Development of new types of retrovirus vectors that modulate epigenetical regulation.

2009 Fiscal Year Final Research Report

• Project/Area Number: 17016015
• Research Category: Grant-in-Aid for Scientific Research on Priority Areas
• Allocation Type: Single-year Grants
• Review Section: Biological Sciences
• Research Institution: The University of Tokyo
• Principal Investigator: IBA Hideo The University of Tokyo, 医科学研究所, 教授 (60111449)
• Co-Investigator(Kenkyū-buntansha): HARAGUCHI Takeshi 東京大学, 医科学研究所, 助教 (10549455) ; MIZUTANI Taketoshi 東京大学, 医科学研究所, 助教 (00376617) ; MINOGUCHI Shigeru 東京大学, 医科学研究所, 助教 (60322757) ; YAMAMICHI Nobutake 東京大学, 医学部附属病院, 非常勤医員 (30463897) ; ITO Taiji 東京大学, 医科学研究所, 助手 (60343109)
• Project Period (FY): 2005 – 2009
• Keywords: レトロウイルスベクター / RelB / requiem / 足場非依存的な増殖

Research Abstract

In this project, we have designed, prepared and improved several retrovirus/lentivirus vectors that carry efficient transcriptional units for the expression of short hairpin (sh) RNA, miRNA, and the newly developed RNA decoy molecule that inhibits specific miRNA (TuD RNA). Using these vector systems, we have studied cancer epigenetics by concentrating on SWI/SNF chromatin remodeling complex.
We have noticed that in human tumor cell lines that are deficient in Brm expression, expression of MLV and HIV vectors that were exogenously transduced into them are rapidly silenced stochastically. We finally demonstrated that Brm-type SWI/SNF complex is essential for the stable expression of retro/lentivirus.
Brm is further shown to have antioncogenic potential because exogenous introduction of Brm reduces oncogenic potential of cell lines deficient in Brm expression. In all these cell lines examined, the functional Brm gene is present and is actively transcribed; Brm expression was suppressed at t he post-transcriptional level. We hypothesized that Brm is targeted by certain miRNAs and screened several miRNA candidates and finally have shown that miR-199a target Brm mRNA. Interestingly, all the cancer cell lines deficient in Brm have high levels of miR-199a, whereas in Brm expressing cells, miR-199a was marginally expressed.
We further show these distinct expression patterns are resulted from double-negativefeedback regulation formed between Brm and miR-199a-5p/-3p via a transcription factor Egr1. We additionally have shown that miR-21 and its target NFIB, a negative transcriptional regulator, also forms a robust feedback regulation in cancer cell lines.
We have also shown human requiem protein functions as an adaptor protein that links SWI/SNF complex and RelB/p52. We further showed that introduction of shRNA against REQ strongly suppresses anchourage-independent growth, but does not affect growth in monolayer culture at all in tumor cell lines such as Panc-1, in which non-canonical NFκB pathway is constitutively activated.

Research Products

• [Journal Article] Loss of the Brm-type SWI/SNF chromatin remodeling complex is a strong barrier to the Tat-independent transcriptional elongation of HIV-1 transcripts. (2009)
  • Author(s): Mizutani T, Ishizaka A, Tomizawa M, Okazaki T, Yamamichi N, Kawana-Tachikawa A, Iwamoto A, Iba H.
  • Journal Title: J of Virol. 83 Pages: 11569-11580
  • Peer Reviewed
• [Journal Article] LNA in situ hybridization analysis of miR-21 expression during colorectal cancer development. (2009)
  • Author(s): Yamamichi N, Shimomura R, Inada K, Sakurai K, Haraguchi T, Ozaki Y, Fujita S, Mizutani T, Furukawa C, Fujishiro M, Ichinose M, Shiogama K, Tsutsumi Y, Omata M, Iba H.
  • Journal Title: Clin. Cancer Res. 15 Pages: 4009-4016
  • Peer Reviewed
• [Journal Article] Cdx2 and the Brm-type SWI/SNF complex cooperatively regulate villin expression in gastrointestinal cells. (2009)
  • Author(s): Yamamichi N, Inada K, Furukawa C, Sakurai K, Tando T, Ishizaka A, Haraguchi T, Mizutani T, Fujishiro M, Shimomura R, Oka M, Ichinose M, Tsutsumi Y, Omata M, Iba H.
