48 results on '"Kanki, Yasuharu"'
Search Results
2. Detection of Gene Doping Using Dried Blood Spots from a Mouse Model with rAAV9 Vector-Mediated Human Erythropoietin Expression as a Pilot Study.
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Otani, Norihiro, Kanki, Yasuharu, Nguyen, Kieu D. M., and Sugasawa, Takehito
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GENE expression , *ERYTHROPOIETIN receptors , *ERYTHROPOIETIN , *LABORATORY mice , *ANIMAL disease models , *PILOT projects - Abstract
Rapid advancements in gene technology have raised concerns regarding the potential abuse of techniques, such as gene doping, for enhancing athletic performance. To identify this possibility, a reliable procedure for detecting doping genes is required. Although detection methods for doping genes have been created, there are still areas for further improvement. One significant challenge is the high storage and transport costs of the test samples. For this issue, the dried blood spot (DBS) method can be a cost-effective solution. This study aimed to assess the practicality of incorporating DBSs into the gene doping detection process as a pilot study. Whole-blood samples were initially collected from mice engineered to express human erythropoietin from the rAAV vector. Then, the blood was placed in filter papers and left to dry at room temperature for five hours to form DBSs. These DBSs were subsequently preserved in sealed plastic bags at room temperature. After the extraction of DNA, DBSs were formed, and TaqMan-qPCR was utilized to detect the presence of rAAV vector-derived DNA. The finding confirmed that doping gene-specific fragments were successfully detected in DBSs. This outcome suggests that the DBS method is an effective approach to be considered when developing a comprehensive protocol for gene doping detection. [ABSTRACT FROM AUTHOR]
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- 2024
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3. Identification and Characterization of Yeast Species Isolated from Cornus kousa Fruits in Japan.
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Otani, Norihiro, Nguyen, Kieu D. M., Hirokawa, Atsushi, Kanki, Yasuharu, Yun, Hyun-Sik, Maeda, Yoshiaki, Gu, Wenchao, Takahashi, Yoichiro, and Sugasawa, Takehito
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FRUIT ,YEAST ,SPECIES ,MICROBIAL ecology ,SACCHAROMYCES cerevisiae - Abstract
The Cornus kousa tree, which is of Asian origin, is often cultivated for ornamental purposes and used in traditional medicine. The tree produces sugar-rich fruits, which are potential habitats for natural yeasts. The identification of new yeast strains has many advantages for the industry and research. This study aimed to isolate and identify yeast species from C. kousa fruits and to understand their microbial ecology. Ripe and rotten fruits, which had fallen on the ground naturally, were collected and soaked in culture media, followed by plate spreading for colony growth. The morphological examination revealed three distinct colony types, including two from the ripe fruits and one from the rotten fruits. The analysis of the internal transcribed spacer 1 region indicated three yeast strains corresponding to the three colony types: Torulaspora delbrueckii and Pichia kluyveri from the ripe fruits and Saccharomyces cerevisiae from the rotten fruits. The metabolic characterizations demonstrated that all three yeasts efficiently consumed glucose and produced alcohol. S. cerevisiae exhibited the strongest fermentation ability and the highest growth rate. These findings showed that Cornus kousa fruit is a source of diverse yeast species, with distinct species associated with different states of fruit decomposition. [ABSTRACT FROM AUTHOR]
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- 2024
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4. Detection Method for Gene Doping in a Mouse Model Expressing Human Erythropoietin from Adeno-Associated Virus Vector-9.
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Sugasawa, Takehito, Hirokawa, Atsushi, Otani, Norihiro, Kanki, Yasuharu, Nguyen, Kieu DM, Takemasa, Tohru, Watanabe, Koichi, Takeuchi, Yoshinori, Yahagi, Naoya, and Takahashi, Yoichiro
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ERYTHROPOIETIN receptors ,ADENO-associated virus ,ERYTHROPOIETIN ,LABORATORY mice ,DOPING agents (Chemistry) ,ANIMAL disease models - Abstract
With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still room for improvement in gene-doping testing methods, and a robust animal model needs to be developed. Therefore, the purposes of this study were to establish a model of gene doping using recombinant adeno-associated virus vector-9, including the human erythropoietin gene (rAAV9-hEPO), and to establish a relevant testing method. First, it was attempted to establish the model using rAAV9-hEPO on mice. The results showed a significant increase in erythrocyte volume accompanied by an increase in spleen weight, confirming the validity of the model. Next, we attempted to detect proof of gene doping by targeting DNA and RNA. Direct proof of gene doping was detected using a TaqMan-qPCR assay with certain primers/probes. In addition, some indirect proof was identified in RNAs through the combination of a TB Green qPCR assay with RNA sequencing. Taken together, these results could provide the foundation for an effective test for gene doping in human athletes in the future. [ABSTRACT FROM AUTHOR]
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- 2024
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5. PERK-Mediated Suppression of microRNAs by Sildenafil Improves Mitochondrial Dysfunction in Heart Failure
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Shimizu, Takashi, Taguchi, Akashi, Higashijima, Yoshiki, Takubo, Naoko, Kanki, Yasuharu, Urade, Yoshihiro, and Wada, Youichiro
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- 2020
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6. Inhibition of cardiac PERK signaling promotes peripartum cardiac dysfunction
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Shimizu, Takashi, Taguchi, Akashi, Higashijima, Yoshiki, Kanki, Yasuharu, Nakaki, Ryo, Urade, Yoshihiro, and Wada, Youichiro
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- 2021
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7. Identification of Cardiomyocyte-Fated Progenitors from Human-Induced Pluripotent Stem Cells Marked with CD82
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Takeda, Masafumi, Kanki, Yasuharu, Masumoto, Hidetoshi, Funakoshi, Shunsuke, Hatani, Takeshi, Fukushima, Hiroyuki, Izumi-Taguchi, Akashi, Matsui, Yusuke, Shimamura, Teppei, Yoshida, Yoshinori, and Yamashita, Jun K.
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- 2018
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8. Lysine demethylase 7a regulates murine anterior-posterior development by modulating the transcription of Hox gene cluster
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Higashijima, Yoshiki, Nagai, Nao, Yamamoto, Masamichi, Kitazawa, Taro, Kawamura, Yumiko K., Taguchi, Akashi, Nakada, Natsuko, Nangaku, Masaomi, Furukawa, Tetsushi, Aburatani, Hiroyuki, Kurihara, Hiroki, Wada, Youichiro, and Kanki, Yasuharu
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- 2020
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9. Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells
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Nakato, Ryuichiro, Wada, Youichiro, Nakaki, Ryo, Nagae, Genta, Katou, Yuki, Tsutsumi, Shuichi, Nakajima, Natsu, Fukuhara, Hiroshi, Iguchi, Atsushi, Kohro, Takahide, Kanki, Yasuharu, Saito, Yutaka, Kobayashi, Mika, Izumi-Taguchi, Akashi, Osato, Naoki, Tatsuno, Kenji, Kamio, Asuka, Hayashi-Takanaka, Yoko, Wada, Hiromi, Ohta, Shinzo, Aikawa, Masanori, Nakajima, Hiroyuki, Nakamura, Masaki, McGee, Rebecca C., Heppner, Kyle W., Kawakatsu, Tatsuo, Genno, Michiru, Yanase, Hiroshi, Kume, Haruki, Senbonmatsu, Takaaki, Homma, Yukio, Nishimura, Shigeyuki, Mitsuyama, Toutai, Aburatani, Hiroyuki, Kimura, Hiroshi, and Shirahige, Katsuhiko
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- 2019
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10. Establishing a Sequencing Method for the Whole Mitochondrial DNA of Domestic Dogs.
