B. F. Vanyushin

DNA methylation in higher plants: past, present and future.

A relatively high degree of nuclear DNA (nDNA) methylation is a specific feature of plant genomes. Targets for cytosine DNA methylation in plant genomes are CG, CHG and CHH (H is A, T, C) sequences. More than 30% total m(5)C in plant DNA is located in non-CG sites. DNA methylation in plants is species-, tissue-, organelle- and age-specific; it is involved in the control of all genetic functions including transcription, replication, DNA repair, gene transposition and cell differentiation. DNA methylation is engaged in gene silencing and parental imprinting, it controls expression of transgenes and foreign DNA in cell. Plants have much more complicated and sophisticated system of the multicomponent genome methylations compared to animals; DNA methylation in plant mitochondria is performed in other fashion as compared to that in nuclei. The nDNA methylation is carried out by cytosine DNA methyltransferases of, at least, three families. In contrast to animals the plants with the major maintenance methyltransferase MET1 (similar to animal Dnmt1) inactivated do survive. One and the same plant gene may be methylated at both adenine and cytosine residues; specific plant adenine DNA methyltransferase was described. Thus, two different systems of the genome modification based on methylation of cytosines and adenines seem to coexist in higher plants. This article is part of a Special Issue entitled: Epigenetic control of cellular and developmental processes in plants.

The content of 5-methylcytosine in animal DNA: The species and tissue specificity

Abstract The base composition of DNA isolated from different organs (tissues) of some invertebrates, fishes, amphibia, reptiles, birds and mammals has been studied. The G + C content in the DNA studied varies from 37.0 to 50.9 mole %. The rare base, 5-methylcytosine, has been found in all animal DNA. The content of 5-methylcytosine in these DNAs varies from 0.65 to 2.61 mole %. A positive correlation between the G + C and 5-methylcytosine contents in the DNA of animals belonging to one class is observed. The content of 5-methylcytosine is species and tissue specific. It is believed that the tissue differences with regard to the 5-methylcytosine content in the DNA of the organism are due to the various levels and specific character of DNA methylation.

DNA Methylation in Plants

DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of siRNA-DNA sequence identity; therefore, it is mainly associated with CNG and non-symmetrical methylations (rare in animals) in coding and promoter regions of silenced genes. Cytoplasmic viral RNA can affect methylation of homologous nuclear sequences and it maybe one of the feedback mechanisms between the cytoplasm and the nucleus to control gene expression.

Enzymatic DNA methylation is an epigenetic control for genetic functions of the cell.

In eukaryotic cells nuclear DNA is subjected to enzymatic methylation resulting in formation of 5-methylcytosine residues mainly in CG and CNG sequences. In plants and animals, this DNA methylation is species-, tissue-, and organelle-specific. It changes (diminishes) with age and is regulated by hormones. On the other hand, genome methylation can control hormonal signal. There are replicative and postreplicative DNA-methylations. They are served by multiple DNA-methyl-transferases with different site specificity. Replication is accompanied by appearance of hemimethylated sites in DNA; pronounced asymmetry of DNA chain methylation disappears at the end of the cell cycle; a model of regulation of replication by DNA methylation is suggested. DNA methylation controls all genetic processes in the cell (replication, transcription, DNA repair, recombination, gene transposition) and it is a mechanism of cell differentiation, gene discrimination, and silencing. Prohibition of DNA methylation stops development (embryogenesis), switches on apoptosis, and is usually lethal. Distortions in DNA methylations result in cancerous cell transformation, and the DNA methylation pattern is one of the safe cancer diagnostics at early stages of carcinogenesis. The malignant cell has a different DNA methylation pattern and a set of DNA-methyltransferase activities expressed as compared with normal cells. Inhibition of DNA methylation in plants is accompanied by induction of genes of seed storage proteins and flowering. In eukaryotes one and the same gene can be methylated both on cytosine and adenine residues; thus, there are, at least, two different and probably interdependent systems of DNA methylation in the cell. First higher eukaryotic adenine DNA-methyltransferase was isolated from plants; this enzyme methylates DNA with formation of N6-methyladenine residues in the sequence TGATCA → TGm6ATCA. Plants have AdoMet-dependent endonucleases sensitive to DNA methylation status; therefore, like microorganisms, plants seem to have a restriction-modification (R-S) system. Revelation of an essential role of DNA methylation in the regulation of genetic processes has laid a foundation for and materialized epigenetics and epigenomics.

