Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
Yasuyuki Ohkawa Last modified date:2019.06.25

Professor / Division of Medical Molecular Cell Biology / Research Center for Transomics Medicine / Medical Institute of Bioregulation

1. Akira Matsuda, Yosuke Asada, Naomasa Suita, Satoshi Iwamoto, Toshiaki Hirakata, Norihiko Yokoi, Yasuyuki Ohkawa, Yukinori Okada, Takehiko Yokomizo, Nobuyuki Ebihara, Transcriptome profiling of refractory atopic keratoconjunctivitis by RNA sequencing, Journal of Allergy and Clinical Immunology, 10.1016/j.jaci.2018.11.007, 143, 4, 1610-1614.e6, 2019.04.
2. Kazu Kobayakawa, Yasuyuki Ohkawa, Shingo Yoshizaki, Tetsuya Tamaru, Takeyuki Saito, Ken Kijima, Kazuya Yokota, Masamitsu Hara, Kensuke Kubota, Yoshihiro Matsumoto, Katsumi Harimaya, Keiko Ozato, Takahiro Masuda, Tsuda Makoto, Tomohiko Tamura, Kazuhide Inoue, V. Reggie Edgerton, Yukihide Iwamoto, Yasuharu Nakashima, Seiji Okada, Macrophage centripetal migration drives spontaneous healing process after spinal cord injury, Science Advances, 10.1126/sciadv.aav5086, 5, 5, 2019.05, Traumatic spinal cord injury (SCI) brings numerous inflammatory cells, including macrophages, from the circulating blood to lesions, but pathophysiological impact resulting from spatiotemporal dynamics of macrophages is unknown. Here, we show that macrophages centripetally migrate toward the lesion epicenter after infiltrating into the wide range of spinal cord, depending on the gradient of chemoattractant C5a. However, macrophages lacking interferon regulatory factor 8 (IRF8) cannot migrate toward the epicenter and remain widely scattered in the injured cord with profound axonal loss and little remyelination, resulting in a poor functional outcome after SCI. Time-lapse imaging and P2X/YRs blockade revealed that macrophage migration via IRF8 was caused by purinergic receptors involved in the C5a-directed migration. Conversely, pharmacological promotion of IRF8 activation facilitated macrophage centripetal movement, thereby improving the SCI recovery. Our findings reveal the importance of macrophage centripetal migration via IRF8, providing a novel therapeutic target for central nervous system injury..
3. Takashi Kozuka, Yoshihiro Omori, Satoshi Watanabe, Etsuko Tarusawa, Haruka Yamamoto, Taro Chaya, Mayu Furuhashi, Makiko Morita, Tetsuya Sato, Shinichi Hirose, Yasuyuki Ohkawa, Yumiko Yoshimura, Takatoshi Hikida, Takahisa Furukawa, miR-124 dosage regulates prefrontal cortex function by dopaminergic modulation, Scientific reports, 10.1038/s41598-019-38910-2, 9, 1, 2019.12, MicroRNA-124 (miR-124) is evolutionarily highly conserved among species and one of the most abundantly expressed miRNAs in the developing and mature central nervous system (CNS). Previous studies reported that miR-124 plays a role in CNS development, such as neuronal differentiation, maturation, and survival. However, the role of miR-124 in normal brain function has not yet been revealed. Here, we subjected miR-124-1
mice, to a comprehensive behavioral battery. We found that miR-124-1
mice showed impaired prepulse inhibition (PPI), methamphetamine-induced hyperactivity, and social deficits. Whole cell recordings using prefrontal cortex (PFC) slices showed enhanced synaptic transmission in layer 5 pyramidal cells in the miR-124-1
PFC. Based on the results of behavioral and electrophysiological analysis, we focused on genes involved in the dopaminergic system and identified a significant increase of Drd2 expression level in the miR-124-1
PFC. Overexpression or knockdown of Drd2 in the control or miR-124-1
PFC demonstrates that aberrant Drd2 signaling leads to impaired PPI. Furthermore, we identified that expression of glucocorticoid receptor gene Nr3c1, which enhances Drd2 expression, increased in the miR-124-1
PFC. Taken together, the current study suggests that miR-124 dosage modulates PFC function through repressing the Drd2 pathway, suggesting a critical role of miR-124 in normal PFC function..
4. Nozomi Sugimoto, Kazumitsu Maehara, Kazumasa Yoshida, Yasuyuki Ohkawa, Masatoshi Fujita, Genome-wide analysis of the spatiotemporal regulation of firing and dormant replication origins in human cells, Nucleic Acids Research, 10.1093/nar/gky476, 46, 13, 6683-6696, 2018.07, In metazoan cells, only a limited number of mini chromosome maintenance (MCM) complexes are fired during S phase, while the majority remain dormant. Several methods have been used to map replication origins, but such methods cannot identify dormant origins. Herein, we determined MCM7-binding sites in human cells using ChIP-Seq, classified them into firing and dormant origins using origin data and analysed their association with various chromatin signatures. Firing origins, but not dormant origins, were well correlated with open chromatin regions and were enriched upstream of transcription start sites (TSSs) of transcribed genes. Aggregation plots of MCM7 signals revealed minimal difference in the efficacy of MCM loading between firing and dormant origins. We also analysed common fragile sites (CFSs) and found a low density of origins at these sites. Nevertheless, firing origins were enriched upstream of the TSSs. Based on the results, we propose a model in which excessive MCMs are actively loaded in a genome-wide manner, irrespective of chromatin status, but only a fraction are passively fired in chromatin areas with an accessible open structure, such as regions upstream of TSSs of transcribed genes. This plasticity in the specification of replication origins may minimize collisions between replication and transcription..
5. Yasuo Takashima, Kenichi Horisawa, Miyako Udono, Yasuyuki Ohkawa, Atsushi Suzuki, Prolonged inhibition of hepatocellular carcinoma cell proliferation by combinatorial expression of defined transcription factors, Cancer Science, 10.1111/cas.13798, 109, 11, 3543-3553, 2018.11, Hepatocellular carcinoma (HCC) accounts for a large proportion of liver cancer cases and has an extremely poor prognosis. Therefore, novel innovative therapies for HCC are strongly desired. As gene therapy tools for HCC, 2 hepatic transcription factors (TF), HNF4A and HNF1A, have been used to suppress proliferation and to extinguish cancer-specific characteristics of target cells. However, our present data demonstrated that single transduction of HNF4A or HNF1A had only a limited effect on suppression of HCC cell proliferation. Thus, in this study, we examined whether combinations of TF could show more effective antitumor activity, and found that combinatorial transduction of 3 hepatic TF, HNF4A, HNF1A and FOXA3, suppressed HCC cell proliferation more stably than single transduction of these TF. The combinatorial transduction also suppressed cancer-specific phenotypes, such as anchorage-independent growth in culture and tumorigenicity after transplantation into mice. HCC cell lines transduced with the 3 TF did not recover their proliferative property after withdrawal of anticancer drugs, indicating that combinatorial expression of the 3 TF suppressed the growth of all cell subtypes within the HCC cell lines, including cancer stem-like cells. Transcriptome analyses revealed that the expression levels of a specific gene set involved in cell proliferation were only decreased in HCC cells overexpressing all 3 TF. Moreover, combined transduction of the 3 TF could facilitate hepatic differentiation of HCC cell lines. Our strategy for inducing stable inhibition and functional differentiation of tumor cells using a defined set of TF will become an effective therapeutic strategy for various types of cancers..
6. Yuichi Shima, Kanako Miyabayashi, Tetsuya Sato, Mikita Suyama, Yasuyuki Ohkawa, Masao Doi, Hitoshi Okamura, Kentaro Suzuki, Fetal leydig cells dedifferentiate and serve as adult leydig stem cells, Development (Cambridge), 10.1242/dev.169136, 145, 23, 2018.12, Previous studies have established that fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) show distinct functional characteristics. However, the lineage relationship between FLCs and ALCs has not been clarified yet. Here, we reveal that a subset of FLCs dedifferentiate at fetal stages to give rise to ALCs at the pubertal stage. Moreover, the dedifferentiated cells contribute to the peritubular myoid cell and vascular pericyte populations in the neonatal testis, and these nonsteroidogenic cells serve as potential ALC stem cells. We generated FLC lineage-specific Nr5a1 (Ad4BP/SF-1) gene-disrupted mice and mice lacking the fetal Leydig enhancer (FLE) of the Nr5a1 gene. Phenotypes of these mice support the conclusion that most of the ALCs arise from dedifferentiated FLCs, and that the FLE of the Nr5a1 gene is essential for both initial FLC differentiation and pubertal ALC redifferentiation..
7. Kazuya Yokota, Kensuke Kubota, Kazu Kobayakawa, Takeyuki Saito, Masamitsu Hara, Ken Kijima, Takeshi Maeda, Hiroyuki Katoh, Yasuyuki Ohkawa, Yasuharu Nakashima, Seiji Okada, Pathological changes of distal motor neurons after complete spinal cord injury, Molecular Brain, 10.1186/s13041-018-0422-3, 12, 1, 2019.01, Traumatic spinal cord injury (SCI) causes serious disruption of neuronal circuits that leads to motor functional deficits. Regeneration of disrupted circuits back to their original target is necessary for the restoration of function after SCI, but the pathophysiological condition of the caudal spinal cord has not been sufficiently studied. Here we investigated the histological and biological changes in the distal part of the injured spinal cord, using a mice model of complete thoracic SCI in the chronic stage (3 months after injury). Atrophic changes were widely observed in the injured spinal cord both rostral and caudal to the lesion, but the decrease in area was mainly in the white matter in the rostral spinal cord while both the white and gray matter decreased in the caudal spinal cord. The number of the motor neurons was maintained in the chronic phase of injury, but the number of presynaptic boutons decreased in the lumbar motor neurons caudal to the lesion. Using laser microdissection, to investigate gene expressions in motor neurons caudal to the lesion, we observed a decrease in the expressions of neuronal activity markers. However, we found that the synaptogenic potential of postsynapse molecules was maintained in the motor neurons after SCI with the expression of acetylcholine-related molecules actually higher after SCI. Collectively, our results show that the potential of synaptogenesis is maintained in the motor neurons caudal to the lesion, even though presynaptic input is decreased. Although researches into SCI concentrate their effort on the lesion epicenter, our findings suggest that the area caudal to the lesion could be an original therapeutic target for the chronically injured spinal cord..
8. 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..
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. 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..
11. Yuko Katoh-Fukui, Takashi Baba, Tetsuya Sato, Hiroyuki Otake, Yuko Nagakui-Noguchi, Miyuki Shindo, Mikita Suyama, Yasuyuki Ohkawa, Hideki Tsumura, Ken-Ichirou Morohashi, Maki Fukami, Mouse polycomb group gene Cbx2 promotes osteoblastic but suppresses adipogenic differentiation in postnatal long bones, Bone, 10.1016/j.bone.2018.10.021, 120, 219-231, 2019.03, A set of key developmental genes is essential for skeletal growth from multipotent progenitor cells at weaning. Polycomb group proteins, which regulate such genes contributes to the cell lineage commitment and subsequent differentiation via epigenetic chromatin modification and remodeling. However, it is unclear which cell lineage and gene sets are targeted by polycomb proteins during skeletal growth. We now report that mice deficient in a polycomb group gene Cbx2cterm/cterm exhibited skeletal hypoplasia in the tibia, femur, and cranium. Long bone cavities in these mice contained fewer multipotent mesenchymal stromal cells. RNA-sequencing of bone marrow cells showed downregulation and upregulation of osteoblastic and adipogenic genes, respectively. Furthermore, the expression levels of genes specifically expressed in B-cell precursors were decreased. Forced expression of Cbx2 in Cbx2cterm/cterm bone marrow stromal cell recovered fibroblastic colony formation and suppressed adipogenic differentiation. Collectively, our results suggest that Cbx2 controls the maintenance and adipogenic differentiation of mesenchymal stromal cells in the bone marrow..
12. 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, 2018.04, 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..
13. Yasuyuki Kita, Yuta Katayama, Taichi Shiraishi, Takeru Oka, Tetsuya Sato, Mikita Suyama, Yasuyuki Ohkawa, Keishi Miyata, Yuichi Oike, Michiko Shirane, Masaaki Nishiyama, Keiichi Nakayama, The Autism-Related Protein CHD8 Cooperates with C/EBPβ to Regulate Adipogenesis, Cell Reports, 10.1016/j.celrep.2018.04.050, 23, 7, 1988-2000, 2018.05, The gene encoding the chromatin remodeler CHD8 is the most frequently mutated gene in individuals with autism spectrum disorder (ASD). Heterozygous mutations in CHD8 give rise to ASD that is often accompanied by macrocephaly, gastrointestinal complaints, and slender habitus. Whereas most phenotypes of CHD8 haploinsufficiency likely result from delayed neurodevelopment, the mechanism underlying slender habitus has remained unknown. Here, we show that CHD8 interacts with CCAAT/enhancer-binding protein β (C/EBPβ) and promotes its transactivation activity during adipocyte differentiation. Adipogenesis was impaired in Chd8-deleted preadipocytes, with the upregulation of C/EBPα and peroxisome-proliferator-activated receptor γ (PPARγ), two master regulators of this process, being attenuated in mutant cells. Furthermore, mice with CHD8 ablation in white preadipocytes had a markedly reduced white adipose tissue mass. Our findings reveal a mode of C/EBPβ regulation by CHD8 during adipogenesis, with CHD8 deficiency resulting in a defect in the development of white adipose tissue. Kita et al. show that autism-related protein CHD8 is essential for adipogenesis and the development of white adipose tissue. Moreover, they demonstrate that CHD8 cooperates with C/EBPβ to regulate transactivation of the genes for C/EBPα and PPARγ during adipogenesis..
14. Teppei Noda, Steven J. Meas, Jumpei Nogami, Yutaka Amemiya, Ryutaro Uchi, Yasuyuki Ohkawa, Koji Nishimura, Alain Dabdoub, Direct reprogramming of spiral ganglion non-neuronal cells into neurons
Toward ameliorating sensorineural hearing loss by gene therapy, Frontiers in Cell and Developmental Biology, 10.3389/fcell.2018.00016, 6, FEB, 2018.02, Primary auditory neurons (PANs) play a critical role in hearing by transmitting sound information from the inner ear to the brain. Their progressive degeneration is associated with excessive noise, disease and aging. The loss of PANs leads to permanent hearing impairment since they are incapable of regenerating. Spiral ganglion non-neuronal cells (SGNNCs), comprised mainly of glia, are resident within the modiolus and continue to survive after PAN loss. These attributes make SGNNCs an excellent target for replacing damaged PANs through cellular reprogramming. We used the neurogenic pioneer transcription factor Ascl1 and the auditory neuron differentiation factor NeuroD1 to reprogram SGNNCs into induced neurons (iNs). The overexpression of both Ascl1 and NeuroD1 in vitro generated iNs at high efficiency. Transcriptome analyses revealed that iNs displayed a transcriptome profile resembling that of endogenous PANs, including expression of several key markers of neuronal identity: Tubb3, Map2, Prph, Snap25, and Prox1. Pathway analyses indicated that essential pathways in neuronal growth and maturation were activated in cells upon neuronal induction. Furthermore, iNs extended projections toward cochlear hair cells and cochlear nucleus neurons when cultured with each respective tissue. Taken together, our study demonstrates that PAN-like neurons can be generated from endogenous SGNNCs. This work suggests that gene therapy can be a viable strategy to treat sensorineural hearing loss caused by degeneration of PANs..
15. Makiko Ueda, Tetsuya Sato, Yasuyuki Ohkawa, Yoshihiro H. Inoue, Identification of miR-305, a microRNA that promotes aging, and its target mRNAs in Drosophila, Genes to Cells, 10.1111/gtc.12555, 23, 2, 80-93, 2018.02, MicroRNAs (miRNAs) are involved in the regulation of important biological processes. Here, we describe a novel Drosophila miRNAs involved in aging. We selected eight Drosophila miRNAs, displaying high homology with seed sequences of aging-related miRNAs characterized in other species, and investigated whether the over-expression of these miRNAs affected aging in Drosophila adult flies. The lifespan of adults over-expressing miR-305, a miRNA showing high homology with miR-239 in C. elegans, was significantly shorter. Conversely, a reduction in miR-305 expression led to a longer lifespan than that in control flies. miR-305 over-expression accelerated the impairment of locomotor activity and promoted the age-dependent accumulation of poly-ubiquitinated protein aggregates in the muscle, as flies aged. Thus, we show that the ectopic expression of miR-305 has a deleterious effect on aging in Drosophila. To identify the targets of miR-305, we performed RNA-Seq. We discovered several mRNAs encoding antimicrobial peptides and insulin-like peptides, whose expression changed in adults upon miR-305 over-expression. We further confirmed, by qRT-PCR, that miR-305 over-expression significantly decreases the mRNA levels of four antimicrobial peptides. As these mRNAs contain multiple sequences matching the seed sequence of miR-305, we speculate that a reduction in target mRNA levels, caused by ectopic miRNA expression, promotes aging..
