Long-read sequencing reveals the molecular landscape of mitochondrial DNA 6mA methylation

Abstract Mitochondria, in addition to being central regulators of metabolism and energy production, transmit essential genetic information to each new generation. Mitochondria contain their own small genome (mtDNA) that encodes for proteins of the electron transport chain. Because of the tight coupling between mtDNA levels and potential for energy production, mtDNA replication and transcription must be very carefully and dynamically regulated. Therefore, it has long been speculated that mitochondrial DNA may be subject to epigenetic modifications. Many efforts to assess mtDNA methylation have focused on detecting 5-methylcytosine (5mC), likely influenced by the important role of 5mC in eukaryotic genomes and the availability of experimental techniques for detecting 5mC. However, mtDNA does not have 5mC methylation, so mtDNA epigenetics research has been stunted by technical limitations, until now. Recent advances in nanopore sequencing technology have made high-throughput study of mtDNA methylation marks other than 5mC possible. Therefore, we applied this modern technology to a classic question and again asked: Is mtDNA epigenetically modified? Using the in vivo model system C. elegans, we find that, indeed, mtDNA contains the epigenetic modification N6-methyldeoxyadenosine (6mA). Single molecule sequencing and modification detection revealed that 6mA is highly heterogenous, with a variety of molecules containing very high methylation or very low methylation. These novel and intriguing sequencing results are complemented by biochemical and genetic experiments that further demonstrate mtDNA 6mA is present and may be an effective means of mediating mitochondrial stress. This is a previously unexplored means by which mitochondrial function may be regulated. Biography Lantana Grub is currently a 5 th year PhD Candidate in the Biological Sciences Department at Vanderbilt University. She received her bachelor’s degree at Tuskegee University and then went on to complete her master’s at UIUC where she investigated the genetic regulation of reproduction. In her PhD thesis work Lantana uses her experience at the intersection of cellular biology and genetics to investigate mitochondrial DNA epigenetics using C. elegans as a model.