Use of an immobilized enzyme and specific antibodies to analyse the accessibility and role of histone tails in chromatin structure.

Using limited proteolysis with subtilisin bound to collagen membranes, the degradation of the histone proteins revealed by specific antibodies was correlated to changes in chromatin conformation and condensation monitored by circular dichroism and electric birefringence. This new approach allows us to detect for the first time a hierarchy of histone tails cleavages. The terminal domains of H1, the NH2-terminal tail of H3 and the carboxy-terminal ends of histones H2A and H2B were found to be cleaved already at the early stages of proteolysis and this led to a decondensation of polynucleosomal chains. Thereafter the C-terminal part of H3 and both NH2-terminal regions of H2A and H2B became rapidly cleaved, resulting in relative reorientation of swinging nucleosomes or partially unfolded segments. Unexpectedly, this removal of tails of H1, H2B, H2A and H3 is not accompanied by significant changes in DNA-protein interactions resulting in free-oriented DNA. This might suggest that histone-histone interactions play a central role in stabilizing the solenoid.