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DNA topoisomerase II sites in the histone H4 gene during the highly synchronous cell cycle of Physarum polycephalum
Introduction
Materials And Methods
Culture of Physarum
Physarum isolated nuclei
Enzymes and drugs
In vitro topoisomerase II cleavage reaction
Mapping strategy and probes
CP-115,953-induced topoisomerase II and DNase I cleavage sites in isolated nuclei
CP-115,953-induced topoisomerase II cleavage analyzed by pulse field gel electrophoresis
Results
Long range topoisomerase II cleavage induced in the chromosomal DNA of Physarum
Topoisomerase II cleavage sites are preferentially induced downstream of the H4 gene in vitro (naked DNA)
Topoisomerase II cleavage sites occur in the histone H4 locus in metaphase nuclei but are not detectable in asynchronous (microplasmodia) nuclei
Topoisomerase II cleavage sites in the histone H4 region are cell cycle dependent but do not correlate with the appearence of DNase I sites
Discussion
Acknowledgements
References
DNA topoisomerase II sites in the histone H4 gene during the highly synchronous cell cycle of Physarum polycephalum
ABSTRACT
INTRODUCTION
A considerable amount of knowledge has been accumulated on DNA topoisomerases during the past two decades (1). Some of the major roles of these enzymes in the living cell were determined by the use of inhibitors and mutants. Using the latter approach, topoisomerase II was found to be essential to perform complete unlinking of newly replicated chromosomal DNA during mitosis in yeast (2-4) and to control the level of DNA supercoiling, at least in prokaryotes (5). However, a number of other functions of topoisomerase II in the eukaryotic cell remain unclear. For instance, their precise roles in initiation of DNA replication and in transcription are not known. These enzymes are thought to participate in control of chromatin condensation-decondensation (6,7) and in anchorage of chromatin loops to the nuclear matrix (8,9), but the mechanisms involved are again not known.
Another approach to the function of topoisomerase II has been to map in vivo the sites of action of this enzyme in a region of DNA at a given time. To do this cells are treated with a variety of topoisomerase II poisons (10,11). This treatment results in trapping of the topoisomerase at its sites, forming complexes in which the DNA is cleaved and the two enzyme subunits are covalently bound to the DNA 5[prime]-ends. Sites of DNA cleavage are subsequently mapped by indirect end-labeling. Using this approach, in vivo topoisomerase II sites were identified near replication origins and termini, where they are supposed to be involved in decatenation of daughter DNA molecules and possibly in initiation of DNA replication (12-14). Topoisomerase II sites were also found in the promoter regions of highly transcribed genes, such as heat shock genes (15), c-myc (16) and globin genes (17), but these sites seem related to the state of condensation or accessibility of chromatin in the region, rather than to the level of transcription itself. For instance, major topoisomerase II sites appear in the c-myc locus only in tumor cell lines in which the gene is transcribed at a high rate (18). Remarkably, these sites persist even when c-myc transcription stops after the first exon, presumably because chromatin remains in an accessible conformation (19). In the case of the HSP70 gene, topoisomerase II sites are profoundly changed upon heat shock, in correlation with decondensation of chromatin occurring at this locus. It was suggested that topoisomerase II is required for re-establishment of condensed chromatin during heat shock recovery (15). Finally, topoisomerase II sites were found in the vicinity of matrix- or scaffold-associated regions (MARs or SARs), suggesting a role for the enzyme in anchorage of chromatin loops (7,9,20-22). For instance, prominent topoisomerase II sites were detected in GC-rich sequences present within the SARs of Drosophila histone repeats (23). However, the main difficulty with these studies is that the experiments were performed on cell populations that are not synchronous (or poorly synchronized): it is therefore not possible to detect sites of topoisomerase II action that appear, increase and rapidly disappear as a function of time at a given locus.
