砂河 孝行 (イサガワ タカユキ)

  • データサイエンスセンター 講師
Last Updated :2021/11/23



  • 博士(理学)


J-Global ID



  • Masamitsu Harada, Jun Nagai, Riho Kurata, Xiaofeng Cui, Takayuki Isagawa, Hiroaki Semba, Yasuhiro Yoshida, Norihiko Takeda, Koji Maemura, Tomo Yonezawa
    International journal of molecular sciences 22 5 2021年02月 
    Repressor element-1 (RE-1) or neural restrictive silencer element (NRSE) bound with a zinc finger transcription repressor, RE-1 silencing transcription factor (REST, also known as neural restrictive silencer factor, NRSF) has been identified as a fundamental repressor element in many genes, including neuronal genes. Genes regulated by REST/NRSF regulate multifaceted neuronal phenotypes, and their defects in the machinery cause neuropathies, disorders of neuron activity), autism and so on. In REST repressions, the N-terminal repressor domain recruits Sin3B via its paired amphipathic helix 1 (PAH1) domain, which plays an important role as a scaffold for histone deacetylase 1 and 2. This machinery has a critical role in maintaining neuronal robustness. In this study, in order to establish protein-protein interaction assays mimicking a binding surface between Sin3B and REST, we selected important amino acids from structural information of the PAH1/REST complex and then tried to reconstitute it using recombinant short peptides derived from PAH1/REST. Initially, we validated whether biotinylated REST interacts with glutathione S-transferase (GST)-tagged PAH1 and whether another PAH1 peptide (PAH1-FLAG) competitively binds with biotinylated REST using surface plasmon resonance (SPR). We observed a direct interaction and competitive binding of two PAH1 peptides. Secondly, in order to establish a high-throughput and high-dynamic-range assay, we utilized an easily performed novel time-resolved fluorescence energy transfer (TR-FRET) assay, and closely monitored this interaction. Finally, we succeeded in establishing a novel high-quality TR-FRET assay and a novel interaction assay based on SPR.
  • Shuoyu Wei, Takayuki Isagawa, Masamichi Eguchi, Daisuke Sato, Hiroto Tsukano, Keishi Miyata, Yuichi Oike, Norihiko Takeda, Satoshi Ikeda, Hiroaki Kawano, Koji Maemura
    Biomedicines 8 11 2020年11月 
    Macrophages in the atheroma region produce matrix metalloproteinases (MMPs) and decrease plaque stability. Tissue oxygen tension decreases in the arterial wall of the atherosclerotic region. Hypoxia inducible factor (HIF)-1α plays a critical role in the transcriptional activation of hypoxia inducible genes. However, the precise roles of HIF-1α independent pathways in hypoxic responses are largely unknown. Xanthine oxidase (XO) is an enzyme that utilizes molecular oxygen and produces reactive oxygen species (ROS). Here, we show that ROS derived from XO increases MMP-3, -10, and -13 expression in murine macrophages. We found that the transcript levels of macrophage MMP-3, -10, and -13 were increased in hypoxic conditions. Hypoxia induced MMP expression in HIF-1α deficient macrophages. N-acetylcysteine (NAC) or febuxostat, an XO inhibitor, suppressed MMP expression in murine macrophages. Febuxostat decreased the incidence of plaque rupture in apolipoprotein-E-deficient mice. Our results indicate that febuxostat stabilized atherosclerotic plaque via suppressing the activities of macrophage MMP-9 and -13. Febuxostat administration is a potential therapeutic option in the management of atherosclerotic patients.
  • ヒトIL-18特異的シグナルを検出する新規リポーター細胞の樹立および独自ライブラリーを用いた創薬への取り組み
    倉田 里穂, 原田 将光, 砂河 孝行, 仙波 宏章, 武田 憲彦, 前村 浩二, 米澤 朋
    別冊Bio Clinica: 慢性炎症と疾患 9 1 96 - 101 (株)北隆館 2020年08月 
  • ヒトIL-18特異的シグナルを検出する新規リポーター細胞の樹立および独自ライブラリーを用いた創薬への取り組み
    倉田 里穂, 原田 将光, 砂河 孝行, 仙波 宏章, 武田 憲彦, 前村 浩二, 米澤 朋
    別冊Bio Clinica: 慢性炎症と疾患 9 1 96 - 101 (株)北隆館 2020年08月 
  • Yukiteru Nakayama, Katsuhito Fujiu, Ryuzaburo Yuki, Yumiko Oishi, Masaki Suimye Morioka, Takayuki Isagawa, Jun Matsuda, Tsukasa Oshima, Takumi Matsubara, Junichi Sugita, Fujimi Kudo, Atsushi Kaneda, Yusuke Endo, Toshinori Nakayama, Ryozo Nagai, Issei Komuro, Ichiro Manabe
    Proceedings of the National Academy of Sciences of the United States of America 117 25 14365 - 14375 2020年06月 [査読有り][通常論文]
    Proper resolution of inflammation is vital for repair and restoration of homeostasis after tissue damage, and its dysregulation underlies various noncommunicable diseases, such as cardiovascular and metabolic diseases. Macrophages play diverse roles throughout initial inflammation, its resolution, and tissue repair. Differential metabolic reprogramming is reportedly required for induction and support of the various macrophage activation states. Here we show that a long noncoding RNA (lncRNA), lncFAO, contributes to inflammation resolution and tissue repair in mice by promoting fatty acid oxidation (FAO) in macrophages. lncFAO is induced late after lipopolysaccharide (LPS) stimulation of cultured macrophages and in Ly6Chi monocyte-derived macrophages in damaged tissue during the resolution and reparative phases. We found that lncFAO directly interacts with the HADHB subunit of mitochondrial trifunctional protein and activates FAO. lncFAO deletion impairs resolution of inflammation related to endotoxic shock and delays resolution of inflammation and tissue repair in a skin wound. These results demonstrate that by tuning mitochondrial metabolism, lncFAO acts as a node of immunometabolic control in macrophages during the resolution and repair phases of inflammation.
  • Masamitsu Harada, Jun Nagai, Riho Kurata, Kenji Shimizu, Xiaofeng Cui, Takayuki Isagawa, Hiroaki Semba, Jun Ishihara, Yasuhiro Yoshida, Norihiko Takeda, Koji Maemura, Tomo Yonezawa
    Marine drugs 18 3 2020年03月 [査読有り][通常論文]
    Toxoplasma gondii is a major protozoan parasite and infects human and many other warm-blooded animals. The infection leads to Toxoplasmosis, a serious issue in AIDS patients, organ transplant recipients and pregnant women. Neospora caninum, another type of protozoa, is closely related to Toxoplasma gondii. Infections of the protozoa in animals also causes serious diseases such as Encephalomyelitis and Myositis-Polyradiculitis in dogs or abortion in cows. Both Toxoplasma gondii and Neospora caninum have similar nucleoside triphosphate hydrolases (NTPase), NcNTPase and TgNTPase-I in Neospora caninum and Toxoplasma gondii, respectively. These possibly play important roles in propagation and survival. Thus, we targeted the enzymes for drug discovery and tried to establish a novel high-standard assay by a combination of original biochemical enzyme assay and fluorescent assay to determine ADP content. We then validated whether or not it can be applied to high-throughput screening (HTS). Then, it fulfilled criterion to carry out HTS in both of the enzymes. In order to identify small molecules having inhibitory effects on the protozoan enzyme, we also performed HTS using two synthetic compound libraries and an extract library derived from marine bacteria and then, identified 19 compounds and 6 extracts. Nagasaki University collected many extracts from over 18,000 marine bacteria found in local Omura bay, and continues to compile an extensive collection of synthetic compounds from numerous drug libraries established by Japanese chemists.
  • Riho Kurata, Kenji Shimizu, Xiaofeng Cui, Masamitsu Harada, Takayuki Isagawa, Hiroaki Semba, Jun Ishihara, Koji Yamada, Jun Nagai, Yasuhiro Yoshida, Norihiko Takeda, Koji Maemura, Tomo Yonezawa
    Marine drugs 18 1 2020年01月 [査読有り][通常論文]
    Very recently, the immunotherapies against cancer, autoimmune diseases, and infection have been feasible and promising. Thus, we have examined the possibility whether or not human gamma delta T cells can be applied for the novel immunotherapies. We previously established the cells stably maintaining NFkB-driven human secreted embryonic alkaline phosphatase (SEAP) expression. The cells can be used to determine the transcription activity of NFkB with high-standard dynamic range and accuracy. Because IL-18 is a kind of cytokines that enhances cytotoxicity and activity of human gamma delta T cells through NFkB activation, we have focused on the activity and signaling of IL-18. In this study, we modified the previous reporter cell that can determine the transcription activity of NFkB to express two subunits consisted of human IL-18 receptor. The modified cells secreted SEAP in response to treatment with human recombinant IL-18 in a concentration-dependent manner. We also observed the concentration-dependently enhancement of NFkB activity in the cells treated with mouse recombinant IL-18 although the affinity was lower compared to human recombinant IL-18. We also previously established the cells stably expressing and secreting human recombinant IL-18 and then validated whether or not the conditioned medium from the cells activate NFkB transcription activity using this assay. Our university has kept collecting many extracts from over 18,000 marine bacteria in our local sea around Omura bay-fungi, plants for Chinese herbal medicine, and so on-and also have kept gathering synthetic compounds from many Japanese chemists as drug libraries. Finally, in order to identify drugs mimicking IL-18 biological activity or possessing inhibitory effects on IL-18-induced NFkB, we demonstrated drug screening using number of extracts derived from marine bacteria and synthetic compounds.
  • 砂河 孝行, 魏 碩俣, 佐藤 大輔, 江口 正倫, 池田 聡司, 河野 浩章, 前村 浩二
    Anti-aging Science 11 1 66 - 66 (株)メディカルレビュー社 2019年12月
  • Kenichi Okamura, Yu Nakagama, Norihiko Takeda, Katsura Soma, Tatsuyuki Sato, Takayuki Isagawa, Yasutoshi Kido, Masaya Sakamoto, Ichiro Manabe, Yasutaka Hirata, Issei Komuro, Minoru Ono
    Journal of pharmacological sciences 141 1 56 - 63 2019年09月 [査読有り][通常論文]
    Concomitant heart failure is associated with poor clinical outcome in dialysis patients. The arteriovenous shunt, created as vascular access for hemodialysis, increases ventricular volume-overload, predisposing patients to developing cardiac dysfunction. The integral function of mitochondrial respiration is critically important for the heart to cope with hemodynamic overload. The involvement, however, of mitochondrial activity or reactive oxygen species (ROS) in the pathogenesis of ventricular-overload-induced heart failure has not been fully elucidated. We herein report that disorganization of mitochondrial respiration increases mitochondrial ROS production in the volume-overloaded heart, leading to ventricular dysfunction. We adopted the murine arteriovenous fistula (AVF) model, which replicates the cardinal features of volume-overload-induced ventricular dysfunction. Enzymatic assays of cardiac mitochondria revealed that the activities of citrate synthase and NADH-quinone reductase (complex Ⅰ) were preserved in the AVF heart. In contrast, the activity of NADH oxidase supercomplex was significantly compromised, resulting in elevated ROS production. Importantly, the antioxidant N-acetylcysteine prevented the development of ventricular dilatation and cardiac dysfunction, suggesting a pathogenic role for ROS in dialysis-related cardiomyopathy. A cardioprotective effect was also observed in metformin-treated mice, illuminating its potential use in the management of heart failure complicating diabetic patients on dialysis.
  • Masaki Wake, Norihiko Takeda, Takayuki Isagawa, Tatsuyuki Sato, Yu Nakagama, Masaki Suimye Morioka, Yasushi Hirota, Masataka Asagiri, Koji Maemura, Ichiro Manabe, Kazuaki Tanabe, Issei Komuro
    International heart journal 60 4 958 - 963 2019年07月 [査読有り][通常論文]
    Myocardial infarction (MI) occurs when the heart muscle is severely damaged due to a decrease in blood flow from the coronary arteries. During recovery from an MI, cardiac fibroblasts become activated and produce extracellular matrices, contributing to the wound healing process in the damaged heart. Inappropriate activation of the fibroblasts leads to excessive fibrosis in the heart. However, the molecular pathways by which cardiac fibroblasts are activated have not yet been fully elucidated.Here we show that serum deprivation, which recapitulates the cellular microenvironment of the MI area, strikingly induces collagen production in C3H/10T1/2 cells. Based on transcriptomic and pharmacological studies, we found that cell cycle perturbation is directly linked to collagen production in fibroblasts. Importantly, collagen synthesis is increased independently of the transcriptional levels of type I collagen genes. These results reveal a novel mode of fibroblast activation in the ischemic area, which will allow us to gain insights into the molecular mechanisms underlying cardiac fibrosis and establish a basis for anti-fibrotic therapy.
