Methyltransferase DnmA is responsible for genome-wide N6-methyladenosine modifications at non-palindromic recognition sites in Bacillus subtilis.

The genomes of organisms from all three domains of life harbor endogenous base modifications in the form of DNA methylation. In bacterial genomes, methylation occurs on adenosine and cytidine residues to include N6-methyladenine (m6A), 5-methylcytosine (m5C), and N4-methylcytosine (m4C). Bacterial DNA methylation has been well characterized in the context of restriction-modification (RM) systems, where methylation regulates DNA incision by the cognate restriction endonuclease. Relative to RM systems less is known about how m6A contributes to the epigenetic regulation of cellular functions in Gram-positive bacteria. Here, we characterize site-specific m6A modifications in the non-palindromic sequence GACGmAG within the genomes of Bacillus subtilis strains. We demonstrate that the yeeA gene is a methyltransferase responsible for the presence of m6A modifications. We show that methylation from YeeA does not function to limit DNA uptake during natural transformation. Instead, we identify a subset of promoters that contain the methylation consensus sequence and show that loss of methylation within promoter regions causes a decrease in reporter expression. Further, we identify a transcriptional repressor that preferentially binds an unmethylated promoter used in the reporter assays. With these results we suggest that m6A modifications in B. subtilis function to promote gene expression.

KMT9 monomethylates histone H4 lysine 12 and controls proliferation of prostate cancer cells.

Histone lysine methylation is generally performed by SET domain methyltransferases and regulates chromatin structure and gene expression. Here, we identify human C21orf127 (HEMK2, N6AMT1, PrmC), a member of the seven-ß-strand family of putative methyltransferases, as a novel histone lysine methyltransferase. C21orf127 functions as an obligate heterodimer with TRMT112, writing the methylation mark on lysine 12 of histone H4 (H4K12) in vitro and in vivo. We characterized H4K12 recognition by solving the crystal structure of human C21orf127-TRMT112, hereafter termed 'lysine methyltransferase 9' (KMT9), in complex with S-adenosyl-homocysteine and H4K12me1 peptide. Additional analyses revealed enrichment for KMT9 and H4K12me1 at the promoters of numerous genes encoding cell cycle regulators and control of cell cycle progression by KMT9. Importantly, KMT9 depletion severely affects the proliferation of androgen receptor-dependent, as well as that of castration- and enzalutamide-resistant prostate cancer cells and xenograft tumors. Our data link H4K12 methylation with KMT9-dependent regulation of androgen-independent prostate tumor cell proliferation, thereby providing a promising paradigm for the treatment of castration-resistant prostate cancer.

Effects of dam and seqA genes on biofilm and pellicle formation in Salmonella.

In this study, the effects of dam and seqA genes on the formation of pellicle and biofilm was determined using five different Salmonella serovars S. Group C1 (DMC2 encoded), S. Typhimurium (DMC4 encoded), S. Virchow (DMC11 encoded), S. Enteritidis (DMC22 encoded), and S. Montevideo (DMC89 encoded). dam and seqA mutants in Salmonella serovars were performed by the single step lambda red recombination method. The mutants obtained were examined according to the properties of biofilm on the polystyrene surfaces and the pellicle formation on the liquid medium. As a result of these investigations, it was determined that the biofilm formation properties on polystyrene surfaces decreased significantly (p < 0.05) in all tested dam and seqA mutants, while the pellicle formation properties were lost in the liquid medium. When pBAD24 vector, containing the dam and seqA genes cloned behind the inducible arabinose promoter, transduced into dam and seqA mutant strains, it was determined that the biofilm formation properties on the polystyrene surfaces reached to the natural strains' level in all mutant strains. Also, the pellicle formation ability was regained in the liquid media. All these data demonstrate that dam and seqA genes play an important role in the formation of biofilm and pellicle structures in Salmonella serovars.

