Histone compostion of chromatin subunits studied by immunosedimentation.

Chromatin subunits were prepared from HeLa cells by in situ digestion of nuclear DNA with micrococcal nuclease followed by sucrose gradient sedimentation. These 11S chromosomal particles (nucleosomes) contain a DNA fragment 140--180 base pairs long and an equal mass of histones, H2A, H2B, H3, and h4. nucleosomes were incubated with purified antibodies to histones H2A and H2B and to hemoglobin A, and the resulting complexes were analyzed by ultracentrifugation. Of these, only anti-H2B bound specifically to nucleosomes. When sufficient antibody was present, all (greater than 98%) the nucleosomes sedimented with increased velocities, indicating that all chromosomal particles contain H2B, as suggested by previous electron microscopic studies (Bustin, M., Goldblatt, D., and Sperling, R. (1976), Cell 7, 297). The amount of antibody reacting with H2B in the nucleosome was quantitated by densitometric scanning of gel electrophoresis patterns of the proteins in various nucleosome-anti-H2B complexes separated by sedimentation on isokinetic sucrose gradients. Under conditions where all particles had increased sedimentation velocities, from 1 to 3 IgG molecules are bound to each nucleosome, the ratio increasing from top to bottom of the sedimenting peak. When nucleosomes are thus dispersed on the basis of reaction with anti-H2B, the ratios of H2A to H4 and of (H2B + H3) to H4 are identical (+/- 8%) for all fractions, suggesting that each nucleosome has an identical histone complement, two each of histones H2A, H2B, H3, and H4. Confidence limits for exclusion of other possible octamers are presented. The variation in ratio of bound antibody to nucleosome probably reflects a normal distribution during the titration, although differential exposure of H2B antigenic determinants in several populations of nucleosomes cannot be excluded as an explanation. The method use should be generally applicable to further studies of the composition and function of nucleosomes.