Nucleosome rearrangement in vitro. 1. Two phases of salt-induced nucleosome migration in nuclei.

Abstract

We have investigated the salt- and temperature-induced rearrangement of nucleosomes in both intact and H1-depleted nuclei from human cells. In agreement with previous reports on the rearrangement of nucleosomes in isolated chromatin or chromatin fragments, we observed a decrease in the average nucleosome repeat length following incubation of nuclei at 37 degrees C in elevated salt concentrations. However, this decrease occurred in two distinct phases. First, incubation of H1-depleted nuclei at 37 degrees C for as little as 10 min in low-salt, isotonic buffer (containing 0.025 M KCl) resulted in a shift in the limiting repeat value from approximately 190 to 168 base pairs (bp). A similar shift was observed for intact nuclei incubated at 37 degrees C for 1 h in buffer containing near-physiological salt concentrations (i.e., 0.175 M KCl). This limiting repeat value was maintained in both intact and H1-depleted nuclei up to a salt concentration of 0.45 M KCl in the incubation buffer. Second, at salt concentrations of 0.625 M KCl, a limiting repeat of approximately 146 bp was obtained, and the nuclei had clearly lysed. During the first shift in repeat length, little additional exchange of nuclear proteins occurred compared to nuclei kept on ice in a low-salt buffer. This was the case even though the conditions used to monitor exchange were optimized by using a high DNA to chromatin ratio. On the other hand, a significant increase in the exchange of nuclear proteins, and formation of nucleosomes on the naked DNA, was observed during the shift in repeat length to 146 bp.(ABSTRACT TRUNCATED AT 250 WORDS)