DNA 6mA Research Articles

Genomewide analysis of 6-methyladenine DNA in peripheral blood mononuclear cells of systemic lupus erythematosus.

The aim of this paper is to explore the expression of 6-methyladenine (6mA) DNA and to elucidate its gene regulation role in systemic lupus erythematosus (SLE). Twenty SLE patients and 20 normal control healthy individuals (HCs) were included in this study. Genomic DNA was isolated from peripheral blood mononuclear cells and subsequently underwent 6mA-immunoprecipitation-sequencing (6mA-IP-Seq) after DNA quality control and 6mA precipitation. Bioinformation analysis was applied to the raw data comparing 6mA levels between SLE patients and HCs. We identified 5462 hypermethylation and 431 hypomethylation genes in PBMCs of individuals with SLE, which indicated that a high level of 6mA participates in the pathogenesis of SLE. Gene ontology analysis revealed that hypermethylation genes might regulate the inflammatory process, which has been well documented in the pathogenesis of SLE. 6mA may be involved in the initial development of SLE, which may lead to its potential use as an early diagnostic marker and therapeutic target.

N6-methyldeoxyadenine is a transgenerational epigenetic signal for mitochondrial stress adaptation.

N6-methyldeoxyadenine (6mA), a major type of DNA methylation in bacteria, represents a part of restriction-modification systems to discriminate host genome from invader DNA1. With the recent advent of more sensitive detection techniques, 6mA has also been detected in some eukaryotes2-8. However, the physiological function of this epigenetic mark in eukaryotes remains elusive. Heritable changes in DNA 5mC methylation have been associated with transgenerational inheritance of responses to a high-fat diet9, thus raising the exciting possibility that 6mA may also be transmitted across generations and serve as a carrier of inheritable information. Using Caenorhabditis elegans as a model, here we report that histone H3K4me3 and DNA 6mA modifications are required for the transmission of mitochondrial stress adaptations to progeny. Intriguingly, the global DNA 6mA level is significantly elevated following mitochondrial perturbation. N6-methyldeoxyadenine marks mitochondrial stress response genes and promotes their transcription to alleviate mitochondrial stress in progeny. These findings suggest that 6mA is a precisely regulated epigenetic mark that modulates stress response and signals transgenerational inheritance in C. elegans.

N6-methyldeoxyadenosine directs nucleosome positioning in Tetrahymena DNA

BackgroundN6-methyldeoxyadenosine (6mA or m6dA) was shown more than 40 years ago in simple eukaryotes. Recent studies revealed the presence of 6mA in more prevalent eukaryotes, even in vertebrates. However, functional characterizations have been limited.ResultsWe use Tetrahymena thermophila as a model organism to examine the effects of 6mA on nucleosome positioning. Independent methods reveal the enrichment of 6mA near and after transcription start sites with a periodic pattern and anti-correlation relationship with the positions of nucleosomes. The distribution pattern can be recapitulated by in vitro nucleosome assembly on native Tetrahymena genomic DNA but not on DNA without 6mA. Model DNA containing artificially installed 6mA resists nucleosome assembling compared to unmodified DNA in vitro. Computational simulation indicates that 6mA increases dsDNA rigidity, which disfavors nucleosome wrapping. Knockout of a potential 6mA methyltransferase leads to a transcriptome-wide change of gene expression.ConclusionsThese findings uncover a mechanism by which DNA 6mA assists to shape the nucleosome positioning and potentially affects gene expression.

