Closely Spaced Nucleosome Cores in Reconstituted Histone . DNA Complexes and Histone-H1-Depleted Chromatin

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Abstract
Digestion studies with micrococcal nuclease showed that reconstitution of complexes from DNA and a mixture of the 4 small calf thymus histones H2A, H2B, H3 and H4 leads to subunits closely spaced in a 137 .+-. 7-nucleotide-pair register. Subunits isolated from the reconstituted complex contain nearly equimolar amounts of the 4 histones and sediment at 11.6S. On DNase I digestion both the reconstituted complex and the separated subunits gave rise to series of single-stranded DNA fragments with a 10 nucleotide periodicity. This indicates that the reconstitution leads to subunits very similar to nucleosome cores. Nucleosome cores closely spaced in a 140-nucleotide-pair register were also obtained upon removal of histone H1 from chromatin by dissociation with 0.63 M NaCl and subsequent untracentrifugation. In reconstitution experiments with all 5 histones (including histone H1) this procedure did not lead to tandemly arranged nucleosomes containing about 200 nucleotide pairs of DNA. In the presence of EDTA, DNase II cleaved calf thymus nuclei and chromatin at about 200 nucleotide-pair intervals whereas in the presence of Mg2+ cleavage at intervals of approximately half this size was observed. The change in the nature of the cleavage pattern was no longer found after removal of histone H1 from chromatin, indicating that H1 influences the accessibility of DNase II cleavage sites in chromatin. This finding is discussed with respect to the influence of histone H1 on chromatin superstructure.