  • Journal Title: Exp. Cell Res. 315 Pages: 1779-1789
  • Peer Reviewed
• [Journal Article] Vectors expressing efficient RNA decoys achieve the long-term suppression of specific microRNA activity in mammalian cells. (2009)
  • Author(s): Haraguchi T, Ozaki Y, Iba H.
  • Journal Title: Nucleic Acids Res. 37 Pages: e43
• [Journal Article] Mir-21 gene expression triggered by AP-1 is sustained through a double negative feedback mechanism. (2008)
  • Author(s): Fujita S, Ito T, Mizutani T, Minoguchi S, Yamamichi N, Sakurai K, Iba, H.
  • Journal Title: J. Mol. Biol 378 Pages: 492-504
• [Journal Article] Instability of retroviral DNA methylation in embryonic stem cells. (2008)
  • Author(s): Minoguchi S, Iba, H.
  • Journal Title: Stem Cells 26 Pages: 1166-1173
• [Journal Article] Class-specific regulation of pro-inflammatory genes by MYD88 pathways and IκBζ. (2008)
  • Author(s): Kayama H, Ramirez-Carrozzi VR, Yamamoto M, Mizutani T, Kuwata H, Iba H., Matsumoto M, Honda, K, Smale ST, Takeda, K.
  • Journal Title: J. Biol. Chem. 283 Pages: 12468-12477
• [Journal Article] Brm transactivates the telomerase reverse transcriptase (TERT) gene and modulates the splicing patterns of its transcripts in concert with p54nrb (2008)
  • Author(s): Ito T, Watanabe H, Yamamichi N, Kondo S, Tando T, Haraguchi T, Mizutani T, Sakurai K, Fujita S, Izumi T, Isobe T, Iba H.
  • Journal Title: Biochemical J. 411 Pages: 201-209
• [Journal Article] Putative promoter regions of miRNA genes involved in evolutionarily conserved regulatory systems among vertebrates. (2008)
  • Author(s): Fujita S, Iba H.
  • Journal Title: Bioinformatics 24 Pages: 303-308
• [Journal Article] SiRNAs do not induce RNA-dependent transcriptional silencing of retrovirus in human cells. (2007)
  • Author(s): Haraguchi T, Mizutani T, Yamamichi N, Ito T, Minoguchi S, Iba, H.
  • Journal Title: FEBS Letter 581 Pages: 4949-4954
• [Journal Article] Frequent loss of Brm expression in gastric cancer correlates with histological features and differentiation state. (2007)
  • Author(s): Haraguchi T, Fujita S, Tsutsumi, Y, Omata M, Iba, H.
  • Journal Title: Cancer Research 67 Pages: 10727-10735
• [Journal Article] Behavior of transplanted bone marrow derived mesenchymal stem cells in periodontal defects. (2006)
  • Author(s): Hasegawa N, Kawaguchi H, Hirachi A, Takeda K, Mizuno N, Nishimura M, Koike C, Tsuji K, Iba, H, Kato Y, Kurihara, H.
  • Journal Title: J. Periodontol. 77 Pages: 1003-7
• [Journal Article] A protein associated with Toll-like Receptor 4 (PRAT4A) regulates cell surface expression of TLR4. (2006)
  • Author(s): Wakabayashi Y, Kobayashi M, Akashi-Takamura S, Tanimura N, Konno K, Takahashi K, Ishii T, Mizutani T, Iba, H, Kouro T, Takaki S, Takatsu K., Oda, Y., Ishihama, Y., Saitoh, S -I., Miyake, K.
  • Journal Title: J. Immunol 177 Pages: 1772-1779
• [Journal Article] SWI/SNF complex is essential for NRSF-mediated suppression of neuronal genes in human nonsmall cell lung carcinoma cell lines. (2006)
  • Author(s): Watanabe H, Mizutani T, Haraguchi T, Yamamichi N, Minoguchi S, Yamamichi-Nishina M, Mori N, Kameda T, Sugiyama T, Iba H.
