All Publications


  • SAR optimization studies on modified salicylamides as a potential treatment for acute myeloid leukemia through inhibition of the CREB pathway. Bioorganic & medicinal chemistry letters Chae, H. D., Cox, N., Capolicchio, S., Lee, J. W., Horikoshi, N., Kam, S., Ng, A. A., Edwards, J., Butler, T. L., Chan, J., Lee, Y., Potter, G., Capece, M. C., Liu, C. W., Wakatsuki, S., Smith, M., Sakamoto, K. M. 2019

    Abstract

    Disruption of cyclic adenosine monophosphate response element binding protein (CREB) provides a potential new strategy to address acute leukemia, a disease associated with poor prognosis, and for which conventional treatment options often carry a significant risk of morbidity and mortality. We describe the structure-activity relationships (SAR) for a series of XX-650-23 derived from naphthol AS-E phosphate that disrupts binding and activation of CREB by the CREB-binding protein (CBP). Through the development of this series, we identified several salicylamides that are potent inhibitors of acute leukemia cell viability through inhibition of CREB-CBP interaction. Among them, a biphenyl salicylamide, compound 71, was identified as a potent inhibitor of CREB-CBP interaction with improved physicochemical properties relative to previously described derivatives of naphthol AS-E phosphate.

    View details for DOI 10.1016/j.bmcl.2019.06.023

    View details for PubMedID 31253529

  • Structure-based design of an H2A.Z.1 mutant stabilizing a nucleosome in vitro and in vivo. Biochemical and biophysical research communications Horikoshi, N., Kujirai, T., Sato, K., Kimura, H., Kurumizaka, H. 2019

    Abstract

    The nucleosome containing the histone H2A.Z.1 variant is unstable, as compared to the canonical nucleosome in vitro, and the incorporation of H2A.Z.1 into chromatin is less stable than that of the canonical H2A in vivo. In the present study, we designed a human H2A.Z.1(S42R) mutant, in which the Ser42 residue is replaced by Arg. In the crystal structure of the nucleosome containing H2A.Z.1(S42R), the Arg residue inserted at the H2A.Z.1-Ser42 position forms additional hydrogen bonds and electrostatic interactions with the DNA backbone phosphates. The Arg42 residue is located in the L1-loop region of H2A.Z.1, but the backbone geometry of the L1-loop is not affected by the H2A.Z.1(S42R) substitution. The nucleosome containing H2A.Z.1(S42R) exhibited enhanced thermal stability, as compared to that containing wild-type H2A.Z.1 in vitro. Fluorescence recovery after photobleaching experiments revealed that H2A.Z.1(S42R) was more stably incorporated in chromatin than wild-type H2A.Z.1 in living cells. Therefore, the H2A.Z.1(S42R) mutant stabilizes the nucleosome in vitro and in vivo, and may be useful as a tool to study the functional significance of the unstable nature of the H2A.Z.1 nucleosome.

    View details for DOI 10.1016/j.bbrc.2019.06.012

    View details for PubMedID 31186139

  • Small-Molecule Activators of Glucose-6-phosephate Dehydrogenase (G6PD) Bridging the Dimer Interface. ChemMedChem Raub, A., Hwang, S., Horikoshi, N., Cunningham, A., Rahighi, S., Wakatsuki, S., Mochly-Rosen, D. 2019

    Abstract

    We have recently identified AG1, a small-molecule glucose-6-phosphate dehydrogenase (G6PD) activator that functions by promoting oligomerization of the enzyme to the catalytically competent forms. Biochemical experiments indicate activation of G6PD by the original hit molecule (AG1) is noncovalent and that one C2-symmetric region of the G6PD homodimer is important for ligand function. Consequently, the disulfide in AG1 is not required for activation of G6PD and a number of analogs were prepared without this reactive moiety. Our Study supports a mechanism of action whereby AG1 bridges the dimer interface at the structural nicotinamide adenine dinucleotide phosphate (NADP+) binding sites of two interacting G6PD monomers. Small molecules that promote G6PD oligomerization have the potential to provide a first-in-class treatment for G6PD deficiency. This general strategy could be applied to other enzyme deficiencies where control of oligomerization can enhance enzymatic activity and/or stability.

    View details for DOI 10.1002/cmdc.201900341

    View details for PubMedID 31183991

  • Cancer-associated mutations of histones H2B, H3.1 and H2A.Z.1 affect the structure and stability of the nucleosome. Nucleic acids research Arimura, Y., Ikura, M., Fujita, R., Noda, M., Kobayashi, W., Horikoshi, N., Sun, J., Shi, L., Kusakabe, M., Harata, M., Ohkawa, Y., Tashiro, S., Kimura, H., Ikura, T., Kurumizaka, H. 2018; 46 (19): 10007–18

    Abstract

    Mutations of the Glu76 residue of canonical histone H2B are frequently found in cancer cells. However, it is quite mysterious how a single amino acid substitution in one of the multiple H2B genes affects cell fate. Here we found that the H2B E76K mutation, in which Glu76 is replaced by Lys (E76K), distorted the interface between H2B and H4 in the nucleosome, as revealed by the crystal structure and induced nucleosome instability in vivo and in vitro. Exogenous production of the H2B E76K mutant robustly enhanced the colony formation ability of the expressing cells, indicating that the H2B E76K mutant has the potential to promote oncogenic transformation in the presence of wild-type H2B. We found that other cancer-associated mutations of histones, H3.1 E97K and H2A.Z.1 R80C, also induced nucleosome instability. Interestingly, like the H2B E76K mutant, the H3.1 E97K mutant was minimally incorporated into chromatin in cells, but it enhanced the colony formation ability. In contrast, the H2A.Z.1 R80C mutant was incorporated into chromatin in cells, and had minor effects on the colony formation ability of the cells. These characteristics of histones with cancer-associated mutations may provide important information toward understanding how the mutations promote cancer progression.

