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

Associate Professor / Department of Stem Cell Biology and Medicine / Department of Stem Cell Biology and Medicine / Faculty of Medical Sciences


Papers
1. Taito Matsuda, Takashi Irie, Shutaro Katsurabayashi, Yoshinori Hayashi, Tatsuya Nagai, Nobuhiko Hamazaki, Aliya Mari D. Adefuin, Fumihito Miura, Takashi Ito, Hiroshi Kimura, Katsuhiko Shirahige, Tadayuki Takeda, Katsunori Iwasaki, Takuya Imamura, Kinichi Nakashima, Pioneer Factor NeuroD1 Rearranges Transcriptional and Epigenetic Profiles to Execute Microglia-Neuron Conversion, Neuron, 10.1016/j.neuron.2018.12.010, 101, 3, 472-485.e7, 2019.02, Matsuda et al. report direct neuronal conversion of microglia induced by the expression of a single transcription factor, NeuroD1, which occupies bivalent epigenetic domains for neuronal gene induction. NeuroD1 can also converts microglia into neurons in the adult mouse striatum..
2. Tomonori Kameda, Takuya Imamura, Kinichi Nakashima, Epigenetic regulation of neural stem cell differentiation towards spinal cord regeneration, Cell and Tissue Research, 10.1007/s00441-017-2656-2, 371, 1, 189-199, 2018.01, Severe spinal cord injury (SCI) leads to almost complete neural cell loss at the injured site, causing the irreversible disruption of neuronal circuits. The transplantation of neural stem or precursor cells (NS/PCs) has been regarded as potentially effective for SCI treatment because NS/PCs can compensate for the injured sites by differentiating into neurons and glial cells (astrocytes and oligodendrocytes). An understanding of the molecular mechanisms that regulate the proliferation, fate specification and maturation of NS/PCs and their progeny would facilitate the establishment of better therapeutic strategies for regeneration after SCI. In recent years, several studies of SCI animal models have demonstrated that the modulation of specific epigenetic marks by histone modifiers and non-coding RNAs directs the setting of favorable cellular environments that promote the neuronal differentiation of NS/PCs and/or the elongation of the axons of the surviving neurons at the injured sites. In this review, we provide an overview of recent progress in the epigenetic regulation/manipulation of neural cells for the treatment of SCI..
3. Tsukasa Sanosaka, Takuya Imamura, Nobuhiko Hamazaki, Muh Chyi Chai, Katsuhide Igarashi, Maky Ideta-Otsuka, Fumihito Miura, Takashi Ito, Nobuyuki Fujii, Kazuho Ikeo, Kinichi Nakashima, DNA Methylome Analysis Identifies Transcription Factor-Based Epigenomic Signatures of Multilineage Competence in Neural Stem/Progenitor Cells, Cell Reports, 10.1016/j.celrep.2017.08.086, 20, 12, 2992-3003, 2017.09, Regulation of the epigenome during in vivo specification of brain stem cells is still poorly understood. Here, we report DNA methylome analyses of directly sampled cortical neural stem and progenitor cells (NS/PCs) at different development stages, as well as those of terminally differentiated cortical neurons, astrocytes, and oligodendrocytes. We found that sequential specification of cortical NS/PCs is regulated by two successive waves of demethylation at early and late development stages, which are responsible for the establishment of neuron- and glia-specific low-methylated regions (LMRs), respectively. The regulatory role of demethylation of the gliogenic genes was substantiated by the enrichment of nuclear factor I (NFI)-binding sites. We provide evidence that de novo DNA methylation of neuron-specific LMRs establishes glia-specific epigenotypes, essentially by silencing neuronal genes. Our data highlight the in vivo implications of DNA methylation dynamics in shaping epigenomic features that confer the differentiation potential of NS/PCs sequentially during development..
4. Takuya Imamura, Evolutionary acquisition of promoterassociated non-coding RNA (pancRNA) repertoires diversifies species-dependent gene activation mechanisms in mammals, BMC GENOMICS, 10.1186/s12864-017-3662-1, 18, 2017.04.
