Naoki Horikoshi

Waseda University, Edo, Tōkyō, Japan

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Publications (11)58.75 Total impact

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    ABSTRACT: The human histone H2B variant TSH2B is highly expressed in testis and may function in the chromatin transition during spermatogenesis. In the present study, the crystal structure of the human testis-specific nucleosome containing TSH2B was determined at 2.8 Å resolution. A local structural difference between TSH2B and canonical H2B in nucleosomes was detected around the TSH2B-specific amino-acid residue Ser85. The TSH2B Ser85 residue does not interact with H4 in the nucleosome, but in the canonical nucleosome the H2B Asn84 residue (corresponding to the TSH2B Ser85 residue) forms water-mediated hydrogen bonds with the H4 Arg78 residue. In contrast, the other TSH2B-specific amino-acid residues did not induce any significant local structural changes in the TSH2B nucleosome. These findings may provide important information for understanding how testis-specific histone variants form nucleosomes during spermatogenesis.
    Acta Crystallographica Section F. 04/2014; 70(4).
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    ABSTRACT: The histone H2A.Z variant is widely conserved among eukaryotes. Two isoforms, H2A.Z.1 and H2A.Z.2, have been identified in vertebrates and may have distinct functions in cell growth and gene expression. However, no structural differences between H2A.Z.1 and H2A.Z.2 have been reported. In the present study, the crystal structures of nucleosomes containing human H2A.Z.1 and H2A.Z.2 were determined. The structures of the L1 loop regions were found to clearly differ between H2A.Z.1 and H2A.Z.2, although their amino-acid sequences in this region are identical. This structural polymorphism may have been induced by a substitution that evolutionally occurred at the position of amino acid 38 and by the flexible nature of the L1 loops of H2A.Z.1 and H2A.Z.2. It was also found that in living cells nucleosomal H2A.Z.1 exchanges more rapidly than H2A.Z.2. A mutational analysis revealed that the amino-acid difference at position 38 is at least partially responsible for the distinctive dynamics of H2A.Z.1 and H2A.Z.2. These findings provide important new information for understanding the differences in the regulation and functions of H2A.Z.1 and H2A.Z.2 in cells.
    Acta Crystallographica Section D Biological Crystallography 12/2013; 69(Pt 12):2431-9. · 12.67 Impact Factor
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    ABSTRACT: The centromere is a specific genomic region upon which the kinetochore is formed to attach to spindle microtubules for faithful chromosome segregation. To distinguish this chromosomal region from other genomic loci, the centromere contains a specific chromatin structure including specialized nucleosomes containing the histone H3 variant CENP-A. In addition to CENP-A nucleosomes, we have found that centromeres contain a nucleosome-like structure comprised of the histone-fold CENP-T-W-S-X complex. However, it is unclear how the CENP-T-W-S-X complex associates with centromere chromatin. Here, we demonstrate that the CENP-T-W-S-X complex binds preferentially to ∼100 bp of linker DNA rather than nucleosome-bound DNA. In addition, we find that the CENP-T-W-S-X complex primarily binds to DNA as a (CENP-T-W-S-X)2 structure. Interestingly, in contrast to canonical nucleosomes that negatively supercoil DNA, the CENP-T-W-S-X complex induces positive DNA supercoils. We found that the DNA-binding regions in CENP-T or CENP-W, but not CENP-S or CENP-X, are required for this positive supercoiling activity and the kinetochore targeting of the CENP-T-W-S-X complex. In summary, our work reveals the structural features and properties of the CENP-T-W-S-X complex for its localization to centromeres.
    Nucleic Acids Research 11/2013; · 8.81 Impact Factor
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    ABSTRACT: Post-translational histone modifications play key roles in gene regulation, development, and differentiation, but their dynamics in living organisms remain almost completely unknown. To address this problem, we developed a genetically encoded system for tracking histone modifications by generating fluorescent modification-specific intracellular antibodies (mintbodies) that can be expressed in vivo. To demonstrate, an H3 lysine 9 acetylation specific mintbody (H3K9ac-mintbody) was engineered and stably expressed in human cells. In good agreement with the localization of its target acetylation, H3K9ac-mintbody was enriched in euchromatin, and its kinetics measurably changed upon treatment with a histone deacetylase inhibitor. We also generated transgenic fruit fly and zebrafish stably expressing H3K9ac-mintbody for in vivo tracking. Dramatic changes in H3K9ac-mintbody localization during Drosophila embryogenesis could highlight enhanced acetylation at the start of zygotic transcription around mitotic cycle 7. Together, this work demonstrates the broad potential of mintbody and lays the foundation for epigenetic analysis in vivo.
