Methylation of adenine residues in DNA of eukaryotes

Like in bacteria, DNA in these organisms is subjected to enzymatic modification (methylation) both at adenine and cytosine residues. There is an indirect evidence that adenine DNA methylation takes place also in animals. In plants m6A was detected in total, mitochondrial and nuclear DNAs; in plants one and the same gene (DRM2) can be methylated both at adenine and cytosine residues. ORF homologous to bacterial adenine DNA-methyltransferases are present in nuclear DNA of protozoa, yeasts, insects, nematodes, higher plants, vertebrates and other eukaryotes. Thus, adenine DNA-methyltransferases can be found in the various evolutionary distant eukaryotes. First N6-adenine DNA-methyltransferase (wadmtase) of higher eukaryotes was isolated from vacuolar fraction of vesicles obtained from aging wheat coleoptiles; in the presence of S-adenosyl-L-methionine this Mg2+ -, Ca2+ -dependent enzyme de novo methylates first adenine residue in TGATCA sequence in single- and double-stranded DNA but it prefers single-stranded DNA structures. Adenine DNA methylation in eukaryotes seems to be involved in regulation of both gene expression and DNA replication including replication of mitochondrial DNA. It can control persistence of foreign DNA in a cell and seems to be an element of R-M system in plants. Thus, in eukaryotic cell there are, at least, two different systems of the enzymatic DNA methylations (adenine and cytosine ones) and a special type of regulation of gene functioning based on the combinatory hierarchy of these interdependent genome modifications.     

A view of an elemental naturalist at the DNA world (Base composition,sequences, methylation)

The pioneering data on base composition and pyrimidine sequences in DNA of pro-and eukaryotes are considered, and their significance for the origin of genosystematics is discussed. The modern views on specificity and functional role of enzymatic DNA methylation in eukaryotes are described. DNA methylation controls all genetic functions and is a mechanism of cellular differentiation and gene silencing. A model of regulation of DNA replication by methylation is suggested. Adenine DNA methylation in higher eukaryotes (higher plants) was first observed, and it was established that one and the same gene can be methylated at both cytosine and adenine moieties. Thus, there are at least two different and seemingly interdependent DNA methylation systems present in eukaryotic cells. The first eukaryotic adenine DNA-methyltransferase is isolated from wheat seedlings and described: the enzyme methylates DNA with formation of N⁶-methyladenine in the sequence TGATCA → TGm⁶ATCA. It is found that higher plants have endonucleases that are dependent on S-adenosyl-L-methionine (SAM) and sensitive to DNA methylation status. Therefore, as in bacteria, plants seem to have a restriction-modification (R-M) system. A system of conjugated up-and down-regulation of SAM-dependent endonucleases by SAM modulations is found in plants. Revelation of an essential role of DNA methylation in regulation of genetic processes is a fundament of materialization of epigenetics and epigenomics. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 12, pp. 1583–1593.     

Adenine Methylation in Eukaryotic DNA

N⁶-Methyladenine (m⁶A) has been found in DNAs of various eukaryotes (algae, fungi, protozoa, and higher plants). Like bacterial DNA, DNAs of these organisms are subject to enzymatic modification (methylation) not only at cytosine, but also at adenine bases. There is indirect evidence that adenine methylation of the genome occurs in animals as well. In plants, m⁶A was detected in total, mitochondrial, and nuclear DNAs. It was observed that both adenines and cytosines can be methylated in one gene (DRM2). Open reading frames coding for homologs of bacterial adenine DNA methyltransferases were revealed in protozoan, yeast, higher plant, insect, nematode, and vertebrate genomes, suggesting the presence of adenine DNA methyltransferases in evolutionarily distant eukaryotes. The first higher-eukaryotic adenine DNA N⁶-methyltransferase (wad-mtase) was isolated from vacuolar vesicles of wheat coleoptiles. The enzyme depends on Mg²⁺ or Ca²⁺ and, in the presence of S-adenosyl-L-methionine, methylates de novo the first adenine of the sequence TGATCA in single- and double-stranded DNAs, preferring the former. Adenine methylation of eukaryotic DNA is probably involved in regulating gene expression and replication, including that of mitochondrial DNA; plays a role in controlling the persistence of foreign DNA in the cell; and acts as a component of a plant restriction— modification system. Thus, the eukaryotic cell has at least two different systems for enzymatic methylation of DNA (at adenines and at cytosines) and a special mechanism regulating the functions of genes via a combinatorial hierarchy of these interdependent modifications of the genome.__________Translated from Molekulyarnaya Biologiya, Vol. 39, No. 4, 2005, pp. 557–566.Original Russian Text Copyright © 2005 by Vanyushin.To the memory of my teacher, Academician Andrei Nikolaevich Belozersky     

