Evidence for nonrandom behavior in 208-12 subsaturated nucleosomal array populations analyzed by AFM.

Atomic force microscopy was used to determine the population distributions in reconstituted, subsaturated 208-12 nucleosomal arrays. The features found in these distributions vary with the average nucleosome loading per template molecule (n(av)): at n(av) < 4, the distributions show a single peak whose breadth is equal to that expected for a random loading process; at n(av) = 4-8, the distributions are broader than random distributions and are complex; i.e., they contain multiple peaks and/or shoulders. Moreover, the peaks/shoulders typically occur at two nucleosome intervals, i.e., 2, 4, 6 or 3, 5, 7 nucleosomes. This two-nucleosome periodicity is statistically significant. The precise cause for such discrete features within the distributions is unknown, but at least these features would seem to indicate some pairwise preference in nucleosome occupation at these loading levels. In these intermediate-level (n(av) = 4-8) distributions, the major peak contains a larger fraction of the total templates than a random nucleosome loading process would produce. This feature indicates that at these intermediate population levels there is some tendency for correlated nucleosome loading among the templates. Hyperacetylated nucleosomal arrays show only subtle differences in their population distributions compared to nonacetylated arrays and demonstrate the above features. AFM allows one to study unfixed chromatin arrays; we find that nucleosomes on the 208-12 template demonstrate significant lability when they are not glutaraldehyde-fixed.