2024/02/25 更新

写真a

フジタ リサ
藤田 理紗
所属
研究院(研究機関) ナノ・ライフ創新研究機構
職名
次席研究員(研究院講師)
学位
博士(理学) ( 早稲田大学 )

経歴

  • 2023年03月
    -
    継続中

    早稲田大学   ナノ・ライフ創新研究機構   次席研究員

  • 2019年04月
    -
    2021年03月

    東京大学   定量生命科学研究所クロマチン構造機能研究分野   特任研究員

学歴

  • 2016年04月
    -
    2019年03月

    早稲田大学   先進理工学研究科   電気・情報生命専攻 博士後期課程  

  • 2014年04月
    -
    2016年03月

    早稲田大学   先進理工学研究科   電気・情報生命専攻 修士課程  

所属学協会

  •  
     
     

    日本エピジェネティクス研究会

  •  
     
     

    日本分子生物学会

研究分野

  • 分子生物学

研究キーワード

  • マイクロ流体デバイス

  • クロマチン

 

論文

  • Cryo-EM structures of RNA polymerase II-nucleosome complexes rewrapping transcribed DNA.

    Munetaka Akatsu, Haruhiko Ehara, Tomoya Kujirai, Risa Fujita, Tomoko Ito, Ken Osumi, Mitsuo Ogasawara, Yoshimasa Takizawa, Shun-Ichi Sekine, Hitoshi Kurumizaka

    The Journal of biological chemistry   299 ( 12 ) 105477 - 105477  2023年11月  [国際誌]

     概要を見る

    RNA polymerase II (RNAPII) transcribes DNA wrapped in the nucleosome by stepwise pausing, especially at nucleosomal superhelical locations -5 and -1 [SHL(-5) and SHL(-1), respectively]. In the present study, we performed cryo-electron microscopy analyses of RNAPII-nucleosome complexes paused at a major nucleosomal pausing site, SHL(-1). We determined two previously undetected structures, in which the transcribed DNA behind RNAPII is sharply kinked at the RNAPII exit tunnel and rewrapped around the nucleosomal histones in front of RNAPII by DNA looping. This DNA kink shifts the DNA orientation toward the nucleosome, and the transcribed DNA region interacts with basic amino acid residues of histones H2A, H2B, and H3 exposed by the RNAPII-mediated nucleosomal DNA peeling. The DNA loop structure was not observed in the presence of the transcription elongation factors Spt4/5 and Elf1. These RNAPII-nucleosome structures provide important information for understanding the functional relevance of DNA looping during transcription elongation in the nucleosome.

    DOI PubMed

    Scopus

  • High-Efficiency Single-Cell Containment Microdevices Based on Fluid Control

    Daiki Tanaka, Junichi Ishihara, Hiroki Takahashi, Masashi Kobayashi, Aya Miyazaki, Satsuki Kajiya, Risa Fujita, Naoki Maekawa, Yuriko Yamazaki, Akiko Takaya, Yuumi Nakamura, Masahiro Furuya, Tetsushi Sekiguchi, Shuichi Shoji

    Micromachines   14 ( 5 ) 1027 - 1027  2023年05月  [査読有り]

     概要を見る

    In this study, we developed a comb-shaped microfluidic device that can efficiently trap and culture a single cell (bacterium). Conventional culture devices have difficulty in trapping a single bacterium and often use a centrifuge to push the bacterium into the channel. The device developed in this study can store bacteria in almost all growth channels using the flowing fluid. In addition, chemical replacement can be performed in a few seconds, making this device suitable for culture experiments with resistant bacteria. The storage efficiency of microbeads that mimic bacteria was significantly improved from 0.2% to 84%. We used simulations to investigate the pressure loss in the growth channel. The pressure in the growth channel of the conventional device was more than 1400 PaG, whereas that of the new device was less than 400 PaG. Our microfluidic device was easily fabricated by a soft microelectromechanical systems method. The device was highly versatile and can be applied to various bacteria, such as Salmonella enterica serovar Typhimurium and Staphylococcus aureus.