Substrate Specificity of the HEMK2 Protein Glutamine Methyltransferase and Identification of Novel Substrates.

Bacterial HEMK2 homologs initially had been proposed to be involved in heme biogenesis or to function as adenine DNA methyltransferase. Later it was shown that this family of enzymes has protein glutamine methyltransferase activity, and they methylate the glutamine residue in the GGQ motif of ribosomal translation termination factors. The murine HEMK2 enzyme methylates Gln(185) of the eukaryotic translation termination factor eRF1. We have employed peptide array libraries to investigate the peptide sequence recognition specificity of murine HEMK2. Our data show that HEMK2 requires a GQX3R motif for methylation activity. In addition, amino acid preferences were observed between the -3 and +7 positions of the peptide substrate (considering the target glutamine as 0), including a preference for Ser, Arg, and Gly at the +1 and a preference for Arg at the +7 position. Based on our specificity profile, we identified several human proteins that contain putative HEMK2 methylation sites and show that HEMK2 methylates 58 novel peptide substrates. After cloning, expression, and purification of the corresponding protein domains, we confirmed methylation for 11 of them at the protein level. Transfected CHD5 (chromodomain helicase DNA-binding protein 5) and NUT (nuclear protein in testis) were also demonstrated to be methylated by HEMK2 in human HEK293 cells. Our data expand the range of proteins potentially subjected to glutamine methylation significantly, but further investigation will be required to understand the function of HEMK2-mediated methylation in proteins other than eRF1.

Dam it's good! DamID profiling of protein-DNA interactions.

The interaction of proteins with chromatin is fundamental for several essential cellular processes. During the development of an organism, genes must to be tightly regulated both temporally and spatially. This is achieved through the action of chromatin-binding proteins such as transcription factors, histone modifiers, nucleosome remodelers, and lamins. Furthermore, protein-DNA interactions are important in the adult, where their perturbation can lead to disruption of homeostasis, metabolic dysregulation, and diseases such as cancer. Understanding the nature of these interactions is of paramount importance in almost all areas of molecular biological research. In recent years, DNA adenine methyltransferase identification (DamID) has emerged as one of the most comprehensive and versatile methods available for profiling protein-DNA interactions on a genomic scale. DamID has been used to map a variety of chromatin-binding proteins in several model organisms and has the potential for continued adaptation and application in the field of genomic biology. WIREs Dev Biol 2016, 5:25-37. doi: 10.1002/wdev.205 For further resources related to this article, please visit the WIREs website.

Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing.

Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle, two orthogonal strategies for reducing off-target cleavage, truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs), could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally, we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR-Cas nucleases and therefore provide a highly specific platform for performing genome editing.

Fidelity index determination of DNA methyltransferases.

DNA methylation is the most frequent form of epigenetic modification in the cell, which involves gene regulation in eukaryotes and protection against restriction enzymes in prokaryotes. Even though many methyltransferases exclusively modify their cognate sites, there have been reports of those that exhibit promiscuity. Previous experimental approaches used to characterize these methyltransferases do not provide the exact concentration at which off-target methylation occurs. Here, we present the first reported fidelity index (FI) for a number of DNA methyltransferases. We define the FI as the ratio of the highest amount of methyltransferase that exhibits no star activity (off-target effects) to the lowest amount that exhibits complete modification of the cognate site. Of the methyltransferases assayed, M.MspI and M.AluI exhibited the highest fidelity of ≥250 and ≥500, respectively, and do not show star activity even at very high concentrations. In contrast, M.HaeIII, M.EcoKDam and M.BamHI have the lowest fidelity of 4, 4 and 2, respectively, and exhibit star activity at concentrations close to complete methylation of the cognate site. The fidelity indexes provide vital information on the usage of methyltransferases and are especially important in applications where site specific methylation is required.

Effects of dam and/or seqA mutations on the fatty acid and phospholipid membrane composition of Salmonella enterica serovar Typhimurium.

