第 37 回日本分子生物学会年会フォーラム国際 FANTOM プロジェクトの最新情報～プロモーター／エンハンサーのダイナミクスでみる細胞活性～日時：11 月 25 日（火）19：15-20：45 会場：パシフィコ横浜第 37 回日本分子生物学会年会オーガナイザー：林崎良英（理化学研究所第 8 会場（会議センター3F 315）予防医療・診断技術開発プログラム） Piero Carninci（理化学研究所ライフサイエンス技術基盤研究センター） FANTOM は、20 か国、114 機関の 261 人の研究者が参加する理化学研究所主催の国際コンソーシアムです。今回のフォーラムでは、細胞分化の過程におけるプロモーター／エンハンサーの動的変化を観測することにより、細胞活性を記述した最近の成果をまとめて紹介します。次世代シーケンサーの一般化にともない、ゲノムワイドなアプローチは、研究の対象にかかわらず重要な意味を持つようになってきました。オミックス研究に携わる研究者はもちろん、「分野が違う」と思う若手の研究者や学生の方にとっても有意義なものとなるでしょう。一部は英語での講演となります。 19：15-19：20 開会にあたって林崎良英（理化学研究所予防医療・診断技術開発プログラム） 19：20-19：25 ヘリスコープ一分子シーケンサーによる CAGE 解析法伊藤昌可（理化学研究所予防医療・診断技術開発プログラム） 19：25-19：45 哺乳類におけるプロモーター単位でのゲノムワイドな発現地図と、これを収録したデータベースシステム川路英哉（理化学研究所予防医療・診断技術開発プログラム） 19：45-20：05 ヒト細胞および組織におけるエンハンサー活性地図 Albin Sandelin（コペンハーゲン大学） 20：05-20：25 哺乳類細胞の活性化および分化の過程におけるエンハンサー/ プロモーター活性のダイナミクス Erik Arner （理化学研究所ライフサイエンス技術基盤研究センター） 20：25-20：35 FANTOM の展望：長鎖 non-coding RNA の機能とネットワーク Piero Carninci （理化学研究所ライフサイエンス技術基盤研究センター） 20：35-20：45 FANTOM の展望：疾患のネットワークの理解とバイオマーカー探索林崎良英（理化学研究所予防医療・診断技術開発プログラム）林崎良英独立行政法人理化学研究所予防医療・診断技術開発プログラムプログラムディレクター Omics for Medical Treatment FANTOM の展望：疾患のネットワークの理解とバイオマーカー探索要旨： The “omics” approach integrates genomics, transcriptomics into single data set and can lead to the identification of unknown genomic elements and their regulatory networks involved in the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. In 2000, RIKEN has launched FANTOM (Functional Annotation of Mammalian Genome), an international consortium, which is leading transcriptome research in the world. Whereas the human genome project mapped every gene in the human genome, the FANTOM project has been mapping which genes are “active”. At the fifth stage of the project, FANTOM has released the data which provides the first overview of the networks regulating transcription across all cell types. Over 250 experts in primary cell biology and bioinformatics from 114 institutions based in more than 20 countries and regions worked as part of FANTOM5. Cap Analysis of Gene Expression (CAGE), the central technology used during FANTOM5, provides genome-wide overview of transcription start sites and their usage in a cell. With this original technology, we identified 180,000 promoters and 44000 enhancers on the genome across over 180 human primary cells. In-depth analysis of the obtained data led to the new knowledge that the activity of the large majority of these transcriptional regulation regions is highly specific to cell type. Our next research target is application of omics data to medical treatment. CAGE analysis of cancer cells enable reclassification of cancer cells and lead to subsequent biomarker discovery to identify patient subgroups with the highest unmet medical need. Furthermore, CAGE data can identify a group of transcription factors that regulates each transcripts. 略歴： Yoshihide Hayashizaki is Director of the Preventive Medicine & Diagnosis Innovation Program. He obtained his M.D. and Ph.D. from Osaka University Medical School in 1982 and 1986, respectively. In 1992, he joined RIKEN, and was appointed Project Director for the RIKEN Genome Project in 1995. Since then he has been taking a transversal data-driven approach to analyse transcriptomes by developing unique technologies including a series of full-length cDNA technologies. This activity was followed by the organization of FANTOM (Functional Annotation of Mammalian Genome), an international consortium, originally to annotate a large number of cDNA and subsequently expanded to transcriptome and network analysis. His research interests are focused on the understanding of biological phenomena as systems at the molecular level and its application to preventive medical care. Piero Carninci 独立行政法人理化学研究所ライフサイエンス技術基盤研究センター機能性ゲノム解析部門部門長 FANTOM Future: long non-coding RNAs functions and networks FANTOM の展望：長鎖 non-coding RNA の機能とネットワーク要旨: We have developed cap-analysis gene expression (CAGE) to simultaneously map mRNAs and non-coding RNAs transcription starting sites (TSSs) and measure their expression at each different promoters. Since CAGE shows single nucleotide resolution, we can use this technology to comprehensively measure gene expression at each TSSs. Due to this unprecedented resolution, we have learned that promoters use different regulatory elements in different cells and tissues. Using CAGE, we can also infer the transcriptional networks that regulate gene expression in each different cell type. For its high resolution to map TSSs, CAGE has been used extensively in the ENCODE and modENCODE projects. In the FANTOM5 project, we have applied CAGE on a comprehensive panel of human and mouse primary cells and other tissues, resulting in a very broad map the promoterome and regulatory networks. Our map reveals the existence of more than 180,000 promoters and 45,000 enhancers, which are often tissue specific. The FANTOM5 database is one of the broadest expression database available to the community (http://fantom.gsc.riken.jp/5/). Additionally, we have determined the pattern of expression of retrotransposon elements (RE), which are likely to have a regulatory role. As example, some families of LTR retrotransposon elements are specifically expressed in ES and iPS cells, where they have a role in maintenance of pluripotency. Future FANTOM projects will be focusing to broadly understand the function and the interaction with cell regulatory networks of these RNAs in several primary cells, with the purpose to create the broadest database of functional lncRNAs. 