Research activity information

• SAMD4 represses VGLL4 mRNA to activate TEAD and promote cancer progression

  Junji Otani, Miki Nishio, Riko Tokita, Hiroki Hikasa, Makoto Nishimori, Shingo Dan, Isao Naguro, Hidenori Ichijo, Masanori Miyanishi, Takehiko Sasaki, Hiroshi Nishina, Tak Wah Mak, Tomohiko Maehama, Akira Suzuki

  Jun. 2026, Oncogene

  Scientific journal


• MOB1 deletion in murine mature adipocytes ameliorates obesity and diabetes.

  Miki Nishio, Keiko Yamaguchi, Junji Otani, Katsuya Yuguchi, Daisuke Kohno, Tsutomu Sasaki, Tadahiro Kitamura, Masakazu Shinohara, Tomoyoshi Soga, Koichi Kawamura, Atsuo T Sasaki, Masashi Oshima, Hiroki Hikasa, Minna Woo, Takehiko Sasaki, Hiroshi Nishina, Kazuwa Nakao, Tomohiko Maehama, Akira Suzuki

  There is currently a global epidemic of obesity and obesity-related diseases such as type 2 diabetes due to decreased physical activity, excessive food intake, and/or genetic predisposition. The Hippo-YAP1 pathway has attracted attention as a potential therapeutic target because YAP1/TAZ activation in murine immature adipocytes in vitro suppresses their differentiation by inhibiting PPARγ activity. However, the role of YAP1 activation in mature adipocytes in vivo remains unclear. MOB1, whose expression is increased in obesity, is the hub of the Hippo core molecule complex and negatively regulates YAP1/TAZ activation. Therefore, we generated aMob1DKO mutant mice, which feature deficiency of Mob1a/b specifically in mature adipocytes. Compared to controls, aMob1DKO mice subjected to a high-fat diet showed beneficial changes consistent with resistance to diet-induced obesity. The mutants exhibited increases in basal lipolysis, "beiging," and energy expenditure, as well as suppression of ROS production and inflammation in white adipose tissue. Insulin sensitivity and glucose tolerance were improved, and ectopic fat accumulation was reduced. Most of these changes were dependent on the YAP1 activation observed in mature white adipose tissue of aMob1DKO mice. FGF21, which improves lipid metabolism, was upregulated directly via YAP1 activation, and many of the phenotypes seen in aMob1DKO mice were also dependent on FGF21. Thus, the aMob1DKO mouse is an interesting model for the study of the metabolic effects of diet-induced obesity and protection against diabetes. Our work suggests that a YAP1-FGF21 axis exists in adipocytes that may be a potential therapeutic target for obesity.

  Apr. 2025, Proceedings of the National Academy of Sciences of the United States of America, 122(17) (17), e2424741122, English, International magazine

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Nucleolar stress: Molecular mechanisms and related human diseases.

  Tomohiko Maehama, Miki Nishio, Junji Otani, Tak Wah Mak, Akira Suzuki

  Ribosome biogenesis in the nucleolus is an important process that consumes 80% of a cell's intracellular energy supply. Disruption of this process results in nucleolar stress, triggering the activation of molecular systems that respond to this stress to maintain homeostasis. Although nucleolar stress was originally thought to be caused solely by abnormalities of ribosomal RNA (rRNA) and ribosomal proteins (RPs), an accumulating body of more current evidence suggests that many other factors, including the DNA damage response and oncogenic stress, are also involved in nucleolar stress response signaling. Cells reacting to nucleolar stress undergo cell cycle arrest or programmed death, mainly driven by activation of the tumor suppressor p53. This observation has nominated nucleolar stress as a promising target for cancer therapy. However, paradoxically, some RP mutations have also been implicated in cancer initiation and progression, necessitating caution. In this article, we summarize recent findings on the molecular mechanisms of nucleolar stress and the human ribosomal diseases and cancers that arise in its wake.

