REDOX-regulation of mitochondrial transcription by mitochondrial DNA topoisomerase I

• Applicant: Professor Dr. Friedrich Boege
• Subject Area: Public Health, Healthcare Research, Social and Occupational Medicine
• Term: from 2015 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 273662364

Mitochondrial topoisomerase I (TOP1MT) removes negative supercoils from mitochondrial DNA (mtDNA) and has been identified as mitochondrial disease candidate gene in humans. However, TOP1MT-/- mice do not exhibit overt mitochondrial dysfunction despite a marked increase in negative mtDNA supercoiling. TOP1MT is thus indicated a regulator of negative mtDNA supercoiling, but the role of that function in mitochondrial homeostasis remains unclear.We have demonstrated that TOP1MT acts as direct negative regulator of mtDNA-transcription. This function can be inhibited in isolated mitochondria by stimulating endogenous ROS production. Upon expression of oxidation-resistant TOP1MT mutants the effect is abolished. Our observations support the model of a double negative loop, in which TOP1MT negatively regulates mtDNA-transcription and is in turn negatively regulated by oxidation. We aim to provide causal proof of the surmised regulatory loop (objective 1), characterise the components and molecular interactions involved (objective 2), and address the role of this mechanism in oxidative stress adaptation and age-associated mitochondrial dysfunction (objective 3). For that pupose, we will demonstrate in isolated mitochondria (i) that REDOX-modulation of mtDNA transcription is lost upon depletion or deletion of TOP1MT and not complemented by inactive or oxidation-resistant TOP1MT mutants, and (ii) that acute increases in mitochondrial ROS diminish the recruitment of TOP1MT to heavy and light strand promoters, decrease nucleoid association of TOP1MT and increase negative mtDNA supercoiling, and that these responses are complemented in TOP1MT0/0 cells by wild type TOP1MT but not inactive or oxidation-resistant TOP1MT mutants. We will then move on to identify TOP1MT-dependent mechanisms in mitochondrial stress responses by subjecting the above complementation models to acute and/or chronic intermittent UVA exposure and monitor the UVA-impact on ROS levels, mtDNA-transcription, mtDNA copy number, nuclear mito-biogenesis, the balance of nuclear and mtDNA-encoded respiratory complexes, and respiratory function. We will finally validate the physiological relevance of TOP1MT-dependent adaptation processes by addressing their state in primary fibroblasts isolated form sun-exposed and sun-protected skin of old and young human donors. Ultimately, we aim at validating our results by inducible TOP1MT knock out in mice, but these experiments are outside the purview of this application.We expect to uncover a mechanism that provides local REDOX-regulation of mtDNA transcription. We expect to elucidate whether this mechanism adjusts respiratory capacity to the REDOX state inside the mitochondrial matrix and serves to fine tune mito-nuclear communication in mito-biogenetic stress responses. We will possibly confirm that disruption of the surmised regulatory mechanism is involved in UVA-induced and age-related mitochondrial dysfunction in human dermal fibroblasts.

• DFG Programme: Research Grants