呂勝春臺灣大學：分子醫學研究所林之立Lin, Chih-LiChih-LiLin2007-11-262018-07-092007-11-262018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/51382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 743438551 bytesapplication/pdfen-US轉錄作用染色質組蛋白修剪作用泛素泛素化接合?transcriptionchromatinhistonesplicingubiquitinE3 ligaseotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51382/1/ntu-95-R93448006-1.pdf