DNA methylation on N6-adenine in lepidopteran Bombyx mori

Increasing knowledge of DNA methylation that occurs on the sixth position of adenine (N6-methyladenine, 6mA) has emerged as a novel epigenetic mark in eukaryotes and plays an important role in regulating gene transcription, DNA replication and repair, transposable activities, and others. Here, we show DNA 6mA methylation is present in Bombyx mori, a lepidopteran model insect, and identify the 6mA methyltransferase, METTL4, and 6mA demethylase, NMAD, which regulate the levels of 6mA in embryogenesis and cultured cells of B. mori. Importantly, RNAi knockdown of METTL4 and NMAD not only induce cell cycle arrest at G1 phase but also result in defects of chromosome alignments at metaphase. We further demonstrate that 6mA methylation is widely distributed across the genome of B. mori by 6mA-Seq and primarily enriched in the regulatory regions as well as gene bodies. Integrated analysis of 6mA-Seq and RNA-Seq reveals that 6mA methylation in B. mori is preferentially related with lowly expressed genes and negatively correlated with active gene transcription, which provides a novel regulatory mechanism of DNA 6mA methylation on target genes. Altogether, these data identify 6mA methylation in B. mori and demonstrate a crucial role of 6mA signaling in controlling cell cycle progression.

N6-Methyladenine DNA Modification in the Human Genome

DNA N6-methyladenine (6mA) modification is the most prevalent DNA modification in prokaryotes, but whether it exists in human cells and whether it plays a role in human diseases remain enigmatic. Here, we showed that 6mA is extensively present in the human genome, and we cataloged 881,240 6mA sites accounting for ∼0.051% of the total adenines. [G/C]AGG[C/T] was the most significantly associated motif with 6mA modification. 6mA sites were enriched in the coding regions and mark actively transcribed genes in human cells. DNA 6mA and N6-demethyladenine modification in the human genome were mediated by methyltransferase N6AMT1 and demethylase ALKBH1, respectively. The abundance of 6mA was significantly lower in cancers, accompanied by decreased N6AMT1 and increased ALKBH1 levels, and downregulation of 6mA modification levels promoted tumorigenesis. Collectively, our results demonstrate that DNA 6mA modification is extensively present in human cells and the decrease of genomic DNA 6mA promotes human tumorigenesis.

Convergence of DNA methylation and phosphorothioation epigenetics in bacterial genomes

Explosive growth in the study of microbial epigenetics has revealed a diversity of chemical structures and biological functions of DNA modifications in restriction-modification (R-M) and basic genetic processes. Here, we describe the discovery of shared consensus sequences for two seemingly unrelated DNA modification systems, 6mA methylation and phosphorothioation (PT), in which sulfur replaces a nonbridging oxygen in the DNA backbone. Mass spectrometric analysis of DNA from Escherichia coli B7A and Salmonella enterica serovar Cerro 87, strains possessing PT-based R-M genes, revealed d(GPS6mA) dinucleotides in the GPS6mAAC consensus representing ∼5% of the 1,100 to 1,300 PT-modified d(GPSA) motifs per genome, with 6mA arising from a yet-to-be-identified methyltransferase. To further explore PT and 6mA in another consensus sequence, GPS6mATC, we engineered a strain of E. coli HST04 to express Dnd genes from Hahella chejuensis KCTC2396 (PT in GPSATC) and Dam methyltransferase from E. coli DH10B (6mA in G6mATC). Based on this model, in vitro studies revealed reduced Dam activity in GPSATC-containing oligonucleotides whereas single-molecule real-time sequencing of HST04 DNA revealed 6mA in all 2,058 GPSATC sites (5% of 37,698 total GATC sites). This model system also revealed temperature-sensitive restriction by DndFGH in KCTC2396 and B7A, which was exploited to discover that 6mA can substitute for PT to confer resistance to restriction by the DndFGH system. These results point to complex but unappreciated interactions between DNA modification systems and raise the possibility of coevolution of interacting systems to facilitate the function of each.

N6-methyladenine DNA modification in the unicellular eukaryotic organism Tetrahymena thermophila

N6-methyladenine DNA (6mA) modification exists in both prokaryotes and eukaryotes in which it plays a crucial role in regulating numerous biological processes. In prokaryotes, 6mA is a marker to distinguish self from foreign DNA. Its role in eukaryotes, however, remains elusive. Ciliates were among the first eukaryotes reported to contain 6mA. In the model organism T. thermophila, cellular localization and features of 6mA have been extensively studied, especially in ribosomal DNA (rDNA) molecules. Here, we summarize the features and potential functions of 6mA in Tetrahymena thermophila and other ciliates, and the major findings and contributions of the Tetrahymena model in studies of 6mA methylation. We also discuss other questions in order to improve understanding of the function and evolution of 6mA in eukaryotes.