略歴： Born and Educated in Italy he obtained his doctoral degree at the University of Trieste in 1989. From 1990 to 1995 he developed technologies for DNA extraction and DNA sequencing at Talent, a spin-off biotech. He moved to Japan in 1995 at RIKEN, Tsukuba Life Science center and became tenure researcher in 1997. He has been developing technologies to capture full-length cDNAs, which were used for the construction of the Fantom projects. Between 2008 and 2013, he was a Team and Unit Leader and a Deputy Project Director at the RIKEN Omics Science Center in Yokohama. He has developed technologies to analyze the transcribed part of the genome (transcriptome), such as the cap-trapper and the CAGE. These technologies have been broadly used in the RIKEN Fantom projects and allowed identifying non-coding RNAs as are the major output of the mammalian genome and providing comprehensive maps of the mammalian promoters. Additionally he developed a miniaturization of CAGE, in order to approach biological problems that for which there is limited amount of starting material. From April in 2013, he is a Director of the Division Genomics Technologies and a Deputy Director of Center for Life Science Technologies, RIKEN. He has published more than 230 papers and book chapters, edited books and is a member of editorial boards of various scientific journals. 伊藤昌可独立行政法人理化学研究所予防医療・診断技術開発プログラムコーディネーター Cap analysis of gene expression on single molecule sequencer, HeliScope genetic analysis system ヘリスコープ一分子シーケンサーによる CAGE 解析法要旨： Cap Analysis of Gene Expression (CAGE) is the method to investigate transcription start sites comprehensively, based on cap-trapper technology. The original protocol involved various problematic steps and difficult to be used widely in research field. We have been working on the simplification of the protocol, and optimization for single molecule sequencer, HeliScope, because the single molecule sequencing technology can eliminate any biases caused by amplification, ligation or another enzymatic processes. The established CAGE protocol for single molecule sequencer, HeliScope CAGE was applied on automatization, then finally we succeeded to achieve high-throughput CAGE library production system. 略歴： Masayoshi Itoh is coordinator of the Preventive Medicine & Diagnosis Innovation Program. He obtained his Ph.D. from Gifu University in 1995. In 1996, he joined RIKEN as a postdoctoral fellow in Genome Science Laboratory. From 1998 to 2000, he was employed as a RIKEN special postdoctoral fellow, then signed as a stuff scientist of Genome Science Laboratory in 2001. He has been working on the technology development for large scale sequencing. The achievements were the large scale plasmid preparation system for full-length cDNA project, and CAGE technology standardization and automatization for single molecule sequencer, HeliScope, for Mouse Encyclopedia and following FANTOM projects. Now, he has intended to apply CAGE technology on various medical uses. 川路英哉独立行政法人理化学研究所予防医療・診断技術開発プログラムコーディネーター A promoter level mammalian expression atlas and its web resource 哺乳類におけるプロモーター単位でのゲノムワイドな発現地図と、これを収録したデータベースシステム要旨：哺乳類の発生経路や生体を構成する数百万の細胞種の空間的構成は、写の制御によってコントロールされる。1 分子 CAGE 法を用いて、 Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly ‘housekeeping’, whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research. To facilitate the exploration of the large-scale data, we assembled it into a centralized data resource with an open-access on-line interface. Descriptions of individual samples are carefully curated manually and an application ontology that uses classes from established ontologies for cell types, anatomy, and diseases is developed to group related samples systematically based on sample types. Web-based databases, and visualization tools (SSTAR, ZENBU, BioLayout Express3D, TET, BioMart) are provided to allow research scientists to search, navigate, and extract data related to samples, genes, promoter activity, and transcription factor gene regulation across the entire FANTOM5 atlas. We also exposed the data via trackHub of the UCSC Genome Browser, plugin of BioGPS, and as nanopublication in the LinkedData community for further integration with other datasets. This combination of software tools, curated databases and systematic sample annotation gives the scientific community powerful tools to explore, examine and extract data in multiple ways. Here we introduce the online resources, their underlying data structure and discuss potential impacts in cell, genome and molecular biology. 