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Mapping Protein/DNA Interactions by Cross-Linking [Internet]. Paris: Institut national de la santé et de la recherche médicale; 2001.

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Analysis of Chromatin Assembly, Chromatin Domains, and DNA Replication Using Xenopus Systems

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Xenopus oocytes or eggs have provided extremely valuable systems for in vitro and in vivo analysis of nuclear processes. Microinjections of defined plasmid molecules in Xenopus oocytes permitted dissection of molecular mechanisms linked to transcription or post-transcriptional regulation (Gurdon and Melton 1981) or chromatin assembly. Microinjections in Xenopus eggs also first permitted us to question the nature of DNA replication origins in multicellular organisms (Harland and Laskey 1981). The development of cell-free systems from Xenopus eggs provided the first chromatin-assembly test tube (Laskey et al. 1977) as well as the first non-viral in vitro systems able to faithfully replicate DNA (Méchali and Harland 1982; Lohka and Masui 1983; Blow and Laskey 1986). A number of aspects of oogenesis in Xenopus explain why Xenopus oocytes or eggs are suitable systems for the molecular analysis of processes such as chromatin assembly, transcription, and DNA replication. The most voluminous organ in Xenopus is the ovary, which harbors several thousand oocytes. Oocytes are cells that are blocked at the prophase stage of the first meiotic division. During a period that lasts 1 to 2 years, they actively transcribe and accumulate a huge stockpile of components that will be used during the early developmental stages that follow fertilization. The maturation of the oocyte into an egg is induced by gonadotropin, and eggs are laid unfertilized outside of the body of the animal. During this complex process, the oocyte's nuclear envelope breaks down, and the cell cycle is arrested at the metaphase of the second division. This is the stage that is usually the source of cell-free extracts, because the egg contains all of the maternal components necessary for the next 12 divisions that occur at an accelerated rate (every 30 min) after fertilization.

Different kinds of extracts can be obtained, depending on the mechanism to be analyzed or the question to be addressed. Figure 1 gives a summary of the main characteristics of the different Xenopus egg extracts. The replication of DNA molecules microinjected into Xenopus eggs was first reported by Gurdon et al. (1969) and demonstrated by Laskey and Gurdon (1973) ,Ford and Woodland (1975) to be a semiconservative mechanism, like that in vivo. DNA replication occurs with no preferred initiation site and under strict cellular control (Harland and Laskey 1980; Méchali and Kearsey 1984; Hyrien and Méchali 1992; Mahbubani et al. 1992). High-speed egg extract was then found to perform complementary strand DNA synthesis in a reaction that reproduces events occurring at the replicating fork in vivo (Méchali and Harland 1982). These extracts convert a single-stranded circular DNA molecule into a complete double-stranded DNA molecule but cannot replicate double-stranded DNA. The production of extracts for genomic DNA replication was reported by Lohka and Masui (1983) and then further demonstrated and characterized by Blow and Laskey (1986) ,Hutchison et al. (1987) , andMurray and Kirschner (1989). Several laboratories are now analyzing DNA replication using these extracts.

Figure 1. Preparation of Xenopus egg extracts. Low-speed and high-speed extracts are prepared from unfertilised Xenopus eggs. See text for details.

Figure 1

Preparation of Xenopus egg extracts. Low-speed and high-speed extracts are prepared from unfertilised Xenopus eggs. See text for details. Different kinds of extracts can be obtained, depending on the mechanism to be analyzed or the question to be addressed. (more...)

These concentrated extracts are also active in transcription in vitro, but because large stores of histones are present in the extract, a dynamic competition between histone assembly and transcription complex assembly occurs, leading to the repression of transcription (Prioleau et al. 1994). This phenomenon can be alleviated in several ways, including addition of transcription factors, or titration of the histone pool by DNA, or dilution of the extract (Wolffe 1989; Almouzni et al. 1990; Toyoda and Wolffe 1992; Prioleau et al. 1994, 1995; Wolffe 1994).

We will describe in the next sections the preparation of cell-free extracts from eggs or early embryos and their use in chromatin assembly chromatin binding and DNA replication in vitro. These protocols as well as other related protocols related to the use of Xenopus eggs and embryos are reported inMenut et al. (1999) and available at www.igh.cnrs.fr./equip/mechali. We will also describe here the use of nuclear matrix purification as a tool to analyze chromatin domains involved in gene regulation. This method was applied with success both in Xenopus embryos and adult tissues (Vassetzky et al. 2000).

Collection of Eggs

Females are injected subcutaneously with human chorionic gonadotropin (HCG; Chorulon). Usually, three females would be injected in their dorsal lymph sacs with 200 units of Chorulon and then 6 hours later with 600 units. After the second injection, frogs should be left in individual tanks containing High Salt Barth solution (HSB). The laying occurs between 8 and 16 hours after the second injection. It is convenient to perform the injections the day before egg collection and to leave the animals overnight in HSB at 20-22°C. If the temperature of the room falls below 18°C, significant delays in the laying can be registered. Frogs also respond to a single injection of 700-800 units of Chorulon, 12 to 14 hours before laying, but less reproducibly.

If difficulties are reproducibly observed in obtaining eggs, the females can be primed first with an injection of 100 units of pregnant mare serum gonadotropin (PMSG), at least 3 days and at most 10 days before HCG injection. A single injection of 300-600 units of Chorulon (HCG) 14-16 hours before egg collection is then sufficient to obtain large amounts of eggs. Although several laboratories including our own use this method with success, we have found that the first method gives eggs of a better quality.

