Hidenori Kiyosawa

Kochi University, Kôti, Kōchi, Japan

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Publications (46)236.36 Total impact

  • Masamitsu Eitoku · Narufumi Suganuma · Hidenori Kiyosawa
    [Show abstract] [Hide abstract] ABSTRACT: In vitro differentiation systems of mouse embryonic stem cells (ESCs) are widely used as tools for studies of cell differentiation, organogenesis, and regenerative medicine. We have studied the regulation of neuron-specific imprinting genes, Ube3a and its antisense transcripts (Ube3a ATS), using in vitro neuronal differentiation of F1 hybrid ESCs. Each different non-adherent plate used for embryoid body (EB) formation during differentiation is associated with different costs; notably, plates coated with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer are more expensive than untreated polystyrene plates. Here, we assessed whether the polymer-coated plates gave better results than the untreated plates. The first stage of differentation was performed in the MPC polymer-coated or untreated plates. The formed EBs were then passaged onto laminin-coated plates for further differentiation into neurons. Neither the neuron-specific imprinting status of Ube3a nor the expression levels of the neuron-specific markers Ube3a ATS and Mtap2 differed between neurons prepared on untreated plates and those prepared on MPC polymer-coated plates. These results suggest that the two non-adherent plates displayed almost the same characteristics for inducing neuronal differentiation of mouse ESCs and EB formation. Our study proved that untreated polystyrene plates are a cost-effective choice for EB formation in in vitro differentiation systems of mouse ESCs.
    No preview · Article · Apr 2016 · Cytotechnology
  • Saya Okui · Chisato Ushida · Hidenori Kiyosawa · Gota Kawai
    [Show abstract] [Hide abstract] ABSTRACT: RNA fragments corresponding to the mirror tRNA that is located upstream of the cytochrome oxidase I (COXI) gene in the mouse mitochondrial genome were found in the sequences obtained from the mouse brain by the next generation sequencing. RNA fragments corresponding to the 5′ terminal of COXI mRNA were also found and it was suggested that the precursor of the COXI mRNA is processed at three residues upstream of the first AUG codon. The mirror tRNA fragment has poly(A) in its 3′ terminal and variable 5′ terminal, suggesting that this RNA is produced during the 5′ processing of COXI mRNA. Secondary structure prediction and NMR analysis indicated that the mirror tRNA is folded into a tRNA-like secondary structure, suggesting that the tRNA-like conformation of the 5′ adjacent sequence of COXI mRNA is involved in the COXI mRNA maturation in the mouse mitochondria.
    No preview · Article · Oct 2015 · Journal of Biochemistry
  • [Show abstract] [Hide abstract] ABSTRACT: Epithelial-to-mesenchymal transition (EMT) is an important mechanism in cancer metastasis and pulmonary fibrosis. Previous studies demonstrated effect of histone H3 and H4 acetylation in cancer and pulmonary fibrosis, so we hypothesized that histone modification might play a crucial role in gene regulation during EMT. In this study, we investigated the mechanism behind EMT by analyzing comprehensive gene expression and the effect of sodium valproate (VPA), a class I histone deacetylase inhibitory drug, on histone modification. EMT was induced in human alveolar epithelial cells (A549) using 5 ng/mL of transforming growth factor (TGF)-β1. Various concentrations of VPA were then administered, and Western blotting was used to analyze histone acetylation or methylation. Comprehensive gene expression analysis was carried out by RNA sequencing, and chromatin immunoprecipitation was performed with an anti-acetyl histone H3 lysine 27 antibody. TGF-β1 stimulation led to a decrease in histone acetylation, especially that of histone H3K27, and H3K27ac localization was decreased at particular gene loci. This decrease was recovered by VPA treatment, which also up-regulated the mRNA expression of genes down-regulated by TGF-β1, and correlated with the localization of H3K27ac. However, genes up-regulated by TGF-β1 stimulation were not suppressed by VPA, with the exception of COL1A1. Histone acetylation was down-regulated by TGF-β1 stimulation in A549 cells. VPA partially inhibited EMT and the decrease of histone acetylation, which plays an important role in the progression of EMT.
    No preview · Article · Aug 2015 · Beiträge zur Klinik der Tuberkulose
  • No preview · Article · Jun 2015
  • Hidenori Kiyosawa · Akio Okumura · Saya Okui · Chisato Ushida · Gota Kawai
    [Show abstract] [Hide abstract] ABSTRACT: In order to find novel structured small RNAs, next-generation sequencing was applied to small RNA fractions with lengths ranging from 40 to 140 nt and secondary structure-based clustering was performed. Sequences of structured RNAs were effectively clustered and analyzed by secondary structure. Although more than 99% of the obtained sequences were known RNAs, 16 candidate mouse structured small non-coding RNAs (MsncRs) were isolated. Based on these results, the merits of secondary structure-based analysis are discussed. Copyright © 2015. Published by Elsevier Inc.
