Characterization of Nuclear Transport Factor 2, DEMETER DNA demethylase-interacting protein in Arabidopsis = DEMETER DNA 탈메틸화효소와 상호작용하는 Nuclear Transport Factor 2의 애기장대에서의 특성 연구

국문 초록 (Abstract)

식물의 발달 과정에서 DNA 메틸화와 탈메틸화에 의한 후성유전적 조절은 유전자 발현 조절의 중요한 메커니즘으로 작용한다. DEMETER (DME)는 애기장대에서 발견된 능동적 DNA 탈메틸화 효소로, 생식세포 형성과 종자 발달에 필수적인 역할을 한다. DME는 수정 전 중심세포(central cell)에서 발현되며, 이 시기에 DME가 발현하지 않으면 수정 후 배유의 세포분열이 비정상적으로 일어나 종자가 치사하게 된다. DME는 생식뿐 아니라 영양생장 발달에도 관여한다. dme-2 돌연변이체에서 뿌리털 신장 이상과 quiescent center(QC)의 비정상적 분열이 관찰되었으며, 이는 DME 매개 DNA 탈메틸화가 두 발달 과정 모두에 필수적임을 보여준다. 그러나 DME가 DNA 메틸화 부위를 어떻게 인식하며, 어떤 보조 인자들과 상호작용하여 탈메틸화를 수행하는지는 명확히 밝혀진 바가 없다. 본 연구에서는 DME와 상호작용하는 단백질을 규명하고, 이러한 상호작용이 DME의 기능 조절에 어떻게 기여하는지를 분석하고자 하였다. Nuclear Transport Factor 2 (NTF2)는 세포질에서 RanGDP와 결합하여 핵 내 물질 수송을 매개하는 단백질로, DME와 물리적 상호작용을 하는 것으로 알려져 있다. ntf2-2 돌연변이체는 dme-2 돌연변이체와 유사하게 뿌리털의 과도한 신장과 QC의 비정상적인 분열을 보이며, 이는 두 단백질이 식물의 체세포 (sporophyte) 발달 과정에서 공통된 경로를 통해 기능함을 시사한다. 본 연구에서 DME-NTF2 복합체와 상호작용하는 인자들을 스크리닝한 결과, 메틸화 부위를 인식하는 Methyl-Reader Protein (MRP)이 DME-NTF2 복합체와 상호작용함을 Bimolecular fluorescence complementation (BiFC), in vitro pull-down, Yeast three-hybrid 분석을 통해 확인하였다. 이는 DME가 DNA 메틸화 부위를 인식하고 탈메틸화 기능을 수행하는 과정에서 NTF2, MRP와의 상호작용이 관여할 가능성을 제시한다. 또한 RNA 시퀀싱 및 전장 유전체 이황화 시퀀싱 (WGBS)을 통해 dme-2 및 ntf2-2 돌연변이체의 전사체 및 DNA 메틸화 패턴 변화를 비교함으로써, 두 단백질이 DNA 메틸화 조절에 미치는 기능적 연관성을 규명하고자 하였다.

다국어 초록 (Multilingual Abstract)

Epigenetic regulation through DNA methylation and demethylation plays a crucial role in controlling gene expression during plant development. DEMETER (DME) is an active DNA demethylase identified in Arabidopsis thaliana that is essential for gametophyte formation and seed development. DME is expressed specifically in the central cell prior to fertilization, and the absence of DME expression at this stage results in abnormal endosperm cell division and seed abortion after fertilization. DME is involved not only in reproductive but also in vegetative development. Abnormal root hair elongation and irregular division of the quiescent center (QC) were observed in dme-2 mutants, indicating that DME-mediated DNA demethylation is essential for both developmental processes. However, the molecular mechanisms by which DME recognizes specific DNA methylation sites and interacts with cofactors to mediate DNA demethylation remain largely unknown. In this study, we aimed to identify proteins interacting with DME and to investigate how these interactions contribute to DME function.
Nuclear Transport Factor 2 (NTF2), which binds RanGDP in the cytoplasm and mediates nuclear transport, has been reported to physically interact with DME. The ntf2-2 mutant displays elongated root hairs and abnormal QC division, phenocopying the dme-2 mutant, suggesting that DME and NTF2 function cooperatively during sporophytic development. Through screening of proteins interacting with the DME–NTF2 complex, we identified a Methyl-Reader Protein (MRP) that recognizes methylated sites and interacts with the DME–NTF2 complex. This interaction was validated by bimolecular fluorescence complementation (BiFC), in vitro pull-down, and yeast three-hybrid assays. These findings suggest that the interaction among DME, NTF2, and MRP may play a critical role in recognizing methylated DNA and mediating active demethylation. Furthermore, RNA sequencing and whole-genome bisulfite sequencing (WGBS) analyses were performed to compare transcriptomic and methylation profiles between dme-2 and ntf2-2 mutants, thereby elucidating the functional relationship of DME and NTF2 in DNA methylation regulation.

목차 (Table of Contents)

• Abstract ⅰ
• Table of Contents ⅳ
• List of Figures vi
• List of Table viii
• Abbreviation ix
• 1. Introduction 1
• 1.1. DNA methylation in Arabidopsis 1
• 1.2. DEMTER DNA demethylase 3
• 1.3. Nuclear Transport Factor 2, a DME-interacting protein 5
• 1.4. Regulation of Sporophyte Growth and Meristem Dynamics by DEMETER 8
• 2. Material and methods 10
• 2.1. Plant Material and Growth Condition 10
• 2.2. BiFC assay 10
• 2.3. in vitro pull-down assay 12
• 2.4. Yeast two hybrid assay 12
• 2.5. Yeast three hybrid assay 13
• 2.6. Intrinsically disordered region prediction 14
• 2.7. Confocal Microscopy 14
• 2.8. Analysis of β-glucuronidase (GUS) staining 14
• 2.9. Observation of Root hair phenotypes 15
• 2.10. RT-qPCR 15
• 2.11. Sample preparation for WGBS and transcriptome analysis 15
• 2.12. WGBS data processing 16
• 2.13. Identification of DMRs 17
• 2.14. RNA-seq analysis 17
• 3. Results 22
• 3.1. NTF2, DME, and MRP physically interact in planta 22
• 3.2. Lack of direct physical interaction between DME and MRP in vitro pull-down assay 24
• 3.3. Yeast three hybrid assay reveals NTF2 mediated DMEMRP Interaction 26
• 3.4. NTF2 mediates DMEMRP interaction via its RRM domain 29
• 3.5. NTF2 expression pattern in various tissue 32
• 3.6. Root phenotypes of ntf2-2 phenocopy dme-2 35
• 3.7. Global DNA methylation dynamic in dme-2 and ntf2-2 mutants in Arabidopsis 39
• 3.8. Genome-wide DNA methylation changes in ntf2-2 and dme-2 seedlings 42
• 3.9. Distinct and broad sporophytic regulatory functions of DME compared with NTF2 47
• 4. Discussion 54
• References 57
• Abstract in Korean 63