Single-nucleotide-resolution sequencing of humanN6-methyldeoxyadenosine reveals strand-asymmetric clusters associated with SSBP1 on the mitochondrial genome

Abstract

N 6-methyldeoxyadenosine (6mA) is a well-characterized DNA modification in prokaryotes but reports on its presence and function in mammals have been controversial. To address this issue, we established the capacity of 6mA-Crosslinking-Exonuclease-sequencing (6mACE-seq) to detect genome-wide 6mA at single-nucleotide-resolution, demonstrating this by accurately mapping 6mA in synthesized DNA and bacterial genomes. Using 6mACE-seq, we generated a human-genome-wide 6mA map that accurately reproduced known 6mA enrichment at active retrotransposons and revealed mitochondrial chromosome-wide 6mA clusters asymmetrically enriched on the heavy-strand. We identified a novel putative 6mA-binding protein in single-stranded DNA-binding protein 1 (SSBP1), a mitochondrial DNA (mtDNA) replication factor known to coat the heavy-strand, linking 6mA with the regulation of mtDNA replication. Finally, we characterized AlkB homologue 1 (ALKBH1) as a mitochondrial protein with 6mA demethylase activity and showed that its loss decreases mitochondrial oxidative phosphorylation. Our results show that 6mA clusters play a previously unappreciated role in regulating human mitochondrial function, despite 6mA being an uncommon DNA modification in the human genome.

Mitochondrial DNA: Epigenetics and environment

“…The distribution of 6mA was found to be uniform throughout mtDNA with no bias toward clusters. The functional implications of 6mA were associated with mtDNA melting and the subsequent recruitment of mtSSB to mtDNA (Koh et al, 2018). The endosymbiotic theory of mitochondrial origin combined with the presence of an~8,000-fold increase in 6mA compared with nDNA indicates the possibility that mtDNA methylation occurs predominantly on adenine.…”

“…DOI 10.1002/em CpG sites; however, non-CpG methylation and adenine methylation within mtDNA have also been observed (Bellizzi et al, 2013;Bianchessi et al, 2016;Blanch et al, 2016;Koh et al, 2018). DOI 10.1002/em CpG sites; however, non-CpG methylation and adenine methylation within mtDNA have also been observed (Bellizzi et al, 2013;Bianchessi et al, 2016;Blanch et al, 2016;Koh et al, 2018).…”

“…To date, most studies aimed at identifying methylation in mtDNA have focused efforts on determining methylation at Environmental and Molecular Mutagenesis. DOI 10.1002/em CpG sites; however, non-CpG methylation and adenine methylation within mtDNA have also been observed (Bellizzi et al, 2013;Bianchessi et al, 2016;Blanch et al, 2016;Koh et al, 2018). The presence of 6mA in the human mitochondrial genome was reported in 2018 using 6mA-crosslinking-exonuclease-sequencing.…”

“…Currently, DNMT1, DNMT3a, DNMT3b, and two TET enzymes (TET1 and TET2) have been identified within the mitochondria using immunofluorescence and western blotting procedures (Chestnut et al, 2011;Shock et al, 2011;Chen et al, 2012;Dzitoyeva et al, 2012;Bellizzi et al, 2013;Wong et al, 2013;reviewed in Maresca et al, 2015). The methyl transferase responsible for adenine methylation in mitochondria has not yet been identified, however, the demethylase ALKBH1 was identified in the mitochondria in 2018 where it plays a role in regulating OXPHOS (Koh et al, 2018). The presence of TET1 and TET2 in the mitochondria indicates that mtDNA demethylation occurs via oxidation-mediated active demethylation (Fig.…”

METTL4 catalyzes m6Am methylation inU2 snRNAto regulate pre-mRNA splicing

“…N 6 -methylation of deoxyadenosine affects the biophysical properties of the modified DNA and also regulates genomic processes (1,2). Likewise, N 6 -methylation of adenosine generates RNA modifications that regulate the expression and metabolism of the host RNA without changing the underlying sequence (3).…”