MethSMRT: an integrative database for DNA N6-methyladenine and N4-methylcytosine generated by single-molecular real-time sequencing.

DNA methylation is an important type of epigenetic modifications, where 5- methylcytosine (5mC), 6-methyadenine (6mA) and 4-methylcytosine (4mC) are the most common types. Previous efforts have been largely focused on 5mC, providing invaluable insights into epigenetic regulation through DNA methylation. Recently developed single-molecule real-time (SMRT) sequencing technology provides a unique opportunity to detect the less studied DNA 6mA and 4mC modifications at single-nucleotide resolution. With a rapidly increased amount of SMRT sequencing data generated, there is an emerging demand to systematically explore DNA 6mA and 4mC modifications from these data sets. MethSMRT is the first resource hosting DNA 6mA and 4mC methylomes. All the data sets were processed using the same analysis pipeline with the same quality control. The current version of the database provides a platform to store, browse, search and download epigenome-wide methylation profiles of 156 species, including seven eukaryotes such as Arabidopsis, C. elegans, Drosophila, mouse and yeast, as well as 149 prokaryotes. It also offers a genome browser to visualize the methylation sites and related information such as single nucleotide polymorphisms (SNP) and genomic annotation. Furthermore, the database provides a quick summary of statistics of methylome of 6mA and 4mC and predicted methylation motifs for each species. MethSMRT is publicly available at http://sysbio.sysu.edu.cn/methsmrt/ without use restriction.

Abundant DNA 6mA methylation during early embryogenesis of zebrafish and pig.

DNA N6-methyldeoxyadenosine (6mA) is a well-known prokaryotic DNA modification that has been shown to exist and play epigenetic roles in eukaryotic DNA. Here we report that 6mA accumulates up to ∼0.1–0.2% of total deoxyadenosine during early embryogenesis of vertebrates, but diminishes to the background level with the progression of the embryo development. During this process a large fraction of 6mAs locate in repetitive regions of the genome.

N6-Methyladenine DNA Modification in Drosophila

DNA N(6)-methyladenine (6mA) modification is commonly found in microbial genomes and plays important functions in regulating numerous biological processes in bacteria. However, whether 6mA occurs and what its potential roles are in higher-eukaryote cells remain unknown. Here, we show that 6mA is present in Drosophila genome and that the 6mA modification is dynamic and is regulated by the Drosophila Tet homolog, DNA 6mA demethylase (DMAD), during embryogenesis. Importantly, our biochemical assays demonstrate that DMAD directly catalyzes 6mA demethylation in vitro. Further genetic and sequencing analyses reveal that DMAD is essential for development and that DMAD removes 6mA primarily from transposon regions, which correlates with transposon suppression in Drosophila ovary. Collectively, we uncover a DNA modification in Drosophila and describe a potential role of the DMAD-6mA regulatory axis in controlling development in higher eukaryotes.

Immunochemical evidence for the presence of 5mC, 6mA and 7mG in human, Drosophila and mealybug DNA

We have reported that production and characterization of antibodies highly specific to 5-methyl-cytosine (5mC) and the development of a sensitive immunochemical method for the detection of 5mC in DNA [FEBS Lett. (1982) 150, 469]. Extension of this method to two other modified bases, 6-methyladenine (6mA) and 7-methylguanine (7mG), is reported here. By use of this immunochemical approach, we are able to detect 5mC, 6mA and 7mG in human and Drosophila DNA and confirm their presence in the DNA of two mealybug species.

Determination of 6-methyladenine in DNA by high-performance liquid chromatography

A method for the determination of 6-methyladenine (6MA) by high-performance liquid chromatography (HPLC) has been developed. DNA bases were separated by using the strong cation-exchange resin Zipax SCX. Purine bases were obtained by hydrolysis and dialysis of DNA and analysed by HPLC. 6MA in DNA from Escherichia coli was determined by the proposed method. It is suggested that the method could be applicable to analyses of 6MA from other biological sources.