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Structure Basis of CENP-A Nucleosome Recognition of Chicken KNL2

Jiang, Honghui 大阪大学 DOI:10.18910/93003

2023.09.25

概要

Title

Structure Basis of CENP-A Nucleosome Recognition
of Chicken KNL2

Author(s)

Jiang, Honghui

Citation

大阪大学, 2023, 博士論文

Version Type VoR
URL

https://doi.org/10.18910/93003

rights
Note

Osaka University Knowledge Archive : OUKA
https://ir.library.osaka-u.ac.jp/
Osaka University

Form 3

Abstract of Thesis
Name

Title





泓慧



Structure Basis of CENP-A Nucleosome Recognition of Chicken KNL2
(KNL2 による CENP-A ヌクレオソーム認識の分子構造基盤)

Abstract of Thesis
Centromere protein-A (CENP-A), a histone H3 variant, is an important epigenetic mark for centromere
specification. For new CENP-A deposition, chicken KINETOCHORE NULL2 (ggKNL2), a component of the licensing
factor Mis18 complex, binds directly to CENP-A-containing nucleosome via CENP-C-like motif to recruit the
CENP-A chaperone Holliday junction recognition protein (HJURP) to the centromere chromatin and ultimately
forming a new CENP-A nucleosome. However, the molecular basis of how the CENP-A nucleosome is recognized by
ggKNL2 remains unclear. To address this question, I performed biochemical and structural biological analyses
on the CENP-A nucleosome in complex with ggKNL2. To understand the centromere binding mode of KNL2 to the
CENP-A nucleosome, the cryo-EM structure of the chicken CENP-A nucleosome in complex with a ggKNL2 fragment
containing the CENP-C-like motif was determined. This structure suggests that ggKNL2 distinguishes between
CENP-A and histone H3 in the nucleosome using the CENP-C-like motif and its downstream region. Both the
C-terminal tail and the RG-loop of the CENP-A nucleosome are simultaneously recognized as CENP-A
characteristics by ggKNL2. Based on the structural findings, the mutational analysis was performed using
chicken DT40 cells and demonstrated that the interaction of KNL2 with the CENP-A nucleosome is critical for
new CENP-A incorporation and cell viability. The CENP-A nucleosome-ggKNL2 interaction is thus essential for
KNL2 functions. It is notable that KNL2 shares the CENP-A binding mode with CENP-C, which is a kinetochore
component. Furthermore, the structural, biochemical, and cell biology data implied the cell cycle dependent
regulation of centromere localization of ggKNL2. ggKNL2 directly binds to the CENP-A nucleosome in interphase,
while in mitosis ggKNL2 localizes to centromere depend on CENP-C.

様式7

論文審査の結果の要旨及び担当者






(職)

論文審査担当者











教 授
教 授
教 授
准教授



泓 慧




深川
上田
廣瀬
岡本


竜郎
昌宏
哲郎
浩二

論文審査の結果の要旨
生物が生命を維持するためには、染色体分配は必須のイベントである。その際に、紡錘体微小管が染色体の特殊領域
を捉え、次世代細胞へ伝える必要がある。この染色体の特殊領域はセントロメアと呼ばれる。興味深いことに、セン
トロメア領域はDNA配列では規定されずに、その領域にCENP-Aという特異的なヒストンが入ることが知られている。し
たがって、CENP-Aがどのようにセントロメア領域に取り込まれるかを理解することは、極めて重要である。そのメカ
ニズムとして、CENP-Aと結合するKNL2タンパク質がセントロメアの目印となり、新しいCENP-Aが取り込まれることが
知られている。しかしながら、KNL2がどのようにCENP-Aと結合するかは明らかでない。江泓慧さんは、KNL2を精製し、
CENP-Aを含むヌクレオソームを再構成した後にCryo-EMでその構造を決定して、結合の分子基盤を明らかにした。また、
その基盤に変異を入れたタンパク質を生化学的、細胞生物学に解析し、その正しさを証明した。これは、セントロメ
ア研究分野における重要な知見であり、博士の学位を授与するに値するものと認められる。
なお、学位論文をチェックツール“iThenticate”を使用し、剽窃、引用漏れ、二重投稿等のチェックを終えているこ
とを申し添えます。

この論文で使われている画像

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Acknowledgement

November 8, 2019 was the day I first came to Japan to meet Prof. Fukagawa. We met

hastily and happily at the elevator at 7:00 p.m., and then the lab students kindly

invited me to dinner. This was my first impression of Japan and the Fukagawa Lab. I

felt the kindness of prof. Fukagawa and the enthusiasm of the lab members, and as a

foreigner coming to Japan for the first time, I was grateful for this warm and

reassuring start.

I was very happy to meet Ariyoshi-sensei, a teacher who took care of me like a family

member in school and life after that, for the first time on the first working day

afterwards, and I started to work on my doctoral thesis under the professional and

kind guidance of Prof. Fukagawa and Ariyoshi-sensei. Every day, the lab members

would chat with me kindly and gently. In such a process of getting along, I am

grateful that people are considerate like friends and make me feel like family.

