| Nucleic Acids Research | Article |
Rapid purification of intact minichromosomes over a glycerol cushion
Introduction
Materials And Methods
Results And Discussion
Acknowledgements
References
Rapid purification of intact minichromosomes over a glycerol cushion
Received April 12, 1999; Revised and Accepted July 1, 1999
ABSTRACT Negatively supercoiled plasmids can be assembled into dynamic minichromosomes using Drosophila embryo extract as a source of histones and chromatin assembly factors. However, analysis of such minichromosomes is often difficult due to the presence in the crude extract of a large excess of macromolecules and low molecular weight molecules including ATP. Several techniques have been used to partially purify the minichromosomes based on either sizing columns or centrifugation on sucrose gradients. We have developed a single-step method employing a 30 min ultracentrifugation through a glycerol cushion. In contrast to chromatin purified in sucrose-containing buffers, the minichromosomes obtained with this method are suitable for transcriptional analysis. This method is fast, quantitative, flexible, can deal with several samples simultaneously and leads to concentration of the chromatin. As centrifugation through glycerol yields chromatin free of ATP and several characterized chromatin remodeling complexes, this method should be useful for structural and functional studies in vitro.
INTRODUCTION
To purify minichromosomes assembled in Drosophila embryo extracts we decided to use ultracentrifugation through dense solutions rather than filtration or spinning through sizing columns, as centrifugation is more flexible in terms of volume and buffer conditions and permits simultaneous processing of several samples under reproducible conditions. Centrifugation through sucrose-containing solutions yielded structurally unaltered chromatin, but these minichromosomes were not suitable for cell-free transcription since a trace amount of sucrose inhibited the transcriptional activity of the HeLa cell extract (see below). Here we report a simple and quantitative method for the purification of small quantities of minichromosomes from the excess of nuclear proteins, including various chromatin remodeling complexes.
MATERIALS AND METHODS
Standard chromatin assembly reactions were performed essentially as described (1). Briefly, 750 ng of plasmid DNA [termed MMTV-77, containing the MMTV promoter truncated upstream of the Nuclear Factor 1 (NF1) site, (2)] were incubated for 6 h at 26°C in the presence of 40 µl of Drosophila preblastoderm embryo extract, 80 µl of EX buffer-82 (82 mM KCl, 10 mM HEPES/KOH, pH 7.6, 1.5 mM MgCl2 and 0.5 mM EGTA), and 13.3 µl of an ATP regenerating system (30 mM creatin phosphate, 3 mM MgCl2, 3 mM ATP, 1 µg/µl creatin phosphokinase, 1 mM DTT). The salt concentration under these standard conditions is 110 mM.
An aliquot of 60 µl of EX buffer-110 (110 mM KCl, 10 mM HEPES/KOH, pH 7.6, 1.5 mM MgCl2 and 0.5 mM EGTA) containing 47% glycerol (vol/vol) was added to the bottom of a Beckman polycarbonate tube (7 × 20 mm), and 30 µl of EX buffer-110 containing 30% glycerol (vol/vol) was gently layered on top of this. An aliquot of 80 µl of the assembly reaction was then diluted with an equal volume of EX buffer-110 and loaded onto the glycerol cushion. After 30 min ultracentrifugation in a Beckman-TLS rotor (55 000 r.p.m., 26°C), the volume consisting of the top and middle layers, as well as the adjacent 10 µl of the bottom layers, was removed. Removal of the upper 10 µl of the bottom layer reproducibly avoided cross-contamination with large chromatin remodeling complexes, which accumulate at the interface between the 30 and 47% layers. The remaining 50 µl of the 47% were recovered and analyzed. If a higher concentration of chromatin (>5 ng/µl) is required for further experiments, a smaller volume for the bottom cushion can be used. For comparison, we used the same purification scheme with EX buffer-110 containing 20 and 10% sucrose instead of 47 and 30% glycerol.
RESULTS AND DISCUSSION
Partial MNase digestion (3) of the glycerol cushion-purified minichromosomes produced a nucleosome ladder spaced by 185 bp and indistinguishable from that of minichromosomes in the crude extract (Fig. 1A). No traces of DNA (either naked or minichromosomes) were found in the top layer fractions, while >85% was recovered in the 50 µl of the 47% glycerol cushion. Protein concentration analysis indicated that >75% of total protein remained in the upper fraction, while 16% was found associated with the minichromosomes (bottom fraction). Western blot analyses demonstrated that neither Brm (4), Brg (5), Gcn5 (6) nor p/CAF (7), and only 15% of the ISWI-containing remodeling complexes [NURF (8), CHRAC (9), ACF (10)] are present in the purified minichromosome fraction (Fig. 1B). This can be lowered to <5% of the input concentration by treating the minichromosomes for 5 min with 0.05% sarcosyl, which is known to inactivate ISWI containing remodeling machines (8). Thus, the glycerol centrifugation method might be useful for the identification and in vitro characterization of chromatin remodeling machineries involved in promoter activation.