    DOI

    Scopus

  • Method for Evaluating Effects of Non-coding RNAs on Nucleosome Stability.

    Mariko Dacher, Risa Fujita, Tomoya Kujirai, Hitoshi Kurumizaka

    Methods in molecular biology (Clifton, N.J.)   2509   195 - 208  2022年  [国際誌]

     概要を見る

    In eukaryotic cells, genomic DNA is stored in the nucleus in a structure called chromatin. The nucleosome, the basic structural unit of chromatin consisting of DNA wound around a histone octamer, regulates access of transcription machinery to DNA. Nucleosome stability is thus tightly associated with gene expression. Recently, a class of non-coding RNAs was found to be directly associated with chromatin. Although these non-coding RNAs are reportedly important in genome regulation, the molecular mechanisms through which these RNAs act remain unclear. Here, we introduce a biochemical method to evaluate the effects of ncRNAs on nucleosome stability, using the breast cancer-associated ncRNA Eleanor2 as an example. This method is useful for assessing the effects of different RNAs on chromatin stability and conformation.

    DOI PubMed

    Scopus

  • Cryo-EM structure of the CENP-A nucleosome in complex with phosphorylated CENP-C.

    Mariko Ariyoshi, Fumiaki Makino, Reito Watanabe, Reiko Nakagawa, Takayuki Kato, Keiichi Namba, Yasuhiro Arimura, Risa Fujita, Hitoshi Kurumizaka, Ei-Ichi Okumura, Masatoshi Hara, Tatsuo Fukagawa

    The EMBO journal   40 ( 5 ) e105671  2021年03月  [査読有り]  [国際誌]

     概要を見る

    The CENP-A nucleosome is a key structure for kinetochore assembly. Once the CENP-A nucleosome is established in the centromere, additional proteins recognize the CENP-A nucleosome to form a kinetochore. CENP-C and CENP-N are CENP-A binding proteins. We previously demonstrated that vertebrate CENP-C binding to the CENP-A nucleosome is regulated by CDK1-mediated CENP-C phosphorylation. However, it is still unknown how the phosphorylation of CENP-C regulates its binding to CENP-A. It is also not completely understood how and whether CENP-C and CENP-N act together on the CENP-A nucleosome. Here, using cryo-electron microscopy (cryo-EM) in combination with biochemical approaches, we reveal a stable CENP-A nucleosome-binding mode of CENP-C through unique regions. The chicken CENP-C structure bound to the CENP-A nucleosome is stabilized by an intramolecular link through the phosphorylated CENP-C residue. The stable CENP-A-CENP-C complex excludes CENP-N from the CENP-A nucleosome. These findings provide mechanistic insights into the dynamic kinetochore assembly regulated by CDK1-mediated CENP-C phosphorylation.

    DOI PubMed

    Scopus

    25
    被引用数
    (Scopus)
  • Nucleosome destabilization by nuclear non-coding RNAs.

    Risa Fujita, Tatsuro Yamamoto, Yasuhiro Arimura, Saori Fujiwara, Hiroaki Tachiwana, Yuichi Ichikawa, Yuka Sakata, Liying Yang, Reo Maruyama, Michiaki Hamada, Mitsuyoshi Nakao, Noriko Saitoh, Hitoshi Kurumizaka

    Communications biology   3 ( 1 ) 60 - 60  2020年02月  [査読有り]  [国際誌]

