1. The retinal progenitor-specific gene Sox11regulates the timing of the differentiation ofearly-born retinal cells, and its expression isregulated by multiple mechanisms during reti-nal development

Ayumi Usui1, Yujin Mochizuki1, Atsumi Iida, To-shiro Iwagawa, Hiroyuki Aburatani2, Sumiko Wa-tanabe: 1Department of Ophthalmology, JuntendoUniversity School of Medicine, 2Genome Science

Division, Research Center for Advanced Scienceand Technology, University of Tokyo

Sry-related HMG box (Sox) proteins play diverseand critical roles in a variety of morphogeneticprocesses during embryonic development. Sox11and Sox4 are members of the SoxC subtype, andwe found that Sox11 was strongly expressed inearly retinal progenitor cells, and that when expres-sion of Sox11 subsided around birth, Sox4 expres-

Donation Laboratories

Department of Molecular and Developmental Biology再生基礎医科学寄付研究部門（SBI，トミー，ロート製薬，慈照会）

Visiting Professor Sumiko Watanabe, Ph.D.Visiting Research Associate Hideto Koso, MD, Ph.D

特任教授 医学博士 渡 辺 すみ子特任助教 医学博士 高 祖 秀 登

Our long-term goal is to understand the molecular mechanisms which coordinatelyregulate growth and differentiation of stem cells as well as differentiated cells withemphasis on intracellular signal transduction. For this purpose we are using mod-els ranging from iPS and various culture cells, zebrafish, mouse, to monkey.Based on our research background on the area of cytokine signals, we now focuson the analysis of development and regeneration of neural retina.The neural retina is a part of the central nervous system (CNS), and regenerationof the retina from retinal stem cells or other sources by transplantation is a criticalissue from both clinical and neurobiological points of view. Although reports ofsuccessful regeneration of the CNS from neural stem cells (NSC) have appearedin the literature, such has not been the case for the vertebrate neural retina. Fur-thermore, the nature of retinal stem cells has not been clarified, making it difficultto attempt regeneration of the retina. Based on the techniques and knowledge thathave been accumulated through work on of haematopoietic systems in our labora-tory, we attempt to identify mammalian retinal stem cells and following develop-mental processes by revealing the expression pattern of cell surface proteins. Wefound that various CD antigens mark spatiotemporally distinct populations of reti-nal cells, and genes specifically expressed in such populations has been revealedby microarray analyses. Various signaling molecules and transcriptional factorsare under investigation for their roles on retinal development. For developmentalbiological analyses, we use zebrafish in addition to mouse as model animals. Wealso continue to work on haematological projects, and bidirectional cooperativeprogress between neurological and haematological works is one of unique fea-tures of our laboratory. Projects, which gave major findings during 2012 are as fol-lows.

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sion began. To analyze the role of Sox11 and Sox4in retinal development, we perturbed their expres-sion pattern by expressing them ectopically in reti-nal explant culture. Overexpression of Sox11 orSox4 in retinal progenitors resulted in similar phe-notypes, that is, increased cone cells and decreasedMüller glia. Sox11-knockout retinas showed de-layed onset and progress of differentiation of early-born retinal cells during the embryonic period. Af-ter birth, retinal differentiation took place relativelynormally, probably because of the redundant activ-ity of Sox4, which starts to differentiate aroundbirth. Neither overexpression nor loss-of-functionanalysis gave any evidence that Sox11 and Sox4 di-rectly regulate transcription of genes critical toearly-born retinal cells. However, histone H3 ace-tylation status of the early neurogenic genes waslowered in knockout retinas, suggesting that Sox11regulates the timing of differentiation in early-bornretinas by creating an epigenetic state that helps toestablish the competency to differentiate. We alsoexamined molecular basis of regulation of expres-sion of Sox11 and Sox4. Gain-of-function and loss-of-function analyses suggested that Notch signalsuppresses Sox11 expression in the early develop-ing retina but not during the later period of devel-opment. The levels of histone H3-acetylation andH3-lysine 4 tri-methylation at the Sox4 and Sox11loci were dramatically changed, as were the levelsof Sox4 and Sox11. In contrast, negative regulationof histone H3-lysine 27 methylation was observed,with reciprocal alterations to the levels of Sox4 andSox11.

2. Differential regulation of expression of thelong/middle wavelength-sensitive opsins ofvertebrates is mediated by thyroid hormonereceptor 2 and COUP-TFII

Toshiro Iwagawa, Yo Tanaka, Atsumi Iida, Sumi-ko Watanabe

Cone photopigments (opsins) are crucial elementsof, and the first detection module in, color vision.Individual opsins have different wavelength sensi-tivity patterns, and the temporal and spatial expres-sion patterns of opsins are unique and stringentlyregulated. Long and middle wavelength-sensitive(L/M) opsins are of the same phylogenetic type. Al-though the roles of thyroid hormone and TR2 inthe transcriptional regulation of L/M opsins havebeen explored, the detailed mechanisms, includingthe target sequence in the enhancer of L/M opsins,have not been revealed. Using several human redopsin enhancer/promoter-luciferase reporter con-structs, we found that TR2 increased luciferase ac-tivities through the 5'-UTR and intron 3-4 region,whereas the presence of T3 affected only the intron3-4 region-dependent luciferase activity. Further-

more, COUP-TFII suppressed intron 3-4 region-de-pendent luciferase activities. However, luciferaseexpression driven by the mouse M opsin intron 3-4region was only slightly increased by TR2, andrather enhanced by COUP-TFII. To determinewhether these differential responses reflect differ-ences between primates and rodents, we examinedthe enhancer/promoter region of the red opsin ofthe common marmoset. Interestingly, while TR2increased 5'-UTR- or intron 3-4 region-driven lu-ciferase expression, as observed for the human redopsin, expression of the latter luciferase was notsuppressed by COUP-TFII. In fact, immunostainingof common marmoset retinal sections revealed co-expression of COUP-TFII and red opsin in the conecells. These results suggest species-specific differen-tial regulation of L/M opsins by TR2 and COUP-TFII.

3. Use of cell type-specific transcritome to iden-tify genes specifically involved in Müller gliadifferentiation during retinal development

Yujin Mochizuki1, Atsumi Iida, Akira Murakami1,Sumiko Watanabe

Retinal progenitor cells alter their properties overthe course of development, and sequentially pro-duce different subpopulations of retinal cells. Wehad previously found that early and late retinalprogenitor cell populations can be distinguished bytheir surface antigens, SSEA-1 and c-kit, respec-tively. Using DNA microarray analysis, we exam-ined the transcriptomes of SSEA-1 positive cells atE14, and c-kit positive and c-kit negative cells at P1.By comparing data, we identified genes specificallyexpressed in c-kit positive late retinal progenitorcells. The previous literature suggests that most ofthe c-kit positive cell-specific genes are related toglia differentiation in brain or are expressed inMüller glia. Since Notch signaling promotes Müllerglia differentiation in retina, we examined the ef-fects of gain- and loss-of-Notch signaling on expres-sion of these genes and found that all the geneswere positively affected by Notch signaling. Finally,we screened the genes for their function in retinaldevelopment by shRNA-based suppression in reti-nal explants. In about half the genes, Müller gliadifferentiation was perturbed when their expressionwas suppressed. Taken together, these results showthat at P1, c-kit positive retinal progenitor cells,which include Müller glia precursor cells, are en-riched for genes related to glial differentiation. Wepropose analysis of purified subsets of retinal cellsas a powerful tool to elucidate the molecular basisof retinal development.

