Choi Yon Sik, Kim Shukho, Kyu Lee Hong, Lee Ki-Up, Pak Youngmi Kim
Asan Institute for Life Sciences, Department of Internal Medicine, College of Medicine, University of Ulsan, Seoul, South Korea.
Biochem Biophys Res Commun. 2004 Jan 30;314(1):118-22. doi: 10.1016/j.bbrc.2003.12.065.
DNA methylation on CpG dinucleotides inactivates the expression of the many genes. The decreased amount of mitochondrial DNA (mtDNA) has been suggested to be an important indicator of mitochondrial biogenesis and the pathogenesis of many human diseases. Since mitochondria transcription factor A (Tfam) is a key molecule to regulate mtDNA replication and its promoter contains many CpG dinucleotides, potential methylation sites, we investigated whether the site-specific methylation would modulate the Tfam promoter-driven transcriptional activity in vitro. The luciferase reporters ligated to Tfam promoter (pGL2-Tfam2378) were in vitro methylated by SssI (CG), HpaII (CCGG), or HhaI (GCGC) methylase and luciferase activities were monitored after transient transfection of HepG2 cells. The SssI or HpaII methylation of pGL2-Tfam2378 or the SV40 promoter-luciferase plasmid (pGL2-Control) decreased the luciferase activities to less than 10% of the unmethylated plasmids, indicating that this inactivation by SssI and HpaII methylation might not be specific for the Tfam promoter. In contrast, HhaI methylation of pGL2-Tfam2378 suppressed the promoter activity to 24.4%, without affecting the control vector. There are two HhaI sites in the nuclear respiratory factor-1 (NRF-1) binding site of the Tfam promoter, whereas HpaII sites are present out of the NRF-1 region. It was concluded that the methylation on the NRF-1 sites might be a route for silencing Tfam promoter resulting in decrease of mitochondrial biogenesis.
CpG二核苷酸上的DNA甲基化会使许多基因的表达失活。线粒体DNA（mtDNA）数量的减少被认为是线粒体生物发生和许多人类疾病发病机制的重要指标。由于线粒体转录因子A（Tfam）是调节mtDNA复制的关键分子，其启动子包含许多CpG二核苷酸，即潜在的甲基化位点，因此我们研究了位点特异性甲基化是否会在体外调节Tfam启动子驱动的转录活性。连接到Tfam启动子的荧光素酶报告基因（pGL2-Tfam2378）在体外被SssI（CG）、HpaII（CCGG）或HhaI（GCGC）甲基化酶甲基化，在瞬时转染HepG2细胞后监测荧光素酶活性。pGL2-Tfam2378或SV40启动子-荧光素酶质粒（pGL2-Control）的SssI或HpaII甲基化使荧光素酶活性降低至未甲基化质粒的10%以下，表明SssI和HpaII甲基化导致的这种失活可能并非Tfam启动子所特有。相比之下，pGL2-Tfam2378的HhaI甲基化将启动子活性抑制至24.4%，而不影响对照载体。Tfam启动子的核呼吸因子-1（NRF-1）结合位点中有两个HhaI位点，而HpaII位点位于NRF-1区域之外。得出的结论是，NRF-1位点上的甲基化可能是使Tfam启动子沉默从而导致线粒体生物发生减少的一条途径。