  • Hajime Abe, Norihiko Takeda, Takayuki Isagawa, Hiroaki Semba, Satoshi Nishimura, Masaki Suimye Morioka, Yu Nakagama, Tatsuyuki Sato, Katsura Soma, Katsuhiro Koyama, Masaki Wake, Manami Katoh, Masataka Asagiri, Michael L Neugent, Jung-Whan Kim, Christian Stockmann, Tomo Yonezawa, Ryo Inuzuka, Yasushi Hirota, Koji Maemura, Takeshi Yamashita, Kinya Otsu, Ichiro Manabe, Ryozo Nagai, Issei Komuro
    Nature communications 10 1 2824 - 2824 2019年06月 [査読有り][通常論文]
    The fibrogenic response in tissue-resident fibroblasts is determined by the balance between activation and repression signals from the tissue microenvironment. While the molecular pathways by which transforming growth factor-1 (TGF-β1) activates pro-fibrogenic mechanisms have been extensively studied and are recognized critical during fibrosis development, the factors regulating TGF-β1 signaling are poorly understood. Here we show that macrophage hypoxia signaling suppresses excessive fibrosis in a heart via oncostatin-m (OSM) secretion. During cardiac remodeling, Ly6Chi monocytes/macrophages accumulate in hypoxic areas through a hypoxia-inducible factor (HIF)-1α dependent manner and suppresses cardiac fibroblast activation. As an underlying molecular mechanism, we identify OSM, part of the interleukin 6 cytokine family, as a HIF-1α target gene, which directly inhibits the TGF-β1 mediated activation of cardiac fibroblasts through extracellular signal-regulated kinase 1/2-dependent phosphorylation of the SMAD linker region. These results demonstrate that macrophage hypoxia signaling regulates fibroblast activation through OSM secretion in vivo.
  • 透析高血圧症の原因として虚血性心疾患が考えられた1例
    内田 真人, 河野 浩章, 砂河 孝行, 山方 勇樹, 米倉 剛, 江口 正倫, 吉牟田 剛, 南 貴子, 古賀 聖士, 恒任 章, 池田 聡司, 前村 浩二
    日本高血圧学会臨床高血圧フォーラムプログラム・抄録集 8回 201 - 201 (NPO)日本高血圧学会 2019年05月
  • Asuka Kumagai, Kenji Shimizu, Riho Kurata, Xiaofeng Cui, Takayuki Isagawa, Masamitsu Harada, Jun Nagai, Yasuhiro Yoshida, Kei-Ichi Ozaki, Norihiko Takeda, Hiroaki Semba, Tomo Yonezawa
    Current pharmaceutical biotechnology 20 1 47 - 55 2019年 [査読有り][通常論文]
    BACKGROUND: The immunotherapies against cancer, autoinmmune diseases or infection are remarkable development. These days programmed cell death (PD)-1 antibody-induced immune checkpoint blockade or chimeric antigen receptor-T cells (CAR-T) have been shown to have eminent therapeutic effects on tumor development. We have focused on adoptive transfer with human gamma delta T cells for novel immunotherapies. Additionally, IL-18 is one of the cytokines that enhances cytokine secretion and cytotoxicity of human gamma delta T cells. METHOD: Thus, we established novel cell lines stably expressing and secreting various types of human recombinant IL-18 proteins to their culture supernatants using episomal vector. We also differentiated primary cultured human gamma delta T cells from peripheral blood mononuclear leukocytes to validate biological activity of the IL-18 proteins using measuring IFN-γ by ELISA. RESULTS AND CONCLUSION: Finally, we demonstrated that the supernatant could activate human gamma delta T cells using monitoring interferon gamma in culture medium.
  • Chijiwa Tsuyoshi, Komura Daisuke, Haraguchi Mizuha, Hashimoto Haruo, Suemizu Hiroshi, Katayama Makoto, Nakamura Yoshiyasu, Furukawa Daisuke, Isagawa Takayuki, Katoh Hiroto, Moriya Takashi, Ishikawa Shumpei, Miyagi Yohei, Nakamura Masato
    CANCER RESEARCH 78 13 2018年07月 [査読有り][通常論文]
  • Chijiwa Tsuyoshi, Noguchi Akira, Komura Daisuke, Katayama Makoto, Haraguchi Mizuha, Hashimoto Haruo, Suemizu Hiroshi, Nakamura Yoshiyasu, Furukawa Daisuke, Isagawa Takayuki, Katoh Hiroto, Moriya Takashi, Ishikawa Shumpei, Nakamura Masato, Miyagi Yohei
    CANCER RESEARCH 78 10 61  2018年05月 [査読有り][通常論文]
  • Mariko Tanaka, Shumpei Ishikawa, Tetsuo Ushiku, Teppei Morikawa, Takayuki Isagawa, Makoto Yamagishi, Hiroyuki Yamamoto, Hiroto Katoh, Kimiko Takeshita, Junichi Arita, Yoshihiro Sakamoto, Kiyoshi Hasegawa, Norihiro Kokudo, Masashi Fukayama
    Oncotarget 8 59 99552 - 99566 2017年11月 [査読有り][通常論文]
    Glypican-1 (GPC1) protein in exosomes was recently identified as a biomarker for the early detection of pancreatic ductal adenocarcinoma (PDAC). Immunohistochemical analyses and in vitro assays were conducted to assess the usefulness of GPC1 as a PDAC biomarker, to reveal the biological role of GPC1 in pancreatic carcinogenesis, and to ascertain the regulation mechanism of GPC1. An aberrant overexpression of GPC1 protein which is usually absent in normal pancreatic duct, was a widespread marker across the full spectrum of human PDAC precursors, PDAC, and pancreatic cancerous stroma. In intraductal papillary-mucinous neoplasms (IPMNs), GPC1 tended to be positive in gastric-type IPMN. KRAS mutations were found in all GPC1-positive IPMN cases and in one-third of GPC1-negative IPMN cases. In pancreatic cell lines, GPC1 depletion caused remarkable inhibition of cell growth and migration, suggesting its oncogenic roles. GPC1 depletion upregulated the molecules associated with cell cycle arrest in pancreatic cell lines. Furthermore, KRAS and ecotropic viral integration site 1 (EVI1) oncoprotein upregulated GPC1 expression. In a clinical cohort, GPC1 overexpression was not correlated with pancreatic cancer prognosis. Taken together, these findings suggest the necessity of establishing a threshold of GPC1 value for detecting pancreatic malignancy because GPC1 is overexpressed even in low-grade PDAC precursors which do not always become malignant. Our study also reveals a new aspect of pancreatic carcinogenesis: KRAS and EVI1, two important molecules in early phases of pancreatic carcinogenesis, positively regulate GPC1 expression and likely promote pancreatic carcinogenesis.
  • Krzywinska E, Kantari-Mimoun C, Kerdiles Y, Sobecki M, Isagawa T, Gotthardt D, Castells M, Haubold J, Millien C, Viel T, Tavitian B, Takeda N, Fandrey J, Vivier E, Sexl V, Stockmann C
    Nature communications 8 1 1597 - 1597 2017年11月 [査読有り][通常論文]
    Productive angiogenesis, a prerequisite for tumour growth, depends on the balanced release of angiogenic and angiostatic factors by different cell types within hypoxic tumours. Natural killer (NK) cells kill cancer cells and infiltrate hypoxic tumour areas. Cellular adaptation to low oxygen is mediated by Hypoxia-inducible factors (HIFs). We found that deletion of HIF-1α in NK cells inhibited tumour growth despite impaired tumour cell killing. Tumours developing in these conditions were characterised by a high-density network of immature vessels, severe haemorrhage, increased hypoxia, and facilitated metastasis due to non-productive angiogenesis. Loss of HIF-1α in NK cells increased the bioavailability of the major angiogenic cytokine vascular endothelial growth factor (VEGF) by decreasing the infiltration of NK cells that express angiostatic soluble VEGFR-1. In summary, this identifies the hypoxic response in NK cells as an inhibitor of VEGF-driven angiogenesis, yet, this promotes tumour growth by allowing the formation of functionally improved vessels.
  • Chijiwa Tsuyoshi, Haraguchi Mizuha, Komura Daisuke, Noguchi Akira, Shiozawa Manabu, Katayama Makoto, Miyao Naoki, Matsui Naruaki, Tateishi Yuichi, Suemizu Hiroshi, Nakamura Yoshiyasu, Isagawa Takayuki, Katoh Hiroto, Ishikawa Shumpei, Nakamura Masato, Miyagi Yohei
    CANCER RESEARCH 77 22 2017年11月 [査読有り][通常論文]
  • Yumiko Oishi, Shinichiro Hayashi, Takayuki Isagawa, Motohiko Oshima, Atsushi Iwama, Shigeki Shimba, Hitoshi Okamura, Ichiro Manabe
    Scientific reports 7 1 7086 - 7086 2017年08月 [査読有り][通常論文]
    Bmal1 (encoded by Arntl gene) is a core circadian clock gene that regulates various genes involved in circadian rhythm. Although Bmal1 is expressed rhythmically in macrophages, the role of Bmal1 in the regulation of their cellular function remains insufficiently understood. Here, we report that Bmal1 regulates time-dependent inflammatory responses following Toll-like receptor 4 (TLR4) activation by modulating enhancer activity. Global transcriptome analysis indicated that deletion of Arntl perturbed the time-dependent inflammatory responses elicited by TLR4 activation by Kdo2-lipid A (KLA). Although the recruitment of NF-κB p65 was unaffected, the acetylation status of lysine 27 of histone 3, which correlates positively with enhancer activity, was globally increased at PU.1-containing enhancers in Arntl -/- macrophages as compared to wild-type cells. Expression of Nr1d1 and Nr1d2, encoding RevErb transcription factors, which repress enhancer RNA expression, was significantly decreased in Arntl -/- macrophages. Moreover, the level of H3K27 acetylation was increased by Arntl deletion at RevErb-dependent eRNA-expressing enhancers. These results suggest that Bmal1 controls KLA-responsive enhancers, in part by regulating RevErb-directed eRNA transcription. Taken together, the results of this study show that the clock transcription factor network containing Bmal1 controls the inflammatory responses of macrophages by regulating the epigenetic states of enhancers.
  • Chijiwa Tsuyoshi, Komura Daisuke, Haraguchi Mizuha, Noguchi Akira, Sato Hidemitsu, Ito Hiroaki, Nakayama Haruhiko, Katayama Makoto, Miyao Naoki, Matsui Naruaki, Tateishi Yuichi, Suemizu Hiroshi, Nakamura Yoshiyasu, Furukawa Daisuke, Isagawa Takayuki, Katoh Hiroto, Ishikawa Shumpei, Nakamura Masato, Miyagi Yohei
    CANCER RESEARCH 77 2017年07月 [査読有り][通常論文]
  • Daisuke Komura, Takayuki Isagawa, Kazuki Kishi, Ryohei Suzuki, Reiko Sato, Mariko Tanaka, Hiroto Katoh, Shogo Yamamoto, Kenji Tatsuno, Masashi Fukayama, Hiroyuki Aburatani, Shumpei Ishikawa
    BMC genomics 17 1 899 - 899 2016年11月 [査読有り][通常論文]
    BACKGROUND: Cancer microenvironment plays a vital role in cancer development and progression, and cancer-stromal interactions have been recognized as important targets for cancer therapy. However, identifying relevant and druggable cancer-stromal interactions is challenging due to the lack of quantitative methods to analyze whole cancer-stromal interactome. RESULTS: We present CASTIN (CAncer-STromal INteractome analysis), a novel framework for the evaluation of cancer-stromal interactome from RNA-Seq data using cancer xenograft models. For each ligand-receptor interaction which is derived from curated protein-protein interaction database, CASTIN summarizes gene expression profiles of cancer and stroma into three evaluation indices. These indices provide quantitative evaluation and comprehensive visualization of interactome, and thus enable to identify critical cancer-microenvironment interactions, which would be potential drug targets. We applied CASTIN to the dataset of pancreas ductal adenocarcinoma, and successfully characterized the individual cancer in terms of cancer-stromal relationships, and identified both well-known and less-characterized druggable interactions. CONCLUSIONS: CASTIN provides comprehensive view of cancer-stromal interactome and is useful to identify critical interactions which may serve as potential drug targets in cancer-microenvironment. CASTIN is available at: http://github.com/tmd-gpat/CASTIN .