All these warm memories are clear and beautiful, and now it seems like it happened

yesterday when I think about it. Time flies, and suddenly I was shocked to realize that

almost four years have passed since the first day I remembered.

There is a Japanese proverb that goes like this, ‘一期一会’. I love this quote and will

carry this state of mind, cherish the fate of meeting and getting along with everyone

here, and be grateful for all the kindness and goodness I encounter here in

Fukagawa-lab.

I would like to thank Prof. Fukagawa for recognizing and accepting me and giving me

the opportunity to receive the most professional guidance and advice in a cutting-edge

laboratory in the field of chromosome biology. I am also very grateful to Prof.

Fukagawa for all the help he has provided me over the years, and I have learned from

him and lab members the traits that a good researcher needs to have.

I would like to express my deep gratitude to Ariyoshi-sensei for her professional

guidance on the details of my experiments, and her and Prof. Fukagawa's excellent

academic ability for supporting me to better grasp the progress of my project.

Thanks to Hara-sensei, Hori-sensei, Takenoshita-sensei, Watanabe-san, Shreyas-san,

97

Yamaguchi-san, Hirano-sansei, Fukui-san, Cao-san, Kong-san, Miao-san, Sha-san,

zhou-san and Li-san et al., for their kind and helpful academic guidance and help.

Thanks to Fukagawa-san, Oshimo-san, Fukuoka-san and Kubota-san for their

professional help in basic experiments, I have more time to focus on experimental

work.

Thanks to my old and new friends who have accompanied me on this journey of

growth over the years.

I would like to express my special thanks to Makino-san and all the seniors of Namba

lab who helped me to learn cryo-electron microscopy.

Finally, I would like to thank Ueda-sensei, Hirose-sensei and Okamoto-sensei for

their valuable comments and guidance on the defense of my doctoral dissertation. And

pecial thanks to the China scholarship council (CSC) scholarship for supporting my

life and research in Japan.

As my four years of valuable doctoral studies in Japan are coming to an end, this is

the last answer that I have seriously completed as a student. I am honored to have all

of you mentioned above or not mentioned above to witness my growth during this

journey.

I will face the rest of my life journey with more maturity and return to my loving

family who have always supported me and loved me deeply. And I look forward to

meeting you all in Japan again in the future!

Here, I look forward to being a contributing researcher in this path of scientific

research.

Grateful to meet, grateful to walk together.

98

Achievement

PUBLICATION

1. Hong Hui Jiang, Mariko Ariyoshi, Tetsuya Hori, Reito Watanabe, Fumiaki

Makino, Keiichi Namba, Tatsuo Fukagawa. The cryo-EM structure of the CENP-A

nucleosome in complex with ggKNL2. EMBO J (Article). 2023;42(6):e111965.

doi:10.15252/embj.2022111965.

2. Hong Hui Jiang, Bo Li, Yue Ma, Su Ying Bai, Thomas D. Dahmer, Adrian Linacre,

Yan Chun Xu. Forensic Validation of a Panel of 12 SNPs for Identification of

Mongolian

Wolf

and

Dog.

Sci

Rep

10,

13249

(2020).

https://doi.org/10.1038/s41598-020-70225-5.

3. Honghui Jiang, Bo Li, Yue Ma, Yanchun Xu. Feasibility Analysis of Species

Identification of Dogs and Wolves by Partial Sequence of mtDNA Control Region.

Chinese Journal of Wildlife, 2018, 039(002):277-285.

POSTER PRESENTATION

1. 2022 The 45th Annual Meeting of the Molecular Biology Society of Japan

(MBSJ)- poster presentation. Member of the Molecular Biology Society of Japan.

2. 2021 The 44th Annual Meeting of the Molecular Biology Society of Japan

(MBSJ)- poster presentation. Member of the Molecular Biology Society of Japan.

ORAL PRESENTATION

2018 Excellent Academic Report, The Ninth Symposium on Animal Research and

Conservation in Northeast provinces, China.

AWARDS

1. 2021-2023 China Scholarship Council (CSC) Chinese Government Scholarship

2. 2018 Excellent Academic Report, The Ninth Symposium on Animal Research and

99

Conservation in Northeast provinces, China.

MAIN CONTRIBUTION AS A CO-AUTHOR

I prepared materials and performed all biochemical experiments under supervision of

Dr. Mariko Ariyoshi; Dr. Mariko Ariyoshi, Dr. Fumiaki Makino and I performed all

cryo-EM experiments and Dr. Keiichi Namba helped cryo-EM single particle image;

Dr. Tetsuya Hori and Dr. Reito Watanabe performed DT 40 experiments; Prof. Tatsuo

Fukagawa supervised the entire project; I wrote this manuscript and Prof. Tatsuo

Fukagawa and Dr. Mariko Ariyoshi supervised my revision.

100

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