We examined the phospholipids (Phls) and the membrane fatty acid (FA) composition in Salmonella enterica serovar Typhimurium dam and/or seqA mutants. Phosphatidylglycerol, phosphatidylethanolamine (PE), and cardiolipin (CL) are the major Phls present in all the strains and accounted for greater than 95% of the total lipid phosphorus. Phosphatidic acid and phosphatidylserine are the minor ones. The seqA mutant showed a decrease in PE and an increase in CL and phosphatidylglycerol proportion compared with the wild-type strain. The same changes were observed with the seqA dam double mutant. However, the dam mutation caused an unusual accumulation of CL with a significant decrease in the PE content, compared with the isogenic wild-type strain. FA composition of the total lipids and the different fractions containing Phls have been determined. The major saturated FAs (SFAs) and unsaturated FAs (UFAs) found were C(14:0), C(16:0) and C(16:1w7), C(18:1w9), respectively. Cyclic FAs, cyc(17:0) and cyc(19:0), were also present in appreciable amounts. Moreover, dam and/or seqA mutations caused a decrease in UFA/SFA ratio and there was a progressive reduction in the content of C(16:1w7) and C(18:1w9), going through the order seqA, dam/seqA, and dam mutants. This decrease in UFA content was compensated for in all strains by an increase in the corresponding C(17-) and C(19-) cyclic FAs. So these UFAs were converted to their cyclopropane derivatives, which resulted in a low UFA/SFA ratio. SeqA and Dam proteins might regulate FA biosynthesis and Phls composition of Salmonella enterica serovar Typhimurium.

Dam and its role in pathogenicity of Salmonella enterica.

Dam methylation is an essential factor involved in the virulence of an increasing number of bacterial pathogens including Salmonella enterica. Lack of Dam methylation causes severe attenuation in animal models. It has been proposed that dysregulation of Dam activity is potentially a general strategy for the generation of vaccines against bacterial pathogens. In this review, we focus our attention on the role of methylation by Dam protein in regulating bacterial gene expression and virulence in Salmonella enterica.

Escherichia coli DNA adenine methyltransferase: intrasite processivity and substrate-induced dimerization and activation.

Methylation of GATC sites in Escherichia coli by DNA adenine methyltransferase (EcoDam) is essential for proper DNA replication timing, gene regulation, and mismatch repair. The low cellular concentration of EcoDam and the high number of GATC sites in the genome (approximately 20000) support the reliance on methylation efficiency-enhancing strategies such as extensive intersite processivity. Here, we present evidence that EcoDam has evolved other unique mechanisms of activation not commonly observed with restriction-modification methyltransferases. EcoDam dimerizes on short, synthetic DNA, resulting in enhanced catalysis; however, dimerization is not observed on large genomic DNA where the potential for intersite processive methylation precludes any dimerization-dependent activation. An activated form of the enzyme is apparent on large genomic DNA and can also be achieved with high concentrations of short, synthetic substrates. We suggest that this activation is inherent on polymeric DNA where either multiple GATC sites are available for methylation or the partitioning of the enzyme onto nonspecific DNA is favored. Unlike other restriction-modification methyltransferases, EcoDam carries out intrasite processive catalysis whereby the enzyme-DNA complex methylates both strands of an unmethylated GATC site prior to dissociation from the DNA. This occurs with short 21 bp oligonucleotides and is highly dependent upon salt concentrations. Kinetic modeling which invokes enzyme activation by both dimerization and excess substrate provides mechanistic insights into key regulatory checkpoints for an enzyme involved in multiple, diverse biological pathways.

Acid pre-adaptation enhances virulence of Salmonella enterica serovar Typhimurium dam mutant.