  • Takeshi Ito, Daisuke Matsubara, Ichidai Tanaka, Kanae Makiya, Zen-Ichi Tanei, Yuki Kumagai, Shu-Jen Shiu, Hiroki J Nakaoka, Shumpei Ishikawa, Takayuki Isagawa, Teppei Morikawa, Aya Shinozaki-Ushiku, Yasushi Goto, Tomoyuki Nakano, Takehiro Tsuchiya, Hiroyoshi Tsubochi, Daisuke Komura, Hiroyuki Aburatani, Yoh Dobashi, Jun Nakajima, Shunsuke Endo, Masashi Fukayama, Yoshitaka Sekido, Toshiro Niki, Yoshinori Murakami
    Cancer science 107 10 1527 - 1538 2016年10月 [査読有り][通常論文]
    YAP1, the main Hippo pathway effector, is a potent oncogene and is overexpressed in non-small-cell lung cancer (NSCLC); however, the YAP1 expression pattern in small-cell lung cancer (SCLC) has not yet been elucidated in detail. We report that the loss of YAP1 is a special feature of high-grade neuroendocrine lung tumors. A hierarchical cluster analysis of 15 high-grade neuroendocrine tumor cell lines containing 14 SCLC cell lines that depended on the genes of Hippo pathway molecules and neuroendocrine markers clearly classified these lines into two groups: the YAP1-negative and neuroendocrine marker-positive group (n = 11), and the YAP1-positive and neuroendocrine marker-negative group (n = 4). Among the 41 NSCLC cell lines examined, the loss of YAP1 was only observed in one cell line showing the strong expression of neuroendocrine markers. Immunostaining for YAP1, using the sections of 189 NSCLC, 41 SCLC, and 30 large cell neuroendocrine carcinoma (LCNEC) cases, revealed that the loss of YAP1 was common in SCLC (40/41, 98%) and LCNEC (18/30, 60%), but was rare in NSCLC (6/189, 3%). Among the SCLC and LCNEC cases tested, the loss of YAP1 correlated with the expression of neuroendocrine markers, and a survival analysis revealed that YAP1-negative cases were more chemosensitive than YAP1-positive cases. Chemosensitivity test for cisplatin using YAP1-positive/YAP1-negative SCLC cell lines also showed compatible results. YAP1-sh-mediated knockdown induced the neuroendocrine marker RAB3a, which suggested the possible involvement of YAP1 in the regulation of neuroendocrine differentiation. Thus, we showed that the loss of YAP1 has potential as a clinical marker for predicting neuroendocrine features and chemosensitivity.
  • Semba H, Takeda N, Isagawa T, Sugiura Y, Honda K, Wake M, Miyazawa H, Yamaguchi Y, Miura M, Jenkins DM, Choi H, Kim JW, Asagiri M, Cowburn AS, Abe H, Soma K, Koyama K, Katoh M, Sayama K, Goda N, Johnson RS, Manabe I, Nagai R, Komuro I
    Nature communications 7 11635 - 11635 2016年05月 [査読有り][通常論文]
    In severely hypoxic condition, HIF-1α-mediated induction of Pdk1 was found to regulate glucose oxidation by preventing the entry of pyruvate into the tricarboxylic cycle. Monocyte-derived macrophages, however, encounter a gradual decrease in oxygen availability during its migration process in inflammatory areas. Here we show that HIF-1α-PDK1-mediated metabolic changes occur in mild hypoxia, where mitochondrial cytochrome c oxidase activity is unimpaired, suggesting a mode of glycolytic reprogramming. In primary macrophages, PKM2, a glycolytic enzyme responsible for glycolytic ATP synthesis localizes in filopodia and lammelipodia, where ATP is rapidly consumed during actin remodelling processes. Remarkably, inhibition of glycolytic reprogramming with dichloroacetate significantly impairs macrophage migration in vitro and in vivo. Furthermore, inhibition of the macrophage HIF-1α-PDK1 axis suppresses systemic inflammation, suggesting a potential therapeutic approach for regulating inflammatory processes. Our findings thus demonstrate that adaptive responses in glucose metabolism contribute to macrophage migratory activity.
  • Hanihara-Tatsuzawa F, Miura H, Kobayashi S, Isagawa T, Okuma A, Manabe I, MaruYama T
    The Journal of biological chemistry 289 45 30925 - 36 2014年11月 [査読有り][通常論文]
    Antibody responses have been classified as being either T cell-dependent or T cell-independent (TI). TI antibody responses are further classified as being either type 1 (TI-1) or type 2 (TI-2), depending on their requirement for B cell-mediated antigen receptor signaling. Although the mechanistic basis of antibody responses has been studied extensively, it remains unclear whether different antibody responses share similarities in their transcriptional regulation. Here, we show that mice deficient in IκB-ζ, specifically in their B cells, have impaired TI-1 antibody responses but normal T cell-dependent and TI-2 antibody responses. The absence of IκB-ζ in B cells also impaired proliferation triggered by Toll-like receptor (TLR) activation, plasma cell differentiation, and class switch recombination (CSR). Mechanistically, IκB-ζ-deficient B cells could not induce TLR-mediated induction of activation-induced cytidine deaminase (AID), a class-switch DNA recombinase. Retroviral transduction of AID in IκB-ζ-deficient B cells restored CSR activity. Furthermore, acetylation of histone H3 in the vicinity of the transcription start site of the gene that encodes AID was reduced in IκB-ζ-deficient B cells relative to IκB-ζ-expressing B cells. These results indicate that IκB-ζ regulates TLR-mediated CSR by inducing AID. Moreover, IκB-ζ defines differences in the transcriptional regulation of different antibody responses.
  • M. Tanaka, H. I. Suzuki, J. Shibahara, A. Kunita, T. Isagawa, A. Yoshimi, M. Kurokawa, K. Miyazono, H. Aburatani, S. Ishikawa, M. Fukayama
    ONCOGENE 33 19 2454 - 2463 2014年05月 [査読有り][通常論文]
    Despite frequent KRAS mutation, the early molecular mechanisms of pancreatic ductal adenocarcinoma (PDAC) development have not been fully elucidated. By tracking a potential regulator of another feature of PDAC precursors, acquisition of foregut or gastric epithelial gene signature, we herein report that aberrant overexpression of ecotropic viral integration site 1 (EVI1) oncoprotein, which is usually absent in normal pancreatic duct, is a widespread marker across the full spectrum of human PDAC precursors and PDAC. In pancreatic cancer cells, EVI1 depletion caused remarkable inhibition of cell growth and migration, indicating its oncogenic roles. Importantly, we found that EVI1 upregulated KRAS expression through suppression of a potent KRAS suppressor, miR-96, in pancreatic cancer cells. Collectively, the present findings suggest that EVI1 overexpression and KRAS mutation converge on activation of the KRAS pathway in early phases of pancreatic carcinogenesis and propose EVI1 and/or miR-96 as early markers and therapeutic targets in this dismal disease.
  • Masashi Yukawa, Tomohiko Akiyama, Vedran Franke, Nathan Mise, Takayuki Isagawa, Yutaka Suzuki, Masataka G Suzuki, Kristian Vlahovicek, Kuniya Abe, Hiroyuki Aburatani, Fugaku Aoki
    PloS one 9 3 e92689  2014年 [査読有り][通常論文]
    Genome-wide distribution of the majority of H2A and H3 variants (H2A, H2AX, H2AZ, macroH2A, H3.1, H3.2 and H3.3) was simultaneously investigated in mouse embryonic stem cells by chromatin immunoprecipitation sequencing. Around the transcription start site, histone variant distribution differed between genes possessing promoters of high and low CpG density, regardless of their expression levels. In the intergenic regions, regulatory elements were enriched in H2A.Z and H3.3, whereas repeat elements were abundant in H2A and macroH2A, and H3.1, respectively. Analysis of H2A and H3 variant combinations composing nucleosomes revealed that the H2A.Z and H3.3 combinations were present at a higher frequency throughout the genome than the other combinations, suggesting that H2A.Z and H3.3 associate preferentially with each other to comprise the nucleosomes independently of genome region. Finally, we found that chromatin was unstable only in regions where it was enriched in both H2A.Z and H3.3, but strongly quantified stable in regions in which only H3.3 was abundant. Therefore, histone variant composition is an important determinant of chromatin structure, which is associated with specific genomic functions.
  • Kobayashi S, Hara A, Isagawa T, Manabe I, Takeda K, MaruYama T
    PloS one 9 10 e110838  2014年 [査読有り][通常論文]
    The nuclear IκB family protein IκBNS is expressed in T cells and plays an important role in Interferon (IFN)-γ and Interleukin (IL)-2 production. IκB-ζ, the most similar homolog of IκBNS, plays an important role in the generation of T helper (Th)17 cells in cooperation with RORγt, a master regulator of Th17 cells. Thus, IκB-ζ deficient mice are resistant to Th17-dependent experimental autoimmune encephalomyelitis (EAE). However, IκB-ζ deficient mice develop the autoimmune-like Sjögren syndrome with aging. Here we found that IκBNS-deficient (Nfkbid-/-) mice show resistance against developing Th17-dependent EAE. We found that Nfkbid-/- T cells have decreased expression of IL-17-related genes and RORγt in response to Transforming Growth Factor (TGF)-β1 and IL-6 stimulation. Thus, IκBNS plays a pivotal role in the generation of Th17 cells and in the control of Th17-dependent EAE.
  • Yuri Yoshida, Megumi Fuchita, Maki Kimura-Koyanagi, Ayumi Kanno, Tomokazu Matsuda, Shun-ichiro Asahara, Naoko Hashimoto, Takayuki Isagawa, Wataru Ogawa, Hiroyuki Aburatani, Tetsuo Noda, Susumu Seino, Masato Kasuga, Yoshiaki Kido
    Diabetology International 5 1 43 - 52 2014年 [査読有り][通常論文]
    Children born with low birth weight have a high risk of developing type 2 diabetes mellitus later in life. In this study, we developed a mouse model for low birth weight induced by maternal caloric restriction and investigated its effects on pancreatic β-cells. At birth, the pancreatic β-cell mass in the restricted diet group (RG) offspring was significantly lower than in the control group (CG) offspring. At 8 weeks of age, the pancreatic β-cell mass was greater in the RG offspring than in the CG offspring. RG offspring showed upregulated expression of insulin receptor substrate 2 in islets at 10 weeks of age. Moreover, the activity of insulin signaling molecules in the pancreatic β-cell mass was increased during the period of catch-up growth during the early stage of life. To investigate the effect of insulin signaling on the regulation of pancreatic β-cell mass, we generated β-cell-specific 3-phosphoinositide-dependent protein kinase 1 (PDK1) heterozygous knockout (βPDK1+/-) mice. We detected delayed catch-up growth in the β-cell mass of βPDK1+/- mice that were undernourished as fetuses. Moreover, the insulin signaling pathway was impaired in the islets of βPDK1+/- mice exposed to fetal undernutrition. These findings indicate that fetal undernutrition affects the regulation of pancreatic β-cell mass through altered insulin signaling. © 2013 The Japan Diabetes Society.
  • Satoshi Goda, Takayuki Isagawa, Yoko Chikaoka, Takeshi Kawamura, Hiroyuki Aburatani
    The Journal of biological chemistry 288 52 36948 - 56 2013年12月 [査読有り][通常論文]
    Post-translational histone methylation is a dynamic and reversible process that is involved in the spatio-temporal regulation of gene transcription and contributes to various cellular phenotypes. Methylation of histone H3 at lysine 9 (H3K9), which is generally a transcriptional repression mark, is demethylated by H3K9-specific demethylases, leading to transcriptional activation. However, how multiple demethylases with the same substrate specificity differ in their chromatin targeting mechanisms has not been well understood. Unlike other H3K9-specific demethylases, it has been reported that JMJD1A likely forms a homodimer, but a detailed mode of dimerization and the possible link between structure and enzymatic activity have remained unresolved. Here, we report the structure-function relationship of JMJD1A in detail. First, JMJD1A forms a homodimer through its catalytic domains, bringing the two active sites close together. Second, increasing the concentration of JMJD1A facilitates efficient production of unmethylated product from dimethyl-H3K9 and decreases the release of the monomethylated intermediate. Finally, substituting one of the two active sites with an inactive mutant results in a significant reduction of the demethylation rate without changing the affinity to the intermediate. Given this evidence, we propose a substrate channeling model for the efficient conversion of dimethylated H3K9 into the unmethylated state. Our study provides valuable information that will help in understanding the redundancy of H3K9-specific demethylases and the complementary activity of their unique structures and enzymatic properties for appropriate control of chromatin modification patterns.
