Nucleic Acids Research, Vol. 18, No. 18 5449
Assembly of correctly spaced chromatin in a nuclear extract from Xenopus laevis oocytes Giovanna Sessa and Ida Ruberti* Centro di studio per gli Acidi Nucleici del CNR, c/o Dipartimento di Genetica e Biologia Molecolare, Universita di Roma La Sapienza, P.le Aldo Moro 5, 00185 Rome, Italy Received June 13, 1990; Revised and Accepted August 20, 1990
ABSTRACT Assembly of nucleosomes on relaxed, covalently closed DNA has been studied in a nuclear extract of Xenopus laevis oocytes. Nucleosomes containing the four histones H3, H4, H2A and H2B but lacking histone Hi are readily assembled on the DNA. The pattern of micrococcal nuclease digestion shows that the nucleosomes assembled in the absence of ATP and Mg (11) are closely packed, with a periodicity of 150 base pairs (bp). In contrast, in the presence of ATP and Mg (11) the spacing of the nucleosomes is 180 bp, similar to that observed for nucleosomes assembled on DNA microinjected into oocyte nuclei. The ATP and Mg (11) requirements for the assembly of correctly spaced nucleosomes are unrelated to the activity of the ATP and Mg (11) dependent DNA topoisomerase 11 in the extract; addition of specific inhibitors of eukaryotic DNA topoisomerase 11 has no effect on the spacing of the reconstituted nucleosomes. The ATP requirement in the assembly of correctly spaced nucleosomes can be substituted by adenosine 5'-0-3'-thiotriphosphate (y-S-ATP) but not by adenyl-5'-yl imidodiphosphate (AMP-P-(NH)-P).
INTRODUCTION There have been a number of attempts to reconstitute chromatin in vitro. One approach involves incubation of DNA and histones in the presence of highly charged molecules such as salts (1), nucleoplasmin (2), and polyglutamic acid (3). Although nucleosomes are reconstituted under these conditions, they are not correctly spaced when compared with nucleosomes assembled in vivo. Another approach uses cell extracts prepared from Xenopus eggs (4) or oocytes (5), both of which contain a large pool of histones (6). Chromatin with nucleosomes regularly spaced at intervals of about 200 base pairs (bp), a spacing similar to that observed in chromatin assembled in vivo, can be obtained in such extracts. The presence of large amounts of ribosomal subunits and other cytoplasmatic contaminants in these extracts make it difficult to analyze the process biochemically (4, 7), though recently several modifications have greatly improved the in vitro system for chromatin assembly from Xenopus oocytes (8). *
To whom correspondence should be addressed
We have recently developed a procedure for the large scale isolation of nuclei from X. laevis oocytes. This nuclear extract was tested for functionality using as assay 5S RNA transcription. The efficiency of transcription in this extract is apparently comparable to that in extracts prepared from manually isolated nuclei (9). In view of the difficulties of using whole cell extracts in the biochemical dissection of chromatin assembly, we have explored the use of this nuclear extract for such a purpose. In this paper, we show that in the presence of ATP and Mg (II), exogenously added DNA is converted into chromatin in the nuclear extract. Furthermore, the spacing of the nucleosomes in the chromatin formed is the same as that formed on DNA microinjected into Xenopus oocyte nuclei (10). ATP and Mg (II) are required for the correct spacing of the in vitro assembled nucleosomes. 7y-S-ATP but not AMP-P-(NH)-P can substitute for ATP in the assembly reaction. Our results are in general agreement with those obtained using whole cell extracts of Xenopus eggs (11), and show that the nuclear extract system may be useful in the study of chromatin assembly in vitro.
METHODS AND MATERIALS Nuclei isolation Nuclei were isolated according to a modification of the method of Scalenghe et al. (12), as described by Ruberti et al. (9). Preparation of nuclear extract Extract was prepared essentially as described by Birkenmeier et al. (13), with slight modifications. Nuclei were disrupted in 0.5 ll/nucleus buffer J (70 mM NH4Cl, 7 mM MgCl2, 0.1 mM EDTA, 2.5 mM DTT, 10 mM Hepes pH 7.4, 10% glycerol) by pipetting with yellow Gilson tips (5-7 times). The resulting lysate was centrifuged twice for 10 min at 500Xg at 40C and the supernatant was stored at -70°C. When necessary, the nuclear extract was passed over a Sephadex G-25 column. 0.2 ml of nuclear extract were brought to 1 M NaCl and passed over a 3 ml column equilibrated with buffer D (300 mM NaCl, 0.1 mM EDTA, 2.5 mM DTT, 10 mM Hepes pH 7.4, 10% glycerol). 20 fractions (0.2 ml) were collected. The fractions containing the proteins were identified with the Biorad microassay
5450 Nucleic Acids Research, Vol. 18, No. 18 (cat. 500-0006), pooled and stored at -70°C. At least 95% of the labelled ATP added to the extract was removed after passage of the extract over the column.
Preparation of DNA Plasmid pXbsF201 carries the 240 bp X. borealis somatic 5S RNA gene inserted in the HindIII-BamHI site of pUC9 (14). Form I pXbsF201 plasmid DNA was isolated according to a conventional procedure (15). Form I pXbsF201 was relaxed by calf thymus DNA topoisomerasi I (BRL), deproteinized with phenol to yield form II DNA (relaxed DNA).
Chromatin assembly reaction The DNA and all other reagents were dissolved in buffer E (20 mM Hepes pH 7.4, 1 mM EGTA, 10% glycerol, 10 mM ,Bglycerophosphate). 1 mM ATP, 30 mM disodium creatine phosphate (Boehringer), 1 ,%g/ml creatine phosphokinase (Boehringer) and nuclear extract to a final reaction volume of 50% were added, and the mixture incubated at 30°C. The assembly was performed at 30°C since we observed that the only difference between 23°C (which is the physiological temperature for Xenopus) and 30°C is the kinetics of the reaction which is faster at 30°C than at 23°C. When nuclear extract passed over a Sephadex G-25 column was used for assembly, unless specified, conditions were as follows: 2 mM MgCl2, 1 mM ATP, 20 mM disodium creatine phosphate, 1 ,ug/ml creatine phosphokinase and nuclear extract to a final reaction volume of 25 % were added, and the mixture was incubated at 30°C. For measurements of DNA linking number, reactions were stopped by the addition of 0.2% SDS and 16 mM EDTA. The DNA was deproteinized with 2 mg of proteinase K per ml, at 37°C for 2 h. Sample dye (5 x contains 25 % glycerol, 0.05 % bromophenol blue) was added and the samples were loaded directly onto 1 % agarose gels in Tris-glycine buffer. DNA was visualized by staining with ethidium bromide. The gels were illuminated with short-wave ultraviolet light and photographed with Polaroid film type 65.
Micrococcal nuclease digestions Analysis of chromatin assembly using MNase was performed by adding, after incubation in the nuclear extract, 3 mM CaCl2 (final concentration) and MNase (3 U/4Lg assembled DNA). The reactions were carried out at 22°C. Aliquots were removed at various times and digestion was stopped by the addition of 0.2% SDS and 16 mM EDTA. The DNA was deproteinized with 2 mg/ml proteinase K, at 37°C for 2 h. The DNA was precipitated with ethanol at -20°C for 2 h. After centrifugation the pellet was dried and dissolved in TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA). Sample dye (5 X contains 50% glycerol, 0.1 % orange G) was added and the samples were electrophoresed in 1.5% agarose gels in Tris-glycine buffer. DNA was either stained with ethidium bromide and the gels photographed as described above, or transferred to a Zeta probe membrane, hybridized to 32p labelled DNA and visualized by autoradiography (15). Isolation of the miniichromosomes Linear 10 to 30% sucrose gradients in 100 mM NaCl, 5 mM Hepes pH 7.4, 0.2 mM EGTA were made in Beckman SW50. 1 centrifuge tubes to a final volume of 4.8 ml. A 0.2 ml sample of the chromatin assembly reaction mixture was layered on top of the gradient and centrifuged at 40.000 rpm for 3 h at 4 OC. 12 fractions (0.4 ml) were collected. Aliquots (25 pl) from each fraction were taken, deproteinized and loaded onto 1 % agarose
gels in Tris glycine buffer to determine the position of the minichromosomes.