  • Journal Title: Oncogene 25 Pages: 470-479
• [Journal Article] The Brm gene suppressed at the post-transcriptional level in various human cell lines is inducible by transient HDAC inhibitor treatment, which exhibits anti-oncogenic potential. (2005)
  • Author(s): Yamamichi N, Yamamichi-Nishina M, Mizutani T, Watanabe H, Minoguchi S, Kobayashi N, Kimura S, Ito T, Yahagi N, Ichinose M, Omata M, Iba, H.
  • Journal Title: Oncogene 24 Pages: 5471-5481
• [Journal Article] Requiem protein links RelB/p52 and the Brm-type SWI/SNF complex in a non-canonical NFκB pathway.
  • Author(s): Tando T, Ishizaka A, Watanabe H, Ito T, Iida S, Haraguchi T, Mizutani T, Izumi T, Isobe T, Akiyama T, Inoue J, Iba H.
  • Journal Title: J Biol. Chem. (in press)
  • Peer Reviewed
• [Journal Article]
  • Author(s): Yamamichi N, Inada K, Ichinose M, Yamamichi-Nishina M, Mizutani T, Watanabe, H., Shiogama K, Okazaki T, Fujishiro M, Yahagi N
• [Presentation] 特定のmicroRNAを長期間、高効率で阻害するDecoy RNA発現ベクター (2009)
  • Author(s): 原口健、伊庭英夫
  • Organizer: 第68回日本癌学会学術総会
  • Place of Presentation: 横浜
  • Year and Date: 20091001-20091003
• [Presentation] Requiem functions in anon-canonical NFκB pathway as a linker protein between Rel/p52 and the SWI/SNF complex (2009)
  • Author(s): 丹藤利夫、石坂彩、原口健、水谷壮利、秋山泰身、井上純一郎、伊庭英夫
  • Organizer: 第32回日本分子生物学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2009-12-09
• [Presentation] 特定のmicroRNAを高効率で阻害するDecoy RNAの設計(Design of efficient RNA decoys that inhibit specific microRNA activity in mammalian cells. ) (2009)
  • Author(s): 原口健、伊庭英夫
  • Organizer: 第32回日本分子生物学会年会
  • Place of Presentation: 横浜
  • Year and Date: 2009-12-09
• [Presentation] Synthesis of 5'-capped sirna for suppression of off-target effects and its capability to achieveprotein expression (2009)
  • Author(s): Ogata, A., Ueno, Y., Furukawa, C., Sakurai, K., Iba, H., Kitamura, Y., Kitade, Y.
  • Organizer: Joint Symposium of 5th Annual Meeting of Oligonucleotide TherapeuticsSociety and the 19th Antisense Symposium
  • Place of Presentation: 福岡
  • Year and Date: 2009-11-03
• [Presentation] レトロウイルスと宿主細胞間の相互作用のエピジェネティクス-ニワトリレトロウイルス研究の魅力とその展開 (2009)
  • Author(s): 伊庭英夫
  • Organizer: 第57回日本ウイルス学会学術集会
  • Place of Presentation: 東京
  • Year and Date: 2009-10-26
• [Presentation] HIV-1 Tat非依存的な発現におけるSWI/SNF複合体の作用点の解析 (2009)
  • Author(s): 水谷壮利
  • Organizer: 第57回日本ウイルス学会学術集会
  • Place of Presentation: 東京
  • Year and Date: 2009-10-26
• [Presentation] EBウイルス感染を制御する宿主転写因子の同定 (2009)
  • Author(s): 浅井理沙、丹藤利夫、伊庭英夫、神田輝、高田賢蔵、上間匡、川口寧
  • Organizer: 第57回日本ウイルス学会学術集会
  • Place of Presentation: 東京
  • Year and Date: 2009-10-26
• [Presentation] クロマチン構造変換因子SWI/SNF複合体の胃癌における発現異常 (2009)
  • Author(s): 山道信毅、稲田健一、一瀬雅夫、藤城光弘、小俣政男、伊庭英夫
  • Organizer: 触媒サブユニットBrmの発現欠失、組織型・分化マーカーとの関係消化器病学会
  • Place of Presentation: 京都
  • Year and Date: 2009-10-14
• [Presentation] Gene regulatory networks formed between miR-21 and its targets during the course of carcinogenesis (2009)
  • Author(s): Iba, H.