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Sugasawa, Takehito, Matsumoto, Yuki, Fang, Hui, Takemasa, Tohru, Komine, Ritsuko, Tamai, Shinsuke, Gu, Wenchao, Tanaka, Kei, Kanki, Yasuharu, and Takahashi, Yoichiro
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MITOCHONDRIAL DNA ,DOGS ,DNA primers ,DOG breeds ,FAMILY structure ,ORAL mucosa ,NUCLEOTIDE sequencing - Abstract
Simple Summary: The present study aims to establish a method of whole mitochondrial DNA (mtDNA) sequencing for domestic dogs. Prior to proceeding with the experiment, the collection of relevant DNA samples was essential. Therefore, we decided to use oral mucosa DNA, which can be collected non-invasively. A polymerase chain reaction (PCR) was performed using the DNA collected from six dogs raised in Japan and four primer pairs as specific to the mtDNA, after which the amplified products were sequenced using next-generation sequencing. As a result, the whole mtDNA obtained from all dogs was correctly sequenced. Thus, we determined that the method we used in our study may be useful for future research on dog-related medical care and welfare. In human beings, whole mitochondrial DNA (mtDNA) sequencing has been widely used in many research fields, including medicine, forensics, and genetics. With respect to the domestic dog (Canis lupus familiaris), which is commonly recognized as being an additional member of the traditional human family structure, research studies on mtDNA should be developed to expand and improve our collective knowledge of dog medicine and welfare as it seems that there is still room for further development in these areas. Moreover, a simple and robust method for sequencing whole mtDNA that can be applied to various dog breeds has not yet been described in the literature. In the present study, we aim to establish such a method for the whole mtDNA sequencing of the domestic dog. In the experiments we conducted, oral mucosa DNA samples obtained from six Japanese domestic dogs were used as a template. We designed four primer pairs that could amplify approximately 5 kbp from each region of the mtDNA and validated several PCR conditions. Subsequently, the PCR amplicons were pooled and subjected to library preparation. The sequencing of the libraries was performed using next-generation sequencing (NGS), followed by bioinformatics analysis. Our results demonstrate that the proposed method can be used to perform highly accurate resequencing. We believe that this method may be useful for future research conducted to better understand dog medicine and welfare. [ABSTRACT FROM AUTHOR]
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- 2023
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11. Abstract 14824: Lysine Demethylase 6A and 7A Regulated by MicroRNA-3679-5p Mediates TNF-α-induced Inflammatory Signaling in Vascular Endothelium
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Higashijima, Yoshiki, Inoue, Tsuyoshi, Nangaku, Masaomi, Wada, Youichiro, Furukawa, Tetsushi, and Kanki, Yasuharu
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- 2017
12. A Wave of Nascent Transcription on Activated Human Genes
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Wada, Youichiro, Ohta, Yoshihiro, Xu, Meng, Tsutsumi, Shuichi, Minami, Takashi, Inoue, Kenji, Komura, Daisuke, Kitakami, Jun'ichi, Oshida, Nobuhiko, Papantonis, Argyris, Izumi, Akashi, Kobayashi, Mika, Meguro, Hiroko, Kanki, Yasuharu, Mimura, Imari, Yamamoto, Kazuki, Mataki, Chikage, Hamakubo, Takao, Shirahige, Katsuhiko, Aburatani, Hiroyuki, Kimura, Hiroshi, Kodama, Tatsuhiko, Cook, Peter R., and Ihara, Sigeo
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- 2009
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13. Potential roles of super enhancers in inflammatory gene transcription.
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Higashijima, Yoshiki and Kanki, Yasuharu
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GENE enhancers , *IMMUNOLOGIC diseases , *NUCLEOTIDE sequencing , *HUMAN body , *COMMUNICABLE diseases , *GENOME editing , *SUPER enhancers - Abstract
Acute and chronic inflammation is a basic pathological event that contributes to atherosclerosis, cancer, infectious diseases, and immune disorders. Inflammation is an adaptive process to both external and internal stimuli experienced by the human body. Although the mechanism of gene transcription is highly complicated and orchestrated in a timely and spatial manner, recent developments in next‐generation sequencing, genome‐editing, cryo‐electron microscopy, and single cell‐based technologies could provide us with insights into the roles of super enhancers (SEs). Initially, SEs were implicated in determining cell fate; subsequent studies have clarified that SEs are associated with various pathological conditions, including cancer and inflammatory diseases. Recent technological advances have unveiled the molecular mechanisms of SEs, which involve epigenetic histone modifications, chromatin three‐dimensional structures, and phase‐separated condensates. In this review, we discuss the relationship between inflammation and SEs and the therapeutic potential of SEs for inflammatory diseases. [ABSTRACT FROM AUTHOR]
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- 2022
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14. Identification of RNA Markers in Red Blood Cells for Doping Control in Autologous Blood Transfusion.
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Sugasawa, Takehito, Kanki, Yasuharu, Komine, Ritsuko, Watanabe, Koichi, and Takekoshi, Kazuhiro
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AUTOTRANSFUSION of blood , *RNA sequencing , *RNA , *DOPING agents (Chemistry) , *LABORATORY rats , *ERYTHROCYTES - Abstract
The World Anti-Doping Agency (WADA) has prohibited the use of autologous blood transfusion (ABT) as a doping method by athletes. It is difficult to detect this doping method in laboratory tests, and a robust testing method has not yet been established. We conducted an animal experiment and used total RNA sequencing (RNA-Seq) to identify novel RNA markers to detect ABT doping within red blood cells (RBCs) as a pilot study before human trials. This study used whole blood samples from Wistar rats. The whole blood samples were mixed with a citrate–phosphate–dextrose solution with adenine (CPDA) and then stored in a refrigerator at 4 °C for 0 (control), 10, or 20 days. After each storage period, total RNA-Seq and bioinformatics were performed following RNA extraction and the purification of the RBCs. In the results, clear patterns of expression fluctuations were observed depending on the storage period, and it was found that there were large numbers of genes whose expression decreased in the 10- and 20-day periods compared to the control. Moreover, additional bioinformatic analysis identified three significant genes whose expression levels were drastically decreased according to the storage period. These results provide novel insights that may allow future studies to develop a testing method for ABT doping. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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15. Gene Expression Profile Provides Novel Insights of Fasting-Refeeding Response in Zebrafish Skeletal Muscle.
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Sugasawa, Takehito, Komine, Ritsuko, Manevich, Lev, Tamai, Shinsuke, Takekoshi, Kazuhiro, and Kanki, Yasuharu
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Recently, fasting has been spotlighted from a healthcare perspective. However, the de-tailed biological mechanisms and significance by which the effects of fasting confer health benefits are not yet clear. Due to certain advantages of the zebrafish as a vertebrate model, it is widely utilized in biological studies. However, the biological responses to nutrient metabolism within zebrafish skeletal muscles have not yet been amply reported. Therefore, we aimed to reveal a gene expression profile in zebrafish skeletal muscles in response to fasting-refeeding. Accordingly, mRNA-sequencing and bioinformatics analysis were performed to examine comprehensive gene expression changes in skeletal muscle tissues during fasting-refeeding. Our results produced a novel set of nutrition-related genes under a fasting-refeeding protocol. Moreover, we found that five genes were dramatically upregulated in each fasting (for 24 h) and refeeding (after 3 h), exhibiting a rapid response to the provided conditional changes. The assessment of the gene length revealed that the gene set whose expression was elevated only after 3 h of refeeding had a shorter length, suggesting that nutrition-related gene function is associated with gene length. Taken together, our results from the bioinformatics analyses provide new insights into biological mechanisms induced by fasting-refeeding conditions within zebrafish skeletal muscle. [ABSTRACT FROM AUTHOR]
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- 2022
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16. GATA factor switching from GATA2 to GATA1 contributes to erythroid differentiation
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Suzuki, Mikiko, Kobayashi-Osaki, Maki, Tsutsumi, Shuichi, Pan, Xiaoqing, Ohmori, Shinʼya, Takai, Jun, Moriguchi, Takashi, Ohneda, Osamu, Ohneda, Kinuko, Shimizu, Ritsuko, Kanki, Yasuharu, Kodama, Tatsuhiko, Aburatani, Hiroyuki, and Yamamoto, Masayuki
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- 2013
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17. ChIP-seq reveals cell type-specific binding patterns of BMP-specific Smads and a novel binding motif
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Morikawa, Masato, Koinuma, Daizo, Tsutsumi, Shuichi, Vasilaki, Eleftheria, Kanki, Yasuharu, Heldin, Carl-Henrik, Aburatani, Hiroyuki, and Miyazono, Kohei
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- 2011
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18. TET1 upregulation drives cancer cell growth through aberrant enhancer hydroxymethylation of HMGA2 in hepatocellular carcinoma.
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Shirai, Kiyokazu, Nagae, Genta, Seki, Motoaki, Kudo, Yotaro, Kamio, Asuka, Hayashi, Akimasa, Okabe, Atsushi, Ota, Satoshi, Tsutsumi, Shuichi, Fujita, Takanori, Yamamoto, Shogo, Nakaki, Ryo, Kanki, Yasuharu, Osawa, Tsuyoshi, Midorikawa, Yutaka, Tateishi, Keisuke, Ichinose, Masao, and Aburatani, Hiroyuki
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Ten‐eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1‐upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast‐like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5‐hmC) profiling and found that 5‐hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4–monomethylated, where the H3K27‐acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast‐like HCC. [ABSTRACT FROM AUTHOR]
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- 2021
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19. PERK inhibition attenuates vascular remodeling in pulmonary arterial hypertension caused by BMPR2 mutation.
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Shimizu, Takashi, Higashijima, Yoshiki, Kanki, Yasuharu, Nakaki, Ryo, Kawamura, Takeshi, Urade, Yoshihiro, and Wada, Youichiro
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BONE morphogenetic protein receptors ,VASCULAR remodeling ,GLYCOLYSIS ,PULMONARY hypertension ,UNFOLDED protein response ,VENTRICULAR remodeling ,HYPERTENSION ,PULMONARY artery - Abstract
Not a PERK for pulmonary arterial hypertension: The vascular remodeling that occurs in pulmonary arterial hypertension (PAH) is eventually fatal because it leads to right ventricular heart failure. PAH induces the unfolded protein response (UPR), and Shimizu et al. focused on the role of the UPR kinase PERK in PAH pathology. Genetic ablation or pharmacological inhibition of PERK limited vascular remodeling in two different mouse models of PAH and prevented mice from developing right ventricular heart failure. These effects involved suppressing the activation of PDGFRβ-STAT1 signaling and glycolysis in pulmonary artery smooth muscle cells. Thus, targeting PERK may help limit the mortality associated with PAH. Pulmonary arterial hypertension (PAH) is a fatal disease characterized by excessive pulmonary vascular remodeling. However, despite advances in therapeutic strategies, patients with PAH bearing mutations in the bone morphogenetic protein receptor type 2 (BMPR2)–encoding gene present severe phenotypes and outcomes. We sought to investigate the effect of PER-like kinase (PERK), which participates in one of three major pathways associated with the unfolded protein response (UPR), on PAH pathophysiology in BMPR2 heterozygous mice. BMPR2 heterozygosity in pulmonary artery smooth muscle cells (PASMCs) decreased the abundance of the antiapoptotic microRNA miR124-3p through the arm of the UPR mediated by PERK. Hypoxia promoted the accumulation of unfolded proteins in BMPR2 heterozygous PASMCs, resulting in increased PERK signaling, cell viability, cellular proliferation, and glycolysis. Proteomic analyses revealed that PERK ablation suppressed PDGFRβ-STAT1 signaling and glycolysis in hypoxic BMPR2 heterozygous PASMCs. Furthermore, PERK ablation or PERK inhibition ameliorated pulmonary vascular remodeling in the Sugen/chronic hypoxia model of PAH, irrespective of BMPR2 status. Hence, these findings suggest that PERK inhibition is a promising therapeutic strategy for patients with PAH with or without BMPR2 mutation. [ABSTRACT FROM AUTHOR]
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- 2021
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20. Molecular mechanistic insights: The emerging role of SOXF transcription factors in tumorigenesis and development.