It is well established that success or failure of bacterial pathogens during infection relies upon its ability to overcome many lethal environments in the host such as acidity, osmolarity and bile salts. In the present study, we have studied the effects of acid adaptation on the virulence of Salmonella enterica serovar Typhimurium dam mutant. Our results indicated that LD(50) of adapted strains were lower than those of control strains. Also, the in vivo assays have shown that the development of a systemic infection is slower for control strains than for adapted strains. In addition, the number of acid-adapted mutants colonizing spleen and liver is higher than control strains. Adhesion and invasion experiments were performed in order to compare the pathogenicity of Salmonella. No significant differences were shown between pre-treated and non-adapted strains. According to these results, we report that acid adaptation of Salmonella enterica serovar Typhimurium dam mutants can increase their in vivo virulence in mice.

EcoR124I: from plasmid-encoded restriction-modification system to nanodevice.

SUMMARY: Plasmid R124 was first described in 1972 as being a new member of incompatibility group IncFIV, yet early physical investigations of plasmid DNA showed that this type of classification was more complex than first imagined. Throughout the history of the study of this plasmid, there have been many unexpected observations. Therefore, in this review, we describe the history of our understanding of this plasmid and the type I restriction-modification (R-M) system that it encodes, which will allow an opportunity to correct errors, or misunderstandings, that have arisen in the literature. We also describe the characterization of the R-M enzyme EcoR124I and describe the unusual properties of both type I R-M enzymes and EcoR124I in particular. As we approached the 21st century, we began to see the potential of the EcoR124I R-M enzyme as a useful molecular motor, and this leads to a description of recent work that has shown that the R-M enzyme can be used as a nanoactuator. Therefore, this is a history that takes us from a plasmid isolated from (presumably) an infected source to the potential use of the plasmid-encoded R-M enzyme in bionanotechnology.

Overproduction of DNA adenine methyltransferase alters motility, invasion, and the lipopolysaccharide O-antigen composition of Yersinia enterocolitica.

DNA adenine methyltransferase (Dam) not only regulates basic cellular functions but also interferes with the proper expression of virulence factors in various pathogens. We showed previously that for the human pathogen Yersinia enterocolitica, overproduction of Dam results in increased invasion of epithelial cells. Since invasion and motility are coordinately regulated in Y. enterocolitica, we analyzed the motility of a Dam-overproducing (Dam(OP)) strain and found it to be highly motile. In Dam(OP) strains, the operon encoding the master regulator of flagellum biosynthesis, flhDC, is upregulated. We show that the increased invasion is not due to enhanced expression of known and putative Y. enterocolitica invasion and adhesion factors, such as Inv, YadA, Ail, Myf fibrils, Pil, or Flp pili. However, overproduction of Dam no longer results in increased invasion for an inv mutant strain, indicating that Inv is necessary for increased invasion after overproduction of Dam. Since we show that overproduction of Dam results in an increased amount of rough lipopolysaccharide (LPS) molecules lacking O-antigen side chains, this implies that reduced steric hindrance by LPS might contribute to increased invasion by a Y. enterocolitica Dam(OP) strain. Our data add an important new aspect to the various virulence-associated phenotypes influenced by DNA methylation in Y. enterocolitica and indicate that Dam targets regulatory processes modulating the composition and function of the bacterial surface.

A putative DNA adenine methyltransferase is involved in Yersinia pseudotuberculosis pathogenicity.

