A model chromatin assembly system: Factors affecting nucleosome spacing

Abstract

Poly[d(A-T)] · poly[d(A-T)], when reconstituted with chicken erythrocyte core histones and subsequently incubated with sufficient histone H5 in a solution containing polyglutamic acid, forms structures resembling chromatin. H5 induces nucleosome alignment in about two hours at physiological ionic strength and 37 °C. The nucleosome spacing and apparent linker heterogeneity in the assembled nucleoprotein are very similar to those in chicken erythrocyte chromatin. Also, condensed chromatin-like fibers on the polynucleotide can be visualized. The binding of one mole of H5 per mole of core octamer is necessary to generate the physiological nucleosome spacing, which remains constant with the addition of more H5. The nucleosome repeat length is not a function of the core histone to poly[d(A-T)] ratio for values lower than the physiological ratio. With increasing ratios, in excess of the physiological value, nucleosome spacing first becomes non-uniform, and then takes on the close packing limit of approximately 165 base-pairs. In addition to eliminating possible base sequence effects on nucleosome positioning, poly[d(A-T)] allows nucleosomes to slide more readily than does DNA, thereby facilitating alignment. Evidence is presented that polyglutamic acid facilitates the nucleosome spacing activity of histone H5, primarily by keeping the nucleoprotein soluble. This model system should be useful for understanding how different repeat lengths arise in chromatin.