Figure 1. (A) Micrococcal nuclease digestion. Chromatin assembled on MMTV-77 DNA was digested with 5 U/ml of MNase for 1 or 3 min, prior to (C, lanes 1 and 2) or after ultracentrifugation (P, lanes 3 and 4). The resulting DNA fragments were separated on a 1.3% agarose gel in 1× TBE buffer and visualized by ethidium bromide staining. Spacing between nucleosomes was evaluated by comparison with the marker (lane 5). The average size of the cleavage products is 185 bp. (B) Western blot analysis of crude assembly reaction (C) and purified minichromosomes (P). Proteins were separated on an 8% SDS gel and transferred to nylon membrane. After incubation with Brg-1 antibodies (top panel), h-BRG1 antibodies (middle panel) or ISWI antibodies (bottom panel), bound antibodies reaction was visualized using ECL kit (Amersham). (C) Binding of NF1 to naked DNA and minichromosomes assayed by DNase I footprinting. Autoradiograms of the sequencing gels illustrate the binding of NF1 to naked MMTV-77 promoter DNA (lanes 1-3), to MMTV-77 minichromosomes prior to (lanes 4-6) or after ultracentrifugation (lanes 7-9), or to the purified minichromosomes containing the WT-MMTV promoter (3) (lanes 10 and 11). Plasmid DNA or assembled chromatin were incubated for 30 min in buffer containing 110 mM NaCl with or without NF1 (90 nM). After incubation the samples were treated with DNase I and analyzed by linear PCR with [alpha]-32P-labeled primer A25 complementary to the region between +25 and +50 of the MMTV-LTR as described (3). (D) Effect of NF1 on transcription from minichromosomes purified through glycerol or sucrose cushion. The chromatin templates were incubated with either buffer or NF1 (90 nM) for 30 min at 25°C. 25 ng of pMMTV-77 minichromosomes were then transcribed for 1 h at 30°C with HeLa nuclear extract (12) in a 54 µl reaction containing 63 mM KCl and 1 mM MgCl2. The conditions of transcription and primer extension were as described (13).
The ability of NF1 to bind the purified minichromosomes was analyzed and compared with the starting material. DNase I footprinting experiments (3) showed no significant differences between the two sets of samples, suggesting that purification through glycerol does not interfere with chromatin structure nor with its analysis by DNase I footprinting (Fig. 1C). NF1 binds with equal affinity to the -77 minichromosomes before and after purification through the glycerol gradient. This affinity is much lower than the affinity of NF1 for naked DNA (lanes 1-3).
A more direct indication of the unaltered structure of the MMTV chromatin after purification was obtained with plasmids containing the wild-type MMTV promoter (WT-MMTV) assembled in minichromosomes (3). Our previous experiments have shown that the NF1 binding site in WT-MMTV minichromosomes is inaccessible for NF1, due to the positioning of the nucleosome over the promoter region (3,11). After purification of these minichromosomes, NF1 is still unable to access its binding site (Fig. 1C, lane 10 versus lane 11), suggesting that nucleosome structure is preserved during purification. Nevertheless, addition of Drosophila embryo extract along with recombinant progesterone receptor allowed the stable binding of NF1 (manuscript in preparation). Thus the purified minichromosomes have preserved the capacity to undergo the structural transitions needed for synergistic binding of hormone receptors and NF1.
Transcriptional analysis of the purified minichromosomes using HeLa nuclear extract revealed important differences between those purified on glycerol and those passed through a sucrose cushion (Fig. 1D). With -77 minichromosomes purified through glycerol, we observed an appreciable basal level of transcription, which was increased by the addition of recombinant NF1. Surprisingly, even upon addition of NF1, no transcription signal was obtained using the chromatin purified through sucrose. Addition of sucrose at concentrations higher than 2% inhibited transcription driven by naked DNA templates (data not shown), precluding the use of a sucrose cushion for purification of chromatin templates to be use for cell-free transcription. Thus, the glycerol centrifugation method provides a useful tool for obtaining purified minichromosomes suitable not only for structural analysis but also for transcription in vitro.
ACKNOWLEDGEMENTS
We thank B. Gross, IMT Marburg, for preparation of recombinant NF1; Ö. Wrange, Karolinska Institute Stockholm, for Brg-1 antibodies; C. Muchardt, Pasteur Institut Paris, for antibodies to h-Brm; Y. Nakatani, NIH Bethesda, and Tony Kouzarides, MRC Cambridge, for antibodies to p/CAF. We also thank P. B. Becker and K. Nightingale, EMBL Heidelberg, for Drosophila embryo extract and for valuable discussions. This work was supported by grants from the European Union, the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. L.D.C was fellow of the Fondazione Pasteur/Cenci-Bolognetti.
REFERENCES
*To whom correspondence should be addressed. Tel: +49 6421 28 6286; Fax: +49 6421 28 5398; Email: beato{at}imt.uni-marburg.de
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