5. Nobuhiko Hamazaki, Kinichi Nakashima, Katsuhiko Hayashi, Takuya Imamura, Detection of bidirectional promoter-derived lncRNAs from small-scale samples using pre-amplification-free directional RNA-seq method, Methods in Molecular Biology, 10.1007/978-1-4939-6988-3_6, 83-103, 2017.01, Development of high-throughput sequencing technologies has uncovered the immensity of the long noncoding RNA (lncRNA) world. Divergently transcribed lncRNAs from bidirectional gene promoters, called promoter-associated noncoding RNAs (pancRNAs), account for ~20% of the total number of lncRNAs, and this major fraction is involved in many biological processes, such as development and cancer formation. Recently, we have found that the pancRNAs activate their partner genes, as represented by the fact that pancIl17d, a pancRNA that is transcribed from the antisense strand of the promoter region of Interleukin 17d (Il17d) at the onset of zygotic gene activation (ZGA), is essential for mouse preimplantation development through Il17d upregulation. The discovery of the expression of a specific set of pancRNAs during ZGA was achieved by using a method that generates directional RNA-seq libraries from small-scale samples. Although there are several methods available for small-scale samples, most of them require a pre-amplification procedure that frequently generates some amplification biases toward a subset of transcripts. We provide here a highly sensitive and reproducible method based on the preparation of directional RNA-seq libraries from as little as 100 mouse oocytes or embryos without pre-amplification for the quantification of lncRNAs as well as mRNAs..
6. Nobuhiko Hamazaki, Kinichi Nakashima, Takuya Imamura, Manipulation of promoter-associated noncoding RNAs in mouse early embryos for controlling sequence-specific epigenetic status, Methods in Molecular Biology, 10.1007/978-1-4939-6716-2_16, 271-282, 2017.01, In mammals, transcription in the zygote begins after fertilization. This transcriptional wave is called zygotic gene activation (ZGA). During ZGA, epigenetic modifications, such as DNA methylation and histone modifications, are dynamically and drastically reconstructed in a sequence-specific manner. However, how such orchestrated gene upregulation is regulated remains unknown. Recently, using microinjection techniques, we have revealed that a class of long noncoding RNAs, named promoter-associated noncoding RNAs (pancRNAs), mediates specific gene upregulation through promoter DNA demethylation during ZGA. Here, we describe the experimental methods available to control the expression levels of pancRNAs and to evaluate epigenetic status after pancRNA manipulation..
7. Takuya Imamura, Reconstitution in vitro of the entire cycle of the mouse female germ line, NATURE, 10.1038/nature20104, 539, 7628, 299-+, 2016.11.
8. Naoki Yamamoto, Kiyokazu Agata, Kinichi Nakashima, Takuya Imamura, Bidirectional promoters link cAMP signaling with irreversible differentiation through promoter-associated non-coding RNA (pancRNA) expression in PC12 cells, Nucleic acids research, 10.1093/nar/gkw113, 44, 11, 5105-5122, 2016.06, Bidirectional promoters are the major source of gene activation-associated noncoding RNA (ncRNA). PC12 cells offer an interesting model for understanding the mechanism underlying bidirectional promoter-mediated cell cycle control. Nerve growth factor (NGF)-stimulated PC12 cells elongate neurites, and are in a reversible cell-cycle-arrested state. In contrast, these cells irreversibly differentiate and cannot re-enter the normal cell cycle after NGF plus cAMP treatment. In this study, using directional RNA-seq, we found that bidirectional promoters for protein-coding genes with promoter-associated ncRNA (pancRNA) were enriched for cAMP response element consensus sequences, and were preferred targets for transcriptional regulation by the transcription factors in the cAMP-dependent pathway. A spindle-formation-associated gene, Nusap1 and pancNusap1 were among the most strictly co-transcribed pancRNA-mRNA pairs. This pancRNA-mRNA pair was specifically repressed in irreversibly differentiated PC12 cells. Knockdown (KD) and overexpression experiments showed that pancNusap1 positively regulated the Nusap1 expression in a sequence-specific manner, which was accompanied by histone acetylation at the Nusap1 promoter. Furthermore, pancNusap1 KD recapitulated the effects of cAMP on cell cycle arrest. Thus, we conclude that pancRNA-mediated histone acetylation contributes to the establishment of the cAMP-induced transcription state of the Nusap1 locus and contributes to the irreversible cell cycle exit for terminal differentiation of PC12 cells..