4. Molecular mechanisms of brain tumor initia-tion

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Hideto Koso, Eli Lyons, Asano Tsuhako, SumikoWatanabe

NSCs are considered to be the cell of origin ofglioblastoma multiforme (GBM). However, the ge-netic alterations that transform NSCs into glioma-initiating cells remain elusive. Using a novel trans-poson mutagenesis strategy that mutagenizes NSCsin culture, followed by additional rounds of muta-genesis to generate tumors in vivo, we have identi-fied genes and signaling pathways that can trans-form NSCs into glioma-initiating cells. Mobilizationof Sleeping Beauty transposons in NSCs induced theimmortalization of astroglial-like cells, which werethen able to generate tumors with characteristics ofthe mesenchymal subtype of GBM upon transplan-tation, consistent with a potential astroglial originfor mesenchymal GBM. Sequence analysis of trans-poson insertion sites from tumors and immortalizedcells identified more than two hundred frequentlymutated genes, including human GBM-associatedgenes such as Met and Nf1, and made it possible todiscriminate between genes that function during as-troglial immortalization versus later stages of tumordevelopment. This novel mutagenesis strategy isfaster and simpler than conventional transposonscreens and can potentially be applied to any tissuestem/progenitor cells that can be grown and differ-entiated in vitro.

5. Temporal regulation of Cre activity in NSCsand rod photoreceptors

Hideto Koso, Asano Tsuhako, Sumiko Watanabe

The RNA-binding protein Musashi1 (Msi1) is oneof two mammalian homologues of Drosophila Musa-shi, which is required for the asymmetric cell divi-sion of sensory organ precursor cells. In the mousecentral nervous system, Msi1 is preferentially ex-pressed in mitotically active NSCs in the ventricularzone of the neural tube during embryonic develop-ment and in the subventricular zone (SVZ) of thepostnatal brain. We described the generation ofMsi1-CreER T2 knock-in mice and showed by celllineage tracing that Msi1-CreER T2-expressing cellsspecifically mark NSCs in both the embryonic andadult brain. We also showed that Msi1-CreER T2 isexpressed in photoreceptor cells of the mature ret-ina. Msi1-CreER T2 mice thus represent a new tool inour arsenal for genetically manipulating NSCs andphotoreceptor cells, which will be essential for un-derstanding the molecular mechanisms underlyingdiseases of the nervous system.

6. Molecular mechanisms regulating differentia-tion and proliferation of retinal stem/progeni-tor cells: Requirement of Fezf2 in Differentia-tion of Cone OFF bipolar cells

Haruna Suzuki-Kerr, Sumiko Watanabe

Cell surface antigens are powerful tools for iso-lating specific subsets of retinal cells during devel-opment from cell mixtures without damaging thecells, which makes it possible to characterize theirproperties and identify genes that regulate theirproliferation and differentiation. By screening reti-nal cells from mice at various developmental stagesfor their reactivity with over 150 different antibod-ies against various cell surface antigens, we identi-fied SSEA-1 and c-kit as early and late progenitormarkers, respectively. SSEA-1 marks retinal pro-genitor cells in the peripheral region of the retina ataround E14-E16. In the later stage of embryogene-sis, SSEA-1 disappears and c-kit expression is ob-served in the retinal progenitor cells in the centralregion of the retina. We compared the gene expres-sion patterns of regionally and temporally differentsubsets of retinal progenitor cells, SSEA-1-positivecells at E14, c-kit positive cells at P1, and differenti-ated c-kit negative cells at P1 using a microarray.We found that several genes are specifically ex-pressed in SSEA-1 positive early retinal progenitorcells. Currently, we are focusing on one of suchgenes, Fezf2. We found that the Fezf2, a transcrip-tional repressor of the Fez zing finger family, to bestrongly expressed in SSEA-1-positive cells. Subse-quent analysis showed that Fezf2 was expressed indeveloping bipolar cells and that there was a sig-nificant reduction in the number of OFF type bipo-lar cells in the Fezf2KO retina, suggesting a roleplayed by Fezf2 during bipolar cell differentiation/maturation. We further examined the role of Fezf2in bipolar differentiation by Electroretinogram(ERG), commonly used to assess the functionalityof the retina in vivo. In 2-month-old Fezf2KO retina,cone ON bipolar response was significantly re-duced, suggesting that these cells are not fullyfunctional in Fezf2KO retina. We then examined thesynaptic structure of ON bipolar cells by transmis-sion electron microscopy (TEM). Ultra-structuralanalysis by TEM revealed that the degree of contactbetween ON bipolar dendrite and cone photorecep-tor terminal was slightly, but significantly reducedin Fezf2KO retina. Since Fezf2 was not expressed inON bipolar cells, these changes to cone ON bipolarcells in Fezf2KO retina may represent an interactionbetween cone OFF and ON bipolar cells duringmaturation of bipolar cells. Next, we analyzed thetime course of Fezf2 expression, and found thatFezf2 expression increased in a subset of retinal bi-polar cells at around P8, around the time of bipolarmaturation. As there are at least 10 morphologicallydistinct subtypes of bipolar cells in the mouse ret-ina, we then investigated the expression of bipolarsubtype specific markers. So far, the use of subtype-specific markers for IHC & qPCR suggests that typeIII OFF bipolar subtype marker is down regulated

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in Fezf2KO. Taken together, our results suggestthat Fezf2 may be involved in OFF bipolar subtypespecification during development of the retina. Thecurrent finding should help clarify molecularmechanisms behind differentiation of retinal pro-genitors and their subsequent maturation into aspecific subtype of neurons.

7. Nucleolar phosphoprotein NPM1 regulatesVsx2 gene expression in retinal progenitorsby binding to evolutionarily conserved enhan-cer elements

Yasuo Ouchi, Yukihiro Baba, Sumiko Watanabe

The homeodomain transcription factor Vsx2 isthe earliest characterized specific marker of retinalprogenitor cells and is known to play an importantrole in their proliferation. To better understand theevolutionarily conserved molecular mechanism ofretinal progenitor cell maintenance, we attemptedto identify Vsx2 promoter and upstream regulatorsusing zebrafish and mice. By database analysis ofVsx2 loci, we identified four evolutionarily con-served sequences in the Vsx2 loci. Through a seriesof transient expression assays conducted with EGFPreporter plasmids, we identified a cis-regulatorymotif located 35 kb upstream of the mouse Vsx2gene as an evolutionarily conserved enhancer inretinal progenitor cells. A proteomic analysis con-ducted using a minimal cis-regulatory motif identi-fied proteins, including NPM1, that bind to this re-gion. The mouse and zebrafish homologs of NPM1were shown to be strongly expressed in retinal pro-genitor cells during development. NPM1 enhancedVsx2 enhancer-luciferase reporter gene expression,and the C-terminal DNA-binding motif of NPM1was essential for this activity. Moreover, downregu-lation of NPM1 expression by shRNA reduced thenumber of Vsx2-positive cells, inhibited cell prolif-eration, and induced apoptosis in retinal progenitorcells. Collectively, these results reveal that NPM1

plays a critical role in retinal development by regu-lating Vsx2 gene expression.

8. In Vitro Cell subtype-specific transduction ofadeno-associated virus in the mouse andmarmoset retinal explant culture

Yukihiro Baba, Sumiko Watanabe

Adeno-associated virus (AAV) is a non-patho-genic human parvovirus that can infect both non-proliferating and proliferating cells. Owing to its fa-vorable safety profile, AAV is regarded as suitablefor clinical purposes such as gene therapy. The tar-get cell types of AAV depend largely on the sero-type. In the retina, AAV has been used to introduceexogenous genes into photoreceptors, and photore-ceptor-specific enhancers/promoters are used inmost cases. Therefore, serotype specificity of AAVin retinal subtypes is unclear, particularly in vitro.We compared its infection profile in mouse andmonkey retinas using EGFP under the control ofthe CAG promoter, which expressed the geneubiquitously and strongly regardless of cell type.AAV1, 8, and 9 infected the horizontal cells whenan embryonic day-17 retina was used as a host.Nearly 100％ of the horizontal cells expressedEGFP. Amacrine cells were also a target of AAVs,and a small number of rod photoreceptors were in-fected. When adult retinas were used as a host, themain target of AAV was Müller glia. A small num-ber of rod photoreceptors were also infected. In theadult common marmoset retina, rod and cone pho-toreceptors were efficiently infected by AAV1, 8,and 9. A portion of the Müller glia and amacrinecells were also infected. In summary, the infectionspecificity of different AAV serotypes did not dif-fer, but was dependent on the stage of the host ret-ina. In addition, infection specificities differed be-tween mature marmoset retinas and mature mouseretinas.