    View details for PubMedID 30053102

  • Correcting glucose-6-phosphate dehydrogenase deficiency with a small-molecule activator. Nature communications Hwang, S., Mruk, K., Rahighi, S., Raub, A. G., Chen, C., Dorn, L. E., Horikoshi, N., Wakatsuki, S., Chen, J. K., Mochly-Rosen, D. 2018; 9 (1): 4045

    Abstract

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic enzymopathies, is caused by over 160 different point mutations and contributes to the severity of many acute and chronic diseases associated with oxidative stress, including hemolytic anemia and bilirubin-induced neurological damage particularly in newborns. As no medications are available to treat G6PD deficiency, here we seek to identify a small molecule that corrects it. Crystallographic study and mutagenesis analysis identify the structural and functional defect of one common mutant (Canton, R459L). Using high-throughput screening, we subsequently identify AG1, a small molecule that increases the activity of the wild-type, the Canton mutant and several other common G6PD mutants. AG1 reduces oxidative stress in cells and zebrafish. Furthermore, AG1 decreases chloroquine- or diamide-induced oxidative stress in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the challenges associated with G6PD deficiency.

    View details for PubMedID 30279493

  • Methods for Preparing Nucleosomes Containing Histone Variants. Methods in molecular biology (Clifton, N.J.) Kujirai, T., Arimura, Y., Fujita, R., Horikoshi, N., Machida, S., Kurumizaka, H. 2018; 1832: 3–20

    Abstract

    Histone variants are key epigenetic players that regulate transcription, repair, replication, and recombination of genomic DNA. Histone variant incorporation into nucleosomes induces structural diversity of nucleosomes, consequently leading to the structural versatility of chromatin. Such chromatin diversity created by histone variants may play a central role in the epigenetic regulation of genes. Each histone variant possesses specific biochemical and physical characteristics, and thus the preparation methods are complicated. Here, we introduce the methods for the purification of human histone variants as recombinant proteins, and describe the preparation methods for histone complexes and nucleosomes containing various histone variants. We also describe the detailed method for the preparation of heterotypic nucleosomes, which may function in certain biological phenomena. These methods are useful for biochemical, structural, and biophysical studies.

    View details for PubMedID 30073519

  • SUMO modification system facilitates the exchange of histone variant H2A.Z-2 at DNA damage sites. Nucleus (Austin, Tex.) Fukuto, A., Ikura, M., Ikura, T., Sun, J., Horikoshi, Y., Shima, H., Igarashi, K., Kusakabe, M., Harata, M., Horikoshi, N., Kurumizaka, H., Kiuchi, Y., Tashiro, S. 2018; 9 (1): 87–94

    Abstract

    Histone exchange and histone post-translational modifications play important roles in the regulation of DNA metabolism, by re-organizing the chromatin configuration. We previously demonstrated that the histone variant H2A.Z-2 is rapidly exchanged at damaged sites after DNA double strand break induction in human cells. In yeast, the small ubiquitin-like modifier (SUMO) modification of H2A.Z is involved in the DNA damage response. However, whether the SUMO modification regulates the exchange of human H2A.Z-2 at DNA damage sites remains unclear. Here, we show that H2A.Z-2 is SUMOylated in a damage-dependent manner, and the SUMOylation of H2A.Z-2 is suppressed by the depletion of the SUMO E3 ligase, PIAS4. Moreover, PIAS4 depletion represses the incorporation and eviction of H2A.Z-2 at damaged sites. These findings demonstrate that the PIAS4-mediated SUMOylation regulates the exchange of H2A.Z-2 at DNA damage sites.

    View details for PubMedID 29095668

  • Crystal Structure and Characterization of Novel Human Histone H3 Variants, H3.6, H3.7, and H3.8 BIOCHEMISTRY Taguchi, H., Xie, Y., Horikoshi, N., Maehara, K., Harada, A., Nogami, J., Sato, K., Arimura, Y., Osakabe, A., Kujirai, T., Iwasaki, T., Semba, Y., Tachibana, T., Kimura, H., Ohkawa, Y., Kurumizaka, H. 2017; 56 (16): 2184-2196

