Analysis of the genome architecture of the hyperthermopholic Title archaeon Thermococcus kodakarensis( Dissertation_全文 ) Author(s) Maruyama, Hugo Citation Kyoto University (京都大学) Issue Date 2011-03-23 URL http://dx.doi.org/10.14989/doctor.k16233 Right Type Thesis or Dissertation Textversion author Kyoto University Analysis of the genome architecture of the hyperthermophilic archaeon Thermococcus kodakarensis Hugo Maruyama 要旨 ゲノム DNA は細胞内で高度に折りたたまれ、この染色体高次構造は転写・複製・染色体分 配といった機構と密接に結びついている。染色体の主要な構成タンパク質は真核生物では ヒストン、バクテリアでは HU と全く異なるが、一様な基本構造を基にゲノム DNA が階層 的に折りたたまれている点で両者の染色体構造は共通している。アーキアは真核生物・バ クテリアと並ぶ生命の第三のドメインであり、遺伝情報の発現（複製・転写・翻訳）の機 構は真核生物に、代謝経路はバクテリアに近い。アーキアには染色体を構成するタンパク 質として真核生物のヒストンに相同なもの、バクテリアの HU に相同なもの、アーキア特有 の Alba と呼ばれるタンパク質などが存在し、種によってゲノムがコードするタンパク質の 組合せが異なる。様々なアーキアのゲノムがどのような高次構造を形成しているかを明ら かにすることで、三つのドメインにわたるゲノム構造の共通性あるいは多様性を明らかに できる。本研究ではその第一歩としてヒストンを持つ超好熱性アーキア Thermococcus kodakarensis の染色体構造を解析した。 T. kodakarensis の染色体に含まれるタンパク質を質量分析により同定した結果、ヒ ストン、Alba、TK0471（TrmBL2）、 RNA ポリメラーゼ等の DNA 結合タンパク質が含まれ ることが分かった。TK0471 は転写因子 TrmB と相同な機能未知の DNA 結合タンパク質で あった。次に、染色体をミクロコッカルヌクレアーゼで部分消化した後、5%-20%のショ糖 密度勾配遠心により構成タンパク質の異なる染色体断片が分離された。原子間力顕微鏡に よる解析から、ヒストンは DNA 上に beads-on-a-string 構造を、TK0471 は線維状の構造を形 成することが示された。また大腸菌で発現させた組換えタンパク質（ヒストンおよび TK0471）を用いて同様の構造が DNA 上に再構成された。ショ糖密度勾配で分離されたそ れぞれの染色体断片に含まれる DNA 配列を超並列シークエンサーで同定した結果、ヒスト ンおよび TK0471 はゲノム上のプロモーター領域にもコーディング領域にも偏りなく存在 するが、両者の存在する領域は重複しない傾向があった。以上の結果から、T. kodakarensis の染色体上には、構成タンパク質および構造の異なる領域が存在することが明らかとなっ た。相同組換えにより TK0471 遺伝子を破壊すると染色体の DNA 消化酵素に対する感受性 が高まった。また、約 100 個の遺伝子の転写産物量が増加した。TK0471 破壊株における各 遺伝子の転写量の上昇と TK0471 タンパク質のプロモーター上の局在とには有意な相関が あった。このことから、TK0471 は染色体に多量に存在してゲノム構造を維持すると同時に、 プロモーターに結合した場合には転写抑制因子として働くと考えられる。TK0471（TrmBL2） は一部のアーキアとバクテリアの種で保存された新しいタイプの染色体構成タンパク質で ある。他の種での機能解析によりその機能がより詳細に明らかになることが期待される。 ii Abstract Genomic DNA is highly organized and compacted inside the cell. The higher-order chromosome architecture is coupled to cellular processes such as DNA replication, transcription, and chromosome segregation. Although the fundamental architectural protein of the chromosome is different for eukaryotes and bacteria (i.e., histone in eukaryotes and HU in bacteria), eukaryotic chromosomes and bacterial nucleoids both exhibit hierarchical architecture. Archaea is the third domain of life, distinct from eukaryotes or bacteria. The information processing machineries (DNA replication, transcription, and translation) of archaea are more similar to those of eukaryotes and the metabolic pathways are more similar to those of bacteria. Interestingly, archaeal species encode diverse chromosome architectural proteins such as eukaryotic histone, bacterial HU, as well as archaea-specific Alba. Each archaeal species encodes different combinations of these proteins. Clarification of the chromosome architecture in archaea will lead to the understanding of the general chromosome folding principle in all three domains. As a first step toward this goal, the chromosome architecture of the hyperthermophilic archaeon Thermococcus kodakarensis was analyzed in this study. Histone, Alba, TK0471, and RNA polymerase subunits were identified through mass spectrometry as the major DNA-binding proteins of the T. kodakarensis chromosome. TK0471 is a protein previously named TrmB-like 2 (TrmBL2) based on its homology to transcription factors TrmB or Tgr/TrmB-like 1 (TrmBL1), but its specific role has been difficult to identify. Sucrose density gradient sedimentation of a partially digested T. kodakarensis chromosome resulted in the separation of chromosome fragments with different protein compositions. Atomic force microscopy revealed that histone forms a “beads-on-a-string” structure, while TK0471 forms a thick fibrous structure on the DNA. High-throughput sequencing of the DNA concentrated with histone or TK0471 showed that these proteins localize to both coding and intergenic regions, but that these areas of localization tend not to overlap. Deletion of the TK0471 gene by homologous recombination results in an alteration of the chromosome into a more nuclease-sensitive state and an increase in the transcript level of approximately 100 genes. The elevation of the transcript level has a significant correlation with the localization of TK0471 to the promoter region of the gene. These results suggest that TK0471 (TrmBL2) is an abundant chromosomal protein that plays dual roles in chromosome architecture and transcriptional regulation. Further analysis in other species may lead to greater understanding of this protein‟s function. iii Acknowledgements First and foremost I offer my sincerest gratitude to my supervisor, Dr. Kunio Takeyasu, who has supported me throughout my study with his patience and knowledge whilst allowing me the room to work in my own way. This