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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15805完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 沈哲鯤(Che-Kun James Shen) | |
| dc.contributor.author | Yu-Szu Huang | en |
| dc.contributor.author | 黃友思 | zh_TW |
| dc.date.accessioned | 2021-06-07T17:52:32Z | - |
| dc.date.copyright | 2012-09-18 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-20 | |
| dc.identifier.citation | 1. Miller, I. J. & Bieker, J. J. A novel, erythroid cell-specific murine transcription factor that binds to the CACCC element and is related to the Kruppel family of nuclear proteins. Molecular and cellular biology 13, 2776-2786 (1993).
2 Nuez, B., Michalovich, D., Bygrave, A., Ploemacher, R. & Grosveld, F. Defective haematopoiesis in fetal liver resulting from inactivation of the EKLF gene. Nature 375, 316-318, (1995). 3 Perkins, A. C., Sharpe, A. H. & Orkin, S. H. Lethal beta-thalassaemia in mice lacking the erythroid CACCC-transcription factor EKLF. Nature 375, 318-322, (1995). 4 Perkins, A. C., Peterson, K. R., Stamatoyannopoulos, G., Witkowska, H. E. & Orkin, S. H. Fetal expression of a human Aγ globin transgene rescues globin chain imbalance but not hemolysis in EKLF null mouse embryos. Blood 95, 1827-1833 (2000). 5 Ouyang, L., Chen, X. & Bieker, J. J. Regulation of erythroid Kruppel-like factor (EKLF) transcriptional activity by phosphorylation of a protein kinase casein kinase II site within its interaction domain. The Journal of biological chemistry 273, 23019-23025 (1998). 6 Zhang, W. & Bieker, J. J. Acetylation and modulation of erythroid Kruppel-like factor (EKLF) activity by interaction with histone acetyltransferases. Proceedings of the National Academy of Sciences of the United States of America 95, 9855-9860 (1998). 7 Siatecka, M., Xue, L. & Bieker, J. J. Sumoylation of EKLF promotes transcriptional repression and is involved in inhibition of megakaryopoiesis. Molecular and cellular biology 27, 8547-8560 (2007). 8 Quadrini, K. J. & Bieker, J. J. EKLF/KLF1 is ubiquitinated in vivo and its stability is regulated by activation domain sequences through the 26S proteasome. FEBS letters 580, 2285-2293 (2006). 9 Chen, X. & Bieker, J. J. Unanticipated repression function linked to erythroid Kruppel-like factor. Molecular and cellular biology 21, 3118-3125 (2001). 10 Chen, X. & Bieker, J. J. Stage-specific repression by the EKLF transcriptional activator. Molecular and cellular biology 24, 10416-10424 (2004). 11 Pilon, A. M. et al. Genome-wide ChIP-Seq reveals a dramatic shift in the binding of the transcription factor erythroid Kruppel-like factor during erythrocyte differentiation. Blood 118, e139-148 (2011). 12 Hodge, D. et al. A global role for EKLF in definitive and primitive erythropoiesis. Blood 107, 3359-3370 (2006). 13 Tallack, M. R. et al. A global role for KLF1 in erythropoiesis revealed by ChIP-seq in primary erythroid cells. Genome research 20, 1052-1063 (2010). 14 Frontelo, P. et al. Novel role for EKLF in megakaryocyte lineage commitment. Blood 110, 3871-3880 (2007). 15 Pilon, A. M. et al. Failure of terminal erythroid differentiation in EKLF-deficient mice is associated with cell cycle perturbation and reduced expression of E2F2. Molecular and cellular biology 28, 7394-7401 (2008). 16 Aplan, P. D., Nakahara, K., Orkin, S. H. & Kirsch, I. R. The SCL gene product: a positive regulator of erythroid differentiation. The EMBO journal 11, 4073-4081 (1992). 17 Robb, L. et al. Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene. Proceedings of the National Academy of Sciences of the United States of America 92, 7075-7079 (1995). 18 Hall, M. A. et al. The critical regulator of embryonic hematopoiesis, SCL, is vital in the adult for megakaryopoiesis, erythropoiesis, and lineage choice in CFU-S12. Proceedings of the National Academy of Sciences of the United States of America 100, 992-997 (2003). 19 Nudel, U. et al. Differential effects of chemical inducers on expression of beta globin genes in murine erythroleukemia cells. Proceedings of the National Academy of Sciences of the United States of America 74, 1100-1104 (1977). 20 Bouilloux, F. et al. EKLF restricts megakaryocytic differentiation at the benefit of erythrocytic differentiation. Blood 112, 576-584 (2008). 