  • Jean-Michel Fustin, Masao Doi, Yoshiaki Yamaguchi, Hayashi Hida, Shinichi Nishimura, Minoru Yoshida, Takayuki Isagawa, Masaki Suimye Morioka, Hideaki Kakeya, Ichiro Manabe, Hitoshi Okamura
    Cell 155 4 793 - 806 2013年11月 [査読有り][通常論文]
    The eukaryotic biological clock involves a negative transcription-translation feedback loop in which clock genes regulate their own transcription and that of output genes of metabolic significance. While around 10% of the liver transcriptome is rhythmic, only about a fifth is driven by de novo transcription, indicating mRNA processing is a major circadian component. Here, we report that inhibition of transmethylation reactions elongates the circadian period. RNA sequencing then reveals methylation inhibition causes widespread changes in the transcription of the RNA processing machinery, associated with m(6)A-RNA methylation. We identify m(6)A sites on many clock gene transcripts and show that specific inhibition of m(6)A methylation by silencing of the m(6)A methylase Mettl3 is sufficient to elicit circadian period elongation and RNA processing delay. Analysis of the circadian nucleocytoplasmic distribution of clock genes Per2 and Arntl then revealed an uncoupling between steady-state pre-mRNA and cytoplasmic mRNA rhythms when m(6)A methylation is inhibited.
  • Shinzo Yamamoto, Keisuke Tateishi, Yotaro Kudo, Keisuke Yamamoto, Takayuki Isagawa, Genta Nagae, Takuma Nakatsuka, Yoshinari Asaoka, Hideaki Ijichi, Yoshihiro Hirata, Motoyuki Otsuka, Tsuneo Ikenoue, Hiroyuki Aburatani, Masao Omata, Kazuhiko Koike
    CARCINOGENESIS 34 10 2380 - 2388 2013年10月 [査読有り][通常論文]
    Alterations in genes coding for histone modifiers are found in human cancers, suggesting that histone modification is involved in malignant features of neoplastic cells. This study showed that a histone demethylase KDM4C is significant for colonosphere formation by mediating the cross talk between oncogenic pathways through a feed-forward mechanism. The expression of KDM4C gene was increased in spheres from colorectal cancer (CRC) cells and the knockdown (KD) of KDM4C eliminated colonosphere formation. We found that the KD of -catenin, an important oncogenic factor in CRC, resulted in not only decreased sphere formation but also impaired upregulation of KDM4C gene in spheres. -Catenin bound to the KDM4C promoter, suggesting that KDM4C is involved in the sphere-forming ability downstream of -catenin in CRC cells. Microarray analysis identified the JAG1 gene that codes for a notch ligand Jagged1 responsible for sphere formation as a target of KDM4C. KDM4C KD decreased the expression of JAG1 gene, and the downregulation of JAG1 gene recapitulated the impaired colonosphere formation. JAG1 is also a target of -catenin, and chromatin immunoprecipitation analysis showed the binding of -catenin and KDM4C onto the JAG1 promoter during colonosphere formation. Importantly, KDM4C KD ruined the recruitment of -catenin onto the JAG1 promoter independently of the H3-K9 methylation status and blunted JAG1 expression during sphere formation. These data indicate that KDM4C maintains the sphere-forming capacity in CRCs by mediating the -catenin-dependent transcription of JAG1 in a feed-forward manner.
  • Tetsuya Saito, Norihiko Takeda, Eisuke Amiya, Tomoko Nakao, Hajime Abe, Hiroaki Semba, Katsura Soma, Katsuhiro Koyama, Yumiko Hosoya, Yasushi Imai, Takayuki Isagawa, Masafumi Watanabe, Ichiro Manabe, Issei Komuro, Ryozo Nagai, Koji Maemura
    FEBS LETTERS 587 14 2179 - 2185 2013年07月 [査読有り][通常論文]
    Vascular endothelial growth factor-A (VEGF-A) is one of the major angiogenic factors, and its actions are primarily mediated through its two membrane receptors, VEGFR-1 and VEGFR-2. A soluble form of VEGFR-1 (sVEGFR-1) sequesters the free form of VEGF-A, and acts as a potent anti-angiogenic factor. While sVEGFR-1 is synthesized as a splice variant of VEGF-R1 gene, the interactions between VEGF-A and sVEGFR-1 remain largely unknown. Here, we show that VEGF-A upregulates sVEGF-R1 expression in human vascular endothelial cells but leaves full-length VEGF-R1 expression unchanged, and that this induction was dependent on the VEGFR-2-protein kinase C-MEK signaling pathway. The VEGF-A-induced sVEGFR-1 upregulation can operate as a negative feedback system, which if modulated can become a novel therapeutic target for regulating pathological angiogenesis. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved.
  • Teruyuki Sato, Atsushi Kaneda, Shingo Tsuji, Takayuki Isagawa, Shogo Yamamoto, Takanori Fujita, Ryota Yamanaka, Yukiko Tanaka, Toshihiro Nukiwa, Victor E. Marquez, Yuichi Ishikawa, Masakazu Ichinose, Hiroyuki Aburatani
    SCIENTIFIC REPORTS 3 1911  2013年05月 [査読有り][通常論文]
    Small cell lung cancer (SCLC) is a subtype of lung cancer with poor prognosis. Expression array analysis of 23 SCLC cases and 42 normal tissues revealed that EZH2 and other PRC2 members were highly expressed in SCLC. ChIP-seq for H3K27me3 suggested that genes with H3K27me3(+) in SCLC were extended not only to PRC2-target genes in ES cells but also to other target genes such as cellular adhesion-related genes. These H3K27me3(+) genes in SCLC were repressed significantly, and introduction of the most repressed gene JUB into SCLC cell line lead to growth inhibition. Shorter overall survival of clinical SCLC cases correlated to repression of JUB alone, or a set of four genes including H3K27me3(+) genes. Treatment with EZH2 inhibitors, DZNep and GSK126, resulted in growth repression of SCLC cell lines. High PRC2 expression was suggested to contribute to gene repression in SCLC, and may play a role in genesis of SCLC.
  • Takase O, Yoshikawa M, Idei M, Hirahashi J, Fujita T, Takato T, Isagawa T, Nagae G, Suemori H, Aburatani H, Hishikawa K
    PloS one 8 2 e56399  2013年02月 [査読有り][通常論文]
    NF-kappa B signaling plays an essential role in maintaining the undifferentiated state of embryonic stem (ES) cells. However, opposing roles of NF-kappa B have been reported in mouse and human ES cells, and the role of NF-kappa B in human induced pluripotent stem (iPS) cells has not yet been clarified. Here, we report the role of NF-kappa B signaling in maintaining the undifferentiated state of human iPS cells. Compared with differentiated cells, undifferentiated human iPS cells showed an augmentation of NF-kappa B activity. During differentiation induced by the removal of feeder cells and FGF2, we observed a reduction in NF-kappa B activity, the expression of the undifferentiation markers Oct3/4 and Nanog, and the up-regulation of the differentiated markers WT-1 and Pax-2. The specific knockdown of NF-kappa B signaling using p65 siRNA also reduced the expression of Oct3/4 and Nanog and up-regulated WT-1 and Pax-2 but did not change the ES-like colony formation. Our results show that the augmentation of NF-kappa B signaling maintains the undifferentiated state of human iPS and suggest the importance of this signaling pathway in maintenance of human iPS cells.
  • Linghua Wang, Shuichi Tsutsumi, Tokuichi Kawaguchi, Koichi Nagasaki, Kenji Tatsuno, Shogo Yamamoto, Fei Sang, Kohtaro Sonoda, Minoru Sugawara, Akio Saiura, Seiko Hirono, Hiroki Yamaue, Yoshio Miki, Minoru Isomura, Yasushi Totoki, Genta Nagae, Takayuki Isagawa, Hiroki Ueda, Satsuki Murayama-Hosokawa, Tatsuhiro Shibata, Hiromi Sakamoto, Yae Kanai, Atsushi Kaneda, Tetsuo Noda, Hiroyuki Aburatani
    GENOME RESEARCH 22 2 208 - 219 2012年02月 [査読有り][通常論文]
    Whole-exome sequencing (Exome-seq) has been successfully applied in several recent studies. We here sequenced the exomes of 15 pancreatic tumor cell lines and their matched normal samples. We captured 162,073 exons of 16,954 genes and sequenced the targeted regions to a mean coverage of 56-fold. This study identified a total of 1517 somatic mutations and validated 934 mutations by transcriptome sequencing. We detected recurrent mutations in 56 genes. Among them, 41 have not been described. The mutation rates varied widely among cell lines. The diversity of the mutation rates was significantly correlated with the distinct MLH1 copy-number status. Exome-seq revealed intensive genomic instability in a cell line with MLH1 homozygous deletion, indicated by a dramatically elevated rate of somatic substitutions, small insertions/deletions (indels), as well as indels in microsatellites. Notably, we found that MLH1 expression was decreased by nearly half in cell lines with an allelic loss of MLH1. While these cell lines were negative in conventional microsatellite instability assay, they showed a 10.5-fold increase in the rate of somatic indels, e.g., truncating indels in TP53 and TGFBR2, indicating MLH1 haploinsufficiency in the correction of DNA indel errors. We further analyzed the exomes of 15 renal cell carcinomas and confirmed MLH1 haploinsufficiency. We observed a much higher rate of indel mutations in the affected cases and identified recurrent truncating indels in several cancer genes such as VHL, PBRM1, and JARIDIC. Together, our data suggest that MLH1 hemizygous deletion, through increasing the rate of indel mutations, could drive the development and progression of sporadic cancers.
  • Takeshi Fujiwara, Miyako Hiramatsu, Takayuki Isagawa, Hironori Ninomiya, Kentaro Inamura, Shumpei Ishikawa, Masaru Ushijima, Masaaki Matsuura, Michael H. Jones, Miyuki Shimane, Hitoshi Nomura, Yuichi Ishikawa, Hiroyuki Aburatani
    LUNG CANCER 75 1 119 - 125 2012年01月 [査読有り][通常論文]
    Background: Lung adenocarcinoma is heterogeneous regarding histology, etiology and prognosis. Although there have been several attempts to find a subgroup with poor prognosis, it is unclear whether or not adenocarcinoma with neuroendocrine (NE) nature has unfavorable prognosis. Materials and methods: To elucidate whether a subtype of adenocarcinoma with NE nature has poor prognosis, we performed gene expression profiling by cDNA microarray for 262 Japanese lung cancer and 30 normal lung samples, including 171 adenocarcinomas, 56 squamous cell carcinomas and 35 NE tumors. A co-expression gene set with ASCL1, an NE master gene, was utilized to classify tumors by non-negative matrix factorization, followed by validation using an ASCL1 knock-down gene set in DMS79 cells as well as an independent cohort (n = 139) derived from public microarray databases as a test set. Results: The co-expression gene set classified the adenocarcinomas into alveolar cell (AL), squamoid, and NE subtypes. The NE subtype, which clustered together almost all the NE tumors, had significantly poorer prognosis than the AL subtype that clustered with normal lung samples (p = 0.0075). The knock-down gene set also classified the 171 adenocarcinomas into three subtypes and this NE subtype also had the poorest prognosis. The co-expression gene set classified the independent database-derived American cohort into two subtypes, with the NE subtype having poorer prognosis. None of the single NE gene expression was found to be linked to survival difference. Conclusion: Co-expression gene set with ASCH, rather than single NE gene expression, successfully identifies an NE subtype of lung adenocarcinoma with poor prognosis. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
  • Takayuki Isagawa, Genta Nagae, Nobuaki Shiraki, Takanori Fujita, Noriko Sato, Shumpei Ishikawa, Shoen Kume, Hiroyuki Aburatani
    PLOS ONE 6 10 e26052  2011年10月 [査読有り][通常論文]
    Embryogenesis is tightly regulated by multiple levels of epigenetic regulation such as DNA methylation, histone modification, and chromatin remodeling. DNA methylation patterns are erased in primordial germ cells and in the interval immediately following fertilization. Subsequent developmental reprogramming occurs by de novo methylation and demethylation. Variance in DNA methylation patterns between different cell types is not well understood. Here, using methylated DNA immunoprecipitation and tiling array technology, we have comprehensively analyzed DNA methylation patterns at proximal promoter regions in mouse embryonic stem (ES) cells, ES cell-derived early germ layers (ectoderm, endoderm and mesoderm) and four adult tissues (brain, liver, skeletal muscle and sperm). Most of the methylated regions are methylated across all three germ layers and in the three adult somatic tissues. This commonly methylated gene set is enriched in germ cell-associated genes that are generally transcriptionally inactive in somatic cells. We also compared DNA methylation patterns by global mapping of histone H3 lysine 4/27 trimethylation, and found that gain of DNA methylation correlates with loss of histone H3 lysine 4 trimethylation. Our combined findings indicate that differentiation of ES cells into the three germ layers is accompanied by an increased number of commonly methylated DNA regions and that these tissue-specific alterations in methylation occur for only a small number of genes. DNA methylation at the proximal promoter regions of commonly methylated genes thus appears to be an irreversible mark which functions to fix somatic lineage by repressing the transcription of germ cell-specific genes.
