Mitochondrial membrane hyperpolarization modulates nuclear DNA methylation and gene expression through phospholipid remodeling [EPIC1]

Maintenance of the mitochondrial inner membrane potential (ΔΨm) is critical for many aspects of mitochondrial function. While ΔΨm loss and its consequences are well studied, little is known about the effects of mitochondrial hyperpolarization. In this study, we used cells deleted of ATP5IF1 (IF1), a natural inhibitor of the hydrolytic activity of the ATP synthase, as a genetic model of increased resting ΔΨm. We found that the nuclear DNA hypermethylates when the ΔΨm is chronically high, regulating the transcription of mitochondrial, carbohydrate and lipid genes. These effects can be reversed by decreasing the ΔΨm and recapitulated in wild-type (WT) cells exposed to environmental chemicals that cause hyperpolarization. Surprisingly, phospholipid changes, but not redox or metabolic alterations, linked the ΔΨm to the epigenome. Sorted hyperpolarized WT and ovarian cancer cells naturally depleted of IF1 also showed phospholipid remodeling, indicating this as an adaptation to mitochondrial hyperpolarization. These data provide a new framework for how mitochondria can impact epigenetics and cellular biology to influence health outcomes, including through chemical exposures and in disease states. We aimed to investigate the DNA methylome of a genetic model of mitochondrial membrane potential hyperpolarization (increased Δψm). DNA from the following treatment groups were isolated from n=4 biologically independent replicates: 1) HEK293T cells containing a doxycycline-inducible dominant negative DNA polymerase gamma (DN-POLG) (WT); 2) the isogenic HEK293T DN-POLG cell line knockout for the gene ATP5IF1 (IF1_KO); 3) the IF1-KO isogenic cell line overexpressing ectopic IF1 (IF1_OE); 4) IF1_KO cells treated with DMSO 1:1000 (IF1_KO_DMSO); 5) IF1_KO cells treated with G6PDi 50 μM, a glucose-6-phosphate dehydrogenase inhibitor (IF1_KO_G6PDi), 6) IF1_KO cells treated with DNP (2,4-dinitrophenol) 25 μM, a mild AAC-dependent uncoupler (IF1_KO_DNP); 7) IF1_KO cells transfected with pcDNA3.1_empty vector (IF1_KO_empty); 8) IF1_KO cells transfected with pcDNA3.1-UCP4(SLC25A27)-NE vector overexpressing UCP4 (IF1_KO_UCP4). DNA was quantified using Nanodrop, and for quality control, samples were re-assessed using Qubit 3.0. DNA methylation was assessed by Infinium Methylation EPIC v1.0.