(D) The list of genes whose expression was reversed by DM–KG supplementation was used to retrieve ENCODE-based ChIP-seq information

(D) The list of genes whose expression was reversed by DM–KG supplementation was used to retrieve ENCODE-based ChIP-seq information. chronic model of mtDNA depletion using biochemical, pharmacological, genomics, and genetic assays. We show that histones are primarily hypoacetylated in both models. We link these effects to decreased histone acetyltransferase activity unrelated to changes in ATP citrate lyase, acetyl coenzyme A synthetase 2, or pyruvate dehydrogenase activities, which can be reversibly modulated by altering the mitochondrial pool of acetyl-coenzyme A. Also, we decided that the accompanying changes in histone acetylation regulate locus-specific gene expression and physiological outcomes, including the production of prostaglandins. These results may be relevant to the pathophysiology of mtDNA depletion syndromes and to understanding the effects of environmental brokers that lead to physical or functional mtDNA loss. Introduction The role of mitochondria in cell biology and organismal health has expanded dramatically in the last decade. From a focus originally on bioenergetics, it is now recognized that mitochondria broadly affect cell physiology in diverse ways. For instance, mitochondria interact with other organelles, such as the endoplasmic reticulum, by close contacts or through the generation of small vesicular carriers, which allows the transport and exchange of lipids, proteins and other small molecules such as calcium (Csordas et al, 2010; Sugiura et al, 2014). Mitochondria are also important players in signaling via reactive oxygen species and other metabolites that impart posttranslational modifications to many proteins, including transcription factors (Chandel, 2015). Most recently, we and others have shown that mitochondria influence the epigenome (Smiraglia et al, 2008; Martinez-Reyes et al, 2016; Liu et al, 2017; Lozoya et al, 2018), yet full mechanistic insights and outcomes of this relationship are still lacking. The relevance of better understanding the impact of mitochondrial function in epigenetics cannot be understated, given the many ways mitochondrial output Rabbit Polyclonal to CRHR2 has been documented to influence gene expression (Durieux et al, 2011; Gomes et al, 2013; Picard et al, 2014). Novel links between mitochondrial function and epigenetics continue to be unveiled and mechanistic understanding of this relationship is emerging. Tricarboxylic acid (TCA) cycle intermediates such as acetyl-coenzyme A (CoA) and -ketoglutarate (-KG) are substrates or cofactors for enzymes that alter the epigenome, AG-494 such as the histone acetyltransferases (HATs) and the demethylases (Smiraglia et al, 2008; Wallace, 2009; Minocherhomji et al, 2012; Meyer et al, 2013). Thus, mitochondrial dysfunction AG-494 could, for example, alter the nuclear epigenome through reduced TCA flux. In fact, we first reported that progressive loss of mitochondrial DNA (mtDNA) and the associated changes in TCA cycle output, by ectopically expressing a dominant-negative mtDNA polymerase (DN-POLG), led to histone hypoacetylation in the nucleus (Martinez-Reyes et al, 2016). Using this same cell system, we also demonstrated a direct link between loss of mtDNA and DNA hypermethylation, which we showed was driven by modulation of methionine salvage and polyamine synthesis, both sensitive to changes in TCA cycle flux. We showed that DNA methylation changes occurred predominantly at the promoters of genes that responded to mitochondrial dysfunction, increased progressively over the course of mtDNA depletion, and could be reversed by maintaining NADH oxidation in the mitochondria, even in the context of complete mtDNA loss (Lozoya et al, 2018). Although our initial work using the DN-POLG system revealed hypoacetylation of histones in the nucleus as a function of progressive mtDNA AG-494 loss (Martinez-Reyes et al, 2016), mechanistic details associated with these effects were not interrogated. Importantly, it remains unknown whether those histone changes are sufficient to alter gene expression and impact functional outcomes. In this work, we used the DN-POLG cells together with a model of chronic mtDNA depletion to establish causeCeffect relationships. Using several biochemical, transcriptomics, epigenomics, genetics, and pharmacological approaches, we found that histone acetylation loss or gain occurred predominantly on the promoters of differentially expressed genes (DEGs), that even chronic transcriptomic changes were amenable to inducible epigenetic manipulation by supplementation with TCA cycle intermediates, and that altered histone acetylation status largely preceded gene expression remodeling. Results Changes in H3K9ac levels by progressive mtDNA depletion occurs early in the course of mtDNA.