  • Genta Nagae, Takayuki Isagawa, Nobuaki Shiraki, Takanori Fujita, Shogo Yamamoto, Shuichi Tsutsumi, Aya Nonaka, Sayaka Yoshiba, Keisuke Matsusaka, Yutaka Midorikawa, Shumpei Ishikawa, Hidenobu Soejima, Masashi Fukayama, Hirofumi Suemori, Norio Nakatsuji, Shoen Kume, Hiroyuki Aburatani
    HUMAN MOLECULAR GENETICS 20 14 2710 - 2721 2011年07月 [査読有り][通常論文]
    Epigenetic regulation is essential in determining cellular phenotypes during differentiation. Although tissue-specific DNA methylation has been studied, the significance of methylation variance for tissue phenotypes remains unresolved, especially for CpG-poor promoters. Here, we comprehensively studied methylation levels of 27 578 CpG sites among 21 human normal tissues from 12 anatomically different regions using an epigenotyping beadarray system. Remarkable changes in tissue-specific DNA methylation were observed within CpG-poor promoters but not CpG-rich promoters. Of note, tissue-specific hypomethylation is accompanied by an increase in gene expression, which gives rise to specialized cellular functions. The hypomethylated regions were significantly enriched with recognition motifs for transcription factors that regulate cell-type-specific differentiation. To investigate the dynamics of hypomethylation events, we analyzed methylation levels of the entire APOA1 gene locus during in vitro differentiation of embryonic stem cells toward the hepatic lineage. A decrease in methylation was observed after day 13, coinciding with alpha-fetoprotein detection, in the vicinity of its transcription start sites (TSSs), and extends up to similar to 200 bp region encompassing the TSS at day 21, equivalent to the hepatoblastic stage. This decrease is even more pronounced in the adult liver, where the entire APOA1 gene locus is hypomethylated. Furthermore, when we compared the methylation status of induced pluripotent stem (iPS) cells with their parental cell, IMR-90, we found that fibroblast-specific hypomethylation is restored to a fully methylated state in iPS cells after reprogramming. These results illuminate tissue-specific methylation dynamics in CpG-poor promoters and provide more comprehensive views on spatiotemporal gene regulation in terminal differentiation.
  • Inowa T, Hishikawa K, Matsuzaki Y, Isagawa T, Takeuchi T, Aburatani H, Kitamura T, Fujita T
    Stem cells international 2010 782967  2010年 [査読有り][通常論文]
    Side population (SP) cells are an enriched population of stem, and the existence of SP cells has been reported in human cancer cell lines. In this study, we performed an SP analysis using 11 human cancer cell lines and confirmed the presence of SP cells in an embryonic carcinoma cell line, NEC8. NEC8 SP cells showed characteristics of cancer stem cells, such as high growth rate, chemoresistance and high invasiveness. To further characterize the NEC8 SP cells, we used DNA microarrays. Among 38,500 genes, we identified 12 genes that were over-expressed in SP cells and 1 gene that was over-expressed in non-SP cells. Among these 13 genes, we focused on GADD45b. GADD45b was over-expressed in non-SP cells, but the inhibition of GADD45b had no effect on non-SP cells. Paradoxically, the inhibition of GADD45b significantly reduced the viability of NEC8 SP cells. The inhibition of ABCG2, which determines the SP phenotype, had no effect on the invasiveness of NEC8 SP cells, but the inhibition of GADD45b significantly reduced invasiveness. These results suggest that GADD45b, but not ABCG2, might determine the cancer stem cell-like phenotype, such as chemoresistance and the high invasiveness of NEC8 SP cells, and might be a good therapeutic target.
  • Keiichi Hishikawa, Toshihiko Inowa, Yumi Matsuzaki, Takayuki Isagawa, Takumi Takeuchi, Hiroyuki Aburatani, Tadaichi Kitamura, Toshiro Fujita
    Stem Cells International 2010年 [査読有り][通常論文]
    Side population (SP) cells are an enriched population of stem, and the existence of SP cells has been reported in human cancer cell lines. In this study, we performed an SP analysis using 11 human cancer cell lines and confirmed the presence of SP cells in an embryonic carcinoma cell line, NEC8. NEC8 SP cells showed characteristics of cancer stem cells, such as high growth rate, chemoresistance and high invasiveness. To further characterize the NEC8 SP cells, we used DNA microarrays. Among 38,500 genes, we identified 12 genes that were over-expressed in SP cells and 1 gene that was over-expressed in non-SP cells. Among these 13 genes, we focused on GADD45b. GADD45b was over-expressed in non-SP cells, but the inhibition of GADD45b had no effect on non-SP cells. Paradoxically, the inhibition of GADD45b significantly reduced the viability of NEC8 SP cells. The inhibition of ABCG2, which determines the SP phenotype, had no effect on the invasiveness of NEC8 SP cells, but the inhibition of GADD45b significantly reduced invasiveness. These results suggest that GADD45b, but not ABCG2, might determine the cancer stem cell-like phenotype, such as chemoresistance and the high invasiveness of NEC8 SP cells, and might be a good therapeutic target. Copyright © 2010 Toshihiko Inowa et al.
  • M. Murohashi, K. Hinohara, M. Kuroda, T. Isagawa, S. Tsuji, S. Kobayashi, K. Umezawa, A. Tojo, H. Aburatani, N. Gotoh
    British Journal of Cancer 102 1 206 - 212 2010年01月 [査読有り][通常論文]
    Background: Tumour-initiating cells (TICs) or cancer stem cells can exist as a small population in malignant tissues. The signalling pathways activated in TICs that contribute to tumourigenesis are not fully understood.Methods: Several breast cancer cell lines were sorted with CD24 and CD44, known markers for enrichment of breast cancer TICs. Tumourigenesis was analysed using sorted cells and total RNA was subjected to gene expression profiling and gene set enrichment analysis (GSEA).Results: We showed that several breast cancer cell lines have a small population of CD24-/low /CD44+ cells in which TICs may be enriched, and confirmed the properties of TICs in a xenograft model. GSEA revealed that CD24-/low /CD44+ cell populations are enriched for genes involved in transforming growth factor-Β, tumour necrosis factor, and interferon response pathways. Moreover, we found the presence of nuclear factor-B (NF-B) activity in CD24 -/low /CD44+ cells, which was previously unrecognised. In addition, NF-B inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) prevented tumourigenesis of CD24-/low /CD44+ cells in vivo.Conclusion: Our findings suggest that signalling pathways identified using GSEA help to identify molecular targets and biomarkers for TIC-like cells. © 2010 Cancer Research UK All rights reserved.
  • Koichi Yagi, Kiwamu Akagi, Hiroshi Hayashi, Genta Nagae, Shingo Tsuji, Takayuki Isagawa, Yutaka Midorikawa, Yoji Nishimura, Hirohiko Sakamoto, Yasuyuki Seto, Hiroyuki Aburatani, Atsushi Kaneda
    CLINICAL CANCER RESEARCH 16 1 21 - 33 2010年01月 [査読有り][通常論文]
    Purpose: Whereas the CpG island methylator phenotype (CIMP) in colorectal cancer associates with microsatellite instability (MSI)-high and BRAF-mutation(+), the existence of an intermediate-methylation subgroup associated with KRAS-mutation(+) is controversial, and suitable markers for the subgroup have yet to be developed. Our aim is to clarify DNA methylation epigenotypes of colorectal cancer more comprehensively. Experimental Design: To select new methylation markers on a genome-wide scale, we did methylated DNA immunoprecipitation-on-chip analysis of colorectal cancer cell lines and re-expression array analysis by 5-aza-2'-deoxycytidine/Trichostatin A treatment. Methylation levels were analyzed quantitatively in 149 colorectal cancer samples using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Colorectal cancer was epigenotyped by unsupervised two-way hierarchical clustering method. Results: Among 1,311 candidate silencing genes, 44 new markers were selected and underwent quantitative methylation analysis in colorectal cancer samples together with 16 previously reported markers. Colorectal cancer was clustered into high-, intermediate-, and low-methylation epigenotypes. Methylation markers were clustered into two major groups: group 1 showing methylation in high- methylation epigenotype, and group 2 showing methylation in high- and intermediate- methylation epigenotypes. A two-step marker panel deciding epigenotypes was developed with 95% accuracy: the 1st panel consisting of three group-1 markers (CACNA1G, LOX, SLC30A10) to extract high- methylation epigenotype, and the 2nd panel consisting of four group-2 markers (ELMO1, FBN2, THBD, HAND1) and SLC30A10 again to divide the remains into intermediate- and low-methylation epigenotypes. The high- methylation epigenotype correlated significantly with MSI-high and BRAF-mutation(+) in concordance with reported CIMP. Intermediate-epigenotype significantly correlated with KRAS-mutation(+). KRAS-mutation(+) colorectal cancer with intermediate- methylation epigenotype showed significantly worse prognosis. Conclusions: Three methylation epigenotypes exist in colorectal cancer, and suitable classification markers have been developed. Intermediate-methylation epigenotype with KRAS-mutation(+) correlated with worse prognosis. Clin Cancer Res; 16(1); 21-33. (C) 2010 AACR.
  • Nobuaki Shiraki, Yuichiro Higuchi, Seiko Harada, Kahoko Umeda, Takayuki Isagawa, Hiroyuki Aburatani, Kazuhiko Kume, Shoen Kume
    Embryonic stem cells differentiated on M15 cells have previously been shown to give rise to cells of the mesendodermal and definitive endodermal lineages. Here we demonstrate that neuroectodermal and mesodermal lineages can be derived from ES cells cultured on M15 cells and subsequently subjected to specific culture conditions, as confirmed by the expression of molecular markers. Prospective isolation and microarray analyses showed that neuroectodermal cells expressed anterior-to-posterior, as well as dorso-ventral regional markers, suggesting that this procedure could be used for the induction of cells belonging to a wide variety of neural lineages. Lateral mesoderm and paraxial mesoderm cells were also produced and their gene expression profiles were confirmed by microarray analyses. These results indicate that the M15 cell system provides a valuable tool for generating ES cell-derived lineage-specific cell types belonging to the three germ layers, namely neuroectoderm, mesoderm, and definitive endoderm. (C) 2009 Elsevier Inc. All rights reserved.
  • Kenji Matsumoto, Takayuki Isagawa, Toshinobu Nishimura, Takunori Ogaeri, Koji Eto, Satsuki Miyazaki, Jun-ichi Miyazaki, Hiroyuki Aburatani, Hiromitsu Nakauchi, Hideo Ema
    PLOS ONE 4 3 e4820  2009年03月 [査読有り][通常論文]
    The cellular ontogeny of hematopoietic stem cells (HSCs) remains poorly understood because their isolation from and their identification in early developing small embryos are difficult. We attempted to dissect early developmental stages of HSCs using an in vitro mouse embryonic stem cell (ESC) differentiation system combined with inducible HOXB4 expression. Here we report the identification of pre-HSCs and an embryonic type of HSCs (embryonic HSCs) as intermediate cells between ESCs and HSCs. Both pre-HSCs and embryonic HSCs were isolated by their c-Kit+ CD41+ CD45 2 phenotype. Pre-HSCs did not engraft in irradiated adult mice. After co-culture with OP9 stromal cells and conditional expression of HOXB4, pre-HSCs gave rise to embryonic HSCs capable of engraftment and long-term reconstitution in irradiated adult mice. Blast colony assays revealed that most hemangioblast activity was detected apart from the pre-HSC population, implying the early divergence of pre-HSCs from hemangioblasts. Gene expression profiling suggests that a particular set of transcripts closely associated with adult HSCs is involved in the transition of pre-HSC to embryonic HSCs. We propose an HSC developmental model in which pre-HSCs and embryonic HSCs sequentially give rise to adult types of HSCs in a stepwise manner.