  Feb. 2023, Cancer science, English, International magazine

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• N-(3,4-dimethoxyphenethyl)-6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine inhibits bladder cancer progression by suppressing YAP1/TAZ

  Yusuke Shiraishi, Tomohiko Maehama, Miki Nishio, Junji Otani, Hiroki Hikasa, Tak Wah Mak, Takehiko Sasaki, Teruki Honma, Yasumitsu Kondoh, Hiroyuki Osada, Minoru Yoshida, Masato Fujisawa, Akira Suzuki

  Oct. 2022, GENES TO CELLS, 27(10) (10), 602 - 612, English

  Scientific journal


• The TIGD5 gene located in 8q24 and frequently amplified in ovarian cancers is a tumor suppressor

  Yuntao Dai, Tetsuya Kawaguchi, Miki Nishio, Junji Otani, Hironori Tashiro, Yoshito Terai, Ryohei Sasaki, Tomohiko Maehama, Akira Suzuki

  Aug. 2022, GENES TO CELLS, English

  Scientific journal


• Hippo-TAZ signaling is the master regulator of the onset of triple-negative basal-like breast cancers

  Hirotoshi Soyama, Miki Nishio, Junji Otani, Toshiko Sakuma, Shintaro Takao, Shigeo Hara, Takaaki Masuda, Koshi Mimori, Shinya Toyokuni, John P. Lydon, Kazuwa Nakao, Hiroshi Nishina, Takumi Fukumoto, Tomohiko Maehama, Akira Suzuki

  Jul. 2022, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 119(29) (29), English

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• 動物モデルによる先端がん研究 Hippoシグナルによるがんの発症進展制御

  西尾 美希, 前濱 朝彦, 大谷 淳二, 曽山 弘敏, 鈴木 聡

  (公社)日本生化学会, Nov. 2021, 日本生化学会大会プログラム・講演要旨集, 94回, [1S04a - 01], Japanese


• Alantolactone is a natural product that potently inhibits YAP1/TAZ through promotion of reactive oxygen species accumulation

  Keisuke Nakatani, Tomohiko Maehama, Miki Nishio, Junji Otani, Keiko Yamaguchi, Miki Fukumoto, Hiroki Hikasa, Shinji Hagiwara, Hiroshi Nishina, Tak Wah Mak, Teruki Honma, Yasumitsu Kondoh, Hiroyuki Osada, Minoru Yoshida, Akira Suzuki

  Wiley, Aug. 2021, Cancer Science

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• TAZ inhibits acinar cell differentiation but promotes immature ductal cell proliferation in adult mouse salivary glands.

  Yosuke Miyachi, Miki Nishio, Junji Otani, Shinji Matsumoto, Akira Kikuchi, Tak Wah Mak, Tomohiko Maehama, Akira Suzuki

  There are currently no treatments for salivary gland diseases, making it vital to understand signaling mechanisms operating in acinar and ductal cells so as to develop regenerative therapies. To date, little work has focused on elucidating the signaling cascades controlling the differentiation of these cell types in adult mammals. To analyze the function of the Hippo-TAZ/YAP1 pathway in adult mouse salivary glands, we generated adMOB1DKO mice in which both MOB1A and MOB1B were TAM-inducibly deleted when the animals were adults. Three weeks after TAM treatment, adMOB1DKO mice exhibited smaller submandibular glands (SMGs) than controls with a decreased number of acinar cells and an increased number of immature dysplastic ductal cells. The mutants suffered from reduced saliva production accompanied by mild inflammatory cell infiltration and fibrosis in SMGs, similar to the Sjogren's syndrome. MOB1-deficient acinar cells showed normal proliferation and apoptosis but decreased differentiation, leading to an increase in acinar/ductal bilineage progenitor cells. These changes were TAZ-dependent but YAP1-independent. Biochemically, MOB1-deficient salivary epithelial cells showed activation of the TAZ/YAP1 and β-catenin in ductal cells, but reduced SOX2 and SOX10 expression in acinar cells. Thus, Hippo-TAZ signaling is critical for proper ductal and acinar cell differentiation and function in adult mice.