9. Takuya Imamura, Bidirectional promoters link cAMP signaling with irreversible differentiation through promoter-associated non-coding RNA (pancRNA) expression in PC12 cells., Nucleic Acids Research, doi:10.1093/nar/gkw113, 2016.03.
10. Keita Tsujimura, Koichiro Irie, Hideyuki Nakashima, Yoshihiro Egashira, Yoichiro Fukao, Masayuki Fujiwara, Masayuki Itoh, Masahiro Uesaka, Takuya Imamura, Yasukazu Nakahata, Yui Yamashita, Takaya Abe, Shigeo Takamori, Kinichi Nakashima, MiR-199a Links MeCP2 with mTOR Signaling and Its Dysregulation Leads to Rett Syndrome Phenotypes, Cell Reports, 10.1016/j.celrep.2015.08.028, 12, 11, 1887-1901, 2015.09, Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Although emerging evidence suggests that MeCP2 deficiency is associated with dysregulation of mechanistic target of rapamycin (mTOR), which functions as a hub for various signaling pathways, the mechanism underlying this association and the molecular pathophysiology of RTT remain elusive. We show here that MeCP2 promotes the posttranscriptional processing of particular microRNAs (miRNAs) as a component of the microprocessor Drosha complex. Among the MeCP2-regulated miRNAs, we found that miR-199a positively controls mTOR signaling by targeting inhibitors for mTOR signaling. miR-199a and its targets have opposite effects on mTOR activity, ameliorating and inducing RTT neuronal phenotypes, respectively. Furthermore, genetic deletion of miR-199a-2 led to a reduction of mTOR activity in the brain and recapitulated numerous RTT phenotypes in mice. Together, these findings establish miR-199a as a critical downstream target of MeCP2 in RTT pathogenesis by linking MeCP2 with mTOR signaling..
11. 今村 拓也, Gene activation-associated long noncoding RNAs function in mouse preimplantation development, DEVELOPMENT, 10.1242/dev.116996, 142, 5, 910-920, 2015.03.
12. Teppei Goto, Junko Tomikawa, Kana Ikegami, Shiori Minabe, Hitomi Abe, Tatsuya Fukanuma, Takuya Imamura, Kenji Takase, Makoto Sanbo, Koichi Tomita, Masumi Hirabayashi, Kei Ichiro Maeda, Hiroko Tsukamura, Yoshihisa Uenoyama, Identification of hypothalamic arcuate nucleus-specific enhancer region of Kiss1 gene in mice, Molecular Endocrinology, 10.1210/me.2014-1289, 29, 1, 121-129, 2015.01, Pulsatile secretion of GnRH plays a pivotal role in follicular development via stimulating tonic gonadotropin secretion in mammals. Kisspeptin neurons, located in the arcuate nucleus (ARC), are considered to be an intrinsic source of the GnRH pulse generator. The present study aimed to determine ARC-specific enhancer(s) of the Kiss1 gene by an in vivo reporter assay. Three green fluorescent protein (GFP) reporter constructs (long, medium length, and short) were generated by insertion of GFP cDNA at the Kiss1 locus. Transgenic female mice bearing the long and mediumlength constructs showed apparent GFP signals in kisspeptin-immunoreactive cells in both the ARC and anteroventral periventricular nucleus, in which another population of kisspeptin neurons are located. On the other hand, transgenic mice bearing 5′-truncated short construct showed few GFP signals in the ARC kisspeptin-immunoreactive cells, whereas they showed colocalization of GFPand kisspeptin-immunoreactivities in the anteroventral periventricular nucleus. In addition, chromatin immunoprecipitation and chromosome conformation capture assays revealed recruitment of unoccupied estrogen receptor-α in the 5′-upstream region and intricate chromatin loop formation between the 5′-upstream and promoter regions of Kiss1 locus in the ARC. Taken together, the present results indicate that 5′-upstream region of Kiss1 locus plays a critical role in Kiss1 gene expression in an ARC-specific manner and that the recruitment of estrogen receptor-α and formation of a chromatin loop between the Kiss1 promoter and the 5′ enhancer region may be required for the induction of ARC-specific Kiss1 gene expression. These results suggest that the 5′-upstream region of Kiss1 locus functions as an enhancer for ARC Kiss1 gene expression in mice..