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Higashijima, Yoshiki and Kanki, Yasuharu
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TRANSCRIPTION factors , *CELLULAR control mechanisms , *SOMATIC cells , *CELL determination , *CARDIOVASCULAR development - Abstract
Over the last decade, the development and progress of next-generation sequencers incorporated with classical biochemical analyses have drastically produced novel insights into transcription factors, including Sry-like high-mobility group box (SOX) factors. In addition to their primary functions in binding to and activating specific downstream genes, transcription factors also participate in the dedifferentiation or direct reprogramming of somatic cells to undifferentiated cells or specific lineage cells. Since the discovery of SOX factors, members of the SOXF (SOX7, SOX17, and SOX18) family have been identified to play broad roles, especially with regard to cardiovascular development. More recently, SOXF factors have been recognized as crucial players in determining the cell fate and in the regulation of cancer cells. Here, we provide an overview of research on the mechanism by which SOXF factors regulate development and cancer, and discuss their potential as new targets for cancer drugs while offering insight into novel mechanistic transcriptional regulation during cell lineage commitment. [ABSTRACT FROM AUTHOR]
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- 2020
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21. Protein kinase A accelerates the rate of early stage differentiation of pluripotent stem cells.
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Minakawa, Tomohiro, Kanki, Yasuharu, Nakamura, Kae, and Yamashita, Jun K.
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CYCLIC-AMP-dependent protein kinase , *EMBRYONIC stem cells , *EPIBLAST , *PLURIPOTENT stem cells , *GERM cells , *MESODERM , *CELL differentiation - Abstract
In normal development, the rate of cell differentiation is tightly controlled and critical for normal development and stem cell differentiation. However, the underlying mechanisms regulating the rate of the differentiation are unknown, and manipulation of the rate of the stem cell differentiation is currently difficult. Here we show that activation of protein kinase A (PKA) accelerates the rate of mouse embryonic stem cell (ESC) differentiation through an early loss of ESC pluripotency markers and early appearance of mesodermal and other germ layer cells. The activation of PKA hastened differentiation by increasing the expression of a histone H3 lysine 9 (H3K9) dimethyltransferase, G9a protein, and the level of a negative epigenetic histone mark, H3K9 dimethylation (H3K9me2), in the promoter regions of the pluripotency markers Nanog and Oct4. These results elucidate a novel role of PKA on ESC differentiation and offer an experimental model for controlling the rate of ESC differentiation. • PKA activation in ESC differentiation accelerated the appearance of the three germ layers, especially mesoderm cells. • PKA activation reciprocally induced the earlier disappearance of pluripotency genes (Oct4, Nanog, Sox2). • PKA activation induced the expression of the H3K9 dimethyltransferase G9a. • PKA activation increased the level of H3K9 dimethylation on the promoter region of the pluripotency genes Nanog and Oct4. [ABSTRACT FROM AUTHOR]
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- 2020
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22. Downregulation of ERG and FLI1 expression in endothelial cells triggers endothelial-to-mesenchymal transition.
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Nagai, Nao, Ohguchi, Hiroto, Nakaki, Ryo, Matsumura, Yoshihiro, Kanki, Yasuharu, Sakai, Juro, Aburatani, Hiroyuki, and Minami, Takashi
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TUMOR microenvironment ,CANCER invasiveness ,DOWNREGULATION ,ENDOTHELIAL cells ,MESENCHYMAL stem cells - Abstract
Endothelial cell (EC) plasticity in pathological settings has recently been recognized as a driver of disease progression. Endothelial-to-mesenchymal transition (EndMT), in which ECs acquire mesenchymal properties, has been described for a wide range of pathologies, including cancer. However, the mechanism regulating EndMT in the tumor microenvironment and the contribution of EndMT in tumor progression are not fully understood. Here, we found that combined knockdown of two ETS family transcription factors, ERG and FLI1, induces EndMT coupled with dynamic epigenetic changes in ECs. Genome-wide analyses revealed that ERG and FLI1 are critical transcriptional activators for EC-specific genes, among which microRNA-126 partially contributes to blocking the induction of EndMT. Moreover, we demonstrated that ERG and FLI1 expression is downregulated in ECs within tumors by soluble factors enriched in the tumor microenvironment. These data provide new insight into the mechanism of EndMT, functions of ERG and FLI1 in ECs, and EC behavior in pathological conditions. [ABSTRACT FROM AUTHOR]
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- 2018
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23. TNFα signals through specialized factories where responsive coding and miRNA genes are transcribed: Specialized transcription factories
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Kodama, Tatsuhiko, Kohro, Takahide, Short, Patrick, Deng, Binwei, Li, Guoliang, Kanki, Yasuharu, Larkin, Joshua D., Aburatani, Hiroyuki, Poh, Huay Mei, Ruan, Xiaoan, Kobayashi, Mika, Papantonis, Argyris, Tsutsumi, Shuichi, Cook, Peter R., Taylor, Stephen, Ruan, Yijun, Baboo, Sabyasachi, and Wada, Youichiro
- Abstract
Tumour necrosis factor alpha (TNFα) is a potent cytokine that signals through nuclear factor kappa B (NFκB) to activate a subset of human genes. It is usually assumed that this involves RNA polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired-end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNFα induces responsive genes to congregate in discrete ‘NFκB factories'. Some factories further specialize in transcribing responsive genes encoding micro-RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories.
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- 2012
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24. Novel lnc RNA regulated by HIF-1 inhibits apoptotic cell death in the renal tubular epithelial cells under hypoxia.
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Mimura, Imari, Hirakawa, Yosuke, Kanki, Yasuharu, Kushida, Natsuki, Nakaki, Ryo, Suzuki, Yutaka, Tanaka, Tetsuhiro, Aburatani, Hiroyuki, and Nangaku, Masaomi
- Subjects
APOPTOSIS ,EPITHELIAL cells ,HYPOXIA-inducible factor 1 ,NON-coding RNA ,TRANSCRIPTION factors - Abstract
Chronic tubulointerstitial hypoxia plays an important role as the final common pathway to end-stage renal disease. HIF-1 (hypoxia-inducible factor-1) is a master transcriptional factor under hypoxia, regulating downstream target genes. Genome-wide analysis of HIF-1 binding sites using high-throughput sequencers has clarified various kinds of downstream targets and made it possible to demonstrate the novel roles of HIF-1. Our aim of this study is to identify novel HIF-1 downstream epigenetic targets which may play important roles in the kidney. Immortalized tubular cell lines (HK2; human kidney-2) and primary cultured cells (RPTEC; renal proximal tubular cell lines) were exposed to 1% hypoxia for 24-72 h. We performed RNA-seq to clarify the expression of mRNA and long non-coding RNA (lncRNA). We also examined ChIP-seq to identify HIF-1 binding sites under hypoxia. RNA-seq identified 44 lncRNAs which are up-regulated under hypoxic condition in both cells. ChIP-seq analysis demonstrated that HIF-1 also binds to the lncRNAs under hypoxia. The expression of novel lncRNA, DARS-AS1 (aspartyl-tRNA synthetase anti-sense 1), is up-regulated only under hypoxia and HIF-1 binds to its promoter region, which includes two hypoxia-responsive elements. Its expression is also up-regulated with cobalt chloride exposure, while it is not under hypoxia when HIF-1 is knocked down by siRNA. To clarify the biological roles of DARS-AS1, we measured the activity of caspase 3/7 using antisense oligo of DARS-AS1. Knockdown of DARS-AS1 deteriorated apoptotic cell death. In conclusion, we identified the novel lncRNAs regulated by HIF-1 under hypoxia and clarified that DARS-AS1 plays an important role in inhibiting apoptotic cell death in renal tubular cells. [ABSTRACT FROM AUTHOR]
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- 2017
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25. Direct Evidence for Pitavastatin Induced Chromatin Structure Change in the KLF4 Gene in Endothelial Cells.
