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. Alterations of the epigenome can change gene expression. We are specifically interested in studying whether environmental agents that target the mitochondria may alter the epigenome. 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, whether changes to the epigenome are a consequence of mitochondrial dysfunction still needs to be established. In order to determine whether environmental agents that target mitochondria also impart their effects through alteration of the epigenome, the first step was to characterize whether mitochondria regulate epigenetic reactions. 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 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, causing 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. Because mitochondria also generate ROS, which are believed to be important signaling molecules, in the 143B model we included a derivative in which a mutated mtDNA was re-introduced into the rho0 cells. The cells carrying this mutated mtDNA (rho-) are not able to generate mitochondrial ATP, but they can still generate ROS at mitochondrial complex III. Therefore, the 143B system relies on 3 different cells that have chronic mtDNA-associated mitochondrial dysfunction: rho+, control cells with their full complement of mtDNA, rho-, cells that carry the mutated mitochondrial genome, and rho0, which are cells that have no mtDNA. With the HEK293DN system we are analyzing 4 time points of acute mitochondrial dysfunction (cells at day 0 are essentially the rho+ controls while those at day 3, 6 and 9 reflect a progressive loss of mtDNA to make them rho0). In this past fiscal year, together with our collaborators we have characterized several biochemical, epigenetic and gene expression parameters of these cells, including levels of different metabolites (ATP, ROS, acetyl-CoA, NAD and TCA intermediates). In both systems we have shown that HAT activity and histone acetylation are diminished upon loss of mitochondrial function, both of which are associated with decreased levels of acetyl-CoA. In the 143B system, we were able to demonstrate that pharmacological supplementation of the mitochondrial acetyl-CoA pool restored HAT activity in the rho0 cells. In the HEK293 model we showed that reconstitution of the TCA cycle by itself allowed for restoration of metabolite (citrate) levels, and, consequently, normalization of histone acetylation levels. DNMT activity is significantly increased in the 143B cells, and we are currently studying whether this reflects changes in SAM levels and in the acetylation of DNMTs shown to be associated with activation of these proteins. Overall the data obtained indicate that loss of mitochondrial function leads to altered activity of some of the enzymes involved in regulating the epigenome. We also studying gene expression using RNAseq in both cell culture models. Additionally, we performed experiments using DNA methylation arrays in the 143B cell system. Collectively we have identified about 1,000 genes that are differentially expressed by loss of mitochondrial function in each cell culture model. We have also identified about 400 loci that are differentially methylated in the cells, many of which are also associated with differentially expressed genes. We are currently working on an in-depth analysis of the data to integrate the biochemical, metabolic, epigenetic and gene expression the data. Given our interest in the expression of repetitive elements (REs), we followed up these experiments by performing quantitative real-time PCR (qRT-PCR) in the RNA samples used for RNAseq. We found that transcription of different types of repeats, including ERVs (endogenous retroviruses) and satellites (minor and major) are modulated by loss of mitochondrial function in the cells; while the pattern is similar in both cell models, the progressive loss of mtDNA in the HEK293DN system revealed that expression of some REs do change between days 3 and 6 but return to basal expression on day 9. We are currently analyzing RE expression in these cells genome-wide using a bioinformatics pipeline recently developed by us (see other project).