    Abstract

    Non-allelic histone variants are considered as epigenetic factors that regulate genomic DNA functions in eukaryotic chromosomes. In this study, we identified three new human histone H3 variants (named H3.6, H3.7, and H3.8), which were previously annotated as pseudogenes. H3.6 and H3.8 conserve the H3.3-specific amino acid residues, but H3.7 shares the specific amino acid residues with H3.1. We successfully reconstituted the nucleosome containing H3.6 in vitro and determined its crystal structure. In the H3.6 nucleosome, the H3.6-specific Val62 residue hydrophobically contacts the cognate H4 molecule, but its contact area is smaller than that of the corresponding H3.3 Ile62 residue. The thermal stability assay revealed that the H3.6 nucleosome is substantially unstable, as compared to the H3.3 nucleosome. Interestingly, mutational analysis demonstrated that the H3.6 Val62 residue is fully responsible for the H3.6 nucleosome instability, probably because of the weakened hydrophobic interaction with H4. We also reconstituted the nucleosome containing H3.8, but its thermal stability was quite low. In contrast, purified H3.7 failed to form nucleosomes in vitro. The identification and characterization of these novel human histone H3 variants provide important new insights into understanding the epigenetic regulation of the human genome.

    View details for DOI 10.1021/acs.biochem.6b01098

    View details for Web of Science ID 000400232900005

    View details for PubMedID 28374988

  • Crystal structure of the overlapping dinucleosome composed of hexasome and octasome SCIENCE Kato, D., Osakabe, A., Arimura, Y., Mizukami, Y., Horikoshi, N., Saikusa, K., Akashi, S., Nishimura, Y., Park, S., Nogami, J., Maehara, K., Ohkawa, Y., Matsumoto, A., Kono, H., Inoue, R., Sugiyama, M., Kurumizaka, H. 2017; 356 (6334): 205-208

    Abstract

    Nucleosomes are dynamic entities that are repositioned along DNA by chromatin remodeling processes. A nucleosome repositioned by the switch-sucrose nonfermentable (SWI/SNF) remodeler collides with a neighbor and forms the intermediate "overlapping dinucleosome." Here, we report the crystal structure of the overlapping dinucleosome, in which two nucleosomes are associated, at 3.14-angstrom resolution. In the overlapping dinucleosome structure, the unusual "hexasome" nucleosome, composed of the histone hexamer lacking one H2A-H2B dimer from the conventional histone octamer, contacts the canonical "octasome" nucleosome, and they intimately associate. Consequently, about 250 base pairs of DNA are left-handedly wrapped in three turns, without a linker DNA segment between the hexasome and octasome moieties. The overlapping dinucleosome structure may provide important information to understand how nucleosome repositioning occurs during the chromatin remodeling process.

    View details for DOI 10.1126/science.aak9867

    View details for Web of Science ID 000399013800041

    View details for PubMedID 28408607

  • Polymorphism of apyrimidinic DNA structures in the nucleosome SCIENTIFIC REPORTS Osakabe, A., Arimura, Y., Matsumoto, S., Horikoshi, N., Sugasawa, K., Kurumizaka, H. 2017; 7

    Abstract

    Huge amounts (>10,000/day) of apurinic/apyrimidinic (AP) sites are produced in genomes, but their structures in chromatin remain undetermined. We determined the crystal structure of the nucleosome containing AP-site analogs at two symmetric sites, which revealed structural polymorphism: one forms an inchworm configuration without an empty space at the AP site, and the other forms a B-form-like structure with an empty space and the orphan base. This unexpected inchworm configuration of the AP site is important to understand the AP DNA repair mechanism, because it may not be recognized by the major AP-binding protein, APE1, during the base excision repair process.

    View details for DOI 10.1038/srep41783

    View details for Web of Science ID 000393080800001

    View details for PubMedID 28139742

    View details for PubMedCentralID PMC5282573

  • Identification of the amino acid residues responsible for stable nucleosome formation by histone H3.Y. Nucleus Kujirai, T., Horikoshi, N., Xie, Y., Taguchi, H., Kurumizaka, H. 2017: 1-10

    Abstract

    Histone H3.Y is conserved among primates. We previously reported that exogenously produced H3.Y accumulates around transcription start sites, suggesting that it may play a role in transcription regulation. The H3.Y nucleosome forms a relaxed chromatin conformation with flexible DNA ends. The H3.Y-specific Lys42 residue is partly responsible for enhancing the flexibility of the nucleosomal DNA. To our surprise, we found that H3.Y stably associates with chromatin and nucleosomes in vivo and in vitro. However, the H3.Y residues responsible for its stable nucleosome incorporation have not been identified yet. In the present study, we performed comprehensive mutational analyses of H3.Y, and determined that the H3.Y C-terminal region including amino acid residues 124-135 is responsible for its stable association with DNA. Among the H3.Y C-terminal residues, the H3.Y Met124 residue significantly contributed to the stable DNA association with the H3.Y-H4 tetramer. The H3.Y M124I mutation substantially reduced the H3.Y-H4 association in the nucleosome. In contrast, the H3.Y K42R mutation affected the nucleosome stability less, although it contributes to the flexible DNA ends of the nucleosome. Therefore, these H3.Y-specific residues, Lys42 and Met124, play different and specific roles in nucleosomal DNA relaxation and stable nucleosome formation, respectively, in chromatin.