To study the sites of topoisomerase II activity during the cell cycle we have chosen the highly synchronous system Physarum polycephalum. This lower eukaryote exists as a plasmodium, whose nuclei divide with nearly perfect synchrony (24). There is no G1 phase, so that S phase immediately follows mitosis and its timing is easy to determine. Moreover, the schedule of replication of individual genes during S phase has been determined with unusual precision (25). The system therefore appears ideal to study precise events occurring in the course of the cell cycle. In a previous work we showed that it was possible to analyze the in vivo topoisomerase II cleavage sites in Physarum ribosomal (r)DNA using the quinolone CP-115,953 as a specific topoisomerase II poison (26). Two classes of sites were identified, as previously defined by Käs and Laemmli (23): those which correlate with DNase I hypersensitive sites and most likely correspond to an open chromatin configuration found in the transcribed region; internucleosomal cleavage sites located near replication origins of the rDNA unit. Drug-induced topoisomerase II cleavage in rDNA was considerably reduced upon Physarum differentiation to a dormant stage of life, the spherule. In contrast, the level of cleavage increased during metaphase, when rDNA is not transcribed. These results suggested a role for topoisomerase II in segregation of rDNA minichromosomes (26). However, since rDNA is extra-chromosomal, its replication is not as precisely scheduled as are chromosomal genes, so that it was not possible to correlate topoisomerase II sites with rDNA replication.
In the present study we took full advantage of the precise synchrony of Physarum to analyze topoisomerase II sites in a chromosomal context during the cell cycle. We chose the region histone H4, whose schedule of replication and transcription is accurately known (27,28). Our results demonstrate that topoisomerase II sites are clustered in the 3[prime]-region of the H4 gene and are present only at certain stages of the cell cycle, restricted to mitosis and the onset of S phase. Remarkably, DNase I hypersensitive sites were detected in the same region, but their schedule was different.
MATERIALS AND METHODS
Culture of Physarum
Microplasmodia of P.polycephalum (strain M3CIV) were grown in shaken cultures at 24°C in semi-defined medium (29). Synchronous macroplasmodia were obtained by coalescence of microplasmodia on filter paper as described (30). Mitosis was followed in ethanol-fixed smears using phase contrast microscopy. After microplasmodia fusion the second (mitosis II) and third mitoses (mitosis III) were observed to take place [sim]16 and 26 h after nutrient medium was added.
Physarum isolated nuclei
Nuclei were prepared by homogenization of a synchronous macroplasmodium in 200 ml buffer A (10 mM Tris-HCl, pH 8.0, 10 mM CaCl2, 0.1% Nonidet P40 and 0.25 M sucrose) at 4°C using a Warring Blendor. Nuclei were filtered, pelleted at 4°C for 5 min at 800 g, washed in 20 ml of the same buffer and pelleted again. For experiments at various times on the same plasmodium a Dounce homogenizer (Potter) was used instead of the Warring Blender: pieces of a synchronous macroplasmodium were harvested at various times in 15 ml buffer A and nuclei isolated after 10 strokes at 4°C. Nucleus isolation was checked under a phase contrast microscope.
Enzymes and drugs
Restriction enzymes were purchased from New England Biolabs. Klenow fragment and DNase I were from Boehringer. mAMSA (amsidine; a gift of J.-F.Riou, Rhône-Poulenc Rorer) and VM-26 (provided by Sandoz) were stored at -20°C as a 10 mM solution in DMSO. CP-115,953 (kindly provided by Pfizer Inc.) was stored at 5 mM concentration in 10 mM Tris-HCl, pH 7.9 at -80°C.
In vitro topoisomerase II cleavage reaction
Two pBR322-derived plasmids containing sequences from the histone H41 locus were provided by F.X.Whilelm (31). They are represented in Figure
Figure 1. Map of the Physarum histone H4 locus. Locus H41 (see Materials and Methods) is represented: the coding region is represented by a black box, with an arrow indicating the direction of transcription. The letters indicate the restriction sites used in this study: B, BamHI; E, EcoRI; H, HindIII; P, PvuI; Sa, SalI; Sm, SmaI. The italic letters correspond to restriction sites located in the vector. (Upper) Fragments derived from plasmids p[Phi]H121 (121) of 4.9 kb length and p[Phi]H125 (125) of 3.4 kb length and labeled at their pBR322 SalI site (asterisk). The hatched boxes represent the pBR322 parts of the plasmids. (Lower) Restriction fragments used for indirect end-labeling. The 3.2 kb HindIII fragment and the 6.4 kb BamHI fragment were probed with probe 2 (400 bp, see Materials and Methods). The 6.5 kb SmaI restriction fragment was probed with probe 1 (335 bp). Probes 1 and 2 are represented by black rectangles. Yeast topoisomerase II was purified as described (32). About 20 ng each labeled fragment were incubated with 1-10 ng purified topoisomerase II in 50 mM Tris-HCl, pH 7.9, 100 mM KCl, 15 mM MgCl2, 0.5 mM EDTA, 0.5 mM dithiothreitol and 15 µg/ml BSA for 10 min at 30°C. The reaction was stopped by addition of SDS (0.5% final) and proteinase K (250 µg/ml final). The sample was further incubated at 55°C for 30 min. Samples were electrophoresed through agarose gels in TEP buffer (36 mM Tris, 30 mM NaH2PO4, 1 mM EDTA, pH 7.8). The gels were dried and autoradiographed with Hyperfilm (Amersham).
