Involvement of Cytoplasmic Soluble Fraction in the Assembly of Nucleosome-Like Materials under Near Physiological Conditions

Abstract

The involvement of cytoplasmic soluble fraction in the assembly of nucleosomes was investigated. MH-134 SC cells were pulse-labeled with [³H] lysine just after the addition of hydroxyurea. This is known to cause accumulation of newly synthesized H 3 and H 4 histones in the cytoplasmic soluble fraction (Senshu & Ohashi (1979) J. Biochem. 86, 1259-1267). The soluble fraction was fractionated by sucrose density gradient centrifugation. Gel electrophoresis of acid-soluble proteins extracted from the fractions obtained showed that newly synthesized H 3 and H 4 histones were present as complexes that sedimented faster than their tetrameric aggregates. After incubation of the soluble fraction with mononucleosome DNA, some of the histones sedimented at a rate comparable to that of native mononucleosomes. In a simplified in vitro system, ¹⁴C-labeled mononucleosome DNA and ³H-labeled histones were mixed into the cytoplasmic soluble fraction derived from normal cells under near physiological conditions. H 3 and H 4 histones added to the soluble fraction formed complexes resembling those found in hydroxyurea-treated cells as determined by sucrose density gradient analyses. After incubation with [¹⁴C] DNA, the labeled histones and DNA co-sedimented as a broad peak at the region of native mononucleosomes. They sedimented as a sharper peak when unlabeled H 2 A and H 2 B histones were included in the incubation mixture. Not only the mixture containing all four histones, but also one devoid of H 2 A and H 2 B histones yielded DNase I digestion products resembling those derived from native nucleosomes. Omission of the soluble fraction resulted in poor formation of nucleosome-like materials. When partially purified complexes of [¹⁴C] DNA-(H 3-H 4) histones were incubated with [³H] (H 2 A-H 2 B) histones, the labeled histones co-sedimented with the preformed complexes.
These data suggest a sequential mode of assembly of nucleosome-like particles in our unique in vitro system. It appears to proceed as follows: (1) formation of soluble complexes between H 3, H 4 histones and cytoplasmic soluble materials, (2) transfer of H 3, H 4 histones from the complexes to mononucleosome DNA to form primary nucleosome-like structures, (3) binding of H 2 A, H 2 B histones without any requirement for the involvement of cytoplasmic materials.