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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蔡懷楨 | |
dc.contributor.author | Po-Chun Wang | en |
dc.contributor.author | 王柏鈞 | zh_TW |
dc.date.accessioned | 2021-06-08T01:55:37Z | - |
dc.date.copyright | 2016-07-26 | |
dc.date.issued | 2016 | |
dc.date.submitted | 2016-07-10 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19366 | - |
dc.description.abstract | Vascular endothelial growth factor A (VEGFA) 是體內重要的血管新生促進因子之一,其調控正常及腫瘤相關的血管新生。已知vegfa的基因表現會受到一些轉錄因子的調控,例如: Sp1、AP-2、STAT-3等轉錄因子皆會與vegfa上游2kb以內相對應的調控序列結合並調節vegfa的轉錄作用。最近的研究則指出Seryl-tRNA synthetase (SARS) 除了胺醯化的活性外,也會進入細胞核並直接結合至hvegfa近端 -62~-36 nt的cis-acting element來抑制hvegfa的轉錄作用。另一方面,SARS也會與Ying-Yang 1 (YY1) 形成蛋白質複合體,且兩者皆參與在hvegfa遠端 -4723~-4184 nt的區域。然而,過去的研究都集中探討hvegfa上游2kb以內的近端調控位置對轉錄作用的影響,但是SARS是否會透過hvegfa上游較遠端的位置 (例如: 4 kb以外) 調節hvegfa的轉錄作用,目前還不清楚。
首先,本研究透過DNA序列的資訊分析,得知在hvegfa -4654~-4623 nt 可能存在一個YY1 / nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) binding cassette。接著,透過Luciferase assay得知hvegfa (-4654~-4623) 為SARS負向調節hvegfa轉錄活性的主導性片段。以及,SARS、YY1皆透過hvegfa (-4654~-4623) 抑制hvegfa的轉錄;而NFκB1則透過此片段活化hvegfa的轉錄。此外,我們利用不同突變的hvegfa (-4654~-4623) 做為報導基因,發現SARS是藉由與YY1形成complex來調控hvegfa的轉錄活性,一旦與YY1或NFκB1的結合序列被突變時,SARS/YY1 complex或 NFκB1 即無法調控hvegfa的轉錄作用。Western blot的分析顯示,過量表現SARS及YY1時,VEGFA的表現會被抑制;而降低SARS及YY1時,VEGFA的表現則增強。相反地,當過量表現或降低NFκB1時,VEGFA的表現則與上述結果相反。最後,於Tg(fli1:EGFP)y1血管綠螢光斑馬魚胚胎in vivo注射的結果顯示,SARS與YY1會協同抑制血管新生,且SARS/ YY1 complex及NFκB1之間藉由陰陽關係來調控體節血管的發育。因此,我們由以上的實驗總結: (1) SARS不會直接結合至hvegfa (-4654~-4623),而是透過與YY1形成SARS/YY1 complex來抑制由hvegfa 遠端 (-4654~-4623) 主導的hvegfa轉錄作用。(2) SARS/YY1 complex透過與NFκB1競爭hvegfa (-4654~-4623) 此遠端位置的cis-acting element,微妙平衡了NFκB1所促進的hvegfa表現,維持血管的正常發育及功能。 | zh_TW |
dc.description.abstract | Vascular endothelial growth factor A (VEGFA) is an endothelial cell-specific mitogen which plays a pivotal role in angiogenesis. The expression of vegfa gene is modulated by some transcription factors, including Sp1, AP-2 and STAT-3. These transcription factors bound at proximal regulatory DNA sequences within the 2kb upstream region and thus regulate transcription activity of vegfa. Recent reports have demonstrated that Seryl-tRNA synthetase (SARS) not only has aminoacylation activity, but also localize into nucleus and directly binds to the proximal -62~-36 cis-element to repress transcription of human vegfa (hvegfa). On the other hand, SARS interacts with Yin-Yang1 (YY1) to form a protein complex. Meanwhile, both of them are bound at the distal -4723~-4184 region of hvegfa. Previous studies have focused on transcriptional regulation modulated by cis-element at proximal region of hvegfa gene. However, it is unclear whether SARS control the expression of hvegfa gene through distal upstream DNA sequences beyond 4 kb.
First, by DNA sequence analysis, we found a potential YY1 / nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) binding cassette at the -4654~-4623 segment of hvegfa gene. Luciferase assy reveal that -4654~-4623 segment is critical for SARS to regulate the hvegfa transcription activity driven by the -5868~+55 segment. Moreover, we indicate that the transcription activity driven by -4654~-4623 segment were repressed by SARS and YY1, but actived by NFκB1. Furthermore, we prove that SARS control the transcriptional activity of hvegfa by form a complex with YY1. Once the YY1- and NFκB1-specific binding sequences within -4654~-4623 segment are mutated, SARS/YY1 and NFκB1 totally lost their ability to influence the promoter activity. Using western blot assay, we demonstrate that the amount of VEGFA protein produced in cells is dependent on competitive binding between SARS/YY1 complex and NFκB1 at the -4654~-4623 segment in vitro. By in vivo assay of zebrafish embryos from transgenic line Tg(fli1:EGFP)y1, we observe that the blood vessel development is reduced by SARS and YY1 synergistically. By contrast, the blood vessel development is enhanced by NFκB1. Taken together, we conclude that (1) SARS binds tightly to YY1 instead of binding to DNA, in that way SARS/YY1 complex function as a negative effector to regulate expression of human vegfa through -4654~-4623 cis-element. (2) NFκB1 serves as a positive effector through competing with SARS/YY1 binding at the same cis-element. Therefore, SARS/YY1 complex and NFκB1 is a pair of “Yin-Yang” regulator for proper development of a functional vasculature. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T01:55:37Z (GMT). No. of bitstreams: 1 ntu-105-R03b43019-1.pdf: 4722625 bytes, checksum: 97f18fefd93a8d4d4452204d355756f2 (MD5) Previous issue date: 2016 | en |
dc.description.tableofcontents | 中文摘要----------------------------------------------------------------------------2
英文摘要----------------------------------------------------------------------------4 文獻回顧----------------------------------------------------------------------------6 前言---------------------------------------------------------------------------------16 材料與方法------------------------------------------------------------------------19 結果---------------------------------------------------------------------------------32 討論---------------------------------------------------------------------------------43 參考文獻---------------------------------------------------------------------------48 圖表---------------------------------------------------------------------------------70 附錄---------------------------------------------------------------------------------80 | |
dc.language.iso | zh-TW | |
dc.title | SARS/YY1 complex藉由與NFκB1競爭vegfa遠端-4654/-4623的cis-element來調控vegfa的轉錄活性與血管的生成 | zh_TW |
dc.title | SARS/YY1 complex compete NFκB1 for binding at vegfa distal -4654/-4623 cis-element to regulate vegfa transcription activity and angiogenesis | en |
dc.type | Thesis | |
dc.date.schoolyear | 104-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 胡清華,陳曜鴻 | |
dc.subject.keyword | 轉錄調控,血管新生, | zh_TW |
dc.subject.keyword | tanscriptional regulation,angiogenesis, | en |
dc.relation.page | 84 | |
dc.identifier.doi | 10.6342/NTU201600778 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2016-07-11 | |
dc.contributor.author-college | 生命科學院 | zh_TW |
dc.contributor.author-dept | 分子與細胞生物學研究所 | zh_TW |
顯示於系所單位: | 分子與細胞生物學研究所 |
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