Transcriptionally competent chromatin assembled with exogenous histone
http://www.100md.com
《核酸研究医学期刊》
CRG, Centre de Regulació Genòmica, Universitat Pompeu Fabra (UPF), Passeig Marítim, 37-49, 08003 Barcelona, Spain and 1 Institut für Molekularbiologie und Tumorforschung, Philipps-Universitt, Emil-Mankopff-Strasse 2, 35037 Marburg, Germany
* To whom correspondence should be addressed. Tel: +34 93 224 09 01; Fax +34 93 224 08 99; Email: miguel.beato@crg.es
ABSTRACT
We describe a cell-free chromatin assembly system derived from the yeast Saccharomyces cerevisiae, which efficiently packages DNA into minichromosomes in a reaction dependent on exogenous core histones and an ATP-regenerating system. Both supercoiled and relaxed plasmid DNA serve as templates for nucleosomal loading in a gradual process that takes at least 6 h for completion at 30°C. Micrococcal nuclease digestion of the assembled minichromosomes displays an extended nucleosomal ladder with a repeat length of 165 bp. The purified minichromosomes contain the four core histones in stoichiometric proportion and exhibit phased nucleosomes over the mouse mammary tumour virus (MMTV) promoter. The progesterone receptor and NF1 synergize on these minichromosomes resulting in efficient cell-free transcription. The ease of manipulation and the potential use of yeast strains carrying mutations in the chromatin handling machinery make this system suitable for detailed mechanistic studies.
INTRODUCTION
The initial level of DNA packaging in the nucleus of eukaryotic cells is a string of repeating units, the nucleosomes, whose subunit structure is common to all eukaryotes and contains 146 bp of left-handed DNA wrapped around a core protein composed by two copies of each of the four histones H3, H4, H2A and H2B. Over the last decade, knowledge of the structural and functional role of chromatin has grown significantly. The nucleosomes, first considered mere packaging structures, are now regarded as essential and active elements in the regulation of all processes that require access to nuclear DNA (1,2). This understanding is supported by the discovery of chromatin remodelling complexes, present in all eukaryotes cells, that participate in the response to stimuli requiring chromatin handling (3,4).
The in vitro biochemical reconstitution of chromatin with physiological nucleosome spacing and positioning is a critical tool in the elucidation of the role of chromatin structure and its chemical modifications in biological phenomena such as transcription and replication. A wide variety of methods have been developed, including those that use purified components (DNA and histones) in the presence of counterions such as NaCl, nucleoplasmin and polyglutamic acid (5–7). These purified systems generate well-defined chromatin templates, but their structure is not dynamic and therefore, these templates are generally not suitable for further functional analysis. In contrast, crude extracts from Xenopus unfertilized eggs (8), Xenopus oocytes (9) or Drosophila embryos (10), which contain a large pool of maternal histones, can assemble under physiological conditions functional minichromosomes with evenly distributed nucleosomes over any exogenously added DNA plasmid. Even though those systems are useful and well scaled-up, the reproducible generation of large amounts of competent extract is expensive in time and requires a temperature-controlled and noise-isolated room or bath for the Drosophila or Xenopus culture. Moreover, the preblastodermic Drosophila system should be made with embryos harvested in a short period of time (0 to 100 min) after egg laying; further delay renders low competent extracts. The Xenopus culture has several drawbacks as the cost of the animals and their sensitivity to thermal, seasonal and feeding changes.
To circumvent these problems, several yeast systems for chromatin assembly have been used based on purified core histones from isolated nuclei and Xenopus laevis nucleoplasmin (11), purified yeast histones and yeast nucleosome assembly protein (NAP-1) (12) or whole-cell extract (13). However, none of these procedures yield an extended regular ladder of DNA fragments after micrococcal nuclease digestion. Furthermore, the reported whole-cell extract can assemble chromatin without exogenous addition of core histones. Based on the grinding method with dry ice (13,14), we describe here a detailed study, including topological and hydrodynamic analysis, protein composition, chromatin structure, nucleosome phasing and transcription, illustrating the development of a very efficient cell-free system for nucleosome assembly with S.cerevisiae cells and isolated native core histones.
MATERIALS AND METHODS
yScS116 cell-free extract preparation
S.cerevisiae (BJ5460 strain) cells were grown overnight at 30°C in 6 l of YPD medium (Gibco) with continuous shaking (180 r.p.m.). After pelleting (in a JLA 9.1000 Beckman rotor) and removing the excess of culture medium as much as possible, cells were extruded with a 20 ml syringe (without the needle) over a Dewar glass containing liquid nitrogen, frozen as ‘noodles’ and stored at –80 °C. Currently, 20–30 g of frozen cells can be obtained from 6 l of a saturated yeast culture. Cell-free extract was made as follows: pellets of dry ice of the best quality were powdered with a home coffee mill; an equal weight of frozen S.cerevisiae noodles was added over the powdered dry ice and then grinded for 2–5 min at maximum speed. Avoiding defrost, the frozen mix was quickly added with a spatula to a plastic tube. One volume of low ionic strength extraction buffer (20 mM HEPES–KOH, pH 7.5. 5 mM KCl, 2.5 mM Mg2Cl, 1 mM EDTA, 0.5 mM dithiothreitol, 10 mM ?-glycerophosphate, 10% glycerol, 10 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin) was added and the mixture thawed without capping to prevent the tube explosion. After CO2 evaporation, the tube was capped and the mixture vortexed for 5 min at room temperature. The cell homogenate was pipetted until it filled completely the 5.2 ml Beckman SW55 tubes and then centrifuged at 35 000 r.p.m. (116 000 g) for 2 h at 4°C. Avoiding the thin lipid layer, the clear supernatant was carefully recovered and aliquots (200–400 μl) were stored at –80 °C. We named our extract as yScS116 (yeast Saccharomyces cerevisiae Supernatant 116 000 g).
Alternatively, we also prepare a very competent extract using commercial dry yeast (Algist Bruggeman N.V., Gent, Belgium). In brief, 8 g of dry cells were grinded in dry ice for 1–2 min, dissolved in 24 ml of extraction buffer and processed as described above.
Histone purification
Native core histones were purified from isolated nuclei from mouse, rat or chicken fresh liver over a hydroxyl apatite (BioGel HTP cDNA grade; Bio-Rad) column (1.5 x 20 cm) (15). H1 and HMG were first eluted at 0.63 M NaCl, 100 mM potassium phosphate, pH 6.7. Core histones were subsequently eluted as octamers with 2 M NaCl, 100 mM potassium phosphate, pH 6.7. Peaks containing stoichiometric amounts of histones were pooled and dialysed overnight in the cold room against 2 l of 0.2 M NaCl, 20 mM Tris, pH 7.5, 0.1 mM EDTA, 20 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin and 2 μg/ml pepstatin. After measuring the protein concentration, histones were aliquoted and stored at –80 °C.
In vitro chromatin assembly
Chromatin assembly was monitored following the supercoiling of a topoisomerase-I-relaxed plasmid. Supercoiled pUC18 (100 ng) was incubated with 0.1 U of calf thymus topoisomerase I (GIBCO) at 37°C for 30 min in a final volume of 10 μl. Meanwhile, the chromatin assembly reaction was made by mixing 20 μl of yeast extract with all the reagents in extraction buffer until the following final concentrations: 500–800 ng of purified core histones (histones/DNA mass ratio = 5 to 8), 20 mM HEPES–KOH, pH 7.5, 5 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol, 10 mM ?-glycerophosphate; 10% glycerol, 40 mM disodium creatin phosphate, 3 mM ATP, 1 ng/μl creatin phosphokinase, 10 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin and 3 mM Mg2Cl in a final volume of 34 μl. As a critical step and to prevent the formation of insoluble histone/DNA complexes, the mixture was well mixed prior to the addition of the relaxing reaction containing the plasmid and then incubated at 30°C for 6–14 h. For the supercoiling assay, reactions were stopped by adding SDS and EDTA of 0.1% and 20 mM, respectively, and the DNA was deproteinized with 1 mg of proteinase K per millilitre at 37°C for 2 h followed by phenol extraction. After pelleting and washing, the DNA was dissolved in a few microlitres of 20 μg/ml DNase-free RNaseA and loaded onto a 1% agarose gel in 1x TBE. After electrophoresis, the gel was stained with ethidium bromide. Chromatin assembly reactions with extracts obtained from commercial dry cells were performed as described before but the amount of extract added was 10% of the final volume of the assembly reaction.