    View details for DOI 10.1080/19491034.2016.1277303

    View details for PubMedID 28118111

  • Testis-Specific Histone Variant H3t Gene Is Essential for Entry into Spermatogenesis CELL REPORTS Ueda, J., Harada, A., Urahama, T., Machida, S., Maehara, K., Hada, M., Makino, Y., Nogami, J., Horikoshi, N., Osakabe, A., Taguchi, H., Tanaka, H., Tachiwana, H., Yao, T., Yamada, M., Iwamoto, T., Isotani, A., Ikawa, M., Tachibana, T., Okada, Y., Kimura, H., Ohkawa, Y., Kurumizaka, H., Yamagata, K. 2017; 18 (3): 593-600

    Abstract

    Cellular differentiation is associated with dynamic chromatin remodeling in establishing a cell-type-specific epigenomic landscape. Here, we find that mouse testis-specific and replication-dependent histone H3 variant H3t is essential for very early stages of spermatogenesis. H3t gene deficiency leads to azoospermia because of the loss of haploid germ cells. When differentiating spermatogonia emerge in normal spermatogenesis, H3t appears and replaces the canonical H3 proteins. Structural and biochemical analyses reveal that H3t-containing nucleosomes are more flexible than the canonical nucleosomes. Thus, by incorporating H3t into the genome during spermatogonial differentiation, male germ cells are able to enter meiosis and beyond.

    View details for DOI 10.1016/j.celrep.2016.12.065

    View details for Web of Science ID 000396470600002

    View details for PubMedID 28099840

  • Synthetic Chromatin Acylation by an Artificial Catalyst System Chem Ishiguro, T., Amamoto, Y., Tanabe, K., Liu, J., Kajino, H., Fujimura, A., Aoi, Y., Osakabe, A., Horikoshi, N., Kurumizaka, H., Yamatsugu, K., Kawashima, S. A., Kanai, M. 2017; 2 (6): 840–859
  • A Genetically Encoded Probe for Live-Cell Imaging of H4K20 Monomethylation JOURNAL OF MOLECULAR BIOLOGY Sato, Y., Kujirai, T., Arai, R., Asakawa, H., Ohtsuki, C., Horikoshi, N., Yamagata, K., Ueda, J., Nagase, T., Haraguchi, T., Hiraoka, Y., Kimura, A., Kurumizaka, H., Kimura, H. 2016; 428 (20): 3885-3902

    Abstract

    Eukaryotic gene expression is regulated in the context of chromatin. Dynamic changes in post-translational histone modification are thought to play key roles in fundamental cellular functions such as regulation of the cell cycle, development, and differentiation. To elucidate the relationship between histone modifications and cellular functions, it is important to monitor the dynamics of modifications in single living cells. A genetically encoded probe called mintbody (modification-specific intracellular antibody), which is a single-chain variable fragment tagged with a fluorescent protein, has been proposed as a useful visualization tool. However, the efficacy of intracellular expression of antibody fragments has been limited, in part due to different environmental conditions in the cytoplasm compared to the endoplasmic reticulum where secreted proteins such as antibodies are folded. In this study, we have developed a new mintbody specific for histone H4 Lys20 monomethylation (H4K20me1). The specificity of the H4K20me1-mintbody in living cells was verified using yeast mutants and mammalian cells in which this target modification was diminished. Expression of the H4K20me1-mintbody allowed us to monitor the oscillation of H4K20me1 levels during the cell cycle. Moreover, dosage-compensated X chromosomes were visualized using the H4K20me1-mintbody in mouse and nematode cells. Using X-ray crystallography and mutational analyses, we identified critical amino acids that contributed to stabilization and/or proper folding of the mintbody. Taken together, these data provide important implications for future studies aimed at developing functional intracellular antibodies. Specifically, the H4K20me1-mintbody provides a powerful tool to track this particular histone modification in living cells and organisms.

    View details for DOI 10.1016/j.jmb.2016.08.010

    View details for Web of Science ID 000385324000001

    View details for PubMedID 27534817

  • Structure and function of human histone H3.Y nucleosome NUCLEIC ACIDS RESEARCH Kujirai, T., Horikoshi, N., Sato, K., Maehara, K., Machida, S., Osakabe, A., Kimura, H., Ohkawa, Y., Kurumizaka, H. 2016; 44 (13): 6127-6141

    Abstract

    Histone H3.Y is a primate-specific, distant H3 variant. It is evolutionarily derived from H3.3, and may function in transcription regulation. However, the mechanism by which H3.Y regulates transcription has not been elucidated. In the present study, we determined the crystal structure of the H3.Y nucleosome, and found that many H3.Y-specific residues are located on the entry/exit sites of the nucleosome. Biochemical analyses revealed that the DNA ends of the H3.Y nucleosome were more flexible than those of the H3.3 nucleosome, although the H3.Y nucleosome was stable in vitro and in vivo Interestingly, the linker histone H1, which compacts nucleosomal DNA, appears to bind to the H3.Y nucleosome less efficiently, as compared to the H3.3 nucleosome. These characteristics of the H3.Y nucleosome are also conserved in the H3.Y/H3.3 heterotypic nucleosome, which may be the predominant form in cells. In human cells, H3.Y preferentially accumulated around transcription start sites (TSSs). Taken together, H3.Y-containing nucleosomes around transcription start sites may form relaxed chromatin that allows transcription factor access, to regulate the transcription status of specific genes.

