Division of Gene Therapy Science
Division of Gene Therapy Science Graduate School of Medicine, Osaka University.
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III. Molecular mechanism of pathogenesis of cancer and congenital heart disease by breakdown of transcription regulation

Regulation of gene expression is necessary to establish cell identity. Transcription factors form huge complex with transcription regulating factors including epigenetic factors and RNA-binding proteins (Nimura et al. Nature 2009, eLife 2016). The transcription factor complex activates or represses genes, and regulates amounts of transcripts and splicing of the target genes. Although mutation in these factors is known to result in several diseases including congenital heart diseases (CHDs) and cancer, the molecular mechanisms of the pathogenesis are remained unclear. Thus, our goal is to understand the mechanisms how the dysregulation of gene expression causes CHDs and contributes the development and progress of cancer.

We continuously introduce and develop state-of-art technologies to elucidate the mechanisms of gene expression. Next-generation sequencer technology was previously introduced to our laboratory. Using this sequencer, we could develop native chromatin immunoprecipitation-sequencing (native ChIPseq). Native ChIPseq method allowed us to find novel binding profile of transcription factors (Nimura et al. eLife 2016). Bioinformatics skills are needed to handle and analyze the next-generation sequencing data. Because we can analyze the sequencing data, we seamlessly connect cell and individual biology with at genome-wide scale gene expression, letting us discover interesting mechanisms of transcription. Indeed, we have discovered that tissue-specific transcription factor regulates not only transcription initiation but also transcription termination, so called alternative polyadenylation (Nimura et al. eLife 2016).

CRISPR/Cas9 quickly becomes very popular technology to edit genome. We immediately tried this technology at the date the papers were shown up. CRISPR/Cas9 allowed us to study the mechanisms how prostate cancer obtains stem cell-like properties (Kawamura et al. Oncotarget). We establishes quick system to generate knock-out and knock-in cell lines using CRISPR/Cas9. Moreover we are now trying to adopt gRNA libraries for genome wide screening.

Super-resolution microscopy can resolve ~20 nm distance, it is 10 times-greater than the resolution in conventional light microscopy. Although conventional light microscopy cannot identify each nucleosome signals, super-resolution microscopy can distinguish each signal. Thus, nucleosome condensation can be target to analyze by super-resolution microscopy. We are now analyzing chromatin dynamics using this microscopy.

Transcription factors and epigenetic factors have a role in the huge protein complex. To elucidate the function of these factors, the transcription factor is needed to identify. We have employed tandem affinity purification (TAP) method to purify the transcription regulating factors-formed complex (Nimura et al. Nature 2009).

