Genome-wide expert annotation of the epigenetic machinery of the plant-parasitic nematodes Meloidogyne spp., with a focus on the asexually reproducing species.

BACKGROUND: The renewed interest in epigenetics has led to the understanding that both the environment and individual lifestyle can directly interact with the epigenome to influence its dynamics. Epigenetic phenomena are mediated by DNA methylation, stable chromatin modifications and non-coding RNA-associated gene silencing involving specific proteins called epigenetic factors. Multiple organisms, ranging from plants to yeast and mammals, have been used as model systems to study epigenetics. The interactions between parasites and their hosts are models of choice to study these mechanisms because the selective pressures are strong and the evolution is fast. The asexually reproducing root-knot nematodes (RKN) offer different advantages to study the processes and mechanisms involved in epigenetic regulation. RKN genomes sequencing and annotation have identified numerous genes, however, which of those are involved in the adaption to an environment and potentially relevant to the evolution of plant-parasitism is yet to be discovered. RESULTS: Here, we used a functional comparative annotation strategy combining orthology data, mining of curated genomics as well as protein domain databases and phylogenetic reconstructions. Overall, we show that (i) neither RKN, nor the model nematode Caenorhabditis elegans possess any DNA methyltransferases (DNMT) (ii) RKN do not possess the complete machinery for DNA methylation on the 6th position of adenine (6mA) (iii) histone (de)acetylation and (de)methylation pathways are conserved between C. elegans and RKN, and the corresponding genes are amplified in asexually reproducing RKN (iv) some specific non-coding RNA families found in plant-parasitic nematodes are dissimilar from those in C. elegans. In the asexually reproducing RKN Meloidogyne incognita, expression data from various developmental stages supported the putative role of these proteins in epigenetic regulations. CONCLUSIONS: Our results refine previous predictions on the epigenetic machinery of model species and constitute the most comprehensive description of epigenetic factors relevant to the plant-parasitic lifestyle and/or asexual mode of reproduction of RKN. Providing an atlas of epigenetic factors in RKN is an informative resource that will enable researchers to explore their potential role in adaptation of these parasites to their environment.

The genome-wide identification and transcriptional levels of DNA methyltransferases and demethylases in globe artichoke.

Changes to the cytosine methylation status of DNA, driven by the activity of C5 methyltransferases (C5-MTases) and demethylases, exert an important influence over development, transposon movement, gene expression and imprinting. Three groups of C5-MTase enzymes have been identified in plants, namely MET (methyltransferase 1), CMT (chromomethyltransferases) and DRM (domains rearranged methyltransferases). Here the repertoire of genes encoding C5-MTase and demethylase by the globe artichoke (Cynara cardunculus var. scolymus) is described, based on sequence homology, a phylogenetic analysis and a characterization of their functional domains. A total of ten genes encoding C5-MTase (one MET, five CMTs and four DRMs) and five demethylases was identified. An analysis of their predicted product's protein structure suggested an extensive level of conservation has been retained by the C5-MTases. Transcriptional profiling based on quantitative real time PCR revealed a number of differences between the genes encoding maintenance and de novo methyltransferases, sometimes in a tissue- or development-dependent manner, which implied a degree of functional specialization.

Mechanism and biological role of Dnmt2 in Nucleic Acid Methylation.

A group of homologous nucleic acid modification enzymes called Dnmt2, Trdmt1, Pmt1, DnmA, and Ehmet in different model organisms catalyze the transfer of a methyl group from the cofactor S-adenosyl-methionine (SAM) to the carbon-5 of cytosine residues. Originally considered as DNA MTases, these enzymes were shown to be tRNA methyltransferases about a decade ago. Between the presumed involvement in DNA modification-related epigenetics, and the recent foray into the RNA modification field, significant progress has characterized Dnmt2-related research. Here, we review this progress in its diverse facets including molecular evolution, structural biology, biochemistry, chemical biology, cell biology and epigenetics.

The DNA methyltransferase DNMT3C protects male germ cells from transposon activity.

