Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Akihito Harada Last modified date:2021.10.27

Associate Professor / Research Center for Transomics Medicine / Medical Institute of Bioregulation

1. Hiroaki Tachiwana, Mariko Dacher, Kazumitsu Maehara, Akihito Harada, Yosuke Seto, Ryohei Katayama, Yasuyuki Ohkawa, Hiroshi Kimura, Hitoshi Kurumizaka, Noriko Saitoh, Chromatin structure-dependent histone incorporation revealed by a genome-wide deposition assay., eLife, 10.7554/eLife.66290, 10, 2021.05, In eukaryotes, histone variant distribution within the genome is the key epigenetic feature. To understand how each histone variant is targeted to the genome, we developed a new method, the RhIP (Reconstituted histone complex Incorporation into chromatin of Permeabilized cell) assay, in which epitope-tagged histone complexes are introduced into permeabilized cells and incorporated into their chromatin. Using this method, we found that H3.1 and H3.3 were incorporated into chromatin in replication-dependent and -independent manners, respectively. We further found that the incorporation of histones H2A and H2A.Z mainly occurred at less condensed chromatin (open), suggesting that condensed chromatin (closed) is a barrier for histone incorporation. To overcome this barrier, H2A, but not H2A.Z, uses a replication-coupled deposition mechanism. Our study revealed that the combination of chromatin structure and DNA replication dictates the differential histone deposition to maintain the epigenetic chromatin states..
2. Atsuko Miyawaki-Kuwakado, Qianmei Wu, Akihito Harada, Kosuke Tomimatsu, Takeru Fujii, Kazumitsu Maehara, Yasuyuki Ohkawa, Transcriptome analysis of gene expression changes upon enzymatic dissociation in skeletal myoblasts., Genes to cells : devoted to molecular & cellular mechanisms, 10.1111/gtc.12870, 2021.05, Single-cell RNA-sequencing analysis is one of the most effective tools for understanding specific cellular states. The use of single cells or pooled cells in RNA-seq analysis requires the isolation of cells from a tissue or culture. Although trypsin or more recently cold-active protease (CAP) has been used for cell dissociation, the extent to which the gene expression changes are suppressed has not been clarified. To this end, we conducted detailed profiling of the enzyme-dependent gene expression changes in mouse skeletal muscle progenitor cells, focusing on the enzyme treatment time, amount, and temperature. We found that the genes whose expression was changed by the enzyme treatment could be classified in a time-dependent manner, and that there were genes whose expression was changed independently of the enzyme treatment time, amount, and temperature. This study will be useful as reference data for genes that should be excluded or considered for RNA-seq analysis using enzyme isolation methods..
3. Qianmei Wu, Takeru Fujii, Akihito Harada, Kosuke Tomimatsu, Atsuko Miyawaki-Kuwakado, Masatoshi Fujita, Kazumitsu Maehara, Yasuyuki Ohkawa, Genome-wide analysis of chromatin structure changes upon MyoD binding in proliferative myoblasts during the cell cycle., Journal of biochemistry, 10.1093/jb/mvab001, 2021.01, MyoD, a myogenic differentiation protein, has been studied for its critical role in skeletal muscle differentiation. MyoD-expressing myoblasts have a potency to be differentiated with proliferation of ectopic cells. However, little is known about the effect on chromatin structure of MyoD binding in proliferative myoblasts. In this study, we evaluated the chromatin structure around MyoD-bound genome regions during the cell cycle by chromatin immunoprecipitation sequencing. Genome-wide analysis of histone modifications was performed in proliferative mouse C2C12 myoblasts during three phases (G1, S, G2/M) of the cell cycle. We found that MyoD-bound genome regions had elevated levels of active histone modifications, such as H3K4me1/2/3, and H3K27ac, compared with MyoD-unbound genome regions during the cell cycle. We also demonstrated that the elevated H3K4me2/3 modification level was maintained during the cell cycle, whereas the H3K27ac and H3K4me1 modification levels decreased to the same level as MyoD-unbound genome regions during the later phases. Immunoblot analysis revealed that MyoD abundance was high in the G1 phase then decreased in the S and G2/M phases. Our results suggest that MyoD binding formed selective epigenetic memories with H3K4me2/3 during the cell cycle in addition to myogenic gene induction via active chromatin formation coupled with transcription..
4. Hiroshi Ochiai, Tetsutaro Hayashi, Mana Umeda, Mika Yoshimura, Akihito Harada, Yukiko Shimizu, Kenta Nakano, Noriko Saitoh, Zhe Liu, Takashi Yamamoto, Tadashi Okamura, Yasuyuki Ohkawa, Hiroshi Kimura, Itoshi Nikaido, Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells., Science advances, 10.1126/sciadv.aaz6699, 6, 25, eaaz6699, 2020.06, Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body-binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9-based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells..
5. Masahiro Oka, Sonoko Mura, Mayumi Otani, Yoichi Miyamoto, Jumpei Nogami, Kazumitsu Maehara, Akihito Harada, Taro Tachibana, Yoshihiro Yoneda, Yasuyuki Ohkawa, Chromatin-bound CRM1 recruits SET-Nup214 and NPM1c onto HOX clusters causing aberrant HOX expression in leukemia cells, eLife, 10.7554/eLife.46667, 8, 2019.11, We previously demonstrated that CRM1, a major nuclear export factor, accumulates at Hox cluster regions to recruit nucleoporin-fusion protein Nup98HoxA9, resulting in robust activation of Hox genes (Oka et al., 2016). However, whether this phenomenon is general to other leukemogenic proteins remains unknown. Here, we show that two other leukemogenic proteins, nucleoporin-fusion SET-Nup214 and the NPM1 mutant, NPM1c, which contains a nuclear export signal (NES) at its C-terminus and is one of the most frequent mutations in acute myeloid leukemia, are recruited to the HOX cluster region via chromatin-bound CRM1, leading to HOX gene activation in human leukemia cells. Furthermore, we demonstrate that this mechanism is highly sensitive to a CRM1 inhibitor in leukemia cell line. Together, these findings indicate that CRM1 acts as a key molecule that connects leukemogenic proteins to aberrant HOX gene regulation either via nucleoporin-CRM1 interaction (for SET-Nup214) or NES-CRM1 interaction (for NPM1c)..
6. Sumiaki Fukuda, Akihiro Kaneshige, Takayuki Kaji, Yu Taro Noguchi, Yusei Takemoto, Lidan Zhang, Kazutake Tsujikawa, Hiroki Kokubo, Akiyoshi Uezumi, Kazumitsu Maehara, Akihito Harada, Yasuyuki Ohkawa, So Ichiro Fukada, Sustained expression of HeyL is critical for the proliferation of muscle stem cells in overloaded muscle, eLife, 10.7554/eLife.48284, 8, 2019.09, In overloaded and regenerating muscle, the generation of new myonuclei depends on muscle satellite cells (MuSCs). Because MuSC behaviors in these two environments have not been considered separately, MuSC behaviors in overloaded muscle remain unexamined. Here, we show that most MuSCs in overloaded muscle, unlike MuSCs in regenerating muscle, proliferate in the absence of MyoD expression. Mechanistically, MuSCs in overloaded muscle sustain the expression of Heyl, a Notch effector gene, to suppress MyoD expression, which allows effective MuSC proliferation on myofibers and beneath the basal lamina. Although Heyl-knockout mice show no impairment in an injury model, in a hypertrophy model, their muscles harbor fewer new MuSCderived myonuclei due to increased MyoD expression and diminished proliferation, which ultimately causes blunted hypertrophy. Our results show that sustained HeyL expression is critical for MuSC proliferation specifically in overloaded muscle, and thus indicate that the MuSCproliferation mechanism differs in overloaded and regenerating muscle..
7. Shoko Sato, Yasuhiro Arimura, Tomoya Kujirai, Akihito Harada, Kazumitsu Maehara, Jumpei Nogami, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Biochemical analysis of nucleosome targeting by Tn5 transposase, Open Biology, 10.1098/rsob.190116, 9, 8, 2019.08, Tn5 transposase is a bacterial enzyme that integrates a DNA fragment into genomic DNA, and is used as a tool for detecting nucleosome-free regions of genomic DNA in eukaryotes. However, in chromatin, the DNA targeting by Tn5 transposase has remained unclear. In the present study, we reconstituted well-positioned 601 dinucleosomes, in which two nucleosomes are connected with a linker DNA, and studied the DNA integration sites in the dinucleosomes by Tn5 transposase in vitro. We found that Tn5 transposase preferentially targets near the entry–exit DNA regions within the nucleosome. Tn5 transposase minimally cleaved the dinucleosome without a linker DNA, indicating that the linker DNA between two nucleosomes is important for the Tn5 transposase activity. In the presence of a 30 base-pair linker DNA, Tn5 transposase targets the middle of the linker DNA, in addition to the entry–exit sites of the nucleosome. Intriguingly, this Tn5-targeting characteristic is conserved in a dinucleosome substrate with a different DNA sequence from the 601 sequence. Therefore, the Tn5-targeting preference in the nucleosomal templates reported here provides important information for the interpretation of Tn5 transposase-based genomics methods, such as ATAC-seq..