16. Yoshimasa Takizawa, Hiroki Tanaka, Shinichi Machida, Masako Koyama, Kazumitsu Maehara, Yasuyuki Ohkawa, Paul A. Wade, Matthias Wolf, Hitoshi Kurumizaka, Cryo-EM structure of the nucleosome containing the ALB1 enhancer DNA sequence, Open Biology, 10.1098/rsob.170255, 8, 3, 2018.01, Pioneer transcription factors specifically target their recognition DNA sequences within nucleosomes. FoxA is the pioneer transcription factor that binds to the ALB1 gene enhancer in liver precursor cells, and is required for liver differentiation in embryos. The ALB1 enhancer DNA sequence is reportedly incorporated into nucleosomes in cells, although the nucleosome structure containing the targeting sites for FoxA has not been clarified yet. In this study, we determined the nucleosome structure containing the ALB1 enhancer (N1) sequence, by cryogenic electron microscopy at 4.0 Å resolution. The nucleosome structure with the ALB1 enhancer DNA is not significantly different from the previously reported nucleosome structure with the Widom 601 DNA. Interestingly, in the nucleosomes, the ALB1 enhancer DNA contains local flexible regions, as compared to the Widom 601 DNA. Consistently, DNaseI treatments revealed that, in the nucleosome, the ALB1 enhancer (N1) DNA is more accessible than the Widom 601 sequence. The histones also associated less strongly with the ALB1 enhancer (N1) DNA than the Widom 601 DNA in the nucleosome. Therefore, the local histone–DNA contacts May be responsible for the enhanced DNA accessibility in the nucleosome with the ALB1 enhancer DNA..
17. 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..
18. 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..
19. Kouhei Shimaji, Ryo Tanaka, Toru Maeda, Mamiko Ozaki, Hideki Yoshida, Yasuyuki Ohkawa, Tetsuya Sato, Mikita Suyama, Masamitsu Yamaguchi, Histone methyltransferase G9a is a key regulator of the starvation-induced behaviors in Drosophila melanogaster, Scientific Reports, 10.1038/s41598-017-15344-2, 7, 1, 2017.12, Organisms have developed behavioral strategies to defend themselves from starvation stress. Despite of their importance in nature, the underlying mechanisms have been poorly understood. Here, we show that Drosophila G9a (dG9a), one of the histone H3 Lys 9-specific histone methyltransferases, functions as a key regulator for the starvation-induced behaviors. RNA-sequencing analyses utilizing dG9a null mutant flies revealed that the expression of some genes relating to gustatory perception are regulated by dG9a under starvation conditions. Reverse transcription quantitative-PCR analyses showed that the expression of gustatory receptor genes for sensing sugar are up-regulated in starved dG9a null mutant. Consistent with this, proboscis extension reflex tests indicated that dG9a depletion increased the sensitivity to sucrose under starvation conditions. Furthermore, the locomotion activity was promoted in starved dG9a null mutant. We also found that dG9a depletion down-regulates the expression of insulin-like peptide genes that are required for the suppression of starvation-induced hyperactivity. Furthermore, refeeding of wild type flies after starvation conditions restores the hyperactivity and increased sensitivity to sucrose as well as dG9a expression level. These data suggest that dG9a functions as a key regulator for the decision of behavioral strategies under starvation conditions..
20. Takuya Kajitani, Hiroaki Kato, Yuji Chikashige, Chihiro Tsutsumi, Yasushi Hiraoka, Hiroshi Kimura, Yasuyuki Ohkawa, Chikashi Obuse, Damien Hermand, Yota Murakami, Ser7 of RNAPII-CTD facilitates heterochromatin formation by linking ncRNA to RNAi, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1714579115, 114, 52, E11208-E11217, 2017.12, Some long noncoding RNAs (ncRNAs) transcribed by RNA polymerase II (RNAPII) are retained on chromatin, where they regulate RNAi and chromatin structure. The molecular basis of this retention remains unknown. We show that in fission yeast serine 7 (Ser7) of the C-terminal domain (CTD) of RNAPII is required for efficient siRNA generation for RNAi-dependent heterochromatin formation. Surprisingly, Ser7 facilitates chromatin retention of nascent heterochromatic RNAs (hRNAs). Chromatin retention of hRNAs and siRNA generation requires both Ser7 and an RNA-binding activity of the chromodomain of Chp1, a subunit of the RNA-induced transcriptional silencing (RITS) complex. Furthermore, RITS associates with RNAPII in a Ser7-dependent manner. We propose that Ser7 promotes cotranscriptional chromatin retention of hRNA by recruiting the RNA-chromatin connector protein Chp1, which facilitates RNAi-dependent heterochromatin formation. Our findings reveal a function of the CTD code: linking ncRNA transcription to RNAi for heterochromatin formation..
21. 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..
22. Junichiro Yuda, Toshihiro Miyamoto, Jun Odawara, Yasuyuki Ohkawa, Yuichiro Semba, Masayasu Hayashi, Koichi Miyamura, Mitsune Tanimoto, Kazuhito Yamamoto, Masafumi Taniwaki, Koichi Akashi, Persistent detection of alternatively spliced BCR-ABL variant results in a failure to achieve deep molecular response, Cancer Science, 10.1111/cas.13353, 108, 11, 2204-2212, 2017.11, Treatment with tyrosine kinase inhibitors (TKI) may sequentially induce TKI-resistant BCR-ABL mutants in chronic myeloid leukemia (CML). Conventional PCR monitoring of BCR-ABL is an important indicator to determine therapeutic intervention for preventing disease progression. However, PCR cannot separately quantify amounts of BCR-ABL and its mutants, including alternatively spliced BCR-ABL with an insertion of 35 intronic nucleotides (BCR-ABLI ns35bp) between ABL exons 8 and 9, which introduces the premature termination and loss of kinase activity. To assess the clinical impact of BCR-ABL mutants, we performed deep sequencing analysis of BCR-ABL transcripts of 409 samples from 37 patients with suboptimal response to frontline imatinib who were switched to nilotinib. At baseline, TKI-resistant mutations were documented in 3 patients, whereas BCR-ABLI ns35bp was detected in all patients. After switching to nilotinib, both BCR-ABL and BCR-ABLI ns35bp became undetectable in 3 patients who attained complete molecular response (CMR), whereas in the remaining all 34 patients, BCR-ABLI ns35bp was persistently detected, and minimal residual disease (MRD) fluctuated at low but detectable levels. PCR monitoring underestimated molecular response in 5 patients whose BCR-ABLI ns35bp was persisted, although BCR-ABLI ns35bp does not definitively mark TKI resistance. Therefore, quantification of BCR-ABLI ns35bp is useful for evaluating “functional” MRD and determining the effectiveness of TKI with accuracy..
23. Yuka Adachi, Mana Umeda, Asako Kawazoe, Tetsuya Sato, Yasuyuki Ohkawa, Sakihito Kitajima, Shingo Izawa, Ikuko Sagami, Shigeru Taketani, The novel heme-dependent inducible protein, SRRD regulates heme biosynthesis and circadian rhythms, Archives of Biochemistry and Biophysics, 10.1016/, 631, 19-29, 2017.10, Heme plays a role in the regulation of the expression of genes related to circadian rhythms and heme metabolism. In order to identify new heme-regulated proteins, an RNA sequence analysis using mouse NIH3T3 cells treated without or with 5-aminolevulinic acid (ALA) was performed. Among the changes observed in the levels of various mRNAs including heme oxygenase-1 (HO-1) and ALA synthase-1 (ALAS1), a mouse homologue of the plant circadian-regulating protein SRR1, SRR1 domain containing (SRRD) was induced by the ALA treatment. The expression of SRRD was dependent on heme biosynthesis, and increased the production of heme. SRRD was expressed under circadian rhythms, and influenced the expression of clock genes including PER2, BMAL1, and CLOCK. The knockout of SRRD arrested the growth of cells, indicating that SRRD plays roles in heme-regulated circadian rhythms and cell proliferation..
24. Fumiyuki Sasaki, Tomoaki Koga, Kazuko Saeki, Toshiaki Okuno, Saiko Kazuno, Tsutomu Fujimura, Yasuyuki Ohkawa, Takehiko Yokomizo, Biochemical and immunological characterization of a novel monoclonal antibody against mouse leukotriene B4 receptor 1, PLoS One, 10.1371/journal.pone.0185133, 12, 9, 2017.09, Leukotriene B4 (LTB4) receptor 1 (BLT1) is a G protein-coupled receptor expressed in various leukocyte subsets; however, the precise expression of mouse BLT1 (mBLT1) has not been reported because a mBLT1 monoclonal antibody (mAb) has not been available. In this study, we present the successful establishment of a hybridoma cell line (clone 7A8) that produces a high-affinity mAb for mBLT1 by direct immunization of BLT1-deficient mice with mBLT1-overexpressing cells. The specificity of clone 7A8 was confirmed using mBLT1-overexpressing cells and mouse peripheral blood leukocytes that endogenously express BLT1. Clone 7A8 did not cross-react with human BLT1 or other G protein-coupled receptors, including human chemokine (C-X-C motif) receptor 4. The 7A8 mAb binds to the second extracellular loop of mBLT1 and did not affect LTB4 binding or intracellular calcium mobilization by LTB4. The 7A8 mAb positively stained Gr-1-positive granulocytes, CD11b-positive granulocytes/monocytes, F4/80-positive monocytes, CCR2-high and CCR2-low monocyte subsets in the peripheral blood and a CD4-positive T cell subset, Th1 cells differentiated in vitro from naïve CD4-positive T cells. This mAb was able to detect Gr-1-positive granulocytes and monocytes in the spleens of naïve mice by immunohistochemistry. Finally, intraperitoneal administration of 7A8 mAb depleted granulocytes and monocytes in the peripheral blood. We have therefore succeeded in generating a high-affinity anti-mBLT1 mAb that is useful for analyzing mBLT1 expression in vitro and in vivo..
25. 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..
26. Kei Fukuda, Yukihiro Inoguchi, Kenji Ichiyanagi, Tomoko Ichiyanagi, Yasuhiro Go, Masashi Nagano, Yojiro Yanagawa, Noboru Takaesu, Yasuyuki Ohkawa, Hiroo Imai, Hiroyuki Sasaki, Evolution of the sperm methylome of primates is associated with retrotransposon insertions and genome instability, Human Molecular Genetics, 10.1093/hmg/ddx236, 26, 18, 3508-3519, 2017.09, Changes in gene expression resulting from epigenetic and/or genetic changes play an important role in the evolutionary divergence of phenotypes. To explore how epigenetic and genetic changes are linked during primate evolution, we have compared the genome-wide DNA methylation profiles (methylomes) of humans and chimpanzees, which have a 1.2% DNA sequence divergence, of sperm, the frontal cortices, B cells, and neutrophils. We revealed that species-specific differentially methylated regions (S-DMRs), ranging from several hundred base pairs (bp) to several kilo base pairs (kb), were frequently associated with sequence changes in transcription factor-binding sites and insertions of Alu and SVA retrotransposons. We then generated a reference macaque sperm methylome map and revealed, in sperm, that both human and chimpanzee S-DMRs arose more frequently owing to methylation loss rather than gain. Moreover, we observed that the sperm methylomes contained many more hypomethylated domains (HMDs), ranging from 20 to 500 kb, than did the somatic methylomes. Interestingly, the sperm HMDs changed rapidly during primate evolution; hundreds of sperm HMDs were specific to humans, whereas most somatic HMDs were highly conserved between humans and chimpanzees. Notably, these human-specific sperm HMDs frequently occurred in regions exhibiting copy number variations. Our findings indicate that primate evolution, particularly in the germline, is significantly impacted by reciprocal changes in the genome and epigenome..
27. Masamitsu Hara, Kazu Kobayakawa, Yasuyuki Ohkawa, Hiromi Kumamaru, Kazuya Yokota, Takeyuki Saito, Ken Kijima, Shingo Yoshizaki, Katsumi Harimaya, Yasuharu Nakashima, Seiji Okada, Interaction of reactive astrocytes with type i collagen induces astrocytic scar formation through the integrin-N-cadherin pathway after spinal cord injury, Nature Medicine, 10.1038/nm.4354, 23, 7, 818-828, 2017.07, Central nervous system (CNS) injury transforms naive astrocytes into reactive astrocytes, which eventually become scar-forming astrocytes that can impair axonal regeneration and functional recovery. This sequential phenotypic change, known as reactive astrogliosis, has long been considered unidirectional and irreversible. However, we report here that reactive astrocytes isolated from injured spinal cord reverted in retrograde to naive astrocytes when transplanted into a naive spinal cord, whereas they formed astrocytic scars when transplanted into injured spinal cord, indicating the environment-dependent plasticity of reactive astrogliosis. We also found that type I collagen was highly expressed in the spinal cord during the scar-forming phase and induced astrocytic scar formation via the integrin-N-cadherin pathway. In a mouse model of spinal cord injury, pharmacological blockade of reactive astrocyte-type I collagen interaction prevented astrocytic scar formation, thereby leading to improved axonal regrowth and better functional outcomes. Our findings reveal environmental cues regulating astrocytic fate decisions, thereby providing a potential therapeutic target for CNS injury..
28. Kanako Miyabayashi, Yuichi Shima, Miki Inoue, Tetsuya Sato, Takashi Baba, Yasuyuki Ohkawa, Mikita Suyama, Ken-Ichirou Morohashi, Alterations in Fetal Leydig Cell Gene Expression during Fetal and Adult Development, Sexual Development, 10.1159/000453323, 11, 2, 53-63, 2017.05, Fetal Leydig cells (FLCs) and adult Leydig cells (ALCs) develop in the mammalian prenatal and postnatal testes, respectively. In mice, FLCs emerge in the interstitial space of the testis as early as embryonic day 12.5 and thereafter increase in number during the fetal stage. We previously established a transgenic mouse line in which FLCs are labeled with EGFP and demonstrated that the EGFP-labeled FLCs were present even in adult testes. However, the characteristics of FLCs during postnatal stages remained unclear. In the present study, a comparison of the transcriptomes of FLCs from prenatal and postnatal testes and of ALCs from adult testes revealed that FLCs gradually alter their characteristics across developmental stages and come to roughly resemble ALCs. Many cholesterogenic genes simultaneously expressed a unique alternation pattern, while many oxidative phosphorylation and β-oxidation (both mitochondrial functions) genes showed a different unique pattern. These metabolic gene expression alterations might be triggered by milieu changes, such as nutrient and oxygen supply, from the prenatal to the postnatal period..
29. Akiyoshi Nakayama, Hirofumi Nakaoka, Ken Yamamoto, Masayuki Sakiyama, Amara Shaukat, Yu Toyoda, Yukinori Okada, Yoichiro Kamatani, Takahiro Nakamura, Tappei Takada, Katsuhisa Inoue, Tomoya Yasujima, Hiroaki Yuasa, Yuko Shirahama, Hiroshi Nakashima, Seiko Shimizu, Toshihide Higashino, Yusuke Kawamura, Hiraku Ogata, Makoto Kawaguchi, Yasuyuki Ohkawa, Inaho Danjoh, Atsumi Tokumasu, Keiko Ooyama, Toshimitsu Ito, Takaaki Kondo, Kenji Wakai, Blanka Stiburkova, Karel Pavelka, Lisa K. Stamp, Nicola Dalbeth, Yutaka Sakurai, Hiroshi Suzuki, Makoto Hosoyamada, Shin Fujimori, Takashi Yokoo, Tatsuo Hosoya, Ituro Inoue, Atsushi Takahashi, Michiaki Kubo, Hiroshi Ooyama, Toru Shimizu, Kimiyoshi Ichida, Nariyoshi Shinomiya, Tony R. Merriman, Hirotaka Matsuo, GWAS of clinically defined gout and subtypes identifies multiple susceptibility loci that include urate transporter genes, Annals of the Rheumatic Diseases, 10.1136/annrheumdis-2016-209632, 76, 5, 869-877, 2017.05, Objective A genome-wide association study (GWAS) of gout and its subtypes was performed to identify novel gout loci, including those that are subtype-specific. Methods Putative causal association signals from a GWAS of 945 clinically defined gout cases and 1213 controls from Japanese males were replicated with 1396 cases and 1268 controls using a custom chip of 1961 single nucleotide polymorphisms (SNPs). We also first conducted GWASs of gout subtypes. Replication with Caucasian and New Zealand Polynesian samples was done to further validate the loci identified in this study. Results In addition to the five loci we reported previously, further susceptibility loci were identified at a genome-wide significance level (p<5.0×10-8): Urate transporter genes (SLC22A12 and SLC17A1) and HIST1H2BF-HIST1H4E for all gout cases, and NIPAL1 and FAM35A for the renal underexcretion gout subtype. While NIPAL1 encodes a magnesium transporter, functional analysis did not detect urate transport via NIPAL1, suggesting an indirect association with urate handling. Localisation analysis in the human kidney revealed expression of NIPAL1 and FAM35A mainly in the distal tubules, which suggests the involvement of the distal nephron in urate handling in humans. Clinically ascertained male patients with gout and controls of Caucasian and Polynesian ancestries were also genotyped, and FAM35A was associated with gout in all cases. A meta-analysis of the three populations revealed FAM35A to be associated with gout at a genome-wide level of significance (p meta =3.58×10-8). Conclusions Our findings including novel gout risk loci provide further understanding of the molecular pathogenesis of gout and lead to a novel concept for the therapeutic target of gout/hyperuricaemia..
30. 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..