    View details for DOI 10.1093/nar/gkw202

    View details for Web of Science ID 000382999300015

    View details for PubMedID 27016736

    View details for PubMedCentralID PMC5291245

  • Structural and biochemical analyses of monoubiquitinated human histones H2B and H4 OPEN BIOLOGY Machida, S., Sekine, S., Nishiyama, Y., Horikoshi, N., Kurumizaka, H. 2016; 6 (6)

    Abstract

    Monoubiquitination is a major histone post-translational modification. In humans, the histone H2B K120 and histone H4 K31 residues are monoubiquitinated and may form transcriptionally active chromatin. In this study, we reconstituted nucleosomes containing H2B monoubiquitinated at position 120 (H2Bub120) and/or H4 monoubiquitinated at position 31 (H4ub31). We found that the H2Bub120 and H4ub31 monoubiquitinations differently affect nucleosome stability: the H2Bub120 monoubiquitination enhances the H2A-H2B association with the nucleosome, while the H4ub31 monoubiquitination decreases the H3-H4 stability in the nucleosome, when compared with the unmodified nucleosome. The H2Bub120 and H4ub31 monoubiquitinations both antagonize the Mg(2+)-dependent compaction of a poly-nucleosome, suggesting that these monoubiquitinations maintain more relaxed conformations of chromatin. In the crystal structure, the H2Bub120 and H4ub31 monoubiquitinations do not change the structure of the nucleosome core particle and the ubiquitin molecules were flexibly disordered in the H2Bub120/H4ub31 nucleosome structure. These results revealed the differences and similarities of the H2Bub120 and H4ub31 monoubiquitinations at the mono- and poly-nucleosome levels and provide novel information to clarify the roles of monoubiquitination in chromatin.

    View details for DOI 10.1098/rsob.160090

    View details for Web of Science ID 000393877700004

    View details for PubMedID 27335322

    View details for PubMedCentralID PMC4929944

  • Crystal structures of heterotypic nucleosomes containing histones H2A.Z and H2A OPEN BIOLOGY Horikoshi, N., Arimura, Y., Taguchi, H., Kurumizaka, H. 2016; 6 (6)

    Abstract

    H2A.Z is incorporated into nucleosomes located around transcription start sites and functions as an epigenetic regulator for the transcription of certain genes. During transcriptional regulation, the heterotypic H2A.Z/H2A nucleosome containing one each of H2A.Z and H2A is formed. However, previous homotypic H2A.Z nucleosome structures suggested that the L1 loop region of H2A.Z would sterically clash with the corresponding region of canonical H2A in the heterotypic nucleosome. To resolve this issue, we determined the crystal structures of heterotypic H2A.Z/H2A nucleosomes. In the H2A.Z/H2A nucleosome structure, the H2A.Z L1 loop structure was drastically altered without any structural changes of the canonical H2A L1 loop, thus avoiding the steric clash. Unexpectedly, the heterotypic H2A.Z/H2A nucleosome is more stable than the homotypic H2A.Z nucleosome. These data suggested that the flexible character of the H2A.Z L1 loop plays an essential role in forming the stable heterotypic H2A.Z/H2A nucleosome.

    View details for DOI 10.1098/rsob.160127

    View details for Web of Science ID 000393877700007

    View details for PubMedID 27358293

    View details for PubMedCentralID PMC4929947

  • Crystal structure of the nucleosome containing ultraviolet light-induced cyclobutane pyrimidine dimer BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Horikoshi, N., Tachiwana, H., Kagawa, W., Osakabe, A., Matsumoto, S., Iwai, S., Sugasawa, K., Kurumizaka, H. 2016; 471 (1): 117-122

    Abstract

    The cyclobutane pyrimidine dimer (CPD) is induced in genomic DNA by ultraviolet (UV) light. In mammals, this photolesion is primarily induced within nucleosomal DNA, and repaired exclusively by the nucleotide excision repair (NER) pathway. However, the mechanism by which the CPD is accommodated within the nucleosome has remained unknown. We now report the crystal structure of a nucleosome containing CPDs. In the nucleosome, the CPD induces only limited local backbone distortion, and the affected bases are accommodated within the duplex. Interestingly, one of the affected thymine bases is located within 3.0 Å from the undamaged complementary adenine base, suggesting the formation of complementary hydrogen bonds in the nucleosome. We also found that UV-DDB, which binds the CPD at the initial stage of the NER pathway, also efficiently binds to the nucleosomal CPD. These results provide important structural and biochemical information for understanding how the CPD is accommodated and recognized in chromatin.

