Nucleosome linking number change controlled by acetylation of histones H3 and H4

High levels of acetylation of lysines in the amino-terminal domains of all four core histones, H2A, H2B, H3, and H4, have been shown to reduce the linking number change per nucleosome core particle in reconstituted minichromosomes (Norton, V. G., Imai, B. S., Yau, P., and Bradbury, E. M. (1989) Cell 57, 449-457). Because there is evidence to suggest that the acetylations of H3 and H4 have functions that are distinct from those of H2A and H2B, we have determined the nucleosome core particle linking number change in minichromosomes containing fully acetylated H3 and H4 and very low levels of acetylation in H2A and H2B. This linking number change was -0.81 +/- 0.05, in close agreement with the linking number change for hyperacetylated nucleosome core particles which contain high levels of acetylation in all four core histones (approximately 70% of full acetylation in H3 and H4). Therefore, high levels of acetylation of H3 and H4 alone are responsible for the reduction in the linking number change per nucleosome core particle.     

Neutron and x-ray scatter studies of the histone octamer and amino and carboxyl domain trimmed octamers

The structure of the nucleosome has been under intense investigation using neutron crystallography, x-ray crystallography, and neutron solution scattering. However the dimension of the histone octamer inside the nucleosome is still a subject of controversy. The radius of gyration (Rg) of the octamer obtained from solution neutron scattering of core particles at 63% 2H2O, 37% 1H2O is 33 A, and x-ray crystallography study of isolated histone octamer gives a Rg of 32.5 A, while the reported values using x-ray crystallography of core particles from two individual studies are 29.7 and 30.4 A, respectively. We report here studies of isolated histone octamer and trypsin-limited digested octamer using both neutron solution scattering and small angle x-ray scattering. The Rg of the octamer obtained is 33 A, whereas that of the trimmed octamer is 29.8 A, similar to the structure obtained from the crystals of the core particles. The N-terminal domains of the core histones in the octamer have been shown by high resolution nuclear magnetic resonance (Schroth, G.P., Yau, P., Imai, B.S., Gatewood, J.M., and Bradbury, E.M. (1990) FEBS Lett. 268, 117-120) to be mobile and flexible; it is likely that these regions are disordered and "not seen" by x-ray crystallography.     

Histone H1 structure probed by Staphylococcus aureus V8-proteinase

Proteolytic digestion of calf thymus histone H1 with Staphylococcus aureus V8-proteinase under structuring conditions generates one major limit peptide P1 which consists of approx. 170 residues. Edman degradation establishes the N-terminal sequence as: Leu-Ile-Thr-Lys-Ala-Val-Ala-Ala-Ser-Lys. Chymotryptic fingerprinting shows that the C-terminal part of the H1 molecule is fully preserved. The peptide therefore comprises the residues H1 (42-210). The Glu-41 cleavage is extremely unusual as it occurs in the structured G-domain which is known to be resistant to proteinases (Hartman, P. G., Chapman, G. E., Moss, T. and Bradbury, E. M. (1977) Eur. J. Biochem. 77, 45-71; B?hm, L., Sautière, P., Cary, P. D. and Crane-Robinson, C. (1982) Biochem. J. 203, 577-582). The V8-proteinase cleavage product H1 (42-210) shows only 20% folding as compared to 95-99% folding shown by the peptides H1 (34-121), H1 (31-210) and H1 (33-210). Folding of the G-domain thus critically depends upon the presence of the eight residues 33-41 amongst which the Gly-Pro-Pro sequence at position 36-38 and a beta-turn predicted at position 35 are considered to be particularly important. The location of the cleavage site in the G-domain renders Staphylococcus aureus V8-proteinase suitable as a structural probe.     

DNA wrapping in nucleosomes. The linking number problem re-examined.

Chromatin was assembled in vitro from relaxed closed circular DNA (SV40) and core histones at histone to DNA ratios of 0.2 to 0.3 (g/g) and incubated with topoisomerase I to relax supercoils in DNA regions not constrained by protein. Addition of histones H1 + H5 to the chromatin at an ionic strength of 0.1 M, in the presence of the solubilizing agent, polyglutamic acid, and topoisomerase I, increased the magnitude of the DNA linking number change, relative to protein-free DNA. No change in the linking number distribution occurred for relaxed protein-free DNA under these conditions. Control experiments indicated that the increase in the absolute value of the DNA linking number change in the chromatin could not be attributed to an increase in the number of nucleosomes per DNA molecule. These data suggest a solution to the linking number problem associated with models of chromatin structure.     

Histone acetylation reduces nucleosome core particle linking number change

Nucleosome core particles differing in their levels of histone acetylation have been formed on a closed circular DNA that contains a tandemly repeated 207 bp nucleosome positioning sequence. The effect of acetylation on the linking number per nucleosome particle has been determined. With increasing levels of acetylation, the negative linking number change per nucleosome decreases from -1.04 +/- 0.08 for control to -0.82 +/- 0.05 for highly acetylated nucleosomes. These results indicate that histone acetylation has the ability to release negative supercoils previously constrained by nucleosomes into a closed chromatin loop and in effect function as a eukaryotic gyrase.