特定MAEL啟動子序列上高度甲基化導致人類細胞中反轉錄轉座子活性增加
鄭裕生1,2、黃詩凱2、林永明2
1國立成功大學醫學院臨床醫學研究所;國立成功大學醫學院附設醫院泌尿部2
The hypermethylation of specific MAEL promoter region induces the increasing LINE-1 retrotransposition activity in human cells
Yu-Sheng Cheng1,2,Shi-Kae Wee2;Yung-Ming Lin2
1Graduate Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan, Taiwan; 2Department of Urology, National Cheng Kung University College of Medicine and Hospital, Tainan, Taiwan
Purpose: MAEL is known for an indispensable role in transposable elements (TEs) control of the male germline. We thus established a two-component EGFP reporter gene system for investigating the effect of methylation of specific MAEL promoter region and downstream LINE-1 retrotransposition activity.
Materials and Methods: We created a targeted DNA methylation (TDM) reporter system in human NCI-H358 cells to investigate the effect of methylation of MAEL promoter on the gene expression and downstream LINE-1 retrotransposition activity. An EGFP reporter gene system consisted of two constructs were established and were co-transfected into human NCI-H358 cells. When the gene promoter is not methylated, Tet repressor is expressed, and then Tet repressor binds to TetO2, thus, in turn, blocks the EGFP expression. The addition of a methylated DNA complementary suppress the Tet repressor expression; therefore, TetO2 is not suppressed and then induce EGFP expression. The validation of expression in MAEL and LINE-1 was performed by using qRT-PCR, Western blot and immunofluorescence staining.
Results: By using this TDM reporter system, we validated the effects of hypermethylation of specific MAEL promoter region and its downstream target LINE-1 expressions. After targeted DNA methylation, both MAEL transcription and protein levels were significantly decreased and the LINE-1 transcriptional and protein levels were significantly increased, compared to unmethylated cells. Also, after adding 5-Aza into TDM cells, the EGFP signal decreased significantly.
Conclusions: TDM is an available tool for studying the effect of methylation of specific promoter region on gene expression. The hypermethylation of specific MAEL promoter regions induces the increasing LINE-1 retrotransposition activity in human cells.