DNA methylation: evolution of a bacterial immune function into a regulator of gene expression and genome structure in higher eukaryotes

The amino acid sequence of mammalian DNA methyltransferase has been deduced from the nucleotide sequence of a cloned cDNA. It appears that the mammalian enzyme arose during evolution via fusion of a prokaryotic restriction methyltransferase gene and a second gene of unknown function. Mammalian DNA methyltransferase currently comprises an N-terminal domain of about 1000 amino acids that may have a regulatory role and a C-terminal 570 amino acid domain that retains similarities to bacterial restriction methyltransferases. The sequence similarities among mammalian and bacterial DNA cytosine methyltransferases suggest a common evolutionary origin. DNA methylation is uncommon among those eukaryotes having genomes of less than 10(8) base pairs, but nearly universal among large-genome eukaryotes. This and other considerations make it likely that sequence inactivation by DNA methylation has evolved to compensate for the expansion of the genome that has accompanied the development of higher plants and animals. As methylated sequences are usually propagated in the repressed, nuclease-insensitive state, it is likely that DNA methylation compartmentalizes the genome to facilitate gene regulation by reducing the total amount of DNA sequence that must be scanned by DNA-binding regulatory proteins. DNA methylation is involved in immune recognition in bacteria but appears to regulate the structure and expression of the genome in complex higher eukaryotes. I suggest that the DNA-methylating system of mammals was derived from that of bacteria by way of a hypothetical intermediate that carried out selective de novo methylation of exogenous DNA and propagated the methylated DNA in the repressed state within its own genome.(ABSTRACT TRUNCATED AT 250 WORDS)     

The DNA-methylation state regulates virulence and stress response of Salmonella

The DNA methylation is a post-replicative event that provides secondary information to that formed by DNA. Addition of this information involves DAM methyltransferase, which methylates DNA on specific sites (5'-GATC-3'). This modification of DNA may play a role in regulating various processes in eukaryote or prokaryote cells. It was well understood that deoxyadenosine methyltransferase (DAM) methylates the adenine of the GATC sequence. Following DNA replication, however, DNA is transiently hemimethylated, and the new strand is then methylated by DAM. In Escherichia coli, removing the dam gene produces several phenotypes indicating multiple functions of methylation: (i) modulation of gene expression, (ii) DNA repair, (iii) initiation of replication, and (iv) stabilising the chromosome.     

The gene for domains rearranged methyltransferase (DRM2) in Arabidopsis thaliana plants is methylated at both cytosine and adenine residues

The methylation patterns of cytosine and adenine residues in the Arabidopsis thaliana gene for domains rearranged methyltransferase (DRM2) were studied in wild-type and several transgene plant lines containing antisense fragments of the cytosine DNA-methyltransferase gene METI under the control of copper-inducible promoters. It was shown that the promoter region of the DRM2 gene is mostly unmethylated at the internal cytosine residue in CCGG sites whereas the 3'-end proximal part of the gene coding region is highly methylated. The DRM2 gene was found to be also methylated at adenine residues in some GATC sequences. Cytosine methylation in CCGG sites and adenine methylation in GATC sites in the DRM2 gene are variable between wild-type and different transgenic plants. The induction of antisense METI constructs with copper ions in transgene plants in most cases leads to further alterations in the DRM2 gene methylation patterns.     

DNA-[Adenine] Methylation in Lower Eukaryotes

DNA methylation in lower eukaryotes, in contrast to vertebrates, can involve modification of adenine to N6-methyladenine (m6A). While DNA-[cytosine] methylation in higher eukaryotes has been implicated in many important cellular processes, the function(s) of DNA-[adenine] methylation in lower eukaryotes remains unknown. I have chosen to study the ciliate Tetrahymena thermophila as a model system, since this organism is known to contain m6A, but not m5C, in its macronuclear DNA. A BLAST analysis revealed an open reading frame (ORF) that appears to encode for the Tetrahymena DNA-[adenine] methyltransferase (MTase), based on the presence of motifs characteristic of the enzymes in prokaryotes. Possible biological roles for DNA-[adenine] methylation in Tetrahymena are discussed. Experiments to test these hypotheses have begun with the cloning of the gene. Orthologous ORFs are also present in three species of the malarial parasite Plasmodium. They are compared to one another and to the putative Tetrahymena DNA-[adenine] MTase. The gene from the human parasite P. falciparum has been cloned.     

The fission yeast gene pmt1+ encodes a DNA methyltransferase homologue.

DNA methylation of cytosine residues is a widespread phenomenon and has been implicated in a number of biological processes in both prokaryotes and eukaryotes. This methylation occurs at the 5-position of cytosine and is catalyzed by a distinct family of conserved enzymes, the cytosine-5 methyltransferases (m5C-MTases). We have cloned a fission yeast gene pmt1+ (pombe methyltransferase) which encodes a protein that shares significant homology with both prokaryotic and eukaryotic m5C-MTases. All 10 conserved domains found in these enzymes are present in the pmt1 protein. This is the first m5C-MTase homologue cloned from a fungal species. Its presence is surprising, given the inability to detect DNA methylation in yeasts. Haploid cells lacking the pmt1+ gene are viable, indicating that pmt1+ is not an essential gene. Purified, bacterially produced pmt1 protein does not possess obvious methyltransferase activity in vitro. Thus the biological significance of the m5C-MTase homologue in fission yeast is currently unclear.     

A DNA adenine methyltransferase of Escherichia coli that is cell cycle regulated and essential for viability

DNA sequence analysis revealed that the putative yhdJ DNA methyltransferase gene of Escherichia coli is 55% identical to the Nostoc sp. strain PCC7120 gene encoding DNA methyltransferase AvaIII, which methylates adenine in the recognition sequence, ATGCAT. The yhdJ gene was cloned, and the enzyme was overexpressed and purified. Methylation and restriction analysis showed that the DNA methyltransferase methylates the first adenine in the sequence ATGCAT. This DNA methylation was found to be regulated during the cell cycle, and the DNA adenine methyltransferase was designated M.EcoKCcrM (for "cell cycle-regulated methyltransferase"). The CcrM DNA adenine methyltransferase is required for viability in E. coli, as a strain lacking a functional genomic copy of ccrM can be isolated only in the presence of an additional copy of ccrM supplied in trans. The cells of such a knockout strain stopped growing when expression of the inducible plasmid ccrM gene was shut off. Overexpression of M.EcoKCcrM slowed bacterial growth, and the ATGCAT sites became fully methylated throughout the cell cycle; a high proportion of cells with an anomalous size distribution and DNA content was found in this population. Thus, the temporal control of this methyltransferase may contribute to accurate cell cycle control of cell division and cellular morphology. Homologs of M.EcoKCcrM are present in other bacteria belonging to the gamma subdivision of the class Proteobacteria, suggesting that methylation at ATGCAT sites may have similar functions in other members of this group.     

Developmentally regulated DNA methylation in Dictyostelium discoideum

Methylation of cytosine residues in DNA plays a critical role in the silencing of gene expression, organization of chromatin structure, and cellular differentiation of eukaryotes. Previous studies failed to detect 5-methylcytosine in Dictyostelium genomic DNA, but the recent sequencing of the Dictyostelium genome revealed a candidate DNA methyltransferase gene (dnmA). The genome sequence also uncovered an unusual distribution of potential methylation sites, CpG islands, throughout the genome. DnmA belongs to the Dnmt2 subfamily and contains all the catalytic motifs necessary for cytosine methyltransferases. Dnmt2 activity is typically weak in Drosophila melanogaster, mouse, and human cells and the gene function in these systems is unknown. We have investigated the methylation status of Dictyostelium genomic DNA with antibodies raised against 5-methylcytosine and detected low levels of the modified nucleotide. We also found that DNA methylation increased during development. We searched the genome for potential methylation sites and found them in retrotransposable elements and in several other genes. Using Southern blot analysis with methylation-sensitive and -insensitive restriction endonucleases, we found that the DIRS retrotransposon and the guaB gene were indeed methylated. We then mutated the dnmA gene and found that DNA methylation was reduced to about 50% of the wild-type level. The mutant cells exhibited morphological defects in late development, indicating that DNA methylation has a regulatory role in Dictyostelium development. Our findings establish a role for a Dnmt2 methyltransferase in eukaryotic development.