31. Daiki Kato, Akihisa Osakabe, Yasuhiro Arimura, Yuka Mizukami, Naoki Horikoshi, Kazumi Saikusa, Satoko Akashi, Yoshifumi Nishimura, Sam Yong Park, Jumpei Nogami, Kazumitsu Maehara, Yasuyuki Ohkawa, Atsushi Matsumoto, Hidetoshi Kono, Rintaro Inoue, Masaaki Sugiyama, Hitoshi Kurumizaka, Crystal structure of the overlapping dinucleosome composed of hexasome and octasome, Science, 10.1126/science.aak9867, 356, 6334, 205-208, 2017.04, 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..
32. Tomohumi Nakamura, Kouichi Murakami, Haruto Tada, Yoshihiko Uehara, Jumpei Nogami, Kazumitsu Maehara, Yasuyuki Ohkawa, Hisato Saitoh, Hideo Nishitani, Tetsuya Ono, Ryotaro Nishi, Masayuki Yokoi, Wataru Sakai, Kaoru Sugasawa, Thymine DNA glycosylase modulates DNA damage response and gene expression by base excision repair-dependent and independent mechanisms, Genes to Cells, 10.1111/gtc.12481, 22, 4, 392-405, 2017.04, Thymine DNA glycosylase (TDG) is a base excision repair (BER) enzyme, which is implicated in correction of deamination-induced DNA mismatches, the DNA demethylation process and regulation of gene expression. Because of these pivotal roles associated, it is crucial to elucidate how the TDG functions are appropriately regulated in vivo. Here, we present evidence that the TDG protein undergoes degradation upon various types of DNA damage, including ultraviolet light (UV). The UV-induced degradation of TDG was dependent on proficiency in nucleotide excision repair and on CRL4CDT 2-mediated ubiquitination that requires a physical interaction between TDG and DNA polymerase clamp PCNA. Using the Tdg-deficient mouse embryonic fibroblasts, we found that ectopic expression of TDG compromised cellular survival after UV irradiation and repair of UV-induced DNA lesions. These negative effects on cellular UV responses were alleviated by introducing mutations in TDG that impaired its BER function. The expression of TDG induced a large-scale alteration in the gene expression profile independently of its DNA glycosylase activity, whereas a subset of genes was affected by the catalytic activity of TDG. Our results indicate the presence of BER-dependent and BER-independent functions of TDG, which are involved in regulation of cellular DNA damage responses and gene expression patterns..
33. 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..
34. Takanari Umegawachi, Hideki Yoshida, Hiromu Koshida, Momoko Yamada, Yasuyuki Ohkawa, Tetsuya Sato, Mikita Suyama, Henry M. Krause, Masamitsu Yamaguchi, Control of tissue size and development by a regulatory element in the yorkie 3'UTR, American Journal of Cancer Research, 7, 3, 673-687, 2017.01, Regulation of the Hippo pathway via phosphorylation of Yorkie (Yki), the Drosophila homolog of human Yes-associated protein 1, is conserved from Drosophila to humans. Overexpression of a non-phosphorylatable form of Yki induces severe overgrowth in adult fly eyes. Here, we show that yki mRNA associates with microsomal fractions and forms foci that partially colocalize to processing bodies in the vicinity of endoplasmic reticulum. This localization is dependent on a stem-loop (SL) structure in the 3' untranslated region of yki. Surprisingly, expression of SL deleted yki in eye imaginal discs also results in severe overgrowth phenotypes. When the structure of the SL is disrupted, Yki protein levels increase without a significant effect on RNA levels. When the SL is completely removed, protein levels drastically increase, but in this case, due to increased RNA stability. In the latter case, we show that the increased RNA accumulation is due to removal of a putative miR-8 seed sequence in the SL. These data demonstrate the function of two novel regulatory mechanisms, both controlled by the yki SL element, that are essential for proper Hippo pathway mediated growth regulation..
35. Yurin Shishido, Takash Baba, Tetsuy Sato, Yuich Shima, Kanak Miyabayashi, Mik Inoue, Haruhik Akiyama, Hirosh Kimura, Yoshiakir Kanai, Yasuhir Ishihara, Shogo Haraguchi, Akir Miyazaki, Damjan Rozman, Takesh Yamazaki, Man Ho Choi, Yasuyuki Ohkawa, Mikit Suyama, Ken-Ichirou Morohashi, Differential lactate and cholesterol synthetic activities in XY and XX Sertoli cells, Scientific Reports, 10.1038/srep41912, 7, 2017.01, SRY, a sex-determining gene, induces testis development in chromosomally female (XX) individuals. However, mouse XX Sertoli cells carrying Sry (XX/Sry Sertoli cells) are incapable of fully supporting germ cell development, even when the karyotype of the germ cells is XY. While it has therefore been assumed that XX/Sry Sertoli cells are not functionally equivalent to XY Sertoli cells, it has remained unclear which specific functions are affected. To elucidate the functional difference, we compared the gene expression of XY and XX/Sry Sertoli cells. Lactate and cholesterol metabolisms, essential for nursing the developing germ cells, were down-regulated in XX/Sry cells, which appears to be caused at least in part by the differential expression of histone modification enzymes SMCX/SMCY (H3K4me3 demethylase) and UTX/UTY (H3K27me3 demethylase) encoded by the sex chromosomes. We suggest that downregulation of lactate and cholesterol metabolism that may be due to altered epigenetic modification affects the nursing functions of XX/Sry Sertoli cells..
36. 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..
37. 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..
38. Shoko Sawano, Yusuke Komiya, Riho Ichitsubo, Yasuyuki Ohkawa, Mako Nakamura, Ryuichi Tatsumi, Yoshihide Ikeuchi, Wataru Mizunoya, A one-step immunostaining method to visualize rodent muscle fiber type within a single specimen, PLoS One, 10.1371/journal.pone.0166080, 11, 11, 2016.11, In this study, we present a quadruple immunostaining method for rapid muscle fiber typing of mice and rats using antibodies specific to the adult myosin heavy chain (MyHC) isoforms MyHC1, 2A, 2X, and 2B, which are common marker proteins of distinct muscle fiber types. We developed rat monoclonal antibodies specific to each MyHC isoform and conjugated these four antibodies to fluorophores with distinct excitation and emission wavelengths. By mixing the four types of conjugated antibodies, MyHC1, 2A, 2X, and 2B could be distinguished within a single specimen allowing for facile delineation of skeletal muscle fiber types. Furthermore, we could observe hybrid fibers expressing MyHC2X and MyHC2B together in single longitudinal muscle sections from mice and rats, that was not attained in previous techniques. This staining method is expected to be applied to study muscle fiber type transition in response to environmental factors, and to ultimately develop techniques to regulate animal muscle fiber types..
39. 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..
40. Maiko Terada, Kenichi Horisawa, Shizuka Miura, Yasuo Takashima, Yasuyuki Ohkawa, Sayaka Sekiya, Kanae Matsuda-Ito, Atsushi Suzuki, Kupffer cells induce Notch-mediated hepatocyte conversion in a common mouse model of intrahepatic cholangiocarcinoma, Scientific Reports, 10.1038/srep34691, 6, 2016.10, Intrahepatic cholangiocarcinoma (ICC) is a malignant epithelial neoplasm composed of cells resembling cholangiocytes that line the intrahepatic bile ducts in portal areas of the hepatic lobule. Although ICC has been defined as a tumor arising from cholangiocyte transformation, recent evidence from genetic lineage-tracing experiments has indicated that hepatocytes can be a cellular origin of ICC by directly changing their fate to that of biliary lineage cells. Notch signaling has been identified as an essential factor for hepatocyte conversion into biliary lineage cells at the onset of ICC. However, the mechanisms underlying Notch signal activation in hepatocytes remain unclear. Here, using a mouse model of ICC, we found that hepatic macrophages called Kupffer cells transiently congregate around the central veins in the liver and express the Notch ligand Jagged-1 coincident with Notch activation in pericentral hepatocytes. Depletion of Kupffer cells prevents the Notch-mediated cell-fate conversion of hepatocytes to biliary lineage cells, inducing hepatocyte apoptosis and increasing mortality in mice. These findings will be useful for uncovering the pathogenic mechanism of ICC and developing prevenient and therapeutic strategies for this refractory disease..
41. Naoki Iwamori, Kaoru Tominaga, Tetsuya Sato, Kevin Riehle, Tokuko Iwamori, Yasuyuki Ohkawa, Cristian Coarfa, Etsuro Ono, Martin M. Matzuk, MRG15 is required for pre-mRNA splicing and spermatogenesis, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1611995113, 113, 37, E5408-E5415, 2016.09, Splicing can be epigenetically regulated and involved in cellular differentiation in somatic cells, but the interplay of epigenetic factors and the splicing machinery during spermatogenesis remains unclear. To study these interactions in vivo, we generated a germline deletion of MORF-related gene on chromosome 15 (MRG15), a multifunctional chromatin organizer that binds to methylated histone H3 lysine 36 (H3K36) in introns of transcriptionally active genes and has been implicated in regulation of histone acetylation, homology-directed DNA repair, and alternative splicing in somatic cells. Conditional KO (cKO) males lacking MRG15 in the germline are sterile secondary to spermatogenic arrest at the round spermatid stage. There were no significant alterations in meiotic division and histone acetylation. Specific mRNA sequences disappeared from 66 germ cell-expressed genes in the absence of MRG15, and specific intronic sequences were retained in mRNAs of 4 genes in the MRG15 cKO testes. In particular, introns were retained in mRNAs encoding the transition proteins that replace histones during sperm chromatin condensation. In round spermatids, MRG15 colocalizes with splicing factors PTBP1 and PTBP2 at H3K36me3 sites between the exons and single intron of transition nuclear protein 2 (Tnp2). Thus, our results reveal that MRG15 is essential for premRNA splicing during spermatogenesis and that epigenetic regulation of pre-mRNA splicing by histone modification could be useful to understand not only spermatogenesis but also, epigenetic disorders underlying male infertile patients..
42. Kosuke Torigata, Okuzaki Daisuke, Satomi Mukai, Akira Hatanaka, Fumiharu Ohka, Daisuke Motooka, Shota Nakamura, Yasuyuki Ohkawa, Norikazu Yabuta, Yutaka Kondo, Hiroshi Nojima, LATS2 positively regulates polycomb repressive complex 2, PLoS One, 10.1371/journal.pone.0158562, 11, 7, 2016.07, LATS2, a pivotal Ser/Thr kinase of the Hippo pathway, plays important roles in many biological processes. LATS2 also function in Hippo-independent pathway, including mitosis, DNA damage response and epithelial to mesenchymal transition. However, the physiological relevance and molecular basis of these LATS2 functions remain obscure. To understand novel functions of LATS2, we constructed a LATS2 knockout HeLa-S3 cell line using TAL-effector nuclease (TALEN). Integrated omics profiling of this cell line revealed that LATS2 knockout caused genome-wide downregulation of Polycomb repressive complex 2 (PRC2) and H3K27me3. Cell-cycle analysis revealed that downregulation of PRC2 was not due to cell cycle aberrations caused by LATS2 knockout. Not LATS1, a homolog of LATS2, but LATS2 bound PRC2 on chromatin and phosphorylated it. LATS2 positively regulates histone methyltransferase activity of PRC2 and their expression at both the mRNA and protein levels. Our findings reveal a novel signal upstream of PRC2, and provide insight into the crucial role of LATS2 in coordinating the epigenome through regulation of PRC2..
43. Tomoya Kujirai, Naoki Horikoshi, Koichi Sato, Kazumitsu Maehara, Shinichi Machida, Akihisa Osakabe, Hiroshi Kimura, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Structure and function of human histone H3.Y nucleosome, Nucleic Acids Research, 10.1093/nar/gkw202, 44, 13, 6127-6141, 2016.07, 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..
44. Toshiki Yabe-Wada, Shintaro Matsuba, Kazuya Takeda, Tetsuya Sato, Mikita Suyama, Yasuyuki Ohkawa, Toshiyuki Takai, Haifeng Shi, Caroline C. Philpott, Akira Nakamura, TLR signals posttranscriptionally regulate the cytokine trafficking mediator sortilin, Scientific Reports, 10.1038/srep26566, 6, 2016.05, Regulating the transcription, translation and secretion of cytokines is crucial for controlling the appropriate balance of inflammation. Here we report that the sorting receptor sortilin plays a key role in cytokine production. We observed interactions of sortilin with multiple cytokines including IFN-α, and sortilin depletion in plasmacytoid dendritic cells (pDCs) led to a reduction of IFN-α secretion, suggesting a pivotal role of sortilin in the exocytic trafficking of IFN-α in pDCs. Moreover, sortilin mRNA was degraded posttranscriptionally upon stimulation with various TLR ligands. Poly-rC-binding protein 1 (PCBP1) recognized the C-rich element (CRE) in the 3' UTR of sortilin mRNA, and depletion of PCBP1 enhanced the degradation of sortilin transcripts, suggesting that PCBP1 can act as a trans-acting factor to stabilize sortilin transcripts. The nucleotide-binding ability of PCBP1 was impaired by zinc ions and alterations of intracellular zinc affect sortilin expression. PCBP1 may therefore control the stability of sortilin transcripts by sensing intracellular zinc levels. Collectively, our findings provide insights into the posttranslational regulation of cytokine production through the posttranscriptional control of sortilin expression by TLR signals..
45. Kazuya Yokota, Takeyuki Saito, Kazu Kobayakawa, Kensuke Kubota, Masamitsu Hara, Masaharu Murata, Yasuyuki Ohkawa, Yukihide Iwamoto, Seiji Okada, The feasibility of in vivo imaging of infiltrating blood cells for predicting the functional prognosis after spinal cord injury, Scientific Reports, 10.1038/srep25673, 6, 2016.05, After a spinal cord injury (SCI), a reliable prediction of the potential functional outcome is essential for determining the optimal treatment strategy. Despite recent advances in the field of neurological assessment, there is still no satisfactory methodology for predicting the functional outcome after SCI. We herein describe a novel method to predict the functional outcome at 12 hours after SCI using in vivo bioluminescence imaging. We produced three groups of SCI mice with different functional prognoses: 50 kdyn (mild), 70 kdyn (moderate) and 90 kdyn (severe). Only the locomotor function within 24 hours after SCI was unable to predict subsequent functional recovery. However, both the number of infiltrating neutrophils and the bioluminescence signal intensity from infiltrating blood cells were found to correlate with the severity of the injury at 12 hours after SCI. Furthermore, a strong linear relationship was observed among the number of infiltrating neutrophils, the bioluminescence signal intensity, and the severity of the injury. Our findings thus indicate that in vivo bioluminescence imaging is able to accurately predict the long-term functional outcome in the hyperacute phase of SCI, thereby providing evidence that this imaging modality could positively contribute to the future development of tailored therapeutic approaches for SCI..
46. Wataru Kobayashi, Motoki Takaku, Shinichi Machida, Hiroaki Tachiwana, Kazumitsu Maehara, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Chromatin architecture may dictate the target site for DMC1, but not for RAD51, during homologous pairing, Scientific Reports, 10.1038/srep24228, 6, 2016.04, In eukaryotes, genomic DNA is compacted as chromatin, in which histones and DNA form the nucleosome as the basic unit. DMC1 and RAD51 are essential eukaryotic recombinases that mediate homologous chromosome pairing during homologous recombination. However, the means by which these two recombinases distinctly function in chromatin have remained elusive. Here we found that, in chromatin, the human DMC1-single-stranded DNA complex bypasses binding to the nucleosome, and preferentially promotes homologous pairing at the nucleosome-depleted regions. Consistently, DMC1 forms ternary complex recombination intermediates with the nucleosome-free DNA or the nucleosome-depleted DNA region. Surprisingly, removal of the histone tails improperly enhances the nucleosome binding by DMC1. In contrast, RAD51 does not specifically target the nucleosome-depleted region in chromatin. These are the first demonstrations that the chromatin architecture specifies the sites to promote the homologous recombination reaction by DMC1, but not by RAD51..
47. 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..
48. 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..
49. Masako Tanaka, Masayuki Shiota, Takafumi Nakao, Ryo Uemura, Satoshi Nishi, Yasuyuki Ohkawa, Masaki Matsumoto, Maki Yamaguchi, Mayuko Osada-Oka, Azusa Inagaki, Katsuyuki Takahashi, Keiichi Nakayama, Min Gi, Yasukatsu Izumi, Katsuyuki Miura, Hiroshi Iwao, Identification of low-abundance proteins in serum via the isolation of HSP72 complexes, Journal of Proteomics, 10.1016/j.jprot.2016.01.008, 136, 214-221, 2016.03, Heat shock protein 72 (HSP72) is an intracellular molecular chaperone that is overexpressed in tumor cells, and has also been detected in extracellular regions such as the blood. HSP72 forms complexes with peptides and proteins that are released from tumors. Accordingly, certain HSP72-binding proteins/peptides present in the blood of cancer patients may be derived from tumor cells. In this study, to effectively identify low-abundance proteins/peptides in the blood as tumor markers, we established a method for isolating HSP72-binding proteins/peptides from serum. Nine HSP72-specific monoclonal antibodies were conjugated to N-hydroxysulfosuccinimide-activated Sepharose beads (NHq) and used to isolate HSP72 complexes from serum samples. Precipitated proteins were then identified by LC-MS/MS analysis. Notably, this approach enabled the isolation of low-abundance proteins from serum without albumin removal. Moreover, by subjecting the serum samples of ten patients with multiple myeloma (MM) to NHq analysis, we identified 299 proteins present in MM HSP72 complexes, including 65 intracellular proteins. Among the intracellular proteins detected, 21 were present in all serum samples tested, while 11 were detected in both the conditioned media from cultured multiple myeloma cells and serum from MM patients. These results suggest that the NHq method can be applied to discover candidate tumor markers..