  Jun. 2021, Genes to cells : devoted to molecular & cellular mechanisms, 26(9) (9), 714 - 726, English, International magazine

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• A natural product shows antitumor activity through the inhibition of YAP1/TAZ

  Miki Nishio, Keisuke Nakatani, Junji Otani, Hiroki Hikasa, Tomohiko Maehama, Akira Suzuki

  Feb. 2021, CANCER SCIENCE, 112, 898 - 898, English


• The role of Hippo-YAP signaling in squamous cell carcinomas.

  Tomohiko Maehama, Miki Nishio, Junji Otani, Tak Wah Mak, Akira Suzuki

  The Hippo-YAP pathway regulates organ size, tissue homeostasis, and tumorigenesis in mammals. In response to cell density, external mechanical pressure, and/or other stimuli, the Hippo core complex controls the translocation of YAP1/TAZ proteins to the nucleus and thereby regulates cell growth. Abnormal upregulation or nuclear localization of YAP1/TAZ occurs in many human malignancies and promotes their formation, progression, and metastasis. A key example is squamous cell carcinoma (SCC) genesis. Many risk factors and crucial signals associated with SCC development in various tissues accelerate YAP1/TAZ accumulation, and mice possessing constitutively activated YAP1/TAZ show immediate carcinoma in situ (CIS) formation in these tissues. Because CIS onset is so rapid in these mutants, we propose that many SCCs initiate and progress when YAP1 activity is sustained and exceeds a certain oncogenic threshold. In this review, we summarize the latest findings on the roles of YAP1/TAZ in several types of SCCs. We also discuss whether targeting aberrant YAP1/TAZ activation might be a promising strategy for SCC treatment.

  Jan. 2021, Cancer science, 112(1) (1), 51 - 60, English, International magazine

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• YAP1/TAZ阻害による抗腫瘍作用を示す天然物の同定

  西尾 美希, 中谷 圭佑, 大谷 淳二, 日笠 弘基, 前濱 朝彦, 鈴木 聡

  (一社)日本癌学会, Oct. 2020, 日本癌学会総会記事, 79回, PJ16 - 5, English


• YAP1/TAZ阻害による抗腫瘍作用を示す天然物の同定

  西尾 美希, 中谷 圭佑, 大谷 淳二, 日笠 弘基, 前濱 朝彦, 鈴木 聡

  (一社)日本癌学会, Oct. 2020, 日本癌学会総会記事, 79回, PJ16 - 5, English


• Endogenous YAP1 activation drives immediate onset of cervical carcinoma in situ in mice.

  Miki Nishio, Yoko To, Tomohiko Maehama, Yukari Aono, Junji Otani, Hiroki Hikasa, Akihiro Kitagawa, Koshi Mimori, Takehiko Sasaki, Hiroshi Nishina, Shinya Toyokuni, John P Lydon, Kazuwa Nakao, Tak Wah Mak, Tohru Kiyono, Hidetaka Katabuchi, Hironori Tashiro, Akira Suzuki

  Cervical cancer (CC) is usually initiated by infection with high-risk types of human papillomavirus (HPV). The HPV E6 and E7 proteins target p53 and RB, respectively, but other cellular targets likely exist. We generated uterus-specific MOB1A/B double KO (uMob1DKO) mice, which immediately developed cervical squamous cell carcinoma in situ. Mutant cervical epithelial cells showed YAP1-dependent hyperproliferation, altered self-renewal, impaired contact inhibition, and chromosomal instability. p53 activation was increased in uMob1DKO cells, and additional p53 loss in uMob1DKO mice accelerated tumor invasion. In human CC, strong YAP1 activation was observed from the precancerous stage. Human cells overexpressing HPV16 E6/E7 showed inactivation of not only p53 and RB but also PTPN14, boosting YAP1 activation. Estrogen, cigarette smoke condensate, and PI3K hyperactivation all increased YAP1 activity in human cervical epithelial cells, and PTPN14 depletion along with PI3K activation or estrogen treatment further enhanced YAP1. Thus, immediate CC onset may initiate when YAP1 activity exceeds an oncogenic threshold, making Hippo-YAP1 signaling a major CC driver.

