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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
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dc.contributor.advisor | 張?仁 | |
dc.contributor.author | Hsin-Pin Lu | en |
dc.contributor.author | 呂欣頻 | zh_TW |
dc.date.accessioned | 2021-06-16T09:17:35Z | - |
dc.date.available | 2020-07-20 | |
dc.date.copyright | 2017-07-20 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-07-11 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/59194 | - |
dc.description.abstract | TRIM28又稱作KAP1或是TIF1β,它是一個調控轉錄因子,會和帶有KRAB domain的鋅指蛋白結合並抑制基因的表現。TRIM28的N端包含了Ring finger、B boxes以及coiled-coil domain,這三個domain稱作RBCC domain,此domain會形成三聚體並與KRAB domain結合,而C端的PHD以及Bromo domain則會和組蛋白去乙醯酶以及甲基轉移酶等複合物結合。TRIM28在發育及分化的過程中是個重要的調控因子。而我們想了解TRIM28在脂肪分化的過程當中所扮演的角色及功能為何。首先,利用shRNA (short hairpin RNA)將3T3-L1脂肪前驅細胞當中的TRIM28進行knockdown,藉由觀察細胞分化型態以及分析基因的表現,結果發現3T3-L1脂肪前驅細胞無法正常分化成脂肪細胞。進一步透過RNA定序分析,結果發現3T3-L1的細胞週期以及PPARγ 訊息路徑受到了knockdown TRIM28影響。另外,我們發現調控脂肪分化的抑制因子Dlk1可能會受到TRIM28的調控。TRIM28的胺基酸上有許多的修飾已經被確定,並且被證明其在基因調控上的功能為何,但詳細的機制仍然不清楚。位於473的絲胺酸之磷酸化在脂肪分化的過程中已被我們偵測到,並建立模擬磷酸化的突變S473E以及不磷酸化的突變S473A;同時,我們也想探究位於305的離胺酸之乙醯化,於是建立了模擬乙醯化的突變K305Q以及不乙醯化的突變K305R,並進行其生化及功能分析。透過電泳遲滯法證明K305Q的突變會降低與Gal4 DBD-KRAB之間的交互作用。我們也利用共免疫沉澱法證明K305Q和S473E這兩個突變會降低與zinc finger 30 (Zfp30)之間的交互作用。另外,將S473A、S473E、K305Q以及K305R這四個突變分別過表現在3T3-L1脂肪前驅細胞當中,試圖探究TRIM28之磷酸化及乙醯化在脂肪分化過程中的功能,並進一步了解他們之間與Zfp30之間的關係,希望能夠找到調控脂肪分化相關的目標基因。 | zh_TW |
dc.description.abstract | The tripartite motif-containing protein 28 (TRIM28), also known as KRAB-associated protein 1 (KAP1) or transcriptional intermediary factor 1-beta (TIF1β), is a transcription corepressor of KRAB-ZFPs (Krüppel-associated box containing zinc finger proteins). TRIM28 contains N-terminal RBCC domain for trimer formation and interacting with KRAB domain, and C-terminal PHD and Bromo domain for interacting with histone deacetylase and methyltransferase complexes. TRIM28 is a crucial regulator in development and differentiation. We would like to investigate its function and regulation in adipogenesis. Knockdown of TRIM28 by transducing lentivirus-carrying shRNAs impairs the differentiation of 3T3-L1 preadipocytes, demonstrated by morphological observation and gene expression analysis. RNA-seq analysis was performed in TRIM28 knockdown 3T3-L1 cells to identify TRIM28-regulated genes. Cell cycle regulators and PPARγ signaling pathway were significantly affected by knockdown of TRIM28. One of adipogenesis repressor, Dlk1, might be downregulated by TRIM28 during adipogenesis. Several amino acid residues of TRIM28 for phosphorylation, sumoylation and acetylation have been identified and play functions in gene regulation. The profiling of serine 473 phosphorylation was monitored during adipogenesis. To demonstrate the function of acetylation at lysine 305 which located in RBCC domain, the acetyl-mimetic K305Q and non-acetyl K305R mutants were generated for biochemical and functional analysis. The electrophoretic mobility shift assay (EMSA) showed that the K305Q mutant decreases its interaction with DNA-bound Gal4 DBD-KRAB. Co-immunoprecipitation also confirmed that K305Q or S473E mutant interacts weakly with a KRAB-ZFP, Zfp30. 3T3-L1 cells ectopically expressed K305Q or K305R mutant was generating to explore the function of TRIM28 acetylation in adipogenesis. We suggest that in response to differentiation signals, TRIM28 might be acetylated and phosphorylated to reduce interaction with KRAB-ZFPs like Zfp30, leading to activation of targeted genes. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T09:17:35Z (GMT). No. of bitstreams: 1 ntu-106-R04b46011-1.pdf: 3213654 bytes, checksum: 015e560c70a82cd429d1263667ed92cf (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | 口試委員會審定書 i