Publications

1. Iguchi-Ishiguro, H., Ouchi, Y., Watanabe, S. andNumabe, Y. Analysis of syndecan-1 gene pro-moter during mouse tooth development. Ar-chives of Oral Biology, 57, 531-538, 2012.

2. Saito, R., Nakauchi, H. and Watanabe, S., Ser-ine/Threonine kinase Melk regulates prolifera-tion and glial differentiation of retinal progeni-tor cells. Can. Sci., Can. Sci., 103, 42-49, 2012.

3. Iwagawa, T., Ohuchi, S., Watanabe, S. and Na-kamura, Y. Selection of RNA aptamers againstmouse embryonic stem cells. Biochimie, 94, 250-257, 2012.

4. Baba, Y., Satoh, S., Otsu, M., Sasaki, E., Okada,

T. and Watanabe, S. In vitro cell subtype-spe-cific transduction of adeno-associated virus inthe mouse and marmoset retinal explant cul-ture, Biochimie, 94, 2716-2722, 2012.

5. Koso, H., Takeda, H., Yew, C., Ward, J.M., Na-riai, N., Ueno, K., Nagasaki, M., Watanabe, S.,Rust, A.G., Adams, D.J., Copeland, N.G. andJenkins, N.A., Transposon mutagenesis identi-fies genes that transform neural stem cells into-glioma-initiating cells, Proc.Natl.Acad.Sci.,USA,109, 2998-3007, 2012.

6. ＊Takeda, H., ＊Koso, H., Tessarollo, L., Cope-land, N.G. and Jenkins, N.A Musashi-CreERT2:

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A new cre line for conditional mutagenesis inneural stem cells. Genesis, 51, 128-134, 2013.＊Equal contribution.

7. Usui, A., Mochizuki, Y., Iida, A., Miyauchi, E.,Satoh, S, Sock, E., Nakauchi, H., Aburatani, H.,Murakami, A., Wegner, M. and Watanabe, S.,The early retinal progenitor-expressed geneSox11 regulates the timing of the differentiationof retinal cells. Development, 140, 740-750, 2013.

8. Usui, A., Iwagawa, T., Mochizuki, Y., Iida, A.,Wegner, M., Murakami, A. and Watanabe, S.,

Expression of Sox4 and Sox11 is regulated bymultiple mechanisms during retinal develop-ment. FEBS Letters, 587, 358-363, 2012.

9. 日本AEM学会誌 Journal of the japan society of ap-plied electromagnetics and mechanicsa, vol ２０，「細胞移植による組織の再生とその実現化をささえる技術開発」５８３―５８８，２０１２．

10. 実験医学 増刊号３０―１心と体のクロストークから解く精神・神経疾患，「サイトカイン：精神・神経病体型制のキープレーヤー」２０３８―２０４４，２０１２．

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A) Zinc-finger-containing transcriptional factor,Kruppel-like factor 2 (KLF2)

The Kruppel-like factor (KLF) proteins are multi-tasked transcriptional regulators with an expandingtumor suppressor function. KLF2 is a member ofthe KLF family of zinc-finger transcription factorsand is involved in maintaining T-cell quiescence,regulating preadipocyte differentiation, endothelialcell function, lung development and the self-re-newal of ES cells. Furthermore, KLF2 is one of theprominent members of the family because of its di-minished expression in malignancies and itsgrowth-inhibitory, pro-apoptotic and anti-angio-genic roles.We indicate that epigenetic silencing of KLF2 oc-

curs in cancer cells through direct transcriptionalrepression mediated by the Polycomb group pro-tein Enhancer of Zeste Homolog 2 (EZH2). Bindingof EZH2 to the 5'-end of KLF2 is also associatedwith a gain of trimethylated lysine 27 histone H3

and a depletion of phosphorylated serine 2 of RNApolymerase.Upon depletion of EZH2 by RNA interference,

short hairpin RNA or use of the small molecule 3-Deazaneplanocin A, the expression of KLF2 is re-stored. The transfection of KLF2 in cells withEZH2-associated silencing showed a significantanti-tumoral effect, both in culture and in xeno-grafted nude mice.In this last setting, KLF2 transfection was also as-

sociated with decreased dissemination and lowermortality rate. In EZH2-depleted cells, which char-acteristically have lower tumorigenicity, the induc-tion of KLF2 depletion `rescued' partially the onco-genic phenotype, suggesting that KLF2 repressionhas an important role in EZH2 oncogenesis.Most importantly, the translation of the described

results to human primary samples demonstratedthat patients with prostate or breast tumors withlow levels of KLF2 and high expression of EZH2had a shorter overall survival.

Donation Laboratories

Division of Antibody, Vaccine & Experimental Therapy抗体・ワクチン治療寄付研究部門

Project Associate Professor Taniguchi Hiroaki, M.D., D.M.Sc.Professor Motoharu Seiki, D.M.Sc.Professor Kohzoh Imai, M.D., D.M.Sc.Professor Kouhei Tsumoto, Ph.D.

特任准教授 医学博士 谷 口 博 昭教授（兼） 医学博士 清 木 元 治教授（兼） 医学博士 今 井 浩 三教授（兼） 工学博士 津 本 浩 平

Tumors contain a small population of putative cancer stem cells (CSC), whichpossess unique self-renewal properties, and survive in a quiescent state for manyyears after remission and result in later relapse and metastasis. Therefore, it isconceivable that targeting CSCs will eradicate tumor-initiating cells, whereas con-ventional chemotherapies will only eradicate the bulk of a tumor.Cancer stem cells and normal tissue stem cells utilize the same self-renewal path-way. However, researchers characterize some of changes, which occur in cancerstem cells, not in normal tissue stem cells. The design of new therapeutic agentsshould be aimed at targeting these unique molecular changes.We have currently focused on studying these unique molecular changes, whichoccur in cancer stem cells, not in normal tissue stem cells. This could be a newtherapeutic target against solid tumors.

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B) PR domain-containing protein, PRDM14

PRDM have been linked to human cancers. Toexplore the role of the PR domain family genes inbreast carcinogenesis, we examined the expressionprofiles of 16 members of the PRDM gene family ina panel of breast cancer cell lines and primarybreast cancer specimens using semiquantitativereal-time PCR.We found that PRDM14 mRNA is overexpressed

in about two thirds of breast cancers. Moreover, im-munohistochemical analysis showed that expressionof PRDM14 protein is also up-regulated. PRDM14are known as a key transcription factor required forthe maintenance of hESC identity and the reacquisi-

tion of pluripotency in human somatic cells.Introduction of PRDM14 into cancer cells reduced

their sensitivity to chemotherapeutic drugs. Con-versely, knockdown of PRDM14 by siRNA inducedapoptosis in breast cancer cells and increased theirsensitivity to chemotherapeutic drugs. Moreover,PRDM14 regulated cancer metastasis, angiogenesis,and stemness of cancer cells.That little or no expression of PRDM14 is seen in

noncancerous tissues suggests that PRDM14 couldbe an ideal therapeutic target for the treatment ofbreast cancer. Now, we also develop new method-logy with nuclear acid medicine and modified anti-body drug against PRDM14.

Publications

Project Associate Professor, Taniguchi Hiroaki:1. Kato N, Yamamoto H, Adachi Y, Ohashi H, Ta-

niguchi H, Suzuki H, Nakazawa M, Kaneto H,Sasaki S, Imai K and Shinomura Y. Cancer detec-tion by ubiquitin carboxyl-terminal esterase L1methylation in pancreatobiliary fluids. World JGastroenterol, in press, 2013.