  Oct. 2020, Cancer science, 111(10) (10), 3576 - 3587, English, International magazine

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• Enhanced processivity of Dnmt1 by monoubiquitinated histone H3.

  Yuichi Mishima, Laura Brueckner, Saori Takahashi, Toru Kawakami, Junji Otani, Akira Shinohara, Kohei Takeshita, Ronald Garingalao Garvilles, Mikio Watanabe, Norio Sakai, Hideyuki Takeshima, Charlotte Nachtegael, Atsuya Nishiyama, Makoto Nakanishi, Kyohei Arita, Kinichi Nakashima, Hironobu Hojo, Isao Suetake

  DNA methylation controls gene expression, and once established, DNA methylation patterns are faithfully copied during DNA replication by the maintenance DNA methyltransferase Dnmt1. In vivo, Dnmt1 interacts with Uhrf1, which recognizes hemimethylated CpGs. Recently, we reported that Uhrf1-catalyzed K18- and K23-ubiquitinated histone H3 binds to the N-terminal region (the replication focus targeting sequence, RFTS) of Dnmt1 to stimulate its methyltransferase activity. However, it is not yet fully understood how ubiquitinated histone H3 stimulates Dnmt1 activity. Here, we show that monoubiquitinated histone H3 stimulates Dnmt1 activity toward DNA with multiple hemimethylated CpGs but not toward DNA with only a single hemimethylated CpG, suggesting an influence of ubiquitination on the processivity of Dnmt1. The Dnmt1 activity stimulated by monoubiquitinated histone H3 was additively enhanced by the Uhrf1 SRA domain, which also binds to RFTS. Thus, Dnmt1 activity is regulated by catalysis (ubiquitination)-dependent and -independent functions of Uhrf1.

  Jan. 2020, Genes to cells : devoted to molecular & cellular mechanisms, 25(1) (1), 22 - 32, English, International magazine

  [Refereed]

  Scientific journal


• Hippo pathway controls cell adhesion and context-dependent cell competition to influence skin engraftment efficiency.

  Miki Nishio, Yousuke Miyachi, Junji Otani, Shoji Tane, Hirofumi Omori, Fumihito Ueda, Hideru Togashi, Takehiko Sasaki, Tak Wah Mak, Kazuwa Nakao, Yasuyuki Fujita, Hiroshi Nishina, Tomohiko Maehama, Akira Suzuki

  Cell competition is involved in mammalian embryogenesis and tumor elimination and progression. It was previously shown that, whereas NIH3T3 mouse fibroblasts expressing high levels of the yes-associated protein 1(YAP1) target TEA domain family (TEAD) transcription factors become "winners" in cell competitions, Madin-Darby canine kidney cells expressing activated YAP1 become "losers" and are eliminated from culture monolayers. Thus, YAP1's role in cell competitions is clearly context dependent. Here, we show that keratinocytes overexpressing a constitutively activated YAP1 mutant lose in in vitro competitions with control cells conducted in standard tissue culture dishes and undergo apical extrusion. Similarly, cells in which endogenous YAP1 is activated by NF2 knockdown become losers. The YAP1-overexpressing cells exhibit a decrease in cell-matrix adhesion because of defective expression of adhesion molecules such as fibronectin-1. Cell adhesion-mediated proliferation is also impaired. However, because of intrinsic factors, YAP1-expressing cells proliferate faster than control cells when cocultured in dishes impeding cell adhesion. In vivo, Mob1a/b-deficient (YAP1-activated) epidermis, which shows decreased expression of type XVII collagen, cannot be engrafted successfully onto donor mice. YAP1-activated skin grafts shrink away from surrounding control skin, and the epidermis peels off the basement membrane. Our data show that YAP1 activation controls cell competition in part by decreasing cell adhesion.-Nishio, M., Miyachi, Y., Otani, J., Tane, S., Omori, H., Ueda, F., Togashi, H., Sasaki, T., Mak, T. W., Nakao, K., Fujita, Y., Nishina, H., Maehama, T., Suzuki, A. Hippo pathway controls cell adhesion and context-dependent cell competition to influence skin engraftment efficiency.

