In the eukaryotic nucleus, DNA is complexed with core histone octamers to form arrays of nucleosomes, which can undergo various conformational transitions to form highly compacted chromatin. These specifically directed conformational changes appear to determine the general functional state of genomic DNA.  In particular, chromatin is targeted for transcriptional control through recognition, binding and post-translational modifications of the core histone N-terminal tail domains.   Previous investigations using in vitro-reconstituted nucleosomal arrays have begun to dissect the mechanistic determinants of chromatin folding transitions.  These studies were able to distinguish between short-range interactions (folding) of nucleosomal arrays correlating to formation of 30nm fibers and long-range interactions (self-association) correlating to higher order folding of “chromonema” fibers.

Recent studies have demonstrated that the requirements for folding and self-association are intrinsic to nucleosomal arrays.  Moreover, these studies have established that the core histone N-termini are the primary mediators of salt-dependent folding and self-association.  Furthermore, experiments utilizing core histone octamers containing selectively trypsinized N-termini, and octamers that were specifically acetylated demonstrated that the mechanisms by which the N-termini mediate chromatin condensation are complex and involve distinct functions and requirements for the N-termini in folding and self-association transitions.

Many questions relating to core histone N-termini function remain to be addressed including the sites of binding of each of the N-termini; the specificity of their interactions; the structure (or lack thereof) of the N-termini when involved in macromolecular condensation interactions; and the ability of individual N-termini to act through distinct multiple mechanisms depending upon their local environment and the exact state of condensation of chromatin in that environment.

Investigations attempting to answer these questions have been limited to date by the use of trypsinized chicken erythrocyte core histones, which dictated that only pairs of H2A/H2B and H3/H4 tail deletions could be proteolytically removed from nucleosomal arrays. The nucleosome core particle crystallized by Luger, et al. (Luger et al., Nature. 1997 Sep 18;389(6648):251-60) was reconstituted entirely from purified bacterially expressed recombinant native Xenopus core histones.  The availability of full length and tailless clones and the means to purify milligram quantities of the recombinant proteins and assemble them into histone octamers has opened the possibilities for study of nucleosomal arrays reconstituted from hybrid tailless octamers missing any combination of the core histone N-terminal tail domains.

The goals of this project were to investigate the individual contributions of the core histone N-termini in folding and self-association using the 208-12 nucleosomal array model system reconstituted with  Xenopus histone octamers lacking all possible combinations of the core histone N-terminal domains.  This system has allowed the study at a very fine level of detail, of the abilities of the individual N-termini to mediate self-association and the mechanisms by which they effect their role.  My results suggest a role for specific tail-tail interactions in condensation of chromatin, and confirm previously reported redundancy in the N-terminal domains.