8. Akihito Harada, Kazumitsu Maehara, Tetsuya Handa, Yasuhiro Arimura, Jumpei Nogami, Yoko Hayashi-Takanaka, Katsuhiko Shirahige, Hitoshi Kurumizaka, Hiroshi Kimura, Yasuyuki Ohkawa, A chromatin integration labelling method enables epigenomic profiling with lower input, Nature Cell Biology, 10.1038/s41556-018-0248-3, 21, 2, 287-296, 2019.02, Chromatin plays a crucial role in gene regulation, and chromatin immunoprecipitation followed by sequencing (ChIP–seq) has been the standard technique for examining protein–DNA interactions across the whole genome. However, it is difficult to obtain epigenomic information from limited numbers of cells by ChIP–seq because of sample loss during chromatin preparation and inefficient immunoprecipitation. In this study, we established an immunoprecipitation-free epigenomic profiling method named chromatin integration labelling (ChIL), which enables the amplification of genomic sequences closely associated with the target molecules before cell lysis. Using ChIL followed by sequencing (ChIL–seq), we reliably detected the distributions of histone modifications and DNA-binding factors in 100–1,000 cells. In addition, ChIL–seq successfully detected genomic regions associated with histone marks at the single-cell level. Thus, ChIL–seq offers an alternative method to ChIP–seq for epigenomic profiling using small numbers of cells, in particular, those attached to culture plates and after immunofluorescence..
9. Yu Taro Noguchi, Miki Nakamura, Nobumasa Hino, Jumpei Nogami, Sayaka Tsuji, Takahiko Sato, Lidan Zhang, Kazutake Tsujikawa, Toru Tanaka, Kohei Izawa, Yoshiaki Okada, Takefumi Doi, Hiroki Kokubo, Akihito Harada, Akiyoshi Uezumi, Manfred Gessler, Yasuyuki Ohkawa, So Ichiro Fukada, Cell-autonomous and redundant roles of Hey1 and HeyL in muscle stem cells
HeyL requires HeS1 to bind diverse DNA sites, Development (Cambridge), 10.1242/dev.163618, 146, 4, 2019.02, The undifferentiated state of muscle stem (satellite) cells (MuSCs) is maintained by the canonical Notch pathway. Although three bHLH transcriptional factors, Hey1, HeyL and Hes1, are considered to be potential effectors of the Notch pathway exerting anti-myogenic effects, neither HeyL nor Hes1 inhibits myogenic differentiation of myogenic cell lines. Furthermore, whether these factors work redundantly or cooperatively is unknown. Here, we showed cell-autonomous functions of Hey1 and HeyL in MuSCs using conditional and genetic null mice. Analysis of cultured MuSCs revealed anti-myogenic activity of both HeyL and Hes1. We found that HeyL forms heterodimeric complexes with Hes1 in living cells. Moreover, our ChIP-seq experiments demonstrated that, compared with HeyL alone, the HeyL-Hes1 heterodimer binds with high affinity to specific sites in the chromatin, including the binding sites of Hey1. Finally, analyses of myogenin promoter activity showed that HeyL and Hes1 act synergistically to suppress myogenic differentiation. Collectively, these results suggest that HeyL and Hey1 function redundantly in MuSCs, and that HeyL requires Hes1 for effective DNA binding and biological activity..
10. Kazu Kobayakawa, Kyleigh Alexis DePetro, Hui Zhong, Bau Pham, Masamitsu Hara, Akihito Harada, Jumpei Nogami, Yasuyuki Ohkawa, V. Reggie Edgerton, Locomotor Training Increases Synaptic Structure With High NGL-2 Expression After Spinal Cord Hemisection, Neurorehabilitation and Neural Repair, 10.1177/1545968319829456, 2019.01, Background. We previously demonstrated that step training leads to reorganization of neuronal networks in the lumbar spinal cord of rodents after a hemisection (HX) injury and step training, including increases excitability of spinally evoked potentials in hindlimb motor neurons. Methods. In this study, we investigated changes in RNA expression and synapse number using RNA-Seq and immunohistochemistry of the lumbar spinal cord 23 days after a mid-thoracic HX in rats with and without post-HX step training. Results. Gene Ontology (GO) term clustering demonstrated that expression levels of 36 synapse-related genes were increased in trained compared with nontrained rats. Many synaptic genes were upregulated in trained rats, but Lrrc4 (coding NGL-2) was the most highly expressed in the lumbar spinal cord caudal to the HX lesion. Trained rats also had a higher number of NGL-2/synaptophysin synaptic puncta in the lumbar ventral horn. Conclusions. Our findings demonstrate clear activity-dependent regulation of synapse-related gene expression post-HX. This effect is consistent with the concept that activity-dependent phenomena can provide a mechanistic drive for epigenetic neuronal group selection in the shaping of the reorganization of synaptic networks to learn the locomotion task being trained after spinal cord injury..
11. Akihito Harada, Kazumitsu Maehara, Yusuke Ono, Hiroyuki Taguchi, Kiyoshi Yoshioka, Yasuo Kitajima, Yan Xie, Yuko Sato, Takeshi Iwasaki, Jumpei Nogami, Seiji Okada, Tetsuro Komatsu, Yuichiro Semba, Tatsuya Takemoto, Hiroshi Kimura, Hitoshi Kurumizaka, Yasuyuki Ohkawa, Histone H3.3 sub-variant H3mm7 is required for normal skeletal muscle regeneration, Nature Communications, 10.1038/s41467-018-03845-1, 9, 1, 2018.12, Regulation of gene expression requires selective incorporation of histone H3 variant H3.3 into chromatin. Histone H3.3 has several subsidiary variants but their functions are unclear. Here we characterize the function of histone H3.3 sub-variant, H3mm7, which is expressed in skeletal muscle satellite cells. H3mm7 knockout mice demonstrate an essential role of H3mm7 in skeletal muscle regeneration. Chromatin analysis reveals that H3mm7 facilitates transcription by forming an open chromatin structure around promoter regions including those of myogenic genes. The crystal structure of the nucleosome containing H3mm7 reveals that, unlike the S57 residue of other H3 proteins, the H3mm7-specific A57 residue cannot form a hydrogen bond with the R40 residue of the cognate H4 molecule. Consequently, the H3mm7 nucleosome is unstable in vitro and exhibited higher mobility in vivo compared with the H3.3 nucleosome. We conclude that the unstable H3mm7 nucleosome may be required for proper skeletal muscle differentiation..
12. K. Shiraishi, A. Shindo, Akihito Harada, H. Kurumizaka, H. Kimura, Yasuyuki Ohkawa, H. Matsuyama, Roles of histone H3.5 in human spermatogenesis and spermatogenic disorders, Andrology, 10.1111/andr.12438, 6, 1, 158-165, 2018.01, Histone H3.5 (H3.5) is a newly identified histone variant highly expressed in the human testis. We have reported the crystal structure, instability of the H3.5 nucleosome and accumulation around transcription start sites, mainly in primary spermatocytes, but its role in human spermatogenesis remains poorly understood. Testicular biopsy specimens from 30 men (mean age: 35 years) with non-obstructive azoospermia (NOA) who underwent microdissection testicular sperm extraction and 23 men with obstructive azoospermia (OA) were included. An H3.5-specific mouse monoclonal antibody recognizing an H3.5-specific synthetic peptide was generated, and immunohistological staining for H3.5 and proliferating cell nuclear antigen (PCNA) was performed on Bouin's solution-fixed sections. Expression and localization of H3.5 were compared with patient background, germinal stage, and PCNA expression. In testes of patients with normal spermatogenesis, differentially expressed H3.5 was specifically localized in either spermatogonia or preleptotene/leptotene-stage primary spermatocytes, especially during germinal stages VI–X. In NOA testes, mRNA expression of H3.5 (H3F3C) was significantly reduced compared with other H3 histone family members, and expression of H3.5 was significantly lower than that in OA. Additionally, the number of H3.5-positive germ cells was higher in hypospermatogenesis or late maturation arrest than in early maturation arrest in NOA testes (p < 0.01). A significant positive correlation was observed between H3.5 and PCNA expression (p < 0.05) but not TUNEL-positive cells, and expression of H3.5 was enhanced after hCG-based salvage hormonal therapy. Different from other testis-specific histones, which are often expressed during the histone-to-protamine transition during meiosis, H3.5 was expressed mainly in immature germ cells. H3.5 may play roles in DNA synthesis, but not apoptosis, and its expression is regulated by gonadotropins, indicating that such epigenetic regulations are important in normal spermatogenesis and spermatogenic disorders..