Some adenine methyltransferases have been shown not only to protect specific DNA restriction sites from cleavage by a restriction endonuclease, but also to play a role in various bacterial processes and sometimes in bacterial virulence. This study focused on a type I restriction-modification system (designated yrmI) of Y. pseudotuberculosis. This system is composed of three adjacent genes which could potentially encode an N6-adenine DNA methylase (YamA), an enzyme involved in site-specific recognition (YrsA) and a restriction endonuclease (YreA). Screening of 85 isolates of Y. pestis and Y. pseudotuberculosis indicated that the yrmI system has been lost by Y. pestis and that yamA (but not yrsA or yreA) is present in all Y. pseudotuberculosis strains tested, suggesting that it may be important at some stages of the epidemiological cycle of this species. To further investigate the role of yamA in Y. pseudotuberculosis survival, multiplication or virulence, a DeltayamA mutant of Y. pseudotuberculosis IP32953 was constructed by allelic exchange with a kanamycin cassette. The fact that DeltayamA mutants were obtained indicated that this gene is not essential for Y. pseudotuberculosis viability. The IP32953DeltayamA mutant strain grew as well as the wild-type in a rich medium at both 28 degrees C and 37 degrees C. It also grew normally in a chemically defined medium at 28 degrees C, but exhibited a growth defect at 37 degrees C. In contrast to the Dam adenine methyltransferase, a mutation in yamA did not impair the functions of DNA repair or resistance to detergents. However, the DeltayamA mutant exhibited a virulence defect in a mouse model of intragastric infection. The in silico analysis indicated that the chromosomal region carrying the Y. pseudotuberculosis yrmI locus has been replaced in Y. pestis by a horizontally acquired region which potentially encodes another methyltransferase. YamA might thus be dispensable for Y. pestis growth and virulence because this species has acquired another gene fulfilling the same functions.

A Dam methylation mutant of Klebsiella pneumoniae is partially attenuated.

In Klebsiella pneumoniae, a chromosomal insertion mutation was constructed in the dam gene, which encodes DNA adenine methylase (Dam), resulting in a mutant unable to methylate specific nucleotides. In some bacteria, the Dam methylase has been shown to play an important role in virulence gene regulation as well as in methyl-directed mismatch repair and the regulation of replication initiation. Disruption of the normal Dam function by either eliminating or greatly increasing expression in several organisms has been shown to cause attenuation of virulence in murine models of infection. In K. pneumoniae, a mutation-eliminating Dam function is shown here to result in only partial attenuation following intranasal and intraperitoneal infection of Balb/C mice.

Altered levels of Salmonella DNA adenine methylase are associated with defects in gene expression, motility, flagellar synthesis, and bile resistance in the pathogenic strain 14028 but not in the laboratory strain LT2.

Comparative genomic analysis has revealed limited strain diversity between Salmonella pathogenic and nonpathogenic isolates. Thus, some of the relative virulence and host-immune response disparities may be credited to differential gene regulation rather than gross differences in genomic content. Here we show that altered levels of Salmonella DNA adenine methylase (Dam) resulted in acute defects in virulence-associated gene expression, motility, flagellin synthesis, and bile resistance in the Salmonella pathogenic strain 14028 but not in avirulent laboratory strain LT2. The defects in motility exhibited by 14028 in response to altered Dam levels was not dependent on the presence of the regulatory protein, RpoS. The transitioning between flagellar types (phase variation) was also differentially regulated in 14028 versus LT2 in response to dam levels, resulting in distinct differences in flagellin expression states. These data suggest that differential gene regulation may contribute to the relative virulence disparities observed between Salmonella serovars that are closely related at the DNA level.

Conformational transitions as determinants of specificity for the DNA methyltransferase EcoRI.

Changes in DNA bending and base flipping in a previously characterized specificity-enhanced M.EcoRI DNA adenine methyltransferase mutant suggest a close relationship between precatalytic conformational transitions and specificity (Allan, B. W., Garcia, R., Maegley, K., Mort, J., Wong, D., Lindstrom, W., Beechem, J. M., and Reich, N. O. (1999) J. Biol. Chem. 274, 19269-19275). The direct measurement of the kinetic rate constants for DNA bending, intercalation, and base flipping with cognate and noncognate substrates (GAATTT, GGATTC) of wild type M.EcoRI using fluorescence resonance energy transfer and 2-aminopurine fluorescence studies reveals that DNA bending precedes both intercalation and base flipping, and base flipping precedes intercalation. Destabilization of these intermediates provides a molecular basis for understanding how conformational transitions contribute to specificity. The 3500- and 23,000-fold decreases in sequence specificity for noncognate sites GAATTT and GGATTC are accounted for largely by an approximately 2500-fold increase in the reverse rate constants for intercalation and base flipping, respectively. Thus, a predominant contribution to specificity is a partitioning of enzyme intermediates away from the Michaelis complex prior to catalysis. Our results provide a basis for understanding enzyme specificity and, in particular, sequence-specific DNA modification. Because many DNA methyltransferases and DNA repair enzymes induce similar DNA distortions, these results are likely to be broadly relevant.

