; most notably, a reduced fidelity of chromosome segregation during both mitosis and meiosis. These findings indicate that the PAT1 gene is necessary for accurate chromosome transmission during cell division in eukaryotic cells and suggest that the interaction of Pat1p and topoisomerase II is an important component of this function.     

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. We have investigated whether the converse is true; namely, if functions responsible for maintenance of chromosome structure play a role in epigenetic control of gene expression. We show that Topoisomerase II (TOPOII) and Barren (BARR) interact in vivo with Polycomb group (PcG) target sequences in the bithorax complex of Drosophila, including Polycomb response elements. In addition, we find that the PcG protein Polyhomeotic (PH) interacts physically with TOPOII and BARR and that BARR is required for Fab-7-regulated homeotic gene expression. Conversely, we find defects in chromosome segregation associated with ph mutations. We propose 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.     

Protein kinase C delta activates topoisomerase IIalpha to induce apoptotic cell death in response to DNA damage

DNA topoisomerase II is an essential nuclear enzyme that modulates DNA processes by altering the topological state of double-stranded DNA. This enzyme is required for chromosome condensation and segregation; however, the regulatory mechanism of its activation is largely unknown. Here we demonstrate that topoisomerase IIalpha is activated in response to genotoxic stress. Concomitant with the activation, the expression of topoisomerase IIalpha is increased following DNA damage. The results also demonstrate that the proapoptotic kinase protein kinase C delta (PKCdelta) interacts with topoisomerase IIalpha. This association is in an S-phase-specific manner and is required for stabilization and catalytic activation of topoisomerase IIalpha in response to DNA damage. Conversely, inhibition of PKCdelta activity attenuates DNA damage-induced activation of topoisomerase IIalpha. Finally, aberrant activation of topoisomerase IIalpha by PKCdelta is associated with induction of apoptosis upon exposure to genotoxic agents. These findings indicate that PKCdelta regulates topoisomerase IIalpha and thereby cell fate in the genotoxic stress response.     

Fbxo28 promotes mitotic progression and regulates topoisomerase IIα-dependent DNA decatenation

Topoisomerase IIα is an essential enzyme that resolves topological constraints in genomic DNA. It functions in disentangling intertwined chromosomes during anaphase leading to chromosome segregation thus preserving genomic stability. Here we describe a previously unrecognized mechanism regulating topoisomerase IIα activity that is dependent on the F-box protein Fbxo28. We find that Fbxo28, an evolutionarily conserved protein, is required for proper mitotic progression. Interfering with Fbxo28 function leads to a delay in metaphase-to-anaphase progression resulting in mitotic defects as lagging chromosomes, multipolar spindles and multinucleation. Furthermore, we find that Fbxo28 interacts and colocalizes with topoisomerase IIα throughout the cell cycle. Depletion of Fbxo28 results in an increase in topoisomerase IIα?dependent DNA decatenation activity. Interestingly, blocking the interaction between Fbxo28 and topoisomerase IIα also results in multinucleated cells. Our findings suggest that Fbxo28 regulates topoisomerase IIα decatenation activity and plays an important role in maintaining genomic stability.     

Structural maintenance of chromosomes protein of Bacillus subtilis affects supercoiling in vivo

Structural maintenance of chromosomes (SMC) proteins are found in nearly all organisms. Members of this protein family are involved in chromosome condensation and sister chromatid cohesion. Bacillus subtilis SMC protein (BsSMC) plays a role in chromosome organization and partitioning. To better understand the function of BsSMC, we studied the effects of an smc null mutation on DNA supercoiling in vivo. We found that an smc null mutant was hypersensitive to the DNA gyrase inhibitors coumermycin A1 and norfloxacin. Furthermore, depleting cells of topoisomerase I substantially suppressed the partitioning defect of an smc null mutant. Plasmid DNA isolated from an smc null mutant was more negatively supercoiled than that from wild-type cells. In vivo cross-linking experiments indicated that BsSMC was bound to the plasmid. Our results indicate that BsSMC affects supercoiling in vivo, most likely by constraining positive supercoils, an activity which contributes to chromosome compaction and organization.     

ScII: an abundant chromosome scaffold protein is a member of a family of putative ATPases with an unusual predicted tertiary structure

Here, we describe the cloning and characterization of ScII, the second most abundant protein after topoisomerase II, of the chromosome scaffold fraction to be identified. ScII is structurally related to a protein, Smc1p, previously found to be required for accurate chromosome segregation in Saccharomyces cerevisiae. ScII and the other members of the emerging family of SMC1-like proteins are likely to be novel ATPases, with NTP-binding A and B sites separated by two lengthy regions predicted to form an alpha-helical coiled-coil. Analysis of the ScII B site predicted that ScII might use ATP by a mechanism similar to the bacterial recN DNA repair and recombination enzyme. ScII is a mitosis-specific scaffold protein that colocalizes with topoisomerase II in mitotic chromosomes. However, ScII appears not to be associated with the interphase nuclear matrix. ScII might thus play a role in mitotic processes such as chromosome condensation or sister chromatid disjunction, both of which have been previously shown to involve topoisomerase II.     