Two-dimensional gel electrophoresis of topoisomers
Minichromosomes were deproteinized as described and two-dimensional electrophoresis (16) was performed in a 20 x 20 cm 1% agarose 1x TBE gel. The first dimension was run at 60 V for 16 h and then the gel was shifted 90°, equilibrated for 8 h in the dark in 1x TBE containing 4 μM chloroquine (Sigma) from a fresh stock solution (10 mg/ml) and electrophoresed in the second dimension at 60 V for 16 h. Chloroquine was extensively removed from the gel with several changes in distilled water and the gel was stained with ethidium bromide and destained with distilled water.
The reference topoisomer ladder was prepared by relaxing negative supercoiled pUC18 plasmid with topoisomerase I in the presence of different amounts of ethidium bromide (17). Both the reference relaxed DNA (L = 0) and the topoisomer ladder were prepared by plasmid relaxation with topoisomerase I at 30°C for 6 h in a reaction mixture containing 20 mM HEPES–KOH, pH 7.5, 5 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol; 10 mM ?-glycerophosphate; 10% glycerol, 40 mM disodium creatin phosphate, 3 mM ATP, 1 ng/μl creatin phosphokinase, 10 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin and 3 mM Mg2Cl; in the reference relaxed DNA, the most abundant topoisomer was considered to have a linking difference (L) = 0.
Micrococcal nuclease digestion
After chromatin assembly, samples were cooled at room temperature and CaCl2 (final concentration of 3 mM) and MNase (0.1–0.3 Boehringer units per microlitre of assembly mixture) were added and incubated at room temperature. At various time intervals (see figure legends), the digestions were stopped with EDTA (50 mM final concentration) and samples deproteinized as in the supercoiling assay or with Wizard Purification Systems (Promega) following the manufacturer's instructions. After DNA precipitation and ethanol washing, the samples were resuspended in 20 μg/ml RNase A, electrophoresed in 1.5% agarose in Tris–glycine buffer at 90–120 V, stained with ethidium bromide and destained in distilled water.
Isolation of minichromosomes
A linear 15–30% sucrose gradients of 11 ml in 100 mM NaCl, 5 mM HEPES–KOH, pH 7.5, 0.2 mM EDTA, 10 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin were made in 12 ml Beckman SW40 tubes. Chromatin assembly reactions were layered on top of the gradient and centrifuged (with the slowest acceleration and without brake at the end of the spin) at 22 000 r.p.m. for 16 h at 4°C in a Beckman-Coulter Optima XL-100K Ultracentrifuge. Fractions (0.8 ml) were collected and one-twentieth of each was deproteinized as described and the DNA electrophoresed in a 1% agarose gel to determine the position of the minichromosome in the gradient. Fractions containing minichromosomes were pooled and a portion was digested by the addition of CaCl2 (3 mM, final concentration) at room temperature for 5 min with 0.03 and 0.06 Boehringer units per microlitre of MNase. The reactions were stopped and processed as described.
Protein analysis
The pooled fractions from the gradient containing the minichromosomes, or the corresponding in the mock-incubated (assembly without plasmid) control, were layered over a 1 ml cushion of 30% sucrose in gradient buffer containing 10 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin, covered with gradient buffer in a 4.2 ml Beckman SW60 tube and centrifuged at 30 000 r.p.m. for 14–16 h at 4°C. The supernatant was very carefully removed and the pellet was dissolved in 1x L?mmli buffer (19) at 90°C and electrophoresed in 15% SDS–PAGE. The gel was stained with silver (20).
Analysis of phased nucleosomes
pMMTV plasmid (30 μg) was digested with BglII and the 5' phosphate removed with alkaline phosphatase. After phenol extraction and DNA pelleting, 10 μl of ATP (3000 Ci/mmol, Amersham Pharmacia biotech) were mixed with 20 U of T4 polynucleotide kinase (Roche) in a final volume of 50 μl and incubated for 30 min at 37°C. The BglII-radiolabelled DNA was then diluted at a final concentration of 0.5 μg/ml in ligation buffer and incubated overnight at 4°C with 30 U of T4 ligase (Roche). This dilution promotes the ligation of intramolecular extremes and prevents the ligation of the plasmid as dimers or higher oligomers. The relaxed, covalently closed plasmid DNA was concentrated carefully with n-butanol, phenol extracted and purified, as described (21). Minichromosomes assembled over the BglII-radiolabeled pMMTV-CAT-BB vector were digested with MNase as described. After DNA pelleting, samples were dissolved, digested with 10 U of XhoI (located at 10 bp of the 3' extreme of the BglII site), ethanol precipitated, dissolved and electrophoresed in 1.5% agarose–Tris–glycine gel. As the free-DNA control, an equal amount of pMMTV-CAT-BB DNA was first digested with 0.0005 Boehringer units per microlitre of MNase and then with XhoI. After electrophoresis, the gel was blotted, UV-crosslinked and exposed.
Transcription assays
Assembled pMMTV minichromosomes were transcribed for 1 h at 30°C with HeLa nuclear extract, recombinant PR and NF1 (from baculovirus-infected Sf9 cells) as described (22).
RESULTS
Our initial attempts to reproduce the supercoiling assay with the previously reported yeast whole-cell-free extract (13) were unsuccessful. We modified the ionic conditions and centrifugation parameters and monitored the ability of the whole extract to assemble nucleosomes by supercoiling analysis of relaxed pUC18. When DNA wraps a nucleosome core, the superhelical density of the covalently closed plasmid DNA increases by one (5). The topoisomerase activity of the extract relaxes this linking difference and, after deproteinization of the reconstituted plasmid, one negative superhelical turn is acquired for each nucleosome introduced (17). Figure 1A shows the supercoiling titration of relaxed pUC18 (lane 1) incubated with yScS116 and increasing amounts of the purified core histones shown in Figure 1B. Importantly, little increase in supercoiling was observed when the incubation was performed with the extract alone (lane 2) suggesting that the yScS116 did not contain a sufficient level of endogenous free histones to assemble nucleosomes onto an exogenously added plasmid. Furthermore, micrococcal nuclease digestion of yScS116 completely failed to detect any endogenous nucleosomal ladder (data not shown). The supercoiling level increased proportionally with the amount of added core histones, reaching its maximum value at a histone/DNA ratio of 6 to 7. The requirement of such a high proportion of histone probably results from the high content of RNA in yeast, which squelches added histones. At a histones/DNA ratio of 7 (lane 6), the minichromosome reached a topological density similar to that of the bacterial supercoiled pUC18 (lane 7). Thus, the yScS116 can efficiently assemble chromatin when supplemented with appropriate amounts of core histones.
Figure 1. Supercoiling assay of core histones-dependent chromatin assembly. (A) Nucleosome assembly with increasing amounts of added core histones. Relaxed pUC18, 75 ng, (R) was incubated with yScS116 and the indicated amounts of purified core histones for 6 h at 30°C. SC indicates the supercoiled pUC18. Migration of relaxed (II) and supercoiled (I) DNA forms are indicated. (B) A 15% SDS–PAGE profile of native core histones purified from mouse liver. (C). An ATP-regenerating system is essential for chromatin assembly. Relaxed pUC18 of 100 ng (lane 1) was assembled in yScS116 with or without ATP (3 mM), creatin phosphokinase (1 ng/μl)/disodium creatin phosphate (40 mM) (CPK/CP), and apyrase (U/ml), as indicated on top. Other symbols are as in (A).