  • K Inamura, T Fujiwara, Y Hoshida, T Isagawa, MH Jones, C Virtanen, M Shimane, Y Satoh, S Okumura, K Nakagawa, E Tsuchiya, S Ishikawa, H Aburatani, H Nomura, Y Ishikawa
    ONCOGENE 24 47 7105 - 7113 2005年10月 [査読有り][通常論文]
    Current clinical and histopathological criteria used to define lung squamous cell carcinomas (SCCs) are insufficient to predict clinical outcome. To make a clinically useful classification by gene expression profiling, we used a 40 386 element cDNA microarray to analyse 48 SCC, nine adenocarcinoma, and 30 normal lung samples. Initial analysis by hierarchical clustering (HC) allowed division of SCCs into two distinct subclasses. An additional independent round of HC induced a similar partition and consensus clustering with the non-negative matrix factorization approach indicated the robustness of this classification. Kaplan-Meier analysis with the log-rank test pointed to a nonsignificant difference in survival (P = 0.071), but the likelihood of survival to 6 years was significantly different between the two groups (40.5 vs 81.8%, P = 0.014, Z-test). Biological process categories characteristic for each subclass were identified statistically and upregulation of cell-proliferation-related genes was evident in the subclass with poor prognosis. In the subclass with better survival, genes involved in differentiated intracellular functions, such as the MAPKKK cascade, ceramide metabolism, or regulation of transcription, were upregulated. This work represents an important step toward the identification of clinically useful classification for lung SCC.
  • H Mukai, T Isagawa, E Goyama, S Tanaka, NF Bence, A Tamura, Y Ono, RR Kopito
    Huntington's disease is a progressive neurodegenerative disorder caused by a polyglutamine repeat expansion in the first exon of the huntingtin (Htt) protein. N-terminal Htt peptides with polyglutamine tracts in the pathological range (51-122 glutamines) form high-molecular-weight protein aggregates with fibrillar morphology in vitro, and they form discrete inclusion bodies in a cell-culture model. However, in some studies, formation of discrete Htt inclusions does not correlate well with cell death. We coexpressed N-terminal Htt fragments containing 91 glutamines fused to different affinity tags in HEK293 cells, and we isolated small aggregates by double sequential-affinity chromatography to assure the isolation of multimeric molecules. Transmission electron microscopy and atomic force microscopy revealed the isolated aggregates as globules or clusters of globules 4-50nm in diameter without any detectable fibrillar species. Because small nonfibrillar oligomers, not mature fibrils, recently have been suggested to be the principal cytotoxic species in neurodegenerative disease, these Htt globular aggregates formed in cells may represent the pathogenic form of mutant Htt.
  • T Isagawa, M Takahashi, T Kato, H Mukai, Y Ono
    MOLECULAR CARCINOGENESIS 43 1 1 - 12 2005年05月 [査読有り][通常論文]
    PKN1 is a serine/threonine protein kinase that has been reported to mediate cellular response to stress. We show here that in response to arsenite exposure, PKN1 kinase activity was stimulated, which was associated with increased binding of PKN1 to Cdc25C and delayed mitotic entry. A role for PKN1 in mediating arsenite-induced G(2)/M delay was supported by the finding that expression of a constitutively active form of PKN1 (PKN1AF3) in HeLa cells delayed the mitotic entry of cell cycle. Further experiments indicate that PKN1 directly phosphorylated serine 216 (Ser216) in Cdc25C, which then facilitated association between Cdc25C and 14-3-3. Significantly, expression of a phosphorylation mutant of Cdc25C (S21 6A) partially abrogated the cell-cycle arrest in response to arsenite. Together, our results suggest that PKN1 mediates arsenite-induced delay of the G2/M transition by binding to and phoshorylating Cdc25C. (c) 2005 Wiley-Liss, Inc.
  • Y Gotoh, K Oishi, H Shibata, A Yamagiwa, T Isagawa, T Nishimura, E Goyama, M Takahashi, H Mukai, Y Ono
    PKN1 is a fatty acid and Rho-activated serine/threonine protein kinase whose catalytic domain is highly homologous to protein kinase C (PKC) family. In yeast two-hybrid screening for PKN1 binding proteins, we identified tumor necrosis factor alpha (TNFalpha) receptor-associated factor 2 (TRAF2). TRAF2 is one of the major mediators of TNF receptor superfamily transducing TNF signal to various functional targets, including activation of NF-kappaB, JNK, and apoptosis. FLAG-tagged PKN1 was co-immunoprecipitated with endogenous TRAF2 from HEK293 cell lysate, and in vitro binding assay using the deletion mutants of TRAF2 showed that PKN1 directly binds to the TRAF domain of TRAF2. PKN1 has the TRAF2-binding consensus sequences PXQX (S/T) at amino acid residues 580-584 (PIQES), and P580AQ582A mutant was not co-immunoprecipitated with TRAF2. Furthermore, the reduced expression of PKN1 by RNA interference (RNAi) down-regulated TRAF2-induced NF-kappaB activation in HEK293T cells. These results suggest that PKN1 is involved in TRAF2-NF-kappaB signaling pathway. (C) 2003 Elsevier Inc. All rights reserved.
  • M Takahashi, Y Gotoh, T Isagawa, T Nishimura, E Goyama, HS Kim, H Mukai, Y Ono
    JOURNAL OF BIOCHEMISTRY 133 2 181 - 187 2003年02月 [査読有り][通常論文]
    PKNalpha is a fatty acid- and Rho-activated serine/threonine protein kinase having a catalytic domain homologous to members of the protein kinase C family. Recently it was reported that PKNalpha is involved in the p38 mitogen-activated protein kinase (MAPK) signaling pathway. To date, however, how PKNalpha regulates the p38gamma MAPK signaling pathway is unclear. Here we demonstrate that PKNalpha efficiently phosphorylates MLTKalpha (MLK-like mitogen-activated protein triple kinase), which was recently identified as a MAPK kinase kinase (MAPKKK) for the p38 MAPK cascade. Phosphorylation of MLTKalpha by PKNalpha enhances its kinase activity in vitro. Expression of the kinase-negative mutant of PKNalpha inhibited the mobility shift of MLTKalpha caused by osmotic shock in SDS-PAGE. Furthermore, PKNalpha associates with each member of the p387gamma MAPK signaling pathway (p38gamma, MKK6, and MLTKalpha). These results suggest that PKNalpha functions as not only an upstream activator of MLTKalpha but also a putative scaffold protein for the p38gamma MAPK signaling pathway.
  • T Taniguchi, T Kawamata, H Mukai, H Hasegawa, T Isagawa, M Yasuda, T Hashimoto, A Terashima, M Nakai, Y Ono, C Tanaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 276 13 10025 - 10031 2001年03月 [査読有り][通常論文]
    For the phosphorylation state of microtubule-associated protein, tau plays a pivotal role in regulating microtubule networks in neurons. Tau promotes the assembly and stabilization of microtubules, The potential for tau to bind to microtubules is down-regulated after local phosphorylation, When we investigated the effects of PKN activation on tau phosphorylation, we found that PKN triggers disruption of the microtubule array both in vitro and in vivo and predominantly phosphorylates tau in microtubule binding domains (MBDs). PKN has a catalytic domain highly homologous to protein kinase C (PI(C), a kinase that phosphorylates Ser-313 (= Ser-324, the number used in this study) in MBDs, Thus, we identified the phosphorylation sites of PKN and PKC subtypes (PKC-alpha, -betaI, -beta II, -gamma, -delta, -epsilon, -xi, and -lambda) in MBDs, PKN phosphorylates Ser-258, Ser-320, and Ser-352, although all PKC subtypes phosphorylate Ser-258, Ser-293, Ser-324, and Ser-352, There is a PKN-specific phosphorylation site, Ser-320, in MBDs. HIA3, a novel phosphorylation-dependent antibody recognizing phosphorylated tau at Ser-320, showed immunoreactivity in Chinese hamster ovary cells expressing tau and the active form of PKN, but not in Chinese hamster ovary cells expressing tan and the inactive form of PKN, The immunoreactivity for phosphorylated tau at Ser-320 increased in the presence of a phosphatase inhibitor, FK506 treatment, which means that calcineurin (protein phosphatase 2B) may be involved in dephosphorylating tau at Ser-320 site. We also noted that PKN reduces the phosphorylation recognized by the phosphorylation-dependent antibodies AT8, AT180, and AT270 in vivo. Thus PKN serves as a regulator of microtubules by specific phosphorylation of tau, which leads to disruption of tubulin assembly.
  • K Misaki, H Mukai, C Yoshinaga, K Oishi, T Isagawa, M Takahashi, K Ohsumi, T Kishimoto, Y Ono
    The role of PKN, a fatty acid- and Rho small GTPase-activated protein kinase, in cell-cycle regulation was analyzed. Microinjection of the active form of PKN into a Xenopus embryo caused cleavage arrest, whereas normal cell division proceeded in the control embryo microinjected with buffer or the inactive form of PKN. Exogenous addition of the active form of PKN delayed mitotic timing in Xenopus egg cycling extracts judging by morphology of sperm nuclei and Cdc2/cyclin B histone H1 kinase activity. The kinase-negative form of PKN did not affect the timing, suggesting that delayed mitotic timing depends on the kinase activity of PKN. The dephosphorylation of Tyr-15 of Cdc2 was also delayed in correlation with Cdc2/cyclin B histone H1 kinase activation in extracts containing active PKN. The Cdc25C activity for the dephosphorylation of Tyr-15 in Cdc2 was suppressed by pretreatment with the active form of PKN. Furthermore, PKN efficiently phosphorylated Cdc25C in vitro, indicating that PKN directly inhibits Cdc25C activity by phosphorylation. These results suggest that PKN plays a significant role in the control of mitotic timing by inhibition of Cdc25C.
  • M Takahashi, H Mukai, K Oishi, T Isagawa, Y Ono
    JOURNAL OF BIOLOGICAL CHEMISTRY 275 44 34592 - 34596 2000年11月 [査読有り][通常論文]
    Protein kinase C (PKC) family requires phosphorylation of itself to become competent for responding to second messengers. Much attention has been focused on elucidating the role of phosphorylation in PKC activity; however, it remains unknown where this modification takes place in the cells. This study examines whether anchoring protein is involved in the regulation of PKC phosphorylation. A certain population of PKC epsilon in rat brain extracts as well as that expressed in COS7 cells was associated with an endogenous anchoring protein CG-NAP (centrosome and Golgi localized PKN-associated protein). Pulse chase experiments revealed that the associated PKC epsilon was an immature species at the hypophosphorylated state. In vitro binding studies confirmed that non- or hypophosphorylated PKC epsilon directly bound to CG-NAP via its catalytic domain, whereas sufficiently phosphorylated PKC epsilon did not. PKC epsilon mutant at a potential phosphorylation site of Thr-566 or Ser-729 to Ala, possessing almost no catalytic activity, was associated and co-localized with CG-NAP at Golgi/centrosome area. On the other hand, wild type and a phosphorylation-mimicking mutant at Thr-566 were mainly distributed in cytosol and represented second messenger-dependent catalytic activation. These results suggest that CG-NAP anchors hypophosphorylated PKC epsilon at the Golgi/centrosome area during maturation and serves as a scaffold for the phosphorylation reaction.