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Maejima, Takashi, Inoue, Tsuyoshi, Kanki, Yasuharu, Kohro, Takahide, Li, Guoliang, Ohta, Yoshihiro, Kimura, Hiroshi, Kobayashi, Mika, Taguchi, Akashi, Tsutsumi, Shuichi, Iwanari, Hiroko, Yamamoto, Shogo, Aruga, Hirofumi, Dong, Shoulian, Stevens, Junko F., Poh, Huay Mei, Yamamoto, Kazuki, Kawamura, Takeshi, Mimura, Imari, and Suehiro, Jun-ichi
- Subjects
MOLECULAR structure of chromatin ,QUINOLINE ,ENDOTHELIAL cells ,STATINS (Cardiovascular agents) ,TRANSCRIPTION factors ,GENETIC regulation - Abstract
Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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26. Revolution of nephrology research by deep sequencing: ChIP-seq and RNA-seq.
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Mimura, Imari, Kanki, Yasuharu, Kodama, Tatsuhiko, and Nangaku, Masaomi
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NEPHROLOGY , *NUCLEOTIDE sequence , *IMMUNOPRECIPITATION , *HISTONE genetics , *GENE expression , *KIDNEY diseases , *GENETICS - Abstract
The recent and rapid advent of next-generation sequencing (NGS) has made this technology broadly available not only to researchers in various molecular and cellular biology fields but also to those in kidney disease. In this paper, we describe the usage of ChIP-seq (chromatin immunoprecipitation with sequencing) and RNA-seq for sample preparation and interpretation of raw data in the investigation of biological phenomenon in renal diseases. ChIP-seq identifies genome-wide transcriptional DNA-binding sites as well as histone modifications, which are known to regulate gene expression, in the intragenic as well as in the intergenic regions. With regard to RNA-seq, this process analyzes not only the expression level of mRNA but also splicing variants, non-coding RNA, and microRNA on a genome-wide scale. The combination of ChIP-seq and RNA-seq allows the clarification of novel transcriptional mechanisms, which have important roles in various kinds of diseases, including chronic kidney disease. The rapid development of these techniques requires an update on the latest information and methods of NGS. In this review, we highlight the merits and characteristics of ChIP-seq and RNA-seq and discuss the use of the genome-wide analysis in kidney disease. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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27. Bivalent-histone-marked immediate-early gene regulation is vital for VEGF-responsive angiogenesis.
- Author
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Kanki, Yasuharu, Muramatsu, Masashi, Miyamura, Yuri, Kikuchi, Kenta, Higashijima, Yoshiki, Nakaki, Ryo, Suehiro, Jun-ichi, Sasaki, Yuji, Kubota, Yoshiaki, Koseki, Haruhiko, Morioka, Hiroshi, Kodama, Tatsuhiko, Nakao, Mitsuyoshi, Kurotaki, Daisuke, Aburatani, Hiroyuki, and Minami, Takashi
- Abstract
Endothelial cells (ECs) are phenotypically heterogeneous, mainly due to their dynamic response to the tissue microenvironment. Vascular endothelial cell growth factor (VEGF), the best-known angiogenic factor, activates calcium-nuclear factor of activated T cells (NFAT) signaling following acute angiogenic gene transcription. Here, we evaluate the global mapping of VEGF-mediated dynamic transcriptional events, focusing on major histone-code profiles using chromatin immunoprecipitation sequencing (ChIP-seq). Remarkably, the gene loci of immediate-early angiogenic transcription factors (TFs) exclusively acquire bivalent H3K4me3-H3K27me3 double-positive histone marks after the VEGF stimulus. Moreover, NFAT-associated Pax transactivation domain-interacting protein (PTIP) directs bivalently marked TF genes transcription through a limited polymerase II running. The non-canonical polycomb1 variant PRC1.3 specifically binds to and allows the transactivation of PRC2-enriched bivalent angiogenic TFs until conventional PRC1-mediated gene silencing is achieved. Knockdown of these genes abrogates post-natal aberrant neovessel formation via the selective inhibition of indispensable bivalent angiogenic TF gene transcription. Collectively, the reported dynamic histone mark landscape may uncover the importance of immediate-early genes and the development of advanced anti-angiogenic strategies. [Display omitted] • Specific bivalent gene body signature regulates VEGF-responsive angiogenic acute TFs • Non-canonical PRC1.3 transiently transactivates PRC2-enriched angiogenic genes • VEGF-NFAT-associated epigenome modifier PTIP directs NFAT-targeted gene activation • Reductions in PRC1.3 or PTIP lead to impaired postnatal angiogenesis Kanki et al. demonstrate that angiogenesis-essential immediate-early genes are silent with canonical PRC-brake in inert endothelium. In a short time, VEGF unlocks the transcription of these genes through bivalent H3K4me3-H3K27me3 histone marks on the gene loci with PTIP/NFAT, MLL3/4, and non-canonical PRC1.3 accumulation. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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28. TNF? signals through specialized factories where responsive coding and miRNA genes are transcribed.
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Papantonis, Argyris, Kohro, Takahide, Baboo, Sabyasachi, Larkin, Joshua D, Deng, Binwei, Short, Patrick, Tsutsumi, Shuichi, Taylor, Stephen, Kanki, Yasuharu, Kobayashi, Mika, Li, Guoliang, Poh, Huay-Mei, Ruan, Xiaoan, Aburatani, Hiroyuki, Ruan, Yijun, Kodama, Tatsuhiko, Wada, Youichiro, and Cook, Peter R
- Subjects
TUMOR necrosis factors ,GENETIC code ,NON-coding RNA ,NF-kappa B ,ENDOTHELIAL cells ,RNA polymerases ,CELLULAR signal transduction - Abstract
Tumour necrosis factor alpha (TNF?) is a potent cytokine that signals through nuclear factor kappa B (NF?B) to activate a subset of human genes. It is usually assumed that this involves RNA polymerases transcribing responsive genes wherever they might be in the nucleus. Using primary human endothelial cells, variants of chromosome conformation capture (including 4C and chromatin interaction analysis with paired-end tag sequencing), and fluorescence in situ hybridization to detect single nascent transcripts, we show that TNF? induces responsive genes to congregate in discrete 'NF?B factories'. Some factories further specialize in transcribing responsive genes encoding micro-RNAs that target downregulated mRNAs. We expect all signalling pathways to contain this extra leg, where responding genes are transcribed in analogous specialized factories. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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29. Dynamic Change of Chromatin Conformation in Response to Hypoxia Enhances the Expression of GLUT3 (SLC2A3) by Cooperative Interaction of Hypoxia-Inducible Factor 1 and KDM3A.
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Mimura, Imari, Nangaku, Masaomi, Kanki, Yasuharu, Tsutsumi, Shuichi, Inoue, Tsuyoshi, Kohro, Takahide, Yamamoto, Shogo, Fujita, Takanori, Shimamura, Teppei, Suehiro, Jun-ichi, Taguchi, Akashi, Kobayashi, Mika, Tanimura, Kyoko, Inagaki, Takeshi, Tanaka, Toshiya, Hamakubo, Takao, Sakai, Juro, Aburatani, Hiroyuki, Kodama, Tatsuhiko, and Wada, Youichiro
- Subjects
CHROMATIN ,HYPOXEMIA ,GENE expression ,ENDOTHELIAL cells ,GLYCOLYSIS ,DNA microarrays ,EPIGENESIS - Abstract
Hypoxia-inducible factor 1 (HIF1) is a master regulator of adaptive gene expression under hypoxia. However, a role for HIF1 in the epigenetic regulation remains unknown. Genome-wide analysis of HIF1 binding sites (chromatin immunoprecipitation [ChIP] with deep sequencing) of endothelial cells clarified that HIF1 mainly binds to the intergenic regions distal from transcriptional starting sites under both normoxia and hypoxia. Next, we examined the temporal profile of gene expression under hypoxic conditions by using DNA microarrays. We clarified that early hypoxia-responsive genes are functionally associated with glycolysis, including GLUT3 (SLC2A3). Acetylated lysine 27 of histone 3 covered the HIF1 binding sites, and HIF1 functioned as an enhancer of SLC2A3 by interaction with lysine (K)-specific demethylase 3A (KDM3A). Knockdown of HIF1α and KDM3A showed that glycolytic genes are regulated by both HIF1 and KDM3A and respond to hypoxia in a manner independent of cell type specificity. We elucidated that both the chromatin conformational structure and histone modification change under hypoxic conditions and enhance the expression of SLC2A3 based on the combined results of chromatin conformation capture (3C) and ChIP assays. KDM3A is recruited to the SLC2A3 locus in an HIF1dependent manner and demethylates H3K9me2 so as to upregulate its expression. These findings provide novel insights into the interaction between HIF1 and KDM3A and also the epigenetic regulation of HIF1. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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30. Epigenetically coordinated GATA2 binding is necessary for endothelium-specific endomucin expression.
