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Chromatin Assembly of DNA Templates Microinjected Into Xenopus Oocytes

Daniele Roche, Genevieve Almouzni, and Jean-Pierre Quivy

Summary The packaging of deoxyribonucleic acid (DNA) into chromatin within the eukaryotic nucleus can affect processes such as DNA replication, transcription, recombination, and repair. Therefore, studies aimed at understanding at the molecular level how these processes are operating have to take into account the chromatin context. We present a method to assemble DNA into chromatin by nuclear microinjection into Xenopus oocytes. This method allows in vivo chromatin formation in a nuclear environment. We provide the experimental procedures for oocyte preparation, DNA injection, and analysis of the assembled chromatin. Key Words: Microinjection; oocytes; chromatin assembly; nucleosomes. 1. Introduction In the nucleus of eukaryotic cells, DNA is packaged into chromatin, a nucleoprotein complex consisting of a basic repeating unit known as the nucleosome. A single nucleosome contains two turns of DNA wrapped around a core histone octamer comprised of the histones H2A, H2B, H3, and H4 (1). Nucleosomes represent the first level of compaction in chromatin, the dynamics of which will influence access to enzymes involved in DNA metabolism (2). In addition to these basic components, linker histones and a variety of nonhistone proteins are incorporated to achieve complete genome organization within a higher-order chromatin structure (3). Thus, studies aimed at understanding mechanisms such as transcription, replication, repair, or recombination at the molecular level have to incorporate the nucleosomes and chromatin components. Although chromatin can be reconstituted using pure histones (4,5), the nucleosomal templates generated in this way do not necessarily possess some physiological characteristics of native chromatin, such as the spacing of the nucleosomes, the diversity of histone posttranslational modifications, or the presence of nonhistone-associated proteins.
From: Methods in Molecular Biology, vol. 322: Xenopus Protocols: Cell Biology and Signal Transduction Edited by: X. J. Liu Humana Press Inc., Totowa, NJ

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germinal vesicle animal pole vegetal pole ssDNA + a-^2pcJCTP

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DNA purification and analysis

(ssDNA I

Fig. 1. Simplified scheme of the experimental strategy. Above: single-stranded circular DNA (ssDNA) molecules with a-^^P-dCTP are injected into the nucleus (germinal vesicle, dashed circle) of a stage VI oocyte. The animal and vegetal poles are indicated. After 3 h at 18C, the DNA is extracted and purified. Below: following its injection, the circular naked ssDNA undergoes a complementary strand synthesis (dashed arrow). During this synthesis, a-^-P-dCTP is incorporated in the synthesized DNA (^^-P), and nucleosomes (N in circles) are deposited concomitantly. After 3 h, the reaction is complete and yields a radioactively labeled doublestranded closed circular DNA molecule (dsDNA) on which nucleosomes are assembled (about 1 nucleosome for 185 bp).

Efficient chromatin assembly can be reproduced in vitro in crude extracts derived from Xenopus oocytes or eggs (6-9), Drosophila embryos (10-12), or human cells (13-16). However, these extracts are usually not simultaneously competent for transcription, repair, and replication. The injection of DNA templates in Xenopus oocytes as a "living test tube" (17) provides a convenient and highly efficient means to assemble in vivo the DNA into chromatin in a nuclear compartment (germinal vesicle) proficient for transcription and DNA synthesis and repair (18-23). Thus, the impact of the chromatin structure on these processes can be studied directly, provided that the DNA template contains adequate features such as sequence control elements, reporter genes, and so on. In this chapter, we describe this powerful approach for chromatin assembly and discuss potential applications. The experimental strategy is summarized in Fig. 1. Following injection of a circular single-stranded DNA (ssDNA; Ml3 derivative) into the nucleus (germinal vesicle) of a stage VI oocyte, complementary-strand DNA synthesis occurs, leading to a closed circular double-stranded DNA (dsDNA) (21). Coupled to this process, nucleosomes are efficiently assembled onto the DNA template. If radioactive deoxycitidine-5'-triphosphate (a-^^P) (a-32P-dCTP) is coinjected with DNA, it will be incorporated during synthesis, enabling the concomitant labeling of the DNA assembled into chromatin. After 3 h, the efficiency of nucleosome assembly can be assessed by purification of injected DNA. Two assays can be performed: (1) a supercoiling assay and (2) a micro-

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supercoiling assay
o'^ 0^ \ ^ fc.

