Abstract
Cytosine methylation is an epigenetic modification of DNA catalyzed by DNA methyltransferases. Cytosine methylation of mitochondrial DNA (mtDNA) is believed to have relative underrepresentation; however, possible tissue and cell differences in mtDNA methylation and relationships to neurodegenerative disease have not been examined. We show by immunoblotting that DNA methyltransferase 3A (Dnmt3a) isoform is present in pure mitochondria of adult mouse CNS, skeletal muscle, and testes, and adult human cerebral cortex. Dnmt1 was not detected in adult mouse CNS or skeletal muscle mitochondria but appeared bound to the outer mitochondrial membrane. Immunofluorescence confirmed the mitochondrial localization of Dnmt3a and showed 5-methylcytosine (5mC) immunoreactivity in mitochondria of neurons and skeletal muscle myofibers. DNA pyrosequencing of two loci (D-loop and 16S rRNA gene) and twelve cytosine-phosphate-guanine (CpG) sites in mtDNA directly showed a tissue differential presence of 5mC. Because mitochondria have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but the disease mechanisms are uncertain, we evaluated mitochondrial Dnmt3a and 5mC levels in human superoxide dismutase-1 (SOD1) transgenic mouse models of ALS. Mitochondrial Dnmt3a protein levels were reduced significantly in skeletal muscle and spinal cord at presymptomatic or early disease. Immunofluorescence showed that 5mC immunoreactivity was present in mitochondria of neurons and skeletal myofibers, and 5mC immunoreactivity became aggregated in motor neurons of ALS mice. DNA pyrosequencing revealed significant abnormalities in 16S rRNA gene methylation in ALS mice. Immunofluorescence showed that 5mC immunoreactivity can be sequestered into autophagosomes and that mitophagy was increased and mitochondrial content was decreased in skeletal muscle in ALS mice. This study reveals a tissue-preferential mitochondrial localization of Dnmt3a and presence of cytosine methylation in mtDNA of nervous tissue and skeletal muscle and demonstrates that mtDNA methylation patterns and mitochondrial Dnmt3a levels are abnormal in skeletal muscle and spinal cord of presymptomatic ALS mice, and these abnormalities occur in parallel with loss of myofiber mitochondria.
Highlights
Methylation of DNA on carbon five of cytosine (5methylcytosine, 5mC) in cytosine-phosphate-guanine (CpG) dinucleotides is an epigenetic modification of DNA used by cells to regulate nuclear gene expression (Jones and Takai, 2001; Bird, 2002; Brenner and Fuks, 2006)
We demonstrate that DNA methyltransferase 3A (Dnmt3a) and 5mC are in mitochondria of adult mouse and human tissue types that are mostly excitable tissues and that tissue Dnmt3a levels are reduced and 5mC immunoreactivity accumulates in skeletal muscle myofibers and spinal cord motor neurons in mouse models of amyotrophic lateral sclerosis (ALS)
Dnmt3a had a robust presence in skeletal muscle mitochondrial fractions (Figure 2A) and was detected with several different antibodies to Dnmt3a that recognize N-terminal, central, and C-terminal domains of the protein (Table 1)
Summary
Methylation of DNA on carbon five of cytosine (5methylcytosine, 5mC) in cytosine-phosphate-guanine (CpG) dinucleotides is an epigenetic modification of DNA used by cells to regulate nuclear gene expression (Jones and Takai, 2001; Bird, 2002; Brenner and Fuks, 2006). Cytosine methylation signals through 5mC-binding proteins to remodel chromatin and downregulate gene expression. Cytosine methylation is mediated by a family of DNA methyltransferase (Dnmt) enzymes (Cheng, 1995). Dnmt ( called tRNA methyltransferase-1) transfers methyl groups to RNA instead of DNA (Schaefer and Lyko, 2010; Motorin and Helm, 2011). DNA methyltransferase 3A (Dnmt3a) and Dnmt3b function as de novo methyltransferases because they methylate hemimethylated DNA and completely unmethylated DNA (Okano et al, 1999; Xie et al, 1999). Nuclear DNA methylation attracts attention because it has been implicated in normal cell and tissue development and differentiation (Geiman and Muegge, 2010) and in human diseases, including cancer
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