[보고서]후성유전체 조절 단백질의 상호작용을 통한 히스톤 탈아세틸화 효소의 환경스트레스 반응 조절 기작 규명

조면행

후성유전체 조절 단백질의 상호작용을 통한 히스톤 탈아세틸화 효소의 환경스트레스 반응 조절 기작 규명
Study on the role of histone deacetylases in response to environmental stress via protein-protein interaction 원문보기

보고서 정보
주관연구기관	연세대학교 Yonsei University
연구책임자	조면행
참여연구자	이원경 , 김안나 , 황희정 , 표영재 , 조성경
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2018-02
과제시작연도	2017
주관부처	농촌진흥청 Rural Development Administration(RDA)
등록번호	TRKO201800043302
과제고유번호	1395049137
사업명	차세대바이오그린21
DB 구축일자	2018-12-15
키워드	후성유전학.염색질.히스톤.DNA 메틸화.환경스트레스.epigenetics.chromatin.histone.DNA methylation.environmental stress.
DOI	https://doi.org/10.23000/TRKO201800043302

초록

○ 히스톤 탈아세틸화 효소와 텔로미어 결합단백질의 결합을 통한 이질염색질 형성과 텔로미어 길이 조절에 대한 후성유전학적 조절 기작 규명: 국제 학술지 게재 (NAR, 2016), 국제학술대회 발표
○ 이질 염색질 형성에 관여하는 히스톤 및 DNA 변형 분자들의 기능적 상호 결합 규명 및 결합지도 작성
○ 후성유전제 조절 단백질들의 유전자 정보, 돌연변이체, 과다발현체 확보를 통한 효율적 연구 체계 구축

(출처 : 보고서 요약서 3p)

Abstract ▼

Purpose&Contents
○ Understanding of epigenetic regulation in chromatin modification, gene expression, and environmental stress response/tolerance in plants is essential for increasing our knowledges and development of desirable crops.

○ It is required to establish and development of the genetic resources related to environmental stress tolerance for developing environment-customized molecular breeding strategies.

Results
○ Histone modifier-involved epigenetic regulation mechanism in heterochromatin formation was characterized and protein-protein interaction map was constructed.

- Histone deacetylases (HDT4, HDA6) are involved in heterochromatin formation and telomere length regulation via interaction with telomere-binding protein, AtTRB2: Published in NAR (2016) and presented in international conferences.

- Genetic and epigentic materials and resources including genes, mutants, and overexpression lines are established for future studies.

Expected Contribution
○ Our knowledges on the epigenome formation and regulation involved in biotic and abiotic stress and tolerance will be useful for redesigning and developing desirable crops.
○ Obtained results will be shared with related research communities and crop industries.
○ Gene information, transformants, and mutants will be available for colleagues and crops breeders.

(출처 : Summary 5p)

목차 Contents

표지 ... 1
제 출 문 ... 2
보고서 요약서 ... 3
국 문 요 약 문 ... 4
Summary ... 5
목차 ... 6
제 1 장 연구 개발 과제의 개요 ... 7
제1절 연구 개발 목적 ... 7
제2절 연구 개발의 필요성 ... 7
제3절 연구 개발 범위 ... 7
제 2 장 국내외 기술개발 현황 ... 9
제 3 장 연구 수행 내용 및 결과 ... 10
제 4 장 목표달성도 및 관련분야 기여도 ... 26
제1절 : 목표대비 달성도 ... 26
제2절 : 정량적 성과(논문게재, 특허출원, 기타)를 기술 ... 27
제 5 장 연구 결과의 활용 계획 ... 28
제 6 장 연구 과정에서 수집한 해외 과학 기술 정보 ... 29
제 7 장 연구 개발 결과의 보안 등급 ... 29
제 8 장 국가과학기술지식정보서비스에 등록한 연구시설·장비 현황 ... 29
제 9 장 연구개발과제 수행에 따른 연구실 등의 안전조치 이행실적 ... 30
제 10 장 연구개발과제의 대표적 연구실적 ... 30
제 11 장 기타사항 ... 30
제 12 장 참고문헌 ... 30
끝페이지 ... 31

표/그림 (24)

