建立並視覺化特定基因啟動子甲基化誘導技術以決定細胞命運

去氧核醣核酸甲基化誘導，是在細胞中透過送入試管外甲基化的特定序列之去氧核醣核酸片段，藉以增加目標位置的甲基化程度。此方法過去曾用於鑑定特定基因甲基化與細胞分化或癌化之間的關係。於本篇論文，我們將此方法應用於兩方面: 一為病毒感染後，融合至人類基因體中的病毒序列；二為癌症發生時，去氧核醣核酸低度甲基化之區域。結果顯示，透過去氧核醣核酸甲基化誘導的技術，我們成功地將帶有報告基因融合至乳癌細胞中的巨細胞病毒啟動子默化，並在以腺病毒5轉型之人類類胚胎腎細胞中，默化病毒之早期區域1A基因 (E1A)啟動子，降低其基因表達並減少細胞被癌化轉型的特徵。為了進一步的驗證去氧核醣核酸甲基化誘導之動能與其所造成之轉錄抑制，我們建立了二元件活細胞甲基化觀察系統，藉由誘導第一元件啟動子的甲基化，即可釋放並表達第二元件的報告基因。當第一元件受到去甲基化藥物移除其啟動子甲基化後，則關閉第二元件報告基因的表達。另一方面，為了能夠以全基因組的方法找出癌細胞與其相對應之正常細胞中去氧核醣核酸低度甲基化的差異區段，我們透過甲基化特異性扣除法，找出全基因組中與癌化相關的低度甲基化區段，並以聚合酶連鎖反應將這些片段擴增。擴增片段可透過生物資訊方法分析其位點，再將試管外甲基化的全基因體擴增片段以細胞轉染的方式送回癌細胞中，進行基因組階層的去氧核醣核酸甲基化誘導，並觀察癌細胞是否因為去氧核醣核酸的甲基化的改變，而降低其癌化程度或存活率。總結而言，去氧核醣核酸甲基化誘導，可以作用於來自病毒感染並融合於細胞中的外源基因，亦可以全基因組的方式增加異常低度甲基化區域的甲基化程度。而異常低度甲基化的區域則可透過甲基化特異性扣除法取得。最後，二元件的報告基因系統的設計，提供了在活細胞中，可直接視覺觀察定序列甲基化的改變，並可同時追蹤甲基化改變後的細胞生理狀態。

關鍵字

二元件報告基因系統；表觀遺傳學；去氧核醣核酸甲基化

並列摘要

Targeted DNA methylation (TDM), a method for increasing DNA methylation at specific loci, has been used to identify genes that participate in cell fate determination and tumorigenesis. Here, we applied TDM to integrated viral genes and hypomethylated cancer-associated regions. Genome-integrated cytomegalovirus (CMV) promoter and adenovirus type 5 early region 1 A (E1A) promoter were hypermethylated in MCF7 and HEK293 cells using TDM; this methylation could be reversed by adding the demethylating agent, 5-aza-2-deoxycytidine (5-Aza). To validate transcriptional silencing and visualize the effects of TDM in live cells, a two-component enhanced green fluorescent protein (EGFP) reporter gene system was established. Expression of EGFP was induced via targeted methylation of the first component and suppressed by adding 5-Aza, allowing the kinetics of TDM to be studied. Furthermore, we established a genome-wide, methylation-specific subtraction polymerase chain reaction method to subtract and amplify DNA loci hypomethylated in cancer cells relative to normal cells. Target loci were deduced from a bioinformatics analysis of the sequence results. Amplified target loci DNA was methylated in vitro and transfected into cancer cells to investigate its effects on tumorigenic properties and cell survival. We report that TDM efficiently silences viral promoters and cancer-associated hypomethylated genes. In addition, the methylation-specific subtraction method is a bottom-up reductionist approach suitable for identifying and targeting hypomethylated loci critical for tumorigenesis. Finally, the development of a DNA methylation visualization system provides a unique opportunity to study DNA methylation changes in live cells.

並列關鍵字

Two-component reporter system ； Epigenomic ； DNA methylatuin

參考文獻

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建立並視覺化特定基因啟動子甲基化誘導技術以決定細胞命運

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