Disruption of Higher-Order Folding by Core Histone Acetylation Dramatically Enhances Transcription of Nucleosomal Arrays by RNA Polymerase III

Reconstitution of underacetylated, moderately acetylated, and highly acetylated 208-12 nucleosomal arrays. (A) Schematic illustration of the 208-12 DNA template used for reconstitution. The 208-12 DNA consists of 12 tandem repeats of a portion of the Lytechinus 5S rRNA gene (51). Each 5S rDNA repeat contains both a preferred nucleosome positioning site (solid box) and a TFIIIA binding site (open box). Initiation of transcription by RNA polymerase III occurs ∼90 bp upstream of the major TFIIIA binding site (+1, arrow). The termination sequence of the 5S rRNA gene was deleted during template construction (51), allowing for production of long read-through transcripts.EcoRI digestion sites are located at sequences 1 and 195 of each 5S rDNA repeat. (B) Micrococcal nuclease digestion. Underacetylated (lanes 1 to 8), highly acetylated (lanes 9 to 16), and moderately acetylated (lanes 17 to 24) reconstitutes were digested with 0.05 U of micrococcal nuclease per μg of DNA in the presence of 1 mM CaCl₂. Digestion was for 0, 0.5, 1.0, 2.5, 5, 10, 20, and 30 min (shown from left to right for each reconstitute type) at room temperature. The reactions were quenched by addition of a 1/5 volume of 5% SDS–25% glycerol–10 mM EDTA–0.3% bromophenol blue. Samples were deproteinated by incubation at 37°C for 30 min and subsequently electrophoresed for 5 h at 2 V/cm in a 1% agarose gel buffered with 40 mM Tris-acetate–1 mM EDTA (pH 8.0). Bands were visualized under UV illumination after incubation of the gel in ethidium bromide. Lambda DNA digested with BstEII (lanes M) was used for the size markers. (C) EcoRI digestion. One microgram (each) of naked 208-12 DNA (lane 1) and underacetylated, highly acetylated, and moderately acetylated nucleosomal arrays (lanes 2 to 4, respectively) was digested with 10 U of EcoRI for 60 min at room temperature in digestion buffer H (Promega). Reactions were quenched by addition of a 1/5 volume of 25% glycerol–10 mM EDTA (pH 8.0). Digestion products were electrophoresed at 4 V/cm for 3 h in a 1% agarose gel buffered with 40 mM Tris-acetate–1 mM EDTA (pH 8.0). Lambda DNA digested with BstEII (lanes M) was used for the size markers. (D) Determination of μ₀. Shown are plots of the mobilities in 0.2 to 0.5% agarose of underacetylated (○) and highly acetylated (▴) nucleosomal arrays in E buffer. The μ₀ was determined from the extrapolated gel-free mobilities as described in Materials and Methods.

Highly acetylated histones enhance transcription and disrupt folding of 208-12 nucleosomal arrays in transcription buffer. (A) In vitro transcription. Underacetylated (U) and highly acetylated (H) nucleosomal arrays were transcribed in Xenopus oocyte nuclear extracts as described in Materials and Methods. A histone-free plasmid containing one copy of the Xenopus 5S RNA gene (which produces a 120-nt transcript) was included in each reaction as an internal control. Transcription reactions were electrophoresed in a denaturing 9% polyacrylamide gel. After electrophoresis, RNA products were visualized with a Molecular Dynamics PhosphorImager.MspI-digested pBR322 was utilized as the size marker. (B) Densitometric trace of the RNA transcripts produced from the highly acetylated arrays shown in panel A. (C) Densitometric quantitation of the total amount of RNA transcripts produced from the highly acetylated nucleosomal arrays (solid bar) expressed as the fold increase over the underacetylated controls. Also shown is the fold increase previously reported by Ura et al. (58) for hyperacetylated dinucleosomes (open bar). (D) Sedimentation velocity analysis of nucleosomal array folding in transcription buffer. Highly acetylated (▴) and underacetylated (○) nucleosomal arrays were incubated in either transcription buffer or transcription buffer containing 50 mM KCl and 7 mM MgCl₂ for 1 h at room temperature. For these experiments, the nucleoside triphosphates in transcription buffer were replaced with 2 mM Na₅PPP_i to avoid interference with the absorbance optical system of the analytical ultracentrifuge as previously described (30, 31). Samples were sedimented at 18,000 rpm in an An-Ti60 rotor, and 20 boundary scans were collected. The temperature of the run was 21°C. Each boundary was divided into 20 equal fractions. The diffusion-corrected sedimentation coefficient at each boundary division was determined by the method of van Holde and Weischet (62), and the data were plotted as boundary fraction versuss _20,w to yield the integral distribution of sedimentation coefficients present in the sample. For both highly acetylated and underacetylated samples, at each boundary fraction the data are expressed as the ratios of thes _20,w in transcription buffer containing 50 mM KCl and 7 mM MgCl₂ divided by thes _20,w in transcription buffer lacking salts (s ^salts/s) to yield the salt-dependent increase in s _20,wacross the entire distribution (30, 31). Thes ^salts/s expected if no folding occurred is 1.0, while a ratio >1.0 is indicative of folding (see text).

Comparison of Mg²⁺-dependent folding of underacetylated, moderately acetylated, and highly acetylated nucleosomal arrays. (A) Illustration of the salt-dependent folding of the 208-12 nucleosomal array as elucidated by sedimentation velocity experiments (27, 48). Shown are schematic representations of array conformations whose extent of compaction would yield the indicated sedimentation coefficients. (B) Sedimentation velocity analysis of underacetylated (U), moderately acetylated (M), and highly acetylated (H), nucleosomal arrays in 2 mM Mg²⁺. Samples were incubated in TE buffer containing 2 mM free Mg²⁺ for 1 h at room temperature. Sedimentation was performed as described in the legend for panel D of Fig. 3. Shown are the sedimentation coefficient distributions obtained after analysis of the data by the method of van Holde and Weischet (62). The inset shows theR_e s of the same samples determined by quantitative agarose gel analysis in E buffer containing 2 mM free Mg²⁺ (see Materials and Methods). The indicated values represent the means ± standard deviations of 18 determinations of the R_e in 0.2 to 0.8% agarose gels (P_e ≥200 nm). At P_e ≥200 nm, the R_e s of 208-12 nucleosomal arrays is constant (20, 26). (C) Sedimentation velocity analysis of moderately acetylated (M) and highly acetylated (H) nucleosomal arrays in 3 mM Mg²⁺. Samples were incubated in TE buffer containing 3 mM free Mg²⁺ for 1 h at room temperature. Sedimentation was performed as described in the legend for panel D of Fig. 3. Shown are the sedimentation coefficient distributions obtained after analysis of the data by the method of van Holde and Weischet (62).