  • T Isagawa, H Mukai, K Oishi, T Taniguchi, H Hasegawa, T Kawamata, C Tanaka, Y Ono
    We analyzed the effects of PKN alpha and protein kinase C (PKC) on phosphorylation of tau protein by glycogen synthase kinase (GSK)-3 beta using monoclonal antibodies (AT8, AT180, and AT270), These antibodies are highly specific for phosphorylated tan in Alzheimer paired helical filaments, and recognize phosphorylated Ser202/Thr205, Thr231, and Thr181 of tan protein, respectively, Immunoblot analysis demonstrated that PKN alpha and PKC did not directly phosphorylate their sites, whereas GSK-3 beta efficiently did so. Incubating GSK-3 beta with PKN alpha or PKC subtypes inhibited subsequent GSK-3 beta-induced AT8 and AT270 immunoreactivity. However, the constitutive active form of the GSK-3 beta(S9A) mutant was almost totally inert to each enzyme. Incubating tau with PKNa increased the GSK-3 beta-induced AT180 immunoreactivity, which was further enhanced when the S9A mutant was used instead of the wild type GSK-3 beta. These results suggest that PKN alpha and PKC directly inhibit GSK-3 beta activity at least in part by phosphorylating Ser9 of GSK-3 beta, and that they indirectly suppress GSK-3 beta-stimulated phosphorylation of tau at amino acids Ser202/Thr205 and Thr181, but enhanced phosphorylation at Thr231 through phosphorylation at other sites of tau. (C) 2000 Academic Press.


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    江口 正倫, 池田 聡司, 砂河 孝行, 河野 浩章, 前村 浩二 血管 41 (1) 27 -27 2018年01月 [査読無し][通常論文]
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    安部 元, 武田 憲彦, 砂河 孝行, 仙波 宏章, 相馬 桂, 小山 雄広, 和氣 正樹, 加藤 愛巳, 中釜 悠, 佐藤 達之, 真鍋 一郎, 小室 一成 血管 41 (1) 35 -35 2018年01月 [査読無し][通常論文]
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    Wei Shuoyu, 砂河 孝行, 江口 正倫, 佐藤 大輔, 池田 聡司, 河野 浩章, 前村 浩二 生命科学系学会合同年次大会 2017年度 [2P -0512] 2017年12月 [査読無し][通常論文]
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    江口 正倫, 池田 聡司, 砂河 孝行, 河野 浩章, 前村 浩二 日本臨床分子医学会学術総会プログラム・抄録集 54回 68 -68 2017年04月 [査読無し][通常論文]
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    河村 大輔, 砂河 孝行, 鈴木 良平, 佐藤 玲子, 加藤 洋人, 田中 麻理子, 山本 尚吾, 深山 正久, 油谷 浩幸, 石川 俊平 日本病理学会会誌 106 (1) 320 -320 2017年03月 [査読無し][通常論文]
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  • T. Chijiwa, M. Haraguchi, T. Isagawa, A. Noguchi, M. Katayama, N. Miyao, M. Yoshioka, N. Matsui, Y. Tateishi, H. Suemizu, R. Yamada, Y. Nakamura, K. Imai, D. Komura, H. Katoh, S. Ishikawa, M. Nakamura, Y. Miyagi ANNALS OF ONCOLOGY 27 2016年12月 [査読無し][通常論文]
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  • 合田哲, 砂河孝行, 近岡洋子, 川村猛, 油谷浩幸 日本分子生物学会年会プログラム・要旨集(Web) 35th 4P-0141 (WEB ONLY) 2012年 [査読無し][通常論文]
  • 大腸癌におけるH3K9脱メチル化酵素KDM4Cの発現異常とその意義の検討
    山本 信三, 立石 敬介, 工藤 洋太郎, 山本 恵介, 浅岡 良成, 伊地知 秀明, 砂河 孝行, 油谷 浩幸, 小池 和彦 日本消化器病学会雑誌 108 (臨増大会) A850 -A850 2011年09月 [査読無し][通常論文]
  • 口腔癌における網羅的DNAメチル化の解析(Epigenomic profiling of oral cancer)
    葭葉 清香, 永江 玄太, 辻 真吾, 砂河 孝行, 金田 篤志, 松坂 恵介, 立川 哲彦, 新谷 悟, 油谷 浩幸 日本癌学会総会記事 70回 131 -131 2011年09月 [査読無し][通常論文]
  • ヒト腎臓由来iPS細胞のDNAメチル化プロファイルと細胞記憶
    菱川 慶一, 高瀬 敦, 宮本 寛治, 阪井 弘治, 山本 直樹, 砂河 孝行, 油谷 浩幸, 藤田 敏郎 日本腎臓学会誌 53 (3) 388 -388 2011年05月 [査読無し][通常論文]
  • 細胞分化過程における低CpGプロモーターの組織特異的な脱メチル化(Tissue-type specific hypomethylation in CpG-poor promoters during cellular differentiation)
    永江 玄太, 砂河 孝行, 白木 伸明, 末盛 博文, 粂 昭苑, 油谷 浩幸 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 3T2 -10 2010年12月 [査読無し][通常論文]
  • マウスES細胞の3胚葉分化におけるDNAメチル化プロファイリング(DNA methylation profiling in differentiation of embryonic stem cells into three germ layers)
    砂河 孝行, 永江 玄太, 白木 伸明, 佐藤 憲子, 粂 昭苑, 油谷 浩幸 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 4P -0733 2010年12月 [査読無し][通常論文]
  • ヒト腎臓由来iPS細胞の包括的DNAメチル化解析と細胞記憶
    菱川 慶一, 高瀬 敦, 上浦 望, 宮本 寛治, 松村 隆紀, 阪井 弘治, 山本 直樹, 砂河 孝行, 油谷 浩幸 日本高血圧学会総会プログラム・抄録集 33回 249 -249 2010年10月 [査読無し][通常論文]
  • EBV関連胃癌におけるDNAメチル化の網羅的解析(Comprehensive DNA methylation analysis in EBV-positive gastric cancer)
    松坂 恵介, 金田 篤志, 永江 玄太, 砂河 孝行, 堤 修一, 石川 俊平, 宇於崎 宏, 高田 賢蔵, 瀬戸 泰之, 油谷 浩幸, 深山 正久 日本癌学会総会記事 69回 151 -152 2010年08月 [査読無し][通常論文]
  • 肺小細胞癌株のヒストン修飾異常に関する検討(Aberrant histone modification pattern in Small cell lung cancer)
    佐藤 輝幸, 砂河 孝行, 石川 俊平, 永江 玄太, 野中 綾, 石川 雄一, 金田 篤志, 貫和 敏博, 油谷 浩幸 日本癌学会総会記事 69回 345 -345 2010年08月 [査読無し][通常論文]
  • 分子生物学を応用した消化器癌の悪性度診断 DNAメチル化情報を用いたエピジェノタイプ分類による大腸癌の予後予測
    八木 浩一, 赤木 究, 林 浩志, 永江 玄太, 辻 真吾, 砂河 孝行, 緑川 泰, 西村 洋治, 坂本 裕彦, 瀬戸 泰之, 油谷 浩幸, 金田 篤志 日本消化器外科学会総会 65回 76 -76 2010年07月 [査読無し][通常論文]
  • ヒト腎臓細胞由来iPS細胞の網羅的解析
    菱川 慶一, 高瀬 敦, 上浦 望, 宮本 寛治, 松村 隆紀, 砂河 孝行, 油谷 浩幸, 藤田 敏郎 日本腎臓学会誌 52 (3) 302 -302 2010年05月 [査読無し][通常論文]
  • M. Murohashi, K. Hinohara, M. Kuroda, T. Isagawa, S. Tsuji, S. Kobayashi, K. Umezawa, A. Tojo, H. Aburatani, N. Gotoh BRITISH JOURNAL OF CANCER 102 (1) 206 -212 2010年01月 [査読無し][通常論文]
    BACKGROUND: Tumour-initiating cells (TICs) or cancer stem cells can exist as a small population in malignant tissues. The signalling pathways activated in TICs that contribute to tumourigenesis are not fully understood. METHODS: Several breast cancer cell lines were sorted with CD24 and CD44, known markers for enrichment of breast cancer TICs. Tumourigenesis was analysed using sorted cells and total RNA was subjected to gene expression profiling and gene set enrichment analysis (GSEA). RESULTS: We showed that several breast cancer cell lines have a small population of CD24(-/low)/CD44(+) cells in which TICs may be enriched, and confirmed the properties of TICs in a xenograft model. GSEA revealed that CD24(-/low)/CD44(+) cell populations are enriched for genes involved in transforming growth factor-beta, tumour necrosis factor, and interferon response pathways. Moreover, we found the presence of nuclear factor-kappa B (NF-kappa B) activity in CD24(-/low)/CD44(+) cells, which was previously unrecognised. In addition, NF-kappa B inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) prevented tumourigenesis of CD24(-/low)/CD44(+) cells in vivo. CONCLUSION: Our findings suggest that signalling pathways identified using GSEA help to identify molecular targets and biomarkers for TIC-like cells. British Journal of Cancer (2010) 102, 206-212. doi:10.1038/sj.bjc.6605468 www.bjcancer.com Published online 8 December 2009 (C) 2010 Cancer Research UK
  • 定量的メチル化解析により大腸癌は3群のエピジェノタイプに分類される(Epigenotyping by quantitative DNA methylation analysis revealed three epigenotypes in colorectal cancer)
    八木 浩一, 赤木 究, 林 浩志, 永江 玄太, 辻 真吾, 砂河 孝行, 緑川 泰, 西村 洋治, 坂本 裕彦, 瀬戸 泰之, 油谷 浩幸, 金田 篤志 日本癌学会総会記事 68回 223 -223 2009年08月 [査読無し][通常論文]
  • 肺腺がんを肺胞、扁平上皮、神経内分泌細胞様の特徴をもつ亜型に分類する遺伝子セットの同定(Identification of a gene set for classifying lung adenocarcinoma into three subtypes with distinct characteristics)
    藤原 大, 平松 美也子, 砂河 孝行, 二宮 浩範, 稲村 健太郎, 石川 俊平, 嶋根 みゆき, 野村 仁, 石川 雄一, 油谷 浩幸 日本癌学会総会記事 68回 454 -454 2009年08月 [査読無し][通常論文]
  • 八木 浩一, 金田 篤志, 赤木 究, 永江 玄太, 砂河 孝行, 西村 洋司, 坂本 裕彦, 瀬戸 泰之, 油谷 浩幸 日本外科学会雑誌 110 (2) 2009年02月 [査読無し][通常論文]
  • MassARRAYを用いた定量的メチル化解析による大腸癌のepigenotyping
