Norihiko Sano

Structure and function of the histone chaperone CIA/ASF1 complexed with histones H3 and H4.

CIA (CCG1-interacting factor A)/ASF1, which is the most conserved histone chaperone among the eukaryotes, was genetically identified as a factor for an anti-silencing function (Asf1) by yeast genetic screening. Shortly after that, the CIA–histone-H3–H4 complex was isolated from Drosophila as a histone chaperone CAF-1 stimulator. Human CIA-I/II (ASF1a/b) was identified as a histone chaperone that interacts with the bromodomain—an acetylated-histone-recognizing domain—of CCG1, in the general transcription initiation factor TFIID. Intensive studies have revealed that CIA/ASF1 mediates nucleosome assembly by forming a complex with another histone chaperone in human cells and yeast, and is involved in DNA replication, transcription, DNA repair and silencing/anti-silencing in yeast. CIA/ASF1 was shown as a major storage chaperone for soluble histones in proliferating human cells. Despite all these biochemical and biological functional analyses, the structure–function relationship of the nucleosome assembly/disassembly activity of CIA/ASF1 has remained elusive. Here we report the crystal structure, at 2.7 Å resolution, of CIA-I in complex with histones H3 and H4. The structure shows the histone H3–H4 dimers mutually exclusive interactions with another histone H3–H4 dimer and CIA-I. The carboxy-terminal β-strand of histone H4 changes its partner from the β-strand in histone H2A to that of CIA-I through large conformational change. In vitro functional analysis demonstrated that CIA-I has a histone H3–H4 tetramer-disrupting activity. Mutants with weak histone H3–H4 dimer binding activity showed critical functional effects on cellular processes related to transcription. The histone H3–H4 tetramer-disrupting activity of CIA/ASF1 and the crystal structure of the CIA/ASF1–histone-H3–H4 dimer complex should give insights into mechanisms of both nucleosome assembly/disassembly and nucleosome semi-conservative replication.

Global analysis of functional surfaces of core histones with comprehensive point mutants

The core histones are essential components of the nucleosome that act as global negative regulators of DNA‐mediated reactions including transcription, DNA replication and DNA repair. Modified residues in the N‐terminal tails are well characterized in transcription, but not in DNA replication and DNA repair. In addition, roles of residues in the core globular domains are not yet well characterized in any DNA‐mediated reactions. To comprehensively understand the functional surface(s) of a core histone, we constructed 320 yeast mutant strains, each of which has a point mutation in a core histone, and identified 42 residues responsible for the suppressor of Ty (Spt‐) phenotypes, and 8, 30 and 61 residues for sensitivities to 6‐azauracil (6AU), hydroxyurea (HU) and methyl‐methanesulfonate (MMS), respectively. In addition to residues that affect one specific assay, residues involved in multiple reactions were found, and surprisingly, about half of them were clustered at either the nucleosome entry site, the surface required for nucleosome–nucleosome interactions in crystal packing or their surroundings. This comprehensive mutation approach was proved to be powerful for identification of the functional surfaces of a core histone in a variety of DNA‐mediated reactions and could be an effective strategy for characterizing other evolutionarily conserved hub‐like factors for which surface structural information is available.

Novel structural and functional mode of a knot essential for RNA binding activity of the Esa1 presumed chromodomain

Chromodomains are methylated histone binding modules that have been widely studied. Interestingly, some chromodomains are reported to bind to RNA and/or DNA, although the molecular basis of their RNA/DNA interactions has not been solved. Here we propose a novel binding mode for chromodomain-RNA interactions. Essential Sas-related acetyltransferase 1 (Esa1) contains a presumed chromodomain in addition to a histone acetyltransferase domain. We initially determined the solution structure of the Esa1 presumed chromodomain and showed it to consist of a well-folded structure containing a five-stranded beta-barrel similar to the tudor domain rather than the canonical chromodomain. Furthermore, the domain showed no RNA/DNA binding ability. Because the N-terminus of the protein forms a helical turn, we prepared an N-terminally extended construct, which we surprisingly found to bind to poly(U) and to be critical for in vivo function. This extended protein contains an additional beta-sheet that acts as a knot for the tudor domain and binds to oligo(U) and oligo(C) with greater affinity compared with other oligo-RNAs and DNAs examined thus far. The knot does not cause a global change in the core structure but induces a well-defined loop in the tudor domain itself, which is responsible for RNA binding. We made 47 point mutants in an esa1 mutant gene in yeast in which amino acids of the Esa1 knotted tudor domain were substituted to alanine residues and their functional abilities were examined. Interestingly, the knotted tudor domain mutations that were lethal to the yeast lost poly(U) binding ability. Amino acids that are related to RNA interaction sites, as revealed by both NMR and affinity binding experiments, are found to be important in vivo. These findings are the first demonstration of how the novel structure of the knotted tudor domain impacts on RNA binding and how this influences in vivo function.