13. Takuya Imamura, Epigenetic setting and reprogramming for neural cell fate determination and differentiation, PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES, 10.1098/rstb.2013.0511, 369, 1652, 2014.09.
14. Naoki Yamamoto, Masahiro Uesaka, Takuya Imamura, Kinichi Nakashima, Roles of Epigenetics in the Neural Stem Cell and Neuron, Epigenetics in Psychiatry, 10.1016/B978-0-12-417114-5.00004-8, 51-78, 2014.06, For higher-order functions of the mammalian brain such as the regulation of motor behavior, consciousness, emotion, learning, and memory, neurons have to establish complicated and elaborate networks. In addition, the functions of neurons are critically supported by glial cells (astrocytes and oligodendrocytes). All of these neural cell types (i.e., neurons, astrocytes, and oligodendrocytes) are generated from common neural stem cells (NSCs), which also have self-renewal activity. Accumulating evidence suggests that the behavior of NSCs is influenced spatiotemporally by both cell-extrinsic factors, including cytokine signaling, and cell-intrinsic epigenetic changes, which together regulate the proliferation and fate decisions of NSCs to produce glial cells or neurons, including different neuronal subtypes, in a spatiotemporal manner. In the first half of this chapter, we summarize recent advances in elucidating the role of epigenetic control in the differentiation of NSCs. In postmitotic neurons, as well as NSCs, several orchestrated epigenetic mechanisms underlie neuronal functioning critical for memory formation. Recent studies have revealed the presence and physiological significance of changes of the epigenetic modifications within a neuron of an already defined cell fate. A dynamic change of epigenetic status induced by neuronal activity can alter synaptic plasticity, which constitutes part of the mechanisms of learning and memory. In the latter half of the chapter, we describe the role of epigenetic plasticity in non-dividing neurons. We also discuss the robust identity of the neuronal cell fate, as exemplified by the extremely poor ability of neurons to be reprogrammed to pluripotent stem cells..
15. Masahiro Uesaka, Osamu Nishimura, Yasuhiro Go, Kinichi Nakashima, Kiyokazu Agata, Takuya Imamura, Bidirectional promoters are the major source of gene activation-associated non-coding RNAs in mammals, BMC Genomics, doi:10.1186/1471-2164-15-35, 15, 35, 2014.01, Background
The majority of non-coding RNAs (ncRNAs) involved in mRNA metabolism in mammals have been believed to downregulate the corresponding mRNA expression level in a pre- or post-transcriptional manner by forming short or long ncRNA-mRNA duplex structures. Information on non-duplex-forming long ncRNAs is now also rapidly accumulating. To examine the directional properties of transcription at the whole-genome level, we performed directional RNA-seq analysis of mouse and chimpanzee tissue samples.

Results
We found that there is only about 1% of the genome where both the top and bottom strands are utilized for transcription, suggesting that RNA-RNA duplexes are not abundantly formed. Focusing on transcription start sites (TSSs) of protein-coding genes revealed that a significant fraction of them contain switching-points that separate antisense- and sense-biased transcription, suggesting that head-to-head transcription is more prevalent than previously thought. More than 90% of head-to-head type promoters contain CpG islands. Moreover, CCG and CGG repeats are significantly enriched in the upstream regions and downstream regions, respectively, of TSSs located in head-to-head type promoters. Genes with tissue-specific promoter-associated ncRNAs (pancRNAs) show a positive correlation between the expression of their pancRNA and mRNA, which is in accord with the proposed role of pancRNA in facultative gene activation, whereas genes with constitutive expression generally lack pancRNAs.

Conclusions
We propose that single-stranded ncRNA resulting from head-to-head transcription at GC-rich sequences regulates tissue-specific gene expression..
