Assembly and Disassembly of Nucleosome Core Particles Containing Histone Variants by Human Nucleosome Assembly Protein I

Abstract

Histone variants play important roles in the maintenance and regulation of the chromatin structure. In order to characterize the biochemical properties of the chromatin structure containing histone variants, we investigated the dynamic status of nucleosome core particles (NCPs) that were assembled with recombinant histones. We found that in the presence of nucleosome assembly protein I (NAP-I), a histone chaperone, H2A-Barr body deficient (H2A.Bbd) confers the most flexible nucleosome structure among the mammalian histone H2A variants known thus far. NAP-I mediated the efficient assembly and disassembly of the H2A.Bbd-H2B dimers from NCPs. This reaction was accomplished more efficiently when the NCPs contained H3.3, a histone H3 variant known to be localized in the active chromatin, than when the NCPs contained the canonical H3. These observations indicate that the histone variants H2A.Bbd and H3.3 are involved in the formation and maintenance of the active chromatin structure. We also observed that acidic histone binding proteins, TAF-I/SET and B23.1, demonstrated dimer assembly and disassembly activity, but the efficiency of their activity was considerably lower than that of NAP-I. Thus, both the acidic nature of NAP-I and its other functional structure(s) may be essential to mediate the assembly and disassembly of the dimers in NCPs.

Nucleosome stability mediated by histone variants H3.3 and H2A.Z

“…On the basis of the present results, we conclude that H3.3-containing nucleosomes are intrinsically less stable than those containing H3, and that this may reduce the energy required to move or displace nucleosomes from promoters, enhancers, and gene-coding regions. This is supported by the observation that nucleosome assembly protein I (NAP-I) mediated assembly and disassembly of the H2A.Bbd-H2B dimers from reconstituted NCPs was accomplished more efficiently when the NCPs contained H3.3 (Okuwaki et al 2005). There has been considerable debate as to whether H3.3 is simply a marker of transcriptionally active regions, deposited on active genes when H3 NCPs are displaced during transcription, or is an active participant in the regulatory process.…”

Epigenetic and replacement roles of histone variant H3.3 in reproduction and development

“…Indeed, nucleosomes containing H3.3, alone or in synergy with the H2A.Z variant, are more prone to loose H2A/H2B dimers in salt-disruption experiments, than regular nucleosomes (Jin and Felsenfeld, 2007). Similarly, assembly and disassembly of nucleosomes containing the mammalian variant H2A.Bbd occur more efficiently in association with H3.3 than with H3 (Okuwaki et al, 2005). If this were true in the context of in vivo chromatin, it would be interesting to see if it has any role in facilitating the replacement of nucleosomes with transition proteins and protamines during spermatid differentiation.…”

Role of Template Activating Factor-I as a chaperone in linker histone dynamics

“…We examined whether TAF-I can facilitate chromatosome (nucleosome core particle-histone H1 complex) assembly, using purified recombinant His-tagged histone H1.1 and GST-tagged TAF-Ib ( Fig. 2A), and nucleosome core particles (NCPs) (Okuwaki et al, 2005). NCPs resolved by polyacrylamide gel electrophoresis were detected by anti-histone H3 antibody (Fig.…”

“…Nucleosome reconstitution and chromatosome assemblydisassembly assay Recombinant chaperone proteins were prepared as described previously (Matsumoto et al, 1999;Miyaji-Yamaguchi et al, 1999;Okuwaki et al, 2005). His-tagged H1.1, GST-tagged H1.1, and its mutant derivatives, were also expressed in E. coli BL21 (DE3).…”

“…For instance, histone H3.3 is suggested to be incorporated into transcription-active chromatin by the HIRA-Asf1 histone chaperone complex in a replicationindependent manner (Ahmad and Henikoff, 2002;Tagami et al, 2004), whereas the canonical histone H3.1 is suggested to be incorporated into chromatin by the chromatin assembly factor 1 p55 subunit (CAF1)-Asf1 histone chaperone complex in a replicationdependent manner (Tagami et al, 2004). In the case of histone H2A-H2B, in vitro studies revealed that Nucleosome Assembly Protein-1 (NAP-1) is involved in the exchange of histone H2A variants such as H2A.Z and H2A-Bbd (Okuwaki et al, 2005;Park et al, 2005).…”