  • Organizer: 第8回日中がんワークショップ
  • Place of Presentation: 大阪
  • Year and Date: 2009-10-05
• [Presentation] The Brm-type SWI/SNF chromatin remodeling complex plays an important role in transcriptional elongation of HIV-1 in the absence of Tat (2009)
  • Author(s): Ishizaka, A., Mizutani, T., Tomizawa, M., Kawana-Tachikawa, A., Iwamoto, A., Iba, H.
  • Organizer: The 9thAwaji International Forum on Infection and Immunity
  • Place of Presentation: 淡路島
  • Year and Date: 2009-09-08
• [Presentation] microRNAの異常発現と発がん (2009)
  • Author(s): 伊庭英夫
  • Organizer: 第41回ケムバイオシンポジウム次世代医療:microRNAとcancer stem cells
  • Place of Presentation: 東京工業大学大岡山キャンパス
  • Year and Date: 2009-03-05
• [Presentation] miR-21と標的遺伝子の発現制御ネットワーク解析(Analysis of regulatory network involving human miR-21 gene and its target genes. ) (2008)
  • Author(s): 藤田修二、山道信毅、原口健、尾崎由佳、伊庭英夫
  • Organizer: 第31回日本分子生物学会年会第81回日本生化学会大会合同大会
  • Place of Presentation: 神戸
  • Year and Date: 20081209-20081212
• [Presentation] ヒトvillin遺伝子の転写制御機構の解析 (2008)
  • Author(s): 古川千尋、山道信毅、丹藤利夫、石坂彩、櫻井浩平、原口健、稲田健一、下村龍一、伊庭英夫
  • Organizer: 第31回日本分子生物学会年会第81回日本生化学会大会合同大会
  • Place of Presentation: 神戸
  • Year and Date: 20081209-20081212
• [Presentation] miRNAが形成する制御ネットワークとがん (2008)
  • Author(s): 伊庭英夫、藤田修二、山道信毅、櫻井浩平、水谷壮利
  • Organizer: 第67回日本癌学会学術総会
  • Place of Presentation: 名古屋
  • Year and Date: 20081028-20081030
• [Presentation] HIV-1のTat非依存的な発現を維持するSWI/SNF複合体の作用機序の解析 (2008)
  • Author(s): 石坂彩、水谷壮利、山道信毅、原口健、富沢麻利子、岩本愛吉、伊庭英夫
  • Organizer: 第5回日本ウイルス学会学術集会
  • Place of Presentation: 岡山
  • Year and Date: 20081026-20081028
• [Presentation] A Catalytic Subunit of the SWI/SNF Complex is Required for Tat-independent Stable Expression of HIV-1 1st China-Japan (2008)
  • Author(s): A Catalytic Subunit of the SWI/SNF Complex is Required for Tat-independent Stable Expression of HIV-1 1st China-Japan
  • Organizer: Joint Workshop on New-Generation Vaccines for Infectious Diseases
  • Place of Presentation: 北京
  • Year and Date: 2008-05-22
• [Presentation] レトロウイルス遺伝子発現の不安定性の解析 (2007)
  • Author(s): 岡崎拓矢、山道信毅、水谷壮利、石坂彩、古川千尋、伊庭英夫
  • Organizer: 第30回日本分子生物学会年会第80回日本生化学会大会合同大会
  • Place of Presentation: 横浜
  • Year and Date: 20071211-20071215
• [Presentation] HIV-1のTat非依存的な発現におけるSWI/SNF複合体の機能解析 (2007)
  • Author(s): 水谷壮利、石坂彩、山道信毅、原口健、伊庭英夫
  • Organizer: 第55回日本ウイルス学会学術集会
  • Place of Presentation: 札幌
  • Year and Date: 20071021-20071023
• [Presentation] Brm, a catalytic subunit of the SWI/SNF complex is required for Tat-independent stable expression of HIV-1 (2007)
  • Author(s): Mizutani T, Ishizaka A, Yamamichi N, Okazaki T, Iba H.
