INVOLVEMENT OF HISTONE-H1 IN THE ORGANIZATION OF THE NUCLEOSOME AND OF THE SALT-DEPENDENT SUPERSTRUCTURES OF CHROMATIN

We describe the results of a systematic study, using electron microscopy, of the effects of ionic strenth on the morphology of chromatin and of H1-depleted chromatin. With increasing ionic strength chromatin folds up progressively from a filament of nucleosomes at ~1 mM monovalent salt through some intermediate higher-order helical structures with a fairly constant pitch but increasing numbers of nucleosomes per turn, until finally at 60 mM (or else in ~0.3 mM Mg++) a thick fiber of 250 ̇A diameter is formed, corresponding to a structurally well-organized but not perfectly regular superhelix or solenoid of pitch ~ 110 ̇A. The number of nucleosomes per turn of the helical structures agree well with those which can be calculated from the light-scattering data of Campbell et al. H1-depleted chromatin also condenses with increasing ionic strength but not so densely as chromatin and not into a definite structure with a well-defined fiber direction. At very low ionic strengths, nucleosomes are present in chromatin but not in H1-depleted chromatin which has the form of an unravelled filament. At somewhat higher ionic strengths (>5 mM triethanolamine chloride), nucleosomes are visible in both types of specimen but the fine details are different. In chromatin containing H1, the DNA enters and leaves the nucleosome on the same side but in chromatin depleted of H1 the entrance and exit points are much more random and more or less on opposite sides of the nucleosome. We conclude that H1 stabilizes the nucleosome and is located in the region of the exit and entry points of the DNA. This result is correlated with biochemical and x-ray crystallographic results on the internal structure of the nucleosome core to give a picture of a nucleosome in which H1 is bound to the unique region on a complete two-turn, 166 base pair particle. In the formation of higher-order structures, these regions on neighboring nucleosomes come closer together so that an H1 polymer may be formed in the center of the superhelical structures.