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Kanki, Yasuharu, Kohro, Takahide, Jiang, Shuying, Tsutsumi, Shuichi, Mimura, Imari, Suehiro, Jun-ichi, Wada, Youichiro, Ohta, Yoshihiro, Ihara, Sigeo, Iwanari, Hiroko, Naito, Makoto, Hamakubo, Takao, Aburatani, Hiroyuki, Kodama, Tatsuhiko, and Minami, Takashi
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CARRIER proteins , *ENDOTHELIUM , *GLYCOPROTEINS , *GENE expression , *CELL differentiation , *CHROMATIN , *NUCLEOTIDE sequence - Abstract
GATA2 is well recognized as a key transcription factor and regulator of cell-type specificity and differentiation. Here, we carried out comparative chromatin immunoprecipitation with comprehensive sequencing (ChIP-seq) to determine genome-wide occupancy of GATA2 in endothelial cells and erythroids, and compared the occupancy to the respective gene expression profile in each cell type. Although GATA2 was commonly expressed in both cell types, different GATA2 bindings and distinct cell-specific gene expressions were observed. By using the ChIP-seq with epigenetic histone modifications and chromatin conformation capture assays; we elucidated the mechanistic regulation of endothelial-specific GATA2-mediated endomucin gene expression, that was regulated by the endothelial-specific chromatin loop with a GATA2-associated distal enhancer and core promoter. Knockdown of endomucin markedly attenuated endothelial cell growth, migration and tube formation. Moreover, abrogation of GATA2 in endothelium demonstrated not only a reduction of endothelial-specific markers, but also induction of mesenchymal transition promoting gene expression. Our findings provide new insights into the correlation of endothelial-expressed GATA2 binding, epigenetic modification, and the determination of endothelial cell specificity. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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31. Proof of Gene Doping in a Mouse Model with a Human Erythropoietin Gene Transferred Using an Adenoviral Vector.
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Sugasawa, Takehito, Nakano, Takuro, Fujita, Shin-ichiro, Matsumoto, Yuki, Ishihara, Genki, Aoki, Kai, Yanazawa, Koki, Ono, Seiko, Tamai, Shinsuke, Manevich, Lev, Ueda, Haruna, Ishibashi, Noriyo, Tamai, Kenshirou, Kanki, Yasuharu, Yoshida, Yasuko, Watanabe, Koichi, Takemasa, Tohru, Kawakami, Yasushi, and Takekoshi, Kazuhiro
- Subjects
LABORATORY mice ,GENETIC transformation ,HUMAN genes ,BLOOD cell count ,ANIMAL disease models - Abstract
Despite the World Anti-Doping Agency (WADA) ban on gene doping in the context of advancements in gene therapy, the risk of EPO gene-based doping among athletes is still present. To address this and similar risks, gene-doping tests are being developed in doping control laboratories worldwide. In this regard, the present study was performed with two objectives: to develop a robust gene-doping mouse model with the human EPO gene (hEPO) transferred using recombinant adenovirus (rAdV) as a vector and to develop a detection method to identify gene doping by using this model. The rAdV including the hEPO gene was injected intravenously to transfer the gene to the liver. After injection, the mice showed significantly increased whole-blood red blood cell counts and increased expression of hematopoietic marker genes in the spleen, indicating successful development of the gene-doping model. Next, direct and potentially indirect proof of gene doping were evaluated in whole-blood DNA and RNA by using a quantitative PCR assay and RNA sequencing. Proof of doping could be detected in DNA and RNA samples from one drop of whole blood for approximately a month; furthermore, the overall RNA expression profiles showed significant changes, allowing advanced detection of hEPO gene doping. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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32. Phosphoethanolamine Accumulation Protects Cancer Cells under Glutamine Starvation through Downregulation of PCYT2.
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Osawa, Tsuyoshi, Shimamura, Teppei, Saito, Kyoko, Hasegawa, Yoko, Ishii, Naoko, Nishida, Miyuki, Ando, Ritsuko, Kondo, Ayano, Anwar, Muyassar, Tsuchida, Rika, Hino, Shinjiro, Sakamoto, Akihisa, Igarashi, Kaori, Saitoh, Kaori, Kato, Keiko, Endo, Keiko, Yamano, Shotaro, Kanki, Yasuharu, Matsumura, Yoshihiro, and Minami, Takashi
- Abstract
Tolerance to severe tumor microenvironments, including hypoxia and nutrient starvation , is a common feature of aggressive cancer cells and can be targeted. However, metabolic alterations that support cancer cells upon nutrient starvation are not well understood. Here, by comprehensive metabolome analyses, we show that glutamine deprivation leads to phosphoethanolamine (PEtn) accumulation in cancer cells via the downregulation of PEtn cytidylyltransferase (PCYT2), a rate-limiting enzyme of phosphatidylethanolamine biosynthesis. PEtn accumulation correlated with tumor growth under nutrient starvation. PCYT2 suppression was partially mediated by downregulation of the transcription factor ELF3. Furthermore, PCYT2 overexpression reduced PEtn levels and tumor growth. In addition, PEtn accumulation and PCYT2 downregulation in human breast tumors correlated with poor prognosis. Thus, we show that glutamine deprivation leads to tumor progression by regulating PE biosynthesis via the ELF3-PCYT2 axis. Furthermore, manipulating glutamine-responsive genes could be a therapeutic approach to limit cancer progression. • Glutamine-deprived cancer cells are rich in phosphoethanolamine (PEtn) • PEtn stimulates tolerance to nutrient starvation and tumor growth • PCYT2 inhibition triggers PEtn accumulation during glutamine deprivation • PCYT2 downregulation is associated with poor prognosis for breast cancer patients Osawa et al. find that accumulation of phosphoethanolamine (PEtn) protects cancer cells under glutamine starvation through the downregulation of PCYT2. Glutamine regulates PE biosynthesis through PCYT2, resulting in pro-tumorigenic metabolite PEtn accumulation. PEtn stimulates the tolerance of cancer cells to starvation, and lowered PCYT2 expression correlates with decreased survival in patients. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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33. Effects of Chronic Low-Dose Radiation on Human Neural Progenitor Cells.
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Katsura, Mari, Cyou-Nakamine, Hiromasa, Zen, Qin, Zen, Yang, Nansai, Hiroko, Amagasa, Shota, Kanki, Yasuharu, Inoue, Tsuyoshi, Kaneki, Kiyomi, Taguchi, Akashi, Kobayashi, Mika, Kaji, Toshiyuki, Kodama, Tatsuhiko, Miyagawa, Kiyoshi, Wada, Youichiro, Akimitsu, Nobuyoshi, and Sone, Hideko
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- 2016
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34. Genome-Wide Approaches Reveal Functional Interleukin-4-Inducible STAT6 Binding to the Vascular Cell Adhesion Molecule 1 Promoter.
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Tozawa, Hideto, Kanki, Yasuharu, Suehiro, Jun-ichi, Tsutsumi, Shuichi, Kohro, Takahide, Wada, Youichiro, Aburatani, Hiroyuki, Aird, William C., Kodama, Tatsuhiko, and Minami, Takashi
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INTERLEUKIN-4 , *CELL adhesion molecules , *VASCULAR endothelium , *TUMOR necrosis factors , *TRANSCRIPTION factors , *THERAPEUTICS - Abstract
Endothelial cell activation and dysfunction underlie many vascular disorders, including atherosclerosis and inflammation. Here, we show that interleukin-4 (IL-4) markedly induced vascular cell adhesion molecule 1 (VCAM-1), both in cultured endothelial cells and in the intact endothelium in mice. Combined treatment with IL-4 and tumor necrosis factor alpha (TNF-α) resulted in further, sustained induction of VCAM-1 expression. IL-4-mediated induction of VCAM-1 and secondary monocyte adhesion was predominantly regulated by the transcription factor STAT6. Genome-wide survey of IL-4-mediated STAT6 binding from sequential chromatin-immunoprecipitation with deep sequencing (chromatin immunoprecipitation sequencing [ChIP-seq]) in endothelial cells revealed regions of transient and sustained transcription factor binding. Through the combination of DNA microarrays and ChIP-seq at the same time points, the majority of IL-4-responsive genes were shown to be STAT6 dependent and associated with direct STAT6 binding to their promoter. IL-4-mediated stable binding of STAT6 led to sustained target gene expression. Moreover, our strategy led to the identification of a novel functionally important STAT6 binding site within 16 kb upstream of the VCAM-1 gene. Taken together, these findings support a critical role for STAT6 in mediating IL-4 signal transduction in endothelial cells. Identification of a novel IL-4-mediated VCAM-1 enhancer may provide a foundation for targeted therapy in vascular disease. [ABSTRACT FROM AUTHOR]
- Published
- 2011
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35. Acute cold stress induces transient MuRF1 upregulation in the skeletal muscle of zebrafish.
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Tamai, Shinsuke, Fujita, Shin-ichiro, Komine, Ritsuko, Kanki, Yasuharu, Aoki, Kai, Watanabe, Koichi, Takekoshi, Kazuhiro, and Sugasawa, Takehito
- Subjects
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SKELETAL muscle , *UBIQUITIN ligases , *BRACHYDANIO , *UBIQUITINATION , *SPORTS medicine , *MUSCULAR atrophy - Abstract
Cryotherapy is one of the most common treatments for trauma or fatigue in the field of sports medicine. However, the molecular biological effects of acute cold exposure on skeletal muscle remain unclear. Therefore, we used zebrafish, which have recently been utilized as an animal model for skeletal muscle, to comprehensively investigate and selectively clarify the time-course changes induced by cryotherapy. Zebrafish were exposed intermittently to cold stimulation three times for 15 min each. Thereafter, skeletal muscle samples were collected after 15 min and 1, 2, 4, and 6 h. mRNA sequencing revealed the involvement of trim63a , fbxo32 , fbxo30a , and klhl38b in "protein ubiquitination" from the top 10 most upregulated genes. Subsequently, we examined the time-course changes of the four genes by quantitative PCR, and their expression peaked 2 h after cryotherapy and returned to baseline after 6 h. Moreover, the proteins encoded by trim63a and fbxo32 (muscle-specific RING finger protein 1 [MuRF1] and muscle atrophy F-box, respectively), which are known to be major genes encoding E3 ubiquitin ligases, were examined by western blotting, and MuRF1 expression displayed similar temporal changes as trim63a expression. These findings suggest that acute cold exposure transiently upregulates E3 ubiquitin ligases, especially MuRF1; thus, cryotherapy may contribute to the treatment of trauma or fatigue by promoting protein processing. • Acute cold stress affected the expression of multiple genes, mainly those related to E3 ubiquitin ligases. • The increase in gene expression was transiently induced, peaking at 2 h after cold exposure and returning to baseline by 6 h. • In particular, the protein expression of MuRF1 (encoded by trim63a) was significantly increased. • Cryotherapy may contribute to recovery from trauma or fatigue by promoting the processing of damaged or unfolded proteins. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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36. Lysine demethylase 2B regulates angiogenesis via Jumonji C dependent suppression of angiogenic transcription factors.
