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 we have been using two genetic-based cell culture models of mitochondrial dysfunction. These systems rely on the chronic loss of mitochondrial DNA based on ethidium bromide treatment of an osteosarcoma cell line (143B) or on the acute loss of mtDNA in HEK293 cells. In the latter, which we call the HEK293DN system, a dominant negative mitochondrial polymerase (polG) is ectopically expressed in an inducible fashion, which results in a progressive loss of mtDNA over a period of 9 days. The cells without mtDNA are termed rho0 and can survive under cell culture conditions that support ATP generation through glycolysis. With the inducible system, we analyzed 4 time points of acute mitochondrial dysfunction (days 0, 3, 6 and 9). The cells are effectively rho0 at day 9. This allowed us to evaluate progressive effects on the epigenome during the time-dependent loss of mtDNA. The chronic 143B system relies on a comparison of control cells containing a full complement of mtDNA (rho+) with cells that are chronically devoid of mtDNA (rho0). We have also used 3 cell lines that harbor mtDNA mutations in either complex III, IV or V. In general, these cell systems switched their metabolism to glutamine, rather than glucose, dependence. Over the past fiscal year, we focused our work on better understanding how changes in mitochondrial function impact the transcriptome and epigenome in the HEK293DN and 143B cell culture systems. Using several NextGen and genomic omics approaches (RNA-seq, ChIP-seq of histone marks, methylation bead arrays and metabolomics), we found that mitochondrial dysfunction results in specific changes to the transcriptome and epigenome that are associated with the specific type of mitochondrial dysfunction. Mechanistically, we found that in the HEK293DN model that loss of mtDNA and the TCA cycle leads to changes in the salvage pathways used to maintain methionine levels. These effects impact the methionine cycle and increases the overall levels of the methyl donor S-adenosyl-methionine, which in turn, resulted in higher DNA methyltransferase activity and hypermethylation of the nuclear genome. In addition, we also found that cells depleted of mtDNA (both under acute and chronic conditions) have hypoacetylated of histone H3, which suggests that mitochondrial-derived acetyl-CoA is necessary and required to maintain this type of histone acetylation in the nucleus. We also found that manipulating the levels of mitochondrial acetyl-CoA results in alterations in bulk histone acetyltransferase (HAT) activity, which correlates with the levels of acetylation of histone H3. Conversely, in cells carrying mutations on the mitochondrial genome, the histone acetylation landscape is instead hyperacetylated and the nuclear genome is hypomethylated, effects that we linked to glutamine metabolism. It appears that the metabolism of glutamine channels glucose into the pentose phosphate pathway, which increases: the pools of NADPH/NADP used for redox reactions, cytosolic acetyl-CoA for histone acetylation and lipid synthesis, and glutathione production. We predict that this channeling limits the amounts of glucose necessary for serine biosynthesis and the de novo methionine cycle. We are currently conducting RNA-seq analyses of these cells to determine the transcriptomic changes associated with these changes in metabolism and the epigenome. Finally, we are finalizing our in vivo work on the analysis of the viable yellow agouti mouse (Avy) treated with rotenone. Rotenone is a well-studied mitochondrial complex I inhibitor pesticide known to contaminate the environment that has been linked to the induction of Parkinsons disease, both in humans and in animal models. The Avy animals have been used in many studies as an epigenetic reporter; they carry a mutation in the promoter of agouti that makes it possible to examine the epigenetic status of the agouti gene by examining the coat color of the mice. 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 totally yellow animals. This result suggested that mitochondrial dysfunction caused by a complex I inhibitor during development has the potential to change the epigenetic status of the nuclear gene, specifically at the agouti gene. We extended this analysis using whole genome bisulfide sequencing and found that many other genes in addition to agouti, including imprinted genes, are differentially methylated in the exposed animals (1,000). Additionally, we found that by 12 months of age, only the animals exposed to rotenone in utero exhibited mitochondrial complex I and II dysfunction as well as impaired antioxidant activities. The latter data reveal that early developmental exposure to a mitochondrial toxicant can lead to phenotypic changes in mitochondrial function that become apparent only much later in life.

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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
Martínez-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