Intellectual Merit: Eukaryotic organisms simultaneously maintain two genomes, the diploid nuclear genome and the mitochondrial genome, which consists of small circular DNA, packaged in protein-containing structures called nucleoids and distributed throughout the mitochondrial network, with hundreds to thousands of copies per cell. Although the mitochondrion is responsible for efficient energy generation, its genome encodes only 13 proteins, each intimately involved in electron transport that is required for ATP production. All other proteins required for mitochondrial function are encoded on the nuclear genome and imported into the mitochondria. Epigenetic modification of cytosine residues to 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in the nuclear genome is critical for regulation of gene expression and, at least in higher eukaryotes, is essential for normal development and survival. Until recently, a role for cytosine methylation in the mitochondrial genome had not been found. However, identification of a mitochondrial form of the mammalian enzyme responsible for conversion of cytosine to 5mC, DNA methyltransferase 1 (mtDNMT1) has revealed the presence of both 5mC and 5hmC in the mitochondrial genome. Moreover, changing the level of this mitochondrial enzyme impacts mitochondrial transcription in a gene-specific fashion. The current project follows on these observations by seeking to uncover the mechanisms generating these modifications on mtDNA cytosine residues, their role in mitochondrial function, and the evolutionary significance of epigenetic modification of the mitochondrial genome. Successful completion of this project is likely to provide a paradigm shift in the current understanding of mechanisms controlling gene transcription within this organelle. This research is expected to uncover the biochemistry involved in mitochondrial cytosine methylation and hydroxymethylation and whether the process differs significantly from that operating in the nucleus. The conservation of epigenetic modification of mitochondrial DNA will allow an understanding of whether this process affords a metabolic advantage to the organisms in which it operates.
Broader Impacts: This project will offer a unique opportunity to both undergraduate and graduate students to participate in the process of discovery of new and exciting biological knowledge. The project will provide students with experience in critical thinking, data analysis, teamwork, oral and written presentation of research results, and participation in a multidisciplinary approach to problem solving in the laboratory environment. The inclusion of undergraduate students in the project will offer mentoring experience to graduate students and postdoctoral fellows, which is essential to their development as active participants in scientific discovery.