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Sasaki, Yuji, Higashijima, Yoshiki, Suehiro, Jun-Ichi, Sugasawa, Takehito, Oguri-Nakamura, Eri, Fukuhara, Shigetomo, Nagai, Nao, Hirakawa, Yosuke, Wada, Youichiro, Nangaku, Masaomi, and Kanki, Yasuharu
- Subjects
- *
VASCULAR endothelial growth factors , *TRANSCRIPTION factors , *DEMETHYLASE , *LYSINE , *ENDOTHELIAL cells - Abstract
Vascular endothelial growth factor (VEGF) signaling plays a central role in vascular development and maintenance of vascular homeostasis. In endothelial cells (ECs), VEGF activates the gene expression of angiogenic transcription factors (TFs), followed by induction of downstream angiogenic responsive genes. Recent findings support that histone modification dynamics contribute to the transcriptional control of genes that are important for EC functions. Lysine demethylase 2B (KDM2B) demethylates histone H3K4me3 and H3K36me2/3 and mediates the monoubiquitination of histone H2AK119. KDM2B functions as a transcriptional repressor in somatic cell reprogramming and tumor development. However, the role of KDM2B in VEGF signaling remains to be elucidated. Here, we show that KDM2B knockdown enhances VEGF-induced angiogenesis in cultured human ECs via increased migration and proliferation. In contrast, ectopic expression of KDM2B inhibits angiogenesis. The function of KDM2B may depend on its catalytic Jumonji C domain. Genome-wide analysis further reveals that KDM2B selectively controls the transcription of VEGF-induced angiogenic TFs that are associated with increased H3K4me3/H3K36me3 and decreased H2AK119ub. These findings suggest an essential role of KDM2B in VEGF signaling in ECs. As dysregulation of VEGF signaling in ECs is involved in various diseases, including cancer, KDM2B may be a potential therapeutic target in VEGF-mediated vasculopathic diseases. ∙ KDM2B has inhibitory effects on VEGF-induced angiogenesis in endothelial cells. ∙The anti-angiogenic functions of KDM2B is mediated via JmjC domain. ∙KDM2B selectively controls transcription of VEGF-responsive angiogenic TFs. ∙Histone modifications linked to KDM2B are dynamically changed during VEGF-signaling. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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37. Critical Role for GATA3 in Mediating Tie2 Expression and Function in Large Vessel Endothelial Cells.
- Author
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Haihua Song, Jun-ichi Suehiro, Kanki, Yasuharu, Kawai, Yoshiko, lnoue, Kenji, Daida, Hiroyuki, Yano, Kiichiro, Ohhashi, Toshio, Oettgen, Peter, Aird, William C., Kodama, Tatsuhiko, and Minami, Takashi
- Subjects
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PHENOTYPES , *TRANSCRIPTION factors , *CELLULAR signal transduction , *DNA microarrays , *GROWTH factors , *TUMOR necrosis factors , *CELL adhesion - Abstract
Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms. There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels. In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited angiopoietin (Ang)1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration, survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown to bind to regulatory regions within the 5'-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF 1, physically interacted and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang1 to attenuate vascular endothelial cell growth factor stimulation of vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial cells to tumor necrosis factor-α resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression. Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial cells. [ABSTRACT FROM AUTHOR]
- Published
- 2009
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38. Endothelial specific LAT1 ablation normalizes tumor vasculature.
- Author
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Suehiro JI, Kimura T, Fukutomi T, Naito H, Kanki Y, Wada Y, Kubota Y, Takakura N, and Sakurai H
- Abstract
Some endothelial cells in the tumor vasculature expressed a system L amino acid transporter LAT1. To elucidate the role of LAT1 in tumor related endothelial cells, tumor cells were injected into endothelial specific LAT1 conditional knockout mice (Slc7a5flox/flox; Cdh5-Cre-ERT2) and found that the shape of the tumor vasculature was normalized and that the size and numbers of lung metastasis was reduced. TNFα-induced expression of VCAM1 and E-selectin at the surface of HUVEC, both of which are responsible for enhanced monocyte attachment and pre-metastatic niche formation, was reduced in the presence of LAT1 inhibitor, nanvuranlat. Deprivation of tryptophan, an LAT1 substrate, mimicked LAT1 inhibition, which led to activation of MEK1/2-ERK1/2 pathway and subsequent cystathionine γ lyase (CTH) induction. Increased production of hydrogen sulfide (H2S) by CTH was at least partially responsible for tumor vascular normalization, leading to decreased leakiness and enhanced delivery of chemotherapeutic agents to the tumor.
- Published
- 2024
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39. Establishing a Sequencing Method for the Whole Mitochondrial DNA of Domestic Dogs.
- Author
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Sugasawa T, Matsumoto Y, Fang H, Takemasa T, Komine R, Tamai S, Gu W, Tanaka K, Kanki Y, and Takahashi Y
- Abstract
In human beings, whole mitochondrial DNA (mtDNA) sequencing has been widely used in many research fields, including medicine, forensics, and genetics. With respect to the domestic dog ( Canis lupus familiaris ), which is commonly recognized as being an additional member of the traditional human family structure, research studies on mtDNA should be developed to expand and improve our collective knowledge of dog medicine and welfare as it seems that there is still room for further development in these areas. Moreover, a simple and robust method for sequencing whole mtDNA that can be applied to various dog breeds has not yet been described in the literature. In the present study, we aim to establish such a method for the whole mtDNA sequencing of the domestic dog. In the experiments we conducted, oral mucosa DNA samples obtained from six Japanese domestic dogs were used as a template. We designed four primer pairs that could amplify approximately 5 kbp from each region of the mtDNA and validated several PCR conditions. Subsequently, the PCR amplicons were pooled and subjected to library preparation. The sequencing of the libraries was performed using next-generation sequencing (NGS), followed by bioinformatics analysis. Our results demonstrate that the proposed method can be used to perform highly accurate resequencing. We believe that this method may be useful for future research conducted to better understand dog medicine and welfare.
- Published
- 2023
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40. Coordinated demethylation of H3K9 and H3K27 is required for rapid inflammatory responses of endothelial cells.
- Author
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Higashijima Y, Matsui Y, Shimamura T, Nakaki R, Nagai N, Tsutsumi S, Abe Y, Link VM, Osaka M, Yoshida M, Watanabe R, Tanaka T, Taguchi A, Miura M, Ruan X, Li G, Inoue T, Nangaku M, Kimura H, Furukawa T, Aburatani H, Wada Y, Ruan Y, Glass CK, and Kanki Y
- Subjects
- Animals, Cell Adhesion, Endothelial Cells cytology, Endothelial Cells metabolism, Gene Expression Regulation, Gene Regulatory Networks, Histones chemistry, Human Umbilical Vein Endothelial Cells, Humans, Lysine metabolism, Male, Methylation, Mice, Signal Transduction, Tumor Necrosis Factor-alpha metabolism, Endothelial Cells immunology, Histone Demethylases metabolism, Histones metabolism, Jumonji Domain-Containing Histone Demethylases metabolism, MicroRNAs genetics
- Abstract
Histone H3 lysine-9 di-methylation (H3K9me2) and lysine-27 tri-methylation (H3K27me3) are linked to repression of gene expression, but the functions of repressive histone methylation dynamics during inflammatory responses remain enigmatic. Here, we report that lysine demethylases 7A (KDM7A) and 6A (UTX) play crucial roles in tumor necrosis factor (TNF)-α signaling in endothelial cells (ECs), where they are regulated by a novel TNF-α-responsive microRNA, miR-3679-5p. TNF-α rapidly induces co-occupancy of KDM7A and UTX at nuclear factor kappa-B (NF-κB)-associated elements in human ECs. KDM7A and UTX demethylate H3K9me2 and H3K27me3, respectively, and are both required for activation of NF-κB-dependent inflammatory genes. Chromosome conformation capture-based methods furthermore uncover increased interactions between TNF-α-induced super enhancers at NF-κB-relevant loci, coinciding with KDM7A and UTX recruitments. Simultaneous pharmacological inhibition of KDM7A and UTX significantly reduces leukocyte adhesion in mice, establishing the biological and potential translational relevance of this mechanism. Collectively, these findings suggest that rapid erasure of repressive histone marks by KDM7A and UTX is essential for NF-κB-dependent regulation of genes that control inflammatory responses of ECs., (© 2020 The Authors.)
