In vitro methylation of nuclear respiratory factor-1 binding site suppresses the promoter activity of mitochondrial transcription factor A
In vitro methylation of nuclear respiratory factor-1 binding site suppresses the promoter activity of mitochondrial transcription factor A
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DOI：
10.1016/j.bbrc.2003.12.065
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发表时间：
2004-01-30
影响因子：
3.1
通讯作者：
Pak, YK
中科院分区：
文献类型：
--
作者：
Choi, YS;Kim, S;Pak, YK
CpG二核苷酸上的DNA甲基化使许多基因的表达失活。线粒体DNA（mtDNA）的数量减少已被认为是线粒体生物合成和许多人类疾病的发病机制的重要指标。由于线粒体转录因子A（Tfam）是调节线粒体DNA复制的关键分子，其启动子中含有大量的CpG二核苷酸，是潜在的甲基化位点，因此我们在体外研究了位点特异性甲基化是否会调节Tfam启动子驱动的转录活性。将连接至Tfam启动子（pGL 2-Tfam 2378）的荧光素酶报告基因在体外用SssI（CG）、HpaII（CCGG）或HhaI（GCGC）甲基化酶甲基化，并在瞬时转染HepG 2细胞后监测荧光素酶活性。pGL 2-Tfam 2378或SV 40启动子-荧光素酶质粒（pGL 2-对照）的SssI或HpaII甲基化使荧光素酶活性降低至低于未甲基化质粒的10%，表明这种由SssI和HpaII甲基化引起的失活可能对Tfam启动子没有特异性。相比之下，pGL 2-Tfam 2378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位点存在于NRF-1区域之外。因此，NRF-1位点的甲基化可能是Tfam启动子沉默导致线粒体生物合成减少的途径之一。(C)2003年爱思唯尔公司All rights reserved.
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. (C) 2003 Elsevier Inc. All rights reserved.