Eggs are normally laid spontaneously, but they can also be squeezed from frogs by gentle pressure on the abdomen immediately before the preparation of the extract. This method has the advantage of providing fresh eggs, but disadvantages such as spontaneous activation and necrosis are increased. Moreover, frogs squeezed in this way often recover with difficulty from this manipulation, and the quality of their laying decreases with time. We do not recommend this method for fertilization and production of embryos. Freshly laid eggs (within the last 4 hours) usually give the best results, but eggs laid overnight can be also used if there is no visible sign of necrosis.

Low-Speed Egg Extract (LSE)

The two main features of this protocol are the disruption of the cells by a simple centrifugation and the preparation and use of the extract in conditions in which no dilution of the egg content occurs (Figure 1).

Low-speed egg extracts replicate sperm nuclei efficiently, and DNA plasmid molecules with a lower efficiency (Blow and Laskey 1986). They form a nuclear envelope around DNA (Blow and Laskey 1986; Newport 1987) and reproduce a number of events linked to the cell cycle (Hutchison et al. 1987; Blow and Laskey 1988; Murray and Kirschner 1989). When freshly prepared egg extracts are used, several cell cycles are obtained in vitro, but this ability is easily lost after freezing, probably because a large decrease (>90%) in the efficiency of translation of the extract occurs (unpublished observations). Nevertheless, frozen extracts efficiently reproduce one complete cell cycle in vitro, especially DNA replication when sperm nuclei are used.

Addition of cycloheximide to the extract blocks protein synthesis. The accumulation of cyclin B and activation of cdc2-cyclin B is thus prevented, and the cell cycle is arrested in vitro in a G2-like state after DNA replication. We prefer to prepare the extract in the absence of cycloheximide, but its addition may help to prevent any progress in the cell cycle during the preparation of the extract itself.

1.

Set the centrifuge at 1°C. Cool all tubes, adaptators, and syringes to 4°C before starting the preparation of the extract.

2.

Transfer the eggs to a glass beaker and rinse with HSB. We do not recommend the pooling of eggs from different frogs.

3.

Add distilled water and leave the external jelly coat to swell for 5 min at room temperature.

4.

Add HSB 0.3X, cysteine 2%, pH 7.9 (the solution should be used within 6 hours of preparation), and dejelly by gentle swirling at intervals. This takes 5 to 10 min, and complete removal of the jelly is obtained when the eggs can be tightly packed together, slightly deformed. It is important to obtain a complete dejellification. At this stage, success depends on both the rapidity with which the preparation is done and the strict observation of the cold temperature conditions after step 10.

5.

Rinse immediately at least five times with 100-200 ml of HSB per ml of eggs. If at this point necrosis is visible in more than 20% of the eggs, do not proceed further. Transfer to a 50-ml glass beaker.

6.

Rinse twice with 0.2X MMR.

7.

Add 0.2X MMR + 0.3 μg/ml calcium ionophore to activate the eggs. Leave 3 to 5 min at room temperature. This step can be avoided because crushing the egg in a buffer containing Ca2+, as in step 8, may be sufficient to activate the extract in interphase. We found, however, that previous activation with Ca2+ ionophore gives more consistent results.

8.

Rinse twice in 0.2X MMR and transfer the eggs to a large glass Petri dish for observation under the microscope. Activation can be monitored by the transient contraction of the animal cortex, which occurs 11 min after addition of calcium ionophore. Using a Pasteur pipette, rapidly remove damaged or abnormal eggs. If more than 20% of eggs need to be removed, do not proceed further.

9.

Transfer to 50-ml Corning tubes. Rinse twice with XB buffer to which 10 μg/ml protease inhibitors have been added.

10.

Transfer the eggs in a Beckman Ultra-Clear tube or equivalent. Add XB containing 10 μg/ml protease inhibitors + 100 μg/ml cytochalasin. Use 1 ml for 3 ml of eggs.

11.

At 12 min post-activation, transfer the tube containing the eggs to ice.

All subsequent steps should be performed at 4°C, with precooled buffer solutions and tubes.

12.

Leave 10 min on ice to chill the eggs before extraction.

13.

Remove the excess buffer and pack the eggs by centrifugation at 150 g, 45 sec, 1°C, in a Sorvall swinging rotor or equivalent.

14.

Rapidly remove the excess buffer and centrifuge at 17,000 g (Sorvall HB4 swinging rotor, 10K), for 10 min at 1°C. The centrifugation crushes the eggs, and the soluble content is thus exuded.

15.

Withdraw the extract by puncturing the side of the tube with a 20-gauge needle inserted into a 1- to 5-ml syringe, depending on the amount of soluble extract. Insert the needle just above the black pigment layer and collect the cytoplasmic layer, avoiding the yellow lipid top layer (Figure 1). Transfer to a cold Ultra-Clear tube. Add 10 μg/ml protease inhibitors, 10 μg/ml cytochalasin B, 1/20 volume Energy Mix 20X, and 5% glycerol. Mix gently.

16.

Centrifuge again in the same conditions. Collect the supernatant in a cold tube. If necessary, add 200 μg/ml cycloheximide to prevent protein synthesis. Store at --80°C in 100- or 200-μl aliquots previously frozen in liquid nitrogen. Protein concentration in low-speed extracts is around 50 mg/ml, and RNA concentration, mainly in ribosomes, is 5-10 mg/ml. Aliquots should be used only once and should not be frozen again after thawing.

