MtDNA copy number is differentially regulated in various types of cancer. The mitochondrial specific DNA polymerase gamma A (POLGA) has been suggested to play a role in regulation of mtDNA in tumorigensis. We have previously shown in non-transformed mouse embryonic stem cells that cell-specific expression of POLGA is negatively correlated with DNA methylation and mtDNA copy number, indicating DNA methylation may have a direct effect on the regulation of mtDNA copy number. However, the relationship between DNA methylation and the regulation of mtDNA copy number has not yet been demonstrated in human.
We examined the enrichment of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) within exon 2 of PolgA of various highly proliferative cells, such as human cancer stem cells and embryonic and adult stem cells as well as in post-mitotic somatic cells and tissues. Immunoprecipitation of methylated DNA (MeDIP) showed that the enrichment of 5mC is at least twice as high as 5hmC in all fast-replicating cells with low mtDNA copy number, whilst in post-mitotic tissues, such as placenta and brain, the ratio is closer to 1 and these tissues contain significantly higher mtDNA copy number. We also observed a significant increase in mtDNA copy number in HepG2 liver cancer cells that have been demethylated with 5-azacytidine. Futhermore, we showed in Glioblastoma multiforme cancer stem cells (HSR-GBM1) that had been progressively depleted of their mtDNA by treatment with 2’3’-dideoxycytidine (ddC) that they had increased levels of 5hmC during replenishment of mtDNA as these cells formed tumours in mice. These data suggest that DNA demethylation of PolgA is essential for up-regulation of mtDNA and thus is important for cellular differentiation. Finally, we demonstrated that enrichment of 5hmC is higher than 5mC within the mtDNA-encoded genes, suggesting that mtDNA is DNA methylated but this is negated by active demethylation.