DomainSieve: a protein domain-based screen that led to the identification of dam-associated genes with potential link to DNA maintenance.

MOTIVATION: The Dam methyltransferase (DamMT) activity, broadly distributed in association with restriction endonucleases, as part of the restriction-modification defense systems, has evolved to become intimately associated with essential biological functions in a few organisms. In Escherichia coli, DamMT is involved in multiple aspects of DNA maintenance, replication initiation, daughter chromosome segregation, DNA mismatch repair, gene expression control, etc. The participation of DamMT in such a diverse set of functions required that other genes adapted, or emerged through evolution, in response to the DamMT-induced modification of the genomic environment. One example is SeqA, a protein that senses the methylation status of the origin of replication of the chromosome to control the timing of replication initiation. Interestingly, seqA is only present in a few DamMT-specifying proteobacteria. This observation led us to hypothesize that other genes, specifying related functions, might also be found in these organisms. To test this hypothesis, we implemented a large-scale comparative genomic screen meant to identify genes specifying DNA methylation sensing domains, probably involved in DNA maintenance functions. RESULTS: We carried out a phylogenetic analysis of DamMT, identifying two contrasting behaviors of the protein. Based on this phylogeny, we defined precisely a set of genomes, in which the protein activity is likely to be involved in DNA maintenance functions, the 'resident' dam genomes. We defined a second set of genomes, in which DamMT is not resident. We developped a new tool, 'DomainSieve', in order to screen these two sets for protein domains that are strictly associated with 'resident' dam genomes. This approach was rewarding and generated a list of genes, among which some, at least, specify activities with clear linkage to DamMT-dependent DNA methylation and DNA maintenance. AVAILABILITY: DomainSieve is implemented as a web resource and is accessible at http://stat.genopole.cnrs.fr/ds/.

HindIII(+/-) polymorphism of the Y chromosome, blood pressure, and serum lipids: no evidence of association in three white populations.

BACKGROUND: Male sex is associated with elevated levels of cardiovascular risk factors, including higher blood pressure (BP). Genetic variants on the Y chromosome may contribute to explain the sexual dimorphism in cardiovascular diseases. Among them, the HindIII(+/-) polymorphism of the male-specific region of the Y chromosome has been associated with BP and serum cholesterol levels, with conflicting results. We evaluated the association between the HindIII(+/-) polymorphism, prevalence of hypertension, BP, and serum lipid levels in a large sample of white men and the previously reported epistatic interaction between HindIII(+/-) and the -344C/T polymorphism of the aldosterone synthase gene (CYP11B2) on BP. METHODS: From three European populations (UK n = 422; Belgium n = 313; Italy n = 1248) 1983 white men were phenotyped for BP and serum lipids and genotyped for HindIII(+/-) site and for -344C/T polymorphism in the promoter of CYP11B2 by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: A higher frequency of the HindIII (+) was found in Italians (63%) as compared to both British (31%) and Belgians (28%) (P < .0001). We found no evidence of association of the HindIII(+/-) site with prevalence of hypertension, BP, and serum lipids in any of the three European populations examined and in the entire sample. Finally, we did not observe any interaction between the HindIII(+/-) polymorphism and the -344C/T variant of CYP11B2 on BP. CONCLUSIONS: Our data do not support the hypothesis that the HindIII(+/-) site of the Y chromosome is a marker of cardiovascular risk in white men, highlighting the need for replication in genetic association studies.

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.