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Recruitment of cdc2 kinase by DNA topoisomerase II is coupled to chromatin remodeling ¹

ALEXANDRE E. ESCARGUEIL^*,2, SERGEI Y. PLISOV^*,2, ANDRZEJ SKLADANOWSKI^*,, ANNIE BORGNE, LAURENT MEIJER, GARY J. GORBSKY^¶ and ANNETTE K. LARSEN^*3

^* Laboratory of Tumor Biology and Pharmacology, CNRS UMR 8532, Institut Gustave Roussy, Villejuif 94805 cedex, France;
Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk, 80–952 Gdansk, Poland;
CNRS, Station Biologique, BP 74, Roscoff 29682 cedex, Bretagne, France; and
^¶ Department of Cell Biology, University of Oklahoma, Oklahoma City, Oklahoma 73104, USA

³Correspondence: CNRS UMR 8532, Institut Gustave-Roussy PR2, Villejuif 94805 cedex, France. E-mail: aklarsen{at}igr.fr

SPECIFIC AIMS

Although the appearance of the mitotic cdc2 cyclin-dependent kinase in the nucleus during early prophase is temporally linked to chromosome condensation, it is not known what targets the kinase to the nucleus and how this is coupled to chromatin remodeling. We wished to establish whether the recruitment of cdc2 kinase is mediated by physical interaction with DNA topoisomerase II and whether the association between the two enzymes leads to altered DNA/topoisomerase interactions.

PRINCIPAL FINDINGS

1. DNA topoisomerase II and cdc2 kinase form stable molecular complexes
Western blot analysis of chromosomes from mitotic HeLa cells shows that topoisomerase II as well as both subunits of cdc2 kinase (including the catalytic p34^cdc2 subunit and the regulatory cyclin B subunit) are constituents of mitotic chromosomes. To determine whether the chromosome-associated cdc2 kinase is associated with topoisomerase II, isolated chromosomes were digested with DNase I and extracted with a high-salt buffer. Chromosome extracts were then immunoprecipitated with a topoisomerase II-directed antibody, followed by Western blot analysis. These experiments demonstrate that both p34^cdc2 and cyclin B coprecipitate with topoisomerase II.

2. DNA binding and the catalytic activity of topoisomerase II are stimulated by binding to cdc2 kinase
Purified enzymes were used to establish how the interaction with cdc2 kinase influences the ability of topoisomerase II to interact with DNA. The presence of cdc2 kinase markedly stimulates the DNA binding properties of topoisomerase II, as shown by both gel retardation assays and binding to nitrocellulose membranes. Binding experiments were carried out in the absence of ATP, suggesting that the influence of cdc2 kinase on topoisomerase II is mediated by physical association and not by phosphorylation.

The catalytic activity of topoisomerase II, as measured by decatenation of interlinked kinetoplast DNA circles, is also stimulated by the presence of cdc2 kinase (Fig. 1 ). To determine whether the activation by cdc2 kinase is mediated by phosphorylation, a series of C-truncated topoisomerase II mutants was constructed (Fig. 1A ). The T3 mutant containing amino acids 1 to 1195 lacks all cdc2 phosphorylation sites but is still stimulated by cdc2 kinase, like wild-type topoisomerase II (Fig. 1 B, C ). This indicates that the stimulation of topoisomerase II by cdc2 kinase is mediated by physical association and not by phosphorylation.

View larger version (51K): [in this window] [in a new window]   Figure 1. Catalytic activity of topoisomerase II is stimulated in the presence of cdc2 kinase independent of topoisomerase II phosphorylation. A series of C-truncated human topoisomerase II mutants was constructed (A, left). The positions of reported cdc2 kinase phosphorylation sites are indicated. The T3 mutant contains no cdc2 kinase phosphorylation sites as revealed by autoradiography after incubation with cdc2 kinase and radiolabeled ATP (A, right). Different concentrations of topoisomerase II were incubated with kinetoplast DNA in the absence or presence of cdc2 kinase, and catalytic activity was determined by agarose gel electrophoresis. Decatenation of kinetoplast DNA (k) by wild-type topoisomerase II (B) or the T3 carboxyl-terminal truncated form of topoisomerase II C) can be followed by liberation of free DNA minicircles (mc).

3. DNA topoisomerase II targets cdc2 kinase to chromatin
Nuclear mitotic events have classically been studied by incubation of isolated nuclei with cell-free mitotic extracts derived from Xenopus eggs. To determine whether the physical interaction with topoisomerase II serves to recruit cdc2 kinase, a new assay was developed in which purified enzymes rather than egg extracts were incubated with isolated erythrocyte nuclei from 14-day chicken embryos that contained neither endogenous topoisomerase II nor cdc2 kinase. The results show that purified topoisomerase II associated strongly with isolated nuclei in a distinct punctuate pattern that is not affected by the presence of cdc2 kinase. In contrast, cdc2 kinase has no affinity for isolated nuclei. However, the simultaneous presence of cdc2 kinase and topoisomerase II results in a clear association of cdc2 kinase with the nuclei in a pattern similar to that observed for topoisomerase II alone.