    八木 浩一, 金田 篤志, 赤木 究, 永江 玄太, 砂河 孝行, 西村 洋司, 坂本 裕彦, 瀬戸 泰之, 油谷 浩幸 日本外科学会雑誌 110 (臨増2) 450 -450 2009年02月 [査読無し][通常論文]
  • Michiko Murohashi, Takayuki Isagawa, Shingo Tsuji, Kazuo Umezawa, Hiroyuki Aburatani, Noriko Gotoh INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 24 S8 -S8 2009年 [査読無し][通常論文]
  • 胚性幹細胞からの造血幹細胞発生に伴う発現遺伝子解析
    西村 聡修, 依馬 秀夫, 砂河 孝行, 油谷 浩幸, 魚返 拓利, 松本 憲二, 中内 啓光 臨床血液 49 (9) 913 -913 2008年09月 [査読無し][通常論文]
  • MassARRAYを用いた定量的メチル化解析による大腸癌のタイピング(Epigenotyping of colorectal cancer by quantitative methylation analysis using MassARRAY)
    八木 浩一, 金田 篤志, 永江 玄太, 砂河 孝行, 赤木 究, 西村 洋司, 坂本 裕彦, 瀬戸 泰之, 油谷 浩幸 日本癌学会総会記事 67回 69 -69 2008年09月 [査読無し][通常論文]
  • 肝癌における異常メチル化に伴った遺伝子発現抑制の網羅的解析(Comprehensive analysis of transcriptionally repressed genes due to aberrant promoter hypermethylation in Liver Cancer)
    とう 穎氷, 金田 篤志, 永江 玄太, 八木 浩一, 砂河 孝行, 油谷 浩幸 日本癌学会総会記事 67回 169 -169 2008年09月 [査読無し][通常論文]
  • 菱川慶一, 井之輪俊彦, 砂河孝行, 松崎有未, 武内巧, 油谷浩幸, 北村唯一, 藤田敏郎 再生医療 7 232 2008年02月 [査読無し][通常論文]
  • 砂河孝行, 白木伸明, 永江玄太, 堤修一, 佐藤憲子, 粂昭苑, 油谷浩幸 生化学 3P-0756 2008年 [査読無し][通常論文]
  • 永江 玄太, 砂河 孝行 医学のあゆみ 223 (11) 884 -888 2007年12月 [査読無し][通常論文]
  • 永江玄太, 砂河孝行 医学のあゆみ 223 (11/12) 884 -888 2007年12月 [査読無し][通常論文]
  • 【Quantitative Biology 定量的生物学】 網羅的メチル化解析
    永江 玄太, 砂河 孝行 医学のあゆみ 223 (11-12) 884 -888 2007年12月 [査読無し][通常論文]
  • マウスES細胞分化に伴うDNAメチル化のプロフィール解析(DNA methylation pro filing along with mouse ES cell differentiation)
    砂河 孝行, 永江 玄太, 金田 篤志, 堤 修一, 佐藤 憲子, 白木 伸明, 粂 昭苑, 油谷 浩幸 日本癌学会総会記事 66回 198 -199 2007年08月 [査読無し][通常論文]
  • 高解像度タイリングアレイを用いた大腸癌の網羅的エピゲノム解析(Comprehensive epigenomic analysis of colorectal cancer using immunoprecipitation technique and tiling array)
    金田 篤志, 堤 修一, 林 浩志, 砂河 孝行, 永江 玄太, 上村 直子, 八木 浩一, トウ 穎氷, 油谷 浩幸 日本癌学会総会記事 66回 267 -267 2007年08月 [査読無し][通常論文]
  • 井之輪俊彦, 菱川慶一, 砂河孝行, 松崎有未, 武内巧, 油谷浩幸, 藤田敏郎, 北村唯一 日本泌尿器科学会雑誌 98 (2) 512 2007年02月 [査読無し][通常論文]
  • 井之輪 俊彦, 菱川 慶一, 砂河 孝行, 松崎 有未, 武内 巧, 油谷 浩幸, 藤田 敏郎, 北村 唯一 日本泌尿器科学会雑誌 98 (2) 512 -512 2007年02月 [査読無し][通常論文]
  • 井之輪 俊彦, 菱川 慶一, 砂河 孝行, 松崎 有未, 武内 巧, 油谷 浩幸, 藤田 敏郎, 北村 唯一 日泌尿会誌 98 (2) 2007年 [査読無し][通常論文]
  • 砂河孝行, 永江玄太, 金田篤志, 堤修一, 佐藤憲子, 白木伸明, 粂昭苑, 油谷浩幸 生化学 3P-0746 2007年 [査読無し][通常論文]
  • 肺神経内分泌腫瘍における高密度ゲノムタイピングアレイを用いた染色体変異の解析
    田中 由紀子, 石川 俊平, 山本 尚吾, 砂河 孝行, 稲村 健太郎, 二宮 浩範, 平松 美也子, 石川 雄一, 橋都 浩平, 油谷 浩幸 日本癌学会総会記事 65回 345 -345 2006年09月 [査読無し][通常論文]
  • cDNAマイクロアレイを用いた神経内分泌遺伝子による肺腺癌分類の検討
    藤原 大, 平松 美也子, 砂河 孝行, 稲村 健太郎, 石川 俊平, 星田 有人, 嶋根 みゆき, 二宮 浩載, 野村 仁, 石川 雄一, 油谷 浩幸 日本癌学会総会記事 65回 382 -383 2006年09月 [査読無し][通常論文]
  • 藤原大, 平松美也子, 砂河孝行, 稲村健太郎, 石川俊平, 星田有人, 嶋根みゆき, 二宮浩載, 野村仁, 石川雄一, 油谷浩幸 日本癌学会学術総会記事 65th 382 -383 2006年08月 [査読無し][通常論文]
  • 田中由紀子, 石川俊平, 山本尚吾, 砂河孝行, 稲村健太郎, 二宮浩範, 平松美也子, 石川雄一, 橋都浩平, 油谷浩幸 日本癌学会学術総会記事 65th 345 2006年08月 [査読無し][通常論文]
  • 網羅的遺伝子発現解析による肺扁平上皮癌の亜型分類の試み
    稲村 健太郎, 二宮 浩範, 佐藤 之俊, 油谷 浩幸, 石川 俊平, 星田 有人, 藤原 大, 野村 仁, 嶋根 みゆき, 砂河 孝行, 深山 正久, 土屋 永寿, 石川 雄一 日本癌学会総会記事 64回 402 -402 2005年09月 [査読無し][通常論文]
  • cDNAマイクロアレイ遺伝子発現プロファイリングに基づく肺腺がんの分類
    藤原 大, 油谷 浩幸, 石川 俊平, 星田 有人, 石川 雄一, 稲村 健太郎, 野村 仁, 嶋根 みゆき, 砂河 孝行 日本癌学会総会記事 64回 402 -403 2005年09月 [査読無し][通常論文]
  • 稲村健太郎, 二宮浩範, 佐藤之俊, 油谷浩幸, 石川俊平, 星田有人, 藤原大, 野村仁, 嶋根みゆき, 砂河孝行, 深山正久, 土屋永寿, 石川雄一 日本癌学会学術総会記事 64th 402 2005年08月 [査読無し][通常論文]
  • 藤原大, 油谷浩幸, 石川俊平, 星田有人, 石川雄一, 稲村健太郎, 野村仁, 嶋根みゆき, 砂河孝行 日本癌学会学術総会記事 64th 402 -403 2005年08月 [査読無し][通常論文]
  • T Taniguchi, T Kawamata, H Mukai, H Hasegawa, T Isagawa, M Nakai, Y Ono, C Tanaka JOURNAL OF NEUROCHEMISTRY 78 136 -136 2001年09月 [査読無し][通常論文]
  • T Taniguchi, T Kawamata, H Mukai, H Hasegawa, T Isagawa, M Yasuda, T Hashimoto, A Terashima, M Nakai, H Mori, Y Ono, C Tanaka JOURNAL OF BIOLOGICAL CHEMISTRY 276 (25) 23212 -23212 2001年06月 [査読無し][通常論文]
  • M Takahashi, H Mukai, K Oishi, T Isagawa, Y Ono FASEB JOURNAL 15 (5) A723 -A723 2001年03月 [査読無し][通常論文]
  • 谷口泰造, 川又敏男, 長谷川浩史, 向井秀幸, 砂河孝行, 小野功貴, 田中千賀子, 三好功峰 日本分子生物学会年会プログラム・講演要旨集 23rd 681 2000年11月 [査読無し][通常論文]



  • 心臓リモデリングにおける抗線維化マクロファージの機能解析
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2019年04月 -2022年03月 
    代表者 : 砂河 孝行, 武田 憲彦, 前村 浩二
  • 循環器疾患における時計遺伝子Clif/Bmal2の役割の解明
    日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 前村 浩二, 池田 聡司, 砂河 孝行
    体内時計の分子機序が明らかになり、その構成因子である時計遺伝子を変異させた動物の解析により、代謝疾患、免疫疾患、循環器疾患などの発症、進展に体内時計の乱れが重要な働きを担っていることが明らかになっている。本研究は、我々が血管内皮細胞から同定した時計遺伝子であるArntl2/Clif/Bmal2のノックアウトマウスを作成し、その循環器疾患における役割を明らかにすることで、循環器疾患の発症、増悪する機序を、サーカディアンリズムの撹乱、体内時計の変調の観点で解析することを目的とする。 まずCRISPR-Cas9システムを用いてArntl2/Clif/Bmal2遺伝子のノックアウトマウスの作成を行った。その結果、Exon3に1塩基のインサーション、2塩基の欠失、及び44塩基インサーションされた3系統のマウスを得ることができた。すべてDNA結合にとって重要なドメインであるbHLH(basic Helix Loop Helix)ドメインの途中でストップコードンが出現しArntl2/Clif/Bmal2の機能は欠失していると考えられる。 これら変異をもつホモ接合体は正常に生まれて来て、組織学的検査では特に問題となる所見は認められなかった。行動のサーカディアンリズムに及ぼす影響を確認するため、Light/Dark10日間-Dark/Dark16日間-6時間のLightPulse-Dark/Drak14日間の条件下で輪回し運動による行動測定を行った。その結果、野生型とノックアウトマウスでphase shiftに有意差は認めなかった。現在、体重増加や糖代謝への影響を検証するとともに、さまざまな循環器疾患モデルを作成した時に異常が無いかを検証しているところである。
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2015年04月 -2018年03月 
    代表者 : 田中 麻理子, 深山 正久, 石川 俊平, 加藤 洋人, 砂河 孝行, 山本 浩之, 阿部 淳, 山岸 誠, 長谷川 潔, 有田 淳一, 阪本 良弘, 國土 貴嗣
    申請者は膵発がん時の分化異常(胃上皮分化)に着目し、胃上皮細胞接着分子CLDN18と転写因子EVI1の高発現、EVI1-miRNA96-KRAS axisを同定し、膵発がん過程の解明をこれまで進めてきた。これに基づき本研究では、膵発がんのbiologicalな背景の解明をEVI1を軸に行い、発がん促進サイクルの一つとしてEVI1-miRNA96-Glypican-1 axisを同定した。また、膵がん細胞と膵前がん細胞のmiRNA発現の比較により、発がん時に包括的変動を示すmiRNA群の解析を進める基盤を作った。本研究の成果は、未解明な部分の多い膵発がん過程を明らかにする重要な結果である。
  • 日本学術振興会:科学研究費助成事業 基盤研究(C)
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 千々和 剛, 宮城 洋平, 砂河 孝行, 加藤 洋人
    がん患者由来ゼノグラフト(Patient-derived xenograft, PDX)の網羅的相互作用解析(Interactome)を行い、抗癌剤の効果予測・個別化医療への応用を検討した。Interactomeにて高依存性の相互作用に関する分子標的薬のin vivo実験では有意な腫瘍縮小効果が見られ、臨床効果とも相関した。PDX樹立過程解析にて、効果的なPDX樹立法を検討した。癌性胸水からの特殊なPDX樹立に成功し、手術検体より簡便に採取でき、より病態を反映するモデルを作成した。PDXを用いたInteractomeから最適な分子標的薬を選択するできる個別化医療システムへの展開が可能となった。
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2014年04月 -2016年03月 
    代表者 : 砂河 孝行, 武田 憲彦
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2013年04月 -2015年03月 
    代表者 : 田中 麻理子, 深山 正久, 石川 俊平, 砂河 孝行, 鈴木 洋
  • 日本学術振興会:科学研究費助成事業 若手研究(B)
    研究期間 : 2012年04月 -2014年03月 
    代表者 : 砂河 孝行
  • 癌における転写ネットワーク変異の体系的解明
    日本学術振興会:科学研究費助成事業 基盤研究(S)
    研究期間 : 2004年 -2007年 
    代表者 : 油谷 浩幸, 堤 修一, 石川 俊平, 緑川 泰, 金城 聖文, 砂河 孝行, 筆宝 義隆
    癌における転写調節異常の体系的解明を行うために、SNPアレイ、タイリングアレイなどのゲノム解析技術を応用した。 1) ゲノムコピー数解析 SNPタイピングアレイを応用してアレル別にコピー数を測定する手法Genome Imbalance Mapを開発した(Ishikawa, 2005)。ヘテロ接合性の消失(LOH)や片親性ダイソミー(UPD)を判定するために有効であり、1アレル分のシグナル差を検出できるので正常組織の混入が避けられない臨床材料からでも効率よくホモ欠失領域を検出できた。肝細胞癌で8p23領域にあるCSMD1遺伝子のホモ欠失が検出された。また、本技術を健常者のコピー数多型(CNV)の解析に応用して初のCNVマップを報告した(Redon, Ishikawa, Nature 2006)。 2) DNAメチル化解析 DNAメチル化は遺伝子の不活性化機構として注目されていたが、ゲノムワイドに検出する手法の確立が望まれていた。抗メチルシトシン抗体を用いてメチル化CpGを含むDNA断片を捕捉し、ゲノムタイリングアレイを用いて検出する手法を開発した。高CpG領域はPCR増幅のバイアスが顕著であるため、in vitro転写法による増幅を行うことによって再現性の高いシグナルが得られた。メチル化領域はヒストンアセチル化と排他的であることを観察できた(Hayashi, 2007)。 3) 転写ネットワークの解析 遺伝子発現プロファイルデータから共発現する遺伝子を抽出して活性化されたネットワークを推定した(Agg arwal, 2006)。さらにタイリングアレイを用いて転写因子が結合部位を検出し、直接標的となる遺伝子を同定した。また、ヒストンアセチル化やメチル化修飾をゲノムワイドに検出することが可能となった。染色体のインスレーターといわれるCTCFがコヒーシンと共局在することを発見し、クロマチンループ形成にかかわると推定さた(Wendt, Nature 2008)。

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