thesis would not have been completed without his support. He has always emphasized the importance of keeping the big picture in mind, and to study something fundamentally novel. These are important attitudes that I will remember throughout my life. I also appreciate the opportunities he provided me with, like to study abroad as an exchange student, and to attend international conferences. These experiences have broadened my vision towards science. Next, I would like to express my gratitude to Dr. Shige H. Yoshimura, who was my first direct supervisor when I started my research in the laboratory as an undergraduate student and has been a great advisor since then. I remember the first experiment I conducted with him was to amplify DNA using PCR. He has given me a lot of helpful suggestions, especially when I was writing the manuscript for my research publication. Working with him, I learned the importance of having solid logic in my study. I sincerely thank all of the current and former members of Takeyasu laboratory for their help and friendship over the years I spent there. The study described in this thesis is based on Dr. Ryosuke L. Ohniwa‟s study and could not have reached this point without him. He has encouraged me throughout my research with his positive attitude toward science. Drs. Chieko Wada, Kohji Hizeme and Masatoshi Yokokawa encouraged me to publish my research, even when it was not going well and seemed as if it would never end. They have been a pillar of support that has encouraged me to complete my study. I learned a lot about computational analysis and programming from Mr. Toshiyuki Oda. Dr. Minsang Shin has given me many insightful discussions about prokaryotic gene regulation. I thank Ms. Abeer Hasan for English proofreading of my manuscript. I would like to thank Drs. Rie Matsumi, Haruyuki Atomi, Tadayuki Imanaka and the members in their laboratory for insightful suggestions and discussions about the study of the archaeal species. This collaborative study would not have been possible without them. I also thank Dr. Katsuhiko Shirahige and Dr. Takehiko Itoh for their support and helpeful suggestions about DNA sequencing, which constitutes an important part of this thesis. Next, I would also like to thank the people I worked with at the Salk Institute for Biological Studies in La Jolla, USA. Professor Takeyasu provided me with the opportunity to study there as an exchange student. There I worked mainly with the iv postdoctoral researcher Dr. Marcela Raices under the supervision of Dr. Andrew Dillin and Dr. Jan Karlseder. I learned to be aware of the biological and/or medical relevance of my own study, and to always think about how I can contribute to move the research field forward. I would like to thank the Graduate School of Biostudies and the “Life Science English Communication Program” for their support to send me to the Gordon Research Conference on Archaea in 2009. I would also like to thank the Ministry of Education, Culture, Sports, Science and Technology of Japan and the “Academic Frontiers Student Exchange Promotion Program” for their support for me to study abroad as an exchange student. Last but not least, I thank my parents, who have educated, supported and encouraged me to pursue my interests. Without their continuous support, it was not possible to complete my work. I also thank all my friends. Without the time I spent with them outside the laboratory, I could not have maintained a healthy mind to concentrate on science. Kyoto, Hugo Maruyama 2011 v Curriculum Vitae Hugo Maruyama Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto 606-8501 Tel. & Fax (075) 753-7906 e-mail: [email protected], [email protected] Born July 31, 1978 in Osaka, Japan Education 4/1994 – 3/1997 Takatsuki High School 4/1998 – 3/2002 Kyoto University, Faculty of Integrated Human Studies 5/2003 – 3/2007 The Salk Institute for biological studies (As an exchange student) 4/2002 – 3/2010 Kyoto University, Graduate School of Biostudies 4/2010 – present Researcher at Kyoto University, Graduate School of Biostudies Fellowship 4/2002 – 3/2007 Fellowship for Graduate Students, The Japan Scholarship Foundation 4/2003 – 3/2004 Exchange student, Academic Frontiers Student Exchange Promotion Program 2003-2004 (Ministry of Education, Culture, Sports, Science and Technology, Japan) Awards Poster Award at The 3rd NTU-Kyoto U student mini-Symposium on molecular and cell biology (2008/5) Second Place Poster Award at Gordon Research Conference, Archaea: Ecology, Metabolism & Molecular Biology (2009/7) Teaching Experience 4/2008 – 3/2010 Teaching assistant at Graduate School of Biostudies, Kyoto University 4/2009 – 3/2010 Part-time lecturer at Shin-Osaka Dental Hygienist College (Biology) Publications (1) Peer-Reviewed