Mapping strategy and probes
In Physarum there are two genes, H41 and H42, encoding histone H4. The two genes were shown to be transcribed and replicated at the same time during the cell cycle and are both linked to a replication origin located in their promoter region (27). A region of at least 6.4 kb containing the H4 gene is duplicated at different loci, since all probes derived from this region hybridize with the two gene loci (not shown). We thus chose restriction enzymes whose sites are conserved in the H41 and H42 loci to map the cleavage sites in both regions by indirect end-labeling. The mapping strategy is shown in Figure
Fragments used as probes were derived by PCR from cloned Physarum DNA. From plasmid p[Phi]H121 we derived probe 2, a 400 bp fragment amplified with primers between BamHI and HindIII sites just upstream of the H41 gene. Probe 1, a 335 bp fragment starting from the SmaI site in p[Phi]H125, was generated with primers located 1.2 kb upstream of probe 2 (Fig.
CP-115,953-induced topoisomerase II and DNase I cleavage sites in isolated nuclei
For determination of topoisomerase II cleavage sites, isolated nuclei were resuspended in 10 ml buffer B (5 mM KH2PO4, 100 mM NaCl, 5 mM MgCl2, 2 mM CaCl2, 1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 0.25 M sucrose, pH 6.2) and, following pelleting at 4°C at 800 g for 5 min, resuspended in buffer B plus 0.2 mM ATP[gamma]S. After 5 min on ice the nucleus suspension was divided into 0.5 ml aliquots and the topoisomerase II poison was added to the final concentration indicated in the text. After 30 min incubation at 26°C the reaction was stopped by sequential addition of 1% SDS, 25 mM EDTA and 500 µg/ml proteinase K. The reaction was further incubated for 16 h at 50°C. The lysate was extracted with phenol/chloroform, treated with 100 µg/ml RNase A for 30 min at 37°C, extracted once with phenol/chloroform, once with chloroform and finally ethanol precipitated. Genomic DNA was resuspended in TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA).
For identification of DNase I sensitive sites, isolated nuclei were washed in 5 ml DNase I buffer (15 mM Tris-HCl, pH 7.4, 60 mM KCl, 15 mM NaCl, 0.5 mM dithiothreitol, 0.05 mM CaCl2, 1 mM MgCl2, 0.25 M sucrose). After pelleting for 5 min at 800 g at 4°C nuclei were resuspended in DNase I buffer, divided into 0.5 ml aliquots and kept at 4°C. DNase I was diluted before use in 25 mM Tris-HCl, pH 7.6, 30% glycerol and added to nuclei for 5 min at 26°C. The DNase I reactions were stopped by addition of 40 µl 0.1 M EDTA and 2.5% SDS. Finally, proteinase K was added to 500 µg/ml and incubation continued for 16 h at 50°C. Genomic DNA was then purified as described above.
About 10 µg genomic DNA were cleaved with restriction enzymes overnight. Samples were then incubated with 200 µg/ml proteinase K for 30 min at 55°C, phenol/chloroform extracted and ethanol precipitated. After resuspension in TE, DNA was electrophoresed through 0.8-1% agarose gels at 1.2 V/cm overnight in TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0). After HCl depurination the DNA was transferred to Hybond N+ (Amersham) in 20× SSC solution (1× SSC = 0.15 M NaCl, 0.015 M sodium citrate) as described (33). Prehybridization and hybridization with the appropriate probe were performed at 42°C in 50% formamide, 5× SSC, 5× Denhardt's reagent, 1% SDS, 25 mM NaH2PO4, pH 6.8, 100 µg/ml denatured salmon sperm DNA. Membranes were washed at a final stringency of 0.2× SSC, 0.1% SDS at 65°C and later exposed for 3-8 days with intensifying screens at -80°C. For removal of the probe, filters were washed twice with boiling 0.5% SDS for 30 min each time.