2. Taniguchi H, Jacinto F.V., Villanueva A, Fernan-dez AF, Yamamoto H, Carmona F.J, Puertas S,Marquez VE, Shinomura Y, Imai K, and EstellerM. Silencing of the Kruppel-like factor 2 by thehistone methyltransferase EZH2 in human can-cer. Oncogene, 31: 1988-1994, 2012.

3. Ohashi H, Adachi Y, Yamamoto H, Taniguchi H,Nosho K, Suzuki H, Arimura Y, Imai K, CarboneDP, Shinomura Y. Insulin-like growth factor re-ceptor expression is associated with aggressivephenotypes and has therapeutic activity in bili-ary tract cancers. Cancer Sci, 103: 251-26, 2012.

4. Fernandez AF, Assenov Y, Martin-Subero JI, Ba-

lint B, Siebert R, Taniguchi H, Yamamoto H, Hi-dalgo M, Tan AC, Galm O, Ferrer I, Sanchez-Cespedes M, Villanueva A, Carmona J, Sanchez-Mut JV, Berdasco M, Moreno V, Capella G,Monk D, Ballestar E, Ropero S, Martinez R,Sanchez-Carbayo M, Prosper F, Agirre X, FragaMF, Grana O, Perez-Jurado L, Mora J, Puig S,Prat J, Badimon L, Puca AA, Meltzer SJ, Len-gauer T, Bridgewater J, Bock C, Esteller M. ADNA methylation fingerprint of 1628 humansamples. Genome Res, 22: 407-419, 2012.

Professor, Motoharu Seiki:Please refer to Division of Cancer Cell Research,Department of Cancer Biology.

Professor, Kohzoh Imai:Please refer to Center for Antibody and VaccineTherapy.

Professor, Kouhei Tsumoto:Please refer to Medical Proteomics Laboratory.

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【Medical Governance】

Tomoko Matsumura, Naoko Murashige, Yuko Ko-dama, Koichiro Yuji1, Masaharu Tsubokura, Ma-sahiro Kami, Tetsuya Tanimoto2, Shuichi Iwa-moto2, Masayoshi Nagata3: 1Department of Medi-cine (Department of Hematology / Oncology), In-stitute of Medical Science, the University of Tokyo,2Navitas Clinic, Internal Medicine, 3Department ofUrology, the University of Tokyo

We performed mathematical simulation of chan-ges in age/sex composition of the population, fatali-ties and the number of physicians in the future topredict the impact of the current government planto increase medical school enrollments. In addition,we also studied the effect of genetic regulations onclinical practice, the system of management, qual-ity-of-life effects of screening, drug approval systemby the regulatory agency, Japan's vaccination pol-icy, information circulation and published the re-sults in scientific journals (Yuji K, et al., MurashigeN, et al., Nagata M, et al., Tanimoto T, et al., Iwa-moto S, et al. Tanimoto T, et al.). These results werewidely published in news papers and popular

magazines.

【Medical support for disaster-stricken area】

Masaharu Tsubokura, Yukio Kanazawa3, To-moyoshi Oikawa3, Kyohei Takahahi4, Akemi Taka-da3, Tomoko Matsumura, Morihito Takita, TamaeHamaki2, Kazuhiko Kobayashi5, Syuichi Iwamoto2,Ginichi Mori6, Yukie Takahashi6, Masaki Miyasa-ka7, Hideki Komatsu8, Makoto Suzuki8, MamikoOhara8, Tsunehiko Komatsu9, Kenji Shibuya10,Amina Sugimoto10, Syuhei Nomura10, Tetsuya Ta-nimoto2, Ken Okai3,Takeaki Ishii11, Shigeaki Ka-to11, Sae Ochi11, Giichiro Oiso12, Yuko Kodama,Masahiro Kami: 3Minamisoma Municipal GeneralHospital, 4Haramachi Central Maternity Clinic,5JR Tokyo General Hospital, 6Tokyo MetropolitanCancer and Infectious diseases Center KomagomeHospital, 7Tokyo Metropolitan Bokutoh Hospital,8Kameda Medical Center, 9Teikyo UniversityChiba Medical Center, 10Pharmaceuticals andMedical Devices Agency, Graduate school of Medi-cine, Department of Global Health Policy, TheUniversity of Tokyo, 11Soma Central Hospital,

Donation Laboratories

Division of Social Communication Systemfor Advanced Clinical Research先端医療社会コミュニケーションシステム社会連携研究部門

Project Professor Masahiro Kami, M.D., Ph.D.Project Assistant Professor Tomoko Matsumura, M.D., Ph.D.

特任教授 医学博士 上 昌 広特任助教 医学博士 松 村 有 子

The aim of our division is to establish and popularize state-of-art medicine and topromote translational research (TR). We investigate medical governance and themethodology to develop national consensus in health care by using media.We also perform individual case studies on economic burden of health care onpatients, medical support for disaster-stricken area by the Great East Japan Earth-quake on March 11, 2011 and physician supply. In each case, we also study thesystem of management, information circulation, and network.

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Collaborating with physicians who work in disas-ter-stricken area and many support physicians, weconducted measurement of internal radiation expo-sure and gave medical guidance to the local peopleand publish the results in scientific journals (Tsu-bokura M, et al., Tanimoto T, et al. Harasawa K, etal., Tsubokura M, et al., Okai K, et al., Ishii T, et al.,Tsubokura M, et al). These results were widelypublished in news papers and popular magazines.

【Economic Burden of Health Care on Patients】

Yuko Kodama, Ryoko Morozumi13, Akihiko Ma-tsui14, Masahiro Kami, Tomoko Matsumura, Nao-ko Murashige, Morihito Takita, Eiji Kusumi2:13Faculty of Economics, University of Toyama,14Faculty of Economics, the University of Tokyo

Imatinib (Glivec), which is the first-line drug forchronic myelogenous leukemia (CML), is highly ef-ficient. We clarified that the cost of Glivec in Japanwas higher than the other countries by internationalresearch. We conduct a collaborate study with Pro-fessor Matsui at Faculty of Economics, the Univer-

sity of Tokyo, to review the utilization of Glivecand its cost in Japan. Because the economic burdenon patients or the government with prevailing ad-vanced medical care including anticancer drugs isan important issue, we continue further investiga-tion. (Kodama Y, et al.)

【Clinically Oriented Research】

Tomoko Matsumura, Tetsuya Tanimoto2, HirotoNarimatsu15, Natsuko Watanabe16 Ginichi Mori6,Yukie Takahashi6, Masaki Miyasaka7: 15GraduateSchool of Medicine / Cohort Management Unit,Yamagata University, 16Ito Hospital

We performed a retrospective cohort study in-volving more than 50 thousand patients withGraves' disease and a retrospective survey involv-ing more than 250 patients with acute lymphoblas-tic leukemia. We also studied in prophylaxis andtreatment in lymphoma. These results were pub-lished in scientific journals (Watanabe N, et al.,Matsumura T, et al., Tanimoto T, et al., Mori J, etal., Takahashi Y, et al.).

Publications

1. Murashige N, Tanimoto T, Kusumi E. Fear ofgenetic discrimination in Japan. Lancet. 380:730, 2012.

2. Iwamoto S, Hagino N, Tanimoto T, Kusumi E.Wheat-dependent exercise-induced anaphylaxisassociated with a facial soap. BMJ Case Rep.:doi: 10.1136 /bcr.01.2012.5641.

3. Yuji K, Imoto S, Yamaguchi R, Matsumura T,Murashige N, Kodama Y, Minayo S, Imai K,Kami M. Forecasting Japan's Physician Shortagein 2035 as the First Full-Fledged Aged Society.PLoS One. 7: e50410, 2012.

4. Nagata M, Tanimoto T, Kami M. Quality-of-lifeeffects of prostate-specific antigen screening. NEngl J Med. 367: 1861-1862; author reply 1862,2012.

