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研究生:林之立
研究生(外文):Chih-Li Lin
論文名稱:EDD及其交互作用蛋白於基因調控中角色的研究與探討
論文名稱(外文):Functional roles of EDD and its associated proteins in regulating gene expression
指導教授:呂勝春
指導教授(外文):Sheng-Chung Lee
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:74
中文關鍵詞:轉錄作用染色質組蛋白修剪作用泛素泛素化接合?
外文關鍵詞:transcriptionchromatinhistonesplicingubiquitinE3 ligase
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利用「與『基因轉錄延伸促進者—FACT複合體』有交互作用之蛋白」的系統性搜尋實驗中,我們找到了 EDD︰一具有HECT區位 (domain) 的細胞泛素E3接合?。雖然目前已知EDD在哺乳類細胞中參與細胞增生、基因轉錄調控和DNA損傷等相關反應,但其在細胞中所扮演的真正角色仍未明瞭。在本論文中,我們證明了大多數的EDD是位於細胞核的基質中,並與胞內的 FACT複合物有交互作用;此結果說明:EDD確實參與在調控核染色質結構的機制中。更進一步的研究指出:EDD能與 AND-1 (另一具有WD40及HMG-BOX區位的蛋白) 交互作用並形成一個複合物,此結果顯示 AND-1為一能與核染色質及核基質相聯的因子,有趣的是在免疫螢光染色的結果發現:AND-1在細胞核中是以特殊的點狀構造散布,此與我們研究AND-1能與前訊息RNA的修剪複合物、核仁蛋白、核骨架蛋白lamin有交互作用的現象相吻合;綜合上述的結果:AND-1可能為一具有多功能的蛋白,並能將轉錄及後轉錄作用聯結在一起。本論文初步揭開了兩個未知蛋白EDD及AND-1可能藉由泛素化的作用來達成調控細胞中的基因轉錄機制,往後的研究方針將著重在EDD 與AND-1交互作用的功能上進行更深入的探討。
EDD, a HECT-domain E3 ubiquitin ligase, was identified to be associated with the transcription elongator – FACT complex. Although mammalian EDD has been implicated in cell proliferation, transcriptional regulation, and DNA damage response, its exact cellular function is largely unknown. Our results demonstrated a predominately nuclear matrix localization of EDD and its in vivo association with FACT, thus suggesting EDD’s involvement in chromatin structure regulation. Further characterization of EDD revealed that EDD forms a stable complex with a WD40 domain- and HMG domain-containing protein AND-1. Experiment evidence indicates that AND-1 is a chromatin and nuclear matrix-associated factor. Interestingly, AND-1 possessed a distinct punctate pattern of subnuclear localization, consistent with its interaction with pre-mRNA splicing complexes, nucleolar proteins, and nuclear lamins. These observations collectively imply that that AND-1 may be a multifunctional protein that links and coordinates transcriptional and post-transcriptional events. Our present study uncovered two key proteins that may be important regulators of gene expression, and putatively linked ubiquitination to such cellular process. Future work on the functional characterization of EDD, AND-1, and their interaction is discussed.
MASTER THESIS I
謝誌 II
CONTENTS 3
ABBREVIATIONS 5
摘要 6
ABSTRACT 7
1. INTRODUCTION 8
2. MATERIALS AND METHODS 13
2.1 PREPARATION OF DNA CONSTRUCTS AND RECOMBINANT PROTEINS 13
Molecular cloning of EDD 13
Molecular cloning of AND-1 13
2.2 GENERATION OF SPECIFIC ANTIBODIES 16
Anti-EDD Antibodies 16
Anti-AND-1 Antibodies 16
2.3 SDS-PAGE 17
Preparation of polyacrylamide gel 17
SDS-PAGE analysis 17
2.4 WESTERN BLOT ANALYSIS 18
2.5 CELL CULTURE 19
2.6 DNA TRANSFECTION 19
2.7 PREPARATION OF CELL EXTRACT 20
2.8 ACID EXTRACTION OF PROTEINS 21
2.9 NUCLEAR PROTEIN EXTRACTION 21
2.10 IMMUNOPRECIPITATION ASSAY 22
Immunoprecipitation with beads-bound antibody 22
Immunoprecipitation with soluble antibody 22
2.11 INDIRECT IMMUNOFLUORESCENCE 22
2.12 SUB-CELLULAR FRACTIONATION AND NUCLEAR MATRIX PREPAPATION 23
2.13 PEPTIDE IDENTIFICATION BY MASS SPECTROMETRY AND BIOIMFORMATICS ANALYSIS 24
3. RESULTS 25
3.1 GENERATION AND CHARACTERIZATION OF EDD-SPECIFIC ANTIBODIES 25
Western blot analysis 25
Immunoprecipitation 25
Indirect immunostaining 26
3.2 IN VIVO INTERACTION BETWEEN EDD AND THE FACT COMPLEX 26
3.3 EDD IS A NUCLEAR MATRIX-ASSOCIATED PROTEIN 27
3.4 INTERACTION BETWEEN EDD AND HISTONE H2A AND H2BS 28
3.5 IDENTIFICATION OF AND-1 AS A NOVEL ASSOCIATED PROTEIN OF EDD 28
3.6 GENERATION AND CHARACTERIZATION OF AND-1-SPECIFIC ANTIBODIES 29
Western blot analysis 29
Immunoprecipitation 29
Indirect immunostaining 30
3.7 MAPPING OF THE PROTEIN DOMAINS RESPONSIBLE FOR THE INTERACTION BETWEEN EDD AND AND-1 30
3.8 AND-1 MAY INTERACT WITH EDD IN VIVO 31
3.9 BOTH EDD AND AND-1 LOCALIZE IN NUCLEUS 31
3.10 IDENTIFICATION OF AND-1-INTERACTING PROTEINS 32
3.11 ASSOCIATION BETWEEN AND-1 AND ACETYLATED-HISTONE H4 34
3.12 INTERACTION BETWEEN EDD AND NUCLEOLIN AND NUCLEOPHOSMIN 34
4. DISCUSSION 36
A NOVEL UBIQUITIN E3 LIGASE CAN INTERACT WITH THE FACT COMPLEX IN VIVO. 36
AND-1, A DNA-BINDING-PROTEIN, CAN INTERACT WITH EDD IN NUCLEAR MATRIX. 38
AND-1 MAY FUNCTION IN POST-TRANSCRIPTIONAL EVENTS 39
CONCLUDING REMARKS 41
5. REFERENCE 43
6. TABLES AND FIGURES 49
TABLE 1. PUTATIVE AND-1-ASSOCIATED PROTEINS 70
TABLE 2. PUTATIVE AND-1-WD-ASSOCIATING PROTEINS 74
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