    View details for DOI 10.1016/j.bbrc.2016.01.170

    View details for Web of Science ID 000372135200020

    View details for PubMedID 26837048

  • Histone H3.5 forms an unstable nucleosome and accumulates around transcription start sites in human testis EPIGENETICS & CHROMATIN Urahama, T., Harada, A., Maehara, K., Horikoshi, N., Sato, K., Sato, Y., Shiraishi, K., Sugino, N., Osakabe, A., Tachiwana, H., Kagawa, W., Kimura, H., Ohkawa, Y., Kurumizaka, H. 2016; 9

    Abstract

    Human histone H3.5 is a non-allelic H3 variant evolutionally derived from H3.3. The H3.5 mRNA is highly expressed in human testis. However, the function of H3.5 has remained poorly understood.We found that the H3.5 nucleosome is less stable than the H3.3 nucleosome. The crystal structure of the H3.5 nucleosome showed that the H3.5-specific Leu103 residue, which corresponds to the H3.3 Phe104 residue, reduces the hydrophobic interaction with histone H4. Mutational analyses revealed that the H3.5-specific Leu103 residue is responsible for the instability of the H3.5 nucleosome, both in vitro and in living cells. The H3.5 protein was present in human seminiferous tubules, but little to none was found in mature sperm. A chromatin immunoprecipitation coupled with sequencing analysis revealed that H3.5 accumulated around transcription start sites (TSSs) in testicular cells.We performed comprehensive studies of H3.5, and found the instability of the H3.5 nucleosome and the accumulation of H3.5 protein around TSSs in human testis. The unstable H3.5 nucleosome may function in the chromatin dynamics around the TSSs, during spermatogenesis.

    View details for DOI 10.1186/s13072-016-0051-y

    View details for Web of Science ID 000368121900001

    View details for PubMedID 26779285

    View details for PubMedCentralID PMC4714512

  • Crystal structure of the nucleosome containing histone H3 with crotonylated lysine 122 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Suzuki, Y., Horikoshi, N., Kato, D., Kurumizaka, H. 2016; 469 (3): 483-489

    Abstract

    The crotonylation of histones is an important post-translational modification, and epigenetically functions in the regulation of genomic DNA activity. The histone modifications in the structured "histone-fold" domains are considered to have an especially important impact on the nucleosome structure and dynamics. In the present study, we reconstituted the human nucleosome containing histone H3.2 crotonylated at the Lys122 residue, and determined its crystal structure at 2.56 Å resolution. We found that the crotonylation of the H3 Lys122 residue does not affect the overall nucleosome structure, but locally impedes the formation of the water-mediated hydrogen bond with the DNA backbone. Consistently, thermal stability assays revealed that the H3 Lys122 crotonylation, as well as the H3 Lys122 acetylation, clearly reduced the histone-DNA association.

    View details for DOI 10.1016/j.bbrc.2015.12.041

    View details for Web of Science ID 000369352800024

    View details for PubMedID 26694698

  • Structural basis of pyrimidine-pyrimidone (6-4) photoproduct recognition by UV-DDB in the nucleosome SCIENTIFIC REPORTS Osakabe, A., Tachiwana, H., Kagawa, W., Horikoshi, N., Matsumoto, S., Hasegawa, M., Matsumoto, N., Toga, T., Yamamoto, J., Hanaoka, F., Thomae, N. H., Sugasawa, K., Iwai, S., Kurumizaka, H. 2015; 5

    Abstract

    UV-DDB, an initiation factor for the nucleotide excision repair pathway, recognizes 6-4PP lesions through a base flipping mechanism. As genomic DNA is almost entirely accommodated within nucleosomes, the flipping of the 6-4PP bases is supposed to be extremely difficult if the lesion occurs in a nucleosome, especially on the strand directly contacting the histone surface. Here we report that UV-DDB binds efficiently to nucleosomal 6-4PPs that are rotationally positioned on the solvent accessible or occluded surface. We determined the crystal structures of nucleosomes containing 6-4PPs in these rotational positions, and found that the 6-4PP DNA regions were flexibly disordered, especially in the strand exposed to the solvent. This characteristic of 6-4PP may facilitate UV-DDB binding to the damaged nucleosome. We present the first atomic-resolution pictures of the detrimental DNA cross-links of neighboring pyrimidine bases within the nucleosome, and provide the mechanistic framework for lesion recognition by UV-DDB in chromatin.

    View details for DOI 10.1038/srep16330

    View details for Web of Science ID 000364787400001

    View details for PubMedID 26573481

    View details for PubMedCentralID PMC4648065

  • Stable complex formation of CENP-B with the CENP-A nucleosome NUCLEIC ACIDS RESEARCH Fujita, R., Otake, K., Arimura, Y., Horikoshi, N., Miya, Y., Shiga, T., Osakabe, A., Tachiwana, H., Ohzeki, J., Larionov, V., Masumoto, H., Kurumizaka, H. 2015; 43 (10): 4909-4922

    Abstract

    CENP-A and CENP-B are major components of centromeric chromatin. CENP-A is the histone H3 variant, which forms the centromere-specific nucleosome. CENP-B specifically binds to the CENP-B box DNA sequence on the centromere-specific repetitive DNA. In the present study, we found that the CENP-A nucleosome more stably retains human CENP-B than the H3.1 nucleosome in vitro. Specifically, CENP-B forms a stable complex with the CENP-A nucleosome, when the CENP-B box sequence is located at the proximal edge of the nucleosome. Surprisingly, the CENP-B binding was weaker when the CENP-B box sequence was located in the distal linker region of the nucleosome. This difference in CENP-B binding, depending on the CENP-B box location, was not observed with the H3.1 nucleosome. Consistently, we found that the DNA-binding domain of CENP-B specifically interacted with the CENP-A-H4 complex, but not with the H3.1-H4 complex, in vitro. These results suggested that CENP-B forms a more stable complex with the CENP-A nucleosome through specific interactions with CENP-A, if the CENP-B box is located proximal to the CENP-A nucleosome. Our in vivo assay also revealed that CENP-B binding in the vicinity of the CENP-A nucleosome substantially stabilizes the CENP-A nucleosome on alphoid DNA in human cells.