For quantitation and positioning of the cleavage sites, membranes were exposed to phosphor screens that were subsequently analyzed using a Molecular Dynamics 400A Phosphorimager and ImageQuant software.
CP-115,953-induced topoisomerase II cleavage analyzed by pulse field gel electrophoresis
Nucleus concentration in buffer B was adjusted to 3.2 × 108 nuclei/ml as described (34). After incubation of nuclei with the quinolone CP-115,953 as described above, 1 vol 1% low melting point agarose (Seaplake, FMC) in buffer B containing the same concentration of drug was added to the nucleus suspension and the mixture was allowed to harden at room temperature. Agarose plugs were dialyzed against 1% SDS at room temperature for 2 h. EDTA (to 0.2 M) and proteinase K (to 500 µg/ml) were added and the plugs were dialyzed again at 50°C for 24 h. Plugs were then dialyzed four times against 0.2 M EDTA at 4°C for 1 h each time and were stored in this solution at 4°C. About 8 µg intact genomic DNA embedded in agarose were loaded on a 1% agarose gel and electrophoresed at 14°C in 0.5× TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8) using a CHEF MAPPER (BioRad) apparatus. Southern blots were then performed as described above.
RESULTS
Long range topoisomerase II cleavage induced in the chromosomal DNA of Physarum
To map topoisomerase II cleavage sites in the chromosomal DNA of Physarum we first checked the long range cleavage induced by incubation of nuclei with a specific topoisomerase II poison, the quinolone CP-115,953 (35). In a previous study this agent appeared to efficiently stimulate in vivo topoisomerase II cleavage in the extra-chromosomal rDNA of Physarum (26).
Incubation of nuclei from a G2 phase plasmodium with increasing amounts of CP-115,953 induced extensive cleavage in bulk chromosomal DNA, as judged by the smear produced in pulse field gel electrophoresis (Fig.
Figure 2. Pulse field gel analysis of long range topoisomerase II cleavage. Nuclei were isolated from a G2 plasmodium (metaphase of mitosis II + 3 h) and incubated with no drug (lanes 1) or 10 µM (lanes 2) or 100 µM (lanes 3) CP-115,953, prior to embedding in agarose. Plugs containing genomic DNA were electrophoresed at 6 V/cm with a switch angle of 120°. (A) Run time 20 h 18 min, with a switch time ramping from 0.47 to 12.91 s. (B) Run time 27 h 12 min, with an initial switch time of 2.91 s and final switch time 44.69 s. In (A) and (B), (left) ethidium bromide stained gels; (right) after transfer and hybridization with the 640 bp HindIII fragment encompassing the H4 gene (see Fig. 1). M is a size marker of a [lambda] DNA ladder. After hybridization with histone H4 probe the pattern of hybridizing DNA fragments was similar to that observed after ethidium bromide staining (Fig. 
Topoisomerase II cleavage sites are preferentially induced downstream of the H4 gene in vitro (naked DNA)
The evidence for cleavage in the region of histone H4 prompted us to more precisely map topoisomerase II sites in this locus. We first looked for drug-induced topoisomerase II cleavage sites on naked DNA within a 6.4 kb region surrounding the H4 gene (Fig.