21 Shyu, Y. C. et al. Subcellular transport of EKLF and switch-on of murine adult beta maj globin gene transcription. Molecular and cellular biology 27, 2309-2323 (2007). 22 Porcher, C. et al. The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 86, 47-57 (1996). 23 Sengupta, T., Chen, K., Milot, E. & Bieker, J. J. Acetylation of Eklf is essential for epigenetic modification and transcriptional activation of the β-globin locus. Molecular and cellular biology 28, 6160-6170 (2008). 24 Murrell, A. M. et al. Discordant regulation of SCL/TAL-1 mRNA and protein during erythroid differentiation. Oncogene 11, 131-139 (1995). 25 Kassouf, M. T. et al. Genome-wide identification of TAL1's functional targets: insights into its mechanisms of action in primary erythroid cells. Genome research 20, 1064-1083 (2010). | |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15805 | - |
| dc.description.abstract | 紅血球是哺乳類動物胚胎發育時期首先形成的細胞，在胚胎時期供給個體所需的氧氣，其分化與成熟對個體的生存是必需的。Erythroid Kruppel-like Factor ( EKLF )是一個表現在紅血球細胞中的轉錄因子，目前已知會在小鼠胚胎發育後期啟動成人時期β-globin (βmaj及βmin)的表現, 並且抑制胎兒時期β-like globin的表現(βh1及εy), 藉此機制達成成人與胎兒時期β-like globin的轉換。以蛋白質結構而言，EKLF會藉由它在C端的三個鋅手指辨認並且結合到它的保守性DNA序列CACCC，藉此調控下游基因的表現。而除了調控β-like globin的表現以外，近幾年的研究更發現EKLF在紅血球分化與成熟的過程中扮演重要的角色，但詳細的分子機制目前還未明瞭。Tal1 ( SCL ) 是另一個與造血功能相關的轉錄因子，目前研究指出它對紅血球的分化與成熟是亦是必需的。以蛋白質結構而言，它是藉由它的bHLH domain與下游基因的啟動子結合並進一步啟動下游基因的表現。為了探討EKLF是藉由何種機制促進紅血球的分化與成熟，我們利用microarray的技術分析Eklf基因剔除小鼠的胚胎在受精後第14.5天的肝臟細胞中，基因表現的情形；結果發現在基因剔除小鼠中，Tal1 mRNA的表現量相較於wild-type小鼠而言有明顯的下降。此外，我們也利用ChIP-chip的技術去找尋Wild-type小鼠中EKLF結合到哪些下游基因的啟動子，結果發現EKLF會結合到Tal1基因的啟動子區域。這些結果暗示著EKLF可能會直接調控Tal1基因並且促進其表現。而利用Real-time PCR及ChIP-PCR的實驗再次確認上述結果，也得到同樣的結論。在得到這些初步結果之後，我們進一步在老鼠紅血球細胞株 (murine erythroleukemia cell, MEL)中進行功能獲得性實驗(gain-of-function study), 由此實驗中我們進一步發現，外加EKLF會造成Tal1 mRNA的表現量上升。此外，在功能缺失性實驗(loss-of-function study)中，我們發現EKLF表現量下降後，Tal1基因的表現也有顯著下降。總結而言，目前的實驗結果暗示著EKLF位於Tal1的上游促進其表現，並且藉由這個調控方式促進紅血球的分化與成熟。 | zh_TW |
| dc.description.abstract | Erythropoiesis is a complex multi-step process that begins with megakaryocytic/erythroid (MEP) progenitors derived from the pluripotent hematopoietic stem cells (HSCs) then terminates with the production of erythrocytes (RBCs). Erythroid Kruppel-like factor, EKLF is essential for the activation of the adult | en |
| dc.description.provenance | Made available in DSpace on 2021-06-07T17:52:32Z (GMT). No. of bitstreams: 1 ntu-101-R99448009-1.pdf: 1371350 bytes, checksum: d9de34109903b376040c281c13716709 (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 1 口試委員會審定書.....................................i
2 誌謝..............................................ii 3 中文摘要..........................................iii 4 英文摘要............................................v 5 Introduction.......................................1 6 Materials and Methods..............................6 7 Results...........................................10 7.1 Real-time quantitative RT-PCR and western blot analysis confirmed the down-regulation of tal1 in eklf-/- E14.5 mice fetal liver....................10 7.2 Eklf -/- E14.5 embryo was anemic and the fetal liver was paler and smaller.............................10 7.3 Analysis of Tal1 promoter structure revealed that EKLF bound to Tal1 promoter region upstream to transcriptional start sites, but not to intron region............................................11 7.4 ChIP-PCR analysis confirmed the binding of EKLF to the tal1 promoter region..........................12 7.5 Luciferase reporter assay demonstrates that the EKLF binding site identified in ChIP-chip analysis was functional in the Tal1 promoter context...........13 7.6 Gain-of-function study demonstrated an up-regulation of tal1 by EKLF...................................13 7.7 Loss-of-function study using EKLF siRNA oligos did not reduce the endogenous eklf mRNA to a significant level. ..........................................14 7.8 The EKLF shRNA stable knockdown clones reveal that EKLF functions upstream of Tal1 and act as a transcriptional activator during erythroid differentiation...................................16 7.9 Analysis of tal1 mRNA and protein level during MEL differentiation reveals that Tal1 might be required for erythroid terminal differentiation at early stage.............................................17 