略歴：理化学研究所予防医療診断技術開発プログラムコーディネーター。情報基盤センター予防医療・ゲノミクス応用開発ユニットユニットリーダーを兼務する。 2003 年大阪大学にて博士号を取得。2007 年、理化学研究所入所。一連の計算科学的手法およびリソース（データベース）の開発、大規模な生物学的データ解析（とくに転写開始、その制御、small RNA、エピジェネティクス）に携わる。研究分野は、計算生物学および分子生物学を基礎とするゲノミックスから、医療に資するトランスレーショナルリサーチにまで及ぶ。 Hideya Kawaji is a coordinator of RIKEN Preventive Medicine and Diagnosis Innovation Program, and the leader of preventive medicine and applied genomics unit in RIKEN Advanced Center for Computing and Communication. He obtained his Ph.D. from Osaka University in 2003. After joining RIKEN in 2007, he developed a series of computational methods and resource (databases), and performed data analysis to interpret a large set of biological data, in particular about transcriptome including transcription initiation, its regulation, small RNAs and epigenetics. His research interests ranges from genomics based on computational and molecular biology to translational research contributing medical care. Albin Sandelin コペンハーゲン大学生物・バイオロジー研究開発センターコンピューター・RNA 生物学部門教授 The FANTOM5 enhancer atlas ヒト細胞および組織におけるエンハンサー活性地図要旨： Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Recent findings indicate that enhancers emit RNAs once they are active. Exploiting this fact, we have used the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas was used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation. I this talk, I will give a brief overview of the FANTOM5 enhancer atlas, but also show our current work in enhancer detection in medical samples and intuitive computational tools for tissue-specific enhancer selection. 略歴： Albin Sandelin is a Professor at the Department of Biology and BRIC, Copenhagen University. He obtained his MSc in Molecular Biology in 2000 from Stockholm University and his PhD from Karolinska Institute in 2004. His most noted work from that period was the JASPAR DNA motif database, now a standard tool in bioinformatics. During his postdoctoral period at RIKEN, he was one of the key analysts of 5’ end data from the FANTOM project. As a principal investigator at Copenhagen University, he has used the same technique in combination with computational methods to investigate the biology of gene regulation, and is now extending this into inflammatory disease. A recent highlight was an atlas of enhancer regions and their usage over the human body. Erik Arner 独立行政法人理化学研究所ライフサイエンス技術基盤研究センター機能性ゲノム解析部門ＬＳＡ要素技術研究グループゲノム情報解析チーム副チームリーダー Dynamics of enhancer and promoter activity during mammalian cellular activation and differentiation 哺乳類細胞の活性化および分化の過程におけるエンハンサー／プロモーター活性のダイナミクス要旨： Cellular differentiation requires the coordinated induction of genes needed in the new cellular state, and down-regulation of those no longer required in the previous one. The dynamic regulation of promoters and enhancers by transcription factors facilitates this state change. Here, using Cap Analysis of Gene Expression (CAGE) we measured enhancer and promoter activity for 20 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. The data further expand the functional identification of enhancers and promoters in the mammalian genome and extend our knowledge of their dynamic regulation and likely function. We demonstrate that enhancer RNAs dominate the earliest expression responses in every time course studied, followed by mRNAs encoding transcription factors and then by other transcripts in successive waves. Early activated genes and enhancers are typically time-course specific. The induction of enhancer RNA expression precedes or occurs concurrently with changes in expression of candidate target genes in their vicinity. In any one system, the binding sites for the same key transcription factors are over-represented in both active enhancers and promoters. These results support a model in which enhancers, like known immediate early promoters, exist in a poised state and are the key targets of early signal transduction in any cellular transition. 略歴： Erik Arner is a Senior Research Scientist at RIKEN, Japan. He is an expert in bioinformatics. After finishing a Master’s degree in engineering biotechnology at Uppsala University he obtained his Ph.D. from Karolinska Institutet in 2006, and joined RIKEN in 2007. His research interest is in applying genome wide technologies to basic questions in biology, aswell as to disease questions with clinical focus.