Preparation of High-Speed Extract (HSE)

High-speed egg extracts (Figure 1) are convenient for chromatin assembly (Laskey et al. 1977; Almouzni and Méchali 1988), and complementary DNA strand synthesis (single-stranded DNA replication; Méchali and Harland 1982). They also assemble chromatin in a process coupled with DNA synthesis (Almouzni and Méchali 1988a). These extracts are inactive in translation, nuclear assembly, and double-stranded DNA replication.

1.

Set the centrifuge to 1°C. Cool all tubes, adaptators, and syringes to 4°C before starting the preparation of the extract.

2.

Transfer the eggs to a glass beaker and rinse with HSB. We do not recommend pooling the eggs from different frogs.

3.

Add distilled water and leave the external jelly coat to swell for 5 min at room temperature.

4.

Add HSB 0.3X, cysteine 2%, pH 7.9 (the solution should be used within 6 hours of preparation). Eggs are dejellied by gentle swirling at intervals. This takes 5 to 10 min, and complete removal of the jelly is obtained when the eggs can be tightly packed together and are slightly deformed. It is important to obtain a complete dejellification.

5.

Rinse immediately at least five times with 100-200 ml HSB per ml egg. Transfer the eggs to a large glass Petri dish for observation under a microscope. If at this point necrosis is visible in more than 20% of the eggs, do not proceed further. Transfer to 50-ml glass beakers.

All subsequent steps are performed at 4°C, with precooled buffers and tubes.

6.

Transfer the eggs to a beaker or 50-ml Corning tube. Rinse very gently twice with cold XB-HS, then with cold XB-HS containing 10 μg/ml protease inhibitors, 100 μg/ml cytochalasin B.

7.

Transfer the eggs to a cold Ultra-Clear 10-ml Beckman tube and leave in ice for 5 to 10 min, depending on the egg volume.

8.

Remove the excess buffer and immediately centrifuge at 17,000 g for 30 min at 1°C in a swinging bucket rotor, to break the eggs. In this protocol, eggs are not packed before crushing, and the first centrifugation lasts longer than that for low-speed extracts. In these conditions, the membranes are more easily removed from the extract.

9.

Remove the supernatant with a 5-ml syringe mounted with a 20-gauge needle. Insert the needle just above the black pigment layer and collect the cytoplasmic extract, leaving the yellow lipid top layer. Transfer to a cold Ultra-Clear tube.

10.

Add 10 μg/ml protease inhibitors, 10 μg/ml cytochalasin B, 1 mM DTT, 10 mM creatine phosphate, and 10 μg/ml creatine kinase.

11.

Transfer the supernatant to a cold 5-ml Ultra-Clear tube. Do not exceed 1/3 of the tube's volume to obtain a clear supernatant (slightly brown is fine). Overlay with paraffin oil. Centrifuge in a SW50 TI Beckman rotor, 40,000 rpm (192,000 g), 60 min at 2°C.

12.

The pellet is composed of two layers: the bottom layer is golden and contains glycogen and ribosomes and is covered by a grey layer of mitochondria and membranes (Figure 1). The high-speed extract should be collected with a 5-ml syringe mounted with a 20-gauge needle. Insert the needle above the fluffy membrane layer and collect the high-speed extract, avoiding the yellow lipid layer.

13.

If the extract is too turbid, centrifuge again, in the same conditions.

14.

Freeze in liquid nitrogen in 50- to 100-μl aliquots in 0.7-ml Eppendorf tubes and store at --80°C. High-speed extracts can be frozen and thawed several times without any detectable loss in their efficiency for chromatin assembly or single-strand DNA replication. They will remain stable for more than 1 year at --80°C.

Preparation of Demembranated Sperm Nuclei

The following protocol includes modifications made by Gurdon (1976) and Murray (1992).

1.

Inject three male frogs with 100 units of human chorionic gonadotropin (Chorulon) 1 to 7 days before the preparation.

2.

Frogs should be anaesthetized in ice water until they no longer respond to manipulation (around 30 min). Lay the frog on its back on an ice bed and cut the skin and the peritoneal wall. The first cut should be made from top to bottom, at 1 cm to the right of the midline (to avoid any blood vessels), and other cuts should made on both sides to expose the body wall. Use a pair of forceps to push aside the intestine and the yellowish fat bodies. Locate the white testes on either side of the backbone. Collect the testes with scissors and wash them in HSB. The remaining fat and blood vessels should be removed from the testes.

3.

Rinse the testes twice in SNB and place them in a Petri dish laid on ice.

4.

Chop the testes with a sterile blade.

5.

Transfer to a Dounce homogenizer (large pestle), together with 2 ml of SNB.

6.

Homogenize with one or two slow strokes.

7.

Filter the homogenate on sterile cheesecloth placed on a small funnel on the top of a 15-ml conical tube. Alternatively, centrifuge briefly (10 sec, 100 g) to remove large pieces of tissue.

8.

Rinse with 5 to 10 ml SNB.

9.

Centrifuge at 1,700 g for 10 min, 4°C. If blood vessels have not been sufficiently removed from the testes, a thin red layer of reticulocytes will be visible. In this case, the pellet can be resuspended in 6 ml of 10 mM Tris (pH 7.6), 1 mM EDTA and left 5 min at 4°C, to break the reticulocyte cytoplasmic membrane.

10.

Centrifuge at 1,700 g, 10 min, 4°C, and resuspend the pellet in SNB. If the pellet is difficult to resuspend, homogenize gently with a Dounce homogenizer (large pestle).

11.

Centrifuge again and resuspend the pellet in 1 ml of SNB at room temperature.

12.