50. Miki Inoue, Yuichi Shima, Kanako Miyabayashi, Kaori Tokunaga, Tetsuya Sato, Takashi Baba, Yasuyuki Ohkawa, Haruhiko Akiyama, Mikita Suyama, Ken-Ichirou Morohashi, Isolation and characterization of Fetal Leydig progenitor cells of male mice, Endocrinology, 10.1210/en.2015-1773, 157, 3, 1222-1233, 2016.03, Fetal and adult Leydig cells develop in mammalian prenatal and postnatal testes, respectively. In mice, fetal Leydig cells (FLCs) emergeintheinterstitial spaceofthe testisatembryonic day 12.5 and thereafter increase in number, possibly through differentiation from progenitor cells. However, the progenitor cells have not yet been identified. Previously, we established transgenic mice in which FLCs are labeled strongly with enhanced green fluorescent protein (EGFP). Interestingly, fluorescence-activated cell sorting provided us with weakly EGFP-labeled cells as well as strongly EGFP-labeled FLCs. In vitro reconstruction of fetal testes demonstrated that weakly EGFP-labeled cells contain FLC progenitors. Transcriptome from the 2 cell populations revealed, as expected, marked differences in the expression of genes required for growth factor/receptor signaling and steroidogenesis. In addition, genes for energy metabolisms such as glycolytic pathways and the citrate cycle were activated in strongly EGFP-labeled cells, suggesting that metabolism is activated during FLC differentiation..
51. Yuta Katayama, Masaaki Nishiyama, Hirotaka Shoji, Yasuyuki Ohkawa, Atsuki Kawamura, Tetsuya Sato, Mikita Suyama, Toru Takumi, Tsuyoshi Miyakawa, Keiichi Nakayama, CHD8 haploinsufficiency results in autistic-like phenotypes in mice, Nature, 10.1038/nature19357, 537, 7622, 675-679, 2016.01, Autism spectrum disorder (ASD) comprises a range of neurodevelopmental disorders characterized by deficits in social interaction and communication as well as by restricted and repetitive behaviours. ASD has a strong genetic component with high heritability. Exome sequencing analysis has recently identified many de novo mutations in a variety of genes in individuals with ASD, with CHD8, a gene encoding a chromatin remodeller, being most frequently affected. Whether CHD8 mutations are causative for ASD and how they might establish ASD traits have remained unknown. Here we show that mice heterozygous for Chd8 mutations manifest ASD-like behavioural characteristics including increased anxiety, repetitive behaviour, and altered social behaviour. CHD8 haploinsufficiency did not result in prominent changes in the expression of a few specific genes but instead gave rise to small but global changes in gene expression in the mouse brain, reminiscent of those in the brains of patients with ASD. Gene set enrichment analysis revealed that neurodevelopment was delayed in the mutant mouse embryos. Furthermore, reduced expression of CHD8 was associated with abnormal activation of RE-1 silencing transcription factor (REST), which suppresses the transcription of many neuronal genes. REST activation was also observed in the brains of humans with ASD, and CHD8 was found to interact physically with REST in the mouse brain. Our results are thus consistent with the notion that CHD8 haploinsufficiency is a highly penetrant risk factor for ASD, with disease pathogenesis probably resulting from a delay in neurodevelopment..
52. Masahiro Oka, Sonoko Mura, Kohji Yamada, Percival Sangel, Saki Hirata, Kazumitsu Maehara, Koichi Kawakami, Taro Tachibana, Yasuyuki Ohkawa, Hiroshi Kimura, Yoshihiro Yoneda, Chromatin-prebound Crm1 recruits Nup98-HoxA9 fusion to induce aberrant expression of Hox cluster genes, eLife, 10.7554/eLife.09540, 5, JANUARY2016, 2016.01, The nucleoporin Nup98 is frequently rearranged to form leukemogenic Nup98-fusion proteins with various partners. However, their function remains largely elusive. Here, we show that Nup98-HoxA9, a fusion between Nup98 and the homeobox transcription factor HoxA9, forms nuclear aggregates that frequently associate with facultative heterochromatin. We demonstrate that stable expression of Nup98-HoxA9 in mouse embryonic stem cells selectively induces the expression of Hox cluster genes. Genome-wide binding site analysis revealed that Nup98-HoxA9 is preferentially targeted and accumulated at Hox cluster regions where the export factor Crm1 is originally prebound. In addition, leptomycin B, an inhibitor of Crm1, disassembled nuclear Nup98-HoxA9 dots, resulting in the loss of chromatin binding of Nup98-HoxA9 and Nup98-HoxA9-mediated activation of Hox genes. Collectively, our results indicate that highly selective targeting of Nup98-fusion proteins to Hox cluster regions via prebound Crm1 induces the formation of higher order chromatin structures that causes aberrant Hox gene regulation..
53. Kazumitsu Maehara, Yasuyuki Ohkawa, Exploration of nucleosome positioning patterns in transcription factor function, Scientific Reports, 10.1038/srep19620, 6, 2016.01, The binding of transcription factors (TFs) triggers activation of specific chromatin regions through the recruitment and activation of RNA polymerase. Unique nucleosome positioning (NP) occurs during gene expression and has been suggested to be involved in various other chromatin functions. However, the diversity of NP that can occur for each function has not been clarified. Here we used MNase-Seq data to evaluate NP around 258 cis-regulatory elements in the mouse genome. Principal component analysis of the 258 elements revealed that NP consisted of five major patterns. Furthermore, the five NP patterns had predictive power for the level of gene expression. We also demonstrated that selective NP patterns appeared around TF binding sites. These results suggest that the NP patterns are correlated to specific functions on chromatin..
54. 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..
55. 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..
56. Kouhei Shimaji, Takahiro Konishi, Shintaro Tanaka, Hideki Yoshida, Yasuko Kato, Yasuyuki Ohkawa, Tetsuya Sato, Mikita Suyama, Hiroshi Kimura, Masamitsu Yamaguchi, Genomewide identification of target genes of histone methyltransferase dG9a during Drosophila embryogenesis, Genes to Cells, 10.1111/gtc.12281, 20, 11, 902-914, 2015.11, Post-translational modification of the histone plays important roles in epigenetic regulation of various biological processes. Among the identified histone methyltransferases (HMTases), G9a is a histone H3 Lys 9 (H3K9)-specific example active in euchromatic regions. Drosophila G9a (dG9a) has been reported to feature H3K9 dimethylation activity in vivo. Here, we show that the time required for hatching of a homozygous dG9a null mutant and heteroallelic combination of dG9a null mutants is delayed, suggesting that dG9a is at least partially responsible for progression of embryogenesis. Immunocytochemical analyses of the wild-type and the dG9a null mutant flies indicated that dG9a localizes in cytoplasm up to nuclear division cycle 7 where it is likely responsible for di-methylation of nucleosome-free H3K9. From cycles 8-11, dG9a moves into the nucleus and is responsible for di-methylating H3K9 in nucleosomes. RNA-sequence analysis utilizing early wild-type and dG9a mutant embryos showed that dG9a down-regulates expression of genes responsible for embryogenesis. RNA fluorescent in situ hybridization analysis further showed temporal and spatial expression patterns of these mRNAs did not significantly change in the dG9a mutant. These results indicate that dG9a controls transcription levels of some zygotic genes without changing temporal and spatial expression patterns of the transcripts of these genes..
57. Yuki Hatanaka, Kimiko Inoue, Mami Oikawa, Satoshi Kamimura, Narumi Ogonuki, Eiichi N. Kodama, Yasuyuki Ohkawa, Yuichi Tsukada, Atsuo Ogura, Histone chaperone CAF-1 mediates repressive histone modifications to protect preimplantation mouse embryos from endogenous retrotransposons, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1512775112, 112, 47, 14641-14646, 2015.11, Substantial proportions of mammalian genomes comprise repetitive elements including endogenous retrotransposons. Although these play diverse roles during development, their appropriate silencing is critically important in maintaining genomic integrity in the host cells. The major mechanism for retrotransposon silencing is DNA methylation, but the wave of global DNA demethylation that occurs after fertilization renders preimplantation embryos exceptionally hypomethylated. Here, we show that hypomethylated preimplantation mouse embryos are protected from retrotransposons by repressive histone modifications mediated by the histone chaperone chromatin assembly factor 1 (CAF-1). We found that knockdown of CAF-1 with specific siRNA injections resulted in significant up-regulation of the retrotransposons long interspersed nuclear element 1, short interspersed nuclear element B2, and intracisternal A particle at the morula stage. Concomitantly, increased histone H2AX phosphorylation and developmental arrest of the majority (>95%) of embryos were observed. The latter was caused at least in part by derepression of retrotransposons, as treatment with reverse transcriptase inhibitors rescued some embryos. Importantly, ChIP analysis revealed that CAF-1 mediated the replacement of H3.3 with H3.1/3.2 at the retrotransposon regions. This replacement was associated with deposition of repressive histone marks, including trimethylation of histone H3 on lysine 9 (H3K9me3), H3K9me2, H3K27me3, and H4K20me3. Among them, H4K20me3 and H3K9me3 seemed to play predominant roles in retrotransposon silencing, as assessed by knockdown of specific histone methyltransferases and forced expression of unmethylatable mutants of H3.1K9 and H4K20. Our data thus indicate that CAF-1 is an essential guardian of the genome in preimplantation mouse embryos by deposition of repressive histone modifications via histone variant replacement..
58. Akihisa Osakabe, Fumiya Adachi, Yasuhiro Arimura, Kazumitsu Maehara, Yasuyuki Ohkawa, Hitoshi Kurumizaka, Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA, Open Biology, 10.1098/rsob.150128, 5, 10, 2015.10, DNA methylation occurs on CpG sites and is important to form pericentric heterochromatin domains. The satellite 2 sequence, containing seven CpG sites, is located in the pericentric region of human chromosome 1 and is highly methylated in normal cells. In contrast, the satellite 2 region is reportedly hypomethylated in cancer cells, suggesting that the methylation status may affect the chromatin structure around the pericentric regions in tumours. In this study, we mapped the nucleosome positioning on the satellite 2 sequence in vitro and found that DNA methylation modestly affects the distribution of the nucleosome positioning. The micrococcal nuclease assay revealed that the DNA end flexibility of the nucleosomes changes, depending on the DNA methylation status. However, the structures and thermal stabilities of the nucleosomes are unaffected by DNA methyl-ation. These findings provide new information to understand how DNA methylation functions in regulating pericentric heterochromatin formation and maintenance in normal and malignant cells..
59. 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..
60. Kazumitsu Maehara, Yasuyuki Ohkawa, Agplus
A rapid and flexible tool for aggregation plots, Bioinformatics, 10.1093/bioinformatics/btv322, 31, 18, 3046-3047, 2015.07, Aggregation plots are frequently used to evaluate signal distributions at user-interested points in ChIP-Seq data analysis. agplus, a new and simple command-line tool, enables rapid and flexible generation of text tables tailored for aggregation plots from which users can easily design multiple groups based on user-definitions such as regulatory regions or transcription initiation sites..
61. Toshiya Nishimura, Tetsuya Sato, Yasuhiro Yamamoto, Ikuko Watakabe, Yasuyuki Ohkawa, Mikita Suyama, Satoru Kobayashi, Minoru Tanaka, Foxl3 is a germ cell-intrinsic factor involved in sperm-egg fate decision in medaka, Science, 10.1126/science.aaa2657, 349, 6245, 328-331, 2015.07, Sex determination is an essential step in the commitment of a germ cell to a sperm or egg. However, the intrinsic factors that determine the sexual fate of vertebrate germ cells are unknown. Here, we show that foxl3, which is expressed in germ cells but not somatic cells in the gonad, is involved in sperm-egg fate decision in medaka fish. Adult XX medaka with disrupted foxl3 developed functional sperm in the expanded germinal epithelium of a histologically functional ovary. In chimeric medaka, mutant germ cells initiated spermatogenesis in female wild-type gonad. These results indicate that a germ cell-intrinsic cue for the sperm-egg fate decision is present in medaka and that spermatogenesis can proceed in a female gonadal environment..
62. Yoko H. Ohnishi, Yoshinori N. Ohnishi, Takanori Nakamura, Ohno Mizuki, Pamela J. Kennedy, Yasuyuki Ohkawa, Akinori Nishi, Rachael Neve, Teruhisa Tsuzuki, Eric J. Nestler, PSMC5, a 19S proteasomal ATPase, regulates cocaine action in the nucleus accumbens, PLoS One, 10.1371/journal.pone.0126710, 10, 6, 2015.06.
63. Brian T. Nasipak, Teresita Padilla-Benavides, Karin M. Green, John D. Leszyk, Wenjie Mao, Silvana Konda, Saïd Sif, Scott A. Shaffer, Yasuyuki Ohkawa, Anthony N. Imbalzano, Opposing calcium-dependent signalling pathways control skeletal muscle differentiation by regulating a chromatin remodelling enzyme, Nature Communications, 10.1038/ncomms8441, 6, 2015.06, Calcium signalling is important for differentiation-dependent gene expression, but is also involved in other cellular functions. Therefore, mechanisms must exist to distinguish calcium signalling relevant to differentiation. Calcineurin is a calcium-regulated phosphatase that is required for myogenic gene expression and skeletal muscle differentiation. Here, we demonstrate that inhibition of calcineurin blocks chromatin remodelling and that the Brg1 ATPase of the SWI/SNF chromatin remodelling enzyme, which is required for the activation of myogenic gene expression, is a calcineurin substrate. Furthermore, we identify the calcium-regulated classical protein kinase C β (PKCβ) as a repressor of myogenesis and as the enzyme that opposes calcineurin function. Replacement of endogenous Brg1 with a phosphomimetic mutant in primary myoblasts inhibits myogenesis, whereas replacement with a non-phosphorylatable mutant allows myogenesis despite inhibition of calcineurin signalling, demonstrating the functionality of calcineurin/PKC-modified residues. Thus, the Brg1 chromatin remodelling enzyme integrates two antagonistic calcium-dependent signalling pathways that control myogenic differentiation..
64. Saori Tomita, Mohamed Osama Ali Abdalla, Saori Fujiwara, Haruka Matsumori, Kazumitsu Maehara, Yasuyuki Ohkawa, Hirotaka Iwase, Noriko Saitoh, Mitsuyoshi Nakao, A cluster of noncoding RNAs activates the ESR1 locus during breast cancer adaptation, Nature Communications, 10.1038/ncomms7966, 6, 2015.04, Estrogen receptor-α (ER)-positive breast cancer cells undergo hormone-independent proliferation after deprivation of oestrogen, leading to endocrine therapy resistance. Up-regulation of the ER gene (ESR1) is critical for this process, but the underlying mechanisms remain unclear. Here we show that the combination of transcriptome and fluorescence in situ hybridization analyses revealed that oestrogen deprivation induced a cluster of noncoding RNAs that defined a large chromatin domain containing the ESR1 locus. We termed these RNAs as Eleanors (ESR1 locus enhancing and activating noncoding RNAs). Eleanors were present in ER-positive breast cancer tissues and localized at the transcriptionally active ESR1 locus to form RNA foci. Depletion of one Eleanor, upstream (u)-Eleanor, impaired cell growth and transcription of intragenic Eleanors and ESR1 mRNA, indicating that Eleanors cis-activate the ESR1 gene. Eleanor-mediated gene activation represents a new type of locus control mechanism and plays an essential role in the adaptation of breast cancer cells..
65. Tetsuya Kawaguchi, Akie Tanigawa, Takao Naganuma, Yasuyuki Ohkawa, Sylvie Souquere, Gerard Pierron, Tetsuro Hirose, Joan A. Steitz, SWI/SNF chromatin-remodeling complexes function in noncoding RNA-dependent assembly of nuclear bodies, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1423819112, 112, 14, 4304-4309, 2015.04, Paraspeckles are subnuclear structures that form around nuclear paraspeckle assembly transcript 1 (NEAT1) long noncoding RNA (lncRNA). Recently, paraspeckles were shown to be functional nuclear bodies involved in stress responses and the development of specific organs. Paraspeckle formation is initiated by transcription of the NEAT1 chromosomal locus and proceeds in conjunction with NEAT1 lncRNA biogenesis and a subsequent assembly step involving >40 paraspeckle proteins (PSPs). In this study, subunits of SWItch/Sucrose NonFermentable (SWI/SNF) chromatin-remodeling complexes were identified as paraspeckle components that interact with PSPs and NEAT1 lncRNA. EM observations revealed that SWI/SNF complexes were enriched in paraspeckle subdomains depleted of chromatin. Knockdown of SWI/SNF components resulted in paraspeckle disintegration, but mutation of the ATPase domain of the catalytic subunit BRG1 did not affect paraspeckle integrity, indicating that the essential role of SWI/SNF complexes in para-speckle formation does not require their canonical activity. Knockdown of SWI/SNF complexes barely affected the levels of known essential paraspeckle components, but markedly diminished the interactions between essential PSPs, suggesting that SWI/SNF complexes facilitate organization of the PSP interaction network required for intact paraspeckle assembly. The interactions between SWI/SNF components and essential PSPs were maintained in NEAT1-depleted cells, suggesting that SWI/SNF complexes not only facilitate interactions between PSPs, but also recruit PSPs during paraspeckle assembly. SWI/SNF complexes were also required for Satellite III lncRNA-dependent formation of nuclear stress bodies under heat-shock conditions. Our data suggest the existence of a common mechanism underlying the formation of lncRNA-dependent nuclear body architectures in mammalian cells..
