Mitochondrial membrane hyperpolarization modulates nuclear DNA methylation and gene expression through phospholipid remodeling [EPICv2]

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 chemical exposure model of mitochondrial membrane potential hyperpolarization (increased Δψm). DNA from the following treatment groups were isolated from n=3 biologically independent replicates for chemical exposures, and n=4 to investigate the role of PEMT in DNA methylation in IF1-KO cells: 1) HEK293T cells containing a doxycycline-inducible dominant negative DNA polymerase gamma (DN-POLG) treated with DMSO (1:1000 v/v) for 10 days (WT); 2) WT cells treated with telmisartan 2 μM for 10 days (TMS); 3) WT cells treated with annatto 10 μM for 10 days (ANTO); 4) the isogenic HEK293T DN-POLG cell line knockout for the gene ATP5IF1 (IF1_KO); 5) IF1_KO cells transfected with pcDNA3.1-PEMT-flag (IF1_KO_PEMT_OE). 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 v2.0.