When the tube is equilibrated at room temperature (20-22°C), add 50 μl of lysolecithin 10 mg/ml (prepared extemporaneously in water) and incubate 5 min. Rapidly check the progress of demembranation with aliquots stained with 1 μg/ml Hoechst.

13.

Add 6 ml of cold SNB containing 30 mg/ml BSA.

14.

Centrifuge 1,700 g, 10 min and resuspend the pellet in 5 ml of SNB, 3 mg/ml BSA.

15.

Centrifuge again and resuspend the pellet in 1 ml of SNB, 3 mg/ml BSA, and 30% glycerol. Measure the concentration of nuclei using a hemocytometer and nuclei stained with Hoechst. Around 108 nuclei can be obtained from one frog.

16.

Freeze in liquid nitrogen in 10-μl aliquots and store at --80°C. Do not re-freeze any thawed aliquots.

Assays for DNA Replication in Xenopus Extracts

Xenopus egg extracts are very efficient in replicating DNA from sperm nuclei or in complementary DNA strand synthesis from a single-strand DNA template. In both cases, 100% replication from the template DNA is routinely observed. With plasmid double-strand DNA molecules, the efficiency is lower; between 2% and 20% input DNA is replicated, and values also obtained in vivo after microinjection. The extent of in vitro replication increases with the size of the DNA template (Blow and Laskey 1986), as observed in vivo (Méchali and Kearsey 1984). It is not yet clear why plasmid DNA molecules do not replicate as well as sperm nuclei, but because DNA replication is dependent on nuclear membrane assembly, it is generally admitted that incorrect assembly will result in less efficient DNA synthesis. The assembly of chromatin in higher order structures may be also less efficient in plasmids than in nuclei.

Like all biochemical reactions, DNA replication is dependent on template concentration. This is an important point to take into consideration when sperm nuclei are used as template, because a limiting quantity of DNA replication factors may change not only the rate of DNA synthesis but also the specificity of the reaction. Assuming that 1 μl of low-speed extract represents the soluble content of two eggs and that DNA replication factors are not limiting up to the mid-blastula transition stage (8,000 cells in 8 hours in vivo), the concentration of nuclei in extract should not exceed 10,000 to 16,000 nuclei/μl. We routinely use 500-2000 nuclei/μl of extract, a concentration sufficient for most analyses.

DNA Replication Assay by TCA Precipitation

1.

Prepare 2.1- or 2.5-cm Whatman GF/C glass filters that have been numbered with India ink.

2.

Thaw immediately before use an aliquot of the Xenopus egg extract (LSE) and put it on ice.

3.

Add the following to a 0.5-ml Eppendorf tube:

Energy mix 20X 2.5 μl

LSE 50 μl

Sperm nuclei 25,000 nuclei (500 nuclei/μl of LSE)

[32P]dATP or [32P]CTP 10 μCi

Because the extract is extremely sensitive to dilution, avoid diluting it by more than 20%. Homogenize very gently and incubate at room temperature (20-23°C). In good extracts, DNA replication starts after 30 to 40 min and reaches 100% by 60 to 90 min (Figure 2).

4.

Spot 2 μl of the mix on a Whatman GF/C glass filter to determine the total radioactivity of the extract (Input). Leave it to dry, put it into scintillation vials, add scintillation fluid, and count.

5.

Take 2-5-μl aliquots of the mix at 0 and then every 10 min. Add the aliquot directly to 25 μl of Stop Mix solution supplemented with 0.6 mg/ml of proteinase K. Vortex vigorously and incubate all the kinetic points at 42°C for at least 1 hour.

6.

Spot each sample on a Whatman GF/C glass filter and drop the filter in a large beaker containing cold 5% TCA, 2% pyrophosphate solution (20 ml/filter). Leave at least 20 min, 4°C. Filters can be accumulated for several hours in the beaker.

7.

Wash the filters four times in cold 5% TCA, 5 min per wash, and once in ethanol. Agitation is not necessary.

8.

Leave all of the filters to dry on tissue paper and count the incorporated TCA precipitable radioactivity (T).

Figure 2. Kinetics of DNA replication after incubation of sperm nuclei in a Xenopus egg extract. Xenopus sperm nuclei were introduced into Low Speed Extract (500 nuclei/μl). At the indicated times, aliquots were removed from the reaction mixture and the percentage of template DNA that replicated was calculated by the TCA precipitation method. Calculation of the activity of the extract.

Figure 2

Kinetics of DNA replication after incubation of sperm nuclei in a Xenopus egg extract. Xenopus sperm nuclei were introduced into Low Speed Extract (500 nuclei/μl). At the indicated times, aliquots were removed from the reaction mixture and the (more...)

Calculation of the Activity of the Extract

To calculate the activity of the extract, it is necessary to evaluate the quantity of deoxynucleotide triphosphates in the extract and the percentage of radioactivity incorporated.

1. Quantity of deoxynucleotides in the reaction mix. The extract contains stockpiles of deoxynucleotide triphosphate, including ATP which is estimated at 50-70 μM by isotope dilution under steady-state replication conditions (Méchali and Harland 1982; Blow and Laskey 1986). We did not observe any significant variations in this value for dATP using different extracts. The value of 60 pmol/μl of extract used in the assay is usually used.