66. 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..
67. Masataka Nakamura, Kensuke Shibata, Shinya Hatano, Tetsuya Sato, Yasuyuki Ohkawa, Hisakata Yamada, Koichi Ikuta, Yasunobu Yoshikai, A genome-wide analysis identifies a Notch-RBP-Jk-IL-7Ra axis that controls IL-17-Producing γδ T cell homeostasis in mice, Journal of Immunology, 10.4049/jimmunol.1401619, 194, 1, 243-251, 2015.01, Notch signaling is an important regulator for the development and function of both ab and γδ T cells, whereas roles of Notch signaling in T cell maintenance remain unclear. We reported previously that the Notch-Hes1 pathway was involved in the intrathymic development of naturally occurring IL-17-producing (IL-17+) γδ T cells. To gain insight into additional roles for the Notch axis in the homeostasis of γδ T cells, we performed a genome-wide analysis of Notch target genes and identified the novel promoter site of IL-7Ra driven by the Notch-RBP-Jk pathway. Constitutive Notch signaling had the potential to induce IL-7Ra expression on γδ T cells in vivo, as well as in vitro, whereas conditional deletion of RBP-Jk abrogated IL-7Ra expression, but not Hes1 expression, by γδ T cells and selectively reduced the pool size of IL-7Rahigh IL-17+ γδ T cells in the periphery. In the absence of IL-7Ra-mediated signaling, IL-17+ γδ T cells were barely maintained in adult mice. Addition of exogenous IL-7 in vitro selectively expanded IL-17+ γδ T cells. Thus, our results revealed a novel role for the Notch-RBP-Jk-IL-7Ra axis that is independent of Hes1 for homeostasis of IL-17+ γδ T cells..
68. Nozomi Sugimoto, Kazumitsu Maehara, Kazumasa Yoshida, Shuhei Yasukouchi, Satoko Osano, Shinya Watanabe, Masahiro Aizawa, Takashi Yugawa, Tohru Kiyono, Hitoshi Kurumizaka, Yasuyuki Ohkawa, Masatoshi Fujita, Cdt1-binding protein GRWD1 is a novel histone-binding protein that facilitates MCM loading through its influence on chromatin architecture, Nucleic Acids Research, 10.1093/nar/gkv509, 43, 12, 5898-5911, 2015.01, Efficient pre-replication complex (pre-RC) formation on chromatin templates is crucial for the maintenance of genome integrity. However, the regulation of chromatin dynamics during this process has remained elusive. We found that a conserved protein, GRWD1 (glutamate-rich WD40 repeat containing 1), binds to two representative replication origins specifically during G1 phase in a CDC6- and Cdt1-dependent manner, and that depletion of GRWD1 reduces loading of MCM but not CDC6 and Cdt1. Furthermore, chromatin immunoprecipitation coupled with high-throughput sequencing (Seq) revealed significant genome-wide co-localization of GRWD1 with CDC6. We found that GRWD1 has histone-binding activity. To investigate the effect of GRWD1 on chromatin architecture, we used formaldehyde-assisted isolation of regulatory elements (FAIRE)-seq or FAIRE-quantitative PCR analyses, and the results suggest that GRWD1 regulates chromatin openness at specific chromatin locations. Taken together, these findings suggest that GRWD1 may be a novel histone-binding protein that regulates chromatin dynamics and MCM loading at replication origins..
69. Kazuya Yokota, Kazu Kobayakawa, Kensuke Kubota, Atsushi Miyawaki, Hideyuki Okano, Yasuyuki Ohkawa, Yukihide Iwamoto, Seiji Okada, Engrafted Neural Stem/Progenitor Cells Promote Functional Recovery through Synapse Reorganization with Spared Host Neurons after Spinal Cord Injury, Stem Cell Reports, 10.1016/j.stemcr.2015.06.004, 5, 2, 264-277, 2015.01, Neural stem/progenitor cell (NSPC) transplantation is a promising therapeutic strategy for spinal cord injury (SCI). However, the efficacy of NSPC transplantation on severe SCI is poorly understood. We herein show that NSPC transplantation promotes functional recovery after mild and moderate SCI, but not after severe SCI. In severe SCI mice, there were few remaining host neurons within the range of NSPC engraftment; thus, we examined whether the co-distribution of transplant and host is a contributory factor for functional improvement. A cellular selective analysis using laser microdissection revealed that drug-induced host neuronal ablation considerably decreased the synaptogenic potential of the engrafted NSPCs. Furthermore, following host neuronal ablation, neuronal retrograde tracing showed less propriospinal relay connections bridging the lesion after NSPC transplantation. Our findings suggest that the interactive synaptic reorganization between engrafted NSPCs and spared host neurons is crucial for functional recovery, providing significant insight for establishing therapeutic strategies for severe SCI..
70. 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..
71. Hidehisa Takahashi, Ichigaku Takigawa, Masashi Watanabe, Delnur Anwar, Mio Shibata, Chieri Tomomori-Sato, Shigeo Sato, Amol Ranjan, Chris W. Seidel, Tadasuke Tsukiyama, Wataru Mizushima, Masayasu Hayashi, Yasuyuki Ohkawa, Joan W. Conaway, Ronald C. Conaway, Shigetsugu Hatakeyama, MED26 regulates the transcription of snRNA genes through the recruitment of little elongation complex, Nature Communications, 10.1038/ncomms6941, 6, 2015.01, Regulation of transcription elongation by RNA polymerase II (Pol II) is a key regulatory step in gene transcription. Recently, the little elongation complex (LEC)-which contains the transcription elongation factor ELL/EAF-was found to be required for the transcription of Pol II-dependent small nuclear RNA (snRNA) genes. Here we show that the human Mediator subunit MED26 plays a role in the recruitment of LEC to a subset of snRNA genes through direct interaction of EAF and the N-terminal domain (NTD) of MED26. Loss of MED26 in cells decreases the occupancy of LEC at a subset of snRNA genes and results in a reduction in their transcription. Our results suggest that the MED26-NTD functions as a molecular switch in the exchange of TBP-associated factor 7 (TAF7) for LEC to facilitate the transition from initiation to elongation during transcription of a subset of snRNA genes..
72. Timothy J. Stasevich, Yoko Hayashi-Takanaka, Yuko Sato, Kazumitsu Maehara, Yasuyuki Ohkawa, Kumiko Sakata-Sogawa, Makio Tokunaga, Takahiro Nagase, Naohito Nozaki, James G. McNally, Hiroshi Kimura, Regulation of RNA polymerase II activation by histone acetylation in single living cells, Nature, 10.1038/nature13714, 516, 7530, 272-275, 2014.12, In eukaryotic cells, post-translational histone modifications have an important role in gene regulation. Starting with early work on histone acetylation, a variety of residue-specific modifications have now been linked to RNA polymerase II (RNAP2) activity, but it remains unclear if these markers are active regulators of transcription or just passive byproducts. This is because studies have traditionally relied on fixed cell populations, meaning temporal resolution is limited to minutes at best, and correlated factors may not actually be present in the same cell at the same time. Complementary approaches are therefore needed to probe the dynamic interplay of histone modifications and RNAP2 with higher temporal resolution in single living cells. Here we address this problem by developing a system to track residue-specific histone modifications and RNAP2 phosphorylation in living cells by fluorescence microscopy. This increases temporal resolution to the tens-of-seconds range. Our single-cell analysis reveals histone H3 lysine-27 acetylation at a gene locus can alter downstream transcription kinetics by as much as 50%, affecting two temporally separate events. First acetylation enhances the search kinetics of transcriptional activators, and later the acetylation accelerates the transition of RNAP2 from initiation to elongation. Signatures of the latter can be found genome-wide using chromatin immunoprecipitation followed by sequencing. We argue that this regulation leads to a robust and potentially tunable transcriptional response..
73. Isao Tamura, Yasuyuki Ohkawa, Tetsuya Sato, Mikita Suyama, Kosuke Jozaki, Maki Okada, Lifa Lee, Ryo Maekawa, Hiromi Asada, Shun Sato, Yoshiaki Yamagata, Hiroshi Tamura, Norihiro Sugino, Genome-wide analysis of histone modifications in human endometrial stromal cells, Molecular Endocrinology, 10.1210/me.2014-1117, 28, 10, 1656-1669, 2014.10, Dramatic changes of gene expressions occur in human endometrial stromal cells (ESCs) during decidualization. The changes in gene expression are associated with changes of chromatin structure, which are regulated by histone modifications. Here we investigated genome-wide changes in histone modifications associated with decidualization in human ESCs using chromatin immunoprecipitation combined with next-generation sequencing. ESCs were incubated with estradiol and medroxyprogesterone acetate for 14 days to induce decidualization. The chromatin immunoprecipitation- sequence data showed that induction of decidualization increased H3K27ac and H3K4me3 signals in many genomic regions but decreased in only a few regions. Most of the H3K27ac-increased regions (80%) and half of the H3K4me3-increased regions were located in the distal promoter regions (more than 3 kb upstream or downstream of the transcription start site). RNA sequence showed that induction of decidualization up-regulated 881 genes, 223 of which had H3K27ac- or H3K4me3-increased regions in the proximal and distal promoter regions. Induction of decidualization increased themRNAlevels of these genes more than it increased themRNA levels of genes without H3K27ac- or H3K4me3-increased regions. Pathway analysis revealed that up-regulated genes with the H3K27ac- or H3K4me3-increased regions were associated with the insulin signaling, which may be involved in glucose uptake that is necessary for ESCs to undergo decidualization. These results show that histone modification statuses on a genome-wide basis change in human ESCs during decidualization. The main changes of histone modifications are increases of H3K27ac and H3K4me3 in both the proximal and distal promoter regions, which are involved in the up-regulation of gene expression that occurs during decidualization..
74. 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..
75. Jun Ueda, Kazumitsu Maehara, Daisuke Mashiko, Takako Ichinose, Tatsuma Yao, Mayuko Hori, Yuko Sato, Hiroshi Kimura, Yasuyuki Ohkawa, Kazuo Yamagata, Heterochromatin dynamics during the differentiation process revealed by the DNA methylation reporter mouse, methylRO, Stem Cell Reports, 10.1016/j.stemcr.2014.05.008, 2, 6, 910-924, 2014.06, In mammals, DNA is methylated at CpG sites, which play pivotal roles in gene silencing and chromatin organization. Furthermore, DNA methylation undergoes dynamic changes during development, differentiation, and in pathological processes. The conventional methods represent snapshots; therefore, the dynamics of this marker within living organisms remains unclear. To track this dynamics, we made a knockin mouse that expresses a red fluorescent protein (RFP)-fused methyl-CpG-binding domain (MBD) protein from the ROSA26 locus ubiquitously; we named it MethylRO (methylation probe in ROSA26 locus). Using this mouse, we performed RFP-mediated methylated DNA immunoprecipitation sequencing (MeDIP-seq), whole-body section analysis, and live-cell imaging. We discovered that mobility and pattern of heterochromatin as well as DNA methylation signal intensity inside the nuclei can be markers for cellular differentiation status. Thus, the MethylRO mouse represents a powerful bioresource and technique for DNA methylation dynamics studies in developmental biology, stem cell biology, as well as in disease states..
76. Atsushi Yokoyama, Katsuhide Igarashi, Tetsuya Sato, Kiyoshi Takagi, Maky I. Otsuka, Yurina Shishido, Takashi Baba, Ryo Ito, Jun Kanno, Yasuyuki Ohkawa, Ken-Ichirou Morohashi, Akira Sugawara, Identification of myelin transcription factor 1 (MyT1) as a subunit of the neural cell type-specific lysine-specific demethylase 1 (LSD1) complex, Journal of Biological Chemistry, 10.1074/jbc.M114.566448, 289, 26, 18152-18162, 2014.06, Regulation of spatiotemporal gene expression in higher eukaryotic cells is critical for the precise and orderly development of undifferentiated progenitors into committed cell types of the adult. It is well known that dynamic epigenomic regulation (including chromatin remodeling and histone modifications by transcriptional coregulator complexes) is involved in transcriptional regulation. Precisely how these coregulator complexes exert their cell type and developing stage-specific activity is largely unknown. In this study we aimed to isolate the histone demethylase lysine-specific demethylase 1 (LSD1) complex from neural cells by biochemical purification. In so doing, we identified myelin transcription factor 1 (MyT1) as a novel LSD1 complex component. MyT1 is a neural cell-specific zinc finger factor, and it forms a stable multiprotein complex with LSD1 through direct interaction. Target gene analysis using microarray and ChIP assays revealed that the Pten gene was directly regulated by the LSD1-MyT1 complex. Knockdown of either LSD1orMyT1 derepressed the expressionofendogenous target genes and inhibited cell proliferation of a neuroblastoma cell line, Neuro2a. We propose that formation of tissue-specific combinations of coregulator complexes is a critical mechanism for tissue-specific transcriptional regulation..
77. 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..
78. Takashi Baba, Hiroyuki Otake, Tetsuya Sato, Kanako Miyabayashi, Yurina Shishido, Chia Yih Wang, Yuichi Shima, Hiroshi Kimura, Mikako Yagi, Yasuhiro Ishihara, Shinjiro Hino, Hidesato Ogawa, Mitsuyoshi Nakao, Takeshi Yamazaki, Dongchon Kang, Yasuyuki Ohkawa, Mikita Suyama, Bon Chu Chung, Ken-Ichirou Morohashi, Glycolytic genes are targets of the nuclear receptor Ad4BP/SF-1, Nature Communications, 10.1038/ncomms4634, 5, 2014.04, Genetic deficiencies in transcription factors can lead to the loss of certain types of cells and tissue. The steroidogenic tissue-specific nuclear receptor Ad4BP/SF-1 (NR5A1) is one such gene, because mice in which this gene is disrupted fail to develop the adrenal gland and gonads. However, the specific role of Ad4BP/SF-1 in these biological events remains unclear. Here we use chromatin immunoprecipitation sequencing to show that nearly all genes in the glycolytic pathway are regulated by Ad4BP/SF-1. Suppression of Ad4BP/SF-1 by small interfering RNA reduces production of the energy carriers ATP and nicotinamide adenine dinucleotide phosphate, as well as lowers expression of genes involved in glucose metabolism. Together, these observations may explain tissue dysgenesis as a result of Ad4BP/SF-1 gene disruption in vivo. Considering the function of estrogen-related receptor α, the present study raises the possibility that certain types of nuclear receptors regulate sets of genes involved in metabolic pathways to generate energy carriers..
79. 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..
80. 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..
81. Kazu Kobayakawa, Hiromi Kumamaru, Hirokazu Saiwai, Kensuke Kubota, Yasuyuki Ohkawa, Junji Kishimoto, Kazuya Yokota, Ryosuke Ideta, Keiichiro Shiba, Hidetoshi Saitoh, Kazuhide Inoue, Yukihide Iwamoto, Seiji Okada, Acute hyperglycemia impairs functional improvement after spinal cord injury in mice and humans, Science Translational Medicine, 10.1126/scitranslmed.3009430, 6, 256, 2014.01, Spinal cord injury (SCI) is a devastating disorder for which the identification of exacerbating factors is urgently needed. We demonstrate that transient hyperglycemia during acute SCI is a detrimental factor that impairs functional improvement in mice and human patients after acute SCI. Under hyperglycemic conditions, both in vivo and in vitro, inflammation was enhanced through promotion of the nuclear translocation of the nuclear factor κB (NF-κB) transcription factor in microglial cells. During acute SCI, hyperglycemic mice exhibited progressive neural damage, with more severe motor deficits than those observed in normoglycemic mice. Consistent with the animal study findings, a Pearson χ2 analysis of data for 528 patients with SCI indicated that hyperglycemia on admission (glucose concentration ≥126 mg/dl) was a significant risk predictor of poor functional outcome. Moreover, a multiple linear regression analysis showed hyperglycemia at admission to be a powerful independent risk factor for a poor motor outcome, even after excluding patients with diabetes mellitus with chronic hyperglycemia (regression coefficient, -1.37; 95% confidence interval, -2.65 to -0.10; P < 0.05). Manipulating blood glucose during acute SCI in hyperglycemic mice rescued the exacerbation of pathophysiology and improved motor functional outcomes. Our findings suggest that hyperglycemia during acute SCI may be a useful prognostic factor with a negative impact on motor function, highlighting the importance of achieving tight glycemic control after central nervous system injury..
82. Masanori Matsumoto, Akemi Baba, Takafumi Yokota, Hiroyoshi Nishikawa, Yasuyuki Ohkawa, Hisako Kayama, Axel Kallies, Stephen L. Nutt, Shimon Sakaguchi, Kiyoshi Takeda, Tomohiro Kurosaki, Yoshihiro Baba, Interleukin-10-producing plasmablasts exert regulatory function in autoimmune inflammation, Immunity, 10.1016/j.immuni.2014.10.016, 41, 6, 1040-1051, 2014.01, B cells can suppress autoimmunity by secreting interleukin-10 (IL-10). Although subpopulations of splenic B lineage cells are reported to express IL-10 invitro, the identity of IL-10-producing B cells with regulatory function invivo remains unknown. By using IL-10 reporter mice, we found that plasmablasts in the draining lymph nodes (dLNs), but not splenic B lineage cells, predominantly expressed IL-10 during experimental autoimmune encephalomyelitis (EAE). These plasmablasts were generated only during EAE inflammation. Mice lacking plasmablasts by genetic ablation of the transcription factors Blimp1 or IRF4 in B lineage cells developed an exacerbated EAE. Furthermore, IRF4 positively regulated IL-10 production that can inhibit dendritic cell functions to generate pathogenic Tcells. Our data demonstrate that plasmablasts in the dLNs serve as IL-10 producers to limit autoimmune inflammation and emphasize the importance of plasmablasts as IL-10-producing regulatory B cells..
