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 directly impacts the epigenome in the nucleus. To this end we have been using two genetic-based cell culture models of mitochondrial dysfunction. These systems rely on chronic loss of mitochondrial DNA based on ethidium bromide treatment of an osteosarcoma cell line (143B) or on 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 acute 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 allows evaluating progressive effects on the epigenome during the time-dependent loss of mtDNA. The chronic 143B system relies on a comparison of control cells with their full complement of mtDNA (rho+) with cells that are chronically devoid of mtDNA (rho0). We had earlier characterized several biochemical parameters in the 143B and HEK293DN models, including levels of different metabolites (ATP, ROS, acetyl-CoA, NAD and TCA intermediates) in addition to epigenetic markers (enzyme activities and bulk histone changes). The picture that emerged in both systems is that mtDNA depletion, whether acute or chronic, decreases levels of acetyl-CoA, which is associated with diminished HAT activity and histone acetylation. In the HEK293DN model we showed that reconstitution of the TCA cycle by re-establishing electron flow allowed for restoration of citrate levels, and normalization of histone acetylation marks. This part of the work on the HEK293DN model has been published earlier this year (Martinez-Reyes et al., 2016). In the 143B system, our data show that pharmacological supplementation of the mitochondrial acetyl-CoA pool is associated with restoration of HAT activity in the rho0 cells while inhibition of the mitochondrial metabolism of acetyl-CoA is associated with a decrease in HAT activity. We also identified increases in DNMT activity and changes in DNA methylation patterns in the rho0 cells. Work on the 143B model is being prepared for submission. Overall the data obtained indicate that loss of mitochondrial function leads to altered activity of some of the enzymes involved in regulating the epigenome, which has the potential to cause changes in epigenetic marks in the nuclear DNA. In this past fiscal year we have focused on understanding whether the histone acetylation changes observed in these systems affect gene expression. We have also embarked on locus-specific analysis of DNA methylation patterns in both cell culture models. To this end, we performed ChIP-seq with the altered histone marks H3K9ac and K3K27ac, RNAseq and DNA methylation arrays in both cell culture models. Collectively we have identified about 2,000-4000 genes that are differentially expressed by loss of mitochondrial function in each cell culture model, many of which parallel changes in either histone mark, DNA methylation or both. It is notable that many changes are common to both models. For instance, in both models one of the first affected metabolic pathways is associated with one carbon metabolism. We are currently working on an in-depth analysis of the data in each model with the goal of integrating the biochemical, metabolic, epigenetic and gene expression data. Finally, we have been exposing the viable yellow agouti mouse (Avy) to rotenone, a mitochondrial complex I inhibitor, through the diet. Rotenone is a 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 since we previously demonstrated that they carry a mutation that makes it possible to determine the epigenetic status of the agouti gene by examining the coat color of the mice. For example, 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 animals are completely yellow. Histone modifications have also been implicated in regulating expression of this allele. Our hypothesis is that animals treated with rotenone will result in a change in the percentage of pseudoagouti offspring as compared to animals fed the control diet.

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3
Fiscal Year
2016
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U.S. National Inst of Environ Hlth Scis
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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