2. Amount of DNA synthesized.

pg DNA synthesized = (% dATP incorporated) x (pmoles dATP in the assay) x 4 x 330

Example:

In a reaction assay of 55 μl containing 50 μl of extract and 25,000 sperm nuclei, 20,000 cpm above the control are detected after TCA precipitation in a 5-μl aliquot. A 2-μl sample of the reaction assay (Input) contains 0.72 x 106 cpm.

pmoles dATP in the assay: 60 x 50 = 3000

Total radioactivity: 0.72 x 106 x 55 = 20 x 106 cpm

Total incorporated: 20,000 x 55 = 220,000 cpm

% incorporated radioactivity: 220,000 x 100/20 x 106 = 1.1%

pg DNA synthesized: 1.1 x 3,000 x 4 x 330/100 = 43,560 pg

Because one sperm nucleus contains 3 pg of DNA (n chromosomes), the DNA in the assay corresponds to 14,520 nuclei. There were 25,000 sperm nuclei in the assay; therefore, the amount of DNA synthesized is 58%.

Detection of Protein Interaction with Chromatin by Immunolocalisation

Interaction of protein with chromatin can be visualised by immunolocalisation of proteins onto purified nuclei. DNA replication can be analyzed in parallel by immunofluorescence with the nucleotide thymidine deoxynucleotide analog biotin-dUTP (biotinylated deoxyuridine triphosphate). This method has several advantages. It is rapid, very sensitive, and detects sites of DNA replication that can be colocalized with replication proteins, for example (Figure 3).

Figure 3. Changes in nuclear morphology in replicating extracts.

Figure 3

Changes in nuclear morphology in replicating extracts. Demembranated sperm nuclei were incubated in a low speed extract and examined by fluorescence with the Hoechst 33342-dye (A). Pre Replication Centers are detected by immunolocalization of RPA protein (more...)

1.

Add to 50 μl of Xenopus egg extract between 1,000 and 2,000 sperm nuclei/μl and 40 μM biotin-dUTP.

2.

Every 30 min, take a 10-μl aliquot of the reaction and fix 1 hour at room temperature in 200 μl of XB containing 4% formaldehyde.

3.

Spin down at 2,500 g through a 0.7 M sucrose cushion in XB onto coverslips.

4.

Post-fix the nuclei 4 min in --20°C ethanol.

5.

Incubate the coverslips in PBS 10 min at room temperature and then 1 hour in PBS, 1% BSA to block nonspecific interactions.

6.

Load on the coverslips 50 μl of PBS 1% BSA containing the specific antibody and incubate at 4°C in a wet chamber.

7.

Wash in PBS, 0.1% Tween 20, 10 min.

8.

Load on the coverslips 50 μl of PBS 1% BSA containing the specific secondary antibody coupled to FITC and streptavidin coupled to Texas Red 1 hour at room temperature.

9.

Wash twice with in PBS, 0.1% Tween 20, 10 min.

10.

Stain DNA 10 min at room temperature with either Hoechst dye 2 μg/ml in PBS or propidium iodide 10-6 M.

11.

Wash twice in PBS, 0.1% Tween 20, 15 min at room temperature.

12.

Mount the coverslips with Citifluor antifading and seal with nail polish or with Mowiol before observation under a fluorescence microscope.

Detection of Protein Interaction with Chromatin by a Purification Assay

Demembranated sperm nuclei were prepared as described. Chromatin reconstituted in Xenopus egg extracts was purified as follows.

1.

Add to 50 μl of Xenopus egg extract adjusted with Energy Mix between 1,000 and 2,000 sperm nuclei/μl.

2.

At each time point, samples were diluted 10-fold in ice-cold XB Buffer containing 0.15% Triton X-100 and protease inhibitors and chilled 2 min on ice.

3.

Chromatin was purified by centrifugation at 6,000 g for 5 minutes at 4°C through a 1.5-ml 0.7 M sucrose cushion made in XB Buffer.

4.

Chromatin pellets were washed once in XB Buffer containing 0.15% Triton X-100, and proteins were eluted with 2X Laemmli buffer, separated by SDS-PAGE, and analysed by immunoblot. An example is shown in Figure 4.

Figure 4. Dynamics of MCMs Binding to Chromatin During the Licensing Reaction.

Figure 4

Dynamics of MCMs Binding to Chromatin During the Licensing Reaction. Kinetics of MCMs binding to chromatin analysed by western blotting of chromatin fractions. A sample of chromatin formed during the licensing reaction (panel A) was purified and bound (more...)

Protocol for Single-stranded DNA Replication and Chromatin Assembly

Single-stranded M13 DNA incubated in the egg extract is entirely converted into a double-stranded DNA form assembled in a minichromosome (Méchali and Harland 1982). Chromatin assembly occurs during the process of DNA replication in the extract (Almouzni and Méchali 1988a). Figure 5 shows the kinetics of replication analyzed by agarose gel electrophoresis. This reaction can be also used to prepare double-stranded labeled DNA by a single addition of [32P]dATP to the reaction medium.

Figure 5. Replication of M13 ss DNA in a high-speed extract.

Figure 5

Replication of M13 ss DNA in a high-speed extract. M13 single stranded DNA was added together with α32P-dATP in High-Speed egg Extract. Aliquots were taken at the indicated times and analyzed by agarose gel electrophoresis. The first complete (more...)

1.

Prepare a reaction medium as follows, scaled to the number of assays to be performed.

Single-stranded DNA 500 μg/ml 0.4 μl

[32P]dATP 0.5 μCi/μl 1 μl (Note A)

H2O 0.6 μl

Total .................................................2 μl Reaction Mix (Note B)

2.

Add to small 0.5-ml Eppendorf tubes:

Reaction Mix 2 μl

Xenopus egg extract 18 μl

Total ..........................20 μl (Note C)

Mix and incubate at room temperature (20 to 25°C). (Note D)

3.

