Chromatin polynucleosomes have been digested with trypsin immobilized on collagen membranes. This method allows the mild removal of the most accessible histone fragments simply by dipping the enzymatic membrane into the chromatin solution, without modification of its ionic and chemical composition. These results demonstrate that the removal of Hl does not affect the higher-order structure of chromatin and that only the elimination of the terminal regions of H3 leads to the unfolding of Hl-depleted fibres. This observation suggests that structural changes reported in many previous works were not due to only the removal of Hl but to a concomitant unbinding from DNA of the N-terminal domain of H3. Chromatin structure Histone HI Histone H3 Trypsin digestion Enzymatic collagen membraneImmobiltzed enzyme

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.

The effectiveness of histone H1 subfractions H1-1 and H1(0) in inducing the ordered condensation of chromatin was examined by thermal denaturation, circular dichroism, electric birefringence, orientation mechanism, and orientational relaxation time measurements. Soluble rat liver chromatin was stripped of H1 by dissociation in 500 mM NaCl and long fragments of chromatin were subsequently reassociated with purified individual H1 subfractions for ratios of 1 and 2 mol of H1 per nucleosome. H1 subfractions behave differently with respect to their interactions with DNA in chromatin: although the orientation mechanisms of reconstituted chromatins are identical, H1(0) induces a less efficient protection of DNA than H1-1, as shown by nuclease digestion and by the length of free extended linker DNA determined by electric birefringence. This corresponds to a more extended structure of H1(0)-reconstituted chromatin as judged by the value of relaxation time. One can imagine that the replacement of H1 by H1(0) leads to a different structure or stability of the chromatin, confering a certain degree of flexibility of this region. This may be related to the functional role of H1(0) in DNA replication or transcription and may explain metabolic and evolutionary differences among H1 subfractions as recently suggested by Lennox [Lennox, R. W. (1984) J. Biol. Chem. 259, 669-672]. The extent of condensation when H1-depleted chromatin is overloaded with histones is probably a function of the electrostatic interactions between the basic C-terminal tails of histones and chromatin. Electric birefringence also reveals differences between native and reconstituted chromatins that are overlooked by several other criteria.