Figure 3. In vitro topoisomerase II cleavage sites in the H4 gene region. (A) In vitro cleavage in fragment 125 with CP-115,953. An aliquot of 20 ng labeled fragment was incubated with 10 ng (lanes 2 and 3) yeast topoisomerase II. Lane 1, control without enzyme and drug; lane 3, in the presence of 50 µM CP-115,953. Electrophoresis was performed at 3.6 V/cm for 5 h. (B) In vitro cleavage in fragment 121 with different topoisomerase II poisons. An aliquot of 20 ng labeled fragment was incubated with 10 ng (lanes 2 and 3), 2 ng (lanes 4, 6 and 7) or 1 ng (lane 5) yeast topoisomerase II in the presence of 50 µM CP-115,953 (lanes 3-5), 50 µM VM-26 (lane 6) or 50 µM mAMSA (lane 7) respectively. Lane 1, control without enzyme and drug. Electrophoresis was performed at 1.2 V/cm overnight. Size marker DNA constituted by DNA restricted with AseI is shown. The asterisk indicates the position of labeling at the DNA extremity and the hatched box corresponds to the vector part from plasmid p[Phi]H121. Arrows numbered 1-3, 5 and 6 represent the CP 115,953-induced cleavage sites found in this region. Cleavage gives rise to various DNA fragments: site 1 corresponds to a 2.7 kb fragment; site 2 to a 2.55 kb fragment; site 3 to a 2.35 kb fragment; site 5 to a 1.53 kb fragment; site 6 to a 0.95 kb fragment. These sites are located at 1.9 (site 1), 1.7 (site 2), 1.5 (site 3), 0.7 (site 5) and 0.13 (site 6) kb from the initiation codon of the H4 gene. The band indicated by the upper arrows without a number has not been considered as a topoisomerase II site, since this band is present in the control and in the other lanes with about the same intensity.
Topoisomerase II cleavage sites occur in the histone H4 locus in metaphase nuclei but are not detectable in asynchronous (microplasmodia) nuclei
In order to determine whether the cleavage sites obtained in vitro could be seen in isolated nuclei and if their appearance was cell cycle dependent, we took advantage of the rigorous synchrony of Physarum plasmodia. We first compared topoisomerase II cleavage sites within the H4 locus in asynchronous microplasmodia and in metaphase plasmodia.
Sites were investigated in the 6.5 kb SmaI restriction fragment encompassing the H4 gene (Fig.
Figure 4. Drug-induced topoisomerase II cleavage within the H4 gene region in nuclei from microplasmodia and metaphase II macroplasmodia. (A) Comparison between CP-115,953-induced topoisomerase II cleavage and DNase I sensitivity in nuclei of asynchronous microplasmodia and of a synchronous macroplasmodium in mitosis (metaphase II). Microplasmodia are asynchronous shaken cultures of Physarum. The synchronous macroplasmodium in metaphase II or microplasmodia were harvested and isolated nuclei were split into two portions and incubated with either CP-115,953, to induce topoisomerase II cleavage, or with DNase I. Genomic DNA was then prepared and digested with SmaI to give a 6.5 kb fragment and hybridized with probe 1 (see Fig. 1). Lanes 1 and 6, controls without drug; lanes 2 and 7, nuclei incubated with 100 µM CP-115,953; lanes 3 and 8, without DNase I; lanes 4 and 9, 0.1 U/ml DNase I; lanes 5 and 10, 1 U/ml DNase I. (B) Cleavage induced by various topoisomerase II poisons in a synchronous macroplasmodium in mitosis (metaphase II). Nuclei of a synchronous macroplasmodium in metaphase II were split into aliquots and incubated with drug for 30 min at 26°C. Genomic DNA was digested with SmaI and probed. Lane 1, no drug control; lanes 2 and 3, 10 and 100 µM mAMSA respectively; lanes 4 and 5, 10 and 100 µM CP-115,953; lanes 6 and 7, 10 and 100 µM VM-26. In (A) and (B) the arrows numbered 1-4 indicate the fragments that correspond to topoisomerase II cleavage sites and arrows 1[prime]-3[prime] refer to DNase I hypersensitive sites described in the text. Sites 1 and 1[prime] correspond to a 3.6 kb fragment; sites 2 and 2[prime] to a 3.44 kb fragment; sites 3 and 3[prime] to a 3.24 kb fragment; site 4 to a 2.94 kb fragment. These sites are located at 1.9 (sites 1 and 1[prime]), 1.7 (sites 2 and 2[prime]), 1.5 (sites 3 and 3[prime]) and 1.24 kb (site 4) from the initiation codon of the H4 gene. To validate the results obtained with the quinolone CP-115,953 we compared these data with the cleavage patterns induced in metaphase nuclei by two other specific topoisomerase II inhibitors, mAMSA and VM-26 (Fig. 