    View details for DOI 10.1093/nar/gkv405

    View details for Web of Science ID 000355929200017

    View details for PubMedID 25916850

    View details for PubMedCentralID PMC4446444

  • Solution structure of variant H2A.Z.1 nucleosome investigated by small-angle X-ray and neutron scatterings Biochemistry and Biophysics Reports Sugiyama, M., Horikoshi, N., Suzuki, Y., Taguchi, H., Kujirai, T., Inoue, R., Oba, Y., Sato, N., Martel, A., Porcar, L., Kurumizaka, H. 2015; 4: 28-32
  • Solution structure of variant H2A.Z.1 nucleosome investigated by small-angle X-ray and neutron scatterings. Biochemistry and biophysics reports Sugiyama, M., Horikoshi, N., Suzuki, Y., Taguchi, H., Kujirai, T., Inoue, R., Oba, Y., Sato, N., Martel, A., Porcar, L., Kurumizaka, H. 2015; 4: 28–32

    Abstract

    Solution structures of nucleosomes containing a human histone variant, H2A.Z.1, were measured by small-angle X-ray and neutron scatterings (SAXS and SANS). SAXS revealed that the outer shape, reflecting the DNA shape, of the H2A.Z.1 nucleosome is almost the same as that of the canonical H2A nucleosome. In contrast, SANS employing a contrast variation technique revealed that the histone octamer of the H2A.Z.1 nucleosome is smaller than that of the canonical nucleosome. The DNA within the H2A.Z.1 nucleosome was more susceptible to micrococcal nuclease than that within the canonical nucleosome. These results suggested that the DNA is loosely wrapped around the histone core in the H2A.Z.1 nucleosome.

    View details for PubMedID 29124184

    View details for PubMedCentralID PMC5668895

  • A method for evaluating nucleosome stability with a protein-binding fluorescent dye METHODS Taguchi, H., Horikoshi, N., Arimura, Y., Kurumizaka, H. 2014; 70 (2-3): 119-126

    Abstract

    Nucleosomes are extremely stable histone-DNA complexes that form the building blocks of chromatin, which accommodates genomic DNA within the nucleus. The dynamic properties of chromatin play essential roles in regulating genomic DNA functions, such as DNA replication, recombination, repair, and transcription. Histones are the protein components of nucleosomes, and their diverse modifications and variants increase the versatility of nucleosome structures and their dynamics in chromatin. Therefore, a technique to evaluate the physical properties of nucleosomes would facilitate functional studies of the various nucleosomes. In this report, we describe a convenient assay for evaluating the thermal stability of nucleosomes in vitro.

    View details for DOI 10.1016/j.ymeth.2014.08.019

    View details for Web of Science ID 000346891000006

    View details for PubMedID 25220913

  • Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3 SCIENTIFIC REPORTS Arimura, Y., Shirayama, K., Horikoshi, N., Fujita, R., Taguchi, H., Kagawa, W., Fukagawa, T., Almouzni, G., Kurumizaka, H. 2014; 4

    Abstract

    The centromere-specific histone H3 variant, CENP-A, is overexpressed in particular aggressive cancer cells, where it can be mislocalized ectopically in the form of heterotypic nucleosomes containing H3.3. In the present study, we report the crystal structure of the heterotypic CENP-A/H3.3 particle and reveal its "hybrid structure", in which the physical characteristics of CENP-A and H3.3 are conserved independently within the same particle. The CENP-A/H3.3 nucleosome forms an unexpectedly stable structure as compared to the CENP-A nucleosome, and allows the binding of the essential centromeric protein, CENP-C, which is ectopically mislocalized in the chromosomes of CENP-A overexpressing cells.

    View details for DOI 10.1038/srep07115

    View details for Web of Science ID 000346175800001

    View details for PubMedID 25408271

    View details for PubMedCentralID PMC4236741

  • Structure of human nucleosome containing the testis-specific histone variant TSH2B ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS Urahama, T., Horikoshi, N., Osakabe, A., Tachiwana, H., Kurumizaka, H. 2014; 70: 444-449

    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.

    View details for DOI 10.1107/S2053230X14004695

    View details for Web of Science ID 000333757800008

    View details for PubMedID 24699735

    View details for PubMedCentralID PMC3976059

  • The centromeric nucleosome-like CENP-T-W-S-X complex induces positive supercoils into DNA NUCLEIC ACIDS RESEARCH Takeuchi, K., Nishino, T., Mayanagi, K., Horikoshi, N., Osakabe, A., Tachiwana, H., Hori, T., Kurumizaka, H., Fukagawa, T. 2014; 42 (3): 1644-1655

    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.

