MNase assay

Intermediates (partial duplexes)

Intermediates (partial duplexes)

Fig. 2. Analysis of chromatin assembly. Left: Autoradiography of a supercoiling gel showing a time-course of chromatin assembly coupled to DNA synthesis. DNA was extracted and purified after 15 min, 30 min, 1 h, 2 h, and 4 h following injection. The positions of the super-coiled form (form I) and the nicked and closed relaxed forms (forms II and Ir, respectively) are indicated on the right. The intermediates reflecting partial duplexes for which complete complementary DNA synthesis is not achieved are indicated. Right: Autoradiography of the agarose gel showing the oligonucleosomal DNA fragments produced by micrococcal nuclease digestion at the end of an assembly reaction (3 h) in the oocyte. Time of digestion in minutes is indicated on the top, and the positions of the fragments corresponding to the mono-, di-, tri-, and tetranucleosome are indicated.

coccal nuclease assay (MNase assay), which are presented on Fig. 2. The supercoiling assay makes use of the topological properties of closed circular DNA molecules. During nucleosome assembly, the progressive deposition of nucleosomes in the presence of topoisomerase activity leads to conformational changes easily detectable on closed circular DNA molecules. Indeed topoisomerase activity allows the absorption of the constraints generated during the process (24). After deproteinization, topo-isomers with an increasing number of negative supercoils correlate with the number of nucleosomes assembled. Resolution and detection of topoisomers is achieved using gel electrophoresis. The accumulation of the supercoiled form (form I) provides a semiquantitative estimation concerning the extent of assembly that can be followed as a function of time (see Fig. 2, left). The MNase assay for chromatin assembly makes use of MNase, which cleaves the most accessible regions in a chromatinized DNA. Cleavage occurs preferentially in the linker region between adjacent core nucleosomes, generating digestion products with sizes that are multiples of the basic nucleosomal unit. The corresponding DNA fragments, when analyzed by gel electrophoresis, give rise to a characteristic profile or nucleosomal ladder. The regularity of the pattern and the spacing between adjacent bands provides information on the quality of the final product obtained in the assembly reaction (see Fig. 2, right).

Importantly, specific pathways are involved in chromatin formation and are coupled and not coupled to DNA synthesis (25-27). The method described here uses a DNA synthesis-coupled chromatin assembly pathway. If dsDNA is injected (instead of ssDNA), chromatin assembly also occurs on the DNA template, but it is not coupled to DNA synthesis and proceeds at a slower rate (21). The methods described here for the ssDNA (injection and analysis) can also be used for the injection of dsDNA. However, the detection of DNA has to be achieved by hybridization procedures, after electrophoresis, as no labeling of the injected DNA occurs in the oocyte, which is not competent to initiate replication on a double-stranded template (no incorporation of a-32P-dCTP).

Perturbations or alterations of specific pathways involved in the regulation of the DNA metabolism can easily be obtained in the oocytes by expression of ectopic proteins, injection of antibodies, drugs, and so on. Following DNA injection, the impact of such perturbations onto DNA metabolism (including transcriptional responses, protein-DNA interactions) can then be monitored in the context of chromatin. The combination of dedicated DNA sequences, functional assays, DNA-protein interactions assays with the approach described herein could thus be further adapted to study specific mechanisms at the level of chromatin.

2. Materials

2.1. Oocytes and Injection of SSDNA

1. Female Xenopus.

2. Surgical kit containing scissors, scalpel, and forceps.

3. OR2 medium: 5 mM HEPES, pH 7.8, 87 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM Na2HPO4-2 H2O, 0.05% polyvinyl pyrollidone.

4. 1X Modified Barth's Saline (MBSH) buffer: 10 mM HEPES, pH 7.6, 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.41 mM CaCl2, 0.33 mM Ca(NO3)2.

5. Collagenase (Sigma, St Louis, MO) solution in OR2 at 2 mg/mL stored at -20°C.

6. 10 mg/mL Gentamicin (Sigma, St Louis, MO), stored at 4°C.

7. M13 derivative ssDNA (see Note 1).

8. Injection buffer: 15 mM HEPES, pH 7.6, 88 mM NaCl.

9. a-32P-dCTP, 3000 Ci/mmol, 10 |Ci/|L (MP Biochemicals, Asse-Relegen, Belgium).