- Published
- 2020
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41. Genome-wide analysis revealed that DZNep reduces tubulointerstitial fibrosis via down-regulation of pro-fibrotic genes.
- Author
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Mimura I, Hirakawa Y, Kanki Y, Nakaki R, Suzuki Y, Tanaka T, Aburatani H, and Nangaku M
- Subjects
- Adenosine pharmacology, Animals, Cells, Cultured, Fibrosis etiology, Fibrosis pathology, Gene Expression Profiling, Humans, Kidney Diseases etiology, Kidney Diseases pathology, Male, Mice, Mice, Inbred C57BL, Adenosine analogs & derivatives, Biomarkers analysis, Fibrosis drug therapy, Gene Expression Regulation drug effects, Genome, Kidney Diseases drug therapy, Reperfusion Injury complications
- Abstract
Tubulointerstitial fibrosis has been recently reported to be caused by the collapse of the epigenetic regulation of kidney diseases. We examined whether pharmacological inhibition of histone modification is effective against renal fibrosis. DZNep (3-deazaneplanocin A) was originally developed as an anti-cancer drug to inhibit the repressive histone mark, H3K27me3. We used a model of chronic tubulointerstitial fibrosis induced by unilateral ischaemia/reperfusion and administered DZNep intravenously to the mice for 8 weeks. We found DZNep contributes to the reduction of tubulointerstitial fibrosis. We selected only tubular cells from in vivo samples using laser-capture microdissection because epigenetic regulation is specific to the cell types, and we focused on the changes in the tubular cells. We performed a genome-wide analysis of tubular cells using high-throughput sequencing (RNA-seq) to identify novel epigenetic factors associated with renal fibrosis. We found that pro-fibrotic genes such as COL3A1 (collagen type 3a1) and TIMP2 (tissue inhibitor of metalloproteinase 2) were suppressed by DZNep in vivo. In addition, pro-fibrotic genes such as COL4A1 (collagen type 4a1), TIMP2 and MMP14 were down-regulated by DZNep in vitro. In conclusion, we found that pharmacological epigenetic modification by DZNep decreased the expression levels of fibrogenic genes in tubular cells and inhibited tubulointerstitial fibrosis.
- Published
- 2018
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42. Dynamically and epigenetically coordinated GATA/ETS/SOX transcription factor expression is indispensable for endothelial cell differentiation.
- Author
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Kanki Y, Nakaki R, Shimamura T, Matsunaga T, Yamamizu K, Katayama S, Suehiro JI, Osawa T, Aburatani H, Kodama T, Wada Y, Yamashita JK, and Minami T
- Subjects
- Animals, Cell Differentiation, Cell Lineage genetics, Endothelial Cells cytology, GATA2 Transcription Factor antagonists & inhibitors, GATA2 Transcription Factor metabolism, Histones metabolism, Mice, Mouse Embryonic Stem Cells cytology, Oligonucleotide Array Sequence Analysis, Primary Cell Culture, Protein Isoforms antagonists & inhibitors, Protein Isoforms genetics, Protein Isoforms metabolism, Proto-Oncogene Protein c-ets-1 antagonists & inhibitors, Proto-Oncogene Protein c-ets-1 metabolism, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, SOXF Transcription Factors antagonists & inhibitors, SOXF Transcription Factors metabolism, Transcription Factors genetics, Transcription Factors metabolism, Endothelial Cells metabolism, Epigenesis, Genetic, GATA2 Transcription Factor genetics, Histones genetics, Mouse Embryonic Stem Cells metabolism, Proto-Oncogene Protein c-ets-1 genetics, SOXF Transcription Factors genetics
- Abstract
Although studies of the differentiation from mouse embryonic stem (ES) cells to vascular endothelial cells (ECs) provide an excellent model for investigating the molecular mechanisms underlying vascular development, temporal dynamics of gene expression and chromatin modifications have not been well studied. Herein, using transcriptomic and epigenomic analyses based on H3K4me3 and H3K27me3 modifications at a genome-wide scale, we analysed the EC differentiation steps from ES cells and crucial epigenetic modifications unique to ECs. We determined that Gata2, Fli1, Sox7 and Sox18 are master regulators of EC that are induced following expression of the haemangioblast commitment pioneer factor, Etv2. These master regulator gene loci were repressed by H3K27me3 throughout the mesoderm period but rapidly transitioned to histone modification switching from H3K27me3 to H3K4me3 after treatment with vascular endothelial growth factor. SiRNA knockdown experiments indicated that these regulators are indispensable not only for proper EC differentiation but also for blocking the commitment to other closely aligned lineages. Collectively, our detailed epigenetic analysis may provide an advanced model for understanding temporal regulation of chromatin signatures and resulting gene expression profiles during EC commitment. These studies may inform the future development of methods to stimulate the vascular endothelium for regenerative medicine., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2017
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43. Genome-wide approaches reveal functional vascular endothelial growth factor (VEGF)-inducible nuclear factor of activated T cells (NFAT) c1 binding to angiogenesis-related genes in the endothelium.
- Author
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Suehiro J, Kanki Y, Makihara C, Schadler K, Miura M, Manabe Y, Aburatani H, Kodama T, and Minami T
- Subjects
- Animals, COS Cells, Cell Movement, Chlorocebus aethiops, Coculture Techniques, Endothelial Cells cytology, Epigenesis, Genetic, Fibroblasts metabolism, Genome-Wide Association Study, HEK293 Cells, Homeostasis, Human Umbilical Vein Endothelial Cells, Humans, Mice, Oligonucleotide Array Sequence Analysis, Phenotype, Receptors, CXCR4 metabolism, Signal Transduction, Transcriptional Activation, rho GTP-Binding Proteins metabolism, Endothelium, Vascular metabolism, NFATC Transcription Factors metabolism, Neovascularization, Pathologic, Vascular Endothelial Growth Factor A metabolism
- Abstract
VEGF is a key regulator of endothelial cell migration, proliferation, and inflammation, which leads to activation of several signaling cascades, including the calcineurin-nuclear factor of activated T cells (NFAT) pathway. NFAT is not only important for immune responses but also for cardiovascular development and the pathogenesis of Down syndrome. By using Down syndrome model mice and clinical patient samples, we showed recently that the VEGF-calcineurin-NFAT signaling axis regulates tumor angiogenesis and tumor metastasis. However, the connection between genome-wide views of NFAT-mediated gene regulation and downstream gene function in the endothelium has not been studied extensively. Here we performed comprehensive mapping of genome-wide NFATc1 binding in VEGF-stimulated primary cultured endothelial cells and elucidated the functional consequences of VEGF-NFATc1-mediated phenotypic changes. A comparison of the NFATc1 ChIP sequence profile and epigenetic histone marks revealed that predominant NFATc1-occupied peaks overlapped with promoter-associated histone marks. Moreover, we identified two novel NFATc1 regulated genes, CXCR7 and RND1. CXCR7 knockdown abrogated SDF-1- and VEGF-mediated cell migration and tube formation. siRNA treatment of RND1 impaired vascular barrier function, caused RhoA hyperactivation, and further stimulated VEGF-mediated vascular outgrowth from aortic rings. Taken together, these findings suggest that dynamic NFATc1 binding to target genes is critical for VEGF-mediated endothelial cell activation. CXCR7 and RND1 are NFATc1 target genes with multiple functions, including regulation of cell migration, tube formation, and barrier formation in endothelial cells., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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44. Direct evidence for pitavastatin induced chromatin structure change in the KLF4 gene in endothelial cells.
- Author
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Maejima T, Inoue T, Kanki Y, Kohro T, Li G, Ohta Y, Kimura H, Kobayashi M, Taguchi A, Tsutsumi S, Iwanari H, Yamamoto S, Aruga H, Dong S, Stevens JF, Poh HM, Yamamoto K, Kawamura T, Mimura I, Suehiro J, Sugiyama A, Kaneki K, Shibata H, Yoshinaka Y, Doi T, Asanuma A, Tanabe S, Tanaka T, Minami T, Hamakubo T, Sakai J, Nozaki N, Aburatani H, Nangaku M, Ruan X, Tanabe H, Ruan Y, Ihara S, Endo A, Kodama T, and Wada Y
- Subjects
- Chromatin genetics, Gene Expression Regulation genetics, Gene Knockdown Techniques, Human Umbilical Vein Endothelial Cells cytology, Humans, Kruppel-Like Factor 4, Kruppel-Like Transcription Factors genetics, MEF2 Transcription Factors genetics, MEF2 Transcription Factors metabolism, Nitric Oxide Synthase Type III biosynthesis, Nitric Oxide Synthase Type III genetics, Response Elements, Thrombomodulin biosynthesis, Thrombomodulin genetics, Chromatin metabolism, Chromatin Assembly and Disassembly drug effects, Gene Expression Regulation drug effects, Human Umbilical Vein Endothelial Cells metabolism, Hydroxymethylglutaryl-CoA Reductase Inhibitors pharmacology, Kruppel-Like Transcription Factors biosynthesis, Quinolines pharmacology
- Abstract
Statins exert atheroprotective effects through the induction of specific transcriptional factors in multiple organs. In endothelial cells, statin-dependent atheroprotective gene up-regulation is mediated by Kruppel-like factor (KLF) family transcription factors. To dissect the mechanism of gene regulation, we sought to determine molecular targets by performing microarray analyses of human umbilical vein endothelial cells (HUVECs) treated with pitavastatin, and KLF4 was determined to be the most highly induced gene. In addition, it was revealed that the atheroprotective genes induced with pitavastatin, such as nitric oxide synthase 3 (NOS3) and thrombomodulin (THBD), were suppressed by KLF4 knockdown. Myocyte enhancer factor-2 (MEF2) family activation is reported to be involved in pitavastatin-dependent KLF4 induction. We focused on MEF2C among the MEF2 family members and identified a novel functional MEF2C binding site 148 kb upstream of the KLF4 gene by chromatin immunoprecipitation along with deep sequencing (ChIP-seq) followed by luciferase assay. By applying whole genome and quantitative chromatin conformation analysis {chromatin interaction analysis with paired end tag sequencing (ChIA-PET), and real time chromosome conformation capture (3C) assay}, we observed that the MEF2C-bound enhancer and transcription start site (TSS) of KLF4 came into closer spatial proximity by pitavastatin treatment. 3D-Fluorescence in situ hybridization (FISH) imaging supported the conformational change in individual cells. Taken together, dynamic chromatin conformation change was shown to mediate pitavastatin-responsive gene induction in endothelial cells.
