Removal of histone tails from nucleosome dissects the physical mechanisms of salt-induced aggregation, linker histone H1-induced compaction, and 30-nm fiber formation of the nucleosome array

Abstract

In order to reveal the roles of histone tails in the formation of higher-order chromatin structures, we employed atomic force microscopy (AFM), and an in vitro reconstitution system to examine the properties of reconstituted chromatin composed of tail-less histones and a long DNA (106-kb plasmid) template. The tail-less nucleosomes did not aggregate at high salt concentrations or with an excess amount of core histones, in contrast with the behavior of nucleosomal arrays composed of nucleosomes containing normal, N-terminal tails. Analysis of our nucleosome distributions reveals that the attractive interaction between tail-less nucleosomes is weakened. Addition of linker histone H1 into the tail-less nucleosomal array failed to promote the formation of 30nm chromatin fibers that are usually formed in the normal nucleosomal array. These results demonstrate that the attractive interaction between nucleosomes via histone tails plays a critical role in the formation of the uniform 30-nm chromatin fiber.

Molecular dynamics of DNA and nucleosomes in solution studied by fast-scanning atomic force microscopy

“…Core histones were purified from HeLa cells according to the method developed by O'Neill et al [14] with slight modifications [34].…”

“…Instead, we used positively charged mica surface that had been treated with spermidine before sample deposition [34,[36][37][38]. The reconstituted nucleosomal array was diluted to a concentration of 0.5 ng/ml in a buffer containing 5 mM HEPESNaOH (pH 7.5) and 50 mM NaCl, and 3 ml of the sample was immediately deposited onto freshly cleaved 1 mm 2 mica discs pretreated with 10 mM spermidine.…”

Atomic Force Microscopy of Chromatin

“…The importance of the N-terminal histone tail in chromatin structure has been also investigated (Hizume et al, 2009). In vitro chromatin reconstitution using tail-less histones is efficient, but interactions between histones are weaker than using full histones.…”

Structural properties of yeast chromatin fiber examined by AFM and in vitro reconstitution system

“…In our previous studies, we analyzed chromatin structures reconstituted from tail-less or hyper-acetylated HeLa core histones [27,28]. In these studies, it was observed that the interaction between nucleosomes decreased both by hyper-acetylation and removal of the tails.…”

“…Salt-dependent precipitation of nucleosomal array revealed that acetylation of histone H4 [20] (or H2B and H4 [21]) is sufficient to inhibit chromatin compaction. AFM observation had also revealed that the reconstituted nucleosomal array using tail-less or hyper-acetylated core histones did not show salt-induced compaction [22].…”