Effect of light quality and 5-azacytidine on genomic methylation and stem elongation in two ecotypes of Stellaria longipes

Changes in cytosine methylation are known to occur in response to various environmental stimuli, therefore, we looked at methylation changes in relation to stem elongation. More specifically, we investigated the response of genomic cytosine methylation to irradiance-mediated plasticity of stem elongation in two ecotypes of Stellaria longipes . Ramets of S. longipes were grown under high and low ratios of red/far-red light (F/FR; 3.7 and 0.7, respectively). Stem elongation and methylated cytosine content were measured over a period of 7 days. Ramets of S. longipes demonstrated the highest level of demethylation after 4 days of long-day warm (LDW) treatment, which coincides with the first day of rapid stem elongation initiation. The extent of demethylation associated with day 4 depended upon the relative ratio of R/FR light. In particular, those plants treated with low R/FR light ratios showed a lower level of methylation, and were taller than the high R/FR light grown counterparts. In addition, prairie ecotype plants demonstrated lower day 4 methylation levels, as well as longer day 7 stem lengths, than the alpine ecotype plants within the same R/FR light treatments. To investigate if the degree of methylation was a crucial factor in controlling the stem elongation response, ramets of both alpine and prairie plants were grown in MS media supplemented with 5-azacytidine (5-AzaC), and grown for 14 days under a R/FR ratio of 3.7 and two different PAR values. 5-AzaC treatments demonstrated that the prairie ecotype plants required greater doses of 5-AzaC, and thus lower methylation levels, than the alpine ecotype plants in order to promote maximal stem elongation. These observations suggest that DNA demethylation is involved in the shade-avoidance response.     

Differentiation of metaxylem cell line in the root ofAllium cepa L.

Summary The differentiation processes of the metaxylem cell line in the root ofAllium cepa are characterized by amplification phenomena of repetitive DNA sequences mainly localized in heterochromatic regions of metaphase chromosomes. Moreover, these sequences are heavily methylated. This paper presents additional results on variation in endogenous DNA methylation in different developing root segments. The results show that methylation is higher in apical meristematic cells than the differentiating segments; contrastingly, total RNA synthesis seems to be correlated with undermethylation. Addition of labelled methyl groups to DNA by eukaryotic methylase, DNA digestions with different restriction enzymes specific for methylated sites and HPLC analysis confirmed the above results. Moreover, variation in methylation levels during differentiation occur not only at the internal cytosine of the-CCG-sites, but also at external cytosine. Furthermore, methylation affects other sites containing the trinucleotides-CXG-. In conclusion, root differentiation inAllium cepa seems to be correlated with gene activation modulated by the methylation/demethylation of particular DNA sequences.     

Characterization of the level,target sites and inheritance of cytosine methylation in tomato nuclear DNA

The tomato nuclear genome was determined to have a G+C content of 37% which is among the lowest reported for any plant species. Non-coding regions have a G+C content even lower (32% average) whereas coding regions are considerably richer in G+C (46%).5-methyl cytosine was the only modified base detected and on average 23% of the cytosine residues are methylated. Immature tissues and protoplasts have significantly lower levels of cytosine methylation (average 20%) than mature tissues (average 25%). Mature pollen has an intermediate level of methylation (22%). Seeds gave the highest value (27%), suggesting de novo methylation after pollination and during seed development.Based on isoschizomer studies we estimate 55% of the CpG target sites (detected by Msp I/Hpa II) and 85% of the CpNpG target sites (detected by Bst NI/Eco RI)are methylated. Unmethylated target sites (both CpG and CpNpG) are not randomly distributed throughout the genome, but frequently occur in clusters. These clusters resemble CpG islands recently reported in maize and tobacco.The low G+C content and high levels of cytosine methylation in tomato may be due to previous transitions of 5mCT. This is supported by the fact that G+C levels are lowest in non-coding portions of the genome in which selection is relaxed and thus transitions are more likely to be tolerated. This hypothesis is also supported by the general deficiency of methylation target sites in the tomato genome, especially in non-coding regions.Using methylation isoschizomers and RFLP analysis we have also determined that polymorphism between plants, for cytosine methylation at allelic sites, is common in tomato. Comparing DNA from two tomato species, 20% of the polymorphisms detected by Bst NI/Eco RII could be attributed to differential methylation at the CpNpG target sites. With Msp I/Hpa II, 50% of the polymorphisms were attributable to methylation (CpG and CpNpG sites). Moreover, these polymorphisms were demonstrated to be inherited in a mendelian fashion and to co-segregate with the methylation target site and thus do not represent variation for transacting factors that might be involved in methylation of DNA. The potential role of heritable methylation polymorphism in evolution of gene regulation and in RFLP studies is discussed.     