MNase assay
o^\ ^ < b

Ir II
Intermediates (partial duplexes)

llo
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 supercoiled 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, topoisomers 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).

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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. 1. 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 MgCU, 1 mM Na2HP04-2 HjO, 0.05% polyvinyl pyrollidone. 4. IX Modified Earth's Saline (MBSH) buffer: 10 mM HEPES, pH 7.6, 88 mM NaCl, 1 mM KCl. 2.4 mM NaHCO,, 0.82 mM MgS04, 0.41 mM CaCh, 0.33 mM Ca(N03)2. 5. Collagenase (Sigma, St Louis, MO) solution in OR2 at 2 mg/mL stored at -20C. 6. 10 mg/mL Gentamicin (Sigma, St Louis, MO), stored at 4C. 7. Ml3 derivative ssDNA (^ee Note 1). 8. Injection buffer: 15 mM HEPES. pH 7.6. 88 mM NaCl. 9. a-32P-dCTP, 3000 Ci/mmol, 10 pCi/^iL (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, I 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 -20C. 4. Ribonuclease A (Roche, Mannheim, Germany): 10 mg/mL in O.OIM sodium acetate, pH 5.2, heated 15 min at 100C and completed with 0.1 vol of Tris-HCl, pH 7.4. Aliquot and store at -20C. 5. Phenol/chloroform/isoamyl alcohol (25/24/1; Invitrogen, Paisley, UK). 6. Glycogen (Roche, Mannheim, Germany): 20 mg/mL. 7. 5M Ammonium acetate. 8. 100% Ethanol stored at -20C.

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9. 10. 11. 12. 13.

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70% Ethanol stored at -20C TE, pH 8.0: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0. 5X Loading buffer: 0.5% bromophenol blue, 5 mM EDTA, 50% glycerol. Agarose (Ultrapure. Sigma, Saint Louis, MO). SOX TAE stock solution buffer: 242 gTris-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 Mictoccocal Digestion Assay

Oocyte homogenization solution (see Subheading 2.2., item 1). MNase (Roche, Mannheim, Germany) solution in water at 15 U/(xL. 100 mM CaClo solution. Items 2 to 6 as in Subheading 2.2. 3 M Sodium acetate, pH 5.2. 100% Ethanol stored at -20C. 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. lOX TBE stock solution buffer: 108 g Tris-base, 55 g boric acid, and 40 mL 0.5MEDTA, pH 8.0, dissolved in deionized water to a final volume of 1 L. 11. See Subheading 2.2., items 14,15. 1. 2. 3. 4. 5. 6. 7. 8.

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 cm^) 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 IX MBSH (see Note 4). Oocytes can be stored for several days at 18C in IX MBSH complemented with 10 |ig/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 |j,L of a solution of the ssDNA to be injected at 100 ng/|iL in the injection buffer, including 3 nL of a-^-P-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).