Dynamic chromatin assembly in cell-free extracts is dependent on the concentration of ATP, Mg2+ and KCl (9,10,23,24). The yScS116 contains a sufficient level of endogenous ATP to generate a certain degree of supercoiling without exogenous addition of ATP (Figure 1C, lane 9). However, as other crude chromatin assembly extracts, the yScS116 requires a constant level of ATP to assemble nucleosomes efficiently (25). In the absence of creatin phosphokinase and creatin phosphate, addition of ATP generated a poor supercoiling (Figure 1C, lane 3) that was further reduced in the presence of apyrase (an enzyme which specifically degrades ATP) (Figure 1C, lanes 4 to 8). Therefore, the chromatin assembly in the yScS116 is a dynamic process that requires hydrolysis of a constant level of ATP.
Chromatin assembly with the yScS116 is a gradual process that takes from 6 to 14 h for completion. As shown in Figure 2A, the endogenous topoisomerases relaxed the negative supercoiled plasmid during the first hour of incubation (lanes 2 to 4). Thereafter, the DNA became more supercoiled in a time-dependent manner. After 14 h of assembly, the supercoiled level of the deproteinized minichromosome and a bacterial negative supercoiled pUC18 were very similar (lane 11). The negative nature of this supercoiling was demonstrated by electrophoresis in two dimensions, the second in the presence of chloroquine (Figure 2B). The deproteinized minichromosome displayed an average linking difference (L) of –14/–16, corresponding to a minichromosome fully loaded with nucleosomes spaced at 160/170 bp. To address whether the assembled nucleosomes were evenly spaced and to determine spacing by a different method, the minichromosomes were digested for different times with micrococcal nuclease and the resulting fragments electrophoresed in an agarose gel. A periodical ladder of oligonucleosome fragments can be distinguished and no subnucleosomal size fragments were detected (Figure 2C). The extended nature of the MNase ladder of oligonucleosomes was revealed by the accumulation of the shortest fragments at longer digestion times (compare band intensities in lanes 4, 5 and 6 in Figure 2C). The electrophoretic migration of the mono, di, tri, tetra and pentanucleosome bands yielded a repeat length average of 165 bp. This spacing is in the range displayed by the native chromatin of S.cerevisiae (26–28) and corresponds to the values determined by topoisomer analysis. Thus, the average minichromosomes are fully loaded with properly spaced nucleosomes.
Figure 2. Topological and enzymatic characterization of the minichromosomes. (A) Time course of chromatin assembly. Supercoiled pMMTV plasmid (1.5 μg) was assembled under standard conditions. At the indicated times, aliquots containing 100 ng of DNA were removed and processed. Samples were co-electrophoresed with topoisomerase-I-relaxed plasmid (lane 1) in a 1% agarose 1x TBE gel at 60 V for 14 h and then stained with ethidium bromide. (B) Two-dimensional electrophoresis. Assembled minichromosome (100 ng) was deproteinized and co-electrophoresed with the standard DNA topoisomer ladder (reference pattern) and the relaxed DNA. To obtain a good matching, the deproteinized minichromosome was first loaded, electrophoresed for 90 min and then, the standard ladder was loaded in the same well. The relaxed DNA was loaded at the same time as the standard but several wells to the right. The gel was electrophoresed and processed as described in Materials and methods. The linking-number change in the minichromosomes was determined by counting in the topoisomer ladder the number of spots from the relaxed DNA (0). Np, Nm and Nr are the nicked form of the reference pattern, minichromosomes and relaxed DNA, respectively. (C) Micrococcal nuclease digestion of assembled minichromosomes. Relaxed pUC18, 1.5 μg (as in lane 1), was incubated with yScS116 and added core histones under standard conditions. After 6 h, 75 ng was removed and deproteinized for topological analysis (lane 3). To the remaining mixture, CaCl2 and MNase were added and incubation was continued at room temperature. At different times, one-third of the digestion mixture was removed and the digestion stopped as described. Lanes 2 and 7, 75 ng of supercoiled DNA and DNA molecular weight marker, respectively. I, II and III indicate the supercoiled, relaxed and linear form of pUC18, respectively. The positions of the mono-, di-, tri-, tetra- and pentanucleosome bands are indicated (arrows).
The protein composition of the assembled minichromosome was analysed after centrifugation through an isotonic linear sucrose gradient, which excludes aggregation and yields purified minichromosomes (25). Figure 3A displays the position of minichromosomes in a 15–30% sucrose gradient (fractions 9 and 10), which yielded the supercoiled form of pMMTV-CAT-BB DNA after deproteinization. The isolated minichromosome generated an MNase digestion ladder (Figure 3B) with the same nucleosomal spacing as the one found in the whole extract (compare the migration of the trinucleosome in Figures 3B and 2C). It should be noted that the sucrose fractionation removed impurities from the extract and a much lower amount of MNase (about one-tenth) was necessary to digest the purified minichromosome to the same extent as with the crude extract. The purified minichromosomes contained comparable amounts of all four nucleosomal core histones as well as lower amounts of additional bands of higher molecular weight corresponding to non-histone proteins present in the extract (Figure 3C).
Figure 3. Purification of the assembled minichromosomes. (A) Minichromosomes assembled under standard conditions on pMMTV-CAT-BB plasmids were loaded in a linear 15–30% sucrose gradient. After the centrifugation and fractionation, one-twentieth of each fraction was deproteinized, purified and electrophoresed. Lanes 3 to 11 contain the corresponding gradient fractions. Lanes 25, 50 and 100 contain 25, 50 and 100 ng of supercoiled pMMTV-CAT-BB plasmid, respectively. The relative position of fractions containing the minichromosome is shown on top. (B) MNase digestion of the gradient-purified minichromosomes. CaCl2 (3 mM, final concentration) and MNase (final concentration in Boehringer units per microlitre on top) were added to one half of the isolated minichromosomes and the mixture was incubated at room temperature for 5 min. After deproteinization, samples were electrophoresed in a 1.5% agarose–Tris–glycine gel. (C) Protein composition of the assembled minichromosomes. Fractions 9 and 10 (asterisks on Figure 4A) were pooled and one half was processed for protein analysis as described in Materials and methods. Mock: assembly performed without added plasmid. pMMTV: minichromosomes assembled over the pMMTV-CAT-BB plasmid. Histones: purified core histones from mouse liver used for chromatin assembly.
Figure 4. Phasing of the nucleosomes over the MMTV promoter and transcription assay. (A) Relaxed closed circular pMMTV-CAT-BB plasmid (1 μg) labelled with 32P at the Bgl II site and circularized was assembled in minichromosomes under standard conditions. CaCl2 (3 mM, final concentration) and MNase (0.1 Boehringer units per microlitre of mixture) were added. At 0.5, 2, 3 and 5 min, one-fourth of the mixture was removed and processed. After deproteinization, each sample was digested at 37°C with 10 U of XhoI, phenol-extracted, pelleted and washed. The naked plasmid was digested at room temperature in 1x extraction buffer (containing 3 mM CaCl2) with 0.0001, 0.00015, 0.0002 and 0.00025 Boehringer units of MNase per microlitre of mixture for 1 min and processed as in chromatin. Samples were electrophoresed at 120 V in a 1.5% agarose–Tris–glycine gel. The gel was blotted, UV-crosslinked and exposed to an X-ray film. Lanes 1 to 4 and 5 to 8 are the naked plasmid and chromatin samples, respectively. Black and grey symbols indicate the frame of both populations. Arrows on the right indicate the major MNase cuts. Fragment size in base pairs are indicated. The deduced estimated positions of nucleosomes in both phases are depicted in the diagram on right; the negative numbers indicate the distance from the start of transcription (+1). The NF1 binding site and the SacI cleavage site are indicated. (B) Transcription on nucleosomal templates. Where indicated, recombinant PR and NF1 (from baculovirus-infected Sf9 cells) were added to the assembled pMMTV minichromosomes and transcribed with HeLa nuclear extract (22). The position of the MMTV transcripts is indicated by an arrow on the right.