  Apr. 2019, FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 33(4) (4), 5548 - 5560, English, International magazine

  Scientific journal


• RFTS-dependent negative regulation of Dnmt1 by nucleosome structure and histone tails.

  Yuichi Mishima, Laura Brueckner, Saori Takahashi, Toru Kawakami, Kyohei Arita, Shota Oka, Junji Otani, Hironobu Hojo, Masahiro Shirakawa, Akira Shinohara, Mikio Watanabe, Isao Suetake

  Oct. 2017, The FEBS journal, 284(20) (20), 3455 - 3469, English, International magazine

  [Refereed]

  Scientific journal

  Link to Kobe Univ. Repository (Kernel)


• NEK6-mediated phosphorylation of human TPP1 regulates telomere length through telomerase recruitment.

  Yugo Hirai, Miki Tamura, Junji Otani, Fuyuki Ishikawa

  Aug. 2016, Genes to cells : devoted to molecular & cellular mechanisms, 21(8) (8), 874 - 89, English, International magazine

  [Refereed]

  Scientific journal


• Nucleosome compaction facilitates HP1γ binding to methylated H3K9.

  Yuichi Mishima, Chanika D Jayasinghe, Kai Lu, Junji Otani, Masahiro Shirakawa, Toru Kawakami, Hironobu Kimura, Hironobu Hojo, Peter Carlton, Shoji Tajima, Isao Suetake

  Dec. 2015, Nucleic acids research, 43(21) (21), 10200 - 12, English, International magazine

  [Refereed]

  Scientific journal


• [Structure basis of versatile base recognition of MBD4].

  Mariko Ariyoshi, Junji Otani, Masahiro Shirakawa

  2015, Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan, 135(1) (1), 3 - 9, Japanese, Domestic magazine

  [Refereed]

  Scientific journal


• Quantitative comparison of protein dynamics in live cells and in vitro by in-cell (19)F-NMR.

  Yousuke Takaoka, Yoshiyuki Kioi, Akira Morito, Junji Otani, Kyohei Arita, Eishi Ashihara, Mariko Ariyoshi, Hidehito Tochio, Masahiro Shirakawa, Itaru Hamachi

  Apr. 2013, Chemical communications (Cambridge, England), 49(27) (27), 2801 - 3, English, International magazine

  [Refereed]

  Scientific journal


• メチル化DNA結合蛋白質MBD4による緩い基質DNA認識

  大谷淳二, 有吉眞理子, 白川昌宏

  Apr. 2013, BioMedサーカス.com http://biomedcircus.com/paper_03_15.html, Japanese

  Scientific journal


• Structural basis of the versatile DNA recognition ability of the methyl-CpG binding domain of methyl-CpG binding domain protein 4.

  Junji Otani, Kyohei Arita, Tsuyoshi Kato, Mariko Kinoshita, Hironobu Kimura, Isao Suetake, Shoji Tajima, Mariko Ariyoshi, Masahiro Shirakawa

  Mar. 2013, The Journal of biological chemistry, 288(9) (9), 6351 - 62, English, International magazine

  [Refereed]

  Scientific journal


• Hinge and chromoshadow of HP1α participate in recognition of K9 methylated histone H3 in nucleosomes.

