title: Mitochondrial DNA copy number is regulated by DNA methylation and demethylation of POLGA in stem and cancer cells and their differentiated progeny.
abstract: Mitochondrial DNA (mtDNA) copy number is strictly regulated during differentiation so that cells with a high requirement for ATP generated through oxidative phosphorylation have high mtDNA copy number, whereas those with a low requirement have few copies. Using immunoprecipitation of DNA methylation on 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), which distinguish between de novo DNA methylation and demethylation, respectively, we set out to determine whether DNA methylation at exon 2 of the human mtDNA-specific polymerase (DNA polymerase gamma A (POLGA)) regulates cell-specific mtDNA copy number in highly proliferative and terminally differentiated cells. Highly proliferative cancer and pluripotent and multipotent cells possessed low mtDNA copy number and were highly methylated at exon 2 of POLGA in contrast to post-mitotic cells. Unlike neural stem cells, cancer cells were unable to differentiate and remained extensively DNA methylated at exon 2 of POLGA. However, mtDNA depletion of cancer cells reduced DNA methylation at exon 2 of POLGA as they replenished mtDNA to form tumours in mice. Glioblastoma cells treated with the DNA demethylation agent 5-azacytidine over 28 days of astrocyte-induced differentiation demethylated exon 2 of POLGA leading to increased mtDNA copy number and expression of the astrocyte endpoint marker glial fibrillary acidic protein (GFAP). However, the demethylation agent vitamin C (VitC) was unable to sustain increased mtDNA copy number and differentiation, as was the case when VitC was withdrawn after short-term treatment. These data demonstrate that DNA demethylation of POLGA is an essential regulator of mtDNA copy number and cellular fate and that cancer cells are only able to modulate DNA methylation of POLGA and mtDNA copy number in the presence of a DNA demethylation agent that inhibits de novo methyltransferase 1 activity.
authors.affiliation: 1] Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia [2] Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia.
authors.forename: W
authors.initials: W
authors.lastname: Lee
authors.affiliation: 1] Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia [2] Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia.
authors.forename: J
authors.initials: J
authors.lastname: Johnson
authors.affiliation: 1] Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia [2] Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia.
authors.forename: D J
authors.initials: DJ
authors.lastname: Gough
authors.affiliation: 1] Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia [2] Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia.
authors.forename: J
authors.initials: J
authors.lastname: Donoghue
authors.affiliation: 1] Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia [2] Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia.
authors.forename: G L M
authors.initials: GL
authors.lastname: Cagnone
authors.affiliation: 1] Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia [2] Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia.
authors.forename: V
authors.initials: V
authors.lastname: Vaghjiani
authors.affiliation: 1] Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia [2] Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia.
authors.forename: K A
authors.initials: KA
authors.lastname: Brown
authors.affiliation: 1] Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia [2] Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia.
authors.forename: T G
authors.initials: TG
authors.lastname: Johns
authors.affiliation: 1] Centre for Genetic Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia [2] Department of Molecular and Translational Science, Monash University, 27-31 Wright Street, Clayton, Victoria 3168, Australia.
authors.forename: J C
authors.initials: JC
authors.lastname: St John
mesh_terms: D001205:Ascorbic Acid / Q000494:pharmacology; D001374:Azacitidine / Q000494:pharmacology; D015153:Blotting, Western; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D056915:DNA Copy Number Variations / Q000235:genetics; D019175:DNA Methylation / Q000187:drug effects / Q000235:genetics*; D000074002:DNA Polymerase gamma; D004272:DNA, Mitochondrial / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004272:DNA, Mitochondrial; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase; C413027:POLG protein, human; D001374:Azacitidine; D001205:Ascorbic Acid