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Disease-associated variants in mitochondrial DNA (mtDNA) are frequently heteroplasmic, a state of co-existence with the wild-type genome. Because heteroplasmy correlates with the severity and penetrance of disease, improvement in the ratio between these genomes in favor of the wild-type, known as heteroplasmy shifting, is potentially therapeutic. We evaluated known pathogenic mtDNA variants and identified those with the potential for allele-specific differences in the formation of non-Watson-Crick G-quadruplex (GQ) structures. We found that the Leigh syndrome (LS)-associated m.10191C variant promotes GQ formation within local sequence in vitro. Interaction of this sequence with a small molecule GQ-binding agent, berberine hydrochloride, further increased GQ stability. The GQ formed at m.10191C differentially impeded the processivity of the mitochondrial DNA polymerase gamma (Pol γ) in vitro, providing a potential means to favor replication of the wild-type allele. We tested the potential for shifting heteroplasmy through the cyclical application of two different mitochondria-targeted GQ binding compounds in primary fibroblasts from patients with m.10191T>C heteroplasmy. Treatment induced alternating mtDNA depletion and repopulation and was effective in shifting heteroplasmy towards the non-pathogenic allele. Similar treatment of pathogenic heteroplasmies that do not affect GQ formation did not induce heteroplasmy shift. Following treatment, heteroplasmic m.10191T>C cells had persistent improvements and heteroplasmy and a corresponding increase in maximal mitochondrial oxygen consumption. This study demonstrates the potential for using small-molecule GQ-binding agents to induce genetic and functional improvements in m.10191T>C heteroplasmy.

The higher order organization of eukaryotic and prokaryotic genomes is pivotal in the regulation of gene expression. Specifically, chromatin accessibility in eukaryotes and nucleoid accessibility in bacteria are regulated by a cohort of proteins to alter gene expression in response to diverse physiological conditions. By contrast, prior studies have suggested that the mitochondrial genome (mtDNA) is coated solely by mitochondrial transcription factor A (TFAM), whose increased cellular concentration was proposed to be the major determinant of mtDNA packaging in the mitochondrial nucleoid. Nevertheless, recent analysis of DNase-seq and ATAC-seq experiments from multiple human and mouse samples suggest gradual increase in mtDNA occupancy during the course of embryonic development to generate a conserved footprinting pattern which correlate with sites that have low TFAM occupancy in vivo (ChIP-seq) and tend to adopt G-quadruplex structures. These findings, along with recent identification of mtDNA binding by known modulators of chromatin accessibility such as MOF, suggest that mtDNA higher order organization is generated by cross talk with the nuclear regulatory system, may have a role in mtDNA regulation, and is more complex than once thought.

Abstract:Pathogenic mitochondrial DNA (mtDNA) variants are typically heteroplasmic, with coexistence of variant and wild type genomes. Because heteroplasmy dictates phenotype, the reduction of heteroplasmy is potentially therapeutic. We identified pathogenic variants that increased the potential for formation of non-canonical G-quadruplexes (GQ) within mtDNA.