Analogs of S-Adenosyl-L-Methionine in Studies of Methyltransferases

Molecular Biology - Methyltransferases (MTases) play an important role in the functioning of living systems, catalyzing the methylation reactions of DNA, RNA, proteins, and small molecules,...     

Effect of in vitro methylation at CpG sites on gene expression in a genome functioning autonomously in a vertebrate host

The effect of in vitro methylation at the HpaII sites in polyoma DNA on viral gene expression and the maintenance of the methyl groups upon replication in vivo were examined. Most of the methylatable sites are located in the early region coding for the viral large T antigen which is essential for the replication and infectivity of the viral DNA. Methylated or mock-methylated polyoma DNA produced the same number of virus plaques appearing at the same time post-transfection in either case. The lack of effect on the infectivity of the viral DNA indicates that the expression of the T antigen gene was not inhibited by methylation. Replication in vivo of the DNA also resulted in a total loss of the methyl groups introduced in vitro. These results underscore basic differences between the behavior of an autonomously functioning papovavirus DNA and the animal cell DNA vis-a-vis methylation at CpG sites. These differences might be due to subtle variations in the mechanism of regulation of gene expression and replication in the two systems.     

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.     

Eukaryotic DNA methylation: facts and problems

Patterns of DNA methylation in complex genomes like those of mammalian cells have been viewed as indicators of different levels of genetic activities. It is as yet unknown how these complicated patterns are generated and maintained during cell replication. There is evidence from many different biological systems that the sequence-specific methylation of promoters in higher eukaryotes is one of the important factors in controlling gene activity at a long-term level. In general, the fifth nucleotide 5-methyldeoxycytidine can be considered as a modulator of protein-DNA interactions. The degree and direction of this modulation has to be assessed experimentally in each individual instance. The establishment of de novo patterns of DNA methylation is characterized by the gradual non-random spreading of DNA methylation by an essentially unknown mechanism. In this review, some of the general concepts of DNA methylation in mammalian systems are presented, and research currently performed in the authors' laboratory has been summarized.     

DNA methylation and cell memory

In this paper we address the question: How do replicating mammalian cells remember with high fidelity their proper state of differentiation? Several possible mechanisms for cell memory are discussed, and it is concluded that only mechanisms involving DNA methylation are supported by strong experimental evidence. This evidence is reviewed. The establishment and modulation of methylation patterns are discussed and a hemimethylation model for stem cells is presented. The overall conclusion is that, although little is yet known about the details, there should be little doubt about the existence of a methylation system functioning at least to aid cell memory.     

Environmental DNA methylation of Lymnaea stagnalis varies with age and is hypermethylated compared to tissue DNA

Environmental DNA (eDNA) approaches contributing to species identifications are quickly becoming the new norm in biomonitoring and ecosystem assessments. Yet, information such as age and health state of the population, which is vital to species biomonitoring, has not been accessible from eDNA. DNA methylation has the potential to provide such information on the state of a population. Here, we measured the methylation of eDNA along with tissue DNA (tDNA) of Lymnaea stagnalis at four life stages. We demonstrate that eDNA methylation varies with age and allows distinguishing among age classes. Moreover, eDNA was globally hypermethylated in comparison to tDNA. This difference was age-specific and connected to a limited number of eDNA sites. This differential methylation pattern suggests that eDNA release with age is partially regulated through DNA methylation. Our findings help to understand mechanisms involved in eDNA release and shows the potential of eDNA methylation analysis to assess age classes. Such age class assessments will encourage future eDNA studies to assess fundamental processes of population dynamics and functioning in ecology, biodiversity conservation and impact assessments.     

Study of Tissue-Specific CpG Methylation of DNA in Extended Genomic Loci

Modern approaches for studies on genome functioning include investigation of its epigenetic regulation. Methylation of cytosines in CpG dinucleotides is an inherited epigenetic modification that is responsible for both functional activity of certain genomic loci and total chromosomal stability. This review describes the main approaches for studies on DNA methylation. Under consideration are site-specific approaches based on bisulfite sequencing and methyl-sensitive PCR, whole-genome approaches aimed at searching for new methylation hot spots, and also mapping of unmethylated CpG sites in extended genomic loci.     

DNA methylation and demethylation in mammals

Cell type-specific DNA methylation patterns are established during mammalian development and maintained in adult somatic cells. Understanding how these patterns of 5-methylcytosine are established and maintained requires the elucidation of mechanisms for both DNA methylation and demethylation. The enzymes involved in the de novo methylation of DNA and the maintenance of the resulting methylation patterns have been fairly well characterized. However, important remaining challenges are to understand how DNA methylation systems function in vivo and in the context of chromatin. In addition, the enzymes and mechanisms for demethylation remain to be elucidated. There is still no consensus as to how active enzymatic demethylation is achieved in mammalian cells, but recent studies implicate base excision repair for genome-wide DNA demethylation in germ cells and early embryos.     

CpG content affects gene silencing in mice: evidence from novel transgenes

Background  

Transgenes are often engineered using regulatory elements from distantly related genomes. Although correct expression patterns are frequently achieved even in transgenic mice, inappropriate expression, especially with promoters of widely expressed genes, has been reported. DNA methylation has been implicated in the aberrant expression, but the mechanism by which the methylation of a CpG-rich sequence can perturb the functioning of a promoter is unknown.     

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.     

The Epigenomic Landscape of Prokaryotes

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.