陳宏文臺灣大學:生化科學研究所梁瀞予Liang, Ching-YeuChing-YeuLiang2010-05-042018-07-062010-05-042018-07-062008U0001-2207200814203600http://ntur.lib.ntu.edu.tw//handle/246246/178832Syncytin 2屬於人類內生性反轉錄病毒（human endogenous retrovirus）HERV-FRD之外鞘蛋白，其專一表現在胎盤滋養層細胞中，已知syncytin 2會促進細胞滋養層細胞進行細胞融合，而形成融合滋養層，故syncytin 2在胎盤發育上扮演重要的角色。而過去研究指出，人類胚胎發育過程中，胎盤組織裡的DNA甲基化程度明顯較胚胎本體組織來的低，且最近有報導發現，syncytin家族另一個成員syncytin 1，在胎盤細胞中，syncytin 1之5’-long terminal repeat（5’-LTR）啟動子上DNA甲基化程度非常低，使得syncytin 1能表現於胎盤細胞中，因此，本篇論文想探討是否細胞能透過改變5’LTR啟動子上DNA甲基化與組蛋白修飾等後基因修飾，而達到syncytin 2表現。實驗結果顯示，胎盤細胞或組織中的syncytin 2 5’LTR上甲基化程度顯著比非胎盤細胞來的低，且5’LTR上的組蛋白修飾皆為活化態的修飾，接著再利用報導基因方式發現syncytin 2 5’LTR若甲激化會影響到其轉錄活性，但有趣的是，含DNA甲基化syncytin 2 5’LTR的報導基因送胎盤細胞中，仍具有一定的轉錄活性，這表示胎盤細胞中具有特殊分子機制可對syncytin 2 5’LTR做去甲基化的動作；接著，發現胎盤組織特定轉錄因子GCMa可調控syncytin 2表現，且在非胎盤細胞MCF-7中表現GCMa後，會促進syncytin 2 5’LTR的DNA去甲基化，故GCMa轉錄因子除了調控基因表現外，同時在syncytin 2 5''LTR啟動子的去甲基化扮演重要角色；最後發現GCMa會與thymine DNA glycosylase（TDG）之間有結合能力，而TDG過去被指出可進行DNA去甲基化的酵素，因此GCMa可能透過吸引TDG至syncytin 2 5’LTR來進行DNA去甲基化的動作。綜合以上實驗結果，證實在胎盤細胞及非胎盤細胞中，syncytin 2 5’LTR上的後基因修飾有所不同，且胎盤特殊轉錄因子GCMa可促進syncytin 2 5’LTR DNA去甲基化以及啟動syncytin 2 mRNA表現。Syncytin 2, an envelope glycoprotein encoded by the human endogenous retrovirus FRD (HERV-FRD), is specifically expressed in placental trophoblasts. It has been reported that syncytin 2 is a fusogenic protein. Therefore, syncytin 2 may play an important role in placental development by regulation of trophoblastic fusion. Previous studies have shown that the placenta exhibits lower overall DNA methylation levels than the embryo. A recent study has shown that the 5’-long terminal repeat (5’LTR) promoter of syncytin 1, which encodes the envelope protein of HERV-W, is hypomehtylated and controls syncytin 1 expression in placenta. n this study, we further investigated whether DNA methylation and histone modification control syncytin 2 expression in placenta. We demonstrated that the 5’LTR of syncytin 2 is hypomethylated and harbors active histone modification in placental cells. In luciferase reporter assay, in vitro DNA methylation inhibited the promoter activity of syncytin 2 5’LTR. Interestingly, the promoter activity of in vitro methylated syncytin 2 5’LTR could be restored in placental cells. To study the mechanism that counteracts the suppressive effect of DNA methylation on syncytin 2 5’LTR promoter in placenta, we demonstrated that placental transcription factor, GCMa, not only regulates syncytin 2 expression but also promotes DNA demethylation on syncytin 2 5’LTR. We further demonstrated that GCMa associates with TDG, an enzyme that involves in DNA demethylation process. This implies that GCMa may recruit TDG to demethylate syncytin 2 5’LTR. Overall, our results reveal an epigenetic regulation of syncytin 2 gene expression and that GCMa is a key factor for DNA demethylation of syncytin 2 promoter and transcription activation of syncytin 2.目錄………………………………………………………………….…………………I文摘要…………………………………………………….………………..……...III文摘要…………………………………………………….……………………….IV一章 緒論. 胎盤.1 人類胎盤發育…………………………………………………………….1.2 胎盤功能………………………………………………………………….3. 後基因調控.1 簡介……………………………………………………………………….4.2去氧核糖酸甲基化………………………………………………….…….4.3 DNA methyltransferases………………………………………………..5.4 去氧核糖酸去甲基化……………………………………………………7.5 組蛋白修飾………………………………………………………………8. 人類內生性反轉錄病毒.1 簡介……………………………………………………………………….9.2 syncytin…………………………………………………………………10. gcm轉錄因子………………………………………………………………...12. 研究動機……………………………………………………………………14二章 材料與方法…………………………………………………………………15三章 結果. syncytin 2 5’LTR啟動子之DNA甲基化狀態...............................................25. syncytin 2 5’LTR啟動子上組蛋白修飾.........................................................26. 核酸甲基化對syncytin 2 5’LTR轉錄活性之影響………………………….26. GCMa調控syncytin2表現並促進syncytin 2 5’LTR區域的去甲基化…..28. GCMa在細胞中會與TDG作結合…………………………………………29四章 討論…………………………………………………………………………31五章 圖表…………………………………………………………………………35六章 參考文獻……………………………………………………………………42application/pdf860497 bytesapplication/pdfen-US後基因胎盤人類融合胎盤蛋白去氧核醣核酸甲基化組蛋白修飾epigeneticssyncytinDNA methylationGCMaTDGhistone modificationhttp://ntur.lib.ntu.edu.tw/bitstream/246246/178832/1/ntu-97-R95b46014-1.pdf