Quantitative analysis of chromatin structure by circular dichroism

Quantitative analysis of the circular dichroism of nucleohistones and protein-free DNA was carried out in order to determine the structure and the role of the linker region DNA in chromatin, in terms of the conformational change of chromatin as a function of the ionic strength. It is shown clearly that the circular dichroism of Hl-depleted chromatin isolated from calf thymus is determined only by the ratio of the core region to the linker region and demonstrated by the linear combination of the spectrum of protein-free DNA and that of the nucleosome core in 5 mm-Tris · HCl, 1 mm-EDTA (pH 7.8). The calculated spectrum for the linker region in the H1-depleted chromatin was in good agreement with that of protein-free DNA. From the difference spectra between nucleohistones and protein-free DNA, it is suggested that the chromatin has an additional winding of DNA other than 146 base-pairs of DNA around the histone core. By decreasing the ionic strength to values lower than 5 mm-Tris · HCl, 1 mm-EDTA, the ellipticity of H1-depleted chromatin increased greatly between 250 nm and 300 nm while the increase was small in the case of chromatin and the nucleosome core. Nucleosomes with linker region DNA but without histone H1 also show great increase in ellipticity in this range of wavelengths as the ionic strength is decreased. Therefore, the linker region in H1-depleted chromatin plays an important role in the conformational changes brought about by changes in the ionic strength, and the conformational changes caused in the DNA of chromatin by decreasing the ionic strength are suppressed by the presence of histone H1.