4. Topoisomerase II-mediated targeting of cdc2 kinase to chromatin is accompanied by altered DNA-topoisomerase II interaction and chromatin remodeling
Next, we wanted to establish whether recruitment of cdc2 kinase influences the interaction between topoisomerase II and DNA in intact nuclei. Covalent binding between DNA and topoisomerase II can be revealed by the addition of a strong topoisomerase II inhibitor such as VM-26, followed by treatment with proteinase K to reveal the topoisomerase II-mediated double-strand breaks. Analysis of DNA fragmentation by pulse-field gel electrophoresis shows that preincubation of nuclei with cdc2 kinase has no detectable effect on the size of the DNA vs. untreated nuclei (Fig. 2A , lanes 1 and 2). In contrast, chromatin was cleaved into high molecular weight DNA fragments of 50 to 150 kb when the nuclei were preincubated with purified topoisomerase II (Fig. 2A , lane 3). Coincubation of nuclei with both cdc2 kinase and topoisomerase II leads to a striking increase in the degree of DNA cleavage well beyond that observed for topoisomerase II alone (Fig. 2A , compare lanes 3 and 4). Next, the influence of topoisomerase II and cdc2 kinase on the nuclear morphology of the erythrocyte nuclei was followed by staining with ethidium bromide (Fig. 2B ). The addition of neither purified topoisomerase II nor purified cdc2 kinase had any noticeable influence on the chromatin structure. In marked contrast, the simultaneous presence of both enzymes induced clear morphological changes in virtually all erythrocyte nuclei. This was associated with the formation of chromatin clumps at the nuclear periphery, giving the nuclei a ruffled appearance. Similar structures have been observed in HeLa cells, where they represent the initial step in the formation of mitotic chromosomes. In contrast, further condensation was not observed even after prolonged incubation, suggesting that additional factors are needed to convert the precondensed structures into prophase chromosomes.

View larger version (41K): [in this window] [in a new window]   Figure 2. Recruitment of cdc2 kinase by topoisomerase II is coupled to altered DNA/topoisomerase interaction and chromatin remodeling. A) Erythrocyte nuclei (lane 1) were incubated with purified cdc2 kinase (lane 2), topoisomerase II (lane 3), or both enzymes (lane 4) in the presence of the specific topoisomerase II inhibitor VM-26, followed by denaturation and proteolysis. Covalent DNA/topoisomerase II interactions were revealed by pulse-field electrophoresis. M, molecular size markers. B) Erythrocyte nuclei were incubated with purified topoisomerase II, cdc2 kinase, or both enzymes; nuclear morphology was revealed by staining with ethidium bromide.

CONCLUSIONS

We report that topoisomerase II and the mitotic kinase cdc2-cyclin B form stable molecular complexes in vitro as well as in mitotic chromosomes. The molecular interaction is required for recruitment of cdc2 kinase to chromatin as shown by incubation of purified enzymes with chicken erythrocyte nuclei, which have neither endogenous topoisomerase II nor cdc2 kinase. This observation is consistent with previous results suggesting a role for topoisomerase II as a chromatin docking protein able to target other proteins to specific subcellular domains, such as the base of the chromatin loops.

Unexpectedly, the simultaneous presence of topoisomerase II and cdc2 kinase, but neither enzyme by itself, was accompanied by extensive chromatin remodeling into precondensed structures with striking resemblance to the first step of chromosome condensation. In contrast, further condensation into prophase- and metaphase-like chromosomes was not observed. This observation can be interpreted in various ways (Fig. 3 ). It is possible that the observed chromatin remodeling is mediated directly by topoisomerase II. Alternatively, topoisomerase II may be required to recruit cdc2 kinase, thereby permitting the kinase to modify other proteins such as the condensins and histone H1, which then mediate the actual chromatin restructuring. Finally, both pathways may occur, with topoisomerase II playing both a direct and an indirect role in chromatin remodeling. Although we are not able to definitively distinguish between the various possibilities at this time, several observations suggest that topoisomerase II contributes directly to chromatin restructuring. First, the association with cdc2 kinase clearly alters the interaction between topoisomerase II and DNA in vitro as well as in intact nuclei. Second, it has previously been shown that topoisomerase II can drive changes in higher order chromosome architecture in chromosomes microsurgically removed from living cells. Finally, recent results suggest that topoisomerase II also contributes directly to a different type of chromatin remodeling that occurs during apoptotic chromosome condensation.

View larger version (25K): [in this window] [in a new window]   Figure 3. Hypothetical model of the influence of the topoisomerase II/cdc2 kinase association on mitotic chromatin remodeling. Recruitment of cdc2 kinase to the nucleus in early prophase is mediated by physical association with topoisomerase II and results in altered DNA/topoisomerase II binding. The recruitment of cdc2 kinase is also coupled to formation of precondensed chromosomes, with striking a resemblance to early prophase structures. The condensation may be a direct result of the altered DNA/topoisomerase II interaction. A different, not necessarily exclusive, possibility is that topoisomerase II serves to target cdc2 kinase to specific nuclear subdomains, such as the base of chromatin loops, thereby enabling the kinase to phosphorylate substrates such as histone H1 and/or the condensins that (also) are required for mitotic chromatin remodeling.

Another unexpected observation was that cdc2 kinase stimulated topoisomerase II through physical interaction. Although there are multiple examples documenting phosphorylation-mediated effects of cyclin-dependent kinases, the results presented here represent the first example of a regulatory effect mediated by direct physical interaction independent of phosphorylation. This may be explained by the symbiotic nature of the interaction between the two enzymes, where cdc2 kinase activates topoisomerase II whereas topoisomerase targets cdc2 kinase to specific sites, such as at the base of chromatin loops where cdc2 kinase is able to phosphorylate additional substrates. It is interesting that activation of the condensins is attributed to phosphorylation by cdc2 kinase.

In summary, the results presented here provide evidence for a physical interaction between the master mitotic kinase cdc2 and topoisomerase II, a key component of the mitotic condensation machinery. The cdc2/topoisomerase II interaction is needed to target cdc2 kinase to chromatin and is associated with initiation of mitotic chromatin remodeling, thereby providing a functional link between the appearance of cdc2 kinase in the nucleus and initiation of chromosome condensation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0726fje; to cite this article, use FASEB J. (August 17, 2001) 10.1096/fj.00-0726fje

² A.E.E. and S.Y.P. contributed equally to the work.

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