The MMTV promoter, located in the long terminal repeat (LTR) of the provirus is organized in positioned nucleosomes in mammalian and yeast cells (29–32) as well as in chromatin assembled in Drosophila embryo extracts (18,22). The so-called nucleosome B is positioned over the hormone responsive region (HRR) containing several binding sites for hormone receptors and a binding site for nuclear factor 1 (NF1). To elucidate whether the yScS116 can assemble phased nucleosomes, we used a low-resolution analysis of chromatin structure on minichromosomes assembled over a closed circular plasmid that contains the pMMTV promoter, radioactively labelled at the unique restriction site BglII (21). After digestion with MNase and subsequent cleavage with XhoI, which cleaves 10 bp 3' of the label, two ladders of periodically spaced chromatin-enhanced cleavage sites separated by 40 ± 10 bp can be observed over a background cleavage pattern similar to that of free DNA due to DNA bending and the sequence preference of MN (Figure 4A lanes 1 to 8). One of them (phase 1) corresponds to the previously reported translational phase with the NF1 binding site included within nucleosome B (31). Furthermore, the strong MNase sensitivity at the 3' border of the nucleosome B sequence (Figure 4A) is in good agreement with previous in vitro (18) and in vivo (31) structural analysis and documents the ability of the yScS116 to interpret the positioning information encompassed in the MMTV nucleotide sequence around the HRR. The second translational phase (phase 2) closely corresponds to previously reported positions (33,34). The less precise positioning of nucleosomes in the yeast (this report) and Drosophila (18) assembly system compared with that observed in metazoan cells may be due to the lack of linker histones, which have been shown to improve translational nucleosome positioning over the MMTV promoter (35).
Correct organization of the MMTV promoter in nucleosomes is essential for the synergistic transcriptional activation mediated by the hormone receptor and NF1 (22,31). Therefore the demonstration of a functional synergism between these two transcription factors is an indirect proof of a well-organized nucleosomal structure. To address the template capacity of yScS116-assembled minichromosomes, baculovirus-expressed recombinant progesterone receptor (PR) and NF1 were incubated with assembled minichromosomes and transcription was assayed in a HeLa cell nuclear extract (22). Addition of PR alone or NF1 alone was not sufficient for transcriptional activation of the MMTV promoter (Figure 4B, lanes 2 and 3). However, both factors added together synergistically activated transcription of the chromatin templates (Figure 4B, lane 4), suggesting that the extract also contains the remodelling machinery necessary for mediating this synergism. Therefore, the yScS116 cell-free system can assemble functional chromatin templates and can catalyse the synergistic transcriptional activation mediated by PR and NF1, that has been observed in vivo (31) and in vitro (22).
DISCUSSION
Based on a reported method (13,14), we have developed a two-component in vitro chromatin assembly system involving a whole-cell-free extract from wild-type Saccharomyces cerevisiae cells, yScS116, and exogenously added native core histones. MNase digestion of the yScS116 failed to detect endogenous chromatin. In the absence of added histones, yScS116 was ineffective in assembling nucleosomes under any condition tested, suggesting a lack of functional free histones. The addition of sufficient amounts of core histones promoted the ordered and evenly distributed deposition of nucleosomes over a covalently closed DNA ring. As observed in other chromatin assembly systems (10,25), yScS116 can assemble a fully loaded minichromosome when the reaction was performed at a constant concentration of ATP, maintained by an ATP-regenerating system. We suggest that the extended centrifugation at 116 000 g, in combination with the addition of MgCl2, minimizes the amount of endogenous chromatin in the final extract. It is noteworthy that a slightly higher centrifugal force (130 000 g) yielded extracts very inefficient in nucleosome assembly even in the presence of added core histones, probably due to pelleting of factors or complexes critical for the assembly process.
Recently, a yeast-derived improved chromatin assembly system dependent on exogenously added histones has been published (36). However, this system differs from the one reported in this paper in three major points: (i) the generation of the extract is much more complicated involving preparation of spheroplasts or crude nuclei; (ii) it requires high salt extraction and DEAE chromatography of the supernatant with a salt elution step; (iii) the assembled chromatin is not characterized with respect to either nucleosome positioning or transcriptional competence.
Till date, only two whole-cell-free systems can efficiently assemble native-like chromatin under physiological conditions. One uses the chromatin assembly machinery and the maternal-inherited pool of histones contained in cleared supernatants of dispersed Xenopus unfertilized eggs (8) or oocytes (25,37); the other is an extension of the Xenopus-based system employing preblastodermic Drosophila embryos (10). Cell-free systems obtained from mammalian tissue culture cells (38,39) are much less efficient in assembling chromatin. The Xenopus and Drosophila systems are reproducible, yield large quantities of competent extracts able to promote chromatin assembly either onto double-stranded DNA or, coupled with DNA synthesis, single-stranded DNA (24) and are prepared at very similar low-ionic and centrifugal (150 000 g) conditions. They can be defined as a single-component system as they rely entirely on the maternal-inherited histones, endogenous chaperones and chromatin assembly machinery. Nevertheless, they have several drawbacks; the activity of Xenopus extracts are sensitive to uncontrolled seasonal and feeding variations (40), and the Drosophila extract is difficult to handle because the embryos should be harvested very quickly after laying; a little delay renders the extracts less competent (41).
Although those single-component systems are very useful, the fact that all the factors (chromatin assembly machinery and histones) are present in the whole extract makes difficult an easy biochemical analysis of their particular contribution to the global assembly process. Furthermore, histone H2A is extensively modified in Xenopus oocytes and eggs as well as in Drosophila embryos (25,42,10) and they contain special HMG proteins, such as HMG-D in Drosophila (43), or linker histone variants, such as B4 in Xenopus (44). The use of postblastodermic Drosophila embryos for the generation of whole cell extracts eliminates some of these problems and makes chromatin assembly partly dependent on exogenously added core histones (41). However, these extracts still contain considerable amounts of endogenous histones that have to be removed by binding to immobilized DNA (45). This step can remove other proteins or complexes with an affinity for DNA, which could be important for physiological chromatin assembly.
In this paper, we describe an assembly system composed of two selectable components. One of them, the histone-defective, whole-cell yeast extract, contributes the nucleosome assembly machinery, which uses the second component, the core histone octamers, to assemble uniformly spaced arrays of nucleosomes on exogenously added DNA, provided a continuous supply of ATP is available. The system is able to properly position nucleosomes over the MMTV promoter and over the URA3 gene (data not shown). Furthermore, the MMTV chromatin templates assembled in this system can be transcribed in vitro in a way that mimics the synergism between transcription factors observed in cultured cells (31,32) and with other chromatin assembly systems (22).
The use of the yeast S.cerevisiae opens the possibility to use as a source of extracts the numerous known mutant strains with specific defects in components of the chromatin assembly and remodelling machinery as well as in other components of the transcriptional apparatus. This strategy will provide extracts with selective defects that could be complemented in vitro and will be a great help for dissecting the components involved in transcriptional regulation of various chromatin templates. The use of synchronized cultures, providing biochemically homogeneous extracts enriched or depleted in the cell-cycle-dependent chromatin assembly factors, introduces a new tool of unexpected possibilities. The final goal is to use wild-type or mutant recombinant yeast core histones for the assembly reaction to reconstitute a homogenous yeast chromatin for the analysis of gene regulation in vitro.