    No preview · Article · May 2015 · Genomics
  • No preview · Conference Paper · May 2015
  • No preview · Article · Dec 2014 · Genes & Genetic Systems
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    [Show abstract] [Hide abstract] ABSTRACT: To investigate the feasibility of developing a method for detection of gene doping in power-athletes, we devised an experimental model system. Myostatin is a potent negative regulator of skeletal muscle development and growth, and myostatin-knockout mice exhibit a double-muscle phenotype. To achieve knockdown, we constructed plasmids expressing short hairpin interfering RNAs (shRNAs) against myostatin. These shRNAs were transfected into C2C12 cultured cells or injected into the tibialis anterior (TA) muscle of adult mice. By performing in vitro and in vivo experiments, we found that some shRNAs effectively reduced the expression of myostatin, and that the TA muscle showed hypertrophy of up to 27.9%. Then, using real-time PCR, we tried to detect the shRNA plasmid in the serum or muscles of mice into which it had been injected. Although we were unable to detect the plasmid in serum samples, it was detectable in the treated muscle at least four weeks after induction. We were also able to detect the plasmid in muscle in the vicinity of the TA. This gene doping model system will be useful for further studies aimed at doping control. Key pointsUsing a myostatin knockdown plasmid, we have succeeded in creating a model system for gene doping using mice that resulted in muscle hypertrophy greater than that reported previously.We confirmed that there was a limit of gene doping detection using real-time PCR, either from serum or muscle smple.This model experimental system can be utilized for examining indirect methods of gene doping detection such as immune responses to gene transfer or a profiling approach using DNA microarray.
    Full-text · Article · Jun 2012 · Journal of sports science & medicine
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    [Show abstract] [Hide abstract] ABSTRACT: To investigate the feasibility of developing a method for detection of gene doping in power-athletes, we devised an experimental model system. Myostatin is a potent negative regulator of skeletal muscle development and growth, and myostatin-knockout mice exhibit a double-muscle phenotype. To achieve knockdown, we constructed plasmids expressing short hairpin interfering RNAs (shRNAs) against myostatin. These shRNAs were transfected into C2C12 cultured cells or injected into the tibialis anterior (TA) muscle of adult mice. By performing in vitro and in vivo experiments, we found that some shRNAs effectively reduced the expression of myostatin, and that the TA muscle showed hypertrophy of up to 27.9%. Then, using real-time PCR, we tried to detect the shRNA plasmid in the serum or muscles of mice into which it had been injected. Although we were unable to detect the plasmid in serum samples, it was detectable in the treated muscle at least four weeks after induction. We were also able to detect the plasmid in muscle in the vicinity of the TA. This gene doping model system will be useful for further studies aimed at doping control.
    Full-text · Article · Jun 2012 · Journal of sports science & medicine
  • [Show abstract] [Hide abstract] ABSTRACT: Mammalian target of rapamycin (mTOR) pathway positively regulates the cell growth through ribosome biogenesis in many cell type. In general, myostatin is understood to repress skeletal muscle hypertrophy through inhibition of mTOR pathway and myogenesis. However, these relationships have not been clarified in skeletal muscle undergoing atrophy. Here, we observed a significant decrease of skeletal muscle mass at 2 weeks after denervation. Unexpectedly, however, mTOR pathway and the expression of genes related to myogenesis were markedly increased, and that of myostatin was decreased. However, de novo ribosomal RNA synthesis and the levels of ribosomal RNAs were dramatically decreased in denervated muscle. These results indicate that ribosome biogenesis is strongly controlled by factors other than the mTOR pathway in denervated atrophic muscle. Finally, we assessed rRNA transcription factors expression and observed that TAFIa was the only factor decreased. TAFIa might be a one of the limiting factor for rRNA synthesis in denervated muscle.
    No preview · Article · Apr 2012 · Journal of Cellular Physiology
  • Koji Numata · Hidenori Kiyosawa
    [Show abstract] [Hide abstract] ABSTRACT: Recent transcriptomic studies revealed that extensive proportions of genomes are transcribed, despite the limited fraction of protein-coding gene loci in the whole genome. Most transcripts are considered to be 'cryptic' output of the genome because of the lack of functional evidence; however, recent progress in molecular analyses has revealed that some of these transcripts at least have functional significance. This review article examines evidence of the functional significance of endogenous cis-antisense transcripts, which are the transcriptional output from the opposite strand of annotated genes. These transcripts are one of the most common types of transcripts that do not correspond to any protein-coding loci. Historical molecular studies revealed the existence of antisense transcripts associated with dozens of gene loci, whereas more recent genome-wide studies have shown that many genes have an antisense counterpart thus stimulating investigations into the functional significance of endogenous antisense transcripts. Here, we summarize the recent progress in the genome-wide characterization of the antisense transcriptome, and discuss the biological mechanisms that underlie the regulatory machinery of eukaryotic gene expression with respect to the potential roles of endogenous cis-antisense transcripts.