16. Masahiro Uesaka, Osamu Nishimura, Yasuhiro Go, Kinichi Nakashima, Kiyokazu Agata, Takuya Imamura, Bidirectional promoters are the major source of gene activation-associated non-coding RNAs in mammals, BMC Genomics, 10.1186/1471-2164-15-35, 15, 1, 2014.01, Background: The majority of non-coding RNAs (ncRNAs) involved in mRNA metabolism in mammals have been believed to downregulate the corresponding mRNA expression level in a pre- or post-transcriptional manner by forming short or long ncRNA-mRNA duplex structures. Information on non-duplex-forming long ncRNAs is now also rapidly accumulating. To examine the directional properties of transcription at the whole-genome level, we performed directional RNA-seq analysis of mouse and chimpanzee tissue samples.Results: We found that there is only about 1% of the genome where both the top and bottom strands are utilized for transcription, suggesting that RNA-RNA duplexes are not abundantly formed. Focusing on transcription start sites (TSSs) of protein-coding genes revealed that a significant fraction of them contain switching-points that separate antisense- and sense-biased transcription, suggesting that head-to-head transcription is more prevalent than previously thought. More than 90% of head-to-head type promoters contain CpG islands. Moreover, CCG and CGG repeats are significantly enriched in the upstream regions and downstream regions, respectively, of TSSs located in head-to-head type promoters. Genes with tissue-specific promoter-associated ncRNAs (pancRNAs) show a positive correlation between the expression of their pancRNA and mRNA, which is in accord with the proposed role of pancRNA in facultative gene activation, whereas genes with constitutive expression generally lack pancRNAs.Conclusions: We propose that single-stranded ncRNA resulting from head-to-head transcription at GC-rich sequences regulates tissue-specific gene expression..
17. Junko Tomikawa, Yoshihisa Uenoyama, Makiko Ozawa, Tatsuya Fukanuma, Kenji Takase, Teppei Goto, Hitomi Abe, Nahoko Ieda, Shiori Minabe, Chikaya Deura, Naoko Inoue, Makoto Sanbo, Koichi Tomita, Masumi Hirabayashi, Satoshi Tanaka, Takuya Imamura, Hiroaki Okamura, Kei Ichiro Maeda, Hiroko Tsukamura, Epigenetic regulation of Kiss1 gene expression mediating estrogen-positive feedback action in the mouse brain, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1114245109, 109, 20, 2012.05, This study aims to determine the epigenetic mechanism regulating Kiss1 gene expression in the anteroventral periventricular nucleus (AVPV) to understand the mechanism underlying estrogen-positive feedback action on gonadotropin-releasing hormone/gonadotropin surge. We investigated estrogen regulation of the epigenetic status of the mouse AVPV Kiss1 gene locus in comparison with the arcuate nucleus (ARC), inwhich Kiss1 expression is down-regulated by estrogen. Histone of AVPV Kiss1 promoter region was highly acetylated, and estrogen receptor α was highly recruited at the region by estrogen. In contrast, the histone of ARC Kiss1 promoter region was deacetylated by estrogen. Inhibition of histone deacetylation upregulated in vitro Kiss1 expression in a hypothalamic non-Kiss1-expressing cell line. Gene conformation analysis indicated that estrogen induced formation of a chromatin loop between Kiss1 promoter and the 3′ intergenic region, suggesting that the intergenic region serves to enhance estrogen-dependent Kiss1 expression in the AVPV. This notion was proved, because transgenic reporter mice with a complete Kiss1 locus sequence showed kisspeptin neuronspecific GFP expression in both the AVPV and ARC, but the deletion of the 3′ region resulted in greatly reduced GFP expression only in the AVPV. Taken together, these results demonstrate that estrogen induces recruitment of estrogen receptor α and histone acetylation in the Kiss1 promoter region of the AVPV and consequently enhances chromatin loop formation of Kiss1 promoter and Kiss1 gene enhancer, resulting in an increase in AVPV-specific Kiss1 gene expression. These results indicate that epigenetic regulation of the Kiss1 gene is involved in estrogen-positive feedback to generate the gonadotropin-releasing hormone/gonadotropin surge..
18. 今村 拓也, Single-stranded Noncoding RNAs Mediate Local Epigenetic Alterations at Gene Promoters in Rat Cell Lines, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M111.275750, 286, 40, 34788-34799, 2011.10.