ACKNOWLEDGEMENTS
We thank Bernhard Gross for the preparation of recombinant progesterone receptor and nuclear factor 1. The experimental work presented in this paper was supported by grants from Spanish MCYT (PM 1999-0030) and from the Deutsche Forschungsgemeinschaft.
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* To whom correspondence should be addressed. Tel: +34 93 224 09 01; Fax +34 93 224 08 99; Email: miguel.beato@crg.es
ABSTRACT
We describe a cell-free chromatin assembly system derived from the yeast Saccharomyces cerevisiae, which efficiently packages DNA into minichromosomes in a reaction dependent on exogenous core histones and an ATP-regenerating system. Both supercoiled and relaxed plasmid DNA serve as templates for nucleosomal loading in a gradual process that takes at least 6 h for completion at 30°C. Micrococcal nuclease digestion of the assembled minichromosomes displays an extended nucleosomal ladder with a repeat length of 165 bp. The purified minichromosomes contain the four core histones in stoichiometric proportion and exhibit phased nucleosomes over the mouse mammary tumour virus (MMTV) promoter. The progesterone receptor and NF1 synergize on these minichromosomes resulting in efficient cell-free transcription. The ease of manipulation and the potential use of yeast strains carrying mutations in the chromatin handling machinery make this system suitable for detailed mechanistic studies.
INTRODUCTION
The initial level of DNA packaging in the nucleus of eukaryotic cells is a string of repeating units, the nucleosomes, whose subunit structure is common to all eukaryotes and contains 146 bp of left-handed DNA wrapped around a core protein composed by two copies of each of the four histones H3, H4, H2A and H2B. Over the last decade, knowledge of the structural and functional role of chromatin has grown significantly. The nucleosomes, first considered mere packaging structures, are now regarded as essential and active elements in the regulation of all processes that require access to nuclear DNA (1,2). This understanding is supported by the discovery of chromatin remodelling complexes, present in all eukaryotes cells, that participate in the response to stimuli requiring chromatin handling (3,4).
The in vitro biochemical reconstitution of chromatin with physiological nucleosome spacing and positioning is a critical tool in the elucidation of the role of chromatin structure and its chemical modifications in biological phenomena such as transcription and replication. A wide variety of methods have been developed, including those that use purified components (DNA and histones) in the presence of counterions such as NaCl, nucleoplasmin and polyglutamic acid (5–7). These purified systems generate well-defined chromatin templates, but their structure is not dynamic and therefore, these templates are generally not suitable for further functional analysis. In contrast, crude extracts from Xenopus unfertilized eggs (8), Xenopus oocytes (9) or Drosophila embryos (10), which contain a large pool of maternal histones, can assemble under physiological conditions functional minichromosomes with evenly distributed nucleosomes over any exogenously added DNA plasmid. Even though those systems are useful and well scaled-up, the reproducible generation of large amounts of competent extract is expensive in time and requires a temperature-controlled and noise-isolated room or bath for the Drosophila or Xenopus culture. Moreover, the preblastodermic Drosophila system should be made with embryos harvested in a short period of time (0 to 100 min) after egg laying; further delay renders low competent extracts. The Xenopus culture has several drawbacks as the cost of the animals and their sensitivity to thermal, seasonal and feeding changes.
To circumvent these problems, several yeast systems for chromatin assembly have been used based on purified core histones from isolated nuclei and Xenopus laevis nucleoplasmin (11), purified yeast histones and yeast nucleosome assembly protein (NAP-1) (12) or whole-cell extract (13). However, none of these procedures yield an extended regular ladder of DNA fragments after micrococcal nuclease digestion. Furthermore, the reported whole-cell extract can assemble chromatin without exogenous addition of core histones. Based on the grinding method with dry ice (13,14), we describe here a detailed study, including topological and hydrodynamic analysis, protein composition, chromatin structure, nucleosome phasing and transcription, illustrating the development of a very efficient cell-free system for nucleosome assembly with S.cerevisiae cells and isolated native core histones.
MATERIALS AND METHODS
yScS116 cell-free extract preparation
S.cerevisiae (BJ5460 strain) cells were grown overnight at 30°C in 6 l of YPD medium (Gibco) with continuous shaking (180 r.p.m.). After pelleting (in a JLA 9.1000 Beckman rotor) and removing the excess of culture medium as much as possible, cells were extruded with a 20 ml syringe (without the needle) over a Dewar glass containing liquid nitrogen, frozen as ‘noodles’ and stored at –80 °C. Currently, 20–30 g of frozen cells can be obtained from 6 l of a saturated yeast culture. Cell-free extract was made as follows: pellets of dry ice of the best quality were powdered with a home coffee mill; an equal weight of frozen S.cerevisiae noodles was added over the powdered dry ice and then grinded for 2–5 min at maximum speed. Avoiding defrost, the frozen mix was quickly added with a spatula to a plastic tube. One volume of low ionic strength extraction buffer (20 mM HEPES–KOH, pH 7.5. 5 mM KCl, 2.5 mM Mg2Cl, 1 mM EDTA, 0.5 mM dithiothreitol, 10 mM ?-glycerophosphate, 10% glycerol, 10 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin) was added and the mixture thawed without capping to prevent the tube explosion. After CO2 evaporation, the tube was capped and the mixture vortexed for 5 min at room temperature. The cell homogenate was pipetted until it filled completely the 5.2 ml Beckman SW55 tubes and then centrifuged at 35 000 r.p.m. (116 000 g) for 2 h at 4°C. Avoiding the thin lipid layer, the clear supernatant was carefully recovered and aliquots (200–400 μl) were stored at –80 °C. We named our extract as yScS116 (yeast Saccharomyces cerevisiae Supernatant 116 000 g).
Alternatively, we also prepare a very competent extract using commercial dry yeast (Algist Bruggeman N.V., Gent, Belgium). In brief, 8 g of dry cells were grinded in dry ice for 1–2 min, dissolved in 24 ml of extraction buffer and processed as described above.
Histone purification
Native core histones were purified from isolated nuclei from mouse, rat or chicken fresh liver over a hydroxyl apatite (BioGel HTP cDNA grade; Bio-Rad) column (1.5 x 20 cm) (15). H1 and HMG were first eluted at 0.63 M NaCl, 100 mM potassium phosphate, pH 6.7. Core histones were subsequently eluted as octamers with 2 M NaCl, 100 mM potassium phosphate, pH 6.7. Peaks containing stoichiometric amounts of histones were pooled and dialysed overnight in the cold room against 2 l of 0.2 M NaCl, 20 mM Tris, pH 7.5, 0.1 mM EDTA, 20 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin and 2 μg/ml pepstatin. After measuring the protein concentration, histones were aliquoted and stored at –80 °C.
In vitro chromatin assembly
Chromatin assembly was monitored following the supercoiling of a topoisomerase-I-relaxed plasmid. Supercoiled pUC18 (100 ng) was incubated with 0.1 U of calf thymus topoisomerase I (GIBCO) at 37°C for 30 min in a final volume of 10 μl. Meanwhile, the chromatin assembly reaction was made by mixing 20 μl of yeast extract with all the reagents in extraction buffer until the following final concentrations: 500–800 ng of purified core histones (histones/DNA mass ratio = 5 to 8), 20 mM HEPES–KOH, pH 7.5, 5 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol, 10 mM ?-glycerophosphate; 10% glycerol, 40 mM disodium creatin phosphate, 3 mM ATP, 1 ng/μl creatin phosphokinase, 10 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin and 3 mM Mg2Cl in a final volume of 34 μl. As a critical step and to prevent the formation of insoluble histone/DNA complexes, the mixture was well mixed prior to the addition of the relaxing reaction containing the plasmid and then incubated at 30°C for 6–14 h. For the supercoiling assay, reactions were stopped by adding SDS and EDTA of 0.1% and 20 mM, respectively, and the DNA was deproteinized with 1 mg of proteinase K per millilitre at 37°C for 2 h followed by phenol extraction. After pelleting and washing, the DNA was dissolved in a few microlitres of 20 μg/ml DNase-free RNaseA and loaded onto a 1% agarose gel in 1x TBE. After electrophoresis, the gel was stained with ethidium bromide. Chromatin assembly reactions with extracts obtained from commercial dry cells were performed as described before but the amount of extract added was 10% of the final volume of the assembly reaction.
