Abstract
Defined oligonucleosome model systems have been used to investigate the molecular mechanisms through which the core histone tail domains modulate chromatin structure. In low salt conditions, the tail domains function at the nucleosome level to facilitate proper organization of nucleosomal DNA, i.e. wrapping of DNA around the histone octamer. Mg2+ ions can substitute for the tail domains to yield a trypsinized oligonucleosome structure that is indistinguishable from that of an intact nucleosomal array in low salt. However, Mg(2+)-dependent formation of highly folded oligonucleosome structures absolutely requires the histone tail domains, and is associated with rearrangement of the tails to a non-nucleosomal location. We conclude that the tail domains mediate oligonucleosome folding and nucleosomal DNA organization through fundamentally different molecular mechanisms.
Highlights
Trypsinized oligonucleosomes lacking their core histone tail domains remain unfolded in the presence of NaCl (8, 13), suggesting that these domains in some way participate in core histone-directed oligonucleosome folding
We have shown previously that the additional decreases in o observed for n Ͼ 6 intact nucleosomal arrays in 2 mM MgCl2 reflect the increased extents of oligonucleosome folding that occur in proportion to the increased extent of nucleosome occupancy of adjacent 5 S repeats (17)
Intact 208-12 nucleosomal arrays by numerous criteria are best modeled as a fully extended structure in which two complete turns of DNA are wrapped around each histone octamer (12, 13, 25)
Summary
Materials—pPOL208-12 plasmid, 208-12 DNA template, and bacteriophage T3 were isolated as described previously (16, 17). Preparation of Trypsinized Histone Octamers—Native oligonucleosomes and core histone octamers were isolated from chicken erythrocytes as described (12). To remove the N-terminal tail domains (and C-terminal tail for histone H2B), native oligonucleosomes were exposed to immobilized trypsin as described by Ausio et al (18). Histones were electrophoresed on a 18% SDS-polyacrylamide gel; only those preparations consisting of the P1-P5 peptides described previously (18, 19) were processed further. Once an appropriate preparation was identified, the trypsinized histone octamers were separated from oligonucleosomal DNA using hydroxylapatite chromatography (20). Trypsinized octamers obtained by this method were used within 1 week of preparation, whereas native octamers were stable for Ͼ6 months under these conditions. Oligonucleosome Reconstitutions—Saturated and subsaturated 208-12 nucleosomal arrays were reconstituted from either intact or trypsinized core histone octamers and DNA by salt dialysis as described (21). 2 P1 is amino acids 27–129 of H3; P2 is amino acids 12–118 and 21–125 of H2A and H2B, respectively; P3 is amino acids 24 –125 of H2B; P4 is amino acids 18 –102 of H4; and P5 is amino acids 20 –102 of H4
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