Abstract
Salt induces aggregation of large chromatin fragments maximally at 150-200 mM NaCl. The soluble fragments are depleted of H1 histones while the aggregated fragments are enriched. H1 histones did not equilibrate between the soluble and insoluble chromatin fractions when they were recycled through the process of salt-induced aggregation. The chromatin fragments that resisted aggregation retained more H1c subtype than they did H1 ab, correlating with previous results which showed complexes of H1c with DNA resisted salt-induced aggregation much more than complexes of DNA with other subtypes. The chromatin that was soluble at physiological concentrations of NaCl was DNase I sensitive and enriched in acetylated core histones. We conclude that H1 histone is nonuniformly distributed in chromatin in a stable pattern that probably correlates with the different degrees of condensation known to exist in vivo.
Highlights
Saltinducesaggregation of largechromatin frag- to show that the differences between H1 subtypes in their ments maximally at 150-200 mM NaCI.The soluble condensing power are expressed in chromatin as well as in fragments are depleted of H1 histownhesile the aggre- H1-DNA complexes
CaClZwas added to a final concentration of 1 mM immediately before digestion, and Chromatin exists in different degrees of condensation, at 45 units/ml of micrococcal nuclease (Worthington, 15,000 units/mg) least some of which are evident in the electron microscope. were added with rapid mixing
Lysed nuclei were centrifuged for 6 min at 3,500 X g, and the supernatantc, ontaining large nucleosome oligomers, was measured spectrophotometrically and stored a t to be the condensation of chromatin, we have considered the 0-4 "C.This material, which is soluble at low salt concentrations, is possibility that the differences in chromatincondensation might correspond to regional differences in H1 histone content, quantitatively or qualitatively
Summary
We began this study because of a n intriguing correlation between the salt-induced aggregation of chromatin and our [NaCI], M earlier observations[13] on salt-induced condensationof H1DNA complexes. Two curves and insoluble chromatin fractions obtained at various salt are presented for H1-DNAcomplexes, one for those contain- concentrations. Ing superhelical DNAand another for those containing linear T h e solubility of chromatin a t various salt concentrations. It should be re-emphasized that the low levels of H1 in the soluble fraction (SI)were not due to a release of this histone into free solution, since no significant H1 displacement was observed until the NaCl concentration exceeded 350 mM. No significant amount of soluble chromatin was produced by reincubation of insoluble fractions; the minor losses in yield of the aggregated fraction were the usual handling losses.
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