mCCG methylation in angiosperms

Two different methods were used to investigate the abundance of cytosine methylation at the outer (5′) position in 5′-CCG-3′ trinucleotides in angiosperm genomes. Mspl is unable to cut its target site if the outer cytosine is methylated (5′-^mCCGG-3′). Using Mspl restriction analysis, it was shown that 5′-^mCCG-3′ is present in all angiosperm genomes examined, and that the amount of cytosine methylation at this site varies between species. Subsequently, direct measurements were made of the amount of methylation at both cytosines in a subset of 5′-CCG-3′ trinucleotides in the Arabidopsis thaliana genome. Based upon these analyses, it was estimated that approximately 20–30% of 5′-CCG-3′ trinucleotides in A. thaliana are methylated at the outer cytosine. Approximately 20% of the 5′-CCG-3′ trinucleotides contain 5-methyl-cytosine at the inner cytosine position, which corresponds to a previous determination of 5′-^mCG-3′ methylation in A. thaliana. The implications of 5′-^mCCG-3′ methylation are discussed.     

Cytosine methylated DNA synthesized by Taq polymerase used to assay methylation sensitivity of restriction endonuclease HinfI.

We have studied the resistance of cytosine methylated DNA to digestion by the restriction endonuclease HinfI, using a simple PCR procedure to synthesize DNA of known sequence in which every cytosine is methylated at the 5 position. We find that HinfI cannot digest cytosine methylated DNA at the concentrations normally used in restriction digests. Complete digestion is possible using a vast excess of enzyme; under these conditions, the rate of HinfI digestion for cytosine methylated DNA is at least 1440-fold slower than for unmethylated DNA. The presence of an additional methylated cytosine at the degenerate position internal to the recognition sequence does not appear to increase the resistance to HinfI digestion. We also tested HhaII, an isoschizomer of HinfI, and found that it is completely inactive on cytosine methylated DNA. The procedure we have used should be of general applicability in determination of the methylation sensitivities of other restiction enzymes, as well as studies of the effects of methylation on gene expression in direct DNA transfer experiments.     

Hypermethylation of tobacco heterochromatic loci in response to osmotic stress

 Plants have to cope with a number of envi-ronmental stresses which may potentially induce genetic and epigenetic changes and thus contribute to genome variability. In the present study we inspected the DNA methylation status of two heterochromatic loci (defined with repetitive DNA sequences HRS60 and GRS) in a tobacco cell culture exposed to osmotic stress. Investigations were performed on a TBY-2 cell suspension culture, and the stress was elicited with NaCl or D-mannitol. Using the restriction enzymes MspI/HpaII and MboI/Sau3AI in combination with Southern hydridization we observed a reversible hypermethylation of the external cytosine at the CpCpG trinucleotides in cells grown under mild osmotic stress equal to a NaCl concentration of 10 g/l. There were no changes in the methylation of the internal cytosine as the CpG dinucleotides within the CCGG motifs (HpaII sites) appeared to be fully methylated in tobacco DNA repetitive sequences under normal physiological conditions. The data suggest epigenetic changes in the plant genome based on de novo methylation of DNA in response to environmental stress. Received: 26 November 1996/Accepted: 20 December 1996     

Detection of DNA methylation pattern in thidiazuron-induced blueberry callus using methylation-sensitive amplification polymorphism

During the normal developmental process, programmed gene expression is an essential phenomenon in all organisms. In eukaryotes, DNA methylation plays an important role in the regulation of gene expression. The extent of cytosine methylation polymorphism was evaluated in leaf tissues collected from the greenhouse grown plants and in in vitro-derived callus of three lowbush and one hybrid blueberry genotypes, using methylation-sensitive amplification polymorphism (MSAP) technique. Callus formation started from the leaf segments after 4 weeks of culture on a thidiazuron (TDZ) containing medium. Maximum callus formation (98 %) was observed in the hybrid blueberry at 1.0 mg dm^-3 TDZ. Although noticeable changes in cytosine methylation pattern were detected within the MSAP profiles of both leaf and callus tissues, methylation events were more polymorphic in calli than in leaf tissues. The number of methylated CCGG sites varied significantly within the genotypes ranging from 75 to 100 in leaf tissues and from 215 to 258 in callus tissues. Differences in the methylation pattern were observed not only in a tissue-specific manner but also within the genotype in a treatment specific manner. These results demonstrated the unique effect of TDZ and the tissue culture process on DNA methylation during callus development.     

Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation‐sensitive amplified polymorphism

Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal‐contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation‐sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal‐contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal‐contaminated site compared to uncontaminated populations. Other genotypes from a different metal‐contaminated site within the same region appear to be recalcitrant to metal‐induced DNA alterations even ≥30 years of tree life exposure to nickel and copper . MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal‐contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.     