표 AtTRB2 interacts physically with the HD2-type histone deacetylase, HDT4, in vivo and in vitro> (A) Yeast two-hybrid assay showing direct interaction between AtTRB2 and HDT4 (left panel). (B) Analysis of AtTRB2 binding to HDT4 in plant cells determined by immunoblot. (C) Bimolecular fluorescence complementation (BiFC) analysis detected fluorescence signals generated by the interaction between AtTRB2 and HDT4 in tobacco epidermal cells (Nicotiana benthamiana). (D) In vitro pull-down assay showing the exclusive interaction of AtTRB2 with HDT4 among HD2 class histone deacetylases in Arabidopsis
표 Mapping the interaction domains on AtTRB2 and HDT4> (A, B) Schematic representation of several truncated AtTRB2 (A) and HDT4 protein constructs (B). AtTRB2 has a Myb DNA-binding domain in the N-terminal and a H1/5 globular domain in the central region. HDT4 has a conserved EFWG motif and an aspartate-rich region in the N-terminal and C-terminal region, respectively. (C) PAGE shows the purified truncated HDT4 recombinant proteins. (D, E) Analysis of HDT4 recombinant proteins binding to the domains of AtTRB2 monitored by immunoblot. (F) Scheme of the regions of HDT4 that interact with AtTRB2
표 HDT4 is a putative histone H3 lysine 27 deacetylase in Arabidopsis> (A) Analysis to map the H3-interacting region on HDT4 in vitro. (B) Scheme of the region of HDT4 involved in the interaction with histone H3. (C) In vitro peptide binding assay showing binding of HDT4 to acetylated lysine residues of histone H3. (D) Acetylated lysine status of histone H3 in two independent HDT4-HA-expressing transgenic plants and hdt4-1 mutant plants determined by various antibodies in vivo
표 HDT4 associates with telomeric repeat DNA in an AtTRB2-dependent manner> (A) Telomeric ChIP (T-ChIP) analysis showing the association between AtTRB2 and telomeric chromatin in tobacco. (B) Quantification of multiple independent T-ChIP experiments
표 AtTRB2 also interacts with HDA6, a RPD3-type histone deacetylase > (A) Western blot analysis showing the different patterns of interaction between AtTRB proteins and various HDAC proteins. (B) Scheme of various HDA6 truncated proteins fused to GST. (C) Pull-down assay to map the regions of HDA6 that interact with AtTRB2. (D) Schematic map indicating the region of HDA6 involved in binding to AtTRB2. (E) Western blots demonstrating the interaction between AtTRB2 and HDA6 in vivo. (F) HDA6 associates with TTTAGGG repeats. (G) HDA6 associates with Arabidopsis-type telomeric chromatin mediated by AtTRB2
표 Telomere-binding protein and epigenetic molecules regulate telomere dynamics in Arabidopsis > (A) Terminal Restriction Fragmentation (TRF) analysis demonstrating the regulation of telomere maintenance by AtTRB2 in Arabidopsis. (B) Level of AtTRB2mRNA in mutants and complemented plants was analyzed by RT-PCR. (C) Telomere lengthening in hdt4-1-/-mutants. (D) Level of HDT4 mRNA in wild-type and hdt4-1mutants. (E, F) TRF assay to analyze the telomere alterations in HDA6, CMT3, and MET1 mutants (ecotype Columbia, axe1-5, cmt3, and met1, respectively)
표 Changes in the pattern of DNA methylation in telomeic and subtelomeric regions> (A) Southern blot analysis showing the pattern of DNA methylation of telomeric repeats in cmt3, met1, axe1-5 (left panel), hdt4-1 and attrb2-1 mutants (right panel). DNA blots of methylation-sensitive restriction enzyme-digested gDNA probed with [TTTAGGG]70 repeat sequences. M, MspI; H, HpaII; B, BstNI; E, EcoRII. (B) Chop-PCR in subtelomeric and ITSs regions using McrBC endonuclease. 4R-300, -600, -800, and -2100 indicate the different regions on the subtelomeric regions closed to telomere in the right arm of chromosome IV. The 60S RPgene was used as a reference for quantitative comparisons.-, +; McrBC-untreated/ -treated
표 Protein-protein interaction between histone methyltransferases and other epigenetic regulators in vitro and in vivo. HMTases (SUVH4/KYP, ATXR5, ATXR6) and LHP1, like-hetrochromatin protein 1 interact with AtTRB2 in vitro and in vivo
표 Telomeric association of LHP1. (A) LHP1 interacts with AtTRB2 in vivo, (B) ATXRs do not associates with TTTAGGG repeats, (C) LHP1 directly associates with Arabidopsis-type telomeric chromatin
표 Effects on the DNA methylation in telomeric chromatin by HMTases and LHP1 by showing Southern blot analysis using methylation-sensitive restriction enzymes. (A) HMTases (SUVH and ATXR proteins) showing non-CpG methylation in telomeric chromatin. (B) LHP1 did not show differences in DNA methylation pattern
표 Pattern of DNA methylation in hetrochromatic region of chromosome IV: McrBC assay. SUVH4/KYP is involved in H3K9 methyation in subtelomeric region and interstitial telomeric sequences
표 HMTases and LHP1 are acting as negative regulators of telomere elongation in Arabidopsis> (A) KYP and SUVH proteins. (B) ATXR5 and ATXR6. (C) LHP1
표 Protein-protein interaction between AtTRBs (AtTRB1, AtTRB2, and AtTRB3) and HDACs (HDA7, HDA9, SRT1, and SRT2) in vitro
표 SRT1 associates with telomeric repeat DNA in an AtTRB2-dependent manner
표 SRT1 and SRT2 directly interact with telomeric repeat binding proteins, AtTRB1, AtTRB2, and AtTRB3 in vitro
표 SRT1 directly interacts with AtTRB2 in vivo shown by Co-IP and BiFC assay
표 SRT1 is a putative histone H3 lysine 9 deacetylase in vitro> (A) Schematic diagram showing the NAD+-dependent deacetylation reaction. (B) Acetylated lysine status of histone H3 in deacetylation reaction in vitro. (C) Acetylated lysine status of histone H3 in SRT1-FLAG-expressing transgenic plants and srt1-1 mutant plants determined by various antibodies in vivo
표 Terminal Restriction Fragmentation (TRF) analysis demonstrating the regulation of telomere maintenance by SRT1 in Arabidopsis
표 Protein-protein interaction between methyl-CpG-binding domain (MBD) proteins and AtTRBs in vitro
표 TERRA (Telomeric repeat-containing RNA) generation in mutants of genes for KYP, ATXR5, ATXR6, and LHP1
표 TERRA (Telomeric repeat-containing RNA) generation in mutants of genes for HDT4, AtTRB2, MET1, CMT3, HDA6
표 Binding specificity of different histone chaperone proteins to AtTRB2 in Arabidopsis
표 Vector map for rice transformation and hygromycin resistance and transgene expression analysis (RT-PCR) of T2 transgenic rice
표 Protein-protein interaction map constructed based on the results obtained thus far in this research project

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연합인증

후성유전체 조절 단백질의 상호작용을 통한 히스톤 탈아세틸화 효소의 환경스트레스 반응 조절 기작 규명
Study on the role of histone deacetylases in response to environmental stress via protein-protein interaction 원문보기