Protein analysis Fractions from two sucrose gradients containing the minichromosomes were pooled. These fractions were layered over a 1.5 ml cushion of 30% sucrose in gradient buffer and pelletted in a Beckman SW50.1 rotor at 32.000 rpm for 16 h. The supernatant was removed and the pellet was suspended in gradient buffer and stored at -20°C (slightly modified from reference 8). To free the histones from the DNA, we followed the procedure described by Shimamura et al. (8). The protein samples were electrophoresed in 15% polyacrylamide tube gels containing 6 M urea (Biorad) and 0.37% Triton X-100 (SIGMA), essentially as described by Alfageme et al. (16). After this electrophoresis, the tube gels were equilibrated for 30 min in several washes of 62.5 mM Tris pH 6.8, 0.1% SDS, 0.1% ,Bmercaptoethanol and electrophoresed in SDS-15% polyacrylamide gels (17). All protein gels were stained with silver using the procedure of Wray et al. (18). Extraction of frog erythrocyte histones The marker histones were obtained from X. laevis erithrocytes. Blood of X. laevis was obtained by puncturing the heart. Citrate (10%) was added to a final concentration of 1%. Erythrocytes (2 ml of blood) were lysed in 50 ml of a solution containing 40 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 0.25 M sucrose, 0.15 mM spermine, 0.5 mM spermidine, 1 mM PMSF, 0.5 % Nonidet P40, and then centrifuged at l000xg for 15 min in the cold. The pellet was washed twice in 40 mM Tris-HCl pH 7.5, 60 mM KCI, 15 mM NaCl, 0.5 mM CaCl2, 0.25 M sucrose, 0.15 mM spermine, 0.5 mM spermidine, 1 mM PMSF, and centrifuged as before (G. Micheli, V. Orlando, personal communication). The histones were extracted from the nuclei essentially according to the method of Adamson and Woodland (6). PMSF (1 mM) was present in all buffers throughout the extraction. Histones were suspended in 10 mM HCI, 10 mM i3mercaptoethanol (19) and stored at -70 'C. DNA catenation reaction The DNA, and the other reagents with the exception of the DNA topoisomerase II inhibitors, were dissolved in buffer E. Coumermycin Al, VP16 and VM26 were dissolved in DMSO and diluted in buffer E. The standard assay was the same as the one described for chromatin assembly except that 6 mM MgCl2 was added to the reaction mixture.
RESULTS Evidence for the assembly of relaxed DNA into chromatin in a nuclear extract from X. laevis oocytes It is well known that the linking number of a relaxed, covalently closed DNA is reduced by nucleosome assembly in the presence of a DNA topoisomerase (1, 4). Figure IA shows that the nuclear extract reduces the linking number of relaxed DNA in a 2 to 4 h reaction, Figure lB shows that the average linking number reduction of the input relaxed DNA becomes smaller as the DNA concentration is increased, indicating that histones are being exhausted by DNA when a large amount of DNA is added to the nuclear extract.
Digestion of the assembled product with micrococcal nuclease
Nucleic Acids Research, Vol. 18, No. 18 5451 A
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(MNase) produces DNA fragments with lengths that are multiples of 180 bp (figure 2, lanes 9-14). DNA devoid of nucleosomes breaks down into a broad spectrum of short fragments lacking discrete sizes when digested under similar conditions (figure 2, lanes 2-7). Incubation of exogenous DNA in the nuclear extract apparently converts it to chromatin displaying a 180 bp periodicity. The spacing observed is essentially the same as that found in vivo after microinjection of DNA into Xenopus oocyte nuclei (10) and close to that observed in chromatin assembled in Xenopus extracts (4, 5, 7, 11, 20). For the particular DNA used in the assembly studies, pXbsF201, the sedimentation coefficient is 18S. Incubation of the DNA for 6 h in the nuclear extract converted it to a 70S form (data not shown). The pXbsF201 minichromosome is estimated to contain about 16 nucleosomes, based on the size of the DNA (2970 bp) and a periodicity of 180 bp per nucleosome repeat; 70S is the sedimentation velocity which is to be expected of a compacted supranucleosomal particle of this size (21). The in vitro assembled minichromosomes were purified by sedimentation in isotonic sucrose gradients, which prevent chromatin unfolding and aggregation (22, 21). In these gradients the 70S minichromosomes are readily separated from the soluble protein components of the nuclear extract remaining at the top of the gradient. Analysis of proteins in fractions containing the 70S particles by SDS-polyacrylamide gel electrophoresis shows that the minichromosomes contain proteins which migrate in the same molecular mass range as the nucleosomal core histones H3, H4, H2A and H2B (results not shown). The presence of these four histones associated in the in vitro assembled minichromosomes was further confirned by two-dimensional gel electrophoresis, using Triton-acid-urea gel in the first dimension,
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Figure 1. DNA linking number reduction in the oocyte nuclear extract. A) Time course of DNA linking number reduction. Relaxed pXbsF201 DNA (250 ng) was incubated in the nuclear extract (5 IlI) in the presence of 3.5 mM MgCI2 and I mM ATP under standard conditions (see Methods and Materials) for 15 min (lane 2), 30 min (lane 3), 1 h (lane 4), 2 h (lane 5), 4 h (lane 6). Samples were treated as described in Methods and Materials and analyzed on a 1 % agarose gel. Lane 1 shows relaxed input DNA and lane 7 shows supercoiled marker DNA. I, supercoiled DNA; II, relaxed DNA. B) Effects of increasing DNA concentration on DNA linking number reduction. Relaxed pXbsF201 DNA was incubated for 4 h in the nuclear extract as described above. The amount of pXbsF201 DNA was 125 ng (lane 2), 187.5 ng (lane 3), 250 ng (lane 4), 375 ng (lane 5), 500 ng (lane 6). After incubation, the deproteinized DNA (125 ng) was electrophoresed in a I% agarose gel. Lane 1 shows relaxed input DNA and lane 7 shows supercoiled marker DNA. I, supercoiled DNA; II, relaxed DNA.
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Figure 2. Micrococcal nuclease digestion pattern of the in vitro assembled chromatin. Relaxed pXbsF201 DNA (2.5 ug) was incubated in the nuclear extract (50
in the presence of 3.5 mM MgCl2 and 1 mM ATP under standard conditions. Two reaction mixtures were incubated at 30 °C for 6 h. After incubation, the Il) 100 pl mM 8 min
contents were pooled, CaCl2 (3 final concentration) and MNase (15 U) were added, and aliquots were taken at 0, 1, 2, 4, and 16 (lanes 9-14). For the protein-free DNA control, relaxed pXbsF201 DNA was incubated at 30 0C for 6 h in the absence of nuclear extract. After incubation, CaC12 and MNase (1.5 U) were added and aliquots were taken at 0, 2, 4, 8, 16 and 32 min (lanes 2-7). The DNA samples were processed as described in Methods and Materials, and electrophoresed in a 1.5% agarose gel with DNA molecular weight markers (lanes 1, 8 and 15). Molecular weight markers were from a pBR322 BamHI-AvaII digest (1746, 1008, 423, 345, 279, 249 and 229 bp).