Topoisomerase II cleavage sites in the histone H4 region are cell cycle dependent but do not correlate with the appearence of DNase I sites
Because of the appearance of topoisomerase sites in metaphase II, we looked more precisely at the behavior of these sites throughout the cell cycle. In Physarum there is no G1 phase. The cell cycle is [sim]10.5 h long, with a 30 min mitosis, immediately followed by a 3 h S phase and then a G2 phase that is 7 h long (30). It is therefore easy to determine the precise schedule of cell cycle events.
For each time point a macroplasmodium was harvested and isolated nuclei were incubated with 100 µM CP-115,953 to induce endogenous topoisomerase II cleavage. Figure
Figure 5. Topoisomerase II cleavage and DNase I sensitivity in the H4 gene region at various times of the cell cycle. For each time of the cell cycle a synchronous macroplasmodium was treated as described in Figure 4A. Lanes 1, 6, 11, 16 and 21, controls without drug; lanes 2, 7, 12, 17 and 22, nuclei incubated with 100 µM CP-115,953; lanes 3, 8, 13, 18 and 23, controls without DNase I; lanes 4, 9, 14, 19 and 24, 0.1 U/ml DNase I; lanes 5, 10, 15, 20 and 25, 1 U/ml DNase I. Times are given relative to the metaphase of mitosis II (metaphase II). The arrows numbered 1-4 indicate the bands that correspond to topoisomerase II cleavage sites described in the text and in Figure 4. Figure 6. Densitometric analysis of cleavage sites downstream of the H4 gene. Phosphorimager profiles. In this experiment DNA was restricted with BamHI and hybridized with probe 2, giving rise to a 6.4 kb fragment (see Fig. 1). The numbers above peaks indicate the DNase I hypersensitive sites (1[prime]-3[prime]) or the CP-115,953-induced topoisomerase II cleavage sites (1-4). The various DNA fragments are: sites 1 and 1[prime] correspond to a 2.5 kb fragment; sites 2 and 2[prime] to a 2.3 kb fragment; sites 3 and 3[prime] to a 2.1 kb fragment; site 4 to a 1.8 kb fragment. These sites are located at 1.9 (sites 1 and 1[prime]), 1.7 (sites 2 and 2[prime]), 1.5 (sites 3 and 3[prime]) and 1.24 (site 4) kb from the beginning of the gene. The asterisk indicates an extra topoisomerase II site during metaphase. Also, as is evident from Figures Phosphorimager quantification of cleavage intensity (Fig. Phosphorimager quantification of the overall CP-115,953-induced topoisomerase II cleavage at each time point of the cell cycle in the 6.5 kb SmaI fragment is shown in Figure Figure 7. Quantification of topoisomerase II cleavage as a function of the cell cycle. The data represent a compilation of results obtained with nuclei from plasmodia taken at various times of their cycle (see legend to Fig. 5). Total cleavage intensity was calculated by quantification of the amount of the various fragments using a Phosphorimager. We observed individual variations of ±2% in cleavage detected from one plasmodium to another. Time +660 min corresponds to metaphase III. (Inset) Expanded scale of the results obtained with a single plasmodium during prophase and onset of S phase. Genomic DNA was digested with SmaI and hybridized with probe 1 (see Fig. 1). These experiments were done using independent plasmodia. Since there is a variation of ± 2% in the intensity of cleavage in samples from different plasmodia, one cannot determine the exact time when cleavage is maximal during the cell cycle. To more precisely measure this time we analyzed topoisomerase II cleavage during mitosis and the beginning of S phase in a single plasmodium. Parts of a single plasmodium were harvested every 5 min to prepare nuclei. Two series of experiments were done. The first experiment was on a plasmodium in prophase, from 20 to 5 min before metaphase. We verified that S phase had not begun in this plasmodium, since no replication intermediates could be detected (not shown). As shown in Figure In the second experiment nuclei were isolated from a macroplasmodium from 1 min before to 12 min after metaphase (Fig. 