2.2. Analysis by Supercoiling Assay

1. Oocyte homogenization solution: 10 mM HEPES, pH 7.5, 5% sucrose, 70 mM KCl, 1 mM dithiothreitol (DTT).

2. 2X Stop mix: 60 mM EDTA (ethylenediaminetetraacetic acid), 1% SDS (sodium dodecyl sulfate).

3. 20 mg/mL Proteinase K (Roche, Mannheim, Germany) stored in water at -20°C.

4. Ribonuclease A (Roche, Mannheim, Germany): 10 mg/mL in 0.01M sodium acetate, pH 5.2, heated 15 min at 100°C and completed with 0.1 vol of Tris-HCl, pH 7.4. Aliquot and store at -20°C.

5. Phenol/chloroform/isoamyl alcohol (25/24/1; Invitrogen, Paisley, UK).

6. Glycogen (Roche, Mannheim, Germany): 20 mg/mL.

7. 5M Ammonium acetate.

10. TE, pH 8.0: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0.

11. 5X Loading buffer: 0.5% bromophenol blue, 5 mM EDTA, 50% glycerol.

12. Agarose (Ultrapure, Sigma, Saint Louis, MO).

13. 50X TAE stock solution buffer: 242 g Tris-base, 57.1 mL glacial acetic acid, and 100 mL 0.5 M EDTA, pH 8.0 dissolved in deionized water to a final volume of 1 L.

14. Intensifying screens for X-ray films.

15. Amersham Hyperfilm MP (Amersham Biosciences, Buckinghamshire, UK).

2.3. Analysis by Microccocal Digestion Assay

Oocyte homogenization solution (see Subheading 2.2., item 1). MNase (Roche, Mannheim, Germany) solution in water at 15 U/||L.

Items 2 to 6 as in Subheading 2.2.

3 M Sodium acetate, pH 5.2. 100% Ethanol stored at -20°C.

TE, pH 8.0: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0.

5X MNase loading buffer: 0.3% Orange G (Sigma, Saint Louis, MO), 5 mM EDTA, 50% glycerol (see Note 2). 9. Agarose (Ultrapure, Sigma, Saint Louis, MO, USA).

10. 10X TBE stock solution buffer: 108 g Tris-base, 55 g boric acid, and 40 mL 0.5M EDTA, pH 8.0, dissolved in deionized water to a final volume of 1 L.

11. See Subheading 2.2., items 14,15.

100 mM CaCl2 solution.

3. Methods

3.1. Oocytes and Injection of Single-Stranded DNA

1. One adult female frog is anesthetized on ice (at least 30 min) and sacrificed.

2. Immediately perform a ventral cut 1 to 2 cm long with a razor blade, pull out the ovaries with pincets, and transfer into a glass Petri dish containing OR2 medium. Quickly rinse several times to eliminate blood. Using two pairs of forceps, tear apart and open up the ovary lobes and cut with scissors until pieces of ovaries homogeneous in size (about 1 cm2) are obtained. Rinse again with OR2 to eliminate blood and lysed or broken oocytes. Transfer about 7.5 to 10 mL into a new 50-mL tube and add the same volume of collagenase solution. Incubate at room temperature to dissociate follicular cells for about 2 h on a rolling shaker. Check regularly to avoid overtreatment (see Note 3). Appearance of individual oocytes dissociated from follicules or follicular membranes indicates that the collagenase treatment is achieved. Rinse thoroughly with OR2 and eliminate by sedimentation the youngest stages (ref. 28; small and nonpigmented, which sediment the slowest) and all debris. Wash again two times with 1X MBSH (see Note 4). Oocytes can be stored for several days at 18°C in 1X MBSH complemented with 10 |g/mL gentamicin (see Note 5).

3. Sort manually under a dissecting microscope healthy stage VI oocytes according to ref. 28 (see Note 6).

4. Make 10 |L of a solution of the ssDNA to be injected at 100 ng/|L in the injection buffer, including 3 |L of a-32P-dCTP (see Note 7).

