Means, mechanisms and consequences of adenine methylation in DNA

Abstract

N⁶-methyl-2'-deoxyadenosine (6mA or m⁶dA) has been reported in the DNA of prokaryotes and eukaryotes ranging from unicellular protozoa and algae to multicellular plants and mammals. It has been proposed to modulate DNA structure and transcription, transmit information across generations and have a role in disease, among other functions. However, its existence in more recently evolved eukaryotes remains a topic of debate. Recent technological advancements have facilitated the identification and quantification of 6mA even when the modification is exceptionally rare, but each approach has limitations. Critical assessment of existing data, rigorous design of future studies and further development of methods will be required to confirm the presence and biological functions of 6mA in multicellular eukaryotes.

Fig. 1 |. Directed epigenetic DnA methylations.

a | The chemical structures, effects on helix stability and reported biological functions are shown for three directed epigenetic modifications of DNA — N⁶-methyladenosine (6mA), C ⁵-methylcytosine (5mC) and N⁴-methylcytosine (4mC)^–. b | Depicted are the enzymatic pathways by which 6mA is added to (in red) and removed from (in blue) the genome. MT-A70 family methylases use S-adenosylmethionine (SAM) to catalyse the methylation of adenines at the sixth position of the purine ring, generating 6mA. 6mA can be removed by AlkB family demethylase enzymes. These enzymes require α-ketoglutarate and Fe²⁺ and use oxygen to oxidize the methyl group to generate 6-hydroxymethyladenine (6hmA), which then releases a formaldehyde group to generate adenine. Alternatively, in prokaryotes, 6mA can be deaminated and excised from the DNA by the base excision repair pathway, whereby a deaminase hydrolyses the methylamine to generate hypoxanthine, which is then recognized by AlkA enzymes as a damaged base. They cleave the glycosyl bond to remove the base, after which apurinic (AP) endonuclease cleaves the phosphodiester backbone at the abasic site, thereby exposing the residual 5′ deoxyribose phosphate group, which is removed by deoxyribose phosphodiesterase. DNA polymerase I (DNA Pol) will repair the DNA by incorporating an unmodified adenine while DNA ligase catalyses the formation of the phosphodiester bond. It remains to be determined whether a similar mechanism exists in eukaryotes. R-M, restriction–modification. Part b reprinted from REF., Springer Nature Limited.

Fig. 2 |. Potential modes of 6mA enrichment.

a | 6mA could be added to and removed from genomes by 6mA-specific methyltransferases (such as Dam in prokaryotes and METTL4, a putative metazoan methyltransferase) and demethylases (such as AlkB in prokaryotes or putative metazoan demethylases ALKBH1 and ALKBH4). b | Adenines in the genome that are methylated at the N1 position (1mA) could undergo a Dimroth rearrangement whereby the methyl group is transferred to the N⁶ position. c | Pre-methylated nucleotides could be incorporated into the genome by DNA polymerase. Pre-methylated nucleotides could be generated by the nucleotide salvage pathway via recycling of RNA N⁶-methyladenosine (m⁶A) or DNA N⁶-methyladenosine (6mA) repurposed from foreign organisms.

Fig. 3 |. Biological roles of DnA adenine methylation potentially conserved in unicellular and multicellular organisms.

a | In prokaryotes (left), DNA adenine methylation provides a basic immune system in the form of modification enzymes that methylate the host DNA and restriction enzymes that recognize and digest foreign unmethylated DNA. Reciprocal examples also exist. Here, the Escherichia coli system is depicted, which uses EcoRI as the restriction enzyme and M.EcoRI as the modification enzyme. In multicellular eukaryotes (right), N⁶-methyladenosine (6mA) has been detected in long interspersed nuclear element (LINE) retrotransposons and is associated with their transcriptional silencing. Thus, 6mA might have a conserved role in recognizing and inhibiting foreign DNA. b | 6mA is postulated to repress transcription in some unicellular and multicellular eukaryotes through repelling some transcription factors and in Saccharomyces cerevisiae, 6mA has been shown to cause RNA polymerase II pausing (left), which would decrease transcription rates. 6mA has also been shown to activate transcription in other unicellular and multicellular eukaryotes by reducing DNA duplex stability to facilitate transcription, and 6mA can also enhance the binding of alternative transcription factors, such as AGP1, to increase transcription (right). c | 6mA is enriched in the linker region between nucleosomes in several protists, raising the possibility that it could actively regulate nucleosome positioning (left). Increasing the separation of nucleosomes increases chromatin accessibility and gene expression. In Oryza sativa, DDM1 could regulate nucleosome positioning through nucleosome remodelling activity and/or through N⁶-adenine methylating linker regions where 6mA is enriched (right). d | As elucidated in E. coli, DNA adenine methylation allows DNA repair proteins to identify the parental strand and replace the sequence of the newly synthesized mutated strand (left). In Caenorhabditis elegans, deletion of the putative 6mA demethylase NMAD-1 causes misregulation of DNA damage repair genes (right), but it remains to be determined whether 6mA has a conserved role in directly regulating DNA damage in eukaryotes. e | In prokaryotes such as E. coli and Caulobacter crescentus, the 6mA binding protein SeqA will bind to hemimethylated DNA at the origin of replication and will prevent the methyltransferase Dam from methylating the newly synthesized strand (left). DNA replication is thereby inhibited until SeqA is released and both strands of DNA are methylated. In vitro studies with Homo sapiens DNA polymerase-η show that 6mA slows the incorporation of thymines during DNA replication (right), raising the possibility that 6mA could have a conserved role in inhibiting DNA replication. C. reinhardtii, Chlamydomonas reinhardtii; D. melanogaster, Drosophila melanogaster; N. tabacum, Nicotiana tabacum; O. trifallax, Oxytricha trifallax; T. thermophila, Tetrahymena thermophila.

Fig. 4 |. emerging potential roles of 6mA in metazoa.

a | Hypoxia induces an increase in the levels of METTL4 and mitochondrial DNA (mtDNA) N⁶-methyladenosine (6mA), which in turn inhibits the binding of the mitochondrial transcription factor TFAM. TFAM facilitates both transcription of mitochondrial genes and mitochondrial replication. Increased 6mA on mtDNA caused by hypoxia stress leads to decreased mtDNA transcription and reduced mtDNA copy number. b | In Drosophila melanogaster, the proposed DNA demethylase Dmad binds to the H3K4 trimethylation complex protein Wds, a component of the protein complex that trimethylates histone H3 at lysine 4 (H3K4Me3, indicated by green circles), thereby suggesting a potential mechanism for chromatin modification crosstalk. c | It has been proposed that 6mA becomes upregulated in glioblastomas and that this increase in 6mA correlates with increased heterochromatin at tumour suppressor genes. Therefore, elevated 6mA could facilitate tumorigenesis. d | It has been shown in Caenorhabditis elegans that levels of 6mA increase in response to the oxidative phosphorylation inhibitor antimycin. This elevated 6mA and the resulting adaptation to mitochondrial stress is inherited by untreated progeny of antimycin-treated individuals, suggesting that 6mA may have a role in transgenerational inheritance.