5452 Nucleic Acids Research, Vol. 18, No. 18 and SDS-polyacrylamide gel in the second (fig. 3). The 2-d gel confirmed that histone HI is absent. Furthermore, it revealed that the minichromosomes contain the H2A variants identified in Xenopus egg extracts by Dilworth et al. (23). The histone composition observed is essentially the same as that found in minichromosomes assembled in cell extracts of Xenopus oocytes
and Mg (H); DNA topoisomerase I, on the other hand, does not require these two cofactors (24). Although nucleosomes in the nuclear extract in the absence of ATP and Mg (II) are assembled, they are not properly spaced
(8).
2
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ATP and Mg (II) are required for the assembly of correctly spaced chromatin In the studies described above, the nuclear extract used in the assembly reactions contained ATP and MgCl2. To determine if ATP and Mg (II) are required for chromatin assembly, the extract was first passed over a Sephadex G-25 column to remove endogenous ATP and Mg(ll) and experiments were then carried out in the absence and presence of exogenous added ATP and MgCl2. Kinetics of nucleosome assembly as monitored by DNA linking number reduction was identical in the absence (fig. 4, lanes 2-5) and in the presence (fig. 4, lanes 6-9) of ATP and MgCl2. These results show that the linking number change of the DNA accompanying nucleosome assembly could be effected by DNA topoisomerase I. DNA topoisomerase II is dependent on ATP A
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Figure 4. Time course of DNA linking number reduction in the absence and presence of ATP and MgCl2. Relaxed pXbsF201 DNA (100 ng) was incubated in the nuclear extract (6 Al) which had been passed over a Sephadex G-25 column either in the absence (lanes 2-5) or in the presence (lanes 6-9) of 2 mM MgCl2 and 1 mM ATP under conditions described in Methods and Materials. The reaction mixtures were incubated at 30 0C for 15 min (lanes 2 and 6), 30 min (lanes 3 and 7), 1 h (lanes 4 and 8), 2 h (lanes 5 and 9). After incubation, the deproteinized DNA was electrophoresed in a 1% agarose gel. Lane 1 shows relaxed input DNA. I, supercoiled DNA; II, relaxed DNA.
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Figure 3. Two dimensional gel electrophoresis of the histones associated in the in vitro assembled minichromosomes. Minichromosomes were assembled for 6 h in the nuclear extract as described in the legend to figure 2. After incubation, the minichromosomes were purified by sucrose gradient centrifugation. The fractions containing the minichromosomes were pooled and processed as described in Methods and Materials. The sample was electrophoresed in the first dimension in Triton-acid-urea gel and in the second dimension in SDS gel. The migration position of the third H2A variant is indicated by the arrow (A). Markers of Xenopus erythrocyte histones are shown in B.
2
Figure 5. Micrococcal nuclease digestion pattern of chromatin assembled in the absence and presence of ATP and MgCl2. Relaxed pXbsF201 DNA (100 ng) was incubated overnight in the nuclear extract (6 pl) which had been passed over a Sephadex G-25 column either in the absence (lanes 1-4) or in the presence (lanes 5-8) of ATP and MgCl2 as described in the legend to figure 4. After incubation, CaC12 (3 mM final concentration) and MNase (0.3 U) were added and aliquots were taken at 0 (lanes 1 and 5), 1 (lanes 2 and 6), 3 (lanes 3 and 7) and 9 min (lanes 4 and 8). The samples were processed as described in Methods and Materials. The deproteinized DNA was electrophoresed in a 1.5% agarose gel, transferred by blotting to a Zeta-probe membrane (Biorad) and hybridized to 32p labelled pXbsF201. The positions of the DNA molecular weight markers coelectrophoresed with the digested fragments are given on the right.
Nucleic Acids Research, Vol. 18, No. 18 5453 (fig. 5, lanes 1-4). Multiples of 150 bp fragments are seen, as revealed by the size distribution of DNA fragments after MNase digestion, indicating that the nucleosomes are closely packed. 2
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Figure 6. Effects of coumermycin Al, VP16 and VM26 on DNA catenation in the oocyte nuclear extract. Relaxed pXbsF201 DNA (100 ng) was incubated for 2 h in the nuclear extract (10 Al) in the presence of 9.5 mM MgC12 and 1 mM ATP, with (lanes 3-7) and without (lane 2) the addition of different topoisomerase II inhibitors: coumermycin A1 (60 Zg/ml) (lane 3), VP16 (20 j.g/ml and 50 jig/ml) (lanes 4 and 5), VM26 (20 ig/ml and 50 yg/ml) (lanes 6 and 7). Lane 8 shows DNA incubated in the presence of 6% DMSO. After incubation, the deproteinized DNA was electrophoresed in a 1% agarose gel. Lane 1 shows relaxed input DNA, lane 9 shows supercoiled marker DNA. I, supercoiled DNA; II, relaxed DNA.
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Minichromosomes assembled in the presence of ATP and MgCl2, on the other hand, display nucleosomes with a spacing of 180 bp. (fig. 5, lanes 5-8). The ATP and Mg (11) requirements are unrelated to DNA topoisomerase II involvement in chromatin assembly To test whether the ATP and Mg (II) requirements are related to the involvement of DNA topoisomerase II, we analyzed the effect of specific inhibitors of the enzyme on nucleosome formation and spacing. The concentration of inhibitors to be used was determined by analyzing their effect on DNA catenation, a reaction catalyzed by DNA topoisomerase H (25). High concentrations of nuclear extract in the presence of ATP and high MgCl2 content convert closed circular DNA into large networks in the span of 5 min. Increasing DNA concentration reduces the percentage of catenated molecules (data not shown), which suggests that an 'aggregating' protein required for DNA catenation, like the one described in Drosophila by Hsieh and Brutlag (26) and in yeast by Goto and Wang (27), is present in our extract in limiting quantity. Under conditions of DNA catenation, relaxed DNA was incubated in the nuclear extract in the absence and presence of three different DNA topoisomerase II specific inhibitors: 1
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7. Micrococcal nuclease
digestion pattern
of chromatin assembled in the
presence of VM26.
Relaxed
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was
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(20 lAg/mld). After incubation, CaCl2 (3 mM concentration) and MNase (0.3 U) were added and aliquots were taken at 0 addition
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8). The analyzed as
4 and
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Figure 8. Micrococcal nuclease digestion pattern of chromatin assembled in the presence of AMP-P-(NH)-P and -y-S-ATP. Relaxed pXbsF201 DNA (100 ng) was incubated overnight in the nuclear extract (6 A1) which had been passed over a Sephadex G-25 column in the absence of the ATP regenerating system with the addition of MgCl2 (2 mM) and AMP-P-(NH)-P (ImM) (lanes 1-3) or MgC12 (2 mM) and -y-S-ATP (1 mM) (lanes 4-6). After incubation, CaC12 (3 mM) and MNase (0.3 U) were added and aliquots were taken at 1 (lanes 1, 4), 3 (lanes 2, 5) and 9 min (lanes 3, 6). The samples were treated as detailed in Methods and Materials and analyzed as described in the legend to figure 5. The positions of the DNA molecular weight markers coelectrophoresed with the digested fragments are given on the right.