13. Yukari Kondo, Shinichiro Higa, Takeshi Iwasaki, Tomoya Matsumoto, Kazumitsu Maehara, Akihito Harada, Yoshihiro Baba, Masatoshi Fujita, Yasuyuki Ohkawa, Sensitive detection of fluorescence in western blotting by merging images, PLoS One, 10.1371/journal.pone.0191532, 13, 1, 2018.01, The western blotting technique is widely used to analyze protein expression levels and protein molecular weight. The chemiluminescence method is mainly used for detection due to its high sensitivity and ease of manipulation, but it is unsuitable for detailed analyses because it cannot be used to detect multiple proteins simultaneously. Recently, more attention has been paid to the fluorescence detection method because it is more quantitative and is suitable for the detection of multiple proteins simultaneously. However, fluorescence detection can be limited by poor image resolution and low detection sensitivity. Here, we describe a method to detect fluorescence in western blots using fluorescence microscopy to obtain high-resolution images. In this method, filters and fluorescent dyes are optimized to enhance detection sensitivity to a level similar to that of the chemiluminescence method..
14. Akihito Harada, Yasuyuki Ohkawa, Anthony N. Imbalzano, Temporal regulation of chromatin during myoblast differentiation, Seminars in Cell and Developmental Biology, 10.1016/j.semcdb.2017.10.022, 72, 77-86, 2017.12, The commitment to and execution of differentiation programmes involves a significant change in gene expression in the precursor cell to facilitate development of the mature cell type. In addition to being regulated by lineage-determining and auxiliary transcription factors that drive these changes, the structural status of the chromatin has a considerable impact on the transcriptional competence of differentiation-specific genes, which is clearly demonstrated by the large number of cofactors and the extraordinary complex mechanisms by which these genes become activated. The terminal differentiation of myoblasts to myotubes and mature skeletal muscle is an excellent system to illustrate these points. The MyoD family of closely related, lineage-determining transcription factors directs, largely through targeting to chromatin, a cascade of cooperating transcription factors and enzymes that incorporate or remove variant histones, post-translationally modify histones, and alter nucleosome structure and positioning via energy released by ATP hydrolysis. The coordinated action of these transcription factors and enzymes prevents expression of differentiation-specific genes in myoblasts and facilitates the transition of these genes from transcriptionally repressed to activated during the differentiation process. Regulation is achieved in both a temporal as well as spatial manner, as at least some of these factors and enzymes affect local chromatin structure at myogenic gene regulatory sequences as well as higher-order genome organization. Here we discuss the transition of genes that promote myoblast differentiation from the silenced to the activated state with an emphasis on the changes that occur to individual histones and the chromatin structure present at these loci..
15. Yuichiro Semba, Akihito Harada, Kazumitsu Maehara, Shinya Oki, Chikara Meno, Jun Ueda, Kazuo Yamagata, Atsushi Suzuki, Mitsuho Onimaru, Jumpei Nogami, Seiji Okada, Koichi Akashi, Yasuyuki Ohkawa, Chd2 regulates chromatin for proper gene expression toward differentiation in mouse embryonic stem cells, Nucleic Acids Research, 10.1093/nar/gkx475, 45, 15, 8758-8772, 2017.09, Chromatin reorganization is necessary for pluripotent stem cells, including embryonic stem cells (ESCs), to acquire lineage potential. However, it remains unclear how ESCs maintain their characteristic chromatin state for appropriate gene expression upon differentiation. Here, we demonstrate that chromodomain helicase DNA-binding domain 2 (Chd2) is required to maintain the differentiation potential of mouse ESCs. Chd2-depleted ESCs showed suppressed expression of developmentally regulated genes upon differentiation and subsequent differentiation defects without affecting gene expression in the undifferentiated state. Furthermore, chromatin immunoprecipitation followed by sequencing revealed alterations in the nucleosome occupancy of the histone variant H3.3 for developmentally regulated genes in Chd2-depleted ESCs, which in turn led to elevated trimethylation of the histone H3 lysine 27. These results suggest that Chd2 is essential in preventing suppressive chromatin formation for developmentally regulated genes and determines subsequent effects on developmental processes in the undifferentiated state..
16. Hiroyuki Taguchi, Yan Xie, Naoki Horikoshi, Kazumitsu Maehara, Akihito Harada, Jumpei Nogami, Koichi Sato, Yasuhiro Arimura, Akihisa Osakabe, Tomoya Kujirai, Takeshi Iwasaki, Yuichiro Semba, Taro Tachibana, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Crystal Structure and Characterization of Novel Human Histone H3 Variants, H3.6, H3.7, and H3.8, Biochemistry, 10.1021/acs.biochem.6b01098, 56, 16, 2184-2196, 2017.04, 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..
17. Koji Shiraishi, Aya Shindo, Akihito Harada, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Hiroshi Kimura, Hideyasu Matsuyama, ROLES OF HISTONE H3.5 IN HUMAN SPERMATOGENESIS AND SPERMATOGENIC DISORDERS, JOURNAL OF UROLOGY, 10.1016/j.juro.2017.02.277, 197, 4, E85-E85, 2017.04.
18. Kazuya Yokota, Kazu Kobayakawa, Takeyuki Saito, Masamitsu Hara, Ken Kijima, Yasuyuki Ohkawa, Akihito Harada, Ken Okazaki, Kohei Ishihara, Shigeo Yoshida, Akira Kudo, Yukihide Iwamoto, Seiji Okada, Periostin Promotes Scar Formation through the Interaction between Pericytes and Infiltrating Monocytes/Macrophages after Spinal Cord Injury, American Journal of Pathology, 10.1016/j.ajpath.2016.11.010, 187, 3, 639-653, 2017.03, Scar formation is a prominent pathological feature of traumatic central nervous system (CNS) injury, which has long been implicated as a major impediment to the CNS regeneration. However, the factors affecting such scar formation remain to be elucidated. We herein demonstrate that the extracellular matrix protein periostin (POSTN) is a key player in scar formation after traumatic spinal cord injury (SCI). Using high-throughput RNA sequencing data sets, we found that the genes involved in the extracellular region, such as POSTN, were significantly expressed in the injured spinal cord. The expression of POSTN peaked at 7 days after SCI, predominantly in the scar-forming pericytes. Notably, we found that genetic deletion of POSTN in mice reduced scar formation at the lesion site by suppressing the proliferation of the pericytes. Conversely, we found that recombinant POSTN promoted the migration capacity of the monocytes/macrophages and increased the expression of tumor necrosis factor-α from the monocytes/macrophages in vitro, which facilitated the proliferation of pericytes. Furthermore, we revealed that the pharmacological blockade of POSTN suppressed scar formation and improved the long-term functional outcome after SCI. Our findings suggest a potential mechanism whereby POSTN regulates the scar formation after SCI and provide significant evidence that POSTN is a promising therapeutic target for CNS injury..
19. Jun Ueda, Akihito Harada, Takashi Urahama, Shinichi Machida, Kazumitsu Maehara, Masashi Hada, Yoshinori Makino, Jumpei Nogami, Naoki Horikoshi, Akihisa Osakabe, Hiroyuki Taguchi, Hiroki Tanaka, Hiroaki Tachiwana, Tatsuma Yao, Minami Yamada, Takashi Iwamoto, Ayako Isotani, Masahito Ikawa, Taro Tachibana, Yuki Okada, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Kazuo Yamagata, Testis-Specific Histone Variant H3t Gene Is Essential for Entry into Spermatogenesis, Cell Reports, 10.1016/j.celrep.2016.12.065, 18, 3, 593-600, 2017.01, 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..
