Differing accessibility in chromatin of the antigenic sites of regions 1-58 and 63-125 of histone H2B.

Abstract

Experiments with antibodies induced by separated fragments 1-58 and 63-125 of H2B histone indicated that the 1-58 portion of the molecule is much more accessible in chromatin than is the 63-125 region . In immunoabsorption and immunoelectron microscopic assays with bovine and chicken chromatins, anti-1-58 antibodies reacted with sheared or unsheared chromatin both at low ionic strength (1 mM Tris-HCI) and in 0.14 M NaCl. Anti-63-125 antibodies were bound only weakly by chromatin at low ionic strength and not at all in 0.14 M NaCl . Antibodies to whole H2B showed intermediate reactivity with chromatin in both assays . In tests of immunofluorescence with unfixed calf liver nuclei in suspension, anti-1-58 caused nucleolar as well as nucleoplasmic fluorescence, whereas anti-63-125 did not lead to detectable fluorescence ; anti-I-1213 showed intermediate staining intensity . In control experiments, anti-H1 antibody was bound by chromatin at low ionic strength but not in 0.14 M NaCl ; anti-H3 antibody was bound poorly under either condition .It has been established that two molecules of each of the histones H2A, H2B, H3, and H4 constitute a core for nucleosome subunits of chromatin and that DNA is wrapped around the outside of the core (19). The precise pathway of the DNA, the nucleosome topography, and the DNA-histone interactions are still under study. In one approach to these questions, exposure of chromatin or nucleosomes to trypsin led to the selective cleavage of the amino terminal 20-30 amino acids of each of the core histories (30), suggesting that these regions are exposed under the conditions of digestion (usually low ionic strength, such as 5 mM Tris-HCI, pH 8). The rate of cleavage was most rapid for H3 . Progressively slower cleavage occurred with H2A, H4, and H2B (31). The interpretation that this susceptibility to trypsin reflects simply selective exposure of the amino terminal regions at the nucleosomal surface or their extension from the core, however, has been questioned (18).Antibodies provide another kind of probe for exploring the organization of histones in chromatin and nucleosomes (7,25) . Studies in which chromatin was used as an immunoabsorbent showed that many of the antigenic sites that are recognized in separated core histories are masked in chromatin; this is especially true for H2A, H3, and H4 determinants, whereas those of H1 and H2B are relatively accessible (6, 10, 11) . Different antigenic determinants, therefore, are exposed to varying ex-THE JOURNAL OF CELL BIOLOGY " VOLUME 91 OCTOBER 1981 135-141 © The Rockefeller University Press " 0021-9525/81/10/0135/07 $1 .00 tents in chromatin or altered in conformation to varying degrees .The accessibility of some antigenic sites of H2B in nucleosomes, at least in solutions of low ionic strength, was also demonstrable by measurement of a specific increase in the sedimentation rate of nucleosomes after their incubation with purified anti-H2B antibodies (1, 24) . Further, anti-112B antibodies that bound to nucleosomes or chromatin have...

Antibodies to histones in systemic lupus erythematosus: localization of prominent autoantigens on histones H1 and H2B.

“…Within the nucleosome core, H2B (with H2A) is also in a relatively exposed position, with one copy being accessible on each face (29), and more readily accessible to H2B antiserum than are H3 and H4 to their antisera (30). Antibodies raised in rabbits against the NH2-terminal half of H2B bind to chromatin, indicating accessibility of the antigenic determinants in this region whereas antigenic determinants in the COOH-terminal half are inaccessible (31). The presence of antigenic determinant(s) on the NH2-terminal [20][21][22][23][24][25][26][27][28][29][30] trypsin-sensitive residues of one or more of the core histones in nucleosomes is also suggested by an earlier study of antibodies in a SLE serum (32).…”

Use of histone antibodies for studying chromatin topography and the phosphorylation of chromatin subunits.

“…The surface location of the first N-terminal residues of H2B in chromatin has previously been demonstrated by various approaches such as limited proteolysis (Rill and Oosterhof, 1982;Diaz and Walker, 1983), cross-linking (DeLange et al, 1979;Suda and Iwai, 1979) and immunochemical studies (Di Padua Mathieu et al, 1981). It is wellestablished that histone HI plays a particular role in the condensation of chromatin and that it stabilizes the core particle Koller, 1977, 1981;Worcel and Benyajati, 1977;Thoma et al, 1979;Stratling, 1979;Butler and Thomas, 1980;Harborne and Allan, 1983).…”

The major core histone antigenic determinants in systemic lupus erythematosus are in the trypsin‐sensitive regions

“…When purified histones (complexed with RNA) are used as immunogens, the antibodies directed against the major determinants often do not bind well to chromatin, suggesting that the determinants are largely masked [23]. For exam-ple, 90% of the antibodies in an anti-H2B serum were directed against the C-terminal 'half' of H2B (residues 63-125) and only 10% against the Nterminal 'half' (residues l-58) [24], but the determinants in the C-terminal 'half' proved to be relatively inaccessible in chromatin, whereas those in the N-terminal half were exposed and bound by antibody [25]. However, although some or all of the region containing residues 30-56 of H2B is accessible in nucleosomes [26], this region is evidently not strongly immunogenic in SLE, in which the antigen(s) is confined to residues l-20.…”