5. This solution is aspirated in a capillary containing mineral oil mounted on the injection system under the microscope (see Note 8).

6. Inject this radioactive DNA solution into the nucleus located one-third deep in the animal part of the oocyte (see Note 9); a volume of 20 to 30 nL corresponding to 2 to 3 ng of DNA is injected.

7. Following injection, transfer the oocytes at 18°C in fresh 1X MBSH medium and incubate for chosen times (usually 3 h for a complete assembly).

3.2. Analysis by Supercoiling Assay

1. Collect 10 healthy oocytes into an Eppendorf tube (see Notes 6 and 10).

2. Remove as much as possible the MBSH buffer and place on dry ice or liquid nitrogen to stop the reaction. This allows the oocytes to be kept for a short time before they are all processed for analysis when several conditions of injections, time-points, or treatments are performed.

3. Homogenize the oocytes by crushing them in 50 ||L of oocyte homogenization buffer with a Pipetman tip and then by pipeting up and down several times.

4. Add the same volume (50 ||L) of 2X stop mix, 3 |L ribonuclease A; incubate 30 min at 37°C.

5. Add 3 |L proteinase K and incubate for at least 2 h at 37°C.

6. Add 100 |L phenol/chloroform/isoamyl alcohol and vortex 10 s minimum each tube. Centrifuge 10 min at 15,000g at room temperature, collect 100 |L of a clear aqueous upper phase, and transfer into a clean tube. Be careful not to take material from the interphase. A second phenol/chloroform/isoamyl alcohol extraction can be performed if the upper phase is not clear.

7. Add 2 |L of glycogen and precipitate DNA with 1 vol of ammonium acetate (100 ||L) and 2 vol (400 |L) of cold 100% ethanol.

8. Centrifuge 30 to 45 min at 15,000g at 4°C to collect the DNA pellet, wash with 800 |L of cold 70% ethanol, centrifuge 5 to 10 min at 15,000g at 4°C, and carefully remove the supernatant, dry the pellet in the Speed Vac, and resuspend in 10 |L TE.

9. Add 2.5 |L of 5X loading buffer, load on a 1% agarose gel in 1X TAE without ethidium bromide, and run at 1.5 V/cm until the dye migrates to the bottom of the gel (see Notes 11-13).

10. Expose the dried gel against an X-ray film for autoradiography to visualize the migration pattern of the radiolabeled DNA. Use two intensifying screens and leave at -80°C for at least 4 h. Exposition time may vary according to the efficiency of DNA synthesis and the number of oocytes used. PhosphorImaging system can also be used (Storm, Amersham Pharmacia Biotech, Uppsala, Sweden)

3.3. Analysis by Microccocal Digestion Assay

1. Homogenize 20 to 30 healthy injected oocytes in 200 |L of homogenization buffer (see Subheading 3.2., item 2, and Notes 6 and 10).

2. Adjust to 3 mM CaCl2 with the 100 mM solution before addition of 60 units of MNase and digest at room temperature.

3. Remove 50-|lL aliquots at 0.5, 1, 2, and 5 min and immediately transfer to a tube containing 50 | L of stop mix to stop the digestion.

4. Add 3 | L of RNase A and incubate at 37°C for 30 min.

5. Proceed to DNA extraction as in Subheading 3.2., steps 4 to 6.

6. Precipitate the DNA by adding 2 |L glycogen, 10 |L 3M sodium acetate, 330 |L 100% ethanol. Vortex and store at -80°C for 30 min.

7. Centrifuge 30 min at 15,000g at 4°C, wash the pellet with 800 ||L 70% ethanol. centrifuge 10 min at 15,000g at 4°C before removing the ethanol. Repeat twice. Dry the DNA pellet in the Speed Vac.

8. Resuspend in 10 |L TE and add 2.5 |L of 5X MNase loading buffer.

9. Load on a 1.3% agarose gel in 0.5X TBE buffer and run at 5 V/cm until the Orange G dye migrates through two-thirds of the gel (see Notes 2 and 12).

10. See Subheading 3.2, step 10.

4. Notes

1. The single-stranded M13 DNA should be of high quality and not contaminated by dsDNA. It is obtained after phage purification and further purification through a CsCl gradient (29).