5454 Nucleic Acids Research, Vol. 18, No. 18 coumermycin Al, VP16 and VM26. Coumermycin Al is assumed to compete with ATP binding to the enzyme. VP16 and VM26 inhibit eukaryotic DNA topoisomerase H by trapping a topoisomerase-DNA reaction intermediate so that subsequent treatment with a protein denaturant results in DNA cleavage (see 28 and 29 for reviews). As shown in figure 6, DNA catenation is completely blocked by coumermycin Al at 60 ,^g/ml (lane 3). Addition of VP16 or VM26 to the DNA catenation assay resulted in DNA breakage when the reaction was stopped by the addition of SDS; the input DNA was converted to the linear form (lanes 4-7). When VM26 was present at a concentration of 20 ,g/ml more than 95 % of the DNA was converted to the linear form (lane 6). We then analyzed the effects of inhibitors on chromatin assembly. Under standard conditions of chromatin assembly, relaxed DNA was incubated in the nuclear extract in the absence and presence of 20 ,ug/ml VM26. As shown in figure 7, DNA linking number reduction is not significantly affected by the inhibitor (compare lanes 1 and 5). Moreover, the MNase digestion pattern of chromatin assembled in the presence of ATP, MgCl2 and VM26 displays correctly spaced nucleosomes (fig. 7, lanes 6-8); compare with samples shown in lanes 2-4, which were MNase digests of chromatin assembled in the presence of ATP and MgCl2. The same result was obtained when the assembly reaction was performed in the presence of 60 jg/ml coumermycin Al (data not shown and fig. 6, lane 3). The ATP requirement in the assembly of correctly spaced chromatin can be substituted by -y-S-ATP but not by
AMP-P-(NH)-P To understand the role of ATP in the chromatin assembly process, we analyzed the effects of ATP analogues on nucleosome spacing. We used two different non-hydrolyzable ATP analogues: AMPP-(NH)-P and -y-S-ATP. MNase digestion pattern of chromatin assembled in the presence of the imido analog and MgCl2 shows that nucleosomes are not correctly spaced (fig. 8, lanes 1-3); the spacing of the nucleosomes is 150 bp, the same as that observed for nucleosomes assembled in the absence of ATP and MgCl2. Chromatin assembled in the presence of -y-S-ATP and MgCl2, on the other hand, displays nucleosomes with a spacing of 180 bp (fig. 8, lanes 4-6).
DISCUSSION We developed an in vitro system for chromatin assembly using an extract prepared from the nuclei of Xenopus oocytes. Our results demonstrate that in the presence of ATP and Mg(II), exogenously added DNA is assembled into correctly spaced chromatin in the nuclear extract. The in vitro assembled chromatin contains the four histones H3, H4, H2A and H2B but no histone HI. The nuclear extract lends itself to a biochemical analysis of the process since relatively large amounts of DNA are assembled into chromatin (50-75 ng DNA per /l nuclear extract) and the nucleoprotein complex can be easily purified. The nuclear extract has been used to investigate the role of ATP and Mg(II) in the assembly of correctly spaced chromatin. The pattern of micrococcal nuclease digestion shows that the nucleosomes assembled in the absence of ATP and Mg(II) are closely packed with a periodicity of 150 bp. In contrast, in the presence of ATP and Mg(II) the spacing of the nucleosomes is 180 bp, essentially the same as that found in vivo after microinjection of DNA into Xenopus oocyte nuclei (10).
Glikin et al. (5) related the ATP and Mg(II) requirements for the assembly of correctly spaced chromatin in cell extracts of Xenopus oocytes to the involvement of the ATP and Mg(II) dependent DNA topoisomerase II in the assembly process. They proposed that DNA topoisomerase II in the presence of other extract components actively supercoils DNA and drives the formation of correctly spaced nucleosomes (5). The involvement of DNA topoisomerase II was proposed on the basis of the observation that novobiocin, an inhibitor of both prokaryotic and eukaryotic DNA topoisomerase H (30, 26), inhibited nucleosome formation in cell extracts of Xenopus oocytes (5). The novobiocin inhibition is difficult to interpret, however, in the light of the recent evidence of secondary effects of the drug (31, 32, 11). Our observations that the rate of nucleosome assembly as monitored by DNA linking number reduction is unaffected by ATP and Mg(ll) and that VM26, a specific inhibitor of eukaryotic DNA topoisomerase H, has no effect on nucleosome formation and spacing clearly indicate that the ATP and Mg(ll) requirements in the process are unrelated to the activity of DNA topoisomerase II. Our results are consistent with the model for chromatin assembly proposed by Almouzni and Mechali (11). They proposed that chromatin is formed in a two-step reaction. In the first step, nucleosome formation occurs independently of ATP and Mg (II). In this step, DNA topoisomerase I activity is involved in the relaxation of the topological constraints generated by nucleosome assembly. The second step, requiring ATP and Mg (II), generates properly spaced chromatin (11). To understand the role of ATP in nucleosome spacing we analyzed the effects of non-hydrolyzable ATP analogues, AMPP-(NH)-P and y-S-ATP. The pattern of micrococcal nuclease digestion of chromatin assembled in the presence of AMP-P(NH)-P and Mg(H) shows that the nucleosomes are closely packed with a periodicity of 150 bp. Chromatin assembled in the presence of -y-S-ATP and Mg(II), on the other hand, displays correctly spaced nucleosomes. The result obtained when we included the imido analog and MgCl2 in the assembly reaction demonstrates that the ATP is essential to the formation of properly spaced chromatin. Moreover, the data show that -y-S-ATP but not AMPP-(NH)-P can substitute for ATP in the process. The observation that 7y-S-ATP, but not AMP-P-(NH)-P, can substitute for ATP in the reaction is reminiscent of recA assembly on duplex DNA. The ATP binding to recA induces a conformational change of the protein and the subsequent formation of a protein-DNA complex. The assembly of recA on duplex DNA can be supported by ATP and -y-S-ATP, but not by AMP-P-(NH)-P or AMP-P-(CH2)-P (33). It should also be noted that the use of y-S-ATP, but not of AMP-P-(NH)-P, can result in thiophosphorilation of proteins since it is readily utilized by protein kinase (34). We suggest that a 'spacing factor' is involved in the assembly of correctly spaced nucleosomes. It is conceivable that in the absence of ATP the 'spacing factor' is inactive and this could cause the observed tight packing of nucleosomes. In the presence of ATP the activation of the factor could generate the correct spacing of the nucleosomes. Experiments will be designed to identify the factor involved in
nucleosome spacing.
ACKNOWLEDGEMENTS We thank Elisabetta Bianchi for the experiment shown in figure 2; Angelo Di Francesco and Roberto Gargamelli for skilled
Nucleic Acids Research, Vol. 18, No. 18 5455 technical assistance; Giorgio Morelli for critical reading of this manuscript. This work has been supported in part by grants from C.N.R., Progetto Bilaterale (I.R.) and the 'Istituto Pasteur-Fondazione Cenci Bolognetti', Universitt di Roma La Sapienza (I.R.).
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66, 299-308. 13. Birkenmeier,E.H., Brown,D. and Jordan,E. (1978) Cell, 15, 1077-1086. 14. Razvi,F., Gargiulo,G. and Worcel,A. (1983) Gene, 23, 175-183. 15. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 16. Alfageme,C.R., Zweidler,A., Mahowald,A. and Cohen,L.H. (1974) J. Biol. Chem., 249, 3729-3736. 17. Laemmli,U.R. (1970) Nature, 227, 680-685. 18. Wray,W., Boulikas,T., Wray,V.P. and Hancock R. (1981) Anal. Biochem., 118, 197-203. 19. Ellison,M.J. and Pulleyblank,D.E. (1983) J. Biol. Chem., 258, 13307-13313. 20. Rodriguez-Campos,A., Shimamura,A. and Worcel,A. (1989) J. Mol. Biol., 209, 135-150. 21. Worcel,A., Han,S. and Wong,M.L. (1978) Cell, 15, 969-977. 22. Christiansen,G. and Griffith, J. (1977) Nucleic Acids Res., 4, 1837- 1851. 23. Dilworth,S.M., Black,S.J. and Laskey,R.A. (1987) Cell, 51, 1009-1018. 24. Wang,J.C. (1985) Annu. Rev. Biochem., 54, 665-697 25. Baldi,M.I., Benedetti,P., Mattoccia,E. and Tocchini-Valentini,G.P. (1980) Cell, 20, 461-467. 26. Hsieh,T.-S. and Brutlag,D. (1980) Cell, 21, 115-125. 27. Goto,T. and Wang,J.C. (1982) J. Bio. Chem., 257, 5866-5872. 28. Drlica,K. and Franco,R.J. (1988) Biochemistry., 27, 2253-2259. 29. Liu,L.F. (1989) Annu. Rev. Biochem., 58, 351-375. 30. Gellert,M., O'Dea,M.H., Itoh,T. and Tomizawa,J.I. (1976) Proc. NatI. Acad. Sci. USA, 73, 3872-3876. 31. Cotten,H., Bresnahan,D., Thompson,S., Sealy,L. and Chalkey,R. (1986) Nucleic Acids Res., 14, 3671-3687. 32. Sealy,L., Cotten,H. and Chalkley,R. (1986) EMBO J., 5, 3305-3311. 33. McEntee,K., Weinstock,G.M. and Lehman,I.R. (1981) J. Biol. Chem. 256, 8835-8844. 34. Burke,B. and Gerace,L. (1986) Cell , 44, 639-652.