19. Takuya Imamura, Epigenetic setting for long-term expression of estrogen receptor α and androgen receptor in cells, Hormones and Behavior, 10.1016/j.yhbeh.2010.05.018, 59, 3, 345-352, 2011.03, Epigenetic regulation of the nuclear estrogen and androgen receptors, ER and AR, constitutes the molecular basis for the long-lasting effects of sex steroids on gene expression in cells. The effects prevail at hundreds of gene loci in the proximity of estrogen- and androgen-responsive elements and many more such loci through intra- and even inter-chromosomal level regulation. Such a memory system should be active in a flexible manner during the early development of vertebrates, and later replaced to establish more stable marks on genomic DNA. In mammals, DNA methylation is utilized as a very stable mark for silencing of the ER α and AR isoform expression during cancer cell and normal brain development. The factors affecting the DNA methylation of the ER α and AR genes in cells include estrogen and androgen. Since testosterone induces brain masculinization through its aromatization to estradiol in a narrow time window of the perinatal stage in rodents, the autoregulation of estrogen receptors, especially the predominant form of ER α, at the level of DNA methylation to set up the "cell memory" affecting the sexually differentiated status of brain function has been attracting increasing attention. The alternative usage of the androgen-AR system for brain masculinization and estrogenic regulation of AR expression in some species imply that the DNA methylation pattern of the AR gene can be established by closely related but different systems for sex steroid-induced phenomena, including brain masculinization..
20. Kinuyo Iwata, Mika Kinoshita, Shunji Yamada, Takuya Imamura, Yoshihisa Uenoyama, Hiroko Tsukamura, Kei Ichiro Maeda, Involvement of brain ketone bodies and the noradrenergic pathway in diabetic hyperphagia in rats, Journal of Physiological Sciences, 10.1007/s12576-010-0127-6, 61, 2, 103-113, 2011.03, Uncontrolled type 1 diabetes leads to hyperphagia and severe ketosis. This study was conducted to test the hypothesis that ketone bodies act on the hindbrain as a starvation signal to induce diabetic hyperphagia. Injection of an inhibitor of monocarboxylate transporter 1, a ketone body transporter, into the fourth ventricle normalized the increase in food intake in streptozotocin (STZ)-induced diabetic rats. Blockade of catecholamine synthesis in the hypothalamic paraventricular nucleus (PVN) also restored food intake to normal levels in diabetic animals. On the other hand, hindbrain injection of the ketone body induced feeding, hyperglycemia, and fatty acid mobilization via increased sympathetic activity and also norepinephrine release in the PVN. This result provides evidence that hyperphagia in STZ-induced type 1 diabetes is signaled by a ketone body sensed in the hindbrain, and mediated by noradrenergic inputs to the PVN..
21. Caroline Bacquet, Takuya Imamura, Claudio A. Gonzalez, Iván Conejeros, Gudrun Kausel, Thi My Anh Neildez-Nguyen, Andras Paldi, Milton H. Gallardo, Epigenetic processes in a tetraploid mammal, Mammalian Genome, 10.1007/s00335-008-9131-z, 19, 6, 439-447, 2008.06, Polyploidy has played a most important role in speciation and evolution of plants and animals. It is thought that low frequency of polyploidy in mammals is due to a dosage imbalance that would interfere with proper development in mammalian polyploids. The first tetraploid mammal, Tympanoctomys barrerae (Octodontidae), appears to be an exception to this rule. In this study we investigated X chromosome inactivation (XCI) and genomic imprinting in T. barrerae, two epigenetic processes usually involved in dosage control in mammalian genomes. The imprinting status of the Peg1 gene was determined by Peg1 allelic expression studies. The inactive X chromosome was identified on interphase nuclei by immunofluorescence using specific antisera raised against Met3H3K27 and macroH2A1. Quantitative PCR was used to compare the Peg1/Dmd ratio in T. barrerae and in its most closely related diploid species, Octomys mimax. Our data demonstrate that parental-specific silencing of at least one gene and normal X chromosomal dosage mechanism are conserved in the tetraploid genome. We hypothesize a concerted action of genetic and epigenetic mechanisms during the process of functional diploidization of this tetraploid genome..
22. 今村 拓也, Dynamic CpG and non-CpG methylation of the Peg1/Mest gene in the mouse oocyte and preimplantation embryo, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M501749200, 280, 20, 20171-20175, 2005.05.