Take a 2-5-μl aliquot to determine the extent of labeling by TCA precipitation. (Note E)

4.

Take another 2-5-μl aliquot. Add 25 μl of Stop Mix and 1 volume of chloroform. Vortex at maximum speed for 20 sec and centrifuge 3 min at maximum speed in an Eppendorf centrifuge at room temperature. Take 18 μl from the aqueous phase and add the sample buffer for gel electrophoresis.

5.

Analyze the reaction product on a 1% agarose gel run with Tris-Acetate-EDTA buffer. (Note F)

Notes

A. A small amount of [32P]dATP (0.5 μCi per reaction) is sufficient to obtain >50,000 cpm incorporated in the assay. The values can be increased by increasing the amount of [32P]dATP.

B. The endogenous ATP/Mg2+ normally present in the extract is sufficient for DNA replication and nucleosome assembly. The addition of exogenous ATP/Mg2+ improves the physiological spacing of the nucleosomes (Almouzni and Méchali 1988b)

C. The reaction medium can be diluted up to at least 10 μl if other components need to be included in the assay. In that case, the final concentration of single-stranded DNA and ATP/Mg2+ should be adjusted accordingly in the reaction mix. We recommend the use of the following dilution medium: 20 mM HEPES pH 7.6, 70 mM potassium acetate, 6 mM MgCl2, 1 mM ATP, 2 mM DTT, 5% sucrose, and 1 mg/ml BSA nuclease free.

D. >95% of DNA is replicated and assembled in a minichromosome after 90 min at 22°C. The physiological spacing of chromatin is slightly improved after 2 hours at 22°C. The reaction can be left for at least 6 hours at room temperature without affecting the final product. Do not incubate below 18°C.

E. The reaction can be quantified by TCA precipitation of aliquots. The extent of incorporation of [32P]dATP in double-stranded DNA is determined by spotting a 2-5-μl aliquot directly on a Whatman GF/C filter, followed by 20 min incubation in cold 5% TCA, 2% sodium pyrophosphate, four washes in 5% cold TCA, and a final wash with ethanol. The filters can be left several hours in cold 5% TCA if necessary. DNA synthesis is estimated as described in the section "Assays for DNA Replication in Xenopus Extracts" above, and the efficiency of DNA replication is calculated in relation to the input single-strand DNA.

F. The protocol of extraction given here is sufficient for an analysis of the DNA products on an agarose gel. It can be improved by treatment with proteinase K, followed by phenol and phenol--chloroform extractions in larger volumes if the DNA has to be extensively purified.

Chromatin Assembly

A double-stranded plasmid DNA molecule incubated in the egg extract is assembled into a minichromosome containing evenly spaced nucleosomes, as occurs in vivo (Laskey et al. 1977; Almouzni and Méchali 1988b). Assembly of spaced chromatin also occurs concomitantly with DNA synthesis (Almouzni and Méchali 1988a). If the plasmid DNA molecule is circular relaxed, it will be converted into a supercoiled form after incubation in the extract and extraction of the DNA.

Chromatin assembly is efficient because a large store of histones is present in the Xenopus egg, estimated at 120 ng, an amount sufficient to assemble 20,000 nuclei (Newport and Kirschner 1982). Using a high-speed extract and a plasmid molecule to titrate histones, the decrease in the supercoiling reaction is not detectable below 50 ng of DNA/μl extract, and at 80 ng of DNA/μl the DNA molecule population is mainly relaxed (Prioleau and Méchali, unpublished data). Thus, 1 μl of extract contains enough histones to assemble 50 to 80 ng of DNA, a value in close agreement with the estimate made by Laskey et al. (1977).

Chromatin assembly can be monitored by a variety of procedures, and we describe here two simple protocols. The first is based on the observation that the assembly of nucleosomes introduces negative superhelical turns into the DNA (Figure 6, supercoiling assay). The second is based on the differential sensitivity to micrococcal nuclease of nucleosomes and linker DNA (microccocal nuclease assay).

Figure 6. Chromatin assembly in a high speed egg extract.

Figure 6

Chromatin assembly in a high speed egg extract. Supercoiled bluescript DNA was added to a high-speed extract and aliquots were processed as described in the text for analysis by agarose gel.

Supercoiling Assay (First Method)

1.

Prepare a reaction medium as follows, scaled to the number of assays to be performed.

Double-stranded DNA 500 μg/ml 0.4 μl

ATP/Mg2+ 1 μl

Sterile H2O 0.6 μl

Total ................................................ 2 μl Reaction Mix

2.

Add to small 0.5-ml Eppendorf tubes:

Reaction Mix 2 μl

Xenopus egg extract 18 μl

Total ......................................20 μl (Note A)

Incubate at room temperature (20 to 25°C). (Note B)

3.

Add 100 μl of Stop Mix containing 500 μg/ml proteinase K. Incubate 1 hour at 37°C.

4.

Add 1 volume of phenol--chloroform saturated with TE. Vortex vigorously and centrifuge 4 min at maximum speed in an Eppendorf centrifuge (room temperature).

5.

Transfer the upper aqueous phase to a new tube and extract with 1 volume of chloroform--isoamylalcohol. Centrifuge 3 min in an Eppendorf centrifuge.

6.

Transfer the aqueous phase to a new tube. Add 1/10th volume 3 M sodium acetate, 2 volumes cold ethanol, and mix.

7.