20. Kensuke Kudou, Tetsuro Komatsu, Jumpei Nogami, Kazumitsu Maehara, Akihito Harada, Hiroshi Saeki, Eiji Oki, Yoshihiko Maehara, Yasuyuki Ohkawa, The requirement of Mettl3-promoted MyoD mRNA maintenance in proliferative myoblasts for skeletal muscle differentiation, Open Biology, 10.1098/rsob.170119, 7, 9, 2017.01, Myogenic progenitor/stem cells retain their skeletal muscle differentiation potential by maintaining myogenic transcription factors such as MyoD. However, the mechanism of how MyoD expression is maintained in proliferative progenitor cells has not been elucidated. Here, we found that MyoD expression was reduced at the mRNA level by cell cycle arrest in S and G2 phases, which in turn led to the absence of skeletal muscle differentiation. The reduction of MyoD mRNA was correlated with the reduced expression of factors regulating RNA metabolism, including methyltransferase like 3 (Mettl3), which induces N6-methyladenosine (m6A) modifications of RNA. Knockdown of Mettl3 revealed that MyoD RNA was specifically downregulated and that this was caused by a decrease in processed, but not unprocessed, mRNA. Potential m6A modification sites were profiled by m6A sequencing and identified within the 50 untranslated region (UTR) of MyoD mRNA. Deletion of the 50 UTR revealed that it has a role in MyoD mRNA processing. These data showed that Mettl3 is required for MyoD mRNA expression in proliferative myoblasts..
21. Yuki Kuniyoshi, Kazumitsu Maehara, Takeshi Iwasaki, Masayasu Hayashi, Yuichiro Semba, Masatoshi Fujita, Yuko Sato, Hiroshi Kimura, Akihito Harada, Yasuyuki Ohkawa, Identification of immunoglobulin gene sequences from a small read number of mRNA-seq using hybridomas, PLoS One, 10.1371/journal.pone.0165473, 11, 10, 2016.10, Identification of immunoglobulin genes in hybridomas is essential for producing antibodies for research and clinical applications. A couple of methods such as RACE and degenerative PCR have been developed for determination of the Igh and Igl/Igk coding sequences (CDSs) but it has been difficult to process a number of hybridomas both with accuracy and rapidness. Here, we propose a new strategy for antibody sequence determination by mRNA-seq of hybridomas. We demonstrated that hybridomas highly expressed the Igh and Igl/Igk genes and that de novo transcriptome assembly using mRNA-seq data enabled identification of the CDS of both Igh and Igl/Igk accurately. Furthermore, we estimated that only 30,000 sequenced reads are required to identify immunoglobulin sequences from four different hybridoma clones. Thus, our approach would facilitate determining variable CDSs drastically..
22. Jun Ya Kaimori, Kazumitsu Maehara, Yoko Hayashi-Takanaka, Akihito Harada, Masafumi Fukuda, Satoko Yamamoto, Naotsugu Ichimaru, Takashi Umehara, Shigeyuki Yokoyama, Ryo Matsuda, Tsuyoshi Ikura, Koji Nagao, Chikashi Obuse, Naohito Nozaki, Shiro Takahara, Toshifumi Takao, Yasuyuki Ohkawa, Hiroshi Kimura, Yoshitaka Isaka, Histone H4 lysine 20 acetylation is associated with gene repression in human cells, Scientific Reports, 10.1038/srep24318, 6, 2016.04, Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression..
23. Masayasu Hayashi, Kazumitsu Maehara, Akihito Harada, Yuichiro Semba, Kensuke Kudo, Hidehisa Takahashi, Shinya Oki, Chikara Meno, Kenji Ichiyanagi, Koichi Akashi, Yasuyuki Ohkawa, Chd5 Regulates MuERV-L/MERVL Expression in Mouse Embryonic Stem Cells Via H3K27me3 Modification and Histone H3.1/H3.2, Journal of Cellular Biochemistry, 10.1002/jcb.25368, 117, 3, 780-792, 2016.03, Chd5 is an essential factor for neuronal differentiation and spermatogenesis and is a known tumor suppressor. H3K27me3 and H3K4un are modifications recognized by Chd5; however, it remains unclear how Chd5 remodels chromatin structure. We completely disrupted the Chd5 locus using the CRISPR-Cas9 system to generate a 52 kbp long deletion and analyzed Chd5 function in mouse embryonic stem cells. Our findings show that Chd5 represses murine endogenous retrovirus-L (MuERV-L/MERVL), an endogenous retrovirus-derived retrotransposon, by regulating H3K27me3 and H3.1/H3.2 function. J. Cell. Biochem. 117: 780-792, 2016..
24. Takashi Urahama, Akihito Harada, Kazumitsu Maehara, Naoki Horikoshi, Koichi Sato, Yuko Sato, Koji Shiraishi, Norihiro Sugino, Akihisa Osakabe, Hiroaki Tachiwana, Wataru Kagawa, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Histone H3.5 forms an unstable nucleosome and accumulates around transcription start sites in human testis, Epigenetics and Chromatin, 10.1186/s13072-016-0051-y, 9, 1, 2016.01, Background: 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. Results: 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. Conclusions: 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..
25. Yoko Hayashi-Takanaka, Kazumitsu Maehara, Akihito Harada, Takashi Umehara, Shigeyuki Yokoyama, Chikashi Obuse, Yasuyuki Ohkawa, Naohito Nozaki, Hiroshi Kimura, Distribution of histone H4 modifications as revealed by a panel of specific monoclonal antibodies, Chromosome Research, 10.1007/s10577-015-9486-4, 23, 4, 753-766, 2015.12, Post-translational histone modifications play a critical role in genome functions such as epigenetic gene regulation and genome maintenance. The tail of the histone H4 N-terminus contains several amino acids that can be acetylated and methylated. Some of these modifications are known to undergo drastic changes during the cell cycle. In this study, we generated a panel of mouse monoclonal antibodies against histone H4 modifications, including acetylation at K5, K8, K12, and K16, and different levels of methylation at K20. Their specificity was evaluated by ELISA and immunoblotting using synthetic peptide and recombinant proteins that harbor specific modifications or amino acid substitutions. Immunofluorescence confirmed the characteristic distributions of target modifications. An H4K5 acetylation (H4K5ac)-specific antibody CMA405 reacted with K5ac only when the neighboring K8 was unacetylated. This unique feature allowed us to detect newly assembled H4, which is diacetylated at K5 and K12, and distinguish it from hyperacetylated H4, where K5 and K8 are both acetylated. Chromatin immunoprecipiation combined with deep sequencing (ChIP-seq) revealed that acetylation of both H4K8 and H4K16 were enriched around transcription start sites. These extensively characterized and highly specific antibodies will be useful for future epigenetics and epigenome studies..
26. Kazumitsu Maehara, Akihito Harada, Yuko Sato, Masaki Matsumoto, Keiichi Nakayama, Hiroshi Kimura, Yasuyuki Ohkawa, Tissue-specific expression of histone H3 variants diversified after species separation, Epigenetics and Chromatin, 10.1186/s13072-015-0027-3, 8, 1, 2015.09, Background: The selective incorporation of appropriate histone variants into chromatin is critical for the regulation of genome function. Although many histone variants have been identified, a complete list has not been compiled. Results: We screened mouse, rat and human genomes by in silico hybridization using canonical histone sequences. In the mouse genome, we identified 14 uncharacterized H3 genes, among which 13 are similar to H3.3 and do not have human or rat counterparts, and one is similar to human testis-specific H3 variant, H3T/H3.4, and had a rat paralog. Although some of these genes were previously annotated as pseudogenes, their tissue-specific expression was confirmed by sequencing the 3′-UTR regions of the transcripts. Certain new variants were also detected at the protein level by mass spectrometry. When expressed as GFP-tagged versions in mouse C2C12 cells, some variants were stably incorporated into chromatin and the genome-wide distributions of most variants were similar to that of H3.3. Moreover, forced expression of H3 variants in chromatin resulted in alternate gene expression patterns after cell differentiation. Conclusions: We comprehensively identified and characterized novel mouse H3 variant genes that encoded highly conserved amino acid sequences compared to known histone H3. We speculated that the diversity of H3 variants acquired after species separation played a role in regulating tissue-specific gene expression in individual species. Their biological relevance and evolutionary aspect involving pseudogene diversification will be addressed by further functional analysis..