Two-dimensional gel electrophoresis of topoisomers
Minichromosomes were deproteinized as described and two-dimensional electrophoresis (16) was performed in a 20 x 20 cm 1% agarose 1x TBE gel. The first dimension was run at 60 V for 16 h and then the gel was shifted 90°, equilibrated for 8 h in the dark in 1x TBE containing 4 μM chloroquine (Sigma) from a fresh stock solution (10 mg/ml) and electrophoresed in the second dimension at 60 V for 16 h. Chloroquine was extensively removed from the gel with several changes in distilled water and the gel was stained with ethidium bromide and destained with distilled water.
The reference topoisomer ladder was prepared by relaxing negative supercoiled pUC18 plasmid with topoisomerase I in the presence of different amounts of ethidium bromide (17). Both the reference relaxed DNA (L = 0) and the topoisomer ladder were prepared by plasmid relaxation with topoisomerase I at 30°C for 6 h in a reaction mixture containing 20 mM HEPES–KOH, pH 7.5, 5 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol; 10 mM ?-glycerophosphate; 10% glycerol, 40 mM disodium creatin phosphate, 3 mM ATP, 1 ng/μl creatin phosphokinase, 10 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin and 3 mM Mg2Cl; in the reference relaxed DNA, the most abundant topoisomer was considered to have a linking difference (L) = 0.
Micrococcal nuclease digestion
After chromatin assembly, samples were cooled at room temperature and CaCl2 (final concentration of 3 mM) and MNase (0.1–0.3 Boehringer units per microlitre of assembly mixture) were added and incubated at room temperature. At various time intervals (see figure legends), the digestions were stopped with EDTA (50 mM final concentration) and samples deproteinized as in the supercoiling assay or with Wizard Purification Systems (Promega) following the manufacturer's instructions. After DNA precipitation and ethanol washing, the samples were resuspended in 20 μg/ml RNase A, electrophoresed in 1.5% agarose in Tris–glycine buffer at 90–120 V, stained with ethidium bromide and destained in distilled water.
Isolation of minichromosomes
A linear 15–30% sucrose gradients of 11 ml in 100 mM NaCl, 5 mM HEPES–KOH, pH 7.5, 0.2 mM EDTA, 10 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin were made in 12 ml Beckman SW40 tubes. Chromatin assembly reactions were layered on top of the gradient and centrifuged (with the slowest acceleration and without brake at the end of the spin) at 22 000 r.p.m. for 16 h at 4°C in a Beckman-Coulter Optima XL-100K Ultracentrifuge. Fractions (0.8 ml) were collected and one-twentieth of each was deproteinized as described and the DNA electrophoresed in a 1% agarose gel to determine the position of the minichromosome in the gradient. Fractions containing minichromosomes were pooled and a portion was digested by the addition of CaCl2 (3 mM, final concentration) at room temperature for 5 min with 0.03 and 0.06 Boehringer units per microlitre of MNase. The reactions were stopped and processed as described.
Protein analysis
The pooled fractions from the gradient containing the minichromosomes, or the corresponding in the mock-incubated (assembly without plasmid) control, were layered over a 1 ml cushion of 30% sucrose in gradient buffer containing 10 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin, covered with gradient buffer in a 4.2 ml Beckman SW60 tube and centrifuged at 30 000 r.p.m. for 14–16 h at 4°C. The supernatant was very carefully removed and the pellet was dissolved in 1x L?mmli buffer (19) at 90°C and electrophoresed in 15% SDS–PAGE. The gel was stained with silver (20).
Analysis of phased nucleosomes
pMMTV plasmid (30 μg) was digested with BglII and the 5' phosphate removed with alkaline phosphatase. After phenol extraction and DNA pelleting, 10 μl of ATP (3000 Ci/mmol, Amersham Pharmacia biotech) were mixed with 20 U of T4 polynucleotide kinase (Roche) in a final volume of 50 μl and incubated for 30 min at 37°C. The BglII-radiolabelled DNA was then diluted at a final concentration of 0.5 μg/ml in ligation buffer and incubated overnight at 4°C with 30 U of T4 ligase (Roche). This dilution promotes the ligation of intramolecular extremes and prevents the ligation of the plasmid as dimers or higher oligomers. The relaxed, covalently closed plasmid DNA was concentrated carefully with n-butanol, phenol extracted and purified, as described (21). Minichromosomes assembled over the BglII-radiolabeled pMMTV-CAT-BB vector were digested with MNase as described. After DNA pelleting, samples were dissolved, digested with 10 U of XhoI (located at 10 bp of the 3' extreme of the BglII site), ethanol precipitated, dissolved and electrophoresed in 1.5% agarose–Tris–glycine gel. As the free-DNA control, an equal amount of pMMTV-CAT-BB DNA was first digested with 0.0005 Boehringer units per microlitre of MNase and then with XhoI. After electrophoresis, the gel was blotted, UV-crosslinked and exposed.
Transcription assays
Assembled pMMTV minichromosomes were transcribed for 1 h at 30°C with HeLa nuclear extract, recombinant PR and NF1 (from baculovirus-infected Sf9 cells) as described (22).
RESULTS
Our initial attempts to reproduce the supercoiling assay with the previously reported yeast whole-cell-free extract (13) were unsuccessful. We modified the ionic conditions and centrifugation parameters and monitored the ability of the whole extract to assemble nucleosomes by supercoiling analysis of relaxed pUC18. When DNA wraps a nucleosome core, the superhelical density of the covalently closed plasmid DNA increases by one (5). The topoisomerase activity of the extract relaxes this linking difference and, after deproteinization of the reconstituted plasmid, one negative superhelical turn is acquired for each nucleosome introduced (17). Figure 1A shows the supercoiling titration of relaxed pUC18 (lane 1) incubated with yScS116 and increasing amounts of the purified core histones shown in Figure 1B. Importantly, little increase in supercoiling was observed when the incubation was performed with the extract alone (lane 2) suggesting that the yScS116 did not contain a sufficient level of endogenous free histones to assemble nucleosomes onto an exogenously added plasmid. Furthermore, micrococcal nuclease digestion of yScS116 completely failed to detect any endogenous nucleosomal ladder (data not shown). The supercoiling level increased proportionally with the amount of added core histones, reaching its maximum value at a histone/DNA ratio of 6 to 7. The requirement of such a high proportion of histone probably results from the high content of RNA in yeast, which squelches added histones. At a histones/DNA ratio of 7 (lane 6), the minichromosome reached a topological density similar to that of the bacterial supercoiled pUC18 (lane 7). Thus, the yScS116 can efficiently assemble chromatin when supplemented with appropriate amounts of core histones.
Figure 1. Supercoiling assay of core histones-dependent chromatin assembly. (A) Nucleosome assembly with increasing amounts of added core histones. Relaxed pUC18, 75 ng, (R) was incubated with yScS116 and the indicated amounts of purified core histones for 6 h at 30°C. SC indicates the supercoiled pUC18. Migration of relaxed (II) and supercoiled (I) DNA forms are indicated. (B) A 15% SDS–PAGE profile of native core histones purified from mouse liver. (C). An ATP-regenerating system is essential for chromatin assembly. Relaxed pUC18 of 100 ng (lane 1) was assembled in yScS116 with or without ATP (3 mM), creatin phosphokinase (1 ng/μl)/disodium creatin phosphate (40 mM) (CPK/CP), and apyrase (U/ml), as indicated on top. Other symbols are as in (A).