Assembly of correctly spaced chromatin in a nuclear extract from Xenopus laevis oocytes Giovanna Sessa and Ida Ruberti* Centro di studio per gli Acidi Nucleici del CNR, c/o Dipartimento di Genetica e Biologia Molecolare, Universita di Roma La Sapienza, P.le Aldo Moro 5, 00185 Rome, Italy Received June 13, 1990; Revised and Accepted August 20, 1990
ABSTRACT Assembly of nucleosomes on relaxed, covalently closed DNA has been studied in a nuclear extract of Xenopus laevis oocytes. Nucleosomes containing the four histones H3, H4, H2A and H2B but lacking histone Hi are readily assembled on the DNA. The pattern of micrococcal nuclease digestion shows that the nucleosomes assembled in the absence of ATP and Mg (11) are closely packed, with a periodicity of 150 base pairs (bp). In contrast, in the presence of ATP and Mg (11) the spacing of the nucleosomes is 180 bp, similar to that observed for nucleosomes assembled on DNA microinjected into oocyte nuclei. The ATP and Mg (11) requirements for the assembly of correctly spaced nucleosomes are unrelated to the activity of the ATP and Mg (11) dependent DNA topoisomerase 11 in the extract; addition of specific inhibitors of eukaryotic DNA topoisomerase 11 has no effect on the spacing of the reconstituted nucleosomes. The ATP requirement in the assembly of correctly spaced nucleosomes can be substituted by adenosine 5'-0-3'-thiotriphosphate (y-S-ATP) but not by adenyl-5'-yl imidodiphosphate (AMP-P-(NH)-P).
INTRODUCTION There have been a number of attempts to reconstitute chromatin in vitro. One approach involves incubation of DNA and histones in the presence of highly charged molecules such as salts (1), nucleoplasmin (2), and polyglutamic acid (3). Although nucleosomes are reconstituted under these conditions, they are not correctly spaced when compared with nucleosomes assembled in vivo. Another approach uses cell extracts prepared from Xenopus eggs (4) or oocytes (5), both of which contain a large pool of histones (6). Chromatin with nucleosomes regularly spaced at intervals of about 200 base pairs (bp), a spacing similar to that observed in chromatin assembled in vivo, can be obtained in such extracts. The presence of large amounts of ribosomal subunits and other cytoplasmatic contaminants in these extracts make it difficult to analyze the process biochemically (4, 7), though recently several modifications have greatly improved the in vitro system for chromatin assembly from Xenopus oocytes (8). *
To whom correspondence should be addressed
We have recently developed a procedure for the large scale isolation of nuclei from X. laevis oocytes. This nuclear extract was tested for functionality using as assay 5S RNA transcription. The efficiency of transcription in this extract is apparently comparable to that in extracts prepared from manually isolated nuclei (9). In view of the difficulties of using whole cell extracts in the biochemical dissection of chromatin assembly, we have explored the use of this nuclear extract for such a purpose. In this paper, we show that in the presence of ATP and Mg (II), exogenously added DNA is converted into chromatin in the nuclear extract. Furthermore, the spacing of the nucleosomes in the chromatin formed is the same as that formed on DNA microinjected into Xenopus oocyte nuclei (10). ATP and Mg (II) are required for the correct spacing of the in vitro assembled nucleosomes. 7y-S-ATP but not AMP-P-(NH)-P can substitute for ATP in the assembly reaction. Our results are in general agreement with those obtained using whole cell extracts of Xenopus eggs (11), and show that the nuclear extract system may be useful in the study of chromatin assembly in vitro.
METHODS AND MATERIALS Nuclei isolation Nuclei were isolated according to a modification of the method of Scalenghe et al. (12), as described by Ruberti et al. (9). Preparation of nuclear extract Extract was prepared essentially as described by Birkenmeier et al. (13), with slight modifications. Nuclei were disrupted in 0.5 ll/nucleus buffer J (70 mM NH4Cl, 7 mM MgCl2, 0.1 mM EDTA, 2.5 mM DTT, 10 mM Hepes pH 7.4, 10% glycerol) by pipetting with yellow Gilson tips (5-7 times). The resulting lysate was centrifuged twice for 10 min at 500Xg at 40C and the supernatant was stored at -70°C. When necessary, the nuclear extract was passed over a Sephadex G-25 column. 0.2 ml of nuclear extract were brought to 1 M NaCl and passed over a 3 ml column equilibrated with buffer D (300 mM NaCl, 0.1 mM EDTA, 2.5 mM DTT, 10 mM Hepes pH 7.4, 10% glycerol). 20 fractions (0.2 ml) were collected. The fractions containing the proteins were identified with the Biorad microassay
5450 Nucleic Acids Research, Vol. 18, No. 18 (cat. 500-0006), pooled and stored at -70°C. At least 95% of the labelled ATP added to the extract was removed after passage of the extract over the column.
Preparation of DNA Plasmid pXbsF201 carries the 240 bp X. borealis somatic 5S RNA gene inserted in the HindIII-BamHI site of pUC9 (14). Form I pXbsF201 plasmid DNA was isolated according to a conventional procedure (15). Form I pXbsF201 was relaxed by calf thymus DNA topoisomerasi I (BRL), deproteinized with phenol to yield form II DNA (relaxed DNA).
Chromatin assembly reaction The DNA and all other reagents were dissolved in buffer E (20 mM Hepes pH 7.4, 1 mM EGTA, 10% glycerol, 10 mM ,Bglycerophosphate). 1 mM ATP, 30 mM disodium creatine phosphate (Boehringer), 1 ,%g/ml creatine phosphokinase (Boehringer) and nuclear extract to a final reaction volume of 50% were added, and the mixture incubated at 30°C. The assembly was performed at 30°C since we observed that the only difference between 23°C (which is the physiological temperature for Xenopus) and 30°C is the kinetics of the reaction which is faster at 30°C than at 23°C. When nuclear extract passed over a Sephadex G-25 column was used for assembly, unless specified, conditions were as follows: 2 mM MgCl2, 1 mM ATP, 20 mM disodium creatine phosphate, 1 ,ug/ml creatine phosphokinase and nuclear extract to a final reaction volume of 25 % were added, and the mixture was incubated at 30°C. For measurements of DNA linking number, reactions were stopped by the addition of 0.2% SDS and 16 mM EDTA. The DNA was deproteinized with 2 mg of proteinase K per ml, at 37°C for 2 h. Sample dye (5 x contains 25 % glycerol, 0.05 % bromophenol blue) was added and the samples were loaded directly onto 1 % agarose gels in Tris-glycine buffer. DNA was visualized by staining with ethidium bromide. The gels were illuminated with short-wave ultraviolet light and photographed with Polaroid film type 65.