The identification and characterisation of a functional interaction between arginyl-tRNA-protein transferase and topoisomerase II

Topoisomerase II is required for the viability of all eukaryotic cells. It plays important roles in DNA replication, recombination, chromosome segregation, and the maintenance of the nuclear scaffold. Proteins that interact with and regulate this essential enzyme are of great interest. To investigate the role of proteins interacting with the N-terminal domain of the Saccharomyces cerevisiae topoisomerase II, we used a yeast two-hybrid protein interaction screen. We identified an interaction between arginyl-tRNA-protein transferase (Ate1) and the N-terminal domain of the S. cerevisiae topoisomerase II, including the potential site of interaction. Ate1 is a component of the N-end rule protein degradation pathway which targets proteins for degradation. We also propose a previously unidentified role for Ate1 in modulating the level of topoisomerase II through the cell cycle.     

Analysis of Scc1-deficient cells defines a key metaphase role of vertebrate cohesin in linking sister kinetochores

Cleavage of the cohesin subunit Scc1p/Mcd1p/Rad21 permits sister chromatid separation and is considered to trigger anaphase onset. It has also been suggested that the cohesin complex is essential for chromosome condensation and for assembling fully functional kinetochores. Here, we used vertebrate cells conditionally deficient in Scc1 to probe cohesin function in mitosis. Cells lacking cohesin arrest in prometaphase, with many chromosomes failing to align at a metaphase plate and high levels of the spindle assembly checkpoint protein, BubR1, at all kinetochores. We show that the structural integrity of chromosomes is normal in the absence of Scc1. Furthermore, specific inhibition of topoisomerase II, which is required for decatenation of replicated chromosomes, can bypass the cohesin requirement for metaphase chromosome alignment and spindle checkpoint silencing. Since the kinetochore effects of Scc1 deficiency can be compensated for by topoisomerase II inhibition, we conclude that Scc1 is not absolutely required for kinetochore assembly or function, and that its principal role in allowing the onset of anaphase is the establishment of sufficient inter-sister tension to allow biorientation.     

SMC proteins in bacteria: condensation motors for chromosome segregation?

SMC proteins are a ubiquitous protein family, present in almost all organisms so far analysed except for a few bacteria. They function in chromosome condensation, segregation, cohesion, and DNA recombination repair in eukaryotes, and can introduce positive writhe into DNA in vitro. SMC proteins and the structurally homologous MukB protein are unusual ATPases that form antiparallel dimers, with long coiled coil segments separating globular ends capable of binding DNA. Recently, SMC proteins have been shown to be essential for chromosome condensation, segregation and cell cycle progression in bacteria. Identification of a suppressor mutation for MukB in topoisomerase I in Escherichia coli suggests that SMC proteins are involved in negative DNA supercoiling in vivo, and by this means organize and compact chromosomes. A model is discussed in which bacterial SMC proteins act after an initial separation of replicated chromosome origins into the future daughter cell, separating sister chromatids by condensing replicated DNA strands within both cell halves. This would be analogous to a pulling of DNA strands into opposite cell halves by a condensation mechanism exerted at two specialised subregions in the cell.     

Topoisomerase II: its functions and phosphorylation

The gene encoding topoisomerase II in yeast is unique and essential, required for both mitotic and meiotic proliferation. The use of temperature-sensitive mutants in topoisomerase II have demonstrated roles in the relaxation of tortional stress, reduction of recombination rates, and in the separation of sister chromatids after replication. In vertebrate cells, topoisomerase II was shown to be the most abundant component of the metaphase chromosomal scaffold, and has been shown to play a role in chromosome condensationin vitro. The cell cycle control of chromosome condensation may well require phosphorylation of topoisomerase II, since the enzyme is more highly phosphorylated in metaphase than in G1. Recent studies have identified casein kinase II as the major enzyme phosphorylating topoisomerase II in intact yeast cells. The target sites of CKII are exclusively in the C-terminal 400 amino acids of topoisomerase II, the region that is most divergent among the eukaryotic type II enzymes and which is absent in the bacterial gyrase homologues.Abbreviations topoII topoisomerase II - CKII Casein Kinase II - SV40 Simian Virus 40     

Inhibition of Hsp90 acts synergistically with topoisomerase II poisons to increase the apoptotic killing of cells due to an increase in topoisomerase II mediated DNA damage

Topoisomerase II plays a crucial role during chromosome condensation and segregation in mitosis and meiosis and is a highly attractive target for chemotherapeutic agents. We have identified previously topoisomerase II and heat shock protein 90 (Hsp90) as part of a complex. In this paper we demonstrate that drug combinations targeting these two enzymes cause a synergistic increase in apoptosis. The objective of our study was to identify the mode of cell killing and the mechanism behind the increase in topoisomerase II mediated DNA damage. Importantly we demonstrate that Hsp90 inhibition results in an increased topoiosmerase II activity but not degradation of topoisomerase II and it is this, in the presence of a topoisomerase II poison that causes the increase in cell death. Our results suggest a novel mechanism of action where the inhibition of Hsp90 disrupts the Hsp90–topoisomerase II interaction leading to an increase in and activation of unbound topoisomerase II, which, in the presence of a topoisomerase II poison leads to the formation of an increased number of cleavable complexes ultimately resulting in rise in DNA damage and a subsequent increase cell death.     