Dynamic chromatin assembly in cell-free extracts is dependent on the concentration of ATP, Mg2+ and KCl (9,10,23,24). The yScS116 contains a sufficient level of endogenous ATP to generate a certain degree of supercoiling without exogenous addition of ATP (Figure 1C, lane 9). However, as other crude chromatin assembly extracts, the yScS116 requires a constant level of ATP to assemble nucleosomes efficiently (25). In the absence of creatin phosphokinase and creatin phosphate, addition of ATP generated a poor supercoiling (Figure 1C, lane 3) that was further reduced in the presence of apyrase (an enzyme which specifically degrades ATP) (Figure 1C, lanes 4 to 8). Therefore, the chromatin assembly in the yScS116 is a dynamic process that requires hydrolysis of a constant level of ATP.
Chromatin assembly with the yScS116 is a gradual process that takes from 6 to 14 h for completion. As shown in Figure 2A, the endogenous topoisomerases relaxed the negative supercoiled plasmid during the first hour of incubation (lanes 2 to 4). Thereafter, the DNA became more supercoiled in a time-dependent manner. After 14 h of assembly, the supercoiled level of the deproteinized minichromosome and a bacterial negative supercoiled pUC18 were very similar (lane 11). The negative nature of this supercoiling was demonstrated by electrophoresis in two dimensions, the second in the presence of chloroquine (Figure 2B). The deproteinized minichromosome displayed an average linking difference (L) of –14/–16, corresponding to a minichromosome fully loaded with nucleosomes spaced at 160/170 bp. To address whether the assembled nucleosomes were evenly spaced and to determine spacing by a different method, the minichromosomes were digested for different times with micrococcal nuclease and the resulting fragments electrophoresed in an agarose gel. A periodical ladder of oligonucleosome fragments can be distinguished and no subnucleosomal size fragments were detected (Figure 2C). The extended nature of the MNase ladder of oligonucleosomes was revealed by the accumulation of the shortest fragments at longer digestion times (compare band intensities in lanes 4, 5 and 6 in Figure 2C). The electrophoretic migration of the mono, di, tri, tetra and pentanucleosome bands yielded a repeat length average of 165 bp. This spacing is in the range displayed by the native chromatin of S.cerevisiae (26–28) and corresponds to the values determined by topoisomer analysis. Thus, the average minichromosomes are fully loaded with properly spaced nucleosomes.
Figure 2. Topological and enzymatic characterization of the minichromosomes. (A) Time course of chromatin assembly. Supercoiled pMMTV plasmid (1.5 μg) was assembled under standard conditions. At the indicated times, aliquots containing 100 ng of DNA were removed and processed. Samples were co-electrophoresed with topoisomerase-I-relaxed plasmid (lane 1) in a 1% agarose 1x TBE gel at 60 V for 14 h and then stained with ethidium bromide. (B) Two-dimensional electrophoresis. Assembled minichromosome (100 ng) was deproteinized and co-electrophoresed with the standard DNA topoisomer ladder (reference pattern) and the relaxed DNA. To obtain a good matching, the deproteinized minichromosome was first loaded, electrophoresed for 90 min and then, the standard ladder was loaded in the same well. The relaxed DNA was loaded at the same time as the standard but several wells to the right. The gel was electrophoresed and processed as described in Materials and methods. The linking-number change in the minichromosomes was determined by counting in the topoisomer ladder the number of spots from the relaxed DNA (0). Np, Nm and Nr are the nicked form of the reference pattern, minichromosomes and relaxed DNA, respectively. (C) Micrococcal nuclease digestion of assembled minichromosomes. Relaxed pUC18, 1.5 μg (as in lane 1), was incubated with yScS116 and added core histones under standard conditions. After 6 h, 75 ng was removed and deproteinized for topological analysis (lane 3). To the remaining mixture, CaCl2 and MNase were added and incubation was continued at room temperature. At different times, one-third of the digestion mixture was removed and the digestion stopped as described. Lanes 2 and 7, 75 ng of supercoiled DNA and DNA molecular weight marker, respectively. I, II and III indicate the supercoiled, relaxed and linear form of pUC18, respectively. The positions of the mono-, di-, tri-, tetra- and pentanucleosome bands are indicated (arrows).
The protein composition of the assembled minichromosome was analysed after centrifugation through an isotonic linear sucrose gradient, which excludes aggregation and yields purified minichromosomes (25). Figure 3A displays the position of minichromosomes in a 15–30% sucrose gradient (fractions 9 and 10), which yielded the supercoiled form of pMMTV-CAT-BB DNA after deproteinization. The isolated minichromosome generated an MNase digestion ladder (Figure 3B) with the same nucleosomal spacing as the one found in the whole extract (compare the migration of the trinucleosome in Figures 3B and 2C). It should be noted that the sucrose fractionation removed impurities from the extract and a much lower amount of MNase (about one-tenth) was necessary to digest the purified minichromosome to the same extent as with the crude extract. The purified minichromosomes contained comparable amounts of all four nucleosomal core histones as well as lower amounts of additional bands of higher molecular weight corresponding to non-histone proteins present in the extract (Figure 3C).
Figure 3. Purification of the assembled minichromosomes. (A) Minichromosomes assembled under standard conditions on pMMTV-CAT-BB plasmids were loaded in a linear 15–30% sucrose gradient. After the centrifugation and fractionation, one-twentieth of each fraction was deproteinized, purified and electrophoresed. Lanes 3 to 11 contain the corresponding gradient fractions. Lanes 25, 50 and 100 contain 25, 50 and 100 ng of supercoiled pMMTV-CAT-BB plasmid, respectively. The relative position of fractions containing the minichromosome is shown on top. (B) MNase digestion of the gradient-purified minichromosomes. CaCl2 (3 mM, final concentration) and MNase (final concentration in Boehringer units per microlitre on top) were added to one half of the isolated minichromosomes and the mixture was incubated at room temperature for 5 min. After deproteinization, samples were electrophoresed in a 1.5% agarose–Tris–glycine gel. (C) Protein composition of the assembled minichromosomes. Fractions 9 and 10 (asterisks on Figure 4A) were pooled and one half was processed for protein analysis as described in Materials and methods. Mock: assembly performed without added plasmid. pMMTV: minichromosomes assembled over the pMMTV-CAT-BB plasmid. Histones: purified core histones from mouse liver used for chromatin assembly.
Figure 4. Phasing of the nucleosomes over the MMTV promoter and transcription assay. (A) Relaxed closed circular pMMTV-CAT-BB plasmid (1 μg) labelled with 32P at the Bgl II site and circularized was assembled in minichromosomes under standard conditions. CaCl2 (3 mM, final concentration) and MNase (0.1 Boehringer units per microlitre of mixture) were added. At 0.5, 2, 3 and 5 min, one-fourth of the mixture was removed and processed. After deproteinization, each sample was digested at 37°C with 10 U of XhoI, phenol-extracted, pelleted and washed. The naked plasmid was digested at room temperature in 1x extraction buffer (containing 3 mM CaCl2) with 0.0001, 0.00015, 0.0002 and 0.00025 Boehringer units of MNase per microlitre of mixture for 1 min and processed as in chromatin. Samples were electrophoresed at 120 V in a 1.5% agarose–Tris–glycine gel. The gel was blotted, UV-crosslinked and exposed to an X-ray film. Lanes 1 to 4 and 5 to 8 are the naked plasmid and chromatin samples, respectively. Black and grey symbols indicate the frame of both populations. Arrows on the right indicate the major MNase cuts. Fragment size in base pairs are indicated. The deduced estimated positions of nucleosomes in both phases are depicted in the diagram on right; the negative numbers indicate the distance from the start of transcription (+1). The NF1 binding site and the SacI cleavage site are indicated. (B) Transcription on nucleosomal templates. Where indicated, recombinant PR and NF1 (from baculovirus-infected Sf9 cells) were added to the assembled pMMTV minichromosomes and transcribed with HeLa nuclear extract (22). The position of the MMTV transcripts is indicated by an arrow on the right.