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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 18C in fresh IX 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 |xL of oocyte homogenization buffer with a Pipetman tip and then by pipeting up and down several times. 4. Add the same volume (50 |J.L) of 2X stop mix, 3 ^L ribonuclease A; incubate 30 min at 37C. 5. Add 3 |a.L proteinase K and incubate for at least 2 h at 37C. 6. Add 100 |J,L phenol/chloroform/isoamyl alcohol and vortex 10 s minimum each tube. Centrifuge 10 min at 15.000g at room temperature, collect 100 |iL 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 |a,L of glycogen and precipitate DNA with 1 vol of ammonium acetate (100 |iL) and 2 vol (400 \ih) of cold 100% ethanol. 8. Centrifuge 30 to 45 min at 15,000^ at 4C to collect the DNA pellet, wash with 800 |a,L of cold 70% ethanol, centrifuge 5 to 10 min at 15,000g at 4C, and carefully remove the supernatant, dry the pellet in the Speed Vac, and resuspend in 10 [xL TE. 9. Add 2.5 |J.L of 5X loading buffer, load on a 1% agarose gel in IX 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 -80C for at least 4 h. Exposition time may vary according to the efficiency of DNA synthesis and the number of oocytes used. Phosphorlmaging 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 nL 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-|xL aliquots at 0.5, t, 2, and 5 min and immediately transfer to a tube containing 50 |iL of stop mix to stop the digestion. 4. Add 3 |iL of RNase A and incubate at 37C for 30 min. 5. Proceed to DNA extraction as in Subheading 3.2., steps 4 to 6. 6. Precipitate the DNA by adding 2 \iL glycogen, 10 |iL 3M sodium acetate. 330 |xL 100% ethanol. Vortex and store at -80C for 30 min.

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7. Centrifuge 30 min at 15,000g at 4C, wash the pellet with 800 |iL 70% ethanol. centrifuge 10 min at 15,000g at 4C before removing the ethanol. Repeat twice. Dry the DNA pellet in the Speed Vac. 8. Resuspend in 10 p-L TE and add 2.5 |iL 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. 5ee 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 Ca-* 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 cytoplasmic 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 doublestranded 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-

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ture is required. soal< tlie gel in a 1 (ig/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 ID gels, but after migration in the first dimension, the gel is rotated by 90, and chloroquine is added at 10 |ig/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 labellisee la Ligue); Euratom (FIGH-CT-1999-00010 and FIGH-CT-2002-00207); the Commissariat a I'Energie Atomique (LRC 26); European Contracts RTN (HPRN-CT-200000078 and HPRN-CT-2002-00238); and Collaborative Programme between the Curie Institute and the Commissariat a I'Energie Atomique (PIC Parametres Epigenetiques). References 1. Luger, K., Mader, A. W., Richmond, R. K., Sargent, D. F., and Richmond, T. J. (1997) Crystal structure of the nucleosome core particle at 2.8 Angstrom resolution. Nature 389, 251-260. 2. Wolffe, A. P. (1997) Chromatin: Structure and Function. Academic Press, New York. 3. Vaquero, A., Loyola, A., and Reinberg, D. (2003) The constantly changing face of chromatin. Available at: http://sageke.sciencemag.org/cgi/content/full/sageke;2003/14/re4. 4. Carruthers, L. M., Tse, C , Walker, K. P., and Hansen, J. C. (1999) Assembly of defined nucleosomal arrays from pure components. Methods Enzymol. 304, 19-35. 5. Clapier, C. R., Langst, G., Corona, D. F., Becker, P. B., and Nightingale, K. P. (2001) Critical role for the histone H4 N terminus in nucleosome remodeling by ISWl. Mol. Cell. Biol. 21, 875-883. 6. Laskey, R. A., Mills, A. D., and Morris, N. R. (1977) Assembly of SV40 chromatin in a cell free system from Xenopus eggs. Cell 10, 237-243. 7. Glikin, G. C , Ruberti, I., and Worcel, A. (1984) Chromatin assembly in Xenopus oocytes: in vitro studies. Cell 37, 33-41. 8. Almouzni, G. and Mechali, M. (1988) Assembly of spaced chromatin involvement of ATP and DNA topoisomerase activity. EMBO J. 7, 4355-4365. 9. Almouzni, G. and Mechali, M. (1988) Assembly of spaced chromatin promoted by DNA synthesis in extracts from Xenopus eggs. EMBO J. 7, 665-672. 10. Nelson, T., Hsieh, T.-S., and Brutlag, D. (1979) Extracts of Drosophila embryos mediate chromatin assembly in vitro. Proc. Natl. Acad. Sci. USA 76, 5510-5514.