5. Tanimoto T, Tsubokura M, Mori J, Pietrek M,Ono S, Kami M. Differences in drug approvalprocesses of 3 regulatory agencies: a case studyof gemtuzumab ozogamicin. Invest New Drugs.2012.

6. Tanimoto T, Murashige N, Hosoda M, KusumiE, Ono S, Kami M, Shibuya K. Vaccination forwhom? Time to reinvigorate Japanese vaccinepolicy. Lancet. 380: 1647, 2012.

7. Tanimoto T, Kusumi E, Ono S. Review of noveltherapeutics by three regulatory agencies. NEngl J Med. 367: 1165-1166; author reply 1166-1167, 2012.

8. Nagata M, Takita M, Kishi Y, Kodama Y, Ma-tsumura T, Murashige N, Homma Y, Kami M.

Cancer articles in weekly magazines - usefulmedia to deliver cancer information to the pub-lic? Japanese Journal of Clinical Oncology, inpress

9. Tanimoto T, Yuji K, Kodama Y, Matsumura T,Yamamoto H, Mori J, Hosoda M, Uchida N,Kami M, Taniguchi S. The long and windingroad for the Fukushima nuclear workers. Lan-cet. 379: e34, 2012.

10. Tsubokura M, Horie S, Komatsu H, Tokiwa M,Kami M. The impact of the Great TohokuEarthquake on the dialysis practice in the disas-ter-stricken area. Hemodial Int. 16: 320-321,2012.

11. Harasawa K, Tanimoto T, Kami M, Oikawa T,Kanazawa Y, Komatsu H. Health problems inthe temporary housing in Fukushima. Lancet.379: 2240-2241, 2012.

12. Tsubokura M, Gilmour S, Takahashi K, OikawaT, Kanazawa Y. Internal radiation exposure af-ter the Fukushima nuclear plant accident.JAMA, 308: 669-670, 2012.

13. Okai K, Tsubokura M, Tanimoto T, KanazawaY. Radiograph abnormalities caused by the nu-clear accident. Intern Med. 51: 2073, 2012.

14. "Sugimoto A, Krull S, Nomura S, Morita T,Tsubokura M. The voice of the most vulnerable:lessons from the nuclear crisis in Fukushima,Japan. Bulletin of the World Health Organiza-tion. 90: 629-630, 2012.

15. Ishii T, Harasawa K, Tanimoto T. Drowning. N

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Engl J Med. 367: 777; author reply 777-778,2012.

16. Tsubokura M, Mori J, Tanimoto T, Oikawa T. Adog bite injury after the Fukushima nuclear ac-cident. Intern Med. 51: 2493, 2012.

17. Mori J, Hasui K, Tanimoto T, Matsumura T,Kami M. Drug Inf J. 46: 607-610, 2012.

18. Tanimoto T, Evacuee camps from the tsunamiand radiation disaster in Fukushima, Japan. Ar-chives of Internal Medicine. 172(3): 236, 2012.

19. Kodama Y, Morozumi R, Matsumura T, KishiY, Murashige N, Tanaka Y, Takita M, HatanakaN, Kusumi E, Kami M, Matsui A. Increased fi-nancial burden among patients with chronicmyelogenous leukaemia receiving imatinib inJapan: a retrospective survey. BMC Cancer. 12:152, 2012.

20. Watanabe N, Narimatsu H, Noh JY, YamaguchiT, Kobayashi K, Kami M, Kunii Y, Mukasa K,Ito K. Antithyroid drug-induced hematopoieticdamage: a retrospective cohort study of agranu-locytosis and pancytopenia involving 50,385 pa-tients with Graves' disease. J Clin EndocrinolMetab. 97: E49-53, 2012.

21. Matsumura T, Kami M, Yamaguchi T, Yuji K,Kusumi E, Taniguchi S, Takahashi S, Okada M,Sakamaki H, Azuma H, Takanashi M, Kodo H,Kai S, Inoue-Nagamura T, Kato K, Kato S. Allo-geneic cord blood transplantation for adultacute lymphoblastic leukemia: retrospective sur-vey involving 256 patients in Japan. Leukemia.26: 1482-1486, 2012.

22. Tanimoto T, Kusumi E, Hosoda K, Tajima F,Oshima Y. CNS prophylaxis in diffuse large B-cell lymphoma. Lancet. 379: 1485-1486; authorreply 1486-1487, 2012.

23. Mori J, Takahashi Y, Tsubokura M, MatsumuraT, Kami M. CNS prophylaxis in diffuse large B-cell lymphoma. Lancet. 379: 1486; author reply1486-1487, 2012.

24. Mori J, Takahashi Y, Tanimoto T. SF3B1 inchronic lymphocytic leukemia. N Engl J Med.366: 1057; author reply 1057-1058, 2012.

25. Takahashi Y, Mori J, Tanimoto T. Treatment ofolder patients with mantle-cell lymphoma. N

Engl J Med. 367: 1765; author reply 1765-1766,2012.

26. Takita M, Matsumoto S, Shimoda M, Chujo D,Itoh T, SoRelle JA, Purcell K, Onaca N, Na-ziruddin B, Levy MF. Safty and tolerability ofthe T-cell depletion protocol coupled with ana-kinra and etanercept for clinical islet cell trans-plantation. Clin Transplant. 26(5): e471-e484,2012.

27. Takita M, Matsumoto S. SUITO index for evalu-ation of clinical islet transplantation. Cell Trans-plant. 21: 341-1347, 2012.

28. 上昌広．電子書籍『震災・原発×医療×上昌広』．上昌広（著）．株式会社キャリアブレイン．２０１２．

29. 上昌広．復興は現場から動き出す 本気で動く個人のネットワークが本当に必要な支援を可能にする．上昌広（著）．東洋経済新報社．２０１２．

30. 上昌広．日本の医療崩壊を招いた構造と再生への提言．上昌広（著）．蕗書房．２０１２．

31. 坪倉正治．浜通り地域での内部被ばくの現状―検査結果の意味するところとその限界．低線量被ばくKEYBOOK．中川恵一編集．㈱メディカルアイ．１０７―１１３，２０１２．

32. 児玉有子，大磯義一郎．医療専門職の役割．介護福祉経営士テキストシリーズ【基礎編（Ⅱ）】【第１巻】医療を知る―介護福祉人材が学ぶべきこと．神津仁編集．日本医療企画．５７―８４，２０１２．

33. 児玉有子．移植医療における患者のQOLから何を学ぶか？ ケアの根拠 第２版―看護の疑問に答える１８０のエビデンス．道又元裕編集．日本看護協会出版会．１８６，２０１２．

34. 児玉有子．がんと医療経済．がん骨転移のバイオロジーとマネージメント．米田俊之編集．医薬ジャーナル社．３８３―３９０，２０１２．

35. 上昌広．住民が求めている内部被ばくデータ―南相馬市立総合病院での５３００例から，科学．８２⑶：２４９―２５１，２０１２．

36. 児玉有子．これからの社会と看護・医療の関係を考える メディアを介した現場からの情報発信．INR（International Nursing Review）．３５（３）：１４０―１４６，２０１２．

37. 永田政義，松村有子，本間之夫，上昌広．週刊誌のがん報道：週刊誌はがん情報を伝える有益な媒体である得るか？ 腎泌尿器学会誌２１⑴in press

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The dynamic whole-cell omics analyses to un-derstand and control cancer stem cells

Shigeo Fujimori1, Takatsune Shimizu1,2, Rui Ya-maguchi3, Hiroyuki Ohashi1, Seiya Imoto3, NaoyaHirai1, Yukiko Kawabata1, Hideyuki Saya2, SatoruMiyano3, Etsuko Miyamoto-Sato1,3: 1Div. Interac-tome Med. Sci., Inst. Med. Sci., Univ. of Tokyo2Div. Gene Reg., IAMR, Keio Univ. Sch. Med.3Human Genome Ctr., Inst. Med. Sci., Univ. of To-kyo