27. Akihito Harada, Chandrashekara Mallappa, Seiji Okada, John T. Butler, Stephen P. Baker, Jeanne B. Lawrence, Yasuyuki Ohkawa, Anthony N. Imbalzano, Spatial re-organization of myogenic regulatory sequences temporally controls gene expression, Nucleic Acids Research, 10.1093/nar/gkv046, 43, 4, 2008-2021, 2015.02, During skeletal muscle differentiation, the activation of some tissue-specific genes occurs immediately while others are delayed. The molecular basis controlling temporal gene regulation is poorly understood. We show that the regulatory sequences, but not other regions of genes expressed at late times of myogenesis, are in close physical proximity in differentiating embryonic tissue and in differentiating culture cells, despite these genes being located on different chromosomes. Formation of these inter-chromosomal interactions requires the lineage-determinant MyoD and functional Brg1, the ATPase subunit of SWI/SNF chromatin remodeling enzymes. Ectopic expression of myogenin and a specific Mef2 isoform induced myogenic differentiation without activating endogenous MyoD expression. Under these conditions, the regulatory sequences of late gene loci were not in close proximity, and these genes were prematurely activated. The data indicate that the spatial organization of late genes contributes to temporal regulation of myogenic transcription by restricting late gene expression during the early stages of myogenesis..
28. Akihito Harada, Kazumitsu Maehara, Yuko Sato, Daijiro Konno, Taro Tachibana, Hiroshi Kimura, Yasuyuki Ohkawa, Incorporation of histone H3.1 suppresses the lineage potential of skeletal muscle, Nucleic Acids Research, 10.1093/nar/gku1346, 43, 2, 775-786, 2015.01, Lineage potential is triggered by lineage-specific transcription factors in association with changes in the chromatin structure. Histone H3.3 variant is thought to play an important role in the regulation of lineage-specific genes. To elucidate the function of H3.3 in myogenic differentiation, we forced the expression of GFP-H3.1 to alter the balance between H3.1 and H3.3 in mouse C2C12 cells that could be differentiated into myotubes. GFP-H3.1 replaced H3.3 in the regulatory regions of skeletal muscle (SKM) genes and induced a decrease of H3K4 trimethylation (H3K4me3) and increase of H3K27 trimethylation (H3K27me3). Similar results were obtained by H3.3 knockdown. In contrast, MyoD-dependent H3.3 incorporation into SKM genes in fibroblasts induced an increase of H3K4me3 and H3K27me3. In mouse embryos, a bivalent modification of H3K4me3 and H3K27me3 was formed on H3.3-incorporated SKM genes before embryonic skeletal muscle differentiation. These results suggest that lineage potential is established through a selective incorporation of specific H3 variants that governs the balance of histone modifications..
29. Masakatsu Usui, Akihito Harada, Shinya Yasumoto, Yoshimasa Sugiura, Anri Nishidai, Maria Ikarashi, Honami Takaba, Taiko Miyasaki, Hiroyuki Azakami, Masakazu Kondo, Relationship between the risk for a shrimp allergy and freshness or cooking, Bioscience, Biotechnology and Biochemistry, 10.1080/09168451.2015.1045830, 79, 10, 1698-1701, 2015.01, Tropomyosins are defined as risk factors for shrimp allergy. However, their concentration in different preparations has not been clarified. We quantified the tropomyosin concentration in shrimp meat, which was cooked using several methods or was stored under various conditions. The results demonstrated that shrimp meat from various preparations and storage conditions maintained tropomyosin concentrations that were sufficient to cause food allergies..
30. Masako Tanaka, Maki Yamaguchi, Masayuki Shiota, Yukiko Kawamoto, Katsuyuki Takahashi, Azusa Inagaki, Mayuko Osada-Oka, Akihito Harada, Hideki Wanibuchi, Yasukatsu Izumi, Katsuyuki Miura, Hiroshi Iwao, Yasuyuki Ohkawa, Establishment of neutralizing rat monoclonal antibodies for fibroblast growth factor-2, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/mab.2013.0085, 33, 4, 261-269, 2014.08, Fibroblast growth factor-2 (FGF-2) plays a critical role in endothelial survival, proliferation, and angiogenesis and is localized on the cell membrane by binding to heparan sulfate proteoglycans. Here we established a neutralizing monoclonal antibody, 1B9B9, against FGF-2 using the rat medial iliac lymph node method. 1B9B9 blocked the binding of FGF-2 to its receptor, inhibiting FGF-2-induced proliferation and corresponding downstream signaling in endothelial cells. Treatment of human umbilical vein endothelial cells with 1B9B9 reduced the basal phosphorylation levels of Akt and MAPK. Furthermore, continued treatment with 1B9B9 induced cell death by apoptosis. Compared with FGF-2 knockdown, 1B9B9 significantly reduced cell survival. In addition, the combination of FGF-2 siRNA and 1B9B9 showed a synergistic effect. The data indicate that 1B9B9 established by the rat iliac lymph node method is a fully compatible neutralizing antibody..
31. Masako Tanaka, Saya Mun, Akihito Harada, Yasuyuki Ohkawa, Azusa Inagaki, Soichi Sano, Katsuyuki Takahashi, Yasukatsu Izumi, Mayuko Osada-Oka, Hideki Wanibuchi, Masayo Yamagata, Tokihito Yukimura, Katsuyuki Miura, Masayuki Shiota, Hiroshi Iwao, Hsc70 contributes to cancer cell survival by preventing Rab1A degradation under stress conditions, PLoS One, 10.1371/journal.pone.0096785, 9, 5, 2014.05, Heat shock cognate protein 70 (Hsc70) acts as a molecular chaperone for the maintenance of intracellular proteins, which allows cancer cells to survive under proteotoxic stress. We attempted to use Hsc70 to identify key molecules in cancer cell survival. Here, we performed mass-spectrometry-based proteomics analysis utilizing affinity purification with anti-Hsc70 antibodies; as a result, 83 differentially expressed proteins were identified under stress conditions. This result implies that there was a change in the proteins with which Hsc70 interacted in response to stress. Among the proteins identified under both serum-depleted and 5-fluorouracil-treated conditions, Rab1A was identified as an essential molecule for cancer cell survival. Hsc70 interacted with Rab1A in a chaperone-dependent manner. In addition, Hsc70 knockdown decreased the level of Rab1A and increased the level of its ubiquitination under stress conditions, suggesting that Hsc70 prevented the degradation of Rab1A denatured by stress exposure. We also found that Rab1A knockdown induced cell death by inhibition of autophagosome formation. Rab1A may therefore contribute to overcoming proteotoxic insults, which allows cancer cells to survive under stress conditions. Analysis of Hsc70 interactors provided insight into changes of intracellular status. We expect further study of the Hsc70 interactome to provide a more comprehensive understanding of cancer cell physiology..
32. Akihito Harada, Masayasu Hayashi, Yuuki Kuniyoshi, Yuichiro Semba, Satoko Sugahara, Taro Tachibana, Yasuyuki Ohkawa, Masatoshi Fujita, Generation of a monoclonal antibody for INI1/hSNF5/BAF47, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/mab.2013.0065, 33, 1, 49-51, 2014.02, INI1/hSNF5/BAF47, which has an SNF5 domain, belongs to the SWI/SNF family. This family is known as ATP-dependent regulators of gene expression by remodeling chromatin structure during cell differentiation. However, the detailed function of INI1/hSNF5/BAF47 is unclear. Here we report the generation of a specific monoclonal antibody for INI1/hSNF5/BAF47 by the mouse iliac lymph node method. The obtained antibody recognized two isoforms of INI1/hSNF5/BAF47 in immunoblotting and precisely recognized the nuclear localization of INI1/hSNF5/BAF47 in immunostaining. This antibody can contribute to further elucidation of the mechanisms of gene expression regulation by INI1/hSNF5/BAF47 during cell differentiation..
33. Akihito Harada, Etsuko Okazaki, Seiji Okada, Taro Tachibana, Yasuyuki Ohkawa, Production of a monoclonal antibody for C/EBPβ
The subnuclear localization of C/EBPβ in mouse L929 cells, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/mab.2013.0069, 33, 1, 34-37, 2014.02, The CCAAT/enhancer-binding protein (C/EBP)β belongs to the C/EBP family of proteins that possesses a basic leucine zipper DNA-binding domain. These proteins bind DNA by dimerization and play a role in the transcriptional regulation of various cells. There are six different types of C/EBPs, and some form isoforms through the use of alternative translation initiation sites. The functional analysis of the C/EBP family is therefore difficult to achieve. Here we report on the production of specific monoclonal antibodies against mouse C/EBPβ using a rat medial iliac lymph node method. Immunoblotting using C/EBPβ monoclonal antibodies identified two types of isoforms, while immunostaining revealed a subnuclear localization for C/EBPβ. Use of this antibody should contribute to the further elucidation of the transcriptional regulatory function of C/EBPβ..