Global analysis of core histones reveals nucleosomal surfaces required for chromosome bi-orientation

The attachment of sister kinetochores to microtubules from opposite spindle poles is essential for faithful chromosome segregation. Kinetochore assembly requires centromere‐specific nucleosomes containing the histone H3 variant CenH3. However, the functional roles of the canonical histones (H2A, H2B, H3, and H4) in chromosome segregation remain elusive. Using a library of histone point mutants in Saccharomyces cerevisiae, 24 histone residues that conferred sensitivity to the microtubule‐depolymerizing drugs thiabendazole (TBZ) and benomyl were identified. Twenty‐three of these mutations were clustered at three spatially separated nucleosomal regions designated TBS‐I, ‐II, and ‐III (TBZ/benomyl‐sensitive regions I–III). Elevation of mono‐polar attachment induced by prior nocodazole treatment was observed in H2A‐I112A (TBS‐I), H2A‐E57A (TBS‐II), and H4‐L97A (TBS‐III) cells. Severe impairment of the centromere localization of Sgo1, a key modulator of chromosome bi‐orientation, occurred in H2A‐I112A and H2A‐E57A cells. In addition, the pericentromeric localization of Htz1, the histone H2A variant, was impaired in H4‐L97A cells. These results suggest that the spatially separated nucleosomal regions, TBS‐I and ‐II, are necessary for Sgo1‐mediated chromosome bi‐orientation and that TBS‐III is required for Htz1 function.

Structure of the histone chaperone CIA/ASF1-double bromodomain complex linking histone modifications and site-specific histone eviction

Nucleosomes around the promoter region are disassembled for transcription in response to various signals, such as acetylation and methylation of histones. Although the interactions between histone-acetylation-recognizing bromodomains and factors involved in nucleosome disassembly have been reported, no structural basis connecting histone modifications and nucleosome disassembly has been obtained. Here, we determined at 3.3 Å resolution the crystal structure of histone chaperone cell cycle gene 1 (CCG1) interacting factor A/antisilencing function 1 (CIA/ASF1) in complex with the double bromodomain in the CCG1/TAF1/TAF(II)250 subunit of transcription factor IID. Structural, biochemical, and biological studies suggested that interaction between double bromodomain and CIA/ASF1 is required for their colocalization, histone eviction, and pol II entry at active promoter regions. Furthermore, the present crystal structure has characteristics that can connect histone acetylation and CIA/ASF1-mediated histone eviction. These findings suggest that the molecular complex between CIA/ASF1 and the double bromodomain plays a key role in site-specific histone eviction at active promoter regions. The model we propose here is the initial structure-based model of the biological signaling from histone modifications to structural change of the nucleosome (hi-MOST model).

Theoretical framework for the histone modification network: modifications in the unstructured histone tails form a robust scale-free network

A rapid increase in research on the relationship between histone modifications and their subsequent reactions in the nucleus has revealed that the histone modification system is complex, and robust against point mutations. The prevailing theoretical framework (the histone code hypothesis) is inadequate to explain either the complexity or robustness, making the formulation of a new theoretical framework both necessary and desirable. Here, we develop a model of the regulatory network of histone modifications in which we encode histone modifications as nodes and regulatory interactions between histone modifications as links. This network has scale‐free properties and subnetworks with a pseudo–mirror symmetry structure, which supports the robustness of the histone modification network. In addition, we show that the unstructured tail regions of histones are suitable for the acquisition of this scale‐free property. Our model and related insights provide the first framework for an overall architecture of a histone modification network system, particularly with regard to the structural and functional roles of the unstructured histone tail region. In general, the post‐translational “modification webs” of natively unfolded regions (proteins) may function as signal routers for the robust processing of the large amounts of signaling information.

Crystal structures of histone chaperone CIA/ASF1-containing complexes

The nucleosome, which is a fundamental repeating unit of chromatin, consists of about 200 base pairs of DNA and a histone octamer. Since the interactions between the histone proteins and DNA in the nucleosome hamper enzymes’ access to DNA, disassembly of nucleosomes is required for nuclear reactions such as transcription. Histone chaperones, which facilitate nucleosome assembly and disassembly, are therefore considered to play a critical role in transcription. Indeed, biological analyses have suggested that the histone chaperone CIA/ASF1, which is the most conserved histone chaperone in eukaryotes, is involved in histone eviction at the promoter regions in the transcriptional activation process. Other biochemical and biological studies have revealed that a critical signal of transcription activation is histone acetylation, which seems to function as a signal for the nucleosome disassembly in transcription. The acetylation signal is therefore likely to be transferred to histone chaperone CIA/ASF1. However, its molecular mechanism has remained elusive. In 2002, the Horikoshi group showed that CIA/ASF1 physically and genetically interacts with the double bromodomain (DBD) of CCG1 in the TFIID complex [1]. This result suggests that the interaction between CIA/ ASF1 and DBD plays a key role in connecting the histone acetylation and site-specific nucleosome disassembly. In order to elucidate this molecular mechanism at the atomic level, we determined the crystal structures of two molecular complexes containing CIA/ASF1. Initially, we determined the crystal structure of the CIA/ASF1–H3–H4 complex at 2.7 Å resolution [2]. This crystal structure showed that CIA/ASF1 interacts with the histone H3–H4 dimer and that the interaction inhibits nucleosome formation, suggesting that this complex occurs in the nucleosome disassembly process. In addition, the genetic analysis suggested that the interaction between CIA/ASF1 and histone H3 is involved in the transcription initiation process in yeast. Next, we determined the crystal structure of the CIA/ASF1–DBD complex at 3.3 Å resolution [3]. The genetic analysis, combined with structural information, showed that the interaction between CIA/ASF1 and DBD is also involved in the transcription initiation process in yeast. A ChIP analysis using a structurally designed DBD mutant suggested that CIA/ASF1 is recruited to promoter regions through the interaction with DBD and induces site-specific histone eviction around the promoter regions, leading to transcriptional activation. Our biochemical results showing that CIA/ASF1 can change its interacting partner from DBD to the histone H3-H4 dimer also supports this model. This is the first structure-based model of the biological signaling from histone modifications to structural change of the nucleosome (hi-MOST model) [3].