Abstract
The accessibility of histone H5 in chromatin was examined with monoclonal antibodies recognizing several epitopes of the globular region (GH5) of the histone (Rózalski, M., Lafleur, L., and Ruiz-Carrillo, A. (1985) J. Biol. Chem. 260, 14379-14385). The stoichiometry of the chromatin-antibody complexes indicated that while 0-86% of the H5 molecules were able to react, depending on the particular epitope, the extent of antibody binding to relaxed chromatin (in 5 mM KCl) and condensed chromatin (in 100 mM KCl or 0.35 mM MgCl2) was virtually identical. This indicates that the topography of H5 does not change during the conformational transition of chromatin. The data suggest that H5 is not completely internalized in the 30-nm fiber or that the fiber is flexible enough to allow full exposure of the GH5 epitopes. Several control experiments, including monoclonal antibody binding, sedimentation analysis, DNase II digestion, and glutaraldehyde cross-linking, showed that epitope accessibility is not due to H5 exchange or to perturbation of the chromatin fiber. The accessibility of GH5 suggests ways in which inactive chromatin may be unfolded in vivo.
Highlights
From the Cancer Research Center and Department of Biochemistry, Laval University, Schooolf Medicine, Quibec, Canada GIK 7P4
Inactive chromatin from the intermitotic cells of higher eukaryotes appears as fibers of approximately 30-nm diameter [1].I n uitro, this structure results from condensation of a relaxed polynucleosome chain by a variety of cations and requires the presence of the linker histone H1 or variants of it like H5 [3,4,5]
Reactivity of Chromatin Fibers Condensed with KCI-The topography of H5 in chromatinwas examined by determining the extent of binding of several mAbs specific for GH5 [10]
Summary
Preparation of Soluble Chromatin-Hen erythrocyte nuclei were obtained in buffer Acontaining 0.5% Nonidet P-40.Calf thymus nuclei were obtained in buffer A. Chromatin-antibody complexes were precipitated by addition of 25 pl of 40 mM MgCL in buffer Bo. After centrifugation, the pellets were quickly rinsed with buffer Bo containing 10 mMMgC12 and 0.05% Nonidet P-40. The results obtained with 5C3 and 3G8, recognizing different epitopes of GH5, are shown in Fig. 1,A and C Binding of these mAbswas essentially independent of the state of chromatin condensation, and the plateau values are comparable for a given mAb, each antibody saturates chromatin to a different level (i.e. about 60 and 30% of the H5 molecules were recognized by 5C3 and 3G8, respectively; see Table I). Binding of the antibodies depended on the presence of H5, since no complex was observed when the reactions were carried out with calf thymus chromatin(not shown; see Ref. 10, Figs. Results of chromatintitration on solid phase, not involving a MgCL precipitation step, were in complete agreement with those obtained in solution (see Fig. 7)
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