34. Masakatsu Usui, Akihito Harada, Takayuki Ishimaru, Emiri Sakumichi, Fumihiko Saratani, Chiho Sato-Minami, Hiroyuki Azakami, Taiko Miyasaki, Ken'ichi Hanaoka, Contribution of structural reversibility to the heat stability of the tropomyosin shrimp allergen, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.120887, 77, 5, 948-953, 2013.06, Tropomyosins are common heat-stable crustacean allergens. However, their heat stability and their effects on antigenicity have not been clarified. We purified tropomyosin in this study from raw kuruma prawns (Marsupenaeus japonicus) without heat processing. SDS-PAGE of the purified protein showed a band at approximately 35 kDa that cross-reacted with IgE from the serum of a shrimp-allergic patient, identifying it as Pen j 1. The circular dichroism spectrum of native Pen j 1 revealed the common α-helical structure of tropomyosins which easily collapsed upon heating to 80 °C. However, there were no insoluble aggregates after heating, and the protein regained its native CD spectral pattern after cooling to 25 °C. There was no significant difference in total IgG production between mice sensitized with native and heated Pen j 1. These results suggest that heat-denatured Pen j 1 refolded upon cooling and maintained its antigenicity following the heat treatment..
35. Kazumitsu Maehara, Jun Odawara, Akihito Harada, Tomohiko Yoshimi, Koji Nagao, Chikashi Obuse, Koichi Akashi, Taro Tachibana, Toshio Sakata, Yasuyuki Ohkawa, A co-localization model of paired ChIP-seq data using a large ENCODE data set enables comparison of multiple samples, Nucleic Acids Research, 10.1093/nar/gks1010, 41, 1, 54-62, 2013.01, Deep sequencing approaches, such as chromatin immunoprecipitation by sequencing (ChIP-seq), have been successful in detecting transcription factor-binding sites and histone modification in the whole genome. An approach for comparing two different ChIP-seq data would be beneficial for predicting unknown functions of a factor. We propose a model to represent co-localization of two different ChIP-seq data. We showed that a meaningful overlapping signal and a meaningless background signal can be separated by this model. We applied this model to compare ChIP-seq data of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation with a large amount of peak-called data, including ChIP-seq and other deep sequencing data in the Encyclopedia of DNA Elements (ENCODE) project, and then extracted factors that were related to RNA polymerase II CTD serine 2 in HeLa cells. We further analyzed RNA polymerase II CTD serine 7 phosphorylation, of which their function is still unclear in HeLa cells. Our results were characterized by the similarity of localization for transcription factor/histone modification in the ENCODE data set, and this suggests that our model is appropriate for understanding ChIP-seq data for factors where their function is unknown..
36. Akihito Harada, Seiji Okada, Daijiro Konno, Jun Odawara, Tomohiko Yoshimi, Saori Yoshimura, Hiromi Kumamaru, Hirokazu Saiwai, Toshiaki Tsubota, Hitoshi Kurumizaka, Koichi Akashi, Taro Tachibana, Anthony N. Imbalzano, Yasuyuki Ohkawa, Chd2 interacts with H3.3 to determine myogenic cell fate, EMBO Journal, 10.1038/emboj.2012.136, 31, 13, 2994-3007, 2012.07, Cell differentiation is mediated by lineage-determining transcription factors. We show that chromodomain helicase DNA-binding domain 2 (Chd2), a SNF2 chromatin remodelling enzyme family member, interacts with MyoD and myogenic gene regulatory sequences to specifically mark these loci via deposition of the histone variant H3.3 prior to cell differentiation. Directed and genome-wide analysis of endogenous H3.3 incorporation demonstrates that knockdown of Chd2 prevents H3.3 deposition at differentiation-dependent, but not housekeeping, genes and inhibits myogenic gene activation. The data indicate that MyoD determines cell fate and facilitates differentiation-dependent gene expression through Chd2-dependent deposition of H3.3 at myogenic loci prior to differentiation..
37. M. Usui, A. Harada, T. Ishimaru, E. Sakumichi, F. Saratani, C. Sato, H. Azakami, T. Miyasaki, K. Hanaoka, Structural reversibility contributes the heat stability of shrimp allergen tropomyosin, Bioscience, Biotechnology, and Biochemistry, 77, 5, 2012.05.
38. Jun Odawara, Akihito Harada, Tomohiko Yoshimi, Kazumitsu Maehara, Taro Tachibana, Seiji Okada, Koichi Akashi, Yasuyuki Ohkawa, The classification of mRNA expression levels by the phosphorylation state of RNAPII CTD based on a combined genome-wide approach, BMC Genomics, 10.1186/1471-2164-12-516, 12, 2011.10, Background: Cellular function is regulated by the balance of stringently regulated amounts of mRNA. Previous reports revealed that RNA polymerase II (RNAPII), which transcribes mRNA, can be classified into the pausing state and the active transcription state according to the phosphorylation state of RPB1, the catalytic subunit of RNAPII. However, genome-wide association between mRNA expression level and the phosphorylation state of RNAPII is unclear. While the functional importance of pausing genes is clear, such as in mouse Embryonic Stem cells for differentiation, understanding this association is critical for distinguishing pausing genes from active transcribing genes in expression profiling data, such as microarrays and RNAseq. Therefore, we examined the correlation between the phosphorylation of RNAPII and mRNA expression levels using a combined analysis by ChIPseq and RNAseq.Results: We first performed a precise quantitative measurement of mRNA by performing an optimized calculation in RNAseq. We then visualized the recruitment of various phosphorylated RNAPIIs, such as Ser2P and Ser5P. A combined analysis using optimized RNAseq and ChIPseq for phosphorylated RNAPII revealed that mRNA levels correlate with the various phosphorylation states of RNAPII.Conclusions: We demonstrated that the amount of mRNA is precisely reflected by the phased phosphorylation of Ser2 and Ser5. In particular, even the most "pausing" genes, for which only Ser5 is phosphorylated, were detectable at a certain level of mRNA. Our analysis indicated that the complexity of quantitative regulation of mRNA levels could be classified into three categories according to the phosphorylation state of RNAPII..
39. Masako Tanaka, Masayuki Shiota, Seiji Okada, Akihito Harada, Jun Odawara, Saya Mun, Hiroshi Iwao, Yasuyuki Ohkawa, Generation of a rat monoclonal antibody specific for Hsp72, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2011.0015, 30, 4, 397-400, 2011.08, The heat shock protein 70 (Hsp70) family members function as ATP-dependent molecular chaperones that assist in the folding of newly synthesized polypeptides and in the refolding of misfolded/aggregated proteins. These heat shock proteins comprise at least eight sets of molecular groups that share high homology, but differ from each other in their expression level and subcellular localization. Hsp72, which is also known as Hsp70 and Hsp70-1, is localized mainly in the cytoplasm but is also found in the nucleus. Stress-induced Hsp72 functions as a chaperone enabling the cells to cope with harmful aggregations of denatured proteins during and following stress. The difference in the function of Hsp72 from that of other Hsp70 members, however, remains unclear. We report the establishment of a monoclonal antibody specific for Hsp72 using the rat medial iliac lymph node method. Immunoblot analysis revealed that our monoclonal antibody against Hsp72 specifically identified the 65 kDa protein. Immunocytochemical staining also revealed that Hsp72 localized in the cytoplasm and nucleus, and aggregated in the nucleus in response to heat stress. This MAb against Hsp72 will allow for further studies to elucidate the mechanism by which Hsp72 is localized in the cell in response to stress stimuli, and aid in the identification of specific interacting molecules..
40. Seiji Okada, Hirokazu Saiwai, Hiromi Kumamaru, Kensuke Kubota, Akihito Harada, Masahiro Yamaguchi, Yukihide Iwamoto, Yasuyuki Ohkawa, Flow cytometric sorting of neuronal and glial nuclei from central nervous system tissue, Journal of Cellular Physiology, 10.1002/jcp.22365, 226, 2, 552-558, 2011.02, Due to the complex cellular heterogeneity of the central nervous system (CNS), it is relatively difficult to reliably obtain molecular descriptions with cell-type specificity. In particular, comparative analysis of epigenetic regulation or molecular profiles is hampered by the lack of adequate methodology for selective purification of defined cell populations from CNS tissue. Here, we developed a direct purification strategy of neural nuclei from CNS tissue based on fluorescence-activated cell sorting (FACS). We successfully fractionated nuclei from complex tissues such as brain, spinal cord, liver, kidney, and skeletal muscle extruded mechanically or chemically, and fractionated nuclei were structurally maintained and contained nucleoproteins and nuclear DNA/RNA. We collected sufficient numbers of nuclei from neurons and oligodendrocytes using FACS with immunolabeling for nucleoproteins or from genetically labeled transgenic mice. In addition, the use of Fab fragments isolated from papain antibody digests, which effectively enriched the specialized cell populations, significantly enhanced the immunolabeling efficacy. This methodology can be applied to a wide variety of heterogeneous tissues and is crucial for understanding the cell-specific information about chromatin dynamics, nucleoproteins, protein-DNA/RNA interactions, and transcriptomes retained in the nucleus, such as non-coding RNAs..