The MMTV promoter, located in the long terminal repeat (LTR) of the provirus is organized in positioned nucleosomes in mammalian and yeast cells (29–32) as well as in chromatin assembled in Drosophila embryo extracts (18,22). The so-called nucleosome B is positioned over the hormone responsive region (HRR) containing several binding sites for hormone receptors and a binding site for nuclear factor 1 (NF1). To elucidate whether the yScS116 can assemble phased nucleosomes, we used a low-resolution analysis of chromatin structure on minichromosomes assembled over a closed circular plasmid that contains the pMMTV promoter, radioactively labelled at the unique restriction site BglII (21). After digestion with MNase and subsequent cleavage with XhoI, which cleaves 10 bp 3' of the label, two ladders of periodically spaced chromatin-enhanced cleavage sites separated by 40 ± 10 bp can be observed over a background cleavage pattern similar to that of free DNA due to DNA bending and the sequence preference of MN (Figure 4A lanes 1 to 8). One of them (phase 1) corresponds to the previously reported translational phase with the NF1 binding site included within nucleosome B (31). Furthermore, the strong MNase sensitivity at the 3' border of the nucleosome B sequence (Figure 4A) is in good agreement with previous in vitro (18) and in vivo (31) structural analysis and documents the ability of the yScS116 to interpret the positioning information encompassed in the MMTV nucleotide sequence around the HRR. The second translational phase (phase 2) closely corresponds to previously reported positions (33,34). The less precise positioning of nucleosomes in the yeast (this report) and Drosophila (18) assembly system compared with that observed in metazoan cells may be due to the lack of linker histones, which have been shown to improve translational nucleosome positioning over the MMTV promoter (35).
Correct organization of the MMTV promoter in nucleosomes is essential for the synergistic transcriptional activation mediated by the hormone receptor and NF1 (22,31). Therefore the demonstration of a functional synergism between these two transcription factors is an indirect proof of a well-organized nucleosomal structure. To address the template capacity of yScS116-assembled minichromosomes, baculovirus-expressed recombinant progesterone receptor (PR) and NF1 were incubated with assembled minichromosomes and transcription was assayed in a HeLa cell nuclear extract (22). Addition of PR alone or NF1 alone was not sufficient for transcriptional activation of the MMTV promoter (Figure 4B, lanes 2 and 3). However, both factors added together synergistically activated transcription of the chromatin templates (Figure 4B, lane 4), suggesting that the extract also contains the remodelling machinery necessary for mediating this synergism. Therefore, the yScS116 cell-free system can assemble functional chromatin templates and can catalyse the synergistic transcriptional activation mediated by PR and NF1, that has been observed in vivo (31) and in vitro (22).
DISCUSSION
Based on a reported method (13,14), we have developed a two-component in vitro chromatin assembly system involving a whole-cell-free extract from wild-type Saccharomyces cerevisiae cells, yScS116, and exogenously added native core histones. MNase digestion of the yScS116 failed to detect endogenous chromatin. In the absence of added histones, yScS116 was ineffective in assembling nucleosomes under any condition tested, suggesting a lack of functional free histones. The addition of sufficient amounts of core histones promoted the ordered and evenly distributed deposition of nucleosomes over a covalently closed DNA ring. As observed in other chromatin assembly systems (10,25), yScS116 can assemble a fully loaded minichromosome when the reaction was performed at a constant concentration of ATP, maintained by an ATP-regenerating system. We suggest that the extended centrifugation at 116 000 g, in combination with the addition of MgCl2, minimizes the amount of endogenous chromatin in the final extract. It is noteworthy that a slightly higher centrifugal force (130 000 g) yielded extracts very inefficient in nucleosome assembly even in the presence of added core histones, probably due to pelleting of factors or complexes critical for the assembly process.
Recently, a yeast-derived improved chromatin assembly system dependent on exogenously added histones has been published (36). However, this system differs from the one reported in this paper in three major points: (i) the generation of the extract is much more complicated involving preparation of spheroplasts or crude nuclei; (ii) it requires high salt extraction and DEAE chromatography of the supernatant with a salt elution step; (iii) the assembled chromatin is not characterized with respect to either nucleosome positioning or transcriptional competence.
Till date, only two whole-cell-free systems can efficiently assemble native-like chromatin under physiological conditions. One uses the chromatin assembly machinery and the maternal-inherited pool of histones contained in cleared supernatants of dispersed Xenopus unfertilized eggs (8) or oocytes (25,37); the other is an extension of the Xenopus-based system employing preblastodermic Drosophila embryos (10). Cell-free systems obtained from mammalian tissue culture cells (38,39) are much less efficient in assembling chromatin. The Xenopus and Drosophila systems are reproducible, yield large quantities of competent extracts able to promote chromatin assembly either onto double-stranded DNA or, coupled with DNA synthesis, single-stranded DNA (24) and are prepared at very similar low-ionic and centrifugal (150 000 g) conditions. They can be defined as a single-component system as they rely entirely on the maternal-inherited histones, endogenous chaperones and chromatin assembly machinery. Nevertheless, they have several drawbacks; the activity of Xenopus extracts are sensitive to uncontrolled seasonal and feeding variations (40), and the Drosophila extract is difficult to handle because the embryos should be harvested very quickly after laying; a little delay renders the extracts less competent (41).
Although those single-component systems are very useful, the fact that all the factors (chromatin assembly machinery and histones) are present in the whole extract makes difficult an easy biochemical analysis of their particular contribution to the global assembly process. Furthermore, histone H2A is extensively modified in Xenopus oocytes and eggs as well as in Drosophila embryos (25,42,10) and they contain special HMG proteins, such as HMG-D in Drosophila (43), or linker histone variants, such as B4 in Xenopus (44). The use of postblastodermic Drosophila embryos for the generation of whole cell extracts eliminates some of these problems and makes chromatin assembly partly dependent on exogenously added core histones (41). However, these extracts still contain considerable amounts of endogenous histones that have to be removed by binding to immobilized DNA (45). This step can remove other proteins or complexes with an affinity for DNA, which could be important for physiological chromatin assembly.
In this paper, we describe an assembly system composed of two selectable components. One of them, the histone-defective, whole-cell yeast extract, contributes the nucleosome assembly machinery, which uses the second component, the core histone octamers, to assemble uniformly spaced arrays of nucleosomes on exogenously added DNA, provided a continuous supply of ATP is available. The system is able to properly position nucleosomes over the MMTV promoter and over the URA3 gene (data not shown). Furthermore, the MMTV chromatin templates assembled in this system can be transcribed in vitro in a way that mimics the synergism between transcription factors observed in cultured cells (31,32) and with other chromatin assembly systems (22).
The use of the yeast S.cerevisiae opens the possibility to use as a source of extracts the numerous known mutant strains with specific defects in components of the chromatin assembly and remodelling machinery as well as in other components of the transcriptional apparatus. This strategy will provide extracts with selective defects that could be complemented in vitro and will be a great help for dissecting the components involved in transcriptional regulation of various chromatin templates. The use of synchronized cultures, providing biochemically homogeneous extracts enriched or depleted in the cell-cycle-dependent chromatin assembly factors, introduces a new tool of unexpected possibilities. The final goal is to use wild-type or mutant recombinant yeast core histones for the assembly reaction to reconstitute a homogenous yeast chromatin for the analysis of gene regulation in vitro.
ACKNOWLEDGEMENTS
We thank Bernhard Gross for the preparation of recombinant progesterone receptor and nuclear factor 1. The experimental work presented in this paper was supported by grants from Spanish MCYT (PM 1999-0030) and from the Deutsche Forschungsgemeinschaft.
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