83. Shinya Oki, Kazumitsu Maehara, Yasuyuki Ohkawa, Chikara Meno, SraTailor
Graphical user interface software for processing and visualizing ChIP-seq data, Genes to Cells, 10.1111/gtc.12190, 19, 12, 919-926, 2014.01, Raw data from ChIP-seq (chromatin immunoprecipitation combined with massively parallel DNA sequencing) experiments are deposited in public databases as SRAs (Sequence Read Archives) that are publically available to all researchers. However, to graphically visualize ChIP-seq data of interest, the corresponding SRAs must be downloaded and converted into BigWig format, a process that involves complicated command-line processing. This task requires users to possess skill with script languages and sequence data processing, a requirement that prevents a wide range of biologists from exploiting SRAs. To address these challenges, we developed SraTailor, a GUI (Graphical User Interface) software package that automatically converts an SRA into a BigWig-formatted file. Simplicity of use is one of the most notable features of SraTailor: entering an accession number of an SRA and clicking the mouse are the only steps required to obtain BigWig-formatted files and to graphically visualize the extents of reads at given loci. SraTailor is also able to make peak calls, generate files of other formats, process users' own data, and accept various command-line-like options. Therefore, this software makes ChIP-seq data fully exploitable by a wide range of biologists. SraTailor is freely available at, and runs on both Mac and Windows machines..
84. Scott E. LeBlanc, Qiong Wu, A. Rasim Barutcu, Hengyi Xiao, Yasuyuki Ohkawa, Anthony N. Imbalzano, The PPARγ locus makes long-range chromatin interactions with selected tissue-specific gene loci during adipocyte differentiation in a protein kinase a dependent manner, PLoS One, 10.1371/journal.pone.0086140, 9, 1, 2014.01, Differentiation signaling results in reprogramming of cellular gene expression that leads to morphological changes and functional specialization of a precursor cell. This global change in gene expression involves temporal regulation of differentiation-specific genes that are located throughout the genome, raising the idea that genome structure may also be re-organized during cell differentiation to facilitate regulated gene expression. Using in vitro adipocyte differentiation as a model, we explored whether gene organization within the nucleus is altered upon exposure of precursor cells to signaling molecules that induce adipogenesis. The peroxisome proliferator-activated receptor gamma (PPARγ) nuclear hormone receptor is a master determinant of adipogenesis and is required for adipose differentiation. We utilized the chromosome conformation capture (3C) assay to determine whether the position of the PPARc locus relative to other adipogenic genes is changed during differentiation. We report that the PPARγ2 promoter is transiently positioned in proximity to the promoters of genes encoding adipokines and lipid droplet associated proteins at 6 hours post-differentiation, a time that precedes expression of any of these genes. In contrast, the PPARγ2 promoter was not in proximity to the EF1α promoter, which drives expression of a constitutively active, housekeeping gene that encodes a translation elongation factor, nor was the PPARγ2 promoter in proximity to the promoter driving the expression of the C/EBPα regulatory protein. The formation of the long-range, intergenic interactions involving the PPARγ2 promoter required the regulatory factor C/EBPβ, elevated cyclic AMP (cAMP) levels, and protein kinase A (PKA) signaling. We conclude that genome organization is dynamically remodeled in response to adipogenic signaling, and we speculate that these transient inter-genic interactions may be formed for the purposes of selecting some of the transcriptionally silent tissue-specific loci for subsequent transcriptional activation..
85. Jun Katahira, Daisuke Okuzaki, Hitomi Inoue, Yoshihiro Yoneda, Kazumitsu Maehara, Yasuyuki Ohkawa, Human TREX component Thoc5 affects alternative polyadenylation site choice by recruiting mammalian cleavage factor I, Nucleic Acids Research, 10.1093/nar/gkt414, 41, 14, 7060-7072, 2013.08, The transcription-export complex (TREX) couples mRNA transcription, processing and nuclear export. We found that CFIm68, a large subunit of a heterotetrameric protein complex mammalian cleavage factor I (CFIm), which is implicated in alternative polyadenylation site choice, co-purified with Thoc5, a component of human TREX. Immunoprecipitation using antibodies against different components of TREX indicated that most likely both complexes interact via an interaction between Thoc5 and CFIm68. Microarray analysis using human HeLa cells revealed that a subset of genes was differentially expressed on Thoc5 knockdown. Notably, the depletion of Thoc5 selectively attenuated the expression of mRNAs polyadenylated at distal, but not proximal, polyadenylation sites, which phenocopied the depletion of CFIm68. Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) indicated that CFIm68 preferentially associated with the 5′ regions of genes; strikingly, the 5′ peak of CFIm68 was significantly and globally reduced on Thoc5 knockdown. We suggest a model in which human Thoc5 controls polyadenylation site choice through the co-transcriptional loading of CFIm68 onto target genes..
86. Hiromi Kumamaru, Hirokazu Saiwai, Kensuke Kubota, Kazu Kobayakawa, Kazuya Yokota, Yasuyuki Ohkawa, Keiichiro Shiba, Yukihide Iwamoto, Seiji Okada, Therapeutic activities of engrafted neural stem/precursor cells are not dormant in the chronically injured spinal cord, Stem Cells, 10.1002/stem.1404, 31, 8, 1535-1547, 2013.08, The transplantation of neural stem/precursor cells (NSPCs) is a promising therapeutic strategy for many neurodegenerative disorders including spinal cord injury (SCI) because it provides for neural replacement or trophic support. This strategy is now being extended to the treatment of chronic SCI patients. However, understanding of biological properties of chronically transplanted NSPCs and their surrounding environments is limited. Here, we performed temporal analysis of injured spinal cords and demonstrated their multiphasic cellular and molecular responses. In particular, chronically injured spinal cords were growth factorenriched environments, whereas acutely injured spinal cords were enriched by neurotrophic and inflammatory factors. To determine how these environmental differences affect engrafted cells, NSPCs transplanted into acutely, subacutely, and chronically injured spinal cords were selectively isolated by flow cytometry, and their whole transcriptomes were compared by RNA sequencing. This analysis revealed that NSPCs produced many regenerative/ neurotrophic molecules irrespective of transplantation timing, and these activities were prominent in chronically transplanted NSPCs. Furthermore, chronically injured spinal cords permitted engrafted NSPCs to differentiate into neurons/oligodendrocytes and provided more neurogenic environment for NSPCs than other environments. Despite these results demonstrate that transplanted NSPCs have adequate capacity in generating neurons/oligodendrocytes and producing therapeutic molecules in chronic SCI microenvironments, they did not improve locomotor function. Our results indicate that failure in chronic transplantation is not due to the lack of therapeutic activities of engrafted NSPCs but the refractory state of chronically injured spinal cords. Environmental modulation, rather modification of transplanting cells, will be significant for successful translation of stem cell-based therapies into chronic SCI patients..
87. Keiko Kitajima, Shinya Oki, Yasuyuki Ohkawa, Tomoyuki Sumi, Chikara Meno, Wnt signaling regulates left-right axis formation in the node of mouse embryos, Developmental Biology, 10.1016/j.ydbio.2013.05.011, 380, 2, 222-232, 2013.08, The node triggers formation of the left-right axis in mouse embryos by establishing local asymmetry of Nodal and Cerl2 expression. We found that Wnt3 is expressed in perinodal crown cells preferentially on the left side. The enhancer responsible for Wnt3 expression was identified and found to be regulated by Foxa2 and Rbpj under the control of Notch signaling. Rbpj binding sites suppress enhancer activity in pit cells of the node, thereby ensuring crown cell-specific expression. In addition, we found that the expression of Gdf1 and Cerl2 is also regulated by Notch signaling, suggesting that such signaling may induce the expression of genes related to left-right asymmetry as a set. Furthermore, Cerl2 expression became symmetric in response to inhibition of Wnt-β-catenin signaling. Our results suggest that Wnt signaling regulates the asymmetry of Cerl2 expression, which likely generates a left-right difference in Nodal activity at the node for further amplification in lateral plate mesoderm..
88. M. Villacorte, K. Suzuki, A. Hirasawa, Yasuyuki Ohkawa, Mikita Suyama, T. Maruyama, D. Aoki, Yukiko Ogino, S. Miyagawa, T. Terabayashi, Y. Tomooka, N. Nakagata, G. Yamada, β-Catenin signaling regulates Foxa2 expression during endometrial hyperplasia formation, Oncogene, 10.1038/onc.2012.376, 32, 29, 3477-3482, 2013.07, The Wnt/β-catenin signaling is essential for various organogenesis and is often implicated during tumorigenesis. Dysregulated β-catenin signaling is associated with the formation of endometrial adenocarcinomas (EACs), which is considered as the common form of endometrial cancer in women. In the current study, we investigate the downstream target of Wnt/β-catenin signaling in the uterine epithelia and the mechanism leading to the formation of endometrial hyperplasia. We report that conditional ablation and activation of β-catenin in the uterine epithelia lead to aberrant epithelial structures and endometrial hyperplasia formation, respectively. We demonstrate that β-catenin regulates Foxa2 with its candidate upstream region for the uterine epithelia. Furthermore, knockdown of Foxa2 leads to defects in cell cycle regulation, suggesting a possible function of Foxa2 in the control of cell proliferation. We also observe that β-catenin and Foxa2 expression levels are augmented in the human specimens of complex atypical endometrial hyperplasia, which is considered to have a greater risk of progression to EACs. Thus, our study indicates that β-catenin regulates Foxa2 expression, and this interaction is possibly essential to control cell cycle progression during endometrial hyperplasia formation. Altogether, the augmented expression levels of β-catenin and Foxa2 are essential features during the formation of endometrial hyperplasia..
89. Tomohiko Yoshimi, Yasuyuki Ohkawa, Masayuki Azuma, Taro Tachibana, A panel of specific monoclonal antibodies directed against various phosphorylated histones H3, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/mab.2012.0105, 32, 2, 119-124, 2013.04, Modification of histone plays a critical role in the epigenetic regulation of gene expression. However, unlike the widely studied roles of histone methylation or acetylation of histone H3, relatively little is known about the molecular mechanisms involved in translating histone phosphorylation into a specific outcome. The present study reports on the development of antibodies (MAbs) directed against phosphorylated histone H3 (S10, T11, S28, S31, and T32), produced by the hybridization of mouse myeloma cells with lymph node cells from an immunized rat or mouse. The MAbs produced specifically recognize different sites of phosphorylation on histone H3. All of these MAbs are suitable for immunoblotting and immunofluorescence analysis. We believe that these antibodies should significantly facilitate our efforts to investigate epigenetic regulation..
90. Hirokazu Saiwai, Hiromi Kumamaru, Yasuyuki Ohkawa, Kensuke Kubota, Kazu Kobayakawa, Hisakata Yamada, Takehiko Yokomizo, Yukihide Iwamoto, Seiji Okada, Ly6C+Ly6G-Myeloid-derived suppressor cells play a critical role in the resolution of acute inflammation and the subsequent tissue repair process after spinal cord injury, Journal of Neurochemistry, 10.1111/jnc.12135, 125, 1, 74-88, 2013.04, Acute inflammation is a prominent feature of central nervous system (CNS) insult and is detrimental to the CNS tissue. Although this reaction spontaneously diminishes within a short period of time, the mechanism underlying this inflammatory resolution remains largely unknown. In this study, we demonstrated that an initial infiltration of Ly6C+Ly6G- immature monocyte fraction exhibited the same characteristics as myeloid-derived suppressor cells (MDSCs), and played a critical role in the resolution of acute inflammation and in the subsequent tissue repair by using mice spinal cord injury (SCI) model. Complete depletion of Ly6C+Ly6G- fraction prior to injury by anti-Gr-1 antibody (clone: RB6-8C5) treatment significantly exacerbated tissue edema, vessel permeability, and hemorrhage, causing impaired neurological outcomes. Functional recovery was barely impaired when infiltration was allowed for the initial 24 h after injury, suggesting that MDSC infiltration at an early phase is critical to improve the neurological outcome. Moreover, intraspinal transplantation of ex vivo-generated MDSCs at sites of SCI significantly reduced inflammation and promoted tissue regeneration, resulting in better functional recovery. Our findings reveal the crucial role of an Ly6C+Ly6G- fraction as MDSCs in regulating inflammation and tissue repair after SCI, and also suggests an MDSC-based strategy that can be applied to acute inflammatory diseases. Myeloid-derived suppressor cells (MDSCs) exert immunosuppressive effects in several inflammatory diseases, including cancer and autoimmune disease. We demonstrated that Ly6C+Ly6G- myeloid cells which infiltrated into injured spinal cord had a typical feature of MDSCs and played a critical role in the attenuation of acute inflammation and the subsequent tissue repair process after spinal cord injury (SCI). Our findings clarified the role of MDSCs after traumatic SCI, and suggested a potential MDSC-based therapeutic strategy for the acute phase of central nervous system injury..
91. 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..
92. Hiromi Kumamaru, Yasuyuki Ohkawa, Hirokazu Saiwai, Hisakata Yamada, Kensuke Kubota, Kazu Kobayakawa, Koichi Akashi, Hideyuki Okano, Yukihide Iwamoto, Seiji Okada, Direct isolation and RNA-seq reveal environment-dependent properties of engrafted neural stem/progenitor cells, Nature Communications, 10.1038/ncomms2132, 3, 2012.11, Neural stem/progenitor cell (NSPC) transplantation is a promising treatment for various neurodegenerative disorders including spinal cord injury, however, no direct analysis has ever been performed on their in vivo profile after transplantation. Here we combined bioimaging, flow-cytometric isolation and ultra-high-throughput RNA sequencing to evaluate the cellular properties of engrafted NSPCs. The acutely transplanted NSPCs had beneficial effects on spinal cord injury, particularly neuroprotection and neurohumoral secretion, whereas their in situ secretory activity differed significantly from that predicted in vitro. The RNA-sequencing of engrafted NSPCs revealed dynamic expression/splicing changes in various genes involved in cellular functions and tumour development depending on graft environments. Notably, in the pathological environment, overall transcriptional activity, external signal transduction and neural differentiation of engrafted NSPCs were significantly suppressed. These results highlight the vulnerability of engrafted NSPCs to environmental force, while emphasizing the importance of in situ analysis in advancing the efficacy and safety of stem cell-based therapies..
93. 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..
94. Kensuke Kubota, Hirokazu Saiwai, Hiromi Kumamaru, Takeshi Maeda, Yasuyuki Ohkawa, Yasuaki Aratani, Tetsuo Nagano, Yukihide Iwamoto, Seiji Okada, Myeloperoxidase exacerbates secondary injury by generating highly reactive oxygen species and mediating neutrophil recruitment in experimental spinal cord injury, Spine, 10.1097/BRS.0b013e31824b9e77, 37, 16, 1363-1369, 2012.07, STUDY DESIGN. An animal study using myeloperoxidase-knockout (MPO-KO) mice to examine the in vivo role of myeloperoxidase (MPO) in spinal cord injury (SCI). OBJECTIVE. To clarify the influence of MPO on inflammatory cell infiltration, tissue damage, and functional recovery after SCI. SUMMARY OF BACKGROUND DATA. MPO is considered to be important in spreading tissue damage after SCI because it generates strong neurotoxic oxidant hypochlorous acid (HOCl). However, the direct involvement of MPO in the pathophysiology of SCI remains to be elucidated. METHODS. To compare the inflammatory reaction, tissue damage, and neurological recovery after SCI, a moderate contusion injury was created at the ninth thoracic level in MPO-KO mice and wild-type mice. A HOCl-specific probe solution was injected into the lesion epicenter to assess the spatiotemporal production of MPO-derived HOCl. Inflammatory reactions were quantified by flow cytometry and quantitative real-time polymerase chain reaction, and tissue damage was evaluated by an immunohistochemical analysis. The motor function recovery was assessed by the open-field locomotor score. RESULTS. Prominent production of HOCl was observed during the hyperacute phase of SCI at the lesion site in the wild-type mice; however, little expression was observed in the MPO-KO mice. In this phase, the number of infiltrated neutrophils was significantly reduced in the MPO-KO mice compared with the wild-type mice. In addition, significant differences were observed in the expression levels of proinflammatory cytokines and apoptosis-related genes between 2 groups. In the histological sections, fewer terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive apoptotic cells and more spared myelin were observed at the lesion site in MPO-KO mice. Consistent with these results, better functional recovery was observed in the MPO-KO mice than in the wild-type mice after SCI. CONCLUSION. These results clearly indicated that MPO exacerbated secondary injury and impaired the functional recovery not only by generating strong oxidant HOCl, but also by enhancing neutrophil infiltration after SCI..