References
Original
  1. Nimura K(corresponding author), Yamamoto M, Takeichi M, Saga K, Takaoka K, Kawamura N, Nitta H, Nagano H, Ishino S, Tanaka T, Schwartz RJ, Aburatani H, Kaneda Y. Regulation of alternative polyadenylation by Nkx2-5 and Xrn2 during mouse heart development. eLife. 2016 Jun 22;5. pii: e16030. doi: 10.7554/eLife.16030.
  2. Kawano F, Nimura K, Ishino S, Nakai N, Nakata K, Ohira Y. Differences in histone modifications between slow- and fast-twitch muscle of adult rats and following overload, denervation or valproic acid administration. J Appl Physiol(1985). 2015, Nov 15;119(10):1042-52.
  3. Kawamura N, Nimura K(corresponding author), Nagano H, Yamaguchi S, Nonomura N, Kaneda Y. CRISPR/Cas9-mediated gene knockout of NANOG and NANOGP8 decreases the malignant potential of prostate cancer cells. Oncotarget. 2015 Sep 8;6(26):22361-74.
  4. Fujita R, Tamai K, Aikawa E, Nimura K, Ishino S, Kikuchi Y, Kaneda Y. Endogenous Mesenchymal Stromal Cells in Bone Marrow Are Required to Preserve Muscle Function in mdx Mice. Stem Cells. 2015 Mar;33(3):962-75.
  5. Lee Y F, Nimura K (corresponding author), Lo W N, Saga K, Kaneda Y. Histone H3 lysine 36 methyltransferase Whsc1 promotes the association of Runx2 and p300 in the activation of bone-related genes. PLoS One. 2014 Sep 4;9(9):e10661.
  6. Umegaki-Arao N, Tamai K, Nimura K, Serada S, Naka T, Nakano H, Katayama I. Karyopherin alpha2 is essential for rRNA transcription and protein synthesis in proliferative keratinocytes. PLoS One. 2013 Oct 3;8(10):e76416.
  7. Sarai N, Nimura K, Tamura T, Kanno T, Patel MC, Heightman TD, Ura K, Ozato K. WHSC1 links transcription elongation to HIRA-mediated histone H3.3 deposition. EMBO J. 2013 Aug 28;32(17):2392-406.
  8. Hatano K, Yamaguchi S, Nimura K, Murakami K, Nagahara A, Fujita K, Uemura M, Nakai Y, Tsuchiya M, Nakayama M, Nonomura N, Kaneda Y. Residual Prostate Cancer Cells after Docetaxel Therapy Increase the Tumorigenic Potential via Constitutive Signaling of CXCR4, ERK1/2 and c-Myc. Mol Cancer Res. 2013 Sep;11(9):1088-100.
  9. Takeichi M, Nimura K, Mori M, Nakagami H, Kaneda Y. The transcription factors Tbx18 and Wt1 control the epicardial epithelial-mesenchymal transition through bi-directional regulation of Slug in murine primary epicardial cells. PLoS One. 2013 8(2):e57829.
  10. Mori M, Nakagami H, Rodriguez-Araujo G, Nimura K, Kaneda Y. Essential role for miR-196a in brown adipogenesis of white fat progenitor cells. PLoS Biol. 2012;10(4):e1001314.
  11. Hatano K, Miyamoto Y, Mori M, Nimura K, Nakai Y, Nonomura N, Kaneda Y. Androgen-regulated transcriptional control of sialyltransferases in prostate cancer cells. PLoS One. 2012;7(2):e31234.
  12. Tamai K, Yamazaki T, Chino T, Ishii M, Otsuru S, Kikuchi Y, Iinuma S, Saga K, Nimura K, Shimbo T, Umegaki N, Katayama I, Miyazaki J, Takeda J, McGrath JA, Uitto J, Kaneda Y. PDGFRalpha-positive cells in bone marrow are mobilized by high mobility group box 1 (HMGB1) to regenerate injured epithelia. Proc Natl Acad Sci U S A. 2011 Apr 19;108(16):6609-14.
  13. Kashiwagi K, Nimura K, Ura K, Kaneda Y. DNA methyltransferase 3b preferentially associates with condensed chromatin. Nucleic Acids Res. 2011 Feb;39(3):874-88.
  14. Matsuda M, Nimura K, Shimbo T, Hamasaki T, Yamamoto T, Matsumura A, Kaneda Y. Immunogene therapy using immunomodulating HVJ-E vector augments anti-tumor effects in murine malignant glioma. J Neurooncol. 2011 May;103(1):19-31.
  15. Nimura K, Ura K, Shiratori H, Ikawa M, Okabe M, Schwartz RJ, Kaneda Y. A histone H3 lysine 36 trimethyltransferase links Nkx2-5 to Wolf-Hirschhorn syndrome. Nature. 2009 Jul 9;460(7252):287-91.
  16. Yuri S, Fujimura S, Nimura K, Takeda N, Toyooka Y, Fujimura Y, Aburatani H, Ura K, Koseki H, Niwa H, Nishinakamura R. Sall4 is essential for stabilization, but not for pluripotency, of embryonic stem cells by repressing aberrant trophectoderm gene expression. Stem Cells. 2009 Apr;27(4):796-805.
  17. Chino T, Tamai K, Yamazaki T, Otsuru S, Kikuchi Y, Nimura K, Endo M, Nagai M, Uitto J, Kitajima Y, Kaneda Y. Bone marrow cell transfer into fetal circulation can ameliorate genetic skin diseases by providing fibroblasts to the skin and inducing immune tolerance. Am J Pathol. 2008 Sep;173(3):803-14.
  18. Kawachi M, Tamai K, Saga K, Yamazaki T, Fujita H, Shimbo T, Kikuchi Y, Nimura K, Nishifuji K, Amagai M, Uitto J, Kaneda Y. Development of tissue-targeting hemagglutinating virus of Japan envelope vector for successful delivery of therapeutic gene to mouse skin. Hum Gene Ther. 2007 Oct;18(10):881-94.
  19. Hayashi M, Nimura K, Kashiwagi K, Harada T, Takaoka K, Kato H, Tamai K, Kaneda Y. Comparative roles of Twist-1 and Id1 in transcriptional regulation by BMP signaling. J Cell Sci. 2007 Apr 15;120(Pt 8):1350-7.
  20. Nimura K, Ishida C, Koriyama H, Hata K, Yamanaka S, Li E, Ura K, Kaneda Y. Dnmt3a2 targets endogenous Dnmt3L to ES cell chromatin and induces regional DNA methylation. Genes Cells. 2006 Oct;11(10):1225-37.
  21. Ito M, Yamamoto S, Nimura K, Hiraoka K, Tamai K, Kaneda Y. Rad51 siRNA delivered by HVJ envelope vector enhances the anti-cancer effect of cisplatin. J Gene Med. 2005 Aug;7(8):1044-52.
Review
  1. Nimura K (corresponding author) Kaneda Y. Elucidating the mechanisms of transcription regulation during heart development by next-generation sequencing. J Hum Genet. 2016 Jan;61(1)5-12.
  2. Nimura K (corresponding author). [Epigenetic regulation of transcription in heart development]. Seikagaku. 2011 Nov;83(11):1043-7. Review. Japanese.
  3. Nimura K (corresponding author), Ura K, Kaneda Y. Histone methyltransferases: regulation of transcription and contribution to human disease. J Mol Med (Berl). 2010 Dec;88(12):1213-20.
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Division of Gene Therapy Science, Graduate School of Medicine, Osaka University
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