Micrococcal nuclease digestions Analysis of chromatin assembly using MNase was performed by adding, after incubation in the nuclear extract, 3 mM CaCl2 (final concentration) and MNase (3 U/4Lg assembled DNA). The reactions were carried out at 22°C. Aliquots were removed at various times and digestion was stopped by the addition of 0.2% SDS and 16 mM EDTA. The DNA was deproteinized with 2 mg/ml proteinase K, at 37°C for 2 h. The DNA was precipitated with ethanol at -20°C for 2 h. After centrifugation the pellet was dried and dissolved in TE (10 mM Tris-HCl pH 7.5, 1 mM EDTA). Sample dye (5 X contains 50% glycerol, 0.1 % orange G) was added and the samples were electrophoresed in 1.5% agarose gels in Tris-glycine buffer. DNA was either stained with ethidium bromide and the gels photographed as described above, or transferred to a Zeta probe membrane, hybridized to 32p labelled DNA and visualized by autoradiography (15). Isolation of the miniichromosomes Linear 10 to 30% sucrose gradients in 100 mM NaCl, 5 mM Hepes pH 7.4, 0.2 mM EGTA were made in Beckman SW50. 1 centrifuge tubes to a final volume of 4.8 ml. A 0.2 ml sample of the chromatin assembly reaction mixture was layered on top of the gradient and centrifuged at 40.000 rpm for 3 h at 4 OC. 12 fractions (0.4 ml) were collected. Aliquots (25 pl) from each fraction were taken, deproteinized and loaded onto 1 % agarose
gels in Tris glycine buffer to determine the position of the minichromosomes.
Protein analysis Fractions from two sucrose gradients containing the minichromosomes were pooled. These fractions were layered over a 1.5 ml cushion of 30% sucrose in gradient buffer and pelletted in a Beckman SW50.1 rotor at 32.000 rpm for 16 h. The supernatant was removed and the pellet was suspended in gradient buffer and stored at -20°C (slightly modified from reference 8). To free the histones from the DNA, we followed the procedure described by Shimamura et al. (8). The protein samples were electrophoresed in 15% polyacrylamide tube gels containing 6 M urea (Biorad) and 0.37% Triton X-100 (SIGMA), essentially as described by Alfageme et al. (16). After this electrophoresis, the tube gels were equilibrated for 30 min in several washes of 62.5 mM Tris pH 6.8, 0.1% SDS, 0.1% ,Bmercaptoethanol and electrophoresed in SDS-15% polyacrylamide gels (17). All protein gels were stained with silver using the procedure of Wray et al. (18). Extraction of frog erythrocyte histones The marker histones were obtained from X. laevis erithrocytes. Blood of X. laevis was obtained by puncturing the heart. Citrate (10%) was added to a final concentration of 1%. Erythrocytes (2 ml of blood) were lysed in 50 ml of a solution containing 40 mM Tris-HCl pH 7.5, 60 mM KCl, 15 mM NaCl, 0.25 M sucrose, 0.15 mM spermine, 0.5 mM spermidine, 1 mM PMSF, 0.5 % Nonidet P40, and then centrifuged at l000xg for 15 min in the cold. The pellet was washed twice in 40 mM Tris-HCl pH 7.5, 60 mM KCI, 15 mM NaCl, 0.5 mM CaCl2, 0.25 M sucrose, 0.15 mM spermine, 0.5 mM spermidine, 1 mM PMSF, and centrifuged as before (G. Micheli, V. Orlando, personal communication). The histones were extracted from the nuclei essentially according to the method of Adamson and Woodland (6). PMSF (1 mM) was present in all buffers throughout the extraction. Histones were suspended in 10 mM HCI, 10 mM i3mercaptoethanol (19) and stored at -70 'C. DNA catenation reaction The DNA, and the other reagents with the exception of the DNA topoisomerase II inhibitors, were dissolved in buffer E. Coumermycin Al, VP16 and VM26 were dissolved in DMSO and diluted in buffer E. The standard assay was the same as the one described for chromatin assembly except that 6 mM MgCl2 was added to the reaction mixture.
RESULTS Evidence for the assembly of relaxed DNA into chromatin in a nuclear extract from X. laevis oocytes It is well known that the linking number of a relaxed, covalently closed DNA is reduced by nucleosome assembly in the presence of a DNA topoisomerase (1, 4). Figure IA shows that the nuclear extract reduces the linking number of relaxed DNA in a 2 to 4 h reaction, Figure lB shows that the average linking number reduction of the input relaxed DNA becomes smaller as the DNA concentration is increased, indicating that histones are being exhausted by DNA when a large amount of DNA is added to the nuclear extract.
Digestion of the assembled product with micrococcal nuclease
Nucleic Acids Research, Vol. 18, No. 18 5451 A
1
2
3
4
5
3
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6
(MNase) produces DNA fragments with lengths that are multiples of 180 bp (figure 2, lanes 9-14). DNA devoid of nucleosomes breaks down into a broad spectrum of short fragments lacking discrete sizes when digested under similar conditions (figure 2, lanes 2-7). Incubation of exogenous DNA in the nuclear extract apparently converts it to chromatin displaying a 180 bp periodicity. The spacing observed is essentially the same as that found in vivo after microinjection of DNA into Xenopus oocyte nuclei (10) and close to that observed in chromatin assembled in Xenopus extracts (4, 5, 7, 11, 20). For the particular DNA used in the assembly studies, pXbsF201, the sedimentation coefficient is 18S. Incubation of the DNA for 6 h in the nuclear extract converted it to a 70S form (data not shown). The pXbsF201 minichromosome is estimated to contain about 16 nucleosomes, based on the size of the DNA (2970 bp) and a periodicity of 180 bp per nucleosome repeat; 70S is the sedimentation velocity which is to be expected of a compacted supranucleosomal particle of this size (21). The in vitro assembled minichromosomes were purified by sedimentation in isotonic sucrose gradients, which prevent chromatin unfolding and aggregation (22, 21). In these gradients the 70S minichromosomes are readily separated from the soluble protein components of the nuclear extract remaining at the top of the gradient. Analysis of proteins in fractions containing the 70S particles by SDS-polyacrylamide gel electrophoresis shows that the minichromosomes contain proteins which migrate in the same molecular mass range as the nucleosomal core histones H3, H4, H2A and H2B (results not shown). The presence of these four histones associated in the in vitro assembled minichromosomes was further confirned by two-dimensional gel electrophoresis, using Triton-acid-urea gel in the first dimension,
7
ii :....
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B
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Figure 1. DNA linking number reduction in the oocyte nuclear extract. A) Time course of DNA linking number reduction. Relaxed pXbsF201 DNA (250 ng) was incubated in the nuclear extract (5 IlI) in the presence of 3.5 mM MgCI2 and I mM ATP under standard conditions (see Methods and Materials) for 15 min (lane 2), 30 min (lane 3), 1 h (lane 4), 2 h (lane 5), 4 h (lane 6). Samples were treated as described in Methods and Materials and analyzed on a 1 % agarose gel. Lane 1 shows relaxed input DNA and lane 7 shows supercoiled marker DNA. I, supercoiled DNA; II, relaxed DNA. B) Effects of increasing DNA concentration on DNA linking number reduction. Relaxed pXbsF201 DNA was incubated for 4 h in the nuclear extract as described above. The amount of pXbsF201 DNA was 125 ng (lane 2), 187.5 ng (lane 3), 250 ng (lane 4), 375 ng (lane 5), 500 ng (lane 6). After incubation, the deproteinized DNA (125 ng) was electrophoresed in a I% agarose gel. Lane 1 shows relaxed input DNA and lane 7 shows supercoiled marker DNA. I, supercoiled DNA; II, relaxed DNA.