    No preview · Article · Jan 2012 · Frontiers in Bioscience
  • [Show abstract] [Hide abstract] ABSTRACT: Genomic imprinting is a phenomenon whereby monoallelic gene expression occurs in a parent-of-origin-specific manner. A subset of imprinted genes acquires a tissue-specific imprinted status during the course of tissue development, and this process can be analyzed by means of an in vitro differentiation system utilizing embryonic stem (ES) cells. In neurons, the gene Ube3a is expressed from the maternal allele only, and a paternally expressed non-coding, antisense RNA has been implicated in the imprinting process in mice and humans. Here, to study the genomic imprinting mechanism, we established F1 hybrid ES cells derived from two sub-species of Mus musculus and established an in vitro neuronal differentiation system in which neuron-specific imprinting of Ube3a was recapitulated. With this system, we revealed that the switch from biallelic expression to maternal, monoallelic expression of Ube3a occurs late in neuronal development, during the neurite outgrowth period, and that the expression of endogenous antisense transcript from the Ube3a locus is up-regulated several hundred-fold during the same period. Our results suggest that evaluation of the quality of ES cells by studying their differentiation in vitro should include evaluation of epigenetic aspects, such as a comparison with the genomic imprinting status found in tissues in vivo, in addition to the evaluation of differentiation gene markers and morphology. Our F1 hybrid ES cells and in vitro differentiation system will allow researchers to investigate complex end-points such as neuron-specific genomic imprinting, and our F1 hybrid ES cells are a useful resource for other tissue-specific genomic imprinting and epigenetic analyses.
    No preview · Article · Dec 2011 · Human Molecular Genetics
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    [Show abstract] [Hide abstract] ABSTRACT: Supplemental tables. Table S1: List of sense-antisense gene pairs having altered expression balances in cancer tissues compared to surrounding normal tissues. Table S2: List of gene categories of sense-antisense transcripts displaying altered expression balances in normal tissues and cancer tissues. Table S3: Percentages of probes detecting positive expression according to criteria supplied by Agilent's platform. Table S4: List of sense-AFAS pairs detecting altered expression balances in normal tissues and cancer tissues. Table S5: Numbers of sense genes belonging to specified functional categories. Table S6: Clinical data of patients.
    Preview · Dataset · May 2011
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    [Show abstract] [Hide abstract] ABSTRACT: Recent studies have identified thousands of sense-antisense gene pairs across different genomes by computational mapping of cDNA sequences. These studies have shown that approximately 25% of all transcriptional units in the human and mouse genomes are involved in cis-sense-antisense pairs. However, the number of known sense-antisense pairs remains limited because currently available cDNA sequences represent only a fraction of the total number of transcripts comprising the transcriptome of each cell type. To discover novel antisense transcripts encoded in the antisense strand of important genes, such as cancer-related genes, we conducted expression analyses of antisense transcripts using our custom microarray platform along with 2376 probes designed specifically to detect the potential antisense transcripts of 501 well-known genes suitable for cancer research. Using colon cancer tissue and normal tissue surrounding the cancer tissue obtained from 6 patients, we found that antisense transcripts without poly(A) tails are expressed from approximately 80% of these well-known genes. This observation is consistent with our previous finding that many antisense transcripts expressed in a cell are poly(A)-. We also identified 101 and 71 antisense probes displaying a high level of expression specifically in normal and cancer tissues respectively. Our microarray analysis identified novel antisense transcripts with expression profiles specific to cancer tissue, some of which might play a role in the regulatory networks underlying oncogenesis and thus are potential targets for further experimental validation. Our microarray data are available at http://www.brc.riken.go.jp/ncrna2007/viewer-Saito-01/index.html.
    Full-text · Article · May 2011 · BMC Medical Genomics