41. Masayuki Shiota, Hirokazu Saiwai, Saya Mun, Akihito Harada, Seiji Okada, Jun Odawara, Masako Tanaka, Hiroshi Iwao, Yasuyuki Ohkawa, Generation of a rat monoclonal antibody specific for heat shock cognate protein 70, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2010.0024, 29, 5, 453-456, 2010.10, Human heat shock cognate protein 70 (Hsc70), also known as Hsp73 and Hsp70-8, is a molecular chaperone. The human Hsp70 family comprises at least eight different molecular groups with strong homology. Among them, Hsc70 and Hsp72 share 86% homology. Both Hsp72 and Hsc70 localize in the cell cytoplasm and the nucleus. While Hsp72 expression is enhanced by stress, Hsc70 is constitutively expressed, suggesting that Hsc70 is critically involved in cell functions other than the stress response. Hsc70 has cell-specific and tissue-specific functions, such as cellular signaling, but its functions are not well understood. To further study the functions of Hsc70, we established a monoclonal antibody specific for Hsc70 using a rat medial iliac lymph node method. Immunoblot analysis with this antibody revealed that it specifically recognizes Hsc70. Immunocytochemical staining using this newly established antibody revealed that Hsc70 localizes predominantly in the cytoplasm in unstressed cells, whereas oxidative stress produced by H2O 2 induces Hsc70 to translocate into the nucleus. This monoclonal antibody will be useful for further studies of Hsc70, including changes in its intracellular location, binding molecules, and functions..
42. Manato Kotani, Akihito Harada, Jun Odawara, Masayuki Azuma, Seiji Okada, Yuko Nishiyama, Mako Nakamura, Taro Tachibana, Yasuyuki Ohkawa, Monoclonal antibody specific for Dhx9/NDHII/RHA, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2009.0107, 29, 3, 259-261, 2010.06, Dhx9/NDHII/RHA is a member of the DEAH family of proteins, which possess a double-stranded RNA-binding domain (dsRBD) and a helicase domain. The DEAH protein family plays a critical role in RNA metabolism. DEAH family members function as ATP-dependent RNA helicases and regulation of transcription. In the present study, we report the establishment of a monoclonal antibody specific for Dhx9 using the rat medial iliac lymph node method. Immunoblot analysis using our antibody against Dhx9 detected full-length Dhx9. In addition, immunocytochemical staining using our antibody against Dhx9 revealed the nuclear localization of Dhx9. This monoclonal antibody against Dhx9 will allow for further detailed studies of Dhx9 expression..
43. Saori Yoshimura, Akihito Harada, Jun Odawara, Masayuki Azuma, Seiji Okada, Mako Nakamura, Yasuyuki Ohkawa, Taro Tachibana, Rat monoclonal antibody specific for the chromatin remodeling factor, CHD1, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2009.0106, 29, 3, 237-240, 2010.06, CHD1 is a subfamily member of the CHD family, which possesses a chromodomain, a helicase domain, and a DNA-binding domain. The CHD family regulates gene expression by contributing to ATP-dependent chromatin remodeling. CHD1 exists in the transcriptionally active region and alters the chromatin structure. Little is known about the function of endogenous CHD1, however, and studies have been hindered by the lack of an antibody specific for CHD1 in mammals. In the present study, we established a monoclonal antibody specifically against CHD1 using the rat medial iliac lymph node method. Immunoblot analysis using our monoclonal antibody showed specific binding to CHD1, allowing us to identify the deduced full-length CHD1. In addition, cell immunostaining clearly revealed the nuclear localization of CHD1. This monoclonal antibody will be useful for further analysis of CHD1 function in mammals..
44. Akihito Harada, Yasuyuki Ohkawa, Shinpei Ao, Jun Odawara, Seiji Okada, Masayuki Azuma, Yuko Nishiyama, Mako Nakamura, Taro Tachibana, Rat monoclonal antibody specific for MyoD, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2009.0117, 29, 3, 255-258, 2010.06, Myogenic determination 1 (MyoD) is a myogenic regulatory factor (MRF) possessing a basic domain and a helix-loop-helix domain. MRFs play a critical role in myoblast fate and terminal differentiation. MyoD is a transcriptional factor that induces transcription by binding with gene regulatory factors expressed in skeletal muscle. As a master gene, MyoD also determines skeletal muscle differentiation. In this study, we established a monoclonal antibody specific for MyoD using the rat medial iliac lymph node method. Immunoblot analysis revealed that our monoclonal antibody against MyoD could identify full-length MyoD. Moreover, immunocytochemical staining revealed a change in the expression of MyoD at the skeletal muscle differentiation stage. This monoclonal antibody against MyoD allows for further studies to elucidate the mechanism by which MyoD influences skeletal muscle differentiation..
45. Akihito Harada, Saori Yoshimura, Jun Odawara, Masayuki Azuma, Seiji Okada, Mako Nakamura, Taro Tachibana, Yasuyuki Ohkawa, Generation of a rat monoclonal antibody specific for Chd2, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2009.0090, 29, 2, 173-177, 2010.04, CHD2 is a member of the CHD family that contains chromodomain, helicase domain as well as DNA-binding domain. The CHD family is involved in gene expression and transcription by ATP-dependent chromatin remodeling. Analysis of mutant mouse revealed that CHD2 is involved in development as well as hematopoiesis, which suggests the involvement of CHD2 in gene expression. However, CHD2 has not yet been analyzed biochemically as there is no specific antibody against it. Here, we report on the establishment of specific monoclonal antibody (MAb) against CHD2 utilizing a rat medial iliac lymph node method. Through cell immunostaining utilizing established MAb to CHD2, we confirmed that CHD2 was localized in euchromatin. Additionally, IP-Western revealed that the expression level of full-length CHD2 did not change during the differentiation stage. Additionally, a specific signal was confirmed around 95 kDa at the undifferentiated stage. This clearly indicated that CHD2 was involved in specific gene expression at this stage. Thus, this antibody can contribute to elucidating the function of CHD2 in cell expression..
46. Akihito Harada, Seiji Okada, Jun Odawara, Hiromi Kumamaru, Hirokazu Saiwai, Mayumi Aoki, Mako Nakamura, Yuko Nishiyama, Yasuyuki Ohkawa, Production of a rat monoclonal antibody specific for Myf5, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2009.0066, 29, 1, 59-62, 2010.02, Myogenic regulatory factors (MRFs) are transcription factors that possess a characteristic basic helix-loop-helix domain. Myf5, MyoD, MRF4, and myogenin are well-known MRF family members that activate muscle-specific genes during differentiation. Myf5 is expressed first among MRFs at the very early phase and plays an important role in myoblast specificity and cell proliferation. Myf5 shares high homology with MyoD, and therefore some commercial Myf5 antibodies are cross-reactive for Myf5 and MyoD. To allow for detailed studies of the function of Myf5, we generated a monoclonal antibody specific for Myf5 utilizing a rat medial iliac lymph node method. Immunoblot analysis using our monoclonal antibody enabled us to identify Myf5 protein from rat myoblast L6E9 cell extract. Moreover, cell immunostaining revealed the nuclear localization of Myf5 in the L6E9 cells. This monoclonal antibody against Myf5 will allow us to perform further detailed studies of Myf5 and Myf5 function..