95. Yoko Fujii, Masayuki Shiota, Yasuyuki Ohkawa, Akemi Baba, Hideki Wanibuchi, Tatsuo Kinashi, Tomohiro Kurosaki, Yoshihiro Baba, Surf4 modulates STIM1-dependent calcium entry, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2012.05.037, 422, 4, 615-620, 2012.06, Store-operated Ca 2+ entry (SOCE) is crucial for various physiological responses in immune cells. Although it is known that STIM1 relocates into discrete puncta juxtaposed to the plasma membrane to initiate SOCE, the machinery modulating the function of STIM1 remains unclear. We explored to find its modulators using affinity purification for STIM1-binding proteins and identified surfeit locus protein 4 (Surf4). Surf4 associated with STIM1 in the endoplasmic reticulum. Deletion of Surf4 in DT40 B cells resulted in marked increase of SOCE and facilitation of STIM1 clustering upon store-depletion. These findings suggest the modulatory function of Surf4 for STIM1-mediated SOCE..
96. Hiromi Kumamaru, Hirokazu Saiwai, Yasuyuki Ohkawa, Hisakata Yamada, Yukihide Iwamoto, Seiji Okada, Age-related differences in cellular and molecular profiles of inflammatory responses after spinal cord injury, Journal of Cellular Physiology, 10.1002/jcp.22845, 227, 4, 1335-1346, 2012.04, Previous experimental and clinical studies have suggested that the behavioral and pathological outcomes of spinal cord injury (SCI) are affected by the individual's age at the time of injury. However, the underlying mechanism responsible for these differences remains elusive because it is difficult to match injuries of similar severities between young and adult animals due to differences in the sizes of their respective spinal cords. In this study, the spinal cord size-matched young (4-week-old) and adult (10-week-old) mice were compared to evaluate their locomotor functions and inflammatory cellular/molecular responses after standardized contusion SCI. During the acute phase of SCI, young mice showed better functional recovery and lower pro-inflammatory cytokines/chemokines compared to adult mice. Flow-cytometric analysis revealed that the time courses of leukocyte infiltration were comparable between both groups, while the number of infiltrating neutrophils significantly decreased from 6h after SCI in young mice. By combining flow-cytometric isolation and gene expression analysis of each inflammatory cell fraction, we found that microglial cells immediately initiate the production of several cytokines in response to SCI, which serve as major sources of IL-6, TNFa, and CXCL1 in injured spinal cord. Interestingly, the secretion of pro-inflammatory cytokines/chemokines but not anti-inflammatory cytokines by microglia was significantly lower in young mice compared to that in adult mice at 3h after SCI, which will be attributed to the attenuation of the subsequent neutrophil infiltration. These results highlight age-related differences in pro-inflammatory properties of microglial cells that contribute to the amplification of detrimental inflammatory responses after SCI..
97. Yasuyuki Ohkawa, Chandrashekara Mallappa, Caroline S Dacwag Vallaster, Anthony N. Imbalzano, An improved restriction enzyme accessibility assay for analyzing changes in chromatin structure in samples of limited cell number, Myogenesis Methods and Protocols, 10.1007/978-1-61779-343-1_32, 798, 531-542, 2012.01, Studies investigating mechanisms that control gene regulation frequently examine the accessibility of specific DNA sequences to nuclease cleavage. In general, sequences that are sensitive to nuclease cleavage are considered to be in an "open" chromatin conformation that is associated with regulatory factor binding, while sequences resistant to nuclease cleavage are considered to be in a "closed" conformation commonly associated with chromatin that is neither poised for transcription nor being actively transcribed. Changes in nuclease accessibility at specific genomic sequences reflect changes in the local chromatin structure that can occur as a result of signaling cues in the extracellular environment. These changes in chromatin structure usually precede or are coincident with changes in gene expression patterns and are therefore a useful marker of regulatory events controlling transcription. We describe a method to perform restriction enzyme accessibility assays (REAA) that utilizes ligation-mediated polymerase chain reaction (LM-PCR) technology and that permits assessment of samples from any source containing as few as 1,000 cells. Use of this modified REAA protocol will enhance analysis of chromatin structural changes at specific DNA sequences of interest by making it possible to analyze samples where unrestricted amounts of sample are not readily available..
98. Yasuyuki Ohkawa, Chandrashekara Mallappa, Caroline S Dacwag Vallaster, Anthony N. Imbalzano, Isolation of nuclei from skeletal muscle satellite cells and myofibers for use in chromatin immunoprecipitation assays, Myogenesis Methods and Protocols, 10.1007/978-1-61779-343-1_31, 798, 517-530, 2012.01, Studies investigating mechanisms controlling gene regulation frequently examine specific DNA sequences using chromatin immunoprecipitation (ChIP) assays to determine whether specific regulatory factors or modified histones are present. While use of primary cells or cell line models for differentiating or differentiated tissue is widespread, the ability to assess factor binding and histone modification in tissue defines the events that occur in vivo and provides corroboration for studies in cultured cells. Many tissues can be analyzed with minimal modification to existing ChIP protocols that are designed for cultured cells; however, some tissues, such as skeletal muscle, are problematic in that accessibility of the cross-linking agent is limited. We describe a method to isolate skeletal muscle tissue nuclei suitable for use in ChIP protocols. Furthermore, we utilize a simple fractionation of digested skeletal muscle tissue that can separate mature myofibers from satellite cells, which are responsible for postnatal skeletal muscle regeneration, thereby allowing simultaneous preparation of nuclei from both cell types..
99. 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..
100. 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..
101. 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..
102. Hengyi Xiao, Scott E. LeBlanc, Qiong Wu, Silvana Konda, Nunciada Salma, Concetta G.A. Marfella, Yasuyuki Ohkawa, Anthony N. Imbalzano, Chromatin accessibility and transcription factor binding at the PPARγ2 promoter during adipogenesis is protein kinase A-dependent, Journal of Cellular Physiology, 10.1002/jcp.22308, 226, 1, 86-93, 2011.01, The nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-activated transcription factor that specifies formation of the adipocyte lineage. PPARγ also serves as a primary target for the treatment of type 2 diabetes, illustrating both its medical relevance as well as the need to understand fundamental aspects of PPARγ expression and function. Here, we characterize molecular changes that occur at the PPARγ2 promoter within the first several hours of adipocyte differentiation in culture. Our results demonstrate that changes in chromatin accessibility at the PPARγ2 promoter and occupancy of the promoter by the c-Fos transcription factor occur within an hour of the onset of differentiation, followed closely by the binding of the CCAAT/enhancer binding protein beta (C/EBPβ) transcription factor. All three events show a remarkable dependency on protein kinase A (PKA) activity. These results reflect novel requirements for the PKA signaling pathway and reinforce the importance of PKA function during the onset of adipocyte differentiation..
103. Seiji Okada, Takeshi Maeda, Hirokazu Saiwai, Yasuyuki Ohkawa, Keiichiro Shiba, Yukihide Iwamoto, Ossification of the posterior longitudinal ligament of the lumbar spine
A case series, Neurosurgery, 10.1227/NEU.0b013e3181ef2806, 67, 5, 1311-1318, 2010.11, Background: Reports on ossification of the posterior longitudinal ligament (OPLL) of the lumbar spine have so far been limited. Objective: To evaluate surgically documented cases of lumbar OPLL at our facility to clarify its characteristics and analyze clinical outcomes. Methods: During the past 27 years, 6192 patients underwent operations for degenerative lumbar spine diseases. Of these, we selected surgically treated lumbar OPLL patients from our database to analyze the clinical and radiological disease characteristics. Surgical results were classified according to the Japanese Orthopaedic Association scale. Results: Only 10 patients underwent surgery for lumbar OPLL: 6 men and 4 women (mean age 44.1 years). Among these, OPLL developed in 4 patients at multiple vertebral body levels and in 6 at a single level. Coexisting lumbar disc herniation was observed in 4 patients. Although the rate of maximum canal stenosis brought about by OPLL was relatively high (mean 45.1%), unilateral radicular symptoms were the most frequently observed, and only 2 patients exhibited typical lumbar claudication caused by the canal stenosis. Two patients underwent surgery via an anterior approach and 8 via a posterior approach. The mean preoperative Japanese Orthopaedic Association scale score was 7.9, which improved to 17.8 postoperatively. No neurological deterioration caused by surgery was observed in any case. Conclusion: Although the frequency of lumbar OPLL requiring surgical treatment was remarkably low, its clinical condition varies greatly among patients depending on the localization and degree of ossification. To achieve a better surgical outcome, precise diagnosis with computed tomography and an appropriate surgical approach are important..
104. 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..
105. Chandrashekara Mallappa, Brian T. Nasipak, Letitiah Etheridge, Elliot J. Androphy, Stephen N. Jones, Charles G. Sagerström, Yasuyuki Ohkawa, Anthony N. Imbalzano, Myogenic microRNA expression requires ATP-dependent chromatin remodeling enzyme function, Molecular and Cellular Biology, 10.1128/MCB.00214-10, 30, 13, 3176-3186, 2010.07, Knockdown of the Brg1 ATPase subunit of SWI/SNF chromatin remodeling enzymes in developing zebrafish caused stunted tail formation and altered sarcomeric actin organization, which phenocopies the loss of the microRNA processing enzyme Dicer, or the knockdown of myogenic microRNAs. Furthermore, myogenic microRNA expression and differentiation was blocked in Brg1 conditional myoblasts differentiated ex vivo. The binding of Brg1 upstream of myogenic microRNA sequences correlated with MyoD binding and accessible chromatin structure in satellite cells and myofibers, and it was required for chromatin accessibility and microRNA expression in a tissue culture model for myogenesis. The results implicate ATP-dependent chromatin remodelers in myogenic microRNA gene regulation..
106. Yasuyuki Ohkawa, Monoclonal antibodies 6D2 and 8H3 against mouse Chd2, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2010.0025.MAb, 29, 3, 270, 2010.06.
107. Yasuyuki Ohkawa, Monoclonal antibody 8E3 against mouse Dhx9/NDHII/RHA, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2010.0026.MAb, 29, 3, 269, 2010.06.
108. 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..
109. 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..
110. 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..
111. 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..
112. Saori Yoshimura, Tomohiko Yoshimi, Yasuyuki Ohkawa, Masayuki Azuma, Taro Tachibana, A rat monoclonal antibody against the chromatin remodeling factor CHD5, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2009.0069, 29, 1, 63-66, 2010.02, CHD5 (chromodomain/helicase/DNA-binding protein 5) is a member of the CHD subfamily of chromatin remodeling Swi/Snf proteins, and has been recently identified as a tumor suppressor in a diverse range of human cancers. We report here on the establishment of a hybridoma cell line for producing a monoclonal antibody against CHD5 by the rat medial iliac lymph node method. Immunoblotting analyses indicated that this antibody, MAb 5A10, specifically recognizes endogenous CHD5. In immunostaining using the antibody, a nuclear staining pattern was observed. The monoclonal antibody will be useful in immunoblotting and immunolocalization experiments in a variety of cells and tissues, as well as in further studies of the biological function and cellular dynamics of this protein..
113. Yasuyuki Ohkawa, Monoclonal antibodies 2F12H4 and 3C3F1 against mouse Pax7, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2009.0076.MAb, 29, 1, 83, 2010.02.
114. Yasuyuki Ohkawa, Monoclonal antibody 1E2G4 against mouse Myf5, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2009.0089.MAb, 29, 1, 74, 2010.02.
115. Yasuyuki Ohkawa, Monoclonal antibody 1H7A10 against mouse Brm, Monoclonal Antibodies in Immunodiagnosis and Immunotherapy, 10.1089/hyb.2009.0077.MAb, 29, 1, 82, 2010.02.
116. 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..
117. 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..
118. 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..
119. 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..
120. 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..
121. Seiji Okada, Takeshi Maeda, Yasuyuki Ohkawa, Katsumi Harimaya, Hirokazu Saiwai, Hiromi Kumamaru, Yoshihiro Matsumoto, Toshio Doi, Takayoshi Ueta, Keiichiro Shiba, Yukihide Iwamoto, Does ossification of the posterior longitudinal ligament affect the neurological outcome after traumatic cervical cord injury?, Spine, 10.1097/BRS.0b013e31819e3215, 34, 11, 1148-1152, 2009.05, STUDY DESIGN.: Retrospective outcome measurement study. OBJECTIVES.: The purpose of this study is to assess whether ossification of the posterior longitudinal ligament (OPLL) affects neurologic outcomes in patients with acute cervical spinal cord injury (SCI). SUMMARY OF BACKGROUND DATA.: There have so far been few reports examining the relationship between OPLL and SCI and there is controversy regarding the deteriorating effects of OPLL-induced canal stenosis on neurologic outcomes. METHODS.: To obtain a relatively uniform background, patients nonsurgically treated for an acute C3-C4 level SCI without any fractures or dislocations of the spinal column were selected, resulting in 129 patients. There were 110 men and 19 women (mean age was 61.1 years), having various neurologic conditions on admission (American Spinal Injury Association [ASIA] impairment scale A, 43; B, 16; C, 58; D, 12). The follow-up period was the duration of their hospital stay and ranged from 50 to 603 days (mean, 233 days). The presence of OPLL, the cause of injury, the degree of canal stenosis (both static and dynamic), and the neurologic outcomes in motor function, including improvement rate, were assessed. RESULTS.: Of the 129 patients investigated in this study, OPLL was identified at the site of the injury in 13 patients (10.1%). In this OPLL+ group, the static and dynamic canal diameters at C3 and C4 were significantly smaller than those of the remaining 116 patients (OPLL- group). However, no significant difference was observed between the 2 groups in terms of ASIA motor score both at the time of administration and discharge, and the mean improvement rate in ASIA motor score was 55.5 ± 9.0% in OPLL+ group, while it was 43.1 ± 2.8% in the OPLL-group. Furthermore, no significant correlation was observed between the static/dynamic canal diameters and neurologic outcome in all 129 patients. CONCLUSION.: No evidence was found for OPLL to have any effect on the initial neurologic status or recovery in motor function after traumatic cervical cord injury, suggesting that the neurologic outcome is not significantly dependent on canal space..
122. Yasuyuki Ohkawa, Saori Yoshimura, Chiduru Higashi, Concetta G A Marfella, Caroline S. Dacwag, Taro Tachibana, Anthony N. Imbalzano, Myogenin and the SWI/SNF ATPase Brg1 maintain myogenic gene expression at different stages of skeletal myogenesis, Journal of Biological Chemistry, 10.1074/jbc.M608898200, 282, 9, 6564-6570, 2007.03, Many studies have examined transcriptional regulation during the initiation of skeletal muscle differentiation; however, there is less information regarding transcriptional control during adult myogenesis and during the maintenance of the differentiated state. MyoD and the mammalian SWI/SNF chromatin-remodeling enzymes containing the Brg1 ATPase are necessary to induce myogenesis in cell culture models and in developing embryonic tissue, whereas myogenin and Brg1 are critical for the expression of the late genes that induce terminal muscle differentiation. Here, we demonstrate that myogenin also binds to its own promoter during the late stages of embryonic muscle development. As is the case during embryonic myogenesis, MyoD and Brg1 co-localize to the myogenin promoter in primary adult muscle satellite cells. However, in mature myofibers, myogenin and Brg1 are preferentially co-localized to the myogenin promoter. Thus, the myogenin promoter is occupied by different myogenic factors at different times of myogenesis. The relevance of myogenin in the continued expression from its own promoter is demonstrated in culture, where we show that myogenin, in the absence of MyoD, is capable of maintaining its own expression by recruiting the Brg1 ATPase to modify promoter chromatin structure and facilitate myogenin expression. Finally, we utilized in vivo electroporation to demonstrate that Brg1 is required for the continued production of the myogenin protein in newborn skeletal muscle tissue. These findings strongly suggest that the skeletal muscle phenotype is maintained by myogenin and the continuous activity of Brg1-based SWI/SNF chromatin-remodeling enzymes..
123. Caroline S. Dacwag, Yasuyuki Ohkawa, Sharmistha Pal, Saíd Sif, Anthony N. Imbalzano, The protein arginine methyltransferase Prmt5 is required for myogenesis because it facilitates ATP-dependent chromatin remodeling, Molecular and Cellular Biology, 10.1128/MCB.01528-06, 27, 1, 384-394, 2007.01, Skeletal muscle differentiation requires the coordinated activity of transcription factors, histone modifying enzymes, and ATP-dependent chromatin remodeling enzymes. The type II protein arginine methyltransferase Prmt5 symmetrically dimethylates histones H3 and H4 and numerous nonchromatin proteins, and prior work has implicated Prmt5 in transcriptional repression. Here we demonstrate that MyoD-induced muscle differentiation requires Prmt5. One of the first genes activated during differentiation encodes the myogenic regulator myogenin. Prmt5 and dimethylated H3R8 (histone 3 arginine 8) are localized at the myogenin promoter in differentiating cells. Modification of H3R8 required Prmt5, and reduction of Prmt5 resulted in the abrogation of promoter binding by the Brg1 ATPase-associated with the SWI/SNF chromatin remodeling enzymes and all subsequent events associated with gene activation, including increases in chromatin accessibility and stable binding by MyoD. Prmt5 and dimethylated H3R8 were also associated with the myogenin promoter in activated satellite cells isolated from muscle tissue, further demonstrating the physiological relevance of these observations. The data indicate that Prmt5 facilitates myogenesis because it is required for Brg1-dependent chromatin remodeling and gene activation at a locus essential for differentiation. We therefore conclude that a histone modifying enzyme is necessary to permit an ATP-dependent chromatin remodeling enzyme to function..