Centrifuge for 10 min. Dry the pellet and resuspend in 15 μl of TE containing 25 μg/ml DNase-free RNase. Incubate 20 min at 37°C.

8.

Analyze the reaction by Southern blot followed by hybridization (Figure 6). (Note C)

Supercoiling Assay (Second Method)

It can be more convenient to analyze chromatin assembly on a molecule that is already radiolabeled, eliminating the need for Southern blot hybridization. 2-5-μl aliquots are then sufficient to follow the reaction. In that case, the labeled DNA can be obtained by labeling DNA through the conversion of single-stranded DNA to the double-stranded form in the extract (see "Protocol for Single-Strand DNA Replication and Chromatin Assembly"). After extraction, a double-stranded, labeled, supercoiled molecule is obtained. Another procedure is to linearize the DNA at one restriction site, dephosphorylate with alkaline phosphatase, label with T4 polynucleotide kinase, and recirculate with T4 DNA ligase (Razvi et al. 1983). If a radiolabeled DNA is used, aliquots of the reaction can be analyzed on an agarose gel after a single chloroform extraction.

Micrococcal Nuclease Digestion Assay

Steps 1 and 2 are the same as steps 1 and 2 of the supercoiling assay.

3.

Add CaCl2 to 3 mM final concentration and 30 units of micrococcal nuclease. Remove aliquots during digestion (2-15 min) at room temperature.

4.

Each aliquot is adjusted to 30 mM EDTA, 0.5 % SDS, and 500 μg/ml proteinase K and extracted as described above (steps 3 to 7 of the supercoiling assay).

5.

Analyze the reaction products on a 1.5-2% agarose gel run in TBE or in acrylamide gels for mononucleosome resolution (Almouzni and Méchali 1988).

Notes

A. The reaction medium can be diluted up to 10 μl if other components have to be included in the assay. In that case, the final concentration of DNA and ATP/Mg2+ should be adjusted accordingly in the Reaction Mix. We suggest the following dilution medium: 20 mM HEPES pH 7.6, 70 mM potassium acetate, 6 mM MgCl2, 1 mM ATP, 2 mM DTT, 5% sucrose, and 1 mg/ml BSA nuclease free.

B. Chromatin assembly is a multistep process. The DNA is first relaxed by the topoisomerase I present in the extract in a reaction that takes place in less than 5 min at 20°C. It is then progressively assembled into chromatin in a reaction that takes place within 2 to 4 hours.

C. It is also possible to directly analyze the reaction products by ethidium bromide staining of the gel. In that case, the amount of DNA and egg extract in the assay must be increased. We suggest using 1 μg of DNA with 25 μl of Xenopus egg extract. A larger amount of DNA may deplete the histone pool.

Studying DNA--Protein Interactions in Vivo Using LM-PCR Coupled with Nuclear Matrix Isolation

Several levels of DNA compaction exist in eukaryotic nucleus. The DNA is packed into nucleosomes, 30-nm fibers, and DNA loop domains (Cook and Brazell 1976; Paulson and Laemmli 1977). The loops can be visualised by extraction of histones from the isolated nuclei or metaphase chromosomes. In this case, they are anchored to the proteinaceous nucleoskeleton, also called a nuclear matrix or scaffold.

The transcription and replication factories are located on the nuclear matrix, and transcribing and replicating DNA is transiently associated with it [for a review see (Vassetzky, Lemaitre et al. 2000)]. In many cases, the study of DNA--protein interaction is hindered by heterogeneity of a substrate. It is known, for example, that in the case of multiple copies of a gene, frequently only one copy is transcriptionally active. In this case, the background of the transcriptionally inactive copies will completely mask the productive DNA--protein interaction. The same is true for cell cycle-regulated genes in a non-synchronous cell population. There is a way to enrich the studied gene population with transcriptionally active copies without leaving the in vivo context. It is based on the nuclear matrix isolation because transcribed genes are transiently associated with the nuclear matrix.

We have developed this new method based on ligation-mediated PCR (LM-PCR) which, in conjunction with already well-established methods for MAR isolation (Gasser and Vassetzky 1998), can provide details of protein--DNA interaction within the transcribed copies of genes. We have designated this method as nuclear matrix footprinting.

This method involves the treatment of isolated permeabilised nuclei with DNase I in a manner similar to genomic footprinting, followed by the isolation of the nuclear matrix-associated DNA by treatment with 2 M NaCl. The DNA component of the nuclear matrix is partially protected from the action of DNase I while non-protein-associated DNA is destroyed. The resulting protected DNA is then isolated and used as a substrate for LM-PCR.

The method of LM-PCR is described in detail elsewhere (Mueller and Wold 1989), and our method is essentially the same [see (Vassetzky et al. 2000a) for an example].

Protocol for the Nuclear Matrix Isolation

Nuclear matrices were prepared by treatment of the isolated nuclei with DNase I or restriction endonucleases, followed by extraction with 2 M NaCl, essentially as described (Gasser and Vassetzky 1998).

1.

105 nuclei were digested with 100 μg/ml DNase I (Sigma) in the digestion buffer (100 mM NaCl, 25 mM KCl, 10 mM Tris-HCl pH 7.5, 0.25 mM spermidine, and 1 mM CaCl2) at 4°C for 2 hours.

2.

Stabilize the nuclear matrices by addition of CuCl2 to a final concentration of 1 mM and incubate for 4°C for 10 min. This optional step increases the stability of the nuclear matrix.

3.

Extract the nuclei by addition of 1 volume of a buffer containing 4 M NaCl, 20 mM EDTA, and 40 mM Tris-HCl, pH 7.5.

