Modulation of the nuclear epigenome as a new strategy for mitochondrial DNA heteroplasmy shift

MAYORGA, LÍA; MORAES, CARLOS T; ROQUÉ, MARÍA

Congreso; 2021 United mitochondrial disease foundation symposium; 2021

UMDF

Introduction: Mitochondrial disorders (MD) are caused by mutations in nuclear or mitochondrial DNA (mtDNA). mtDNA disease-causing mutations co-exist with wild-type molecules and the percentage of mutated mtDNA, called heteroplasmy, defines the emergence of disease when higher than a threshold. So, reducing the mutant to wild-type proportion or ?heteroplasmy shift? is a valuable strategy for treatment. Gene therapy for nuclear genes has reached the clinic for many diseases. mtDNA editing has been more difficult and although there have been major achievements in the lab, they have not yet attained the clinic. There is evidence that different degrees of mitochondrial stress modify the nuclear epigenome in a specific manner. Particularly, nuclear DNA methylation patterning has been proposed by us as an adaptive response to intense chronic mitochondrial stress. Considering that high-heteroplasmy is associated with intense mitochondrial dysfunction, we hypothesized that these highly stressed cells have this epigenetic mechanism turned on. We propose to disrupt this epigenetic rearrangement to favor the survival of low-heteroplasmy and wild-type cells resulting in an ?epigenetically-mediated cellular heteroplasmy shift?. Methods: We characterized the nuclear DNA methylation of cybrid cells with the m.13513G>A disease-causing mutation at different heteroplasmy levels using Infinium 850k Methylation EPIC array®. Gene enrichment analysis of the differentially methylated regions (DMR) was performed with ENRICHR®. We explored the effect of the general nuclear DNA-methylation inhibitor 5-Aza-2′-deoxycytidine (Aza) on heteroplasmy and mitochondrial function of these cells and the m.5024C>T mice embryonic fibroblasts (MEFs). Heteroplasmy was tested using RT-qPCR. Mitochondrial function was explored with an oxygen flux analyzer (Seahorse®).Results: The nuclear DNA methylation pattern of the m.13513G>A cybrids distinctively separated the samples according to their mutation load. Enrichment analysis of the DMR (wild-type versus mutated) showed significant hypermethylation of apoptotic and tissue differentiation pathways and hypomethylation of proliferation-related routes. When comparing low and high-heteroplamsy cybrids, the latter showed hypermethylation of differentiation and anti-autophagy pathways and hypomethylation of cancer-related genes. Treatment with Aza decreased the m.13513G>A heteroplasmy (~20% reduction, p