Cytosine methylation of mitochondrial DNA (mtDNA) was first described several decades ago, but neither the mechanism generating this modification nor its functional significance was known. Because mitochondrial dysfunction is a hallmark characteristic of numerous human diseases, including neurological and cardiovascular disease, aging and cancer, this dissertation addressed whether epigenetic modification of mtDNA regulates mitochondrial function. We show that mtDNA contains not only 5-methylcytosine (5mC), but also 5-hydroxymethylcytosine (5hmC), suggesting that previous reports likely underestimated the degree of epigenetic modification within the mitochondrial genome. We questioned how these modifications were generated by looking for mitochondrial isoforms of the nuclear-encoded DNA methyltransferases. We found that an isoform of the most abundant mammalian methyltransferase, DNA methyltransferase 1 (DNMT1) translocates to mitochondria, driven by an in-frame mitochondrial targeting sequence (MTS) located upstream of the nuclear DNMT1 translational start site. This MTS is highly conserved across mammalian species, and directs a heterologous protein to the mitochondria. To investigate the function of mitochondrial DNMT1 (mtDNMT1), we created a cell line that carries a tandem-affinity purification (TAP) tag at the C-terminus of a single endogenous human DNMT1 allele. Using the DNMT1-TAP cell line, we showed that mtDNMT1 specifically binds mtDNA in a manner that is proportional to CpG density, proving its presence in the mitochondrial matrix. mtDNMT1 exhibits CpG-specific methyltransferase activity in vitro that is resistant to trypsin-treatment of intact mitochondria, but moderately susceptible to pharmacologic inhibition by the nucleoside analog 5-aza-2’-deoxycytidine (5-aza-dC). NRF1 and PGC1α, transcription factors that activate nuclear-encoded mitochondrial proteins in response to oxidative stress, were observed to up-regulate expression of mtDNMT1. Loss of p53, a tumor suppressor gene known to help control mitochondrial metabolism, also results in a striking increase in mtDNMT1 expression, and this up-regulation of mtDNMT1 appears to modify mitochondrial transcription in a gene-specific fashion. Our data suggests roles for mtDNMT1 in both the establishment and maintenance of cytosine methylation (from which 5hmC is presumably derived) and in the regulation of mitochondrial transcription. We propose that the enzymes responsible for epigenetic modification of mtDNA have potential as therapeutic targets, with relevance to a broad spectrum of human disorders.
| Identifer | oai:union.ndltd.org:vcu.edu/oai:scholarscompass.vcu.edu:etd-1270 |
| Date | 01 January 2011 |
| Creators | Shock, Lisa |
| Publisher | VCU Scholars Compass |
| Source Sets | Virginia Commonwealth University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
| Rights | © The Author |
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