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11. Becker, P. B. and Wu, C. (1992) Cell-free system for assembly of transcriptionally repressed chromatin from Drosophila embryos. Mol. Cell. Biol. 12, 2241-2249. 12. Kamakaka, R. T., Bulger, M., and Kadonaga, J. T. (1993) Potentiation of RNA polymerase II transcription by Gal4-VP16 during but not after DNA replication and chromatin assembly. Genes Dev. 7, 1779-1795. 13. Stillman, B. (1986) Chromatin assembly during SV40 DNA replication in vitro. Cell 45, 555-565. 14. Banerjee, S. and Cantor, C. R. (1990) Nucleosome assembly of simian virus 40 DNA in a mammalian cell extract. Mol. Cell. Biol. 10, 2863-2873. 15. Gruss. C , Gutierrez, C , Burhnans, W. C , DePamphilis, M. L., Koller, T., and Sogo, J. M. (1990) Nucleosome assembly in mammalian cell extracts before and after DNA replication. EMBO J. 9, 2911-2922. 16. Krude, T. and Knippers, R. (1991) Transfer of nucleosomes from parental to replicated chromatin. Mol. Cell. Biol. 11, 6257-6267. 17. Brown, D. D. and Gurdon, J. B. (1977) High fidelity transcription of 5S DNA injected into Xenopus oocytes. Proc. Natl. Acad. Sci. USA 74, 2064-2068. 18. McKnight, S. L. and Kingsbury, R. (1982) Transcriptional control signals of a eucaryotic protein coding gene. Science 217, 316-325. 19. Wyllie, A., H., Laskey, R., A., Finch, J., and Gurdon, J., B. (1978) Selective DNA conservation and chromatin assembly after injection of SV40 into Xenopus oocytes. Dev. Biol. 64, 178-188. 20. Ryoji, M. and Worcel. A. (1984) Chromatin assembly in Zenop.s oocytes: in vivo studies. CeH 37, 21-32. 21. Almouzni, G. and Wolffe, A. P. (1993) Replication-coupled chromatin assembly is required for the repression of basal transcription in vivo. Genes Dev. 7, 2033-2047. 22. Gaillard, P.-H., Martini, E., Kaufman, P. D., Stilman, B., Moustacchi, E., and Almouzni, G. (1996) Chromatin assembly coupled to DNA repair: a new role for chromatin assembly factor I. Cell 86, 887-896. 23. Belikov, S., Gelius B., Almouzni, G., and Wrange, O. (2000) Hormone activation induces nucleosome positioning in vivo. EMBO J. 16, 1023-1033. 24. Germond, J. E., Rouviere-Yaniv, J., Yaniv, M., and Brutlag, D. (1979) Nicking-closing enzyme assembles nucleosome-like structures in vitro. Proc. Natl. Acad. Sci. USA 76, 3779-3783. 25. Ray-Gallet, D., Quivy J.-P., Scamps, C , Martini, E. M., Lipinski, M., and Almouzni, G. (2002). HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis. Mo/. Cell9, 1091-100. 26. Ahmad, K. and Henikoff, S. (2002). The histone variant H3.3 marks active chromatin by replication-independent nucleosome assembly. Mol. Cell 9, 1191-200. 27. Tagami, H., Ray-Gallet, D., Almouzni, G., and Nakatani, Y. (2004). Histone H3.1 and H3.3 complexes mediate nucleosome assembly pathways dependent or independent of DNA synthesis. Cell 116, 51-61. 28. Dumont. J. N. (1972) Oogenesis in Xenopus laevis (Daudin). J. Morphol. 136, 153-180. 29. Sambrok. J., Fritsch, E. F., and Maniatis, T. (1989). Molecular CloningA Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 30. Peck, L. J. and Wang, J. C. (1985) Transcriptional block caused by a negative supercoiling induced structural change in an alternating CG sequence. Cell 40, 129-137.