- Published
- 2014
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45. Cross-enhancement of ANGPTL4 transcription by HIF1 alpha and PPAR beta/delta is the result of the conformational proximity of two response elements.
- Author
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Inoue T, Kohro T, Tanaka T, Kanki Y, Li G, Poh HM, Mimura I, Kobayashi M, Taguchi A, Maejima T, Suehiro J, Sugiyama A, Kaneki K, Aruga H, Dong S, Stevens JF, Yamamoto S, Tsutsumi S, Fujita T, Ruan X, Aburatani H, Nangaku M, Ruan Y, Kodama T, and Wada Y
- Subjects
- Angiopoietin-Like Protein 4, Angiopoietins genetics, Cell Hypoxia, Chromatin chemistry, Chromatin genetics, Chromatin Assembly and Disassembly, Human Umbilical Vein Endothelial Cells metabolism, Humans, Hypoxia-Inducible Factor 1, alpha Subunit genetics, PPAR delta genetics, PPAR-beta genetics, Angiopoietins metabolism, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, PPAR delta metabolism, PPAR-beta metabolism, Response Elements
- Abstract
Background: Synergistic transcriptional activation by different stimuli has been reported along with a diverse array of mechanisms, but the full scope of these mechanisms has yet to be elucidated., Results: We present a detailed investigation of hypoxia-inducible factor (HIF) 1 dependent gene expression in endothelial cells which suggests the importance of crosstalk between the peroxisome proliferator-activated receptor (PPAR) β/δ and HIF signaling axes. A migration assay shows a synergistic interaction between these two stimuli, and we identify angiopoietin-like 4 (ANGPTL4) as a common target gene by using a combination of microarray and ChIP-seq analysis. We profile changes of histone marks at enhancers under hypoxia, PPARβ/δ agonist and dual stimulations and these suggest that the spatial proximity of two response elements is the principal cause of the synergistic transcription induction. A newly developed quantitative chromosome conformation capture assay shows the quantitative change of the frequency of proximity of the two response elements., Conclusions: To the best of our knowledge, this is the first report that two different transcription factors cooperate in transcriptional regulation in a synergistic fashion through conformational change of their common target genes.
- Published
- 2014
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46. Inhibition of histone demethylase JMJD1A improves anti-angiogenic therapy and reduces tumor-associated macrophages.
- Author
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Osawa T, Tsuchida R, Muramatsu M, Shimamura T, Wang F, Suehiro J, Kanki Y, Wada Y, Yuasa Y, Aburatani H, Miyano S, Minami T, Kodama T, and Shibuya M
- Subjects
- Animals, Cell Hypoxia, Cell Line, Tumor, Drug Resistance, Neoplasm, Humans, Jumonji Domain-Containing Histone Demethylases physiology, Mice, Mice, Inbred C57BL, Neoplasms blood supply, Neoplasms pathology, Neovascularization, Pathologic etiology, Receptors, Vascular Endothelial Growth Factor antagonists & inhibitors, Vascular Endothelial Growth Factor A antagonists & inhibitors, Angiogenesis Inhibitors therapeutic use, Jumonji Domain-Containing Histone Demethylases antagonists & inhibitors, Macrophages drug effects, Neoplasms drug therapy
- Abstract
Antiangiogenic strategies can be effective for cancer therapy, but like all therapies resistance poses a major clinical challenge. Hypoxia and nutrient starvation select for aggressive qualities that may render tumors resistant to antiangiogenic attack. Here, we show that hypoxia and nutrient starvation cooperate to drive tumor aggressiveness through epigenetic regulation of the histone demethylase JMJD1A (JHDM2A; KDM3A). In cancer cells rendered resistant to long-term hypoxia and nutrient starvation, we documented a stimulation of AKT phosphorylation, cell morphologic changes, cell migration, invasion, and anchorage-independent growth in culture. These qualities associated in vivo with increased angiogenesis and infiltration of macrophages into tumor tissues. Through expression microarray analysis, we identified a cluster of functional drivers such as VEGFA, FGF18, and JMJD1A, the latter which was upregulated in vitro under conditions of hypoxia and nutrient starvation and in vivo before activation of the angiogenic switch or the prerefractory phase of antiangiogenic therapy. JMJD1A inhibition suppressed tumor growth by downregulating angiogenesis and macrophage infiltration, by suppressing expression of FGF2, HGF, and ANG2. Notably, JMJD1A inhibition enhanced the antitumor effects of the anti-VEGF compound bevacizumab and the VEGFR/KDR inhibitor sunitinib. Our results form the foundation of a strategy to attack hypoxia- and nutrient starvation-resistant cancer cells as an approach to leverage antiangiogenic treatments and limit resistance to them., (©2013 AACR.)
- Published
- 2013
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47. Alternative splicing of CD44 mRNA by ESRP1 enhances lung colonization of metastatic cancer cell.
- Author
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Yae T, Tsuchihashi K, Ishimoto T, Motohara T, Yoshikawa M, Yoshida GJ, Wada T, Masuko T, Mogushi K, Tanaka H, Osawa T, Kanki Y, Minami T, Aburatani H, Ohmura M, Kubo A, Suematsu M, Takahashi K, Saya H, and Nagano O
- Subjects
- Animals, Cell Line, Tumor, Chromatin Immunoprecipitation, Female, Flow Cytometry, Immunoblotting, Mice, Mice, Inbred BALB C, RNA-Binding Proteins genetics, Alternative Splicing genetics, Breast Neoplasms complications, Breast Neoplasms genetics, Hyaluronan Receptors genetics, Lung Neoplasms genetics, Lung Neoplasms secondary, RNA, Messenger genetics, RNA-Binding Proteins metabolism
- Abstract
In cancer metastasis, various environmental stressors attack the disseminating cells. The successful colonization of cancer cells in secondary sites therefore requires the ability of the cells to avoid the consequences of such exposure to the stressors. Here we show that orthotopic transplantation of a CD44 variant isoform-expressing (CD44v(+)) subpopulation of 4T1 breast cancer cells, but not that of a CD44v(-) subpopulation, in mice results in efficient lung metastasis accompanied by expansion of stem-like cancer cells. Such metastasis is dependent on the activity of the cystine transporter xCT, and the stability of this protein is controlled by CD44v. We find that epithelial splicing regulatory protein 1 regulates the expression of CD44v, and knockdown of epithelial splicing regulatory protein 1 in CD44v(+) cells results in an isoform switch from CD44v to CD44 standard (CD44s), leading to reduced cell surface expression of xCT and suppression of lung colonization. The epithelial splicing regulatory protein 1-CD44v-xCT axis is thus a potential therapeutic target for the prevention of metastasis.
- Published
- 2012
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48. Critical role for GATA3 in mediating Tie2 expression and function in large vessel endothelial cells.
- Author
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Song H, Suehiro J, Kanki Y, Kawai Y, Inoue K, Daida H, Yano K, Ohhashi T, Oettgen P, Aird WC, Kodama T, and Minami T
- Subjects
- Angiopoietin-1 metabolism, Endothelial Cells cytology, Endothelium, Vascular cytology, Endothelium, Vascular metabolism, GATA3 Transcription Factor metabolism, Humans, Immunoprecipitation, Oligonucleotide Array Sequence Analysis, Phenotype, Promoter Regions, Genetic, RNA, Small Interfering metabolism, Receptor, TIE-2 chemistry, Tumor Necrosis Factor-alpha metabolism, Umbilical Veins cytology, Vascular Endothelial Growth Factor A metabolism, GATA3 Transcription Factor physiology, Gene Expression Regulation, Receptor, TIE-2 biosynthesis
- Abstract
Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms. There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels. In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited angiopoietin (Ang)-1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration, survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown to bind to regulatory regions within the 5'-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF1, physically interacted and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang-1 to attenuate vascular endothelial cell growth factor stimulation of vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial cells to tumor necrosis factor-alpha resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression. Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial cells.
- Published
- 2009
- Full Text
- View/download PDF
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