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研究生:畢文潔
研究生(外文):Wen-Chieh Pi
論文名稱:E2A-PBX1誘導B細胞淋巴白血病的機轉
論文名稱(外文):Mechanism of E2A-PBX1-mediated B-cell leukemogenesis
指導教授:陳威儀
指導教授(外文):Wei-Yi Chen
學位類別:博士
校院名稱:國立陽明大學
系所名稱:生化暨分子生物研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2020
畢業學年度:108
語文別:英文
論文頁數:105
中文關鍵詞:急性淋巴性白血病融合蛋白轉錄體共同活化子
外文關鍵詞:E2A-PBX1RUNX1Acute Lymphoblastic LeukemiaTranscriptionco-activator
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誌謝 i
Table of Contents ii
摘要 v
Abstract vii
List of Figures ix
Abbreviation table xii
Chapter 1. Introduction 1
1. Hematopoiesis 1
1.1 Lymphopoiesis 1
1.2 Acute lymphoblastic leukemia (ALL) 2
1.3 Leukemogenesis 2
1.4 Leukemic stem cell theory 3
2 Leukemogenic chromosomal abnormalities 4
2.1 Chromosome translocation 4
2.2 Aberrant gene activation: MYC 5
2.3 Philadelphia chromosome: BCR-ABL 6
2.4 Transcription aberration fusion 6
2.4.1 Epigenetic regulator: MLL-rearrangements 6
2.4.2 Transcription factor: AML1-ETO 7
3. Components of transcriptional control 8
3.1 Cis-acting elements 8
3.2 Trans-acting factors 8
3.3 General transcription factors (GTFs) 9
3.4 Transcriptional activators and co-activators 9
3.5 Activator-dependent transactivation 10
3.6 Chromatin dynamics 11
3.7 Transcriptional misregulation in cancer 12
4. Hematopoietic transcription factors 12
4.1 E2A and E2A-fusions 13
4.1.1 E2A 13
4.1.2 E2A fusions 13
4.2 PBX1 and E2A-PBX1 14
4.2.1 PBX1 14
4.2.2 E2A-PBX1 14
4.3 RUNX1 15
4.3.1 RUNX1 overexpression in AML and MLL 15
Chapter 2. Materials and Methods 17
1. Materials 17
1.1 Reagents 17
1.2 Buffers 18
1.3 Antibodies 20
1.4 shRNA clone ID 20
1.5 RT-QPCR and ChIP-QPCR primers 21
2. Methods 22
2.1 Cell lines and Cell culture 22
2.2 Generate Tet-On E2A/E2APBX1 697 stable lines 22
2.3 Molecular cloning 23
2.4 Bacmid construction 23
2.5 Baculovirus preparation 24
2.6 Plaque assay 25
2.7 Baculoviral protein expression and purification 25
2.8 Bacterial recombinant protein expression and purification 26
2.9 RNA interference 26
2.10 Cell proliferation assays 27
2.11 Cell formation assays 27
2.12 Gene expression analysis 27
2.13 Western blotting 28
2.14 Luciferase reporter assay 28
2.15 GST pull down assay 29
2.16 Co-immunoprecipitation assay 29
2.17 Chromatin immunoprecipitation (ChIP) assay 30
Chapter 3. Results 31
1. Generation of a stable line inducibly express HA-Flag-tagged E2A-PBX1 31
2. Genome-wide profiling of E2A-PBX1 regulating locus 33
3. E2A-PBX1-bound regions are enriched by RUNX1 and EBF motifs rather than PBX motif. 34
4. PBX1 response sequence is likely not required for the DNA binding of E2A-PBX1 34
5. HOX proteins are not co-regulators of E2A-PBX1 in 697 leukemic line 35
6. PLS3 is a direct target of E2A-PBX1, but not essential for leukemic maintenance 36
7. E2A-PBX1 was recruited by RUNX1 to the RUNX1-controlling genes. 36
8. E2A-PBX1 recruits p300 to the RUNX1-bound regions, and concomitantly enhances the H3K27Ac mark and the binding of MED1. 38
9. E2A-PBX1 controls the RUNX1 transcriptome to maintain the leukemic cell growth 39
10. RUNX1 is a direct target of E2A-PBX1 40
11. The IH-HD domain within E2A-PBX1 homeodomain is essential for both interactions with RUNX1 and E2A-PBX1-mediated transactivation 41
Chapter 4. Discussion 44
1. The DNA-binding function of E2A-PBX1 44
2. The targets selectivity of E2A-PBX1 to a small subset of RUNX1 binding locus 45
3. PLS3, a direct target of E2A-PBX1 itself, is dispensable for leukemia cell maintenance 46
4. Druggable targets under the mechanism of E2A-PBX1 controlling RUNX1 transcriptome 47
5. It remains elusive why E2A-PBX1 driven leukemogenesis in murine model initiates either T-ALL or AML but not B-ALL 48
6. RUNX1 as a general target in leukemogenesis 49
References 51
Figures 59
Appendix: Figures 101
Appendix: Publications 106
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