23. Takuya Imamura, Nanami Miyauchi-Senda, Satoshi Tanaka, Kunio Shiota, Identification of genetic and epigenetic similarities of SPHK1/Sphk1 in mammals, Journal of Veterinary Medical Science, 10.1292/jvms.66.1387, 66, 11, 1387-1393, 2004.11, In normal tissues, methylation of CpG islands is generally accepted to be limited to the inactive X-chromosome and imprinting clusters. Gene Sphk1 has shown complex organization, indicated by multiple alternative splicing and tissue-dependent DNA methylation within the limited area (T-DMR) of the CpG island in the rat. Comparisons among human, mouse and rat SPHK1/Sphk1 genomic DNA revealed five coding exons and association of a CpG island at the 5′ end in common. We also found two novel subtypes, for a total of eight mRNA subtypes generated through selective usage of untranslated first exons. A 38-bp region at the 5′-end of T-DMR is highly conserved. This restricted area is specifically hypomethylated in the brain. Here, we examine the complex genetic/epigenetic features of the SPHK1/Sphk1 CpG island, and suggest that the T-DMR is the core target for tissue-dependent CpG island methylation..
24. Takuya Imamura, Non-coding RNA directed DNA demethylation of Sphk1 CpG island, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 10.1016/j.bbrc.2004.07.159, 322, 2, 593-600, 2004.09.
25. Takuya Imamura, Thi My Anh Neildez, Catherine Thenevin, Andras Paldi, Essential role for poly (ADP-ribosyl)ation in mouse preimplantation development, BMC Molecular Biology, 10.1186/1471-2199-5-4, 5, 2004.06, Background: Poly (ADP-ribosyl)ation is a covalent modification of many nuclear proteins. It has a strong chromatin modifying potential involved in DNA repair, transcription and replication. Its role during preimplantation development is unknown. Results: We have observed strong but transient synthesis of poly ADP-ribose polymers on decondensing chromosomes of fertilized and parthenogenetically activated mouse oocytes. Inhibition of this transient upregulation with a specific enzyme inhibitor, 3-aminobenzamide, has long-term effects on the postimplantation development of the embryos. In addition, inhibition of poly (ADP-ribosyl)ation at the 4-8 cell stage selectively blocks morula compaction. Conclusion: These observations suggest that poly (ADP-ribosyl)ation is involved in the epigenetic chromatin remodeling in the zygote..
26. Kunio Shiota, Yasushi Kogo, Jun Ohgane, Takuya Imamura, Atsushi Urano, Koichiro Nishino, Satoshi Tanaka, Naka Hattori, Epigenetic marks by DNA methylation specific to stem, germ and somatic cells in mice, Genes to Cells, 10.1046/j.1365-2443.2002.00574.x, 7, 9, 961-969, 2002.09, Background: DNA methylation is involved in many gene functions such as gene-silencing, X-inactivation, imprinting and stability of the gene. We recently found that some CpG islands had a tissue-dependent and differentially methylated region (T-DMR) in normal tissues, raising the possibility that there may be more CpG islands capable of differential methylation. Results: We investigated the genome-wide DNA methylation pattern of CpG islands by restriction landmark genomic scanning (RLGS) in mouse stem cells (ES, EG and trophoblast stem) before and after differentiation, and sperm as well as somatic tissues. A total of 247 spots out of 1500 (16%) showed differences in the appearance of their RLGS profiles, indicating that CpG islands having T-DMR were numerous and widespread. The methylation pattern was specific, and varied in a precise manner according to cell lineage, tissue type and during cell differentiation. Conclusions: Genomic loci with altered methylation status seem to be more common than has hitherto been realized. The formation of DNA methylation patterns at CpG islands is one of the epigenetic events which underlies the production of various cell types in the body. These findings should have implications for the use of embryonic stem cells and cells derived from them therapeutically, and also for the cloning of animals by the transfer of somatic cell nuclei..
27. 今村 拓也, CpG island of rat sphingosine kinase-1 gene: tissue-dependent DNA methylation status and multiple alternative first exons, GENOMICS, 76, 1-3, 117-125, 2001.08.