DNA methylation is prevalent in mammalian genomes and plays a central role in the epigenetic control of development. The mammalian DNA methylation machinery is thought to be composed of three DNA methyltransferase enzymes (DNMT1, DNMT3A, and DNMT3B) and one cofactor (DNMT3L). Here, we describe the discovery of Dnmt3C, a de novo DNA methyltransferase gene that evolved via a duplication of Dnmt3B in rodent genomes and was previously annotated as a pseudogene. We show that DNMT3C is the enzyme responsible for methylating the promoters of evolutionarily young retrotransposons in the male germ line and that this specialized activity is required for mouse fertility. DNMT3C reveals the plasticity of the mammalian DNA methylation system and expands the scope of the mechanisms involved in the epigenetic control of retrotransposons.

Genome-wide profiling of DNA methylation provides insights into epigenetic regulation of fungal development in a plant pathogenic fungus, Magnaporthe oryzae.

DNA methylation is an important epigenetic modification that regulates development of plants and mammals. To investigate the roles of DNA methylation in fungal development, we profiled genome-wide methylation patterns at single-nucleotide resolution during vegetative growth, asexual reproduction, and infection-related morphogenesis in a model plant pathogenic fungus, Magnaporthe oryzae. We found that DNA methylation occurs in and around genes as well as transposable elements and undergoes global reprogramming during fungal development. Such reprogramming of DNA methylation suggests that it may have acquired new roles other than controlling the proliferation of TEs. Genetic analysis of DNA methyltransferase deletion mutants also indicated that proper reprogramming in methylomes is required for asexual reproduction in the fungus. Furthermore, RNA-seq analysis showed that DNA methylation is associated with transcriptional silencing of transposable elements and transcript abundance of genes in context-dependent manner, reinforcing the role of DNA methylation as a genome defense mechanism. This comprehensive approach suggests that DNA methylation in fungi can be a dynamic epigenetic entity contributing to fungal development and genome defense. Furthermore, our DNA methylomes provide a foundation for future studies exploring this key epigenetic modification in fungal development and pathogenesis.

Faithful inheritance of cytosine methylation patterns in repeated sequences of the allotetraploid tobacco correlates with the expression of DNA methyltransferase gene families from both parental genomes.

The widespread occurrence of epigenetic alterations in allopolyploid species deserves scrutiny that DNA methylation systems may be perturbed by interspecies hybridization and polyploidization. Here we studied the genes involved in DNA methylation in Nicotiana tabacum (tobacco) allotetraploid containing S and T genomes inherited from Nicotiana sylvestris and Nicotiana tomentosiformis progenitors. To determine the inheritance of DNA methyltransferase genes and their expression patterns we examined three major DNA methyltransferase families (MET1, CMT3 and DRM) from tobacco and the progenitor species. Using Southern blot hybridization and PCR-based methods (genomic CAPS), we found that the parental loci of these gene families are retained in tobacco. Homoeologous expression was found in all tissues examined (leaf, root, flower) suggesting that DNA methyltransferase genes were probably not themselves targets of uniparental epigenetic silencing for over thousands of generations of allotetraploid evolution. The level of CG and CHG methylation of selected high-copy repeated sequences was similar and high in tobacco and its diploid progenitors. We speculate that natural selection might favor additive expression of parental DNA methyltransferase genes maintaining high levels of DNA methylation in tobacco, which has a repeat-rich heterochromatic genome.

Sequence-structure-function studies of tRNA:m5C methyltransferase Trm4p and its relationship to DNA:m5C and RNA:m5U methyltransferases.