    Scientific Reports 08/2013; 3:2436. · 5.08 Impact Factor
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    ABSTRACT: The Epstein-Barr virus (EBV) genome is episomally maintained in latently infected cells. The viral protein EBNA1 is a bridging molecule that tethers EBV episomes to host mitotic chromosomes as well as to interphase chromatin. EBNA1 localizes to cellular chromosomes (chromatin) via its chromosome binding domains (CBDs), which are rich in glycine and arginine residues. However, the molecular mechanism by which the CBDs of EBNA1 attach to cellular chromatin is still under debate. Mutation analyses revealed that stepwise substitution of arginine residues within the CBD1 [amino acids (a.a.) 40-54] and CBD2 [a.a. 328-377] regions with alanines progressively impaired chromosome binding activity of EBNA1. The complete arginine-to-alanine substitutions within the CBD1 and 2 regions abolished the ability of EBNA1 to stably maintain EBV-derived oriP plasmids in dividing cells. Importantly, replacing the same arginines with lysines had minimal effect, if any, on chromosome binding of EBNA1 as well as on its ability to stably maintain oriP plasmids. Furthermore, a glycine-arginine rich peptide derived from the CBD1 region bound to reconstituted nucleosome core particles in vitro, as did a glycine-lysine rich peptide, while a glycine-alanine rich peptide did not. These results support the idea that the chromosome binding of EBNA1 is mediated by electrostatic interactions between the basic amino acids within the CBDs and negatively charged cellular chromatin.
    Journal of Biological Chemistry 07/2013; · 4.65 Impact Factor
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    ABSTRACT: The Athe_0614 protein is a component of the extracellular proteins secreted by the anaerobic, extremely thermophilic and cellulolytic bacterium Caldicellulosiruptor bescii. The recombinant protein was expressed in Escherichia coli, purified to near-homogeneity and crystallized using polyethylene glycol 2000 monomethyl ether as a precipitant. The crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 48.4, b = 42.2, c = 97.8 Å, β = 96.1°, and diffracted to 2.7 Å resolution using synchrotron radiation.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 04/2013; 69(Pt 4):438-40. · 0.55 Impact Factor
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    ABSTRACT: Histones are the protein components of the nucleosome, which forms the basic architecture of eukaryotic chromatin. Histones H2A, H2B, H3, and H4 are composed of two common regions, the "histone fold" and the "histone tail". Many efforts have been focused on the mechanisms by which the post-translational modifications of histone tails regulate the higher-order chromatin architecture. On the other hand, previous biochemical studies have suggested that histone tails also affect the structure and stability of the nucleosome core particle itself. However, the precise contributions of each histone tail are unclear. In the present study, we determined the crystal structures of four mutant nucleosomes, in which one of the four histones, H2A, H2B, H3, or H4, lacked the N-terminal tail. We found that the deletion of the H2B or H3 N-terminal tail affected histone-DNA interactions and substantially decreased nucleosome stability. These findings provide important information for understanding the complex roles of histone tails in regulating chromatin structure.
    FEBS open bio. 01/2013; 3:363-9.
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    ABSTRACT: The nucleosome is the basic repeating unit of chromatin. During the nucleosome assembly process, DNA is wrapped around two H3-H4 dimers, followed by the inclusion of two H2A-H2B dimers. The H3-H4 dimers provide the fundamental architecture of the nucleosome. Many non-allelic variants have been found for H3, but not for H4, suggesting that the functions of chromatin domains may, at least in part, be dictated by the specific H3 variant that is incorporated. A prominent example is the centromeric H3 variant, CENP-A, which specifies the function of centromeres in chromosomes. In this review, we survey the current progress in the studies of nucleosomes containing H3 variants, and discuss their implications for the architecture and dynamics of chromatin domains.