     概要を見る

    In the nucleus, genomic DNA is wrapped around histone octamers to form nucleosomes. In principle, nucleosomes are substantial barriers to transcriptional activities. Nuclear non-coding RNAs (ncRNAs) are proposed to function in chromatin conformation modulation and transcriptional regulation. However, it remains unclear how ncRNAs affect the nucleosome structure. Eleanors are clusters of ncRNAs that accumulate around the estrogen receptor-α (ESR1) gene locus in long-term estrogen deprivation (LTED) breast cancer cells, and markedly enhance the transcription of the ESR1 gene. Here we detected nucleosome depletion around the transcription site of Eleanor2, the most highly expressed Eleanor in the LTED cells. We found that the purified Eleanor2 RNA fragment drastically destabilized the nucleosome in vitro. This activity was also exerted by other ncRNAs, but not by poly(U) RNA or DNA. The RNA-mediated nucleosome destabilization may be a common feature among natural nuclear RNAs, and may function in transcription regulation in chromatin.

    DOI PubMed

    Scopus

    6
    被引用数
    (Scopus)
  • Cancer-associated mutations of histones H2B, H3.1 and H2A.Z.1 affect the structure and stability of the nucleosome

    Arimura, Yasuhiro, Ikura, Masae, Fujita, Risa, Noda, Mamiko, Kobayashi, Wataru, Horikoshi, Naoki, Sun, Jiying, Shi, Lin, Kusakabe, Masayuki, Harata, Masahiko, Ohkawa, Yasuyuki, Tashiro, Satoshi, Kimura, Hiroshi, Ikura, Tsuyoshi, Kurumizaka, Hitoshi

    Nucleic acids research   46 ( 19 ) 10007 - 10018  2018年11月  [査読有り]

     概要を見る

    Mutations of the Glu76 residue of canonical histone H2B are frequently found in cancer cells. However, it is quite mysterious how a single amino acid substitution in one of the multiple H2B genes affects cell fate. Here we found that the H2B E76K mutation, in which Glu76 is replaced by Lys (E76K), distorted the interface between H2B and H4 in the nucleosome, as revealed by the crystal structure and induced nucleosome instability in vivo and in vitro. Exogenous production of the H2B E76K mutant robustly enhanced the colony formation ability of the expressing cells, indicating that the H2B E76K mutant has the potential to promote oncogenic transformation in the presence of wild-type H2B. We found that other cancer-associated mutations of histones, H3.1 E97K and H2A.Z.1 R80C, also induced nucleosome instability. Interestingly, like the H2B E76K mutant, the H3.1 E97K mutant was minimally incorporated into chromatin in cells, but it enhanced the colony formation ability. In contrast, the H2A.Z.1 R80C mutant was incorporated into chromatin in cells, and had minor effects on the colony formation ability of the cells. These characteristics of histones with cancer-associated mutations may p

    DOI

    Scopus

    61
    被引用数
    (Scopus)
  • Cancer-associated mutations of histones H2B, H3.1 and H2A.Z.1 affect the structure and stability of the nucleosome.

    Arimura Y, Ikura M, Fujita R, Noda M, Kobayashi W, Horikoshi N, Sun J, Shi L, Kusakabe M, Harata M, Ohkawa Y, Tashiro S, Kimura H, Ikura T, Kurumizaka H

    Nucleic acids research   46 ( 19 ) 10007 - 10018  2018年11月  [査読有り]  [国際誌]

    DOI PubMed

    Scopus

    61
    被引用数
    (Scopus)
  • Methods for Preparing Nucleosomes Containing Histone Variants

    Kujirai, Tomoya, Arimura, Yasuhiro, Fujita, Risa, Horikoshi, Naoki, Machida, Shinichi, Kurumizaka, Hitoshi

    Methods in molecular biology (Clifton, N.J.)   1832   3 - 20  2018年04月  [査読有り]  [国際誌]

     概要を見る

    Histone variants are key epigenetic players that regulate transcription, repair, replication, and recombination of genomic DNA. Histone variant incorporation into nucleosomes induces structural diversity of nucleosomes, consequently leading to the structural versatility of chromatin. Such chromatin diversity created by histone variants may play a central role in the epigenetic regulation of genes. Each histone variant possesses specific biochemical and physical characteristics, and thus the preparation methods are complicated. Here, we introduce the methods for the purification of human histone variants as recombinant proteins, and describe the preparation methods for histone complexes and nucleosomes containing various histone variants. We also describe the detailed method for the preparation of heterotypic nucleosomes, which may function in certain biological phenomena. These methods are useful for biochemical, structural, and biophysical studies.

