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研究生:蔡宗樺
研究生(外文):Chung-huaTsai
論文名稱:神經細胞分化時Necdin基因甲基化調控之研究
論文名稱(外文):DNA Methylation Status of Necdin Gene during Neuronal Differentiation
指導教授:曾淑芬曾淑芬引用關係
指導教授(外文):Shun-Fen Tzeng
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生命科學系碩博士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:34
中文關鍵詞:甲基化神經分化
外文關鍵詞:necdinmethylationneuronal differentiation
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Necdin由325個胺基酸所構成,為一與神經細胞分化有關的蛋白質。其主要表現在神經細胞或骨骼肌細胞等後分裂時期細胞 (postmitotic cells)。Necdin基因 (NDN) 的母系對偶基因具有基因印痕 (gene imprinting),只有父系的對偶基因能夠被轉錄成mRNA。雖然Necdin被認為對於神經細胞的分化和成熟扮演著重要的角色,但是NDN本身是如何被調控的,目前還不是非常了解。NDN位於人類染色體15q11-q13,此段基因如發生缺失 (deletion),會造成稱做小胖威利症 (Prader-Willi syndrome, PWS)的神經發育不正常遺傳疾病,此疾病也是一典型的與基因印痕有關的疾病。基因印痕是藉由甲基化基因上CpG雙核?酸上的胞嘧啶,使得基因表現被抑制的機制 (gene silencing)。
亞硫酸鹽轉換 (bisulfite conversion)是一種能夠讓胞嘧啶 (C)轉換成尿嘧啶 (U)的方法,然而被甲基的胞嘧啶不會進行轉換,因此可以區分該胞嘧啶是否被甲基化。利用這個方法,我分析了老鼠ndn CpG-rich區域的甲基化狀態。DNA樣品取自E14.5、ndn突變老鼠大腦皮層的神經幹細胞,其ndn父系對偶基因插入了neomycin耐受基因 (neomycin-resistant gene cassette),因此定序時可以利用Necdin和neomycin專一的引子區別父系和母系對偶基因。我分析了23個介於 -299到 +93 (ATG轉譯起點為 +1)的CpG位置 (CpG site),而這23個CpG位置位於 -264到 +470的CpG島 (CpG island)。同時,這23個CpG位置包含了轉錄和轉譯起點。
我預期大部分母系ndn對偶基因的CpG位置會被甲基化。然而,定序結果顯示其未分化與已分化的大腦皮層神經幹細胞,CpG位置甲基化程度分別約為60%和53%。父系ndn對偶基因方面,未分化與已分化的甲基化程度分別為62%與40%。這顯示了不論是父系或是母系ndn對偶基因,在神經細胞分化時都會受到甲基化機制的調控,而且父系對偶基因受到的影響比母系還大。此外,有些CpG位置在神經細胞分化時,其甲基化程度變動很大。這些結果顯示在神經分化時,ndn的表現可能與某些特定的CpG位置甲基化有關。

NECDIN is a neural differentiation-associated protein composed of 325 amino acids. NECDIN is predominantly expressed in postmitotic cells such as neuron and skeletal muscle. The NECDIN gene (NDN) is maternally imprinted, and only the paternal allele is transcribed into mRNA. Although NECDIN is thought to play an important role in neuronal differentiation and maturation, the regulatory mechanism of NDN expression is not well understood. NDN is located in human chromosome 15q11-q13, whose deletion causes the neurodevelopmental disorder Prader-Willi syndrome (PWS), a classic genomic imprinting-associated disorder. Genomic imprinting is a gene silencing process through cytosine-specific methylation at CpG sites.
I analyzed the methylation status of CpG-rich region of mouse ndn using bisulfite conversion method based on the principle that methylated cytosine is protected from conversion to uracil. Therefore, the difference between methylated and unmethylated cytosine can be detected by DNA sequence analysis. Genomic DNA samples were prepared from neural stem cells (NSCs) of the neocortex of E14.5 ndn- mutated mice. The paternal allele of ndn-mutated mice is mutated by insertion with a neomycin-resistant gene cassette. Using primers that specifically recognize Necdin and neomycin-resistant gene sequences, the difference between paternal and maternal Necdin alleles can be distinguished. Among CpG islands from -264 to +470 (ATG translation start site=+1), 23 CpG sites from -229 to +93, in which the transcription and translation start sites are included, were analyzed.
We found that DNA sequence data indicated that 60% and 53% of CpG sites were methylated in undifferentiated and differentiated cortical NSCs, respectively. In contrast, 62% and 40% of the CpG sites in the paternal allele were methylated in undifferentiated and differentiated NSCs, respectively. The results also indicated that the methylation status is regulated in both the paternal and maternal alleles during neural differentiation of NSCs, but the methylation status of the paternal allele is more affected than that of the maternal allele. Furthermore, some specific CpG sites in the paternal and maternal alleles are profoundly affected by neuronal differentiation. These results suggest that ndn expression is regulated through methylation of specific CpG sites during neuronal differentiation.

中文摘要…………………………………………………………………………………..01
Abstract……………………………………………………………………………………03
致謝……………………………………………………………………………………......05
Content………………………………………………………………………………….....06
List of Figures…………………………………………………………………………......07
Introduction………………………………………………………………………………..08
I. Neural stem cells…………………………………...………………………..08
II. Molecules involved in neuronal differentiation……………………………..09
III. Necdin gene (NDN)…………………………………………………………09
IV. Epigenetic regulation………………………………………………………..11
V. Purpose of the study…………………………………………………………12
Materials and Methods…………………………………………………………………….13
I. Neural stem cell culture……………………………………………………..13
II. P19 cell culture………………………………………………………………13
III. Isolation of genomic DNA…………………………………………………..14
IV. Bisulfite conversion………………………………………………………....14
V. PCR………………………………………………………………………….14
VI. Cloning………………………………………………………………………15
VII. DNA sequencing…………………………………………………………….16
VIII. CpG island analysis and CpG methylation analysis………………………..16
Results……………………………………………………………………………………..17
I. CpG islands in Necdin gene…………………………………………………17
II. DNA methylation status of paternal Necdin in Ndn-KO cortical NSCs……17
III. DNA methylation status of maternal Necdin in Ndn-KO cortical NSCs……17
IV. Specific CpG sites demethylated during neuronal differentiation…………..18
V. DNA methylation status of Necdin in p19 cell line………………………....18
Discussion…………………………………………………………………………………20
I. Methylation status of Necdin………………………………………………..20
II. Necdin and DNA methylation machinery………………………………..…21
III. Tissue-specific expression and functions of Necdin…………………..……22
IV. DNA methylation of MAGE family genes………………………………….23
V. Conclusions………………………………………………………………….24
References………………………………………………………………………………....25

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