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    [Show abstract] [Hide abstract] ABSTRACT: Supplemental figures. Figure S1: Clustering analysis of the expression ratio of the sense and antisense pair of transcripts. Figure S2: Changes in expression for sense-antisense gene pairs in cancer tissues, compared with surrounding normal tissues. Figure S3: Changes in expression for sense-AFAS gene pairs in cancer tissues, compared with surrounding normal tissues. Figure S4: Clustering analysis of the expression ratio of well-known genes (sense transcripts) and their putative antisense transcripts. Figure S5: Expression intensities detected by probes designed for the sense gene U69611 and its corresponding AFAS transcript. Figure S6: Average Expression levels detected by AFAS probes within every 500 bases with respect to the both termini of sense transcripts.
    Preview · Dataset · May 2011
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    [Show abstract] [Hide abstract] ABSTRACT: The functionality of sense-antisense transcripts (SATs), although widespread throughout the mammalian genome, is largely unknown. Here, we analyzed the SATs expression and its associated promoter DNA methylation status by surveying 12 tissues of mice to gain insights into the relationship between expression and DNA methylation of SATs. We have found that sense and antisense expression positively correlate in most tissues. However, in some SATs with tissue-specific expression, the expression level of a transcript from a CpG island-bearing promoter is low when the promoter DNA methylation is present. In these circumstances, the expression level of its opposite-strand transcript, especially when it is poly(A)-negative was coincidentally higher. These observations suggest that, albeit the general tendency of sense-antisense simultaneous expression, some antisense transcripts have coordinated expression with its counterpart sense gene promoter methylation. This cross-strand relationship is not a privilege of imprinted genes but seems to occur widely in SATs.
    Full-text · Article · Dec 2010 · Genomics
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    Koji Numata · Chihiro Kohama · Kuniya Abe · Hidenori Kiyosawa
    [Show abstract] [Hide abstract] ABSTRACT: We investigated the allele- and strand-specific transcriptional landscape of a megabase-wide genomic region of mouse Ube3a (ubiquitin protein ligase E3A) by means of a highly parallel SNP genotyping platform. We have successfully identified maternal-specific expression of Ube3a and its antisense counterpart (Ube3a-ATS) in brain, but not in liver. Because of the use of inter-subspecies hybrid mice, this megabase-wide analysis provided high-resolution picture of the transcriptional patterns of this region. First, we showed that brain-specific maternal expression of Ube3a is restricted to the second half part of the locus, but is absent from the first half part. Balance of allelic expression is altered in the middle of the locus. Second, we showed that expression of the brain-specific Ube3a-ATS appeared to be terminated in the region upstream to the Ube3a transcription start site. The present study highlights the importance of locus-wide competition between sense and antisense transcripts.
    Preview · Article · Dec 2010 · Nucleic Acids Research
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    [Show abstract] [Hide abstract] ABSTRACT: Recent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs). NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes. First, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Acaa1b and Aard) confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation of cellular events and in pathological conditions. Our microarray platform targeting the complementary strand of annotated genes successfully identified novel NATs that could not be identified by publically available cDNA data, and as such could not be detected by the usual "sense-targeting" microarray approach. Differentially expressed NATs monitored by this platform may provide candidates for investigations of gene function. An advantage of our microarray platform is that it can be applied to any genes and target samples of interest.
    Preview · Article · Sep 2009 · BMC Genomics
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    [Show abstract] [Hide abstract] ABSTRACT: Changes in expression of Pdcd6 and Drd4.
    Preview · Dataset · Aug 2009
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    [Show abstract] [Hide abstract] ABSTRACT: Expression analyses of sense and antisense transcripts of Acaa1b. Quantitative RT-PCR, ISH and microarray analyses were able to detect this transcript within the testis and kidney.
    Preview · Dataset · Aug 2009

Publication Stats

2k Citations
236.36 Total Impact Points

Institutions

  • 2015
    • Kochi University
      • Department of Environmental Medicine
      Kôti, Kōchi, Japan
  • 2012
    • The University of Tokyo
      • Department of Animal Resource Sciences
      Edo, Tōkyō, Japan
  • 2011-2012
    • National Institute of Genetics
      Мисима, Shizuoka, Japan
  • 2005-2012
    • University of Tsukuba
      Tsukuba, Ibaraki, Japan
  • 2003-2009
    • RIKEN
      • Biometal Science Laboratory
      Вако, Saitama, Japan
    • Yokohama City University
      Yokohama, Kanagawa, Japan