Differential DNA methylation between two wing phenotypes adults of Sogatella furcifera

Sogatella furcifera is a major rice pest with wing dimorphism . DNA methylation is an important epigenetic modification that plays a role in gene regulation and phenotype variation in most organisms. The objective of the current research was to survey the frequencies and variation of cytosine methylation at CCGG sequences in macropterous female adults (MFA) and brachypterous female adults (BFA) of S. furcifera, and to determine the occurrence of methylation changes associated with wing phenotypes by using methylation‐sensitive amplification polymorphism (MSAP). No differences were found in the average proportions of methylated CCGG sites between MFA and BFA, but there were significant differences for methylation patterns between MFA and BFA. The fully methylated ratio was 5.81% in BFA, much higher than 2.40% in MFA; while the hemi‐methylated ratio was 4.35% in BFA, much lower than 8.35% in MFA. These results provide circumstantial evidence that DNA methylation might be related to wing phenotype variation in S. furcifera. We also cloned and got 14 satisfactory sequences, which displayed variable cytosine methylation patterns between MFA and BFA. All these data will facilitate the researches on the epigenetic mechanisms of insect wing polymorphism. genesis 51:819–826. © 2013 Wiley Periodicals, Inc.     

Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification.

Bisulfite converts non-methylated cytosine in DNA to uracil leaving 5-methylcytosine unaltered. Here, predicted changes in restriction enzyme sites following reaction of genomic DNA with bisulfite and amplification of the product by the polymerase chain reaction (PCR) were used to assess the methylation of CpG sites. This procedure differs from conventional DNA methylation analysis by methylation-sensitive restriction enzymes because it does not rely on an absence of cleavage to detect methylated sites, the two strands of DNA produce different restriction enzyme sites and may be differentially analyzed, and closely related sequences may be separately analyzed by using specific PCR primers.     

Detection of DNA Methylation by Dnmt3a Methyltransferase using Methyl-Dependent Restriction Endonucleases

DNA methylation at cytosine residues in CpG sites by DNA methyltransferases (MTases) is associated with various cell processes. Eukaryotic MTase Dnmt3a is the key enzyme that establishes the de novo methylation pattern. A new in vitro assay for DNA methylation by murine MTase Dnmt3a was developed using methyl-dependent restriction endonucleases (MD-REs), which specifically cleave methylated DNA. The Dnmt3a catalytic domain (Dnmt3a-CD) was used together with KroI and PcsI MD-REs. The assay consists in consecutive methylation and cleavage of fluorescently labeled DNA substrates, then the reaction products are visualized in polyacrylamide gel to determine the DNA methylation efficiency. Each MD-RE was tested with various substrates, including partly methylated ones. PcsI was identified as an optimal MDRE. PcsI recognizes two methylated CpG sites located 7 bp apart, the distance roughly corresponding to the distance between the active centers of the Dnmt3a-CD tetramer. An optimal substrate was designed to contain two methylated cytosine residues and two target cytosines in the orientation suitable for methylation by Dnmt3a-CD. The assay is reliable, simple, and inexpensive and, unlike conventional methods, does not require radioactive compounds. The assay may be used to assess the effectiveness of Dnmt3a inhibitors as potential therapeutic agents and to investigate the features of the Dnmt3a-CD function.     

Novel methylation at GpC dinucleotide in the fish Sparus aurata genome

To date, vertebrate DNA has been found methylated at the 5 position of cytosine exclusively in dinucleotide CpG or CpNpG stretches. On the the other hand, we determined that cytosine was methylated unusually in dinucleotide GpC at 5-GGCC-3 sequences in the teleost Sparus aurata EcoRI satellite DNA family. This finding is the first example of methylated GpC sequences in the eukaryotic genomes. At this regard, we have examined the relative methylation levels at this site of the highly repetitive EcoRI satellite DNA family from Sparus aurata different tissues. The EcoRI repeat was remarkably more methylated in male germ cells but hypomethylated in female germ cells at the Hae III restriction site ( GpC). The novel modification and the differential methylation pattern suggest that EcoRI satellite could have a structural and/or functional role at the centromeres of Sparus aurata.     

Identification of restriction endonucleases sensitive to 5-cytosine methylation at non-CpG sites,including expanded (CAG)n/(CTG)n repeats

Most epigenetic studies assess methylation of 5'-CpG-3' sites but recent evidence indicates that non-CpG cytosine methylation occurs at high levels in humans and other species. This is most prevalent at 5'-CHG-3', where H = A, C or T, and it preferentially occurs at 5'-CpA-3' and 5'-CpT-3' sites. With the goal of facilitating the detection of non-CpG methylation, the restriction endonucleases ApeKI, BbvI, EcoP15I, Fnu4HI, MwoI and TseI were assessed for their sensitivity to 5-methylcytosine at GpCpA