Recent scientific discoveries resulted from thenext-generation sequencing (NGS) highlight thestriking impact of massively parallel sequencingdata on genetics. Thus, the interest of basic researchin the medical care has changed from the conven-tional post-genomic to the personal genomic re-search, especially for cancer therapy. To understandthe individuality of cancer, we have to conductvarious dynamic omics analyses and comprehendthe individual biomolecular networks for each celltype. Our research focuses on diversity of cancerstem cells (CSCs), and is aimed at discerning andregulation of CSCs by the collaboration of research-ers, who have the experiment system for studyingCSCs, the technology for omics analysis, and the

network analysis technique. We have model CSCsderived from bone-marrow stromal cells of Ink4a/Arf KO mice (Shimizu et al., Oncogene, 2010). Tocompare those CSCs having distinct characters, weattempt to conduct "dynamic whole-cell omics"analyses, especially for interactome sequencing. Tocollect comprehensive protein level's data fromNGS, we have developed IVV-HiTSeq (Fujimori etal., Scientific Reports, 2012) standing for the in vitrovirus method (Miyamoto-Sato et al., PLoS ONE,2010) coupled with High-Throughput Sequencing.IVV-HiTSeq can obtain reliable interactome datasuitable for the medical field due to low false posi-tives. Moreover, we attempt to develop the bait-freeIVV method as IVV square (IVV2) to obtain not par-tial but a whole-cell interactome. IVV2 also detects'interacting regions (IR)', allowing us to analyze re-lations between interactome and aberrance (e.g.,DNA mutation) observed in cancer. Accordingly,we have developed the database to investigate suchrelations.

IRView: a database and viewer for protein inter-acting regions

Shigeo Fujimori1, Naoya Hirai1, Tomohiro Oshi-kubo1,2, Tatsuhiro Yamashita1,3, Takanori Washio1,4,

Donatinon Laboratories

Interactome Medical Sciences Laboratoryインタラクトーム医科学社会連携研究部門

Project Associate Professor Etsuko Miyamoto-Sato, Ph.D. 特任准教授 工学博士 宮 本 悦 子

In our lab, the interactome (protein-protein interactions) is analyzed using technol-ogy developed in Japan and referred to as "puromycin technology". Toward the"personalized medicine" era, we will apply these tools to medical science. To de-velop the "interactome analysis pipeline", we are collaborating with the HumanGenome Center, where large amounts of sequence data are generated from next-generation sequencers and analyzed using a supercomputer. In particular, our re-search focuses on "cancer", and we collaborate with cancer researchers. For acooperative study involving society and academia, our research involves industrialpartnerships.

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Ayumu Saito5, Satoru Miyano5, Etsuko Miyamoto-Sato1,5: 1Div. Interactome Med. Sci., Inst. of Med.Sci., Univ. of Tokyo 2FAE Ltd. 3Fujitsu Ltd.4RIKEN GENESIS Co., Ltd. 5Human Genome Ctr,Inst. of Med. Sci., Univ. of Tokyo

Protein-protein interactions (PPIs) and their net-works play central roles in governing cellular proc-esses. In recent years, the interest of researchers hasshifted from the conventional genomics to the per-sonal genomics, bringing a new challenge to exam-ine the impact of small differences in genomesamong individuals, cells and tissues on the interac-tomes. Although basic relationships between pro-teins have been compiled in public databases at theprotein level, proteins interact with other proteinsthrough regions (i.e. the interacting regions; IRs)specific to each interactant, thereby simultaneousinteractions with multiple proteins become possible.Furthermore, some IRs are competitively used fordifferent proteins. Therefore, to comprehend suchcomplex relations underlying protein interactionsand to reveal differences of networks among indi-viduals further refined interaction data are needed.The IRView (http://ir.hgc.jp/) is our developed data-base for IRs, which focuses on regions required forPPIs (Fujimori et al, Bioinformatics 2012). IRViewcurrently contains data for over 3,000 IRs deter-mined mainly with mRNA-display, known as the invitro virus (IVV) method (Miyamoto-Sato et al,PLoS ONE 2010). The IVV method employs thecDNA library created by the random priming RT-PCR for poly(A)＋RNA library for screening, allow-ing us to detect not only interacting proteins withbait, but also their regions. The IR data stored inthe IRView are combined with annotated regiondata such as InterPro, SNPs and variable regionsowing to alternative mRNA splicing.

PRD: A protein-RNA interaction database

Shigeo Fujimori1, Katsuya Hino2, Ayumu Saito3,Satoru Miyano3, Etsuko Miyamoto-Sato1,3: 1Div. In-teractome Med. Sci., Inst. Med. Sci., Univ. of To-kyo 2Dept. Biosci. & Info., Keio Univ. 3HumanGenome Ctr, Inst. of Med. Sci., Univ. of Tokyo

Although protein-RNA interactions (PRIs) are in-volved in various important cellular processes,compiled data on PRIs are still limited. This con-trasts with protein-protein interactions, which havebeen intensively recorded in public databases andsubjected to network level analysis. We developedPRD, an online database of PRIs, dispersed acrossseveral sources, including scientific literature. Cur-rently, over 10,000 interactions have been stored inPRD using PSI-MI 2.5, which is a standard modelfor describing detailed molecular interactions, withan emphasis on gene level data. Users can browse

all recorded interactions and execute flexible key-word searches against the database via a web inter-face. Our database is not only a reference of PRIs,but will also be a valuable resource for studyingcharacteristics of PRI networks. AVAILABILITY:PRD can be freely accessed at http://pri.hgc.jp/

A whole-cell interactome mapping using IVVsquare toward personal genomics

Hiroyuki Ohashi1, Shigeo Fujimori1, Naoya Hirai1,Takatsune Shimizu2, Hideyuki Saya2 and EtsukoMiyamoto-Sato1: 1Div. Interactome Med. Sci., Inst.Med. Sci., Univ. of Tokyo 2Div. Gene Reg., IAMR,Keio Univ. Sch. Med.

In the last decade, huge amount of genomic se-quence data have been determined and gathered.The Human Genome Project provided the humangenome sequence. Understanding the genetic se-quences of individual patients is becoming a centralfeature of medical care. It is very important that theaccumulation of genetic alterations and the interac-tome networks in cancer cells represents a universalfeature of the disease. The knowledge can contrib-ute to determining optimal therapy for the disease.Since protein-protein interactions (PPIs) are at thecore of the biomolecule network, we have devel-oped in vitro virus (IVV) system, which is a mRNAdisplay, to detect PPIs toward personal genomics.We have succeeded to obtain significant results ofinteractome analysis by using the system. On theother hand, the system has a limitation in the high-throughput screening and identification of interac-tion pairs of proteins, due to the time consumingpreparation of bait proteins and the low ability ofthe conventional sequencing method. To overcomethe problem, we are trying to develop "a bait-freeIVV", termed IVV Square (IVV2), which enablesgenes encoding interacting protein pairs to belinked. This will facilitate archiving of the interac-tome mapping of a whole-cell library. IVV2 librarieswill be subjected to the high-throughput sequenc-ing with the next-generation sequencers to generateinteractome information. We believe that the newsystem will contribute to understand the interac-tome networks in cancer cells, and to develop phar-maceutical drugs to treat intractable diseases.

Efficiency of puromycin-based technologies me-diated by release factors and a ribosome recy-cling factor

Hiroyuki Ohashi1, Naoya Hirai1 and Etsuko Miya-moto-Sato1: 1Div. Interactome Med. Sci., Inst.Med. Sci., Univ. of Tokyo

Two useful puromycin-based techniques, in vitrovirus (IVV) and C-terminal labelling of proteins,

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were developed based on the observation thatpuromycin can bind to the C-terminus of a full-length protein. Puromycin technology is a usefultool for the detection of proteins and analysis ofprotein-protein interactions (PPIs); however, prob-lems arise due to the existence of stop codons inthe native mRNAs. Release factors (RFs) enter theA site of the ribosome at stop codons, which inevi-tably compete with the puromycin. To overcomethis difficulty, we have used a highly controllablereconstituted cell-free system for puromycin-based

techniques and observed efficient IVV formationand C-terminal labelling using templates possessinga stop codon. The optimal conditions of IVV forma-tion using templates possessing a stop codon wasRF (－), while that of C-terminal labelling was RF(－) and the ribosome recycling factor (RRF) (＋).Thus, we have overcome the experimental limita-tions of conventional IVV. In addition, we discov-ered that RRF significantly increases the efficiencyof C-terminal protein labelling, but not IVV forma-tion.