  Yuichi Mishima, Makoto Watanabe, Toru Kawakami, Chanika D Jayasinghe, Junji Otani, Yusuke Kikugawa, Masahiro Shirakawa, Hiroshi Kimura, Osamu Nishimura, Saburo Aimoto, Shoji Tajima, Isao Suetake

  Jan. 2013, Journal of molecular biology, 425(1) (1), 54 - 70, English, International magazine

  [Refereed]

  Scientific journal


• 2SBP-01 Structural insight into versatile DNA recognition of methyl CpG binding protein MBD4(2SBP Transient macromolecular complexes involved in multilevel biological phenomena,Symposium,The 51th Annual Meeting of the Biophysical Society of Japan)

  Ariyoshi Mariko, Otani Jyunji, Shirakawa Masahiro

  The Biophysical Society of Japan General Incorporated Association, 2013, Seibutsu Butsuri, 53(1) (1), S97, English


• Cell cycle-dependent turnover of 5-hydroxymethyl cytosine in mouse embryonic stem cells.

  Junji Otani, Hironobu Kimura, Jafar Sharif, Takaho A Endo, Yuichi Mishima, Toru Kawakami, Haruhiko Koseki, Masahiro Shirakawa, Isao Suetake, Shoji Tajima

  2013, PloS one, 8(12) (12), e82961, English, International magazine

  [Refereed]

  Scientific journal


• Structural basis for recognition of H3K4 methylation status by the DNA methyltransferase 3A ATRX-DNMT3-DNMT3L domain.

  Junji Otani, Toshiyuki Nankumo, Kyohei Arita, Susumu Inamoto, Mariko Ariyoshi, Masahiro Shirakawa

  Nov. 2009, EMBO reports, 10(11) (11), 1235 - 41, English, International magazine

  [Refereed]

  Scientific journal

• HP1γによるヌクレオソーム構造依存的なヒストンH3K9me3の認識機構

  三島優一, 川上徹, 大谷淳二, 木村博信, 白川昌宏, 白川昌宏, 田嶋正二, 末武勲, 末武勲

  2014, 日本分子生物学会年会プログラム・要旨集(Web), 37th


• Structure basis for regulation of DNA methylation.

  Ariyoshi, M, Otani, J, Arita, K, Kinoshita, M, Shirakawa, M

  Dec. 2011, 第34回 分子生物学会年会 Workshop “Molecular basis of gene regulation and genome maintenance in chromosomes”, English


• Structural Basis of versatile DNA recognition of MBD4.

  Ariyoshi, M, Otani, J, Arita, K, Kinoshita, M, Shirakawa, M

  Aug. 2011, 第12回 国際結晶学会 Mini Symposium “Nucleosome Processing and Epigenetics”, English

• YAP転写複合体制御機構の解明とその応用

  大谷 淳二, 西尾 美希

  日本学術振興会, 科学研究費助成事業, 基盤研究(C), 神戸大学, 01 Apr. 2023 - 31 Mar. 2026

  多種類のがんで転写共役因子YAP/TAZの異常な活性化が認められ、その活性を抑制するHippo経路の構成因子欠損マウスにおいて、種々のがんが早期から高率に発症することから、Hippo-YAP経路のがんの発症、進展における役割に注目が集まっている。本研究では、ゲノムワイドsiRNAスクリーニングにより、YAP依存的な遺伝子転写活性に強力に作用する薬剤標的分子を同定し、YAP活性を標的とした抗腫瘍薬の開発に貢献することを目指している。 YAPによる遺伝子転写の活性を感度良くモニターするレポーター細胞を用いて、ヒト全遺伝子に対するsiRNAライブラリーを用いたスクリーニングを行った。このスクリーニングにおいて、配列特異的RNA結合ドメインを持ち、mRNAの分解、翻訳抑制に働く蛋白質であるRNABPのノックダウンにより、最も強くYAPによる遺伝子転写活性が抑制された。CRISPR/Cas9を用いてRNABPを欠損させた細胞株では、YAP依存的な遺伝子転写が顕著に不活化され、RNABPの過剰発現により、YAP依存的な遺伝子転写が活性化した。ヌードマウスを用いたin vivoでの腫瘍形成実験において、RNABPの過剰発現は腫瘍形成を促進し、RNABPの欠失により腫瘍形成が阻害されたことから、生体内での腫瘍形成にRNABPが寄与することが明らかになった。また、RNABPのYAP活性抑制機構として、RNABPが、YAP抑制因子であるVGLL4の発現を抑制し、YAPを活性化していることを明らかにした。組換え蛋白質を用いたRNABP-RNA結合可視化系確立し、結合阻害剤のスクリーニングを行なっている。


