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研究生:蔡易叡
研究生(外文):Yi-Jui Tsai
論文名稱:轉錄因子EGR1調控P1-及P2-A4GALT血型基因型的差異性表現
論文名稱(外文):The differential expression of blood group P1-and P2-A4GALT alleles is stimulated by transcription factor EGR1
指導教授:余榮熾
口試委員:朱善德涂玉青
口試日期:2017-06-01
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:35
中文關鍵詞:P1/P2血型系統α-14-半乳糖基轉移酶單一核苷酸多型性早期生長反應蛋白1(EGR1)
外文關鍵詞:P1/P2 blood groupsA4GALTSNPEGR-1
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P1PK血型系統是人類血型系統中的一種,在此系統中P1及Pk是表現在紅血球細胞上的醣類抗原,這兩種抗原的形成與A4GALT基因調控有關。A4GALT基因能轉錄轉譯成α-1,4-Galactosyltransferase(α4Gal-T),為一種半乳糖機轉移酶,是主要合成P1及Pk抗原的主要酵素。在過去研究指出P1PK血型系統中有兩種主要的表現型:P1、P2。P1表現型中含P1及P抗原(Pk抗原為P抗原的前驅物),P2表現型則只有P抗原。目前已知在P1表現型的紅血球中A4GALT的表現量高於P2表現型的紅血球,因此若能找出調控A4GALT表現量的原因,便能進一步了解形成P1/P2表現型形成的分子機制。
在先前實驗室的研究裡已經發現,A4GALT SNP的不同是形成P1/P2表現型的主要原因。SNP (single nucleotide polymorphism單核苷酸多型性)指的是由單個核苷酸的改變而引起DNA序列的改變,造成物種之間染色體基因組的多樣性。我們實驗室在先前的研究發現,SNP rs2143918 (SNP5)及SNP rs5751348 (SNP6)這兩個SNP,對於A4GALT表現量高低與P1/P2表型的形成有很重要的關聯。因此在本研究中我們用電腦軟體預測這兩個SNP位置可能結合哪些轉錄因子,預測結果有RUNX1、KLF1、EGR3、KLF6A、ETS2、CEBPD。我們接著利用K562細胞模型以及報導基因分析法(reporter assay)進行實驗,結果發現EGR3在A4GALT SNP6 P1基因型的報導基因載體有較高的轉錄活性。之後我們再擴大以EGR家族系列(EGR1、EGR2、EGR4他們與EGR3有類似的結合能力)進行實驗,發現EGR家族系列的轉錄因子都能刺激A4GALT SNP6高表現基因型(P1 allele)的報導基因載體的轉錄活性。接著經由基因表現分析發現在類似紅血球的細胞中EGR1轉錄因子的表現量遠高於EGR3轉錄因子,且在K-562細胞若以(sodium butyrate)進行誘導紅血球分化,發現EGR1轉錄因子上升的量也遠高於EGR3轉錄因子,因此我們推測EGR1轉錄因子為主要在紅血球中最有可能刺激P1-A4GALT基因型等位基因的表現量。
為進一步證實轉錄因子EGR1與A4GALT SNP6 P1基因型的關係,我們將EGR1表現於SNP6 P1/P2異型核子HT-29細胞中,並觀察HT-29細胞核中A4GALT SNP6 P1及P2基因型hnRNA的表現量。實驗結果發現EGR1轉錄因子確實會與細胞核中A4GALT帶有SNP6 P1基因型結合,增加其hnRNA的表現量,最後造成A4GALT基因SNP6 P1及P2基因型的差異性表現。
The P1/P2 phenotypic polymorphism is one of the earliest blood groups discovered in humans. These blood groups have been connected to presence of different levels of expression of the A4GALT gene in P1 and P2 red cells; however, the detailed molecular genetic mechanism that leads to these two phenotypes has not been established. Following our previous identification of an association between the SNPs rs2143918 and rs5751348 in A4GALT gene and the P1/P2 phenotype, we conduct a survey of transcription factors that might connect these SNPs with the differential expression of the P1-A4GALT and P2-A4GALT alleles. An in silico analysis of potential transcription factor binding motifs within the polymorphic rs2143918 and rs5751348 genomic regions was performed, and this was followed by reporter assays examining the candidate transcription factors, gene expression profiling, electrophoretic mobility shift assays, and P1-A4GALT and P2-A4GALT allelic expression analysis.
The results revealed that the differential binding of transcription factor EGR1 to the polymorphic SNP rs5751348 genomic region leads to differential activation of P1-A4GALT and P2-A4GALT expression and this consequently results in the quantitative difference of P1 antigen expression levels in P1 and P2 red cells. The present investigation has elucidated the molecular genetic details associated with the P1/P2 blood groups and revealing the first human blood group whose formation involves the transcriptional activity of a specific transcription factor and its interaction with genetic polymorphism within the gene regulatory region.
口試委員審定書 i
摘要 ii
Abstract iv
縮寫表 vi
目錄 viii
圖目錄 xi
第一章 緒論 1
1.1 人類P1PK血型系統 1
1.2 α-1,4-Galactosyltransferase (α4Gal-T) 2
1.3 A4GALT基因的SNP與P1/P2血型之關聯 3
1.4 研究構想與目的 4
第二章 實驗材料與方法 5
2.1 細胞培養與分化 5
2.2 報導基因分析法(Reporter assay) 5
2.3 RNA表現之分析 7
2.3.1 RNA萃取 7
2.3.2 反轉錄聚合酶連鎖反應 (Reverse Transcription-Polymerase Chain Reaction,RT-PCR) 7
2.3.3 即時定量聚合酶連鎖反應 (Real-Time Polymerase Chain Reaction) 7
2.4 脂質體轉染法(Liposome Transfection) 8
2.5 蛋白質表現之分析 8
2.5.1 蛋白質萃取 8
2.5.2 SDS聚丙烯醯胺凝膠電泳(SDS-Polyacrylamide gel electrophoresis, SDS-PAGE) ……………………………………………………………………………...8
2.5.3 西方墨點法(Western blotting) 8
2.6 電泳遷移率變動分析 (Electrophoretic Mobility Shift Assay, EMSA) 9
2.6.1 核蛋白質萃取 9
2.6.2 探針(probe)製備 9
2.6.3 蛋白質與探針反應 9
2.6.4 偵測 10
2.7 細胞核hnRNA(heterogenous nuclear)萃取及放大 10
第三章 結果 11
3.1 轉錄因子與A4GALT基因上SNP5及SNP6的轉錄活性調控關係 11
3.1.1 利用電腦預測及分析可能會和A4GALT基因SNP5及SNP6基因型不同而導致結合親和性差別之轉錄因子 11
3.1.2 分析有可能有結合能力的轉錄因子對A4GALT基因SNP5及SNP6之P1及P2基因型轉錄活性的影響 12
3.2 轉錄因子EGR家族對A4GALT基因SNP5及SNP6之P1及P2基因型轉錄活性的關係 13
3.3 轉錄因子EGR家族在類紅血球細胞(erythroid lineage)中的表現 13
3.4 轉錄因子EGR1對A4GALT基因SNP6 P1基因型的影響 14
3.5 轉錄因子EGR1與A4GALT基因SNP6 P1及P2基因型之間的蛋白質與DNA的交互作用(interaction) 14
3.6 轉錄因子EGR1在A4GALT SNP6為異型核子HT-29細胞中對P1-及P2-A4GALT基因表現的影響 15
第四章 討論 17
第五章 圖表 20
參考文獻 26
附錄 31
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