Characterization of monoclonal antibodies to histone 2B. Localization of epitopes and analysis of binding to chromatin.
Two mouse monoclonal IgM antibodies have been isolated which bind to histone 2B (H2B), as shown by protein blotting and immunostaining and by solid-phase radioimmunoassay (RIA). One of these (HBC-7) was specific for H2B by both techniques whereas the other (2F8) cross-reacted with histone H1 by RIA. Both antibodies failed to recognize H2B limit peptides from trypsin-digested chromatin and did not bind to Drosophila H2B, which differs extensively from vertebrate H2B only in the N-terminal region. These findings indicate that both antibodies recognize epitopes within the trypsin-sensitive, N-terminal region comprising residues 1-20. Binding of antibody HBC-7 was inhibited by in vitro ADP-ribosylation of H2B at glutamic acid residue 2. This strongly suggests that the epitope recognized by HBC-7 is located at the N-terminus of H2B, probably between residues 1 and 8. We have used solid-phase radioimmunoassay to investigate factors which influence the accessibility of this epitope in chromatin. Removal of H1 ('stripping') from high-molecular-mass chromatin had no effect on HBC-7 binding, nor was any difference observed between binding to stripped chromatin and to 146-base-pair (bp) core particles derived from it by nuclease digestion. These results suggest that accessibility of the N-terminal region of H2B is not influenced by H1 itself or by the size or conformation of linker DNA. In contrast, binding of antibody HBC-7 to 146-bp core particles derived from unstripped chromatin was reduced by up to 70%. Binding was restored by exposure of these core particles to the conditions used for stripping. Analysis of the protein content of core particle preparations from stripped and unstripped chromatin suggests that these findings may be attributable to redistribution of non-histone proteins during nuclease digestion. Pre-treatment of high-molecular-mass chromatin or 146-bp core particles with the intercalating dye ethidium bromide resulted in a severalfold increase in binding of HBC-7. The major changes in nucleosome morphology induced by ethidium are therefore accompanied by an increase in accessibility of the N-terminal region of H2B, possibly as a direct result of changes in the spatial relationship between H2B and core DNA.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D002843:Chromatin / Q000276:immunology*; D004331:Drosophila melanogaster / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology*; D004996:Ethidium / Q000494:pharmacology; D006657:Histones / Q000276:immunology* / Q000378:metabolism; D007075:Immunoglobulin M / Q000276:immunology; D007158:Immunologic Techniques; D008099:Liver / Q000032:analysis; D051379:Mice; D008836:Micrococcal Nuclease / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology; D051381:Rats
D000911:Antibodies, Monoclonal; D002843:Chromatin; D000939:Epitopes; D006657:Histones; D007075:Immunoglobulin M; D010446:Peptide Fragments; D000246:Adenosine Diphosphate Ribose; D008836:Micrococcal Nuclease; D004996:Ethidium