    DOI PubMed

    Scopus

    37
    被引用数
    (Scopus)
  • Methods for Preparing Nucleosomes Containing Histone Variants.

    Kujirai T, Arimura Y, Fujita R, Horikoshi N, Machida S, Kurumizaka H

    Methods in molecular biology (Clifton, N.J.)   1832   3 - 20  2018年  [査読有り]  [国際誌]

    DOI PubMed

    Scopus

    37
    被引用数
    (Scopus)
  • Association of M18BP1/KNL2 with CENP-A Nucleosome Is Essential for Centromere Formation in Non-mammalian Vertebrates

    Tetsuya Hori, Wei-Hao Shang, Masatoshi Hara, Mariko Ariyoshi, Yasuhiro Arimura, Risa Fujita, Hitoshi Kurumizaka, Tatsuo Fukagawa

    DEVELOPMENTAL CELL   42 ( 2 ) 181 - +  2017年07月  [査読有り]

     概要を見る

    Centromeres are specified and maintained by sequence-independent epigenetic mechanisms through the incorporation of CENP-A into centromeres. Given that CENP-A incorporation requires the Mis18 complex to be in the centromere region, it is necessary to precisely understand how the Mis18 complex localizes to the centromere region. Here, we showed that centromere localization of the Mis18 complex depends on CENP-A, but not CENP-C or CENP-T, in chicken DT40 cells. Furthermore, we demonstrated that M18BP1/KNL2, a member of the Mis18 complex, contained the CENP-C-like motif in chicken and other vertebrates, which is essential for centromere localization and M18BP1/KNL2 function in DT40 cells. We also showed that in vitro reconstituted CENP-A nucleosome, but not H3 nucleosome, bound to the CENP-C-like motif containing M18BP1/KNL2. Based on these results, we conclude thatM18BP1/KNL2 is essential for centromere formation through direct binding to CENP-A nucleosome in non-mammalian vertebrates. This explains how new CENP-A recognizes the centromere position.

    DOI PubMed

    Scopus

    43
    被引用数
    (Scopus)
  • Stable complex formation of CENP-B with the CENP-A nucleosome

    Risa Fujita, Koichiro Otake, Yasuhiro Arimura, Naoki Horikoshi, Yuta Miya, Tatsuya Shiga, Akihisa Osakabe, Hiroaki Tachiwana, Jun-ichirou Ohzeki, Vladimir Larionov, Hiroshi Masumoto, Hitoshi Kurumizaka

    NUCLEIC ACIDS RESEARCH   43 ( 10 ) 4909 - 4922  2015年05月  [査読有り]

     概要を見る

    CENP-A and CENP-B are major components of centromeric chromatin. CENP-A is the histone H3 variant, which forms the centromere-specific nucleosome. CENP-B specifically binds to the CENP-B box DNA sequence on the centromere-specific repetitive DNA. In the present study, we found that the CENP-A nucleosome more stably retains human CENP-B than the H3.1 nucleosome in vitro. Specifically, CENP-B forms a stable complex with the CENP-A nucleosome, when the CENP-B box sequence is located at the proximal edge of the nucleosome. Surprisingly, the CENP-B binding was weaker when the CENP-B box sequence was located in the distal linker region of the nucleosome. This difference in CENP-B binding, depending on the CENP-B box location, was not observed with the H3.1 nucleosome. Consistently, we found that the DNA-binding domain of CENP-B specifically interacted with the CENP-A-H4 complex, but not with the H3.1-H4 complex, in vitro. These results suggested that CENP-B forms a more stable complex with the CENP-A nucleosome through specific interactions with CENP-A, if the CENP-B box is located proximal to the CENP-A nucleosome. Our in vivo assay also revealed that CENP-B binding in the vicinity of the CENP-A nucleosome substantially stabilizes the CENP-A nucleosome on alphoid DNA in human cells.