• 配列特異的翻訳抑制蛋白質によるHippo-YAP経路の制御

  大谷 淳二

  日本学術振興会, 科学研究費助成事業 基盤研究(C), 基盤研究(C), 神戸大学, 01 Apr. 2020 - 31 Mar. 2023

  多種類のがんで転写共役因子YAP/TAZの異常な活性化が認められ、その活性を抑制するHippo経路の構成因子欠損マウスにおいて、種々のがんが早期から高率に発症することから、Hippo-YAP経路のがんの発症、進展における役割に注目が集まっている。本研究では、ゲノムワイドsiRNAスクリーニングにより、YAP依存的な遺伝子転写活性に強力に作用する薬剤標的分子を同定し、YAP活性を標的とした抗腫瘍薬の開発に貢献することを目指している。 YAPによる遺伝子転写の活性を感度良くモニターするレポーター細胞を用いて、ヒト全遺伝子に対するsiRNAライブラリーを用いたスクリーニングを行った。このスクリーニングにおいて、配列特異的RNA結合ドメインを持ち、mRNAの分解、翻訳抑制に働く蛋白質であるRNABPのノックダウンにより、最も強くYAPによる遺伝子転写活性が抑制された。CRISPR/Cas9を用いてRNABPを欠損させた細胞株では、YAP依存的な遺伝子転写が顕著に不活化され、RNABPの過剰発現により、YAP依存的な遺伝子転写が活性化した。また、RNABPのRNA結合能を欠いた変異体ではYAPを活性化できないことから、RNABPのRNA結合能がYAP活性化に必要であることが明らかになった。さらに、RNABPの標的遺伝子Xを同定し、RNABPによるYAP活性化が標的遺伝子Xの蛋白質発現の抑制を介していることを示した。また、結合阻害剤の探索に向け、組換え蛋白質として調製したRNABPのRNA結合ドメインと基質RNAの結合を、Alphaスクリーンにより可視化する実験系を確立した。


• Elucidation of molecular mechanisms of tumor initiation and progression by ribosomal stresses

  Otani Junji

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Early-Career Scientists, Grant-in-Aid for Early-Career Scientists, Kobe University, 01 Apr. 2018 - 31 Mar. 2020

  During the last decade, increasing attention has been focused on the nucleolar stress pathway that releases ribosomal proteins from the nucleolus to the nucleoplasm when ribosome biosynthesis is abnormal and suppresses cell proliferation through p53 activation. On the other hand, it has been suggested that an abnormality in the ribosomal protein gene may promote canceration in studies of ribosomal protein mutants with Zebrafish model system and in epidemiological studies of ribosomopathy patients. In this study, we found that transcriptional inhibition of ribosomal RNA by an RNA polymerase inhibitor actinomycin D enhances the expression of down-stream target genes of the oncogenic transcriptional co-factors YAP1/TAZ and nuclear accumulation of YAP1 protein. Promoting effect of actinomycin D on the oncogenic growth signaling pathway may underlie the cancer promoting effect of ribosomal stress conditions and ribosomopathies.