Publications

Shigeo Fujimori, Naoya Hirai, Hiroyuki Ohashi,Kazuyo Masuoka, Akihiko Nishikimi, YoshinoriFukui, Takanori Washio, Tomohiro Oshikubo, Ta-tsuhiro Yamashita, Etsuko Miyamoto-Sato: Next-generation sequencing coupled with a cell-freedisplay technology for high-throughput produc-tion of reliable interactome data. Scientific Re-ports 2: 691 (2012)

Shigeo Fujimori, Naoya Hirai, Kazuyo Masuoka,Tomohiro Oshikubo, Tatsuhiro Yamashita, Taka-nori Washio, Ayumu Saito, Masao Nagasaki, Sa-toru Miyano, Etsuko Miyamoto-Sato: IRView: adatabase and viewer for protein interacting re-gions. Bioinformatics 28(14): 1949-1950 (2012)

Shigeo Fujimori, Katsuya Hino, Ayumu Saito, Sa-toru Miyano, Etsuko Miyamoto-Sato: PRD: Aprotein-RNA interaction database. Bioinforma-tion, 8 (15): (2012)

Hiroyuki Ohashi, Etsuko Miyamoto-Sato: A nextgeneration evolutionary molecular engineeringsystem and the future prospects. Viva origino 40suppl (2012)

柳川弘志，宮本悦子，高嶋秀昭：遺伝子型と表現型の対応付け分子とその構成要素，およびそれらの製法と利用方法，国立大学法人東京大学，特許第４９６３１４２号（２０１２）

宮本悦子，藤森茂雄：標的物質と相互作用するタンパク質の検出方法，国立大学法人東京大学，世界知的所有権機関（WIPO），出願番号：PCT／JP２０１２／０７５１５２（２０１２）宮本悦子，辻 融，藤森茂雄，石坂正道：生体分子相互作用解析ツールの構成とそれを用いた解析方法，国立大学法人東京大学，出願番号：特願２０１２―５０９５７０（２０１２）

Etsuko Miyamoto, Toru Tsuji, Shigeo Fujimori, Ma-samichi Ishizaka: CONSTITUTION OF TOOLFOR ANALYZING BIOMOLECULAR INTERAC-TION AND ANALYSIS METHOD USINGSAME. PCT/ European Patent/11765733.8 (2012)

Etsuko Miyamoto, Toru Tsuji, Shigeo Fujimori, Ma-samichi Ishizaka: CONSTITUTION OF TOOLFOR ANALYZING BIOMOLECULAR INTERAC-TION AND ANALYSIS METHOD USINGSAME. PCT/US Patent/ 13/638203 (2012)

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1. Therapeutic Development of RNA Aptameragainst Interleukin-17

Akira Ishiguro, Shigeyuki Mori1, Natsuki Otaki1,Haruta Kazuhiko1, Shoichiro Shibata, MichiruOzawa, Tomoyo Kimijima1, and Yoshikazu Naka-mura.: 1Zenyaku Kogyo Co. Ltd.

Interleukin-17A (IL-17A) is a pro-inflammatorycytokine produced primarily by a subset of CD4＋Tcells, called Th17 cells, that is involved in host de-fense, inflammation and autoimmune disorders. Wehave reported previously the successful isolation ofRNA aptamer against IL-17A, named Apt21-2, andits antagonistic activity against the in vitro and invivo functionality of IL-17A, implicating its potenttherapeutic potential (Ishiguro et al. 2011). Towardclinical application, we improved the activity andpharmacokinetic property of Apt21-2 by ribose 2'modifications and 5' PEGylation or a conjugation of

inverted dT. Through screening more than 300chemically synthesized derivatives of Apt21-2, thebest candidate showed the IC50 value 40 foldsmaller than that of Apt21-2 in the cell-based in vi-tro assay. We trust that this candidate aptamershould be applicable to the clinical trial after toxic-ity test.

2. Selection of RNA Aptamer against FibroblastGrowth Factor 2 and its Therapeutic Applica-tion

Akira Ishiguro, Maiko Sakamoto, Michiru Ozawa,Shoichiro Shibata, Yoshikazu Nakamura

Fibroblast growth factor is a class of heparinbinding proteins that mediate a variety of cellularresponses during embryonic development and inthe adult organism. Fibroblast growth factor 2(FGF2 or bFGF for basic fibroblast growth factor)

Donatinon Laboratories

Division of RNA Medical Science「RNA医科学」社会連携研究部門

Project Assoiate Professor Akira Ishiguro, Ph.D.Project Assistant Professor Takahiro Negishi, Ph.D.

特任准教授 理学博士 石 黒 亮特任助教 薬学博士 根 岸 崇 大

Protein non-cording RNAs can be classified into two types: one, like antisenseand microRNA, those function with the sequence complementarity to the targetmRNA or DNA, while the other, like aptamer, those function independent of thesequence complementarity.RNA aptamer is a biochemical or therapeutic agent that targets a given moleculethat is selected by a process known as SELEX (systematic evolution of ligands byexponential enrichment) from a complex library of random RNA sequences of typi-cally 1014 different molecules. The concept is based on the ability of short (20-80mer) sequences to fold, in the presence of a target, into unique three-dimensionalstructures that bind the target with high affinity and specificity. This binding, inmany cases, leads to a blockade of protein activity. Therefore, aptamers can bethought of as nucleic acid analogs to antibodies.By studying RNA aptamers, we hope to clarify superior potential of RNA, whichwould be highly beneficial to the development of RNA medicine and the compre-hensive understanding of RNA and protein functions.

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has an important role in proliferation, migration,angiogenesis and morphogenesis. Secreted FGF2protein activates several cell signaling such as JNK,MAPK, via through FGF receptors on the cell sur-face. Recent reports suggested that enhanced ex-pression in human RA patients, and also elevatedin rat joints of adjuvant-induced arthritis (AIA).By screening of a large library of nuclease-resis-

tant RNA oligonucleotides by SELEX, we selectedan RNA aptamer that bind human and mouseFGF2 proteins with high affinity and specificity.The selected aptamer binds strongly to FGF2, butnot to FGF1, and inhibits the interaction betweenFGF2 and its receptor when examined by the SPR(surface plasmon resonance) analysis. Consistently,the aptamer prevented efficient phosphorylation ofFGF2 signaling factors, FRS2 and MAPK, in thecell-based assay with NIH3T3. Furthermore, the ap-tamer inhibited the FGF2-dependent repression ofOPG (osteoprotegerin) secretion in HFLS-RA (Hu-man Fibroblast-Like Synoviocytes cells from RA pa-tient).We then demonstrated that in vivo efficacy of the

aptamer using GPI-induced rheumatoid arthritismice model. When administrated immediately afterimmunization with GPI, the aptamer inhibited thedevelopment of arthritic symptoms in a dose-de-pendent manner. Significantly, the aptamer slowedthe progression of arthritis when administered afterthe onset of GPI induced arthritis. Our findings in-dicate that the chemically processed anti-FGF2 ap-tamer inhibits FGF2 action and the development ofRA in mouse models. These results offer, for thefirst time an aptamer-based therapeutic approachfor FGF2 related disorders.