  • Organizer: 第7回淡路島感染・免疫フォーラム
  • Place of Presentation: 兵庫
  • Year and Date: 20070901-20070905
• [Presentation] AP-1 triggered transcription of miR-21 through an evolutionally conserved mechanism Systems Biology (2007)
  • Author(s): Fujita S, Ito T, Mizutani T, Minoguchi S, Yamamichi N, Iba H.
  • Organizer: Global Regulation of Gene Expression
  • Place of Presentation: New York, USA
  • Year and Date: 20070300-20070400
• [Presentation] The Novel Role of Dnmt3L in Maintenance of DNA Methylation-Dependent Retroviral Gene Silencing (2007)
  • Author(s): 箕口滋、伊庭英夫
  • Organizer: 理化学研究所発生再生科学総合研究センターCDB Symposium 2007
  • Place of Presentation: 神戸
  • Year and Date: 20070300
• [Presentation] 幹細胞におけるDNAメチル化の動態制御機構 (2007)
  • Author(s): 箕口滋
  • Organizer: 熊本大学発生医学研究センター21世紀COEリエゾンラボ研究会
  • Place of Presentation: 熊本
  • Year and Date: 20070200
• [Presentation] AP-1 triggers the sustained transcription of miR-21 through an evolutionarily conserved feedback mechanism (2007)
  • Author(s): Fujita S, Ito T, Mizutani T, Minoguchi S, Yamamichi N, Sakurai K, Iba H.
  • Organizer: The 11th German-Japanese Cancer Workshop
  • Place of Presentation: 京都
  • Year and Date: 2007-11-30
• [Presentation] Frequent Loss of Brm Expression in Gastric Cancer Correlates with Histological Features and Differentiation State (2007)
  • Author(s): Yamamichi N, Inada K, Iba H.
  • Organizer: 第14回The East Asian Joint Symposium (EAJS14): Expanding Scientific Extending Therapeutic Horizons
  • Place of Presentation: 東京
  • Year and Date: 2007-11-13
• [Presentation] High expression of miR-21 was detected in colorectal carcinoma but not in adenoma by LNA-based in situhybridization (2007)
  • Author(s): 山道信毅、下村龍一、水谷壮利、稲田健一、藤田修二、櫻井浩平、一瀬雅夫、堤寛、小俣政男、伊庭英夫
  • Organizer: 第66回日本癌学会総会
  • Place of Presentation: 横浜
  • Year and Date: 2007-10-05
• [Presentation] SWI/SNF複合体の触媒サブユニットBrmはTERT遺伝子の転写と選択的スプライシングに関与する (2006)
  • Author(s): 伊藤太二、渡部博貴、山道信毅、近藤俊輔、板垣千春、丹藤利夫、水谷壮利、原口健、櫻井浩平、藤田修二、泉友則、礒辺俊明、伊庭英夫
  • Organizer: 日本分子生物学会2006フォーラム(分子生物学の未来)
  • Place of Presentation: 名古屋
  • Year and Date: 20061206-20061208
• [Presentation] SWI/SNF chromatin remodeling complex and cancer (2006)
  • Author(s): Iba, H.