124. Concetta G A Marfella, Yasuyuki Ohkawa, Andrew H. Coles, David S. Garlick, Stephen N. Jones, Anthony N. Imbalzano, Mutation of the SNF2 family member Chd2 affects mouse development and survival, Journal of Cellular Physiology, 10.1002/jcp.20718, 209, 1, 162-171, 2006.10, The chromodomain helicase DNA-binding domain (Chd) proteins belong to the SNF2-like family of ATPases that function in chromatin remodeling and assembly. These proteins are characterized by the presence of tandem chromodomains and are further subdivided based on the presence or absence of additional structural motifs. The Chd1-Chd2 subfamily is distinguished by the presence of a DNA-binding domain that recognizes AT-rich sequence. Currently, there are no reports addressing the function of the Chd2 family member. Embryonic stem cells containing a retroviral gene-trap inserted at the Chd2 locus were utilized to generate mice expressing a Chd2 protein lacking the DNA-binding domain. This mutation in Chd2 resulted in a general growth delay in homozygous mutants late in embryogenesis and in perinatal lethality. Animals heterozygous for the mutation showed decreased neonatal viability and increased susceptibility to non-neoplastic lesions affecting most primary organs. In particular, approximately 85% of the heterozygotes showed gross kidney abnormalities. Our results demonstrate that mutation of Chd2 dramatically affects mammalian development and long-term survival..
125. Ivana L. De La Serna, Yasuyuki Ohkawa, Chiduru Higashi, Chaitali Dutta, Jules Osias, Naveen Kommajosyula, Taro Tachibana, Anthony N. Imbalzano, The microphthalmia-associated transcription factor requires SWI/SNF enzymes to activate melanocyte-specific genes, Journal of Biological Chemistry, 10.1074/jbc.M512052200, 281, 29, 20233-20241, 2006.07, The microphthalmia transcription factor (Mitf) activates melanocyte-specific gene expression, is critical for survival and proliferation of melanocytes during development, and has been described as an oncogene in malignant melanoma. SWI/SNF complexes are ATP-dependent chromatin-remodeling enzymes that play a role in many developmental processes. To determine the requirement for SWI/SNF enzymes in melanocyte differentiation, we introduced Mitf into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, Brahma or Brahma-related gene 1 (BRG1). These dominant negative SWI/SNF components have been shown to inhibit gene activation events that normally require SWI/SNF enzymes. We found that Mitf-mediated activation of a subset of endogenous melanocyte-specific genes required SWI/SNF enzymes but that cell-cycle regulation occurred independently of SWI/SNF function. Activation of tyrosinase-related protein 1, a melanocyte-specific gene, correlated with SWI/SNF-dependent changes in chromatin accessibility at the endogenous locus. Both BRG1 and Mitf could be localized to the tyrosinase-related protein 1 and tyrosinase promoters by chromatin immunoprecipitation, whereas immunofluorescence and immunoprecipitation experiments indicate that Mitf and BRG1 co-localized in the nucleus and physically interacted. Together these results suggest that Mitf can recruit SWI/SNF enzymes to melanocyte-specific promoters for the activation of gene expression via induced changes in chromatin structure at endogenous loci..
126. Ivana L. De La Serna, Yasuyuki Ohkawa, Anthony N. Imbalzano, Chromatin remodelling in mammalian differentiation
Lessons from ATP-dependent remodellers, Nature Reviews Genetics, 10.1038/nrg1882, 7, 6, 461-473, 2006.06, The initiation of cellular differentiation involves alterations in gene expression that depend on chromatin changes, at the level of both higher-order structures and individual genes. Consistent with this, chromatin-remodelling enzymes have key roles in differentiation and development. The functions of ATP-dependent chromatin-remodelling enzymes have been studied in several mammalian differentiation pathways, revealing cell-type-specific and gene-specific roles for these proteins that add another layer of precision to the regulation of differentiation. Recent studies have also revealed a role for ATP-dependent remodelling in regulating the balance between proliferation and differentiation, and have uncovered intriguing links between chromatin remodelling and other cellular processes during differentiation, including recombination, genome organization and the cell cycle..
127. Yasuyuki Ohkawa, Concetta G A Marfella, Anthony N. Imbalzano, Skeletal muscle specification by myogenin and Mef2D via the SWI/SNF ATPase Brg1, EMBO Journal, 10.1038/sj.emboj.7600943, 25, 3, 490-501, 2006.02, Myogenin is required not for the initiation of myogenesis but instead for skeletal muscle formation through poorly understood mechanisms. We demonstrate in cultured cells and, for the first time, in embryonic tissue, that myogenic late genes that specify the skeletal muscle phenotype are bound by MyoD prior to the initiation of gene expression. At the onset of muscle specification, a transition from MyoD to myogenin occurred at late gene loci, concomitant with loss of HDAC2, the appearance of both the Mef2D regulator and the Brg1 chromatin-remodeling enzyme, and the opening of chromatin structure. We further demonstrated that ectopic expression of myogenin and Mef2D, in the absence of MyoD, was sufficient to induce muscle differentiation in a manner entirely dependent on Brg1. These results indicate that myogenin specifies the muscle phenotype by cooperating with Mef2D to recruit an ATP-dependent chromatin-remodeling enzyme that alters chromatin structure at regulatory sequences to promote terminal differentiation..
128. Ivana L. De La Serna, Yasuyuki Ohkawa, Charlotte A. Berkes, Donald A. Bergstrom, Caroline S. Dacwag, Stephen J. Tapscott, Anthony N. Imbalzano, MyoD targets chromatin remodeling complexes to the myogenin locus prior to forming a stable DNA-bound complex, Molecular and Cellular Biology, 10.1128/MCB.25.10.3997-4009.2005, 25, 10, 3997-4009, 2005.05, The activation of muscle-specific gene expression requires the coordinated action of muscle regulatory proteins and chromatin-remodeling enzymes. Microarray analysis performed in the presence or absence of a dominant-negative BRG1 ATPase demonstrated that approximately one-third of MyoD-induced genes were highly dependent on SWI/SNF enzymes. To understand the mechanism of activation, we performed chromatin immunoprecipitations analyzing the myogenin promoter. We found that H4 hyperacetylation preceded Brg1 binding in a MyoD-dependent manner but that MyoD binding occurred subsequent to H4 modification and Brg1 interaction. In the absence of functional SWI/SNF enzymes, muscle regulatory proteins did not bind to the myogenin promoter, thereby providing evidence for SWI/SNF-dependent activator binding. We observed that the homeodomain factor Pbx1, which cooperates with MyoD to stimulate myogenin expression, is constitutively bound to the myogenin promoter in a SWI/SNF-independent manner, suggesting a two-step mechanism in which MyoD initially interacts indirectly with the myogenin promoter and attracts chromatin-remodeling enzymes, which then facilitate direct binding by MyoD and other regulatory proteins..
129. Daniel W. Young, Jitesh Pratap, Amjad Javed, Brian Weiner, Yasuyuki Ohkawa, Andre Van Wijnen, Martin Montecino, Gary S. Stein, Janet L. Stein, Anthony N. Imbalzano, Jane B. Lian, SWI/SNF chromatin remodeling complex is obligatory for BMP2-induced, Runx2-dependent skeletal gene expression that controls osteoblast differentiation, Journal of Cellular Biochemistry, 10.1002/jcb.20332, 94, 4, 720-730, 2005.03, Development of bone tissue requires maturation of osteoblasts from mesenchymal precursors. BMP2, a member of the TGFβ superfamily, and the Runx2 (AML3/Cbfa1) transcription factor, a downstream BMP2 effector, are regulatory signals required for osteoblast differentiation. While Runx2 responsive osteogenic gene expression has been functionally linked to alterations in chromatin structure, the factors that govern this chromatin remodeling remain to be identified. Here, we address the role of the SWI/SNF chromatin remodeling enzymes in BMP2-induced, Runx2-dependent development of the osteoblast phenotype. For these studies, we have examined cal varial cells from wild-type (WT) mice and mice that are homozygous for the Runx2 null allele, as well as the C2C12 model of BMP2-induced osteogenesis. By the analysis of microarray data, we find that several components of the SWI/SNF complex are regulated during BMP2-mediated osteoblast differentiation. Brg1 is an essential DNA dependent ATPase subunit of the SWI/SNF complex. Thus, functional studies were carried out using a fibroblast cell line that conditionally expresses a mutant Brg1 protein, which exerts a dominant negative effect on SWI/SNF function. Our findings demonstrate that SWI/SNF is required for BMP2-induced expression of alkaline phosphatase (APase), an early marker reflecting Runx2 control of osteoblast differentiation. In addition, Brg1 is expressed in cells with in the developing skeleton of the mouse embryo as well as in osteoblasts ex vivo. Taken together these results support the concept that BMP2-mediated osteogenesis requires Runx2, and demonstrates that initiation of BMP2-induced, Runx2-dependent skeletal gene expression requires SWI/SNF chromatin remodeling complexes..
130. Masanori Takahashi, Ken'ichiro Hayashi, Kenji Yoshida, Yasuyuki Ohkawa, Toshi Komurasaki, Akira Kitabatake, Akira Ogawa, Wataru Nishida, Masahiko Yano, Morito Monden, Kenji Sobue, Epiregulin as a Major Autocrine/Paracrine Factor Released from ERK- and p38MAPK-Activated Vascular Smooth Muscle Cells, Circulation, 10.1161/01.CIR.0000096482.02567.8C, 108, 20, 2524-2529, 2003.11, Background - The coordinated activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) is critical for the induction of vascular and visceral smooth muscle cell (SMC) dedifferentiation. We previously reported that on the forced activation of both MAPKs, visceral SMCs secrete a non-heparin-binding protein factor(s) that is involved in the dedifferentiation of neighboring SMCs. In this study, we sought to identify the dedifferentiation factor(s) derived from vascular SMCs (VSMCs). Methods and Results - We fractionated the VSMC dedifferentiation factor(s) in the conditioned medium obtained from differentiated VSMCs in which both ERK and p38MAPK were forcedly activated and identified epiregulin as a major autocrine/paracrine factor for VSMC dedifferentiation. The epiregulin-induced VSMC dedifferentiation was mediated through the coordinated activation of ERK and p38MAPK. Unsaturated lysophosphatidic acid and platelet-derived growth factor-BB, which are potent VSMC dedifferentiation factors, rapidly upregulated epiregulin mRNA expression in an ERK- and p38MAPK-dependent manner. Reverse transcriptase-polymerase chain reaction and/or immunohistological analyses revealed the restricted expression of epiregulin in human atherosclerotic and balloon-injured rat arteries, in which the phenotypic modulation of medial VSMCs occurred in vivo. Conclusions - Epiregulin is released from VSMCs primed by atherogenic factors and acts as a major autocrine/paracrine factor for VSMC dedifferentiation. It may be involved in the progression of vascular remodeling such as atherosclerosis..
131. Kenji Yoshida, Wataru Nishida, Ken'ichiro Hayashi, Yasuyuki Ohkawa, Akira Ogawa, Junken Aoki, Hiroyuki Arai, Kenji Sobue, Vascular remodeling induced by naturally occurring unsaturated lysophosphatidic acid in vivo, Circulation, 10.1161/01.CIR.0000089374.35455.F3, 108, 14, 1746-1752, 2003.10, Background - We previously identified unsaturated (16:1, 18:1, and 18:2) but not saturated (12:0, 14:0, 16:0, and 18:0) lysophosphatidic acids (LPAs) as potent factors for vascular smooth muscle cell (VSMC) dedifferentiation. Unsaturated LPAs strongly induce VSMC dedifferentiation via the coordinated activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK), resulting in the proliferation and migration of dedifferentiated VSMCs. Here, we investigated the effects of 18:1 and 18:0 LPAs (as representative unsaturated and saturated LPAs, respectively) on the vasculature in vivo. Methods and Results - Rat common carotid arteries (CCAs) were treated transiently with 18:1 or 18:0 LPA and then examined by histological and biochemical analyses. The 18:1 but not 18:0 LPA potently induced vascular remodeling that was composed primarily of neointima. The incorporation of [3H]18:1 LPA into the CCAs revealed that a sufficient amount of unmetabolized [3H]18:1 LPA to induce VSMC dedifferentiation was present in the vascular wall. The 18:1 LPA-induced neointimal formation in vivo was also dependent on the coordinated activation of ERK and p38MAPK. Unlike balloon-injured CCAs, the 18:1 LPA-treated CCAs showed a histological similarity to human atherosclerotic arteries. Conclusions - This is the first report demonstrating a role for a naturally occurring unsaturated LPA in inducing vascular remodeling in vivo and provides a novel animal model for neointimal formation..
132. Yasuyuki Ohkawa, Ken'ichiro Hayashi, Kenji Sobue, Calcineurin-mediated pathway involved in the differentiated phenotype of smooth muscle cells, Biochemical and Biophysical Research Communications, 10.1016/S0006-291X(02)02965-0, 301, 1, 78-83, 2003.01, The calcineurin-mediated pathway is involved in skeletal and cardiac hypertrophy and vascular development in vivo, but the relationship between this pathway and the phenotype of smooth muscle cells (SMCs) remains unknown. Using visceral SMCs in culture as a model system of differentiated SMCs, we investigated the role of the calcineurin-mediated pathway in maintaining the differentiated phenotype of SMCs, which depends on the insulin-like growth factor (IGF-I)-triggered activation of the phosphatidylinositol 3-kinase (PI3-K)/protein kinase B (PKB(Akt)) pathway. Treatment with calcineurin inhibitors, cyclosporin A or FK506, or the forced expression of the natural calcineurin inhibitor, CAIN, induced SMC dedifferentiation. Notably, suppression of the promoter activities of the SMC molecular markers caldesmon and α1 integrin by blocking the PI3-K/PKB(Akt) pathway was rescued by the forced expression of constitutively active calcineurin Aα, suggesting that the calcineurin-mediated pathway is critical for maintaining the differentiated phenotype of SMCs and works downstream of the PI3-K/PKB(Akt) pathway..
133. Wataru Nishida, Mako Nakamura, Syunsuke Mori, Masanori Takahashi, Yasuyuki Ohkawa, Satoko Tadokoro, Kenji Yoshida, Kunio Hiwada, Ke N.Ichiro Hayashi, Kenji Sobue, A triad of serum response factor and the GATA and NK families governs the transcription of smooth and cardiac muscle genes, Journal of Biological Chemistry, 10.1074/jbc.M111824200, 277, 9, 7308-7317, 2002.03, Serum response factor and the (CC(A/T)6GG) (CArG) box interact to promote the transcription of c-fos and muscle genes; this tissue-specific activity may require co-regulators for serum response factor. The α1 integrin promoter contains two cis-elements besides the CArG box: a TAAT sequence, a consensus binding site for homeoproteins, and a GATA-binding box. As a candidate TAAT-binding factor, we cloned an NK family homeobox gene, Nkx-3.2, which is expressed mainly in smooth muscle tissues and skeletal structures. Nkx-3.2, serum response factor, and GATA-6 were co-expressed only in the medial smooth muscle layer of arteries. These three transcription factors formed a complex with their corresponding cis-elements and cooperatively transactivated smooth muscle genes, including α1 integrin, SM22α, and caldesmon. Cardiac muscle-specific members of the NK and GATA families exist, and the triad of Nkx-2.5, serum response factor, and GATA-4 also transactivated the cardiac atrial natriuretic factor gene, which contains a CArG-like box, a GATA-binding box, and an NK-binding element. Our findings demonstrate that smooth and cardiac muscle have a shared transcriptional machinery and that the GATA and NK families confer muscle specificity on the serum response factor/CArG interaction..
134. Ken'ichiro Hayashi, Masanori Takahashi, Wataru Nishida, Kenji Yoshida, Yasuyuki Ohkawa, Akira Kitabatake, Junken Aoki, Hiroyuki Arai, Kenji Sobue, Phenotypic modulation of vascular smooth muscle cells induced by unsaturated lysophosphatidic acids, Circulation Research, 10.1161/hh1501.094265, 89, 3, 251-258, 2001.08, The phenotypic modulation of vascular smooth muscle cells (VSMCs) from the differentiated state to the dedifferentiated one is critically involved in the development and progression of atherosclerosis. Although many cytokines and growth factors have been reported as atherogenic factors, the critical pathogens for inducing atherosclerosis remain unknown, largely because proper examining systems of them have not been developed. We recently established primary culture systems for visceral SMCs and VSMCs in which both SMCs, when cultured on laminin with insulin-like growth factor-I, show a differentiated phenotype, as indicated by a spindle-like shape, ligand-induced contractility, and a high level of SMC differentiation marker gene expression. In this study, we searched for critical dedifferentiation factors for these SMCs using our culture system. We found that polar lipids extracted from human serum markedly induced VSMC dedifferentiation, and this activity was solely present in the lysophosphatidic acid (LPA) fraction. Among several LPA species detected in human serum lipids, unsaturated LPAs were identified as major contributors to the induction of VSMC dedifferentiation. Signaling and phenotype analyses revealed that unsaturated LPA-induced VSMC dedifferentiation is mediated through the coordinated activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase. Thus, this report demonstrates the first finding that unsaturated LPAs, but not saturated LPAs, specifically induce VSMC phenotypic modulation, suggesting that these molecules could function as atherogenic factors..