Three types of methyltransferases (MTases) generate 5-methylpyrimidine in nucleic acids, forming m5U in RNA, m5C in RNA and m5C in DNA. The DNA:m5C MTases have been extensively studied by crystallographic, biophysical, biochemical and computational methods. On the other hand, the sequence-structure-function relationships of RNA:m5C MTases remain obscure, as do the potential evolutionary relationships between the three types of 5-methylpyrimidine-generating enzymes. Sequence analyses and homology modeling of the yeast tRNA:m5C MTase Trm4p (also called Ncl1p) provided a structural and evolutionary platform for identification of catalytic residues and modeling of the architecture of the RNA:m5C MTase active site. The analysis led to the identification of two invariant residues that are important for Trm4p activity in addition to the conserved Cys residues in motif IV and motif VI that were previously found to be critical. The newly identified residues include a Lys residue in motif I and an Asp in motif IV. A conserved Gln found in motif X was found to be dispensable for MTase activity. Locations of essential residues in the model of Trm4p are in very good agreement with the X-ray structure of an RNA:m5C MTase homolog PH1374. Theoretical and experimental analyses revealed that RNA:m5C MTases share a number of features with either RNA:m5U MTases or DNA:m5C MTases, which suggested a tentative phylogenetic model of relationships between these three classes of 5-methylpyrimidine MTases. We infer that RNA:m5C MTases evolved from RNA:m5U MTases by acquiring an additional Cys residue in motif IV, which was adapted to function as the nucleophilic catalyst only later in DNA:m5C MTases, accompanied by loss of the original Cys from motif VI, transfer of a conserved carboxylate from motif IV to motif VI and sequence permutation.

Specificity of restriction endonucleases and methylases--a review.

The properties and sources of all known restriction endonucleases and methylases are listed. The enzymes are cross-indexed (Table I), classified according to their recognition sequence homologies (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the double-stranded DNA of the bacteriophages lambda, phi X174 and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328, and the microorganisms from which they originate. Other tabulated properties of the restriction endonucleases include relaxed specificities (integrated into Table II), the structure of the generated fragment ends (Table III), and the sensitivity to different kinds of DNA methylation (Table V). In Table IV the conversion of two- and four-base 5'-protruding ends into new recognition sequences is compiled which is obtained by the fill-in reaction with Klenow fragment of the Escherichia coli DNA polymerase I or additional nuclease S1 treatment followed by ligation of the modified fragment termini [P3]. Interconversion of restriction sites generates novel cloning sites without the need of linkers. This should improve the flexibility of genetic engineering experiments. Table VI classifies the restriction methylases according to the nature of the methylated base(s) within their recognition sequences. This table also comprises restriction endonucleases which are known to be inhibited or activated by the modified nucleotides. The detailed sequences of those overlapping restriction sites are also included which become resistant to cleavage after the sequential action of corresponding restriction methylases and endonucleases [N11, M21]. By this approach large DNA fragments can be generated which is helpful in the construction of genomic libraries. The data given in both Tables IV and VI allow the design of novel sequence specificities. These procedures complement the creation of universal cleavage specificities applying class IIS enzymes and bivalent DNA adapter molecules [P17, S82].

Recognition sequences of restriction endonucleases and methylases--a review.

The properties and sources of all known endonucleases and methylases acting site-specifically on DNA are listed. The enzymes are crossindexed (Table I), classified according to homologies within their recognition sequences (Table II), and characterized within Table II by the cleavage and methylation positions, the number of recognition sites on the DNA of the bacteriophages lambda, phi X174 and M13mp7, the viruses Ad2 and SV40, the plasmids pBR322 and pBR328 and the microorganisms from which they originate. Other tabulated properties of the restriction endonucleases include relaxed specificities (Table III), the structure of the restriction fragment ends (Table IV), and the sensitivity to different kinds of DNA methylation (Table V). Table VI classifies the methylases according to the nature of the methylated base(s) within their recognition sequences. This table also comprises those restriction endonucleases, which are known to be inhibited by the modified nucleotides. Furthermore, this review includes a restriction map of bacteriophage lambda DNA based on sequence data. Table VII lists the exact nucleotide positions of the cleavage sites, the length of the generated fragments ordered according to size, and the effects of the Escherichia coli dam- and dcmI-coded methylases M X Eco dam and M X Eco dcmI on the particular recognition sites.