    View details for DOI 10.1093/nar/gkt1124

    View details for Web of Science ID 000331138800025

    View details for PubMedID 24234442

    View details for PubMedCentralID PMC3919578

  • Structural polymorphism in the L1 loop regions of human H2AZ1 and H2AZ2 ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY Horikoshi, N., Sato, K., Shimada, K., Arimura, Y., Osakabe, A., Tachiwana, H., Hayashi-Takanaka, Y., Iwasaki, W., Kagawa, W., Harata, M., Kimura, H., Kurumizaka, H. 2013; 69: 2431-2439

    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.

    View details for DOI 10.1107/S090744491302252X

    View details for Web of Science ID 000328370400016

    View details for PubMedID 24311584

    View details for PubMedCentralID PMC3852653

  • Interaction between Basic Residues of Epstein-Barr Virus EBNA1 Protein and Cellular Chromatin Mediates Viral Plasmid Maintenance JOURNAL OF BIOLOGICAL CHEMISTRY Kanda, T., Horikoshi, N., Murata, T., Kawashima, D., Sugimoto, A., Narita, Y., Kurumizaka, H., Tsurumi, T. 2013; 288 (33): 24189-24199

    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 40-54) and CBD2 (amino acids 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, whereas 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.

    View details for DOI 10.1074/jbc.M113.491167

    View details for Web of Science ID 000330611400057

    View details for PubMedID 23836915

    View details for PubMedCentralID PMC3745364

  • Genetically encoded system to track histone modification in vivo SCIENTIFIC REPORTS Sato, Y., Mukai, M., Ueda, J., Muraki, M., Stasevich, T. J., Horikoshi, N., Kujirai, T., Kita, H., Kimura, T., Hira, S., Okada, Y., Hayashi-Takanaka, Y., Obuse, C., Kurumizaka, H., Kawahara, A., Yamagata, K., Nozaki, N., Kimura, H. 2013; 3

    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.

    View details for DOI 10.1038/srep02436

    View details for Web of Science ID 000323097900004

    View details for PubMedID 23942372

    View details for PubMedCentralID PMC3743053

  • Crystallization and preliminary X-ray diffraction analysis of the secreted protein Athe_0614 from Caldicellulosiruptor bescii ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS Yokoyama, H., Yamashita, T., Horikoshi, N., Kurumizaka, H., Kagawa, W. 2013; 69: 438-440

    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 P2(1), 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.

    View details for DOI 10.1107/S174430911300554X

    View details for Web of Science ID 000316949100020

    View details for PubMedID 23545654

    View details for PubMedCentralID PMC3614173

  • Current progress on structural studies of nucleosomes containing histone H3 variants CURRENT OPINION IN STRUCTURAL BIOLOGY Kurumizaka, H., Horikoshi, N., Tachiwana, H., Kagawa, W. 2013; 23 (1): 109-115

    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.

    View details for DOI 10.1016/j.sbi.2012.10.009

    View details for Web of Science ID 000315832700015

    View details for PubMedID 23265997

  • Contribution of histone N-terminal tails to the structure and stability of nucleosomes FEBS OPEN BIO Iwasaki, W., Miya, Y., Horikoshi, N., Osakabe, A., Taguchi, H., Tachiwana, H., Shibata, T., Kagawa, W., Kurumizaka, H. 2013; 3: 363-369

    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.

    View details for DOI 10.1016/j.fob.2013.08.007

    View details for Web of Science ID 000339569800057

    View details for PubMedID 24251097

    View details for PubMedCentralID PMC3821030

  • Purification of the Human SMN-GEMIN2 Complex and Assessment of Its Stimulation of RAD51-Mediated DNA Recombination Reactions BIOCHEMISTRY Takaku, M., Tsujita, T., Horikoshi, N., Takizawa, Y., Qing, Y., Hirota, K., Ikura, M., Ikura, T., Takeda, S., Kurumizaka, H. 2011; 50 (32): 6797-6805

    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.

    View details for DOI 10.1021/bi200828g

    View details for Web of Science ID 000293665500007

    View details for PubMedID 21732698

  • Structural and biochemical analyses of the human PAD4 variant encoded by a functional haplotype gene ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY Horikoshi, N., Tachiwana, H., Saito, K., Osakabe, A., Sato, M., Yamada, M., Akashi, S., Nishimura, Y., Kagawa, W., Kurumizaka, H. 2011; 67: 112-118

    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.

    View details for DOI 10.1107/S0907444910051711

    View details for Web of Science ID 000287765100005

    View details for PubMedID 21245532

  • Holliday junction-binding activity of human SPF45 GENES TO CELLS Horikoshi, N., Morozumi, Y., Takaku, M., Takizawa, Y., Kurumizaka, H. 2010; 15 (4): 373-383

    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.

    View details for DOI 10.1111/j.1365-2443.2010.01383.x

    View details for Web of Science ID 000276010300006

    View details for PubMedID 20236180