28. Takuya Imamura, Jun Ohgane, Seiichiro Ito, Tomoya Ogawa, Naka Hattori, Satoshi Tanaka, Kunio Shiota, CpG island of rat sphingosine kinase-1 gene
Tissue-dependent DNA methylation status and multiple alternative first exons, Genomics, 10.1006/geno.2001.6607, 76, 1-3, 117-125, 2001.07, It is generally recognized that CpG islands are not methylated in normal tissues. SPHK1 is a key enzyme catalyzing the production of sphingosine 1-phosphate, a novel signaling molecule for the proliferation and differentiation of various cells, including neural cells. Sequencing of genomic DNA and cDNA reveals that rat Sphk1a consists of six exons encoding 383 amino acids. Furthermore, we identified six alternative first exons for mRNA subtypes (Sphk1a, -b, -c, -d, -e, and -f) within a 3.7-kb CpG island. The CpG island contains a tissue-dependent, differentially methylated region (T-DMR; ∼ 200 bp), which is located ∼ 800 bp upstream of the first exon of Sphk1a. T-DMR is hypomethylated in the adult brain where Sphk1a is expressed, whereas it is hypermethylated in the adult heart where the gene is not expressed. In fetal tissues, hypomethylation of T-DMR is not associated with expression of Sphk1a, which suggests that differential availability of transcription factors is also likely to be involved in the mechanism of its expression. Here, we identify rat Sphk1, using multiple alternative first exons for the subtypes, and demonstrate that there is a CpG island bearing T-DMR..
29. Takuya Imamura, Naka Hattori, Kunio Shiota, Tomoya Ogawa, Generation and characterization of a monoclonal antibody recognizing a petal brain enriched O-linked sialoglycoprotein, FOG100, Journal of Biochemistry, 10.1093/oxfordjournals.jbchem.a022145, 124, 3, 534-539, 1998.01, With the aim of identifying molecules that are expressed specifically in the brain during neurogenesis, we tried to generate monoclonal antibodies which recognize molecules showing unique temporal expression patterns and molecular characteristics. We used a homogenate of the rat fetal forebrain (day 12 of fetal life, E12) as an immunogen, and antibodies which reacted with this preparation were screened by immunoblotting. One of the antibodies, Mab3C8, recognized a 100-kDa antigen that is enriched in fetal brain. This 100-kDa antigen was constantly expressed during fetal life (from E12 to E20) and became scarcely detectable two days after birth. The antigen was detected in the insoluble fraction of fetal brain and its isoelectric point ranged from 6 to 7, suggesting that it was a membrane-coupled glycoprotein. Analysis by glycosidase treatment and lectin blotting suggested that it was an O-linked glycoprotein with an α2,6 sialyl linkage. Thus, a molecule unique to the fetal brain, an O-linked sialoglycoprotein with a molecular mass of 100 kDa (FOG100), was found by generating an antibody..
30. Jun ichi Aikawa, Sakae Ikeda-Naiki, Jun Ohgane, Kwan Sik Min, Takuya Imamura, Ken Sasai, Kunio Shiota, Tomoya Ogawa, Molecular cloning of rat leukemia inhibitory factor receptor α-chain gene and its expression during pregnancy, Biochimica et Biophysica Acta - Gene Structure and Expression, 10.1016/S0167-4781(97)00079-1, 1353, 3, 266-276, 1997.09, Leukemia inhibitory factor (LIF) is a secreted glycoprotein and a pluripotent growth factor that acts on diverse cell systems. LIF transmits its effects via binding to transmembrane receptors, of which both high- and low-affinity forms have been identified. In this study, we analyzed the structure and expression of rat LIF receptor α-chain (rLIFR α) cDNA. A full-length clone of the cDNA encoding the membrane-bound form of rLIFR α protein was prepared by a combination of LA-PCR and 5' RACE using DNA reverse-transcribed from total RNA isolated from the livers of day-12 and day-14 pregnant rats as templates. The nucleotide sequence of a full-length clone was determined and further confirmed by analysis of shorter DNA fragment prepared by PCR using pfu polymerase. The gene for rLIFR α encodes a 1093 amine acid residue protein. The rLIFR α protein shows a high degree of similarity to mouse and human LIF receptor α-chain protein (89% and 76% amino acid sequence identifies, respectively). Only one molecular species of mRNA for the rLIFR α gene was detected in the liver and placenta. rLIFR α was expressed in liver of both non-pregnant and pregnant rats. The level of mRNA for the rLIFR α gene in placenta was maximum on day 16 of pregnancy..