Abstract
Concatameric 5 S rDNA templates reconstituted in vitro into nucleosomal arrays provide very popular chromatin models for many kinds of studies. Here, atomic force microscopy is used to determine the population distributions for one such nucleosomal array, the 172-12, reconstituted to various subsaturated levels with nonacetylated or hyperacetylated HeLa histones. This array is a model for short linker length genomes and transcriptionally active and newly replicated chromatins. The analysis shows that as input histone levels increase, template occupation increases progressively as discrete population distributions. The distributions are random at low (n(av) < 4) and high (n(av) > 8) loadings but display specific nonrandom features, such as a deficit of molecules with one nucleosome more or less than the peak species in the distribution and enhanced distribution breadths, in the mid-range (n(av) = 4-8). Thus, the mid-range of occupation on polynucleosomal arrays may be a special range for chromatin structure and/or assembly. The mid-range nonrandom features are enhanced in distributions from short repeat (172-12) arrays, particularly for unacetylated chromatin, and in distributions from hyperacetylated chromatin, particularly for long repeat (208-12) arrays. Thus, short repeat length and acetylation can affect basic chromatin properties, like population tendencies, in very similar ways and therefore may cause similar changes in chromatin structure. Some possible effects are suggested. The data also indicate that it is thermodynamically more difficult for hyperacetylated nucleosomes to assemble onto the 172-12 templates, a result having implications for in vivo chromatin assembly.
Highlights
A characteristic feature of eukaryotic chromatins is their significant variability in average nucleosome repeat length, a feature that arises from variations in the average lengths of linker DNA that separate the conserved core nucleosomal particles in vivo [1, 2]
The 5 S templates were made with a choice of DNA lengths between the basic positioning sequences [8], e.g.172-12, 190-12, and 208-12, creating templates that effectively differ in average nucleosome repeat length
The 172-12 provides a model for short linker length chromatins like yeast, and the 208-12 provides a model for long linker length chromatins such as chicken erythrocyte
Summary
Plasmid and Histone Isolation—The plasmid (p5S172-12) containing the 172-12 was a generous gift of J. DNA and histone octamers were mixed together on ice at a final concentration of 2 M NaCl (plus TE (10 mM Tris-1 mM EDTA, pH 8.0)) and a final volume of 30 l. We counted molecules containing closely contacting nucleosomes so long as we could be certain, by measuring widths for example, of the number of nucleosomes present. Nav is the average number of nucleosomes present on the templates in a sample distribution. The experimental nucleosome loading distributions from AFM analysis were compared with theoretical distributions calculated, for the same average number of nucleosomes (nav), by assuming that nucleosomes bind randomly and with equal frequencies to each of the twelve 172-bp sites, regardless of the histone octamer to DNA ratio. For the theoretical (random) distributions, the fraction of DNA fragments with n nucleosomes bound (Fn) is given by Equation 3. (nav/12)n(1 Ϫ nav/12)(12 Ϫ n) is the total probability that any given arrangement of n nucleosomes will exist, and 12!(12 Ϫ n)!n! is the total number of arrangements, assuming that nucleosomes are indistinguishable
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