  • Organizer: The 10th Korea-Japan Cancer Research Workshop
  • Place of Presentation: Tokyo, Japan
  • Year and Date: 20061200
• [Presentation] クロマチン構造変換因子SWI/SNF複合体のHIV-1発現維持機構の機能解析 (2006)
  • Author(s): 水谷壮利、石坂彩、山道信毅、岡崎拓矢、伊庭英夫
  • Organizer: 日本分子生物学会2006フォーラム
  • Place of Presentation: 名古屋
  • Year and Date: 20061200
• [Presentation] レトロウイルスプロモーターサイレンシングにおけるRNAi機構の役割の検証 (2006)
  • Author(s): 原口健、水谷壮利、伊庭英夫
  • Organizer: 第54回日本ウイルス学会学術集会
  • Place of Presentation: 名古屋
  • Year and Date: 20061119-20061121
• [Presentation] 多くのヒト癌で高レベルに発現するmir-21遺伝子のプロモーター解析とmir-21の標的遺伝子の同定 (2006)
  • Author(s): 伊庭英夫、藤田修二、伊藤太二、水谷壮利
  • Organizer: 第65回日本癌学会学術総会
  • Place of Presentation: 横浜
  • Year and Date: 20060928-20060930
• [Presentation] 胃癌におけるクロマチン構造変換因子SWI/SNF複合体の触媒サブユニットBrm欠失:癌化におけるBrm欠失の役割 (2006)
  • Author(s): 山道信毅、稲田健一、渡部博貴、山道(仁科)光恵、水谷壮利、矢作直久、一瀬雅夫、堤寛、小俣政男、伊庭英夫
  • Organizer: 第65回日本癌学会学術総会
  • Place of Presentation: 横浜
  • Year and Date: 20060928-20060930
• [Presentation] Structure of human miR-21 promoter that is driven by AP-1 Computational prediction of promoter regions of vertebrate miRNAs (2006)
  • Author(s): Fujita S, Ito T, Yamamichi-Nishina M, Iba H.
  • Organizer: 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress
  • Place of Presentation: Kyoto, Japan
  • Year and Date: 20060600
• [Presentation] ES細胞に感染したレトロウイルスに見られるDNAメチル化に依存した可逆的ジーンサイレンシング (2005)
  • Author(s): 箕口滋、渡部博貴、水谷壮利、伊庭英夫
  • Organizer: 第28回日本分子生物学会年会
  • Place of Presentation: 福岡
  • Year and Date: 20051207-20051210
• [Presentation] p54nrbとSWI/SNF複合体の相互作用とその生化学的意義 (2005)
  • Author(s): 伊藤太二、渡部博貴、山道信毅、近藤俊輔、板垣千春、丹藤利夫、水谷壮利、原口健、藤田修二、泉友則、礒辺俊明、伊庭英夫
  • Organizer: 第28回日本分子生物学会年会
  • Place of Presentation: 福岡
  • Year and Date: 20051207-20051210
• [Presentation] Reversible switching of MSCV Proviral Expression Reflects Dynamics of DNA Methylation in Embryonic Stem Cells (2005)
  • Author(s): 箕口滋、渡部博貴、水谷壮利、伊庭英夫
  • Organizer: 文部科学省特定領域研究「幹細胞の可塑性と未分化性維持機構」国際シンポジウム
  • Place of Presentation: 京都
  • Year and Date: 20051100
• [Presentation] ヒト癌細胞株で転写後抑制を受けているSWI/SNF複合体の触媒サブユニットBrmは、HDAC阻害剤により発現誘導され抗癌活性を示す (2005)
  • Author(s): 山道信毅、水谷壮利、渡部博貴、伊藤太二、矢作直久、一瀬雅夫、小俣政男、伊庭英夫
  • Organizer: 第64回日本癌学会学術総会
  • Place of Presentation: 札幌
  • Year and Date: 20050914-20050916
• [Presentation] BrmとBRG1の発現を共に欠失するヒト非小細胞肺がん細胞株における神経特異的遺伝子群の発現誘導とその機構 (2005)
  • Author(s): 渡部博貴、水谷壮利、山道信毅、伊庭英夫
  • Organizer: 第64回日本癌学会学術総会
  • Place of Presentation: 札幌
  • Year and Date: 20050914-20050916
• [Presentation] Unique expression mechanisms of Brm protein, a key regulatory host factor for continuous retroviral expression (2005)
  • Author(s): Yamamichi N, Mizutani T, Yamamichi-Nishina M, Haraguchi T, Kameda T, Iba H.
  • Organizer: 第5回あわじしま感染症・免疫フォーラム
  • Place of Presentation: 兵庫
  • Year and Date: 20050900
• [Presentation] レトロウイルスの発現維持に必須な宿主因子Brmの特異な生合成過程 (2005)
  • Author(s): 伊庭英夫、山道信毅、水谷壮利、原口健
  • Organizer: 第53回日本ウイルス学会学術集会
  • Place of Presentation: 横浜
  • Year and Date: 2005-11-22
• [Remarks]
  • URL: http://www.ims.u-tokyo.ac.jp/div-host-parasite/Version1.html