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Sequencing of N6-methyl-deoxyadenosine at single-base resolution across the mammalian genome

ABSTRACT: While DNA N6-methyl-deoxyadenosine (6mA) is abundant in bacteria and protists, its presence and function in mammalian genomes have been less clear. We present Direct-Read 6mA sequencing (DR-6mA-seq), an antibody-independent method to measure 6mA at base-resolution with high sensitivity. DR-6mA-seq employs a unique mutation-based strategy to reveal 6mA sites as misincorporation signatures without any chemical or enzymatic modulation of 6mA. We validated DR-6mA-seq through successful mapping of the well-characterized G(6mA)TC motif in the E. coli DNA and identified 6mA sites in the mammalian mitochondrial DNA. As expected, when applying DR-6mA-seq to mammalian systems, we found that genomic DNA (gDNA) 6mA abundance is in general low in most mammalian tissues and cells; however, we did observe distinct gDNA 6mA sites in mouse testis and glioblastoma cells. DR-6mA-seq provides an enabling tool to detect 6mA at single-base resolution with high sensitivity for a comprehensive understanding of DNA 6mA in eukaryotes.

ORGANISM(S): Mus musculus Escherichia coli K-12 synthetic construct Escherichia coli

PROVIDER: GSE213876 | GEO | 2023/12/28

REPOSITORIES: GEO

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High-precision mapping reveals rare N6-deoxyadenosine methylation in the mammalian genome

Project description:DNA modification has been characterized as a critical epigenetic regulator in gene expression. N6-deoxyadenosine methylation (6mA) is the most widespread type in prokaryotes and some unicellular eukaryotes. Nevertheless, the lack of a precise and comprehensive mapping method greatly hinders studying the presence and biological significance of 6mA in mammals. Here, we develop a new method MM-seq (Modification-induced Mismatch Sequencing) for genome-wide 6mA mapping in single-base resolution. This method balances accuracy and sensitivity based on a novel detection principle. We find modified DNA bases are likely to flip out of the DNA duplex, forming a mismatch-like structure that an antibody can capture. The mismatch-like structure can be recognized by endonuclease or exonuclease, which marks the exact modified site for readout via sequencing. Using this method, we examine the genomic positions of 6mA in bacteria, green algae, and mammalian cells. We find that human and mouse genomes contain rare and randomly distributed 6mA sites, with almost no overlap between different cell types. After knock-out of the RNA m6A methyltransferase Mettl3, limited 6mA sites in gDNA are diminished. Our results support the perspective that rare 6mA in the mammalian genome is caused by the misincorporation of N6-methyladenine via the nucleotide-salvage pathway.

2023-01-11 | GSE171698 | GEO

N6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas

Project description:N6-methyldeoxyadenosine (6mA or m6A) is a DNA modification preserved in prokaryotes to eukaryotes. It is widespread in bacteria, and functions in DNA mismatch repair, chromosome segregation, and virulence regulation. In contrast, the distribution and function of 6mA in eukaryotes have been unclear. Here we present a comprehensive analysis of the 6mA landscape in the genome of Chlamydomonas using new sequencing approaches. We identified the 6mA modification in 84% of genes in Chlamydomonas. We found that 6mA mainly locates at ApT dinucleotides around transcription start sites (TSS) with a bimodal distribution, and appears to mark active genes. A periodic pattern of 6mA deposition was also observed at base resolution, which is associated with nucleosome distribution near the TSS, suggesting a possible role in nucleosome positioning. The new genome-wide mapping of 6mA and its unique distribution in the Chlamydomonas genome suggest potential regulatory roles of 6mA in gene expression in eukaryotic organisms. Multiple sequencing methods are developed to profile the distribution of 6mA in Chlamydomonas including MeDIP-Seq, enzyme-treated DNA-Seq, MNase-Seq and RNA-Seq.

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Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing

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2016-04-12 | E-GEOD-74430 | biostudies-arrayexpress

Prevalent N6-methyldeoxyadenosine methylation in mammalian mitochondrial DNA

Project description:N6-methyldeoxyadenosine (6mA or m6dA), a well-known prokaryotic DNA modification has recently been shown to exist and play regulatory roles in eukaryotic genomic DNA. However, biological functions of 6mA in mammals remain to be explored largely due to its low abundance in most mammalian genomes. Here, we report a significant enrichment of 6mA in mammalian mitochondrial DNA (mtDNA), and identify METTL4 as a methyltransferase that installs 6mA. The level of 6mA in mtDNA is elevated up to over 0.02% of the total deoxyadenosines under hypoxia, representing ~7,00-fold enrichment over 6mA in the genomic DNA (gDNA) under normal growth conditions. The presence of 6mA negatively regulates mitochondrial transcription and balances mitochondrial function for cellular adaption to hypoxic stress. While DNA 5-methylcytosine (5mC) is a well-established, prevalent epigenetic mark in mammalian genomic DNA, our study reveals for the first time DNA 6mA methylation as a regulatory mark in mammalian mtDNA.

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2016-10-10 | GSE76740 | GEO

Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing

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N6-Methyldeoxyadenosine Marks Active Transcription Start Sites in Chlamydomonas

2015-04-30 | GSE62690 | GEO

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2020-04-22 | GSE149060 | GEO

single-nucleotide resolution sequencing of N6-methyladenine in E. coli genomes and mammalian mitochondrial DNA

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