Acknowledgement ii 摘要 iii Abstract iv Contents vi Abbreviations ix 1. Introduction 1 1.1 Tripartite motif-containing protein 28 1 1.2 Zinc finger proteins family 3 1.3 Molecular regulation of adipogenesis 4 1.4 Specific aims 6 2. Materials and Methods 8 2.1 Plasmid constructs 8 2.2 Cell culture 8 2.3 Oil red O staining 9 2.4 Western blot 10 2.5 Transfection 11 2.6 Immunoprecipitation assay 12 2.7 Antibodies and chemicals 13 2.8 Dual luciferase reporter assay 13 2.9 Lentivirus production and infection of 3T3-L1 cells 14 2.10 Protein purification 15 2.11 GST pull-down assay 16 2.12 Indirect immunofluorescence microscopy 16 2.13 RNA extraction and reverse transcription 17 2.14 Real-time PCR 18 2.15 Electrophoretic mobility shift assay 18 2.16 In vitro acetylation 20 2.17 RNA sequencing (quantification) 21 2.18 Chromatin immunoprecipitation assay 22 2.19 Statistical analysis 24 3. Results 25 3.1 TRIM28 protein expression and phosphorylation during adipogenesis of 3T3-L1 preadipocytes 25 3.2 Knockdown of TRIM28 in 3T3-L1 preadipocytes 25 3.3 RNA-seq analysis in TRIM28 knockdown 3T3-L1 cells 26 3.4 KRAB domain-containing Zfp30 interacts with TRIM28 28 3.5 The functional regulation of TRIM28 by protein acetylation 29 3.6 Overexpression of TRIM28 mutants in 3T3-L1 preadipocytes 32 4. Discussion 33 5. Figures 38 Figure 1. TRIM28 protein expression and phosphorylation during adipogenesis of 3T3-L1 preadipocytes 39 Figure 2. Lentiviral shRNA-mediated knockdown of TRIM28 in 3T3-L1 cells 43 Figure 3. RNA-seq analysis in knockdown TRIM28 3T3-L1 cells 48 Figure 4. KRAB domain-containing Zfp30 interacts with TRIM28 50 Figure 5. Co-Immunoprecipitation of flag-TRIM28 constructs and Zfp30 51 Figure 6. The corepressor activity analysis of TRIM28 mutants 53 Figure 7. TRIM28 targets on DNA through KRAB-DNA-binding protein 56 Figure 8. TRIM28 interacts with histone acetyltransferase p300 58 Figure 9. Overexpression of TRIM28 mutants in 3T3-L1 preadipocytes 60 6. Tables 61 Table1. Primers for generating constructs 61 Table2. Primers for real-time PCR 62 Table3.1 Pathway analysis with TRIM28 knock down vs. control 3T3-L1 at 0 day by using KEGG pathway database 63 Table 4. Primers for ChIP 71 Table 5.1 TRIM28-associated proteins identified by MS/MS analysis 72 7. References 75 | |
dc.language.iso | en | |
dc.title | TRIM28在脂肪前驅細胞分化過程中之調控 | zh_TW |
dc.title | The functional regulation of TRIM28 in adipogenesis | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 張震東,張茂山,呂勝春 | |
dc.subject.keyword | TRIM28,乙醯化,脂肪前驅細胞,脂肪分化,鋅指蛋白, | zh_TW |
dc.subject.keyword | TRIM28,acetylation,3T3-L1,adipogenesis,ZFPs, | en |
dc.relation.page | 81 | |
dc.identifier.doi | 10.6342/NTU201701319 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-07-12 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 生化科學研究所 | zh_TW |
顯示於系所單位: | 生化科學研究所 |
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