Accessibility and Structural Role of Histone Domains in Chromatin. Biophysical and Immunochemical Studies of Progressive Digestion with Immobilized Proteases

Abstract

The accessibility and role of histone regions in chromatin fibres were investigated using limited proteolysis with enzymes covalently bound to collagen membranes. The changes in chromatin conformation and condensation monitored by various biophysical methods, were correlated to the degradation of the histone proteins revealed by antibodies specific for histones and histone peptides. Upon digestion with trypsin and subtilisin, chromatin undergoes successive structural transitions. The cleavage of the C-terminal domains of H1, H2A and H2B, and of the N-terminal tail of H3 led to a decondensation of chromatin fibres, indicated by increases in electric birefringence and orientational relaxation times. It corresponds to a 15% increase in linear dimensions. The degradation of the other terminal regions of histones H3, H2A and H2B resulted in the appearance of hinge points between nucleosomes without alteration of the overall orientation of polynucleosome chains. Despite the loss of all the basic domains of H1, H3, H2A and H2B, no significant change in DNA-protein interactions occurred, suggesting that most of these protease-accessible regions interact weakly, if at all, with DNA in chromatin. Further proteolysis led to H4 degradation and other additional cleavages of H1, H2B and H3. This caused the relaxation of no more than 8% of the total DNA but resulted in changes in the ability of chromatin to condense at high ionic strength. More extensive digestion resulted in a total unravelling of nucleosomal chains which acquired properties similar to those of H1-depleted chromatin, although the globular part of H1 was still present. The data suggest that histone-histone interactions between H1 and core histone domains play a central role in stabilizing the chromatin fibres, and cuts in H3, H2A and H2B as well as H1, seem necessary for chromatin expansion. On the contrary, H4 might be involved in the stabilization of nucleosomes only.

Temporal and spatial association of histone H2A variant hv1 with transcriptionally competent chromatin during nuclear development in Tetrahymena thermophila.

“…The core domain is involved largely in histone-histone interactions necessary for nucleosome stability, whereas the amino terminus is likely to interact with DNA (Hill and Thomas 1990) or with extranucleosomal proteins (Johnson et al 1990) and serves as a site for acetylation (Bonner and Stedman 1979). The carboxy-terminal tail of histone H2A can be cross-linked to histone HI (Bonner and Stedman 1979), and the digestion of this domain leads to decondensation of nucleosomal fibers (Hacques et al 1990). Thus, this domain may be important for higher order chromatin structure because the presence of H1 in vitro can transform 10-nm nucleosomal fibers into 30-nm filaments (Van Holde 1989).…”

Structural analysis of Trypanosoma brucei brucei chromatin by limited proteolysis

“…The unfolding of chromatin upon proteolytic attack by either soluble or immobilized proteases has already been reported (see [12], for a discussion). In this study, we have described limited protease digestion of 7: brucei brucei chromatin fragments using immobilized trypsin and subtilisin.…”

“…brucei brucei chromatin was very low ( B = -0.076 e.s.u. ), as compared to that of rat liver or chicken erythocyte chromatin [12,23,241. Indeed, by assuming the values of Kerr's constant determined previously for the core particle and for H1-depleted nucleosomes [24], the amplitude of birefringence of 7: brucei brucei chromatin only corresponds to that of an H1-depleted rat liver nucleosome (-0.0715 e.s.u.).…”

“…The last point is particularly important because, as discussed previously, we are fully aware of the problems raised by our approach which requires different and possibly conflicting experimental conditions for the results to be made accessible. Indeed, the correlation between structural changes and the change in protein composi- tion of chromatin requires, on the one hand, low ionic strength to make it possible for electric birefringence to detect small changes in conformation and, on the other hand, a sufficient concentration of salt to make it possible to visualize the degradation of proteins in the absence of immunochemical detection (as in [11] and [12]). The use of specific protein-reactive and peptide-reactive antibodies will be the subject of future studies, as soon as the determination of histone-like sequence has been achieved.…”

“…Since similar experiments on eukaryotic chromatin have been described in detail in previous studies [9,12,23,241, it will be sufficient to mention here that measurements were made at chromatin concentration and field strength where electro-optical signals are not dependent upon these conditions, and with repeated applications of the electric field. The absence of additional bands in control samples shows that no proteolytic degradation occurred [13, 141. The optical anisotropy of chromatin has been the subject of many studies in recent years, with contradictory results regarding both its sign and magnitude.…”