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系統識別號 U0026-0812200915282506
論文名稱(中文) 探討ZNF179啟動子: 辨識在神經分化過程中可以調控ZNF179表現的轉錄因子
論文名稱(英文) Characterization of the ZNF179 promoter: identification of transcription factors that regulate ZNF179 expression during neural differentiation
校院名稱 成功大學
系所名稱(中) 藥理學研究所
系所名稱(英) Department of Pharmacology
學年度 97
學期 2
出版年 98
研究生(中文) 王怡方
研究生(英文) I-Fang Wang
電子信箱 e_fung0207@hotmail.com
學號 s2696112
學位類別 碩士
語文別 中文
論文頁數 80頁
口試委員 指導教授-李宜釗
指導教授-張文昌
口試委員-王育民
中文關鍵字 啟動子  轉錄因子 
英文關鍵字 ZNF179  pura  promoter 
學科別分類
中文摘要 腦指蛋白 (brain finger protein, BFP),又名 ZNF179,是一個功能未知的新基因,屬於RING finger 家族中的一員。過去的文獻指出 ZNF179 基因的表現會伴隨老鼠胚胎發育的過程逐漸增加,並且於成鼠的腦中維持高度的表現。從 Gene Expression Omnibus (GEO) 的資料分析結果中發現 ZNF179 基因的表現在一些神經退化性疾病之基因轉殖鼠體內明顯下降,例如亨丁頓疾病 (huntington’s disease) 與肌萎縮性側索硬化症 (amyotrophic lateral sclerosis, ALS),顯示 ZNF179 基因的表現可能與這些神經退化性疾病有關連。本實驗室先前的研究顯示 ZNF179 基因的表
現,在利用維他命 A 酸刺激 P19 細胞進行神經分化的過程中有逐漸增加的現象,而降低 ZNF179 基因的表現則可明顯地抑制神經細胞分化。因此,探討在神經分化的過程中,ZNF179 基因受到誘導表現的分子機制將有助於瞭解其可能參與之生物功能。已知的研究結果顯示,雖然在 P19 細胞進行神經分化的過程中有許多基因會受到 DNA 去甲基化的表觀遺傳調控 (epigenetic regulation) 作用而誘發表現,但 ZNF179 基因的表現並沒有受到這樣的調控。進一步地分析 ZNF179 基因啟動區序列,並利用不同長度的 ZNF179 基因啟動區片段進行報導基因活性分析後,發現ZNF179 基因轉譯起始點上游 - 5 至 - 533 bp 的片段足以在P19 細胞分化過程中誘發 ZNF179 基因啟動區的活性。而利用軟體預測出此片段上可能的轉錄因子結合位點,例如:Gata2、Purα,並進一步使用定點突變、報導基因活性分析及染色質體免疫沉澱分析的方法來進行測試,確認出轉錄調控因子 Purα 在進行神經分化的P19 細胞中誘導 ZNF179 基因的表現上扮演了相當重要的角色。
英文摘要 ZNF179, a novel and function uncertain gene, belongs to the RING finger protein family. The expression of ZNF179 was shown to be accompanied with the embryogenesis and sustains high expression level in the adult brain. From the Gene Expression Omnibus (GEO), a public gene expression profiles database, the expression of ZNF179 was significantly decreased in the neurodegenerative diseases such as Huntington’s disease and amyotrophic lateral sclerosis (ALS), indicating expression level of ZNF179
might play functional role in these diseases. Previously, we have demonstrated that the expression of ZNF179 was increased in the retinoic acid-induced neural differentiation of P19 cells, and knockdown of ZNF179
can significantly attenuate neural differentiation. Therefore investigating the molecular mechanism of ZNF179 gene expression can provide useful information in revealing its function in cells. During the neural differentiation process of P19 cells, the expression of many genes can be regulated through the epigenetic controls, such as DNA demethylation. However, the promoter
methylation of ZNF179 is consistent during the differentiation process. Next, we analyzed the promoter region of ZNF179 gene, by using various fragments of 5’-upstream region to verify the promoter activity of ZNF179 during P19 cells neural differentiation, the region containing -533/-5 fragment of ZNF179 promtor is sufficient for its transcriptional activity. Meanwhile, several interesting transcription factor binding motifs were predicted, such as Gata2 and Purα. By using site-directed mutagenesis, reporter assay and chromatin immunoprecipitation analysis, we demonstrated that Purα plays an important role in induction of ZNF179 expression during the neural differentiation process of P19 cells.
論文目次 中文摘要 ---------------------------------------------------------------------------- Ⅰ
英文摘要 ---------------------------------------------------------------------------- Ⅲ
致謝 ---------------------------------------------------------------------------------- Ⅴ
目錄 ---------------------------------------------------------------------------------- Ⅵ
圖目錄 ------------------------------------------------------------------------------- Ⅶ
附錄目錄 ---------------------------------------------------------------------------- Ⅷ
縮寫檢索 ---------------------------------------------------------------------------- Ⅸ
第一章 緒論 ------------------------------------------------------------------------ 1
第二章 實驗材料與方法 --------------------------------------------------------- 9
第三章 實驗結果 ------------------------------------------------------------------- 51
第四章 討論 ------------------------------------------------------------------------- 59
第五章 參考文獻 ------------------------------------------------------------------- 64
附圖 ---------------------------------------------------------------------------------- 67
附錄 ---------------------------------------------------------------------------------- 75
自述 ---------------------------------------------------------------------------------- 80
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