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 foun d in these distributions vary with the average nucleosome loading per templ ate molecule (n(av)): at n(av) < 4, the distributions show a single peak wh ose 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 co mplex; i.e., they contain multiple peaks and/or shoulders. Moreover, the pe aks/shoulders typically occur at two nucleosome intervals, i.e., 2, 4, 6 or 3, 5, 7 nucleosomes. This two-nucleosome periodicity is statistically sign ificant. The precise cause for such discrete features within the distributi ons is unknown, but at least these features would seem to indicate some pai rwise 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 pro cess would produce. This feature indicates that at these intermediate popul ation levels there is some tendency for correlated nucleosome loading among the templates. Hyperacetylated nucleosomal arrays show only subtle differe nces 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 signifi cant lability when they are not glutaraldehyde-fixed.