N6-Adenine DNA-methyltransferase in wheat seedlings

The N 6‐adenine DNA‐methyltransferase was isolated from the vacuolar vesicle fraction of wheat coleoptiles. In the presence of S‐adenosyl‐L‐methionine the enzyme de novo methylates the first adenine residue in the TGATCA sequence in the single‐ or double‐stranded DNA substrates but it prefers single‐stranded structures. Wheat adenine DNA‐methyltransferase (wadmtase) is a Mg2+‐ or Ca2+‐dependent enzyme with a maximum activity at pH 7.5–8.0. Wadmtase seems to be responsible for mitochondrial DNA modification that might be involved in the regulation of replication of mitochondria in plants.

Penetration of short fluorescence-labeled peptides into the nucleus in HeLa cells and in vitro specific interaction of the peptides with deoxyribooligonucleotides and DNA

Marked fluorescence in cytoplasm, nucleus, and nucleolus was observed in HeLa cells after incubation with each of several fluorescein isothiocyanate-labeled peptides (epithalon, Ala-Glu-Asp-Gly; pinealon, Glu-Asp-Arg; testagen, Lys-Glu-Asp-Gly). This means that short biologically active peptides are able to penetrate into an animal cell and its nucleus and, in principle they may interact with various components of cytoplasm and nucleus including DNA and RNA. It was established that various initial (intact) peptides differently affect the fluorescence of the 5,6-carboxyfluorescein-labeled deoxyribooligonucleotides and DNA-ethidium bromide complexes. The Stern-Volmer constants characterizing the degree of fluorescence quenching of various single- and double-stranded fluorescence-labeled deoxyribooligonucleotides with short peptides used were different depending on the peptide primary structures. This indicates the specific interaction between short biologically active peptides and nucleic acid structures. On binding to them, the peptides discriminate between different nucleotide sequences and recognize even their cytosine methylation status. Judging from corresponding constants of the fluorescence quenching, the epithalon, pinealon, and bronchogen (Ala-Glu-Asp-Leu) bind preferentially with deoxyribooligonucleotides containing CNG sequence (CNG sites are targets for cytosine DNA methylation in eukaryotes). Epithalon, testagen, and pinealon seem to preferentially bind with CAG- but bronchogen with CTG-containing sequences. The site-specific interactions of peptides with DNA can control epigenetically the cell genetic functions, and they seem to play an important role in regulation of gene activity even at the earliest stages of life origin and in evolution.

DNA methylation and epigenetics

In eukaryotic cells, nuclear DNA is subject to enzymatic methylation with the formation of 5-methylcytosine residues, mostly within the CG and CNG sequences. In plants and animals this DNA methylation is species-, tissue-, and organelle-specific. It changes (decreases) with age and is regulated by hormones. On the other hand, genome methylation can control hormonal signal. Replicative and post-replicative DNA methylation types are distinguished. They are mediated by multiple DNA methyltransferases with different site-specificity. Replication is accompanied by the appearance of hemimethylated DNA sites. Pronounced asymmetry of the DNA strand methylation disappears to the end of the cell cycle. A model of methylation-regulated DNA replication is proposed. DNA methylation controls all genetic processes in the cell (replication, transcription, DNA repair, recombination, and gene transposition). It is the mechanism of cell differentiation, gene discrimination and silencing. In animals, suppression of DNA methylation stops development (embryogenesis), switches on apoptosis, and is usually lethal. Disruption of DNA methylation pattern results in the malignant cell transformation and serves as one of the early diagnostic features of carcinogenesis. In malignant cell the pattern of DNA methylation, as well as the set of DNA methyltransferase activities, differs from that in normal cell. In plants inhibition of DNA methylation is accompanied by the induction of seed storage and florescence genes. In eukaryotes one and the same gene can be simultaneously methylated both at cytosine and adenine residues. It can be thus suggested, that the plant cell contains at least two different, and probably, interdependent systems of DNA methylation. The first eukaryotic adenine DNA methyltransferase was isolated from plants. This enzyme methylates DNA with the formation of N6-methyladenine residues in the sequence TGATCA (TGATCA → TGm6ATCA). Plants possess AdoMet-dependent endonucleases sensitive to DNA methylation. It seems likely that plants, similarly to microorganisms and some lower eukaryotes, have restriction-modification (R-M) system. Discovery of the essential role of DNA methylation in regulation of genetic processes served as a principle basis and materialization of epigenetics and epigenomics.

