藉由建立目標DNA序列甲基化系統來研究MAEL基因啟動子甲基化對LINE1基因表現的影響
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 investigation of MAEL promoter methylation pattern in LINE1 expression by establishing targeted DNA methylation reporter system in human cell line
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:
Male MAEL knockout mice are found sterile with meiotic arrest and concordant with derepression of retrotransposon activity. In this study, we aimed to investigate if methylation pattern of specific MAEL promoter segment had impact on transposable elements (TEs) expression in human cell line.
Materials and methods:
We established the targeted DNA methylation (TDM) reporter system in human H358 cell line to study the impact of methylation pattern on retrotransposon activity.
Initially, we validated the two-component reporter gene system after 3 consecutive transfection of in-vitro methylated MAEL promoter DNA at days 1, 3, and 5. Then, we determined both MAEL transcript level and LINE1 mRNA by quantitative real-time RT-PCR in TDM cells versus un-methylated cells. Moreover, western blotting of MAEL was done to check the protein level in TDM cells. We detected the LINE1 protein by using the immunostain method in TDM cells.
Results:
The targeted DNA methylation (TDM) reporter system to MAEL promoter segment in human H358 cell line was validated by EGFP intensity. In TDM cells, both MAEL transcript and protein levels decreased significantly after targeted DNA methylation. Nevertheless, LINE-1 transcripts and immunostaining intensity of LINE1 protein were both significantly higher in hypermethylated TDM cells than in un-methylated cells.
Conclusions:
TDM reporter system is an useful tool to study the epigenetic mechanism of specific gene promoter segment. Methylation of MAEL promoter region results in gene silencing and further increasing LINE1 expression responsible for genome instability. This might implicate one of the causes of male infertility by interfering piRNA-mediated defense mechanism from transposable elements.