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 first characterized whether changes in mitochondrial function result in epigenetic changes in the nucleus. To this end, our initial work in cell culture models demonstrated that mitochondrial dysfunction results in modulation of nuclear DNA methylation and histone acetylation, which are paralleled by transcriptomic changes. More importantly, we found that these effects are reversible by genetic or pharmacological modulation of the TCA cycle, establishing a causal-effect relationship between mitochondrial dysfunction and modulation of the transcriptome through epigenetics. To define whether these in vitro effects are relevant in vivo, we have utilized 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. Using this model, we have determined 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. In this past year we extended this analysis to the liver DNA methylome 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 did not identify any pathology in the livers of the rotenone treated animals as they aged, but we did find that the animals exposed to rotenone in utero exhibited mitochondrial complex I and II dysfunction as well as impaired antioxidant activities at 12 months of age. 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.
| Lozoya, Oswaldo A; Martinez-Reyes, Inmaculada; Wang, Tianyuan et al. (2018) Mitochondrial nicotinamide adenine dinucleotide reduced (NADH) oxidation links the tricarboxylic acid (TCA) cycle with methionine metabolism and nuclear DNA methylation. PLoS Biol 16:e2005707 |
| Lozoya, Oswaldo A; Santos, Janine H; Woychik, Richard P (2018) A Leveraged Signal-to-Noise Ratio (LSTNR) Method to Extract Differentially Expressed Genes and Multivariate Patterns of Expression From Noisy and Low-Replication RNAseq Data. Front Genet 9:176 |
| MartÃnez-Reyes, Inmaculada; Diebold, Lauren P; Kong, Hyewon et al. (2016) TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions. Mol Cell 61:199-209 |
| Carlin, Danielle J; Rider, Cynthia V; Woychik, Rick et al. (2013) Unraveling the health effects of environmental mixtures: an NIEHS priority. Environ Health Perspect 121:A6-8 |
