title: In vitro methylation of nuclear respiratory factor-1 binding site suppresses the promoter activity of mitochondrial transcription factor A.
abstract: 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.
authors.affiliation: Asan Institute for Life Sciences, Department of Internal Medicine, College of Medicine, University of Ulsan, Seoul, South Korea.
authors.forename: Yon Sik
authors.initials: YS
authors.lastname: Choi
authors.forename: Shukho
authors.initials: S
authors.lastname: Kim
authors.forename: Hong
authors.initials: H
authors.lastname: Kyu Lee
authors.forename: Ki-Up
authors.initials: KU
authors.lastname: Lee
authors.forename: Youngmi Kim
authors.initials: YK
authors.lastname: Pak
mesh_terms: D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation / Q000502:physiology; D020868:Gene Silencing / Q000502:physiology; D018197:Hepatoblastoma / Q000235:genetics* / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D051265:NF-E2-Related Factor 1; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D051266:Nuclear Respiratory Factor 1; D051264:Nuclear Respiratory Factors; D011401:Promoter Regions, Genetic / Q000235:genetics*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D024101:Mitochondrial Proteins; D051265:NF-E2-Related Factor 1; C495633:NRF1 protein, human; D009687:Nuclear Proteins; D051266:Nuclear Respiratory Factor 1; D051264:Nuclear Respiratory Factors; D015534:Trans-Activators; D014157:Transcription Factors; C422562:mitochondrial transcription factor A