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Please use this identifier to cite or link to this item: http://ir.hwai.edu.tw:8080/ir/handle/310996100Q/1648

Title: γ-tubulin―FAM134C 啟動子的雙向調控和γ-tubulin在神經分化的功能
Bidirectional regulation of γ-tubulin―FAM134C promoter and function of γ-tubulin in neuronal differentiation
Authors: 王偉信
Wang, Wei Hsing
Keywords: 微小管
γ-微管蛋白
神經突生長
轉錄因子
microtubulin
γ-tubulin
neurite outgrowth
NRF-1
Date: 2011
Issue Date: 2013-11-01 11:49:19 (UTC+8)
Abstract: 微小管動態的組合與分離與細胞分裂、細胞移動以及蛋白質與囊泡的運送有關,並且在這個過程裡須要γ-tubulin形成微小管核化以及誘導神經突的延伸。在發展中的神經系統中,對於神經突、生長、分枝,微小管的形成扮演一個關鍵的作用,可是在神經細胞中γ-tubulin作用的分子機轉還不清楚。根據我們先前的研究,NRF-1這個轉錄因子能調控與神經突生長有關的很多基因蛋白,例如CD47與synapsin I。為了研究神經的細胞型態我們在神經纖瘤維母細胞中大量表現γ-tubulin觀察是否能促進神經突生長。此外先前的研究顯示出NRF-1能調控GPAT與AIRC基因的雙向轉錄作用。有趣的是,根據生物資訊學的工具發現,γ-tubulin 與 FAM134C之間的共同啟動子也包含一個NRF-1結合位,我們為了研究NRF-1在這個位子的結合能力,使用gel electrophoretic mobility shift assay證明NRF-1會結合在該基因啟動子上。研究發現突變NRF-1結合部位可以降低γ-tubulin基因啟動子活性,大量表現NRF-1則可促進該基因啟動子活性,表現競爭型抑制的NRF-1則會抑制。為進一步釐清NRF-1在雙向轉錄調控的角色,我們建構不同長度雙向啟動子報導基因表現載體,發現這些雙向啟動子確實具有轉錄活性,突變雙向啟動子上的NRF-1結合部位時,轉錄活性也會下降。在神經纖維瘤母細胞中大量表現γ-tubulin不會增加具有神經突的細胞比例,但可以促進神經突長度。根據本篇研究,NRF-1可以調控γ-tubulin與FAM134C之間的共同啟動子活性,且γ-tubulin具有促進神經突生長的活性。
Dynamic assembly and disassembly of microtubules associate with cell division, motility, and directional transport of proteins and vesicles, and require γ-tubulin to form microtubule nucleation in these processes as well as induce axonal elongation. In the developing nervous system, microtubule formation plays a key role in neurite outgrowth, elongation, and branching, whereas the molecular mechanisms of γ-tubulin in neurons remain unknown. According to our previous studies, nuclear respiratory factor (NRF)-1 is a transcription factor and regulates many genes associated with neurite outgrowth, such as CD47 and synapsin I. To understand the role of γ-tubulin in neurons, we overexpressed γ-tubulin in neuroblastoma cells to observe cellular morphology, such as neurite outgrowth. In addition, a previous study showed that NRF-1 regulates bidirectional transcription in intergenic promoter of GPAT and AIRC genes. Interestingly, according to the prediction using bioinformatics tools, there also is a NRF-1 site on intergenic promoter between γ-tubulin and FAM134C genes. Therefore, we proposed that NRF-1 may regulate bidirectional transactivation of these two genes. To address this issue, we used gel electrophoretic mobility shift assay to study NRF-1 binding activity in this site and site-directed mutagenesis, wild type and dominant-negative NRF-1 expression and promoter assays to determine regulation of NRF-1 on this bidirectional promoter. We found that NRF-1 binds to this site and mutation of this site on γ-tubulin promoter decreased its promoter activity. Furthermore, overexpression of NRF-1 increased the promoter activity but that of dominant-negative NRF-1 decreased that. To clarify the role of NRF-1 in regulating bidirectional intergenic promoter activity, we constructed bidirectional promoter reporter vectors, including various lengths of wild type and mutant promoters, and found these wild type promoters exerted transcriptional activity but mutation of NRF-1 binding site decreased that. We also overexressed γ-tubulin in neuroblastoma cells and found that γ-tubulin did not affected percentage of neurite-bearing cells but increased neurite length. According to these results, NRF-1 involves in regulating intergenic promoter between γ-tubulin and FAM134C genes and γ-tubulin increases neurite outgrowth.
Appears in Collections:[生物科技系暨生物醫學研究所] 博碩士論文

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