3. Selection of Aptamer Antagonists againstMouse Shh Protein and its Application as aNovel Silencing Tool in Developmental Biol-ogy

Akira Ishiguro

We aim to apply aptamer technology to the de-velopment of a novel protein-silencing tool, whichis applicable to dissection and/or discovery of therole of protein of interest in developmental biology.As a first model case, we selected RNA aptamersthat specifically inhibit mouse sonic hedgehog pro-tein, mShh. S9 trim-3 is one such aptamer raisedagainst mShh protein, specifically binds to mShhwith high affinity.During vertebrate development, morphogen gra-

dients generate different cell types in distinct spa-tial order. Secreted proteins of this class, such asHH, BMP, Wnt, FGF family proteins, play essentialroles in normal cell differentiation, axis determina-tion and patterning many systems. Shh is a well es-tablished morfogen, and plays various roles in de-velopment such as, patterning the central nervoussystem, during limb development, and formation offacial structures. The Shh signaling pathway in-volves two transmembrane proteins, Patched (Ptc)and Smoothened (Smo): Shh binds Ptc, whereasSmo acts as a signal transducer.In this program, aptamers were selected against

mShh by SELEX from RNA pools randomized over30 nucleotides with 2'-fluoro pyrimidine modifica-tions to resist ribonucleases. Isolated aptamer can-didates were examined for the binding affinity tomShh protein by the SPR assay. The highest affinityof the aptamer to mShh showed the dissociationconstant 15nM. The mShh protein has been shownto control osteoblast differentiation in vertebrates.The differentiated C3H10T1/2 embryonic fibroblastcells produce vascular endothelial growth factor(VEGF). The selected aptamer, S9 trim-3, inhibitedthe mShh-dependent differentiation of C3H10T1/2cells and blocked secretion of VEGF. Thus, the invivo efficacy of anti-mShh aptamer remains to beinvestigated in mouse model.

Publications

1. Nakamura Y, Ishiguro A & Miyakawa S. RNAplasticity and selectivity applicable to therapeu-tics and novel biosensor development. GenesCells. 17, 344-364, 2012.

2. 石黒亮：アプタマー創薬と国内外の開発状況，バイオインダストリー，２９：４―９．２０１２．

3. 石黒亮，中村義一：関節炎モデルマウスに対する抗IL-１７Ａアプタマーの効果，リウマチ科，４７：２９４―２９９．２０１２．

4. 石黒亮，中村義一：RNAアプタマーの分子標的への応用，医薬ジャーナル，４８：８５―８８．２０１２．

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1. A bacterial effector deamidates Ubc13 todampen the inflammatory response.

Sanada T, Kim M, Mimuro H, Suzuki M, OgawaM, Oyama A, Ashida H, Kobyashi T, Koyama T1,Nagai S1, Shibata Y2, Gohda J2, Inoue J2, Mizu-shima T3, and Sasakawa C.: 1Nippon Institute forBiological Science, Shinmachi, Ome, Tokyo, Ja-pan, 2Division of Cellular and Molecular Biology;Department of Cancer Biology; Institute of Medi-cal Science, University of Tokyo, Tokyo, Japan,3Picobiology Institute; Department of Life Science;Graduate School of Life Science; University ofHyogo, Hyogo, Japan.

Upon infection of many bacterial pathogens, bac-terial invasion is quickly sensed by the innate im-mune system and triggers acute inflammatory re-sponses. However, it is still unclear how pathogensmodulate host inflammatory responses. We foundthat a Shigella OspI effector delivered via the typeIII secretion system dampens acute inflammatoryresponses during bacterial invasion by targetingTNF receptor-associated factor 6 (TRAF6). OspI wasa glutamine deamidase and selectively deamidatedGln100 to Glu100 in Ubc13. Consequently, the E2ubiquitin-conjugating activity that is required forTRAF6 activation was inhibited, allowing Shigella

OspI to modulate the diacylglycerol-CBM complex-TRAF6-NF-B signaling pathway. We determinedthe 2.0 Å crystal structure of OspI, which containsa putative Cys-His-Asp catalytic triad. A mutationalanalysis showed that this catalytic triad was essen-tial for deamidation activity. Our results suggestthat Shigella inhibits acute inflammatory responsesat the initial stage of infection by targeting theUbc13-TRAF6 complex.

2. Shigella Targets Epithelial Tricellular Junc-tionsand Uses a Noncanonical Clathrin-De-pendent Endocytic Pathway to Spread Be-tween Cells.

Makoto Fukumatsu, Michinaga Ogawa, SatokoArakawa1, Masato Suzuki, Kazuhisa Nakayama2,Shigeomi Shimizu1, Minsoo Kim, Hitomi Mimuro,and Chihiro Sasakawa: 1Department of Pathologi-cal Cell Biology, Medical Research Institute, To-kyo Medical and Dental University, 1-5-45 Yu-shima, Bunkyo-ku, Tokyo 113-8510, Japan 2De-partment of Physiological Chemistry, GraduateSchool of Pharmaceutical Sciences, Kyoto Univer-sity, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku,Kyoto 606-8501, Japan

Bacteria move between cells in the epithelium us-

Donatinon Laboratories

Division of Bacterial Infection Biology細菌感染生物学社会連携研究部門（ヤクルト本社，日本生物化学研究所）

Project Associate Professor Minsoo Kim, Ph.D.Project Assistant Professor Hiroshi Ashida, Ph.D.

特任准教授 理学博士 金 玟 秀特任助教 医学博士 芦 田 浩

Many pathogenic bacteria, including Shigella, enteropathogenic Escherichia coli(EPEC), and enterohemorrhagic E.coli (EHEC), are associated with diarrheal dis-eases and are an important cause of death in many countries. Our current interestis to understand the complex interactions among pathogenic bacteria, the gastro-intestinal epithelium and microbiota during pathogenic bacteria infection. The maingoal of our research is to develop new therapeutic tools or vaccines that will targetthese bacterial infections.

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ing a sequential pseudopodium-mediated processbut the underlying mechanisms remain unclear. Weshow that during cell-to-cell movement, Shigel-lacontaining pseudopodia target epithelial tricellu-larjunctions, the contact point where three epithelialcells meet. The bacteria-containing pseudopodiawere engulfed by neighboring cells only in thepresence of tricellulin, a protein essential for tricel-lular junction integrity. Shigella cell-to-cell spread,but not pseudopodium protrusion, also depended

on phosphoinositide 3-kinase, clathrin, Epsin-1, andDynamin-2, which localized beneath the plasmamembrane of the engulfing cell. Depleting tricel-lulin, Epsin-1, clathrin, or Dynamin-2 expression re-duced Shigella cell-to-cell spread, whereas AP-2,Dab2, and Eps15 were not critical for this process.Our findings highlight a mechanism for Shigelladissemination into neighboring cells via targeting oftricellular junctions and a noncanonical clathrin-de-pendent endocytic pathway.

Publications

Sanada, T., Kim, M., Mimuro, H., Suzuki, M., Oga-wa, M., Oyama, A., Ashida, H., Kobyashi, T., Ko-yama, T., Nagai, S., Shibata, Y., Gohda, J., Inoue,J., Mizushima, T., and Sasakawa, C.A The Shigellaflexneri effector OspI deamidates Ubc13 todampen the inflammatory response. Nature, 483,623-626, 2012.

Fukumatsu M, Ogawa M, Arakawa S, Ahida H, Su-zuki M, Nakayama K, Shimizu S, Kim M, Mimu-ro H, Sasakawa C. Shigella targets epithelial tricel-lular junctions and uses a noncanonical Clathrin-dependent endocytic pathway to spread between

cells. Cell Host and Microbe, 11, 325-336, 2012Keiko Kawauchi, Wee Wee Tan, Keigo Araki,Farhana Binte Abu Bakar, Minsoo Kim, HideakiFujita, Hiroaki Hirata and Yasuhiro Sawada. p130Cas-dependent actin remodeling regulates myo-genic differentiation" Biochemical Journal, 445, 323-332, 2012

Ashida, H., Ogawa, M., Kim, M., Mimuro, H., andSasakawa C. Bacteria and host interactions in thegut epithelial barrier. Nat. Chem. Biol. 8: 36-45,2012.

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