Apoptosis in plants: specific features of plant apoptotic cells and effect of various factors and agents.

Apoptosis is an integral part of plant ontogenesis; it is controlled by cellular oxidative status, phytohormones, and DNA methylation. In wheat plants apoptosis appears at early stages of development in coleoptile and initial leaf of 5- to 6-day-old seedlings. Distinct ultrastructural features of apoptosis observed are (1). compaction and vacuolization of cytoplasm in the apoptotic cell, (2). specific fragmentation of cytoplasm and appearance in the vacuole of unique single-membrane vesicles containing active organelles, (3). cessation of nuclear DNA synthesis, (4). condensation and margination of chromatin in the nucleus, (5). internucleosomal fragmentation of nuclear DNA, and (6). intensive synthesis of mitochondrial DNA in vacuolar vesicles. Peroxides, abscisic acid, ethylene releaser ethrel, and DNA methylation inhibitor 5-azacytidine induce and stimulate apoptosis. Modulation of the reactive oxygen species (ROS) level in seedling by antioxidants and peroxides results in tissue-specific changes in the target date for the appearance and the intensity of apoptosis. Antioxidant butylated hydroxytoluene (BHT) reduces the amount of ROS and prevents apoptosis in etiolated seedlings, prolongs coleoptile life span, and prevents the appearance of all apoptotic features mentioned. Besides, BHT induces large structural changes in the organization of all cellular organelles and the formation of new unusual membrane structures in the cytoplasm. BHT distorts mitosis and this results in the appearance of multiblade polyploid nuclei and multinuclear cells. In roots of etiolated wheat seedlings, BHT induces differentiation of plastids with the formation of chloro(chromo)plasts. Therefore, ROS controlled by BHT seems to regulate mitosis, trigger apoptosis, and control plastid differentiation and the organization of various cellular structures formed by endocytoplasmic reticulum.

N 6-Methyladenine in mitochondrial DNA of higher plants

After incubation of etiolated wheat seedlings in the presence of [8‐14C]adenine the radioactive N 6‐methyladenine (m6A) has been detected in the newly synthesized mitochondrial DNA (mtDNA) (ML = 100·m6A/(m6A+A)=0.4–0.6). This DNA is a low molecular mass (7.7 S) fraction of the mtDNA population. The detection of N 6‐methyladenine in the mtDNA of wheat seedlings indicates the presence of adenine DNA methylase in mitochondria of higher plants. The presence of m6A in plant mtDNAs makes them distinct from animal mtDNAs in which, as we know, this additional base has not been found.

Age-Related Genomic Hypomethylation

Aging is a multi-factorial process of the progressive gradual decline of cellular functions with the passage of time. It is clear that aging affects the mammalian epigenome, including hypomethylation of DNA. DNA methylation is a crucial biological process that controls maintenance of genomic integrity and an accurate expression of genetic information. The accurate status of DNA methylation is balanced in mature cells, but with age this balance is strongly shifted in favor of DNA demethylation. Therefore, DNA hypomethylation that occurs during normal aging appears to be a critical risk factor contributing to the development of chronic age-related human pathological states. This review describes the involvement of DNA hypomethylation in the pathogenesis of several major age-related human diseases, including cancer, atherosclerosis, Alzheimer’s disease, psychiatric disorders, and autoimmune pathologies.