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, in turn changing 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 affect mitochondrial function may impart their effects through altered regulation of gene expression. Whether and how mitochondrial metabolism affects the epigenome is currently unknown. Existing evidence indicates that mitochondrial dysfunction can lead to altered DNA methylation patterns in nuclear DNA and hypermethylation of histones. Mitochondrial impairment can also affect gene expression. However, whether the changes in gene expression observed are caused by, or a consequence of, mitochondrial dysfunction and whether they result from alterations in the epigenome remain to be elucidated. In order to determine whether environmental agents that target mitochondria also impart their effects through alteration of the epigenome and gene expression, the first step was to characterize whether mitochondria regulate epigenetic reactions. To this end we have been using a cell culture model of mitochondrial dysfunction. This system relies on ethidium bromide-induced loss of mitochondrial DNA (mtDNA) in an osteosarcoma cell line (143B). These cells, without any mtDNA, are termed rho0 and can survive under cell culture conditions that support ATP generation through glycolysis. Because mitochondria also generate ROS that are believed to be important signaling molecules, our model also includes a cell line in which a mutated mtDNA was re-introduced into rho0 cells. The cells carrying this mutated mtDNA (rho-) are also unable to generate mitochondrial ATP and are thus glycolytic. However, they can still generate ROS at mitochondrial complex III. Therefore, our system relies on 3 different cells that are in the isogenic 143B background: rho+ are control cells with their full complement of mtDNA, rho- carry the mutated mitochondrial genome, rho0 that have no mtDNA. In this past fiscal year we have characterized several parameters of these cells, including their mtDNA content, levels of ATP generation, free radical production and their NAD+/NADH ratio (which informs about the redox status of the cells). We have also interrogated the activity of several enzymes that are involved in regulation of the epigenome such as HATs, HDACs, Dnmts and TETs. Our analyses have shown that HAT activity is diminished upon loss of mitochondrial function, and that HDAC and Dnmt activity is significantly increased. We did not find any changes in TET activity. Accordingly, we also found decreased levels of acetylated histones as well as increased levels of methylated DNA. We did not identify changes in the amounts of hydroxymethylated DNA in the cells. Overall these data indicate that loss of mitochondrial function leads to altered function of some of the enzymes involved in regulating the epigenome, which has the potential to cause changes in epigenetic marks in the nuclear DNA. To determine whether these changes affect gene expression, we recently performed DNA microarrays and have identified about 2,000 genes that are differentially expressed by loss of mitochondrial function. We are currently working with staff from the bioinformatics core to analyze the data. Preliminary analysis indicates that protein coding genes, long non-coding RNAs (lincRNAs) and some retroviral-like elements are differentially expressed. 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 same RNA samples used for the microarrays. We found that transcription of both ERVs (endogenous retroviruses) and satellites (minor and major) are upregulated by loss of mitochondrial function. Whether these REs can play a role in the biological outcome of mitochondrial dysfunction is part of current work with a different model system (see below). While the data gathered thus far have established a link between loss of mitochondrial function and the epigenetic regulation of gene expression, the mechanism linking the two has yet to be established. Thus, we are currently performing experiments that are measuring the levels of mitochondrial metabolites (those involved in the activity of the enzymes regulating the epigenome) in the cells by NMR. We are also performing DNA methylation arrays to interrogate whether there is differential methylation of the genome in response to changes in mitochondrial function. As a complementary approach, we have been working in collaboration with Dr. Navdeep Chandel at Northwestern University using a different model system of mitochondrial dysfunction. In this case mtDNA and mitochondrial function are lost progressively over a period of days. This is accomplished by using a transgene model of a dominant-negative mtDNA polymerase, which is under the control of doxycycline. Induction of the transgene leads to the progressive loss of mtDNA over the course of 9 days. The advantage of this system is that it more closely recapitulates mitochondrial dysfunction induced by environmental agents or in disease states where total loss of mtDNA and mitochondrial function are not normally detected. Under these conditions partial and chronic changes in mitochondrial function are observed. Because of the progression of the events, this system will also allow us to more accurately track the sequence of events initiating with mitochondrial dysfunction leading to any epigenetic changes. Dr. Chandels group is performing all the biochemical characterization of the system, including metabolic profiling and non-biased analysis of histone modifications through mass spectrometry. Our group is performing RNA-seq, ChIP-seq, DNA methylation and other analyses to examine gene expression and changes in the epigenome. We have recently received the RNA-seq data and we are currently working with staff from the bioinformatics core to analyze the results.
| 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 |