2. If an ethidium bromide picture is required, it is important not to use a loading buffer containing bromophenol blue as this dye will migrate at about the same position as the mononucleosomal DNA and will interfere with the analysis of the migration pattern. Thus, as an alternative to bromophenol blue, Orange G is used.

3. The collagenase treatment is a critical step for successful injections. Overtreatment will result in very fragile oocytes that will not support storage and the injection injury. Undertreatment will result in oocytes difficult to pierce. Therefore, frequent monitoring of the oocytes during collagenase treatment is strongly advised. Different treatment times can be performed and the corresponding defolliculated oocytes stored. The best time treatment can be quickly determined at the time of injection.

4. The MBSH contains Ca2+ ions that inhibit the action of collagenase, therefore preventing overtreatment on storage.

5. Do not keep oocytes too concentrated. They should not be in contact with each other.

6. The quality of the oocyte is crucial. Avoid taking any oocyte that does not look healthy and that is not stage VI (28). Stage VI oocytes should have a white band separating the upper brown animal pole from the whitish lower vegetal pole (see scheme of the oocyte in Fig. 1). They should not be grayish or have white spots on the animal pole.

7. Follow safety rules concerning the handling of radioactivity. Plexiglas screens and protection devices should be implemented in the microinjection system. Check after use that the injector is free of contamination.

8. We use a nanoject injector (Drummond) mounted on a Leica micromanipulator, which we found most convenient for these types of injections. The volume of injection can be adjusted within a range of 4.6 to 73.6 nL. Capillaries used are first beveled (with the beveler model EG-40, Narishige) to ensure the best penetration into the oocyte as well as reproducible injection without clotting the tip of the capillary.

9. An increase in size of the oocyte is visualized by quick swelling, which is the sign of a successful nuclear injection. When removing the needle, only limited leakage of the cyto-plasmic material should occur.

10. In these assays, the DNA is radioactive and may be subjected to radiolysis. Analysis should thus be performed rapidly to avoid degradation of DNA, which could lead to loss of detectable supercoiled forms or to smeary MNase digestion patterns.

11. Because intercalation of ethidium bromide modifies the topology of closed double-stranded molecules, its presence must be avoided during the electrophoresis to ensure good separation of the supercoiled form (form I) and the nicked and closed relaxed forms (forms II and Ir).

12. We use gels that are 20 cm long (model SGE, VWR International) to obtain good resolution of topoisomers and oligonucleosomal DNA fragments. If an ethidium bromide pic ture is required, soak the gel in a 1 |lg/mL ethidium bromide solution and rinse 30 min in water at room temperature. Visualize the DNA by placing the gel on an ultraviolet transilluminator. Note that, to be visible, at least 100 ng of DNA should have been loaded on the gel.

13. The absolute number of superhelical turns, corresponding to each assembled nucleosome, can be determined by visualization of the plasmid topoisomers on a two-dimensional (2D) agarose gel (30). Furthermore, closed circular relaxed (Ir) and nicked (II) forms of the plasmid can only be separated in a 2D gel. In some cases, it can be informative to determine the amount of nicked plasmids because these may correspond to plasmids assembled into chromatin in which there are nicks. The 2D gels are set up as classical 1D gels, but after migration in the first dimension, the gel is rotated by 90°, and chloroquine is added at 10 |g/mL to the electrophoresis buffer. The gel is then left to equilibrate for 45 min in the dark. The second dimension electrophoresis is then run in the dark under the same conditions as for run. Under those conditions, the closed circular relaxed form (Ir), indicating that no nucleosomes are assembled, migrates faster than the nicked form (II). During the second run, it is important to recirculate the running buffer to maintain an even distribution of chloroquine.

Acknowledgments

This work was supported by la Ligue Nationale contre le Cancer (Equipe labellisée la Ligue); Euratom (FIGH-CT-1999-00010 and FIGH-CT-2002-00207); the Commissariat à l'Energie Atomique (LRC 26); European Contracts RTN (HPRN-CT-2000-00078 and HPRN-CT-2002-00238); and Collaborative Programme between the Curie Institute and the Commissariat à l'Energie Atomique (PIC Paramètres Epigénétiques).

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