Cytosine methylation of mitochondrial DNA at CpG sequences impacts transcription factor A DNA binding and...

In eukaryotes, cytosine methylation of nuclear DNA at CpG sequences (^5mCpG) regulates epigenetic inheritance through alterations in chromatin structure. However, mitochondria lack nucleosomal chromatin, therefore the molecular mechanisms by which ^5mCpG influences mitochondria must be different and are as yet unknown. Mitochondrial Transcription Factor A (TFAM) is both the primary DNA-compacting protein in the mitochondrial DNA (mtDNA) nucleoid and a transcription-initiation factor. TFAM must encounter hundreds of CpGs in mtDNA, so the occurrence of ^5mCpG has the potential to impact TFAM-DNA recognition. We used biophysical approaches to determine whether ^5mCpG alters any TFAM-dependent activities. ^5mCpG in the heavy strand promoter (HSP1) increased the binding affinity of TFAM and induced TFAM multimerization with increased cooperativity compared to nonmethylated DNA. However, ^5mCpG had no apparent effect on TFAM-dependent DNA compaction. Additionally, ^5mCpG had a clear and context-dependent effect on transcription initiating from the three mitochondrial promoters. Taken together, our findings demonstrate that ^5mCpG in the mitochondrial promoter region does impact TFAM-dependent activities in vitro.