8 Discussions.......................................19 8.1 Tal1 mRNA and protein expression was reduced but not abolished in E14.5 eklf-/- mice fetal liver, indicating that there may exist other upstream factors for Tal1 activation.......................19 8.2 Gain-of-function studies revealed genetic interactions between Eklf and Tal1................19 8.3 The unsuccessful knockdown by transient transfection of siRNA oligos suggests a possible role of EKLF on cell viability or proliferation...................20 8.4 Loss-of-function studies by stable clones identified the epistasis interaction between EKLF and Tal1...21 8.5 Discordant expression of Tal1 mRNA and protein during erythroid differentiation indicates a post- transcriptional modification......................21 9 References........................................23 10 Figures...........................................27 10.1 Figure 1. Tal1 mRNA and protein level are down- regulated in eklf-/- mice.........................27 10.2 Figure 2. Phenotypes and typing results of eklf +/+ and -/- E14.5 fetuses.............................28 10.3 Figure 3. Promoter structure analysis of tal1 gene reveals the existence of isoform 2, 3, and 4 in MEL cell line.........................................29 10.4 Figure 4. tal1 isoform 1 is not exists in cell lines or tissues expressing tal1 gene...................30 10.5 Figure 5. tal1 isoform 2, 3, 4 exist in the tissues and the cell lines tested in figure 4.............31 10.6 Figure 6. ChIP-PCR of the ChIP-chip binding region confirms the binding of EKLF on Tal1 gene promoter. ..................................................32 10.7 Figure 7. EKLF transactivates wild-type but not deleted tal1 promoter, which lacks the EKLF binding box...............................................33 10.8 Figure 8. Gain-of-function study shows that EKLF activates tal1 gene at transcriptional level......34 10.9 Figure 9. Quantitative data of the gain-of-function experiments in figure 7...........................35 10.10 Figure 10. siRNA knockdown of eklf did not reduce the endogenous eklf mRNA to a significant level...36 10.11 Figure 11. eklf siRNA targets to eklf mRNA in 293T system............................................37 10.12 Figure 12. tal1 mRNA level reduces after doxycycline-induced EKLF knockdown under DMSO treatment.........................................38 10.13 Figure 13. Quantitative data of the loss-of- function experiments in figure 11.................39 10.14 Figure 14. Doxycycline reduces EKLF expression to the translational level in two independent shRNA stable clones, 4D7 and 2M12.......................40 10.15 Figure 15. mRNA levels of tal1 isoforms 2, 3, and 4 increase during erythroid differentiation.........41 10.16 Figure 16. TAL1 protein level increases from D0 to D2 and subsequently decreased from D2 to D4.......42 10.17 Supplementary Figure 1. Transient siRNA knockdown of Tal1 reduces the tal1 mRNA to a significant level.............................................43 10.18 Supplementary Figure 2. Corresponding stages between MEL differentiation and primary erythroblast......44 | |
| dc.language.iso | en | |
| dc.subject | 功能缺失性實驗 | zh_TW |
| dc.subject | 小鼠胚胎發育 | zh_TW |
| dc.subject | 基因交互作用 | zh_TW |
| dc.subject | 功能獲得性實驗 | zh_TW |
| dc.subject | 紅血球 | zh_TW |
| dc.subject | 細胞分化 | zh_TW |
| dc.subject | reporter assay | en |
| dc.subject | EKLF | en |
| dc.subject | Tal1 | en |
| dc.subject | erythroid differentiation | en |
| dc.subject | gene interaction | en |
| dc.subject | ChIP-chip | en |
| dc.subject | microarray | en |
| dc.title | EKLF在紅血球分化過程中對Tal1/SCL的調控 | zh_TW |
| dc.title | EKLF-Mediated Gene Expression of Tal1/SCL During Differentiation of Erythroid Cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 阮麗蓉(Li-Jung Juan),呂勝春(Sheng-Chung Lee) | |
| dc.subject.keyword | 紅血球,基因交互作用,小鼠胚胎發育,細胞分化,功能獲得性實驗,功能缺失性實驗, | zh_TW |
| dc.subject.keyword | EKLF,Tal1,erythroid differentiation,gene interaction,ChIP-chip,microarray,reporter assay, | en |
| dc.relation.page | 44 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2012-08-20 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 分子醫學研究所 | zh_TW |
| 顯示於系所單位： | 分子醫學研究所 | |
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