4.

Precipitate the nuclear matrices by centrifugation at 4000 g for 5 min. Discard the supernatant.

5.

Wash three times with 20 mM Tris-HCl pH 7.5, 2 M NaCl, and 10 mM EDTA. Centrifuge at 4,000 g for 5 min. Discard the supernatant.

6.

Digest the nuclear matrices with proteinase K and extract with phenol--chloroform. The size range of the nuclear matrix-attached DNA should be 200-1000 bp.

7.

Treat the DNA associated with the nuclear matrix with RNase A and use it as a substrate for LM-PCR.

Ligation-mediated PCR

Ligation-mediated PCR (LM-PCR) is carried out essentially by the method of Mueller and Wold 1989. Initial extension was with Vent (exo-) DNA polymerase (New England Biolabs), and amplification/labeling was with Gold Star DNA polymerase (Eurogentec). 5' primer labeling was with T4 polynucleotide kinase (Life Technologies), following the manufacturer's instructions.

Appendix: Buffers and Solutions

Extracts

High Salt Barth (HSB)

15 mM HEPES, pH 7.6

110 mM NaCl

2 mM KCl

1 mM MgSO4

0.5 mM Na2HPO4

2 mM NaHCO3

Prepare an 8X stock and store at 4°C.

XB

10 mM HEPES, pH 7.7

100 mM KCl

0.1 mM CaCl2

1 mM MgCl2

5% sucrose

1 mM DTT

Prepare a 2X stock and store at --20°C.

Cytochalasin B, 10 mg/ml

10 mg/ml cytochalasin B in DMSO

Store at --20°C.

Protease inhibitors, 10 mg/ml

10 mg/ml leupeptin

10 mg/ml pepstatin

10 mg/ml aprotinin in DMSO

Store in aliquots at --20°C.

Energy Mix, 20 X

200 μg/ml creatine kinase

200 mM creatine phosphate

20 mM ATP

20 mM MgCl2

Store in aliquots at --20°C.

Creatine phosphate, 1 M

1 M creatine phosphate in water

Store in aliquots at --20°C.

Creatine kinase, 10 mg/ml

10 mg/ml creatine kinase in HEPES, 10 mM, pH 7.6

20% glycerol

Store in aliquots at --20°C.

XB-HS

10 mM HEPES, pH 7.7

10 mM potassium acetate

5 mM MgCl2

1 mM DTT

5% sucrose

Store at 4°C or --20°C.

MMR

5 mM HEPES, pH 7.8

0.1 M NaCl

2 mM KCl

0.1 mM EDTA (added before Ca2+ and Mg2+)

1 mM MgCl2

2 mM CaCl2

Prepare a 10X stock and store at 4°C.

HSB-CSF

Same as HSB + 2 mM EGTA

Energy Mix-CSF 20X

Same as Energy Mix + 2 mM EGTA

XB-CSF

10 mM HEPES, pH 7.7

100 mM KCl

1 mM MgCl2

5% sucrose

1 mM DTT

5 mM EGTA

Store at 4°C or --20°C.

Calcium ionophore

5 mM in DMSO and store at --20°C.

Single-Strand DNA and Chromatin Assembly

Single-stranded M13 DNA or any other single-stranded molecule, 200 μg/ml.

Double-stranded M13 DNA, 500 μg/ml in TE.

ATP/Mg2+

60 mM ATP

100 mM MgCl2

pH 7.5 (check the pH of the solution with a few μl spotted on a pH paper) Store at --20°C.

Stop Mix

40 mM EDTA

1% SDS

0.5% Triton X-100

ATP/Mg2+

30 mM ATP

50 mM MgCl2

pH 7.5 (check the pH of the solution with a few μl spotted on a pH paper)

Store at --20°C.

Preparation of Nuclei

Sperm Nuclei

Lysolecithin

10 mg/ml lysolecithin in water

Prepare extemporaneously.

SNB

15 mM HEPES, pH 7.4

0.25 M sucrose

75 mM NaCl

0.5 mM spermidine

0.15 mM spermine

1 mM DTT

BSA

BSA (nuclease-free) 100 mg/ml in Tris 10 mM, pH 7.6

20 mM NaCl

Store at --20°C.

PBS

137 mM NaCl

2.7 mM KCl

4.3 mM Na2HPO4

1.4 mM KH2PO4, pH 7.4

Embryonic Nuclei

Nuc A

10 mM HEPES, pH 7.6

15 mM KCl

1 mM EDTA

0.5 mM spermidine

0.15 mM spermine

0.5 mM DTT

10 μg/ml protease inhibitors

0.4% Triton N-101 (This latter is not essential, although it improves the yield. The point at which it is added should also be considered. Its addition or omission will depend on the requirements of the experiment.)

Nuc B

Part B1

10 mM HEPES, pH 7.6

10% glycerol

2 M sucrose

15 mM KCl

1 mM EDTA

Part B2

0.5 mM spemidine

0.15 mM spermine

0.5 M DTT

Protease inhibitors, 10 μg/ml

Analysis of DNA Replication

TCA precipitation

Stop Mix

20 mM Tris, pH 7.9

30 mM EDTA

1% Sarkosyl

5% TCA (keep at 4°C)

5% TCA , 2% sodium pyrophosphate (keep at 4°C)

Glass fiber filters (e.g., Whatman GF/C)

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Copyright © 2001, Institut national de la santé et de la recherche médicale (INSERM)
Bookshelf ID: NBK7096PMID: 21413355

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