DISCUSSION
The experiments described in this paper provide the first demonstration that topoisomerase II sites in a chromosomal locus are strongly cell cycle dependent. This was made possible by the nearly perfect synchrony of nuclear division in a Physarum plasmodium. Short range cleavage sites were detected within a 6.6 kb region in conventional agarose gels. These sites are clustered downstream of the H4 gene, four of them in a 500 bp segment, and occur exclusively in the 30 min period that contains mitosis and the very beginning of S phase. The shortness of this period relative to the 10.5 h length of the complete cell cycle explains why these topoisomerase II sites are not detected in asynchronous microplasmodia: indeed, in an exponentially growing culture of microplasmodia there are [sim]67% nuclei in G2 phase, 28% in S phase and 5% in mitosis. Cleavage sites, which are absent in G2, reappear precisely at the next metaphase (metaphase III). This illustrates the high degree of synchrony of the system. It is worth distinguishing this short range cleavage seen in the vicinity of H4 from the long range cleavage detected by pulse field electrophoresis in a region of 20-200 kb encompassing the H4 probe. This cleavage may represent clusters of sites corresponding to anchorage points for chromatin loops, as suggested by Razin (9). Contrary to short range sites, they are detected during all the cell cycle stages and may thus be cell cycle independent.
One of the main questions addressed in studies of topoisomerase II sites is whether or not they are related to gene expression (17,18). In Physarum plasmodia, histone H4 genes are transcribed with a precise schedule (28,37). Two peaks of transcription have been described: one is coupled to S phase, with a peak at the middle of S phase; the other begins after mid-G2, peaks at about 75 min before mitosis and ends before it. Our experiments with DNase I are consistent with this scheme: hypersensitive sites in the H4 region appear during S phase and persist in G2 phase, but disappear at each mitosis. Remarkably, both topoisomerase II and DNase I sites seem to occur at the same locations downstream of the H4 gene (given the precision of [sim]100 bp of the agarose gel analysis), but their timing is completely different. This strongly suggests that topoisomerase II cleavage is not related to H4 transcription. Instead, we suggest that topoisomerase II is required in metaphase and during replication of the H4 gene. The appearance of topoisomerase II sites at prophase, increasing towards metaphase, coordinated with the disappearance of DNase I sensitivity, suggests a role for topoisomerase II in remodeling of chromatin structure during chromosome condensation and allowing their correct decatenation. The same conclusion of a role for topoisomerase II in decatenation was proposed in the case of rDNA minichromosomes, for which we found enhanced topoisomerase II cleavage although transcription was off (26). It is noteworthy that early experiments using alkaline elution suggested that overall DNA cleavage induced by topoisomerase II poisons in mammalian cells was considerably increased during mitosis (38,39). In the same way, since the gene is replicated during the first 10 min of S phase, it is tempting to speculate that occurrence of topoisomerase II sites at this time correlates with rapid chromosome decondensation after metaphase, to allow replication fork passage throughout this region. Supporting this view is the recent finding that topoisomerase II is part of a chromatin remodeling factor (40). Evidence that topoisomerase II cleavage in H4 is not related to transcription is also substantiated by the absence of topoisomerase II sites in the promoter region of the gene, contrary to several highly transcribed genes.
Also remarkable is the finding that the topoisomerase II sites detected downstream of the H4 gene during the cell cycle seem identical to the sites found on naked DNA with purified heterologous topoisomerase II in the presence of drugs. No additional major site occurs in vitro that was not also detected in nuclei. This finding suggests that sequences specific for topoisomerase II were maintained during evolution, since these sequences were effectively used by the enzyme in vivo. Thus the idea that in vivo topoisomerase II sites are selected from a vast repertoire of in vitro specific sequences on the basis of their accessibility within chromatin (41,42) has to be completed by the possibility that the sequences that were not accessible were presumably not conserved in evolution, explaining the high similarity between in vitro and in vivo results.
From these different results comes the notion of functional sites for topoisomerase II. In contrast to DNase I sites, these sites do not simply reflect accessibility of the chromatin, but functioning of the enzyme on its sites to fulfill specific roles at precise times of the cell cycle.
ACKNOWLEDGEMENTS
We are indebted to Marianne Bénard and Gérard Pierron for help and advice concerning the biology of Physarum and for sharing unpublished information and to Xavier Wilhelm for the gift of plasmids containing the H4 sequences. We thank Christine Jaxel and Marc Nadal for their important work on the manuscript and Michael Lichten for helpful comments on the manuscript. This work was supported by grants from the CNRS (URA 2225) and Association pour la Recherche sur le Cancer (6198).
REFERENCES
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