1
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DNA
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Figure 2. Micrococcal nuclease digestion pattern of the in vitro assembled chromatin. Relaxed pXbsF201 DNA (2.5 ug) was incubated in the nuclear extract (50
in the presence of 3.5 mM MgCl2 and 1 mM ATP under standard conditions. Two reaction mixtures were incubated at 30 °C for 6 h. After incubation, the Il) 100 pl mM 8 min
contents were pooled, CaCl2 (3 final concentration) and MNase (15 U) were added, and aliquots were taken at 0, 1, 2, 4, and 16 (lanes 9-14). For the protein-free DNA control, relaxed pXbsF201 DNA was incubated at 30 0C for 6 h in the absence of nuclear extract. After incubation, CaC12 and MNase (1.5 U) were added and aliquots were taken at 0, 2, 4, 8, 16 and 32 min (lanes 2-7). The DNA samples were processed as described in Methods and Materials, and electrophoresed in a 1.5% agarose gel with DNA molecular weight markers (lanes 1, 8 and 15). Molecular weight markers were from a pBR322 BamHI-AvaII digest (1746, 1008, 423, 345, 279, 249 and 229 bp).
5452 Nucleic Acids Research, Vol. 18, No. 18 and SDS-polyacrylamide gel in the second (fig. 3). The 2-d gel confirmed that histone HI is absent. Furthermore, it revealed that the minichromosomes contain the H2A variants identified in Xenopus egg extracts by Dilworth et al. (23). The histone composition observed is essentially the same as that found in minichromosomes assembled in cell extracts of Xenopus oocytes
and Mg (H); DNA topoisomerase I, on the other hand, does not require these two cofactors (24). Although nucleosomes in the nuclear extract in the absence of ATP and Mg (II) are assembled, they are not properly spaced
(8).
2
1
ATP and Mg (II) are required for the assembly of correctly spaced chromatin In the studies described above, the nuclear extract used in the assembly reactions contained ATP and MgCl2. To determine if ATP and Mg (II) are required for chromatin assembly, the extract was first passed over a Sephadex G-25 column to remove endogenous ATP and Mg(ll) and experiments were then carried out in the absence and presence of exogenous added ATP and MgCl2. Kinetics of nucleosome assembly as monitored by DNA linking number reduction was identical in the absence (fig. 4, lanes 2-5) and in the presence (fig. 4, lanes 6-9) of ATP and MgCl2. These results show that the linking number change of the DNA accompanying nucleosome assembly could be effected by DNA topoisomerase I. DNA topoisomerase II is dependent on ATP A
0
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I., 1
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5 6 7 low .."t_i-
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a
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Figure 4. Time course of DNA linking number reduction in the absence and presence of ATP and MgCl2. Relaxed pXbsF201 DNA (100 ng) was incubated in the nuclear extract (6 Al) which had been passed over a Sephadex G-25 column either in the absence (lanes 2-5) or in the presence (lanes 6-9) of 2 mM MgCl2 and 1 mM ATP under conditions described in Methods and Materials. The reaction mixtures were incubated at 30 0C for 15 min (lanes 2 and 6), 30 min (lanes 3 and 7), 1 h (lanes 4 and 8), 2 h (lanes 5 and 9). After incubation, the deproteinized DNA was electrophoresed in a 1% agarose gel. Lane 1 shows relaxed input DNA. I, supercoiled DNA; II, relaxed DNA.
0b
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Figure 3. Two dimensional gel electrophoresis of the histones associated in the in vitro assembled minichromosomes. Minichromosomes were assembled for 6 h in the nuclear extract as described in the legend to figure 2. After incubation, the minichromosomes were purified by sucrose gradient centrifugation. The fractions containing the minichromosomes were pooled and processed as described in Methods and Materials. The sample was electrophoresed in the first dimension in Triton-acid-urea gel and in the second dimension in SDS gel. The migration position of the third H2A variant is indicated by the arrow (A). Markers of Xenopus erythrocyte histones are shown in B.
2
Figure 5. Micrococcal nuclease digestion pattern of chromatin assembled in the absence and presence of ATP and MgCl2. Relaxed pXbsF201 DNA (100 ng) was incubated overnight in the nuclear extract (6 pl) which had been passed over a Sephadex G-25 column either in the absence (lanes 1-4) or in the presence (lanes 5-8) of ATP and MgCl2 as described in the legend to figure 4. After incubation, CaC12 (3 mM final concentration) and MNase (0.3 U) were added and aliquots were taken at 0 (lanes 1 and 5), 1 (lanes 2 and 6), 3 (lanes 3 and 7) and 9 min (lanes 4 and 8). The samples were processed as described in Methods and Materials. The deproteinized DNA was electrophoresed in a 1.5% agarose gel, transferred by blotting to a Zeta-probe membrane (Biorad) and hybridized to 32p labelled pXbsF201. The positions of the DNA molecular weight markers coelectrophoresed with the digested fragments are given on the right.
Nucleic Acids Research, Vol. 18, No. 18 5453 (fig. 5, lanes 1-4). Multiples of 150 bp fragments are seen, as revealed by the size distribution of DNA fragments after MNase digestion, indicating that the nucleosomes are closely packed. 2
3
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5
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DNA NETWORl
II
I
Figure 6. Effects of coumermycin Al, VP16 and VM26 on DNA catenation in the oocyte nuclear extract. Relaxed pXbsF201 DNA (100 ng) was incubated for 2 h in the nuclear extract (10 Al) in the presence of 9.5 mM MgC12 and 1 mM ATP, with (lanes 3-7) and without (lane 2) the addition of different topoisomerase II inhibitors: coumermycin A1 (60 Zg/ml) (lane 3), VP16 (20 j.g/ml and 50 jig/ml) (lanes 4 and 5), VM26 (20 ig/ml and 50 yg/ml) (lanes 6 and 7). Lane 8 shows DNA incubated in the presence of 6% DMSO. After incubation, the deproteinized DNA was electrophoresed in a 1% agarose gel. Lane 1 shows relaxed input DNA, lane 9 shows supercoiled marker DNA. I, supercoiled DNA; II, relaxed DNA.
3
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Minichromosomes assembled in the presence of ATP and MgCl2, on the other hand, display nucleosomes with a spacing of 180 bp. (fig. 5, lanes 5-8). The ATP and Mg (11) requirements are unrelated to DNA topoisomerase II involvement in chromatin assembly To test whether the ATP and Mg (II) requirements are related to the involvement of DNA topoisomerase II, we analyzed the effect of specific inhibitors of the enzyme on nucleosome formation and spacing. The concentration of inhibitors to be used was determined by analyzing their effect on DNA catenation, a reaction catalyzed by DNA topoisomerase H (25). High concentrations of nuclear extract in the presence of ATP and high MgCl2 content convert closed circular DNA into large networks in the span of 5 min. Increasing DNA concentration reduces the percentage of catenated molecules (data not shown), which suggests that an 'aggregating' protein required for DNA catenation, like the one described in Drosophila by Hsieh and Brutlag (26) and in yeast by Goto and Wang (27), is present in our extract in limiting quantity. Under conditions of DNA catenation, relaxed DNA was incubated in the nuclear extract in the absence and presence of three different DNA topoisomerase II specific inhibitors: 1
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7. Micrococcal nuclease
digestion pattern
of chromatin assembled in the
presence of VM26.