47. Hirokazu Saiwai, Yasuyuki Ohkawa, Hisakata Yamada, Hiromi Kumamaru, Akihito Harada, Hideyuki Okano, Takehiko Yokomizo, Yukihide Iwamoto, Seiji Okada, The LTB4-BLT1 axis mediates neutrophil infiltration and secondary injury in experimental spinal cord injury, American Journal of Pathology, 10.2353/ajpath.2010.090839, 176, 5, 2352-2366, 2010.01, Traumatic injury in the central nervous system induces inflammation; however, the role of this inflammation is controversial. Precise analysis of the inflammatory cells is important to gain a better understanding of the inflammatory machinery in response to neural injury. Here, we demonstrated that leukotriene B4 plays a significant role in mediating leukocyte infiltration after spinal cord injury. Using flow cytometry, we revealed that neutrophil and monocyte/macrophage infiltration peaked 12 hours after injury and was significantly suppressed in leukotriene B4 receptor 1 knockout mice. Similar findings were observed in mice treated with a leukotriene B4 receptor antagonist. Further, by isolating each inflammatory cell subset with a cell sorter, and performing quantitative reverse transcription-PCR, we demonstrated the individual contributions of more highly expressed subsets, ie, interleukins 6 and 1β, tumor necrosis factor-α, and FasL, to the inflammatory reaction and neural apoptosis. Inhibition of leukotriene B4 suppressed leukocyte infiltration after injury, thereby attenuating the inflammatory reaction, sparing the white matter, and reducing neural apoptosis, as well as inducing better functional recovery. These findings are the first to demonstrate that leukotriene B4 is involved in the pathogenesis of spinal cord injury through the amplification of leukocyte infiltration, and provide a potential therapeutic strategy for traumatic spinal cord injury..
48. Akihito Harada, Seiji Okada, Hirokazu Saiwai, Mayumi Aoki, Mako Nakamura, Yasuyuki Ohkawa, Generation of a rat monoclonal antibody specific for Pax7, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2009.0039, 28, 6, 451-453, 2009.12, Pax7 is a nuclear localization protein, well known as a member of the paired box family. It is expressed at a very early stage of muscle differentiation and is also found in muscle satellite cells that are recognized as muscle stem cells. Pax7 is also recognized as a tumor cell marker since it is greatly expressed in various types of tumor cells. Pax7 has homology among other paired family members and is not easy to distinguish one from the others. In this study, we report on the establishment of monoclonal antibodies (MAb) against Pax7 using a rat medial iliac lymph node method. The quality of the antibody was examined by immunoblotting analysis. It was confirmed that the antibody can specifically recognize the Pax7 protein. It was also revealed that the MAb antibody successfully recognizes the nuclear localized Pax7 protein in Ewing's sarcoma cells by immunocytochemistry. The antibody can clearly show the regions of euchromatin and heterochromatin where hoechst is positive..
49. Seiji Okada, Akihito Harada, Hirokazu Saiwai, Mako Nakamura, Yasuyuki Ohkawa, Generation of a rat monoclonal antibody specific for Brm, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2009.0044, 28, 6, 455-458, 2009.12, Brm is a subunit of the SWI/SNF complex that has a ATPase activity. It is well known that the complex plays a major role in cell processes, such as proliferation, differentiation, and DNA repair of cells. Here we report the production of monoclonal antibody (1H7A10) against Brm by rat medial iliac lymph node method. Immunoblot analysis with the antibody revealed the specific recognition of Brm and increase of Brm protein level in skeletal muscle differentiation. Immunocytochemistry analysis shows nuclear localization in myoblast C2C12 and involvement of transcription in the late stages of differentiation..
50. Yasuyuki Ohkawa, Akihito Harada, Mako Nakamura, Saori Yoshimura, Taro Tachibana, Production of a rat monoclonal antibody against Brg1, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2009.0041, 28, 6, 463-466, 2009.12, Brm-related gene-1 (Brg1) is a catalytic subunit of the SWI/SNF chromatin remodeling enzyme complex that has ATPase activity. This complex facilitates chromatin remodeling for gene expression by utilizing energy for ATP hydrolysis. It is well known that the SWI/SNF chromatin remodeling enzyme complex is essential for cell differentiation, cell cycle regulation, and embryogenesis. Here we report the establishment of a hybridoma cell line for producing an antibody against Brg1 subunit by the rat medial iliac lymph node method. Immunoblot analysis showed that our antibody can specifically recognize Brg1. It was revealed by immunocytochemistry that Brg1 is located in euchromatin of C2C12 myoblast nuclei. These data suggested this antibody is useful for analyzing molecular function of Brg1 protein in cells..
51. Akihito Harada, Hiroyuki Azakami, Akio Kato, Amyloid fibril formation of hen lysozyme depends on the instability of the C-helix (88-99), Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.80032, 72, 6, 1523-1530, 2008.07, Stable and unstable mutant lysozymes in long helices B and C were constructed to evaluate the effect of the helices on amyloid fibril formation at pH 2. Stable mutant N27D and unstable mutant K33D in the B-helix did not change in amyloid fibril formation. In contrast, stable mutant N93D and unstable mutant K97D in the C-helix showed big differences in behavior as to amyloid fibril formation. Stable mutant N93D showed a longer lag phase of aggregation and suppressed the amyloid fibril formation, whereas unstable mutant K97D showed a shorter lag phase of aggregation and accelerated amyloid fibril formation. These results suggest that the long C-helix is involved mainly in the α-helix to β-sheet transition during amyloid formation of lysozyme..
52. Akihito Harada, Hiroshi Yagi, Akira Saito, Hiroyuki Azakami, Akio Kato, Relationship between the stability of hen egg-white lysozymes mutated at sites designed to interact with α-helix dipoles and their secretion amounts in yeast, Bioscience, Biotechnology and Biochemistry, 10.1271/bbb.70354, 71, 12, 2952-2961, 2007.12, The positively charged lysine at the C-terminals of three long α-helices (5-15, 25-35, and 88-99) was replaced with alanine (K13A, K33A, K97A) or aspartic acid (K13D, K33D, K97D) in hen lysozyme by genetic engineering. The denaturation transition point (Tm) and Gibbs energy change ΔG of the mutant lysozymes decreased remarkably, suggesting that the positive charge at the C-terminals of helices is involved in the stabilization of the helix dipole. On the other hand, the non-charged asparagine at the N-terminal of the long α-helices (25-35 and 88-99) was replaced with negatively charged aspartic acid (N27D and N93D). The Tm and ΔG of N27D increased, suggesting that the dipole moment of the N-terminal of the helices is diminished by replacement with negatively charged amino acid strengthening the stability of the helices. The stabilities of those hen egg white lysozymes mutated at the N- or Cterminal sites of the three long α-helices were related with their secretion amounts in yeast (Pichia pastoris). The secretion amounts of these mutant lysozymes in yeast were closely correlated with their stability..
53. Jianwei He, Youtao Song, Nobuhiro Ueyama, Akihito Harada, Hiroyuki Azakami, Akio Kato, Characterization of recombinant amyloidogenic chicken cystatin mutant I66Q expressed in yeast, Journal of Biochemistry, 10.1093/jb/mvi064, 137, 4, 477-485, 2005.04, Amyloidogenic chicken cystatin mutant I66Q (cC I66Q) was successfully secreted by yeasts Pichia pastoris and Saccharomyces cerevisiae. The soluble monomer and dimer forms of amyloidogenic cC I66Q were found in the culture medium, while large amounts of insoluble aggregate and polymeric form cC I66Q besides the monomer and dimer forms were secreted into the culture medium. The amyloidogenic cC I66Q showed a comparable circular dichroism spectrum to that of the wild cystatin, and the monomer form exhibited a similar level of inhibitory activity toward papain, but the dimmer form did not. During storage of amyloidogenic cC I66Q under physiological and acidic conditions, typical binding with Congo red and thioflavin T, and the formation of amyloid fibrils were observed, whereas the characteristic of similar amyloidosis was hardly detected for the wild recombinant cystatin..
54. Jianwei He, Takashi Sakamoto, Youtao Song, Akira Saito, Akihito Harada, Hiroyuki Azakami, Akio Kato, Effect of EPS1 gene deletion in Saccharomyces cerevisiae on the secretion of foreign proteins which have disulfide bridges, FEBS Letters, 10.1016/j.febslet.2005.03.019, 579, 11, 2277-2283, 2005.04, Both amyloid-prone cystatin and unstable mutant C94A lysozyme were secreted in wild-type and Δeps1 Saccharomyces cerevisiae cells. Amyloid-prone cystatin secreted at much higher level in Δeps1 cells than that in wild-type yeast. In parallel, the secretion amount of disulfide bond disrupted mutant C94A lysozyme greatly increased in Δeps1 cells although that was apparently low in wild-type yeast cells compared with the secretion amount of wild-type lysozyme. It is interesting that neither the unstable mutant C94A lysozyme nor amyloid-prone cystatin secreted in Δeps1 cells maintained their specific activities. These observations lead to the supposition that yeast cells deficient for the protein disulfide isomerase-family-member EPS1 locus secrete more of labile disulfide-containing model proteins..