PICH and Cotargeted Plk1 Coordinately Maintain Prometaphase Chromosome Arm Architecture

To maintain genomic stability, chromosome architecture needs to be tightly regulated as chromosomes undergo condensation during prophase and separation during anaphase, but the mechanisms remain poorly understood. Here, we show that the Plk1-binding protein PICH and Plk1 kinase coordinately maintain chromosome architecture during prometaphase. PICH knockdown results in a loss of Plk1 from the chromosome arm and an increase in highly disorganized “wavy” chromosomes that exhibit an “open” or “X-shaped” configuration, consistent with a loss of chromosome arm cohesion. Such chromosome disorganization occurs with essentially no change in the localization of condensin or cohesin on chromosomes. Interestingly, the chromosome disorganization could be prevented by treatment with a topoisomerase II inhibitor ICRF-193, suggesting that the PICH–Plk1 complex normally maintains chromosome architecture in a manner that involves topoisomerase II activity. PICH knockdown does not affect initial chromosome compaction at prophase but causes anaphase DNA bridge formation and failed abscission. Our studies suggest that the PICH–Plk1 complex plays a critical role in maintaining prometaphase chromosome architecture.     

Teniposide, a topoisomerase II inhibitor, prevents chromosome condensation and separation but not decondensation in fertilized surf clam (Spisula solidissima) oocytes

DNA topoisomerase II has been implicated in regulating chromosome interactions. We investigated the effects of the specific DNA topoisomerase II inhibitor, teniposide on nuclear events during oocyte maturation, fertilization, and early embryonic development of fertilized Spisula solidissima oocytes using DNA fluorescence. Teniposide treatment before fertilization not only inhibited chromosome separation during meiosis, but also blocked chromosome condensation during mitosis; however, sperm nuclear decondensation was unaffected. Chromosome separation was selectively blocked in oocytes treated with teniposide during either meiotic metaphase I or II indicating that topoisomerase II activity may be required during oocyte maturation. Teniposide treatment during meiosis also disrupted mitotic chromosome condensation. Chromosome separation during anaphase was unaffected in embryos treated with teniposide when the chromosomes were already condensed in metaphase of either first or second mitosis; however, chromosome condensation during the next mitosis was blocked. When interphase two- and four-cell embryos were exposed to topoisomerase II inhibitor, the subsequent mitosis proceeded normally in that the chromosomes condensed, separated, and decondensed; in contrast, chromosome condensation of the next mitosis was blocked. These observations suggest that in Spisula oocytes, topoisomerase II activity is required for chromosome separation during meiosis and condensation during mitosis, but is not involved in decondensation of the sperm nucleus, maternal chromosomes, and somatic chromatin.     

Extracellular signal-regulated kinase activates topoisomerase IIalpha through a mechanism independent of phosphorylation

The mitogen-activated protein (MAP) kinases, extracellular signal-related kinase 1 (ERK1) and ERK2, regulate cellular responses by mediating extracellular growth signals toward cytoplasmic and nuclear targets. A potential target for ERK is topoisomerase IIalpha, which becomes highly phosphorylated during mitosis and is required for several aspects of nucleic acid metabolism, including chromosome condensation and daughter chromosome separation. In this study, we demonstrated interactions between ERK2 and topoisomerase IIalpha proteins by coimmunoprecipitation from mixtures of purified enzymes and from nuclear extracts. In vitro, diphosphorylated active ERK2 phosphorylated topoisomerase IIalpha and enhanced its specific activity by sevenfold, as measured by DNA relaxation assays, whereas unphosphorylated ERK2 had no effect. However, activation of topoisomerase II was also observed with diphosphorylated inactive mutant ERK2, suggesting a mechanism of activation that depends on the phosphorylation state of ERK2 but not on its kinase activity. Nevertheless, activation of ERK by transient transfection of constitutively active mutant MAP kinase kinase 1 (MKK1) enhanced endogenous topoisomerase II activity by fourfold. Our findings indicate that ERK regulates topoisomerase IIalpha in vitro and in vivo, suggesting a potential target for the MKK/ERK pathway in the modulation of chromatin reorganization events during mitosis and in other phases of the cell cycle.     

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

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