Role of environmental agents targeting mitochondria in epigenetic regulation of nuclear gene expression

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The purpose of this project is to determine the role that mitochondria have in the regulation of enzymes responsible for maintenance of the epigenome. The role of mitochondria in generating ATP and reactive oxygen species (ROS) is well recognized. However, less appreciated is the fact that these organelles are also involved in various biochemical pathways in the cells that give rise to a diverse range of metabolic products, including co-factors of proteins that epigenetically regulate the nuclear genome. For instance, mitochondria participate in the metabolism of S-adenosyl-methionine (SAM), which is the substrate used by DNA and histone methyltransferases to methylate CpG dinucleotides and histones, respectively, in the nucleus. Likewise, the production of acetyl-CoA and NAD+ occurs primarily in mitochondria, and these are co-factors of histone acetyltransferases (HATs) and deacetylases (HDACs), respectively, to modify histones. ATP is used by various protein kinases to phosphorylate substrates, including histones, which can change the composition of nucleosomes. Alpha-ketoglutarate, a metabolite from the tricarboxylic acid (TCA) cycle is a co-factor for the Ten-Eleven Translocation (TET) family of hydroxylases involved in hydroxyl-methylation of cytosines. Finally, mitochondrial-generated ROS can inhibit the jumonji (Jmj) demethylases leading to global histone hypermethylation. As modulation of the epigenome regulates gene expression, it follows that environmental agents that target the mitochondria may alter the regulation of gene expression by changing mitochondrial metabolism. Existing evidence indicates that mitochondrial dysfunction can lead to altered DNA methylation patterns in nuclear DNA and hyper-methylation of histones. Mitochondrial impairment can also affect gene expression. However, it still needs to be established whether epigenetic-driven changes in gene expression in the nucleus are a consequence of environmentally-mediated changes in mitochondrial function. In order to determine whether environmental agents that target mitochondria also impart their effects through alteration of the epigenome and gene expression, we exposed a mouse model to a pesticide, rotenone, which is a well-studied mitochondrial toxicant. Specifically, we used the viable yellow agouti mouse (Avy), a powerful epigenetics model that reports on the DNA methylation status of a mutant agouti locus based on the coat color of the animals. When the promoter in the Avy allele is methylated, the animals have a normal agouti coat color (called pseudoagouti). On the other extreme, when the promoter is unmethylated, the coat color of the animals is completely yellow. We found that the offspring of dams exposed to rotenone throughout pregnancy and lactation have an increased frequency of yellow animals, which is indicative of demethylation of the locus. We went ahead and extended this analysis to the liver using whole genome bisulfide sequencing, and found that perinatal rotenone exposure altered the DNA methylation status of thousands of loci throughout the life of the animals. In parallel, we found that gene expression was also altered by this exposure, with some genes having altered expression 6, 12 and even 18 months after rotenone exposure ceased. By employing several approaches, we have been able to establish a strong correlation between differential methylation and changes in gene expression. Finally, we found mitochondrial complex I and II dysfunction as well as impaired antioxidant activities in animals at 12 months of age although we did not identify any pathology in the livers. Collectively, these results show that developmental mitochondrial dysfunction results in changes in the nuclear methylome, including the Avy locus, in a way that remodels the normal aging DNA methylome of the liver. These changes are not only accompanied by altered gene expression profiles but also facilitate mitochondrial dysfunction later in life. These data raise fundamental questions about the long-term impact of changes in mitochondrial metabolism to health and disease, including those induced by environmental exposures. Having confirmed that mitochondrial dysfunction can impact the epigenome in vivo, we have gone back to some in vitro cell culture models in which specific mutations of mitochondrial genes have been engineered. As these mutations affect distinct biochemical processes within the organelle, we aim to better define the extent to which these specific types of mitochondrial dysfunction impact the epigenome.