• Role of YAP1/TAZ in cell competition

  Nishio Miki, MIYACHI Yosuke, OTANI Junji, TANE Shoji, OMORI Hirofumi, UEDA Fumihito, MAEHAMA Tomohiko, SUZUKI Akira

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Scientific Research (C), Grant-in-Aid for Scientific Research (C), Kobe University, 01 Apr. 2016 - 31 Mar. 2019

  In mammalian cells, the cell with constitutively active YAP became "loser" and eliminated by the cell competition. This type of cell competition was observed in NIH3T3 cell and PAM cell in addition to previously reported MDCK cell. YAP-activated cells were prone to extrude and form a multi-stacked cell layer, when cultured with wild-type cells that were tightly attached to culture vessels and grew within flat monolayer. In contrast, when cultured on low-binding culture vessels, the YAP-activated cells turned to be "winner", because YAP-activated cells retained proliferation ability in anchorage-independent manner but wild-type cells did not. Intriguingly, in both cases, ECM coating of culture vessels strengthened binding of "loser" cells to the vessels and restored their proliferation, blunting "loser" cell elimination by cell competition. These observations suggest that the binding of cells to culture vessels and ECM may play a role in cell competition of mammalian cells.


• Biochemical study of a splicing factor Sde2

  Otani Junji

  Japan Society for the Promotion of Science, Grants-in-Aid for Scientific Research Grant-in-Aid for Young Scientists (B), Grant-in-Aid for Young Scientists (B), Kobe University, 01 Apr. 2016 - 31 Mar. 2018

  Post-translational modification by ubiquitin and several ubiquitin like protein modifiers controls diverse cellular processes. There are many proteins carrying the protein domains structurally similar to ubiquitin without the ability to form covalent linkage. Sde2 was first identified as a factor involved in gene silencing at telomere in Schizosaccharomyces pombe and later found to be required for mRNA splicing. In this study, we showed that the evolutionary conserved ubiquitin like domain (Ubl) of Sde2 at its N-terminus is cleaved off in S. pombe and also in human cells. We found that the Ubl itself is dispensable but the newly exposed neo N-terminus after removal of the Ubl is important for mRNA splicing.


• テロメア特異的蛋白質複合体シェルタリンによるテロメラーゼの制御機構解明

  大谷 淳二

  日本学術振興会, 科学研究費助成事業 特別研究員奨励費, 特別研究員奨励費, 京都大学, 2011 - 2012

  前年度までに、分裂酵母のテロメラーゼ制御において重要な蛋白質である、Ccq1の493-735アミノ酸の領域の蛋白質結晶を調製する事に成功していたため、本年度は、X線データの位相情報を得ることを目指し、重原子置換体結晶の調製に取り組んだ。しかし、蛋白質の結晶化実験の再現性が悪いことが問題となった。そこで、表面残基エントロピー減少法(SERp server)を用いて蛋白質表面にアミノ酸変異を導入した所、再現性よく、同型の結晶が得られるようになった。さらに得られた結晶は、元の野生型の蛋白質を用いて得られていた結晶よりも対称性が高く、X線回折強度データの精密な測定が必要になる位相決定に有利なものとなっていた。今後、これらの変異体結晶を用いて結晶構造解析を進める。 また、先行研究の結果などから、ヒトなどの高等真核生物において、シェルタリン蛋白質、TPP1のリン酸化依存的な二量体化がテロメア伸長に重要であるとの仮説を立て、それを立証するための実験を行った。293T細胞において、FLAGタグ融合およびHAタグ融合TPP1蛋白質を強制発現し、免疫沈降実験を行った。この実験から、二種のタグが融合された蛋白質どうしの相互作用が検出され、TPP1が実際に多量体化していることが示唆された。ところが、予想される二量体表面のアミノ酸残基に種々の変異を導入しても、この相互作用を壊す事ができないことから、TPP1は予想された二量体結合表面を介して結合しているのではないことが示唆された。本年度の研究によって予想された形式での多量体化はみられなかったものの、TPP1どうしの相互作用が確認された。この相互作用の生物学的意義については引き続き検討したい。