Transcriptionally competent chromatin assembled with exogenous histones in a yeast whole cell extract

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 degrees 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.

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).

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 1× 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). N_p, N_m and N_r 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).

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 1× 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.