    DOI PubMed

    Scopus

    48
    被引用数
    (Scopus)
  • Crystal structure and stable property of the cancer-associated heterotypic nucleosome containing CENP-A and H3.3

    Yasuhiro Arimura, Kazuyoshi Shirayama, Naoki Horikoshi, Risa Fujita, Hiroyuki Taguchi, Wataru Kagawa, Tatsuo Fukagawa, Genevieve Almouzni, Hitoshi Kurumizaka

    SCIENTIFIC REPORTS   4   7115  2014年11月  [査読有り]

     概要を見る

    The centromere-specific histone H3 variant, CENP-A, is overexpressed in particular aggressive cancer cells, where it can be mislocalized ectopically in the form of heterotypic nucleosomes containing H3.3. In the present study, we report the crystal structure of the heterotypic CENP-A/H3.3 particle and reveal its "hybrid structure", in which the physical characteristics of CENP-A and H3.3 are conserved independently within the same particle. The CENP-A/H3.3 nucleosome forms an unexpectedly stable structure as compared to the CENP-A nucleosome, and allows the binding of the essential centromeric protein, CENP-C, which is ectopically mislocalized in the chromosomes of CENP-A overexpressing cells.

    DOI PubMed

    Scopus

    61
    被引用数
    (Scopus)
  • Distinct Features of the Histone Core Structure in Nucleosomes Containing the Histone H2A.B Variant

    Masaaki Sugiyama, Yasuhiro Arimura, Kazuyoshi Shirayama, Risa Fujita, Yojiro Oba, Nobuhiro Sato, Rintaro Inoue, Takashi Oda, Mamoru Sato, Richard K. Heenan, Hitoshi Kurumizaka

    BIOPHYSICAL JOURNAL   106 ( 10 ) 2206 - 2213  2014年05月  [査読有り]

     概要を見る

    Nucleosomes containing a human histone variant, H2A.B, in an aqueous solution were analyzed by small-angle neutron scattering utilizing a contrast variation technique. Comparisons with the canonical H2A nucleosome structure revealed that the DNA termini of the H2A.B nucleosome are detached from the histone core surface, and flexibly expanded toward the solvent. In contrast, the histone tails are compacted in H2A.B nucleosomes compared to those in canonical H2A nucleosomes, suggesting that they bind to the surface of the histone core and/or DNA. Therefore, the histone tail dynamics may function to regulate the flexibility of the DNA termini in the nucleosomes.

    DOI PubMed

    Scopus

    20
    被引用数
    (Scopus)

▼全件表示

講演・口頭発表等

  • 非コードRNAがヌクレオソームに直接与える影響の生化学的解析

    藤田 理紗

    第12回日本エピジェネティクス研究会  

    発表年月: 2018年05月

  • 非コード RNA はヌクレオソーム中の H2A-H2B の解離を促進する

    藤田 理紗

    第35回染色体ワークショップ・第16回核ダイナミクス研究会  

    発表年月: 2017年12月

  • 非コードRNA Eleanorはヌクレオソーム中のヒストンの交換を促進する

    藤田 理紗

    2017年度生命科学系学会合同年次大会  

    発表年月: 2017年12月

  • Nucleosome destabilization and histone exchange mediated by noncoding RNA

    Risa Fujita

    1st HMGU-Japan Mini Symposium “Epigenetics and Chromatin”  

    発表年月: 2017年09月

  • Biochemical analysis of the effect of noncoding RNA on the nucleosome

    Risa Fujita

    EMBO CONFERENCE “The Nucleosome: From Atoms to Genomes”  