Relaxed
(6
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pXbsF20i
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MgCl2
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was
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(20 lAg/mld). After incubation, CaCl2 (3 mM concentration) and MNase (0.3 U) were added and aliquots were taken at 0 addition
and
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8). The analyzed as
4 and
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Figure 8. Micrococcal nuclease digestion pattern of chromatin assembled in the presence of AMP-P-(NH)-P and -y-S-ATP. Relaxed pXbsF201 DNA (100 ng) was incubated overnight in the nuclear extract (6 A1) which had been passed over a Sephadex G-25 column in the absence of the ATP regenerating system with the addition of MgCl2 (2 mM) and AMP-P-(NH)-P (ImM) (lanes 1-3) or MgC12 (2 mM) and -y-S-ATP (1 mM) (lanes 4-6). After incubation, CaC12 (3 mM) and MNase (0.3 U) were added and aliquots were taken at 1 (lanes 1, 4), 3 (lanes 2, 5) and 9 min (lanes 3, 6). The samples were treated as detailed in Methods and Materials and analyzed as described in the legend to figure 5. The positions of the DNA molecular weight markers coelectrophoresed with the digested fragments are given on the right.
5454 Nucleic Acids Research, Vol. 18, No. 18 coumermycin Al, VP16 and VM26. Coumermycin Al is assumed to compete with ATP binding to the enzyme. VP16 and VM26 inhibit eukaryotic DNA topoisomerase H by trapping a topoisomerase-DNA reaction intermediate so that subsequent treatment with a protein denaturant results in DNA cleavage (see 28 and 29 for reviews). As shown in figure 6, DNA catenation is completely blocked by coumermycin Al at 60 ,^g/ml (lane 3). Addition of VP16 or VM26 to the DNA catenation assay resulted in DNA breakage when the reaction was stopped by the addition of SDS; the input DNA was converted to the linear form (lanes 4-7). When VM26 was present at a concentration of 20 ,g/ml more than 95 % of the DNA was converted to the linear form (lane 6). We then analyzed the effects of inhibitors on chromatin assembly. Under standard conditions of chromatin assembly, relaxed DNA was incubated in the nuclear extract in the absence and presence of 20 ,ug/ml VM26. As shown in figure 7, DNA linking number reduction is not significantly affected by the inhibitor (compare lanes 1 and 5). Moreover, the MNase digestion pattern of chromatin assembled in the presence of ATP, MgCl2 and VM26 displays correctly spaced nucleosomes (fig. 7, lanes 6-8); compare with samples shown in lanes 2-4, which were MNase digests of chromatin assembled in the presence of ATP and MgCl2. The same result was obtained when the assembly reaction was performed in the presence of 60 jg/ml coumermycin Al (data not shown and fig. 6, lane 3). The ATP requirement in the assembly of correctly spaced chromatin can be substituted by -y-S-ATP but not by
AMP-P-(NH)-P To understand the role of ATP in the chromatin assembly process, we analyzed the effects of ATP analogues on nucleosome spacing. We used two different non-hydrolyzable ATP analogues: AMPP-(NH)-P and -y-S-ATP. MNase digestion pattern of chromatin assembled in the presence of the imido analog and MgCl2 shows that nucleosomes are not correctly spaced (fig. 8, lanes 1-3); the spacing of the nucleosomes is 150 bp, the same as that observed for nucleosomes assembled in the absence of ATP and MgCl2. Chromatin assembled in the presence of -y-S-ATP and MgCl2, on the other hand, displays nucleosomes with a spacing of 180 bp (fig. 8, lanes 4-6).
DISCUSSION We developed an in vitro system for chromatin assembly using an extract prepared from the nuclei of Xenopus oocytes. Our results demonstrate that in the presence of ATP and Mg(II), exogenously added DNA is assembled into correctly spaced chromatin in the nuclear extract. The in vitro assembled chromatin contains the four histones H3, H4, H2A and H2B but no histone HI. The nuclear extract lends itself to a biochemical analysis of the process since relatively large amounts of DNA are assembled into chromatin (50-75 ng DNA per /l nuclear extract) and the nucleoprotein complex can be easily purified. The nuclear extract has been used to investigate the role of ATP and Mg(II) in the assembly of correctly spaced chromatin. The pattern of micrococcal nuclease digestion shows that the nucleosomes assembled in the absence of ATP and Mg(II) are closely packed with a periodicity of 150 bp. In contrast, in the presence of ATP and Mg(II) the spacing of the nucleosomes is 180 bp, essentially the same as that found in vivo after microinjection of DNA into Xenopus oocyte nuclei (10).
Glikin et al. (5) related the ATP and Mg(II) requirements for the assembly of correctly spaced chromatin in cell extracts of Xenopus oocytes to the involvement of the ATP and Mg(II) dependent DNA topoisomerase II in the assembly process. They proposed that DNA topoisomerase II in the presence of other extract components actively supercoils DNA and drives the formation of correctly spaced nucleosomes (5). The involvement of DNA topoisomerase II was proposed on the basis of the observation that novobiocin, an inhibitor of both prokaryotic and eukaryotic DNA topoisomerase H (30, 26), inhibited nucleosome formation in cell extracts of Xenopus oocytes (5). The novobiocin inhibition is difficult to interpret, however, in the light of the recent evidence of secondary effects of the drug (31, 32, 11). Our observations that the rate of nucleosome assembly as monitored by DNA linking number reduction is unaffected by ATP and Mg(ll) and that VM26, a specific inhibitor of eukaryotic DNA topoisomerase H, has no effect on nucleosome formation and spacing clearly indicate that the ATP and Mg(ll) requirements in the process are unrelated to the activity of DNA topoisomerase II. Our results are consistent with the model for chromatin assembly proposed by Almouzni and Mechali (11). They proposed that chromatin is formed in a two-step reaction. In the first step, nucleosome formation occurs independently of ATP and Mg (II). In this step, DNA topoisomerase I activity is involved in the relaxation of the topological constraints generated by nucleosome assembly. The second step, requiring ATP and Mg (II), generates properly spaced chromatin (11). To understand the role of ATP in nucleosome spacing we analyzed the effects of non-hydrolyzable ATP analogues, AMPP-(NH)-P and y-S-ATP. The pattern of micrococcal nuclease digestion of chromatin assembled in the presence of AMP-P(NH)-P and Mg(H) shows that the nucleosomes are closely packed with a periodicity of 150 bp. Chromatin assembled in the presence of -y-S-ATP and Mg(II), on the other hand, displays correctly spaced nucleosomes. The result obtained when we included the imido analog and MgCl2 in the assembly reaction demonstrates that the ATP is essential to the formation of properly spaced chromatin. Moreover, the data show that -y-S-ATP but not AMPP-(NH)-P can substitute for ATP in the process. The observation that 7y-S-ATP, but not AMP-P-(NH)-P, can substitute for ATP in the reaction is reminiscent of recA assembly on duplex DNA. The ATP binding to recA induces a conformational change of the protein and the subsequent formation of a protein-DNA complex. The assembly of recA on duplex DNA can be supported by ATP and -y-S-ATP, but not by AMP-P-(NH)-P or AMP-P-(CH2)-P (33). It should also be noted that the use of y-S-ATP, but not of AMP-P-(NH)-P, can result in thiophosphorilation of proteins since it is readily utilized by protein kinase (34). We suggest that a 'spacing factor' is involved in the assembly of correctly spaced nucleosomes. It is conceivable that in the absence of ATP the 'spacing factor' is inactive and this could cause the observed tight packing of nucleosomes. In the presence of ATP the activation of the factor could generate the correct spacing of the nucleosomes. Experiments will be designed to identify the factor involved in
nucleosome spacing.
ACKNOWLEDGEMENTS We thank Elisabetta Bianchi for the experiment shown in figure 2; Angelo Di Francesco and Roberto Gargamelli for skilled
Nucleic Acids Research, Vol. 18, No. 18 5455 technical assistance; Giorgio Morelli for critical reading of this manuscript. This work has been supported in part by grants from C.N.R., Progetto Bilaterale (I.R.) and the 'Istituto Pasteur-Fondazione Cenci Bolognetti', Universitt di Roma La Sapienza (I.R.).
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