Mitochondrial DNA copy number is regulated by DNA methylation and demethylation of POLGA in stem and cancer cells and their differentiated progeny.

W Lee,K A Brown,V Vaghjiani,J Donoghue,J Johnson,J C St John,D J Gough,T G Johns,G L M Cagnone

doi:10.1038/cddis.2015.34

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vitamin C (VitC) was unable to sustain increased mtDNA copy number and differentiation, as was the case when VitC was withdrawn after short-term treatment.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.

Highlights

MtDNA copy number is cell type specific and dependent on the strict regulation of mtDNA replication during development.[3,4] Copy number increases progressively during oogenesis and reaches maximal levels in mature fertilisable oocytes.[4,5] Copy number is significantly reduced during preimplantation development before gastrulation.[3]
These findings demonstrate that mtDNA copy number can be reset in cancer cells, which results in the expansion of mtDNA copy number during differentiation. This is only maintained in the presence of a DNA demethylation agent. Pluripotent cells, such as ESCs, establish the mtDNA set point, which is defined as maintaining low mtDNA copy number in the presence of the OCT4-SOX2-NANOG network.[6,7,13]
We show that the CpG islands in exon 2 of polymerase gamma A (POLGA) were highly methylated in cancer, pluripotent and multipotent cells

Summary

Introduction

MtDNA copy number is cell type specific and dependent on the strict regulation of mtDNA replication during development.[3,4] Copy number increases progressively during oogenesis and reaches maximal levels in mature fertilisable oocytes.[4,5] Copy number is significantly reduced during preimplantation development before gastrulation.[3]. Human embryonic stem cells (hESCs) are extensively DNA methylated, which is reduced during differentiation.[14,15,16] They progressively increase mtDNA copy number in a cellspecific manner, as do human neural stem cells (hNSCs).[17] For example, at the completion of astrocyte differentiation, hNSCs possess significantly more copies of mtDNA, downregulate expression of multipotent neural genes, such as NESTIN, MUSASHI1 and CD133, and upregulate expression of the endpoint marker glial fibrillary acidic protein (GFAP) Cancer cells, such as those giving rise to Glioblastoma multiforme, the most aggressive malignant primary brain tumour,[18,19] are extensively DNA methylated.[20] When induced to differentiate, they fail to increase mtDNA copy number and continue to express NESTIN, MUSASHI1 and CD133.17 The maintenance of low mtDNA copy number promotes aerobic glycolysis for the generation of ATP, a combination of glycolysis with a small contribution from OXPHOS.[21,22] This promotes cell proliferation and self-renewal and prevents differentiation.[23,24]. DNA demethylation agents, such as 5-azacytidine (5azaC), have been used as therapeutic agents for cancer patients.[25,26] By demethylating the tumour genome, these agents promote cell differentiation to deplete the cancer cell pool.[27,28,29] withdrawal results in cells returning to their semi-undifferentiated state, known as the ‘rebound’ effect.[30,31] treatment of induced pluripotent stem cells with 5-azaC induces differentiation and synchronised increases in mtDNA copy number similar to ESCs.[13]

Methods

Results

Conclusion