Abstract
Histone chaperones are a group of histone-binding proteins that facilitate the assembly of nucleosomes, the fundamental structural units of chromatin in eukaryotes. In nucleosome assembly, deposition of a histone H3-H4 tetramer onto DNA is the first and critical step, which is mediated by the histone chaperones HIRA and CAF-1. HIRA and CAF-1 are reportedly involved in DNA replication independent (RI) and replication coupled nucleosome assembly, respectively. However, the mechanisms by which they mediate histone deposition remain unclear. In this study, we focused on the mechanism by which HIRA induces RI-nucleosome assembly. We looked for HIRA domains that are required for nucleosome assembly and its localization to chromatin. We used cell-free extracts from Xenopus eggs that carry out RI-nucleosome assembly of plasmid DNA. We confirmed that HIRA formed stable complexes with Asf1, another histone H3-H4 chaperone, and the HIRA-Asf1 complex was solely responsible for RI-nucleosome assembly in egg extracts. We further demonstrated that the HIRA N-terminus containing the WD40 domain, which comprises seven WD40 repeats, and the B domain, to which Asf1 binds, were essential for RI-nucleosome assembly; the three WD40 repeats from the N-terminus were especially critical. Using egg extracts that reproduce nuclear formation accompanying the duplication of chromatin, we also demonstrated that the Hir domain was indispensable for the binding of HIRA to chromatin. Thus, the WD40 and B domains are the core elements for inducing RI-nucleosome assembly. Hir domain regulates the binding to chromatin. Based on these findings, similarities and differences between HIRA and CAF-1 are discussed.
Highlights
Introduction ofXenopus laevis eggs, that are able to induce nucleosome assembly of plasmid DNA in vitro (Dilworth et al, 1987; Kleinschmidt et al, 1986; Laskey et al, 1978)
To investigate the mechanism by which histone H3-H4 chaperones induce tetrasome assembly, we focused on HIRA, because nucleosome assembly mediated by this chaperone can be analyzed independently of DNA replication, and was expected to be simpler than that mediated by CAF-1
B domain of HIRA independently of the cell cycle, but bound to the B-like domain of p60 to a lesser extent, only in I-HSE (Fig. 1C, D). These results indicate that in Xenopus eggs, HIRA forms a stable complex with Asf1 throughout the cell cycle, while CAF-1 is partly associated with
Summary
Low-speed extract (LSE) from unfertilized Xenopus eggs, which were arrested in M phase (M-LSE), was prepared as described by Yamamoto et al (Yamamoto et al, 2005). To confirm the requirement of histone chaperone complexes for RI-nucleosome assembly, we examined the supercoil formation in I-HSE from which Asf, HIRA, or CAF-1 had been selectively immunodepleted. Consistent with previous results (RayGallet et al, 2007), supercoil formation was severely impaired in I-HSE that had been immunodepleted of Asf and HIRA, but not CAF-1 (Fig. 2A, lanes 5–13) This result confirmed that the RI-nucleosome assembly activity in IHSE is dependent on the HIRA-Asf complex but not on the CAF-1-Asf complex. In complex with Asf was added to HIRA-depleted I-HSE, supercoil formation was successfully restored (Fig. 2B, lanes 5–7) in a dose-dependent manner (Fig. 2C, D) This result demonstrates that the RI-nucleosome assembly activity in I-HSE can exclusively be ascribed to the HIRA-Asf complex.
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