    発表年月: 2017年08月

  • 非コードRNAがヌクレオソームの安定性に及ぼす影響の生化学解析

    藤田 理紗

    新学術領域「生殖細胞エピゲノム」「ステムセルエイジング」「クロマチン動構造」3領域合同若手勉強会2017  

    発表年月: 2017年06月

▼全件表示

共同研究・競争的資金等の研究課題

  • 非コードRNAによるクロマチン転写制御機構

    日本学術振興会  科学研究費助成事業 研究活動スタート支援

    研究期間:

    2019年08月
    -
    2021年03月
     

    藤田 理紗

     概要を見る

    真核生物のゲノムDNAはクロマチン構造を形成して核内に収納されている。本研究では、非コードRNAがクロマチン上の転写を制御する機構の解明を目的とした。そこで、非コードRNAが引き起こすヌクレオソーム構造の変化および非コードRNAによるRNAポリメラーゼIIの転写活性への影響を構造生物学的および生化学的解析を行った。その結果として、非コードRNAがヌクレオソームの安定性を変化させることを明らかにした。また、非コードRNAがヌクレオソーム上での転写活性や転写中のヌクレオソーム構造に影響を及ぼす可能性を見出した。

  • セントロメアタンパク質CENP-Bのセントロメア構築における機能の解析

    日本学術振興会  科学研究費助成事業 特別研究員奨励費

    研究期間:

    2016年04月
    -
    2019年03月
     

    藤田 理紗

     概要を見る

    細胞分裂期における染色体の均等分配には、セントロメアの正常な形成が必要である。セントロメアの形成領域は、セントロメア特異的に形成されるCENP-Aを含むヌクレオソームによって決定づけられている。その際、CENP-Aと多数のセントロメアタンパク質の相互作用を介してセントロメアが構築される。本研究では、CENP-Aヌクレオソームに結合するCENP-Bのセントロメア構築における機能解明を目的とした。CENP-Bの機能として、CENP-BがCENP-Aヌクレオソームに結合することで、CENP-Aヌクレオソームの構造を変化させるのではないかと考えた。その点を明らかにするため、構造生物学的手法による解析に取り組んだ。本年度は、CENP-BのN末端領域のDNA結合ドメイン(DBD:DNA Binding Domain)を用いて、CENP-B DBD結合CENP-Aヌクレオソームのクライオ電子顕微鏡による単粒子解析に着手した。この手法では溶液中の構造を捉えることが可能である。観察に用いる複合体を急速凍結する際に、因子同士の結合を保持する必要があるため、架橋剤の種類の検討を行い、複合体の調製方法を確立した。
    また昨年度には、CENP-B DBDがCENP-AのN末端領域と結合することを見出している。この結果を受けて、この相互作用がCENP-B DBDのDNA結合の安定化に寄与する可能性を考えた。その点を検証するために、CENP-AのN末端領域のペプチドを添加した際のCENP-B DBDとDNAの複合体の熱安定性を解析した。本解析では、CENP-AのN末端領域はCENP-B DBDのDNA結合の安定化に寄与しないという結果が得られた。したがって、CENP-AとCENP-B DBDの相互作用には別の意義があると考えられる。今後、異なる解析手法によってその相互作用の意義の解明が期待される。

Misc

  • 非コードRNA Eleanorはヌクレオソーム中のヒストンの交換を促進する

    藤田 理紗, 有村 泰宏, 山本 達郎, 浜田 道昭, 斉藤 典子, 胡桃坂 仁志

    生命科学系学会合同年次大会   2017年度   [3PT18 - 0555)]  2017年12月

  • セントロメアタンパク質CENP-BとCENP-Aヌクレオソームの相互作用解析

    藤田理紗, 大竹興一郎, 有村泰宏, 宮優太, 越阪部晃永, 立和名博昭, 大関淳一郎, 舛本寛, 胡桃坂仁志

    日本生化学会大会(Web)   88th  2015年

    J-GLOBAL