LINKER HISTONES STABILIZE THE INTRINSIC SALT-DEPENDENT FOLDING OF NUCLEOSOMAL ARRAYS - MECHANISTIC RAMIFICATIONS FOR HIGHER-ORDER CHROMATINFOLDING

Defined nucleosomal arrays reconstituted from core histone octamers an d twelve 208 bp tandem repeats of Lytechinus 5S rDNA (208-12 nucleosom al arrays) possess the ability to form an unstable folded species in M gCl2 whose extent of compaction equals that of canonical higher-order 30 nm diameter chromatin structures [Schwarz, P. M,, and Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. To address the mechanistic f unctions of linker histones in chromatin condensation, purified histon e 1-15 has been assembled with 208-12 nucleosomal arrays in 50 mM NaCl . Novel purification procedures subsequently were developed that yield ed preparations of 208-12 chromatin model systems in which a majority of the sample contained both one histone octamer per 5S rDNA repeat an d one molecule of histone 1-15 per histone octamer. The integrity of t he purified 208-12 chromatin has been extensively characterized under low-salt conditions using analytical ultracentrifugation, quantitative agarose gel electrophoresis, electron cryomicroscopy, and nuclease di gestion. Results indicate that histone 1-15 binding to 208-12 nucleoso mal arrays constrains the entering and exiting linker DNA in a way tha t produces structures that are indistinguishable from native chicken e rythrocyte chromatin, Folding experiments performed in NaCl and MgCl2 have shown that 1-15 binding markedly stabilizes both the intermediate and extensively folded states of nucleosomal arrays without fundament ally altering the intrinsic nucleosomal array folding pathway These re sult provide new insight into the mechanism of chromatin folding by de monstrating for the first time that distinctly different macromolecula r determinants are required for formation and stabilization of higher- order chromatin structures.