Regulation of mitochondrial DNA packaging and gene expression

About This Grant

PROJECT SUMMARY/ABSTRACT Our lab aims to understand the fundamental mechanisms of mitochondrial gene regulation to advance our understanding of metabolic adaptation and mitochondrial-associated diseases. Over the next five years, we will develop and apply cutting-edge sequencing technologies to reveal how cells regulate their energy production through mitochondrial DNA (mtDNA) packaging, expression, and replication. Mitochondria generate ATP through oxidative phosphorylation (OXPHOS) and contain their own genome, which encodes 13 core OXPHOS subunits, exists in hundreds to thousands of copies per cell, and is packaged into “nuceloids” by TFAM. The high ploidy of mtDNA has posed challenges to previous functional genomics techniques that rely on Illumina short- read sequencing, as population averaging effects obscure any relevant information. We previously developed mtFiber-seq, an approach that measures mtDNA accessibility at single-genome resolution. We discovered that most nucleoids are inaccessible but can change state during OXPHOS dysfunction or cellular generation. Many questions remain regarding how mitochondrial gene expression is regulated in response to perturbations to rewire metabolism and maintain homeostasis. The high ploidy of mtDNA allows for mechanisms unique from those in the nucleus. Using a variety of perturbations and systems, we will determine how gene expression is regulated on both short and long time scales. TFAM acts as both a repressor and activator through poorly understood mechanisms. We showed that varying TFAM levels can shift the active nucleoid population and developed an in vitro version of mtFiber-seq with reconstituted nucleoids which allowed us to understand new binding properties of TFAM and recapitulated cell culture observations. How TFAM behavior is regulated and how nucleoids shift between inactive and active states is not understood. This is likely done through TFAM post- translational modifications, but these modified PTMs remain understudied. We expect to clarify how these changes change the propensity of TFAM to repress and activate transcription and replication. We are developing an expanded mtFiber-seq approach that will allow us to simultaneously map DNA accessibility and the positions of specifically modified TFAM on single genomes. There are also many outstanding questions regarding mtDNA replication, and this process is critical for maintaining a healthy population of mtDNA. The signals controlling replication are not understood. Using mtFiber-seq with metabolic labeling, we will study differences in replicating and non-replicating populations to understand how these signals control replication and copy number. Overall, we seek to better understand the mechanisms of nucleoid regulation, and mitochondrial gene expression more broadly, by applying state-of-the-art technologies that are allowing for previously impossible levels of resolution. This work will provide fundamental insights into how cells maintain energy homeostasis and adapt to changing metabolic demands, with implications for understanding mitochondrial diseases and developing therapeutic strategies.