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Single-nucleotide-resolution sequencing of human N6-methyldeoxyadenosine reveals strand-asymmetric clusters associated with SSBP1 in the mitochondrial genome

ABSTRACT: N6-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 homolog 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.

ORGANISM(S): Escherichia coli Salmonella enterica subsp. enterica serovar Typhimurium Homo sapiens

PROVIDER: GSE119094 | GEO | 2018/11/09

REPOSITORIES: GEO

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Project description:BACKGROUND: Mitochondrial dysfunction is a key factor in the development of atherogenesis. Methyltransferase-like protein 4 (METTL4) mediates N6-deoxyadenosine (6mA) methylation of mammalian mitochondrial DNA (mtDNA). However, the role of METTL4-mediated mitoepigenetic regulation in atherosclerosis is still unknown. This study aims to investigate the potential involvement of METTL4 in atherosclerosis, explore the underlying mechanism, and develop targeted strategies for treating atherosclerosis. METHODS: Expression levels of mtDNA 6mA and METTL4 were determined in atherosclerotic lesions. We explored the mechanism of METTL4 involvement in atherosclerosis using Mettl4Mac-KO-Apoe-/- and Mettl4MUT-Apoe-/- mice and cell models, as well as bone marrow transplantation. Natural compound libraries were screened to identify potent METTL4 antagonists. In addition, bioinspired proteolysis-targeting (PROTAC) technology targeting macrophages within plaques was used to increase the efficacy of the METTL4 antagonist. RESULTS: The expression levels of mtDNA 6mA and METTL4 were significantly increased in plaque macrophages. Mettl4Mac-KO-Apoe-/- mice displayed suppressed mtDNA 6mA levels and atherosclerotic progression, which were reversed by METTL4 restoration via bone marrow transplantation (n = 6). Mechanistically, elevated METTL4 expression reduces MT-ATP6 expression by suppressing its transcription, thereby impairing the activity of mitochondrial respiration chain complex V. This disruption leads to the accumulation of excess protons in the mitochondrial intermembrane space, causing mitochondrial dysfunction. Consequently, mtDNA is released into the cytoplasm, ultimately triggering inflammasome activation. All results were reversed by the mutation in the METTL4 methyltransferase active site. Mettl4MUT-Apoe-/- mice showed suppressed mtDNA 6mA levels and atherosclerotic progression, and repaired mitochondrial function of macrophage, which were reversed by METTL4 restoration via bone marrow transplantation (n = 6). Pemetrexed (PEM) was identified as the first METTL4 antagonist to effectively alleviate atherosclerotic progression. Furthermore, we generated a proteolysis-targeting chimera drug based on PEM (PROTAC-PEM) that specifically targeting METTL4 in macrophages within plaques, showing a promising therapeutic effect on atherosclerosis. CONCLUSIONS: This study revealed a novel mechanism by which mtDNA 6mA orchestrated mitochondrial function-related gene expression in macrophages, thereby promoting atherosclerosis. Through various experimental techniques, such as gene manipulation, pharmacological inhibition and PROTAC, this study demonstrated that mtDNA 6mA and its specific enzyme METTL4 hold potential as therapeutic targets for atherosclerosis.

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Single-nucleotide-resolution sequencing of human N6-methyldeoxyadenosine reveals strand-asymmetric clusters associated with SSBP1 in the mitochondrial genome

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