    Current Opinion in Structural Biology 12/2012; · 8.74 Impact Factor
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    ABSTRACT: A deficiency in the SMN gene product causes the motor neuron degenerative disease spinal muscular atrophy. GEMIN2 was identified as an SMN-interacting protein, and the SMN-GEMIN2 complex constitutes part of the large SMN complex, which promotes the assembly of the spliceosomal small nuclear ribonucleoprotein (snRNP). In addition to its splicing function, we previously found that GEMIN2 alone stimulates RAD51-mediated recombination in vitro, and functions in DNA double-strand-break (DSB) repair through homologous recombination in vivo. However, the function of SMN in homologous recombination has not been reported. In the present study, we successfully purified the SMN-GEMIN2 complex as a fusion protein. The SMN-GEMIN2 fusion protein complemented the growth-defective phenotype of GEMIN2-knockout cells. The purified SMN-GEMIN2 fusion protein enhanced the RAD51-mediated homologous pairing much more efficiently than GEMIN2 alone. SMN-GEMIN2 possessed DNA-binding activity, which was not observed with the GEMIN2 protein, and significantly stimulated the secondary duplex DNA capture by the RAD51-single-stranded DNA complex during homologous pairing. These results provide the first evidence that the SMN-GEMIN2 complex plays a role in homologous recombination, in addition to spliceosomal snRNP assembly.
    Biochemistry 08/2011; 50(32):6797-805. · 3.38 Impact Factor
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    ABSTRACT: PAD4 is a peptidylarginine deiminase that catalyzes citrullination, a type of post-translational modification. In this reaction, arginine residues in proteins are converted to citrulline. PAD4 promotes the deimination of arginine residues in histones and may regulate transcription in the context of the chromatin. Single-nucleotide polymorphisms (SNP) in the gene encoding PAD4 identified it as one of the genes associated with susceptibility to rheumatoid arthritis. The PAD4 SNP involve three amino-acid substitutions: Ser55 to Gly, Ala82 to Val and Ala112 to Gly. Autoantibodies for improperly citrullinated proteins have been found in rheumatoid arthritis patients, suggesting that the PAD4(SNP) mRNA is more stable than the conventional PAD4 mRNA and/or the PAD4(SNP) protein possesses a higher citrullination activity than the PAD4 protein. In order to study the effects of the three amino-acid substitutions found in PAD4(SNP), the crystal structure of PAD4(SNP) was determined and it was found that the amino-acid substitutions in PAD4(SNP) only induced conformational changes within the N-terminal domain, not in the active centre for citrullination located in the C-terminal domain. Biochemical analyses also suggested that the citrullination activity of PAD4(SNP) may not substantially differ from that of conventional PAD4. These structural and biochemical findings suggested that the improper protein citrullination found in rheumatoid arthritis patients is not caused by defects in the citrullination activity of PAD4(SNP) but by other reasons such as enhanced PAD4(SNP) mRNA stability.
    Acta Crystallographica Section D Biological Crystallography 02/2011; 67(Pt 2):112-8. · 12.67 Impact Factor
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    ABSTRACT: SPF45 is considered to be a bifunctional protein that functions in splicing and DNA repair. A previous genetic study reported that Drosophila SPF45 participates in the DNA-repair pathway with a RAD51-family protein, RAD201, suggesting that SPF45 may function in DNA repair by the homologous-recombination pathway. To study the function of SPF45 in homologous recombination, we purified human SPF45 and found that it preferentially binds to the Holliday junction, which is a key DNA intermediate in the homologous-recombination pathway. Deletion analyses revealed that the RNA recognition motif, which is located in the C-terminal region of human SPF45, is not involved in DNA binding. On the other hand, alanine-scanning mutagenesis identified the N-terminal lysine residues, which may be involved in Holliday junction binding by human SPF45. We also found that human SPF45 significantly binds to a RAD51 paralog, RAD51B, although it also binds to RAD51 and DMC1 with lower affinity. These biochemical results support the idea that human SPF45 functions in DNA repair by homologous recombination.
    Genes to Cells 03/2010; 15(4):373-83. · 2.73 Impact Factor