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.     

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     

Processing character of the action of wheat endonucleases WEN1 and WEN2. Kinetic parameters

The wheat seedling endonucleases WEN1 and WEN2 dependent on Mg²⁺, Ca²⁺, and S-adenosyl-L-methionine (SAM) and sensitive to the substrate DNA methylation status have an expressed processing action. The enzymes hydrolyze DNA at a few subsequent stages: first, they split λ phage DNA specifically at CNG-sites (WEN1) with liberation of large fragments; second, they hydrolyze these fragments to 120–140 bp oligonucleotides that finally are hydrolyzed to very short fragments and mononucleotides. Initial stages of DNA hydrolysis may proceed in the absence of Mg²⁺, but subsequent hydrolysis stages are very strongly stimulated by Mg²⁺. It cannot be ruled out that modulation of enzymatic activity with Mg²⁺ and probably with DNA fragments formed is associated with reorganization of the structure of eukaryotic (wheat) endonucleases with respective changes in their catalytic properties and site specificity of action. Michaelis constant value for WEN1 endonuclease on hydrolysis of methylated λ phage DNA containing Cm⁵CWGG and Gm⁶ATC sites is four-fold lower compared with that observed on hydrolysis of unmethylated λ phage DNA. This may indicate that affinity of WEN1 enzyme to methylated DNA is higher than that to unmethylated DNA. In the presence of SAM, the Michaelis constant for WEN2 on the DNA hydrolysis stage characterized by formation of 120–140 bp fragments is decreased, but for WEN1 it is increased by 1.5–2.0-fold. This means that SAM inhibits WEN1 but stimulates WEN2. Thus, wheat endonucleases WEN1 and WEN2 differ significantly in affinities to substrate DNAs with different methylation status, in velocities of DNA hydrolysis, and time of production of DNA fragments of similar length. It seems that the investigated plant endonucleases can hydrolyze DNA in the nucleus as well to both large and very short fragments including mononucleotides, that is, in particular, essential for utilization of cell nucleic acid material during apoptosis.     

Endonucleases and their involvement in plant apoptosis

This review considers modern data about the set, nature, specificity of action, and other properties of plant endonucleases involved in various forms of programmed cell death (PCD) in various plant tissues (organs). Apoptosis is an obligatory component of plant development; plant development is impossible without apoptosis. In dependence on the conditions of plant growth, this process can be induced by various biotic and abiotic factors, including stressors. Endonucleases accomplishing apoptotic degradation of nuclear material in the plant cell play one of the main roles in PCD. Plant endonucleases belong to at least two classes: (1) Ca²⁺- and Mg²⁺-dependent and (2) Zn²⁺-dependent nucleases. The set and activities of endonucleases change with plant age and during apoptosis in a tissue-specific manner. Apoptosis is accompanied by the induction of specific endonucleases hydrolyzing DNA in chromatin with the formation firstly of large domains and then internucleosomal DNA fragments; the products produced are of about 140 nucleotides in length with their subsequent degradation to low-molecular-weight oligonucleotides and mononucleotides. About 30 enzymes are involved in apoptotic DNA degradation. Histone H1 modulates endonuclease activity; separate (sub)fractions of this nuclear protein can stimulate or inhibit corresponding plant endonucleases. In the nucleus and cytoplasm of the plant cells, Ca²⁺/Mg²⁺-dependent endonucleases recognizing substrate DNA methylation status were revealed and described for the first time; their action resembles that of bacterial restrictases, which activity is modulated by the donor of methyl groups, S-adenosylmethionine. This indicates that higher eukaryotes (higher plants) might possess the system of restriction-modification to some degree analogous to that of prokaryotes.     

Conserved dual-mode gene regulation programs in higher eukaryotes

Recent genomic data analyses have revealed important underlying logics in eukaryotic gene regulation, such as CpG islands (CGIs)-dependent dual-mode gene regulation. In mammals, genes lacking CGIs at their promoters are generally regulated by interconversion between euchromatin and heterochromatin, while genes associated with CGIs constitutively remain as euchromatin. Whether a similar mode of gene regulation exists in non-mammalian species has been unknown. Here, through comparative epigenomic analyses, we demonstrate that the dual-mode gene regulation program is common in various eukaryotes, even in the species lacking CGIs. In cases of vertebrates or plants, we find that genes associated with high methylation level promoters are inactivated by forming heterochromatin and expressed in a context-dependent manner. In contrast, the genes with low methylation level promoters are broadly expressed and remain as euchromatin even when repressed by Polycomb proteins. Furthermore, we show that invertebrate animals lacking DNA methylation, such as fruit flies and nematodes, also have divergence in gene types: some genes are regulated by Polycomb proteins, while others are regulated by heterochromatin formation. Altogether, our study establishes gene type divergence and the resulting dual-mode gene regulation as fundamental features shared in a broad range of higher eukaryotic species.     

Conservation and divergence of DNA methylation in eukaryotes

DNA methylation is one of the most important heritable epigenetic modifications of the genome and is involved in the regulation of many cellular processes. Aberrant DNA methylation has been frequently reported to influence gene expression and subsequently cause various human diseases, including cancer. Recent rapid advances in next-generation sequencing technologies have enabled investigators to profile genome methylation patterns at single-base resolution. Remarkably, more than 20 eukaryotic methylomes have been generated thus far, with a majority published since November 2009. Analysis of this vast amount of data has dramatically enriched our knowledge of biological function, conservation and divergence of DNA methylation in eukaryotes. Even so, many specific functions of DNA methylation and their underlying regulatory systems still remain unknown to us. Here, we briefly introduce current approaches for DNA methylation profiling and then systematically review the features of whole genome DNA methylation patterns in eight animals, six plants and five fungi. Our systematic comparison provides new insights into the conservation and divergence of DNA methylation in eukaryotes and their regulation of gene expression. This work aims to summarize the current state of available methylome data and features informatively.     

Adenine methylation in zein genes

This paper reports the novel finding of adenine methylation in higher plants. Comparison of restriction patterns of genomic maize DNA digested with enzymes MboI and Sau3A enabled us to detect the existence of adenine methylation in zein genes. Adenine methylation within or around zein genes turned out to be similar in endosperm (where zeins are actively synthesized) and in seedling tissue (where zein genes are not expressed). Furthermore, adenine methylation patterns were found to be similar both in wild-type and opaque-2 mutant plants. These lines of evidence suggest that adenine methylation is unrelated to the regulation of gene expression.     

Conservation and divergence of DNA methylation in eukaryotes: New insights from single base-resolution DNA methylomes

DNA methylation is one of the most important heritable epigenetic modifications of the genome and is involved in the regulation of many cellular processes. Aberrant DNA methylation has been frequently reported to influence gene expression and subsequently cause various human diseases, including cancer. Recent rapid advances in next-generation sequencing technologies have enabled investigators to profile genome methylation patterns at singlebase resolution. Remarkably, more than 20 eukaryotic methylomes have been generated thus far, with a majority published since November 2009. Analysis of this vast amount of data has dramatically enriched our knowledge of biological function, conservation and divergence of DNA methylation in eukaryotes. Even so, many specific functions of DNA methylation and their underlying regulatory systems still remain unknown to us. Here, we briefly introduce current approaches for DNA methylation profiling and then systematically review the features of whole genome DNA methylation patterns in eight animals, six plants and five fungi. Our systematic comparison provides new insights into the conservation and divergence of DNA methylation in eukaryotes and their regulation of gene expression. This work aims to summarize the current state of available methylome data and features informatively.Key words: DNA methylation, methylome, single-base resolution, CpG, gene body, broadness, deepness, promoter     

DNA甲基化与植物抗逆性研究进展

DNA甲基化是真核细胞基因组重要修饰方式之一.DNA甲基化通过与转录因子相互作用或通过改变染色质结构来影响基因的表达,从表观遗传水平对生物遗传信息进行调节,在生长发育过程中起着重要的作用,而且植物DNA甲基化还参与了环境胁迫下的基因表达调控过程.本文对植物DNA甲基化的产生机制、功能,以及DNA甲基化在植物应对逆境胁迫中的作用进行综述,以更好地理解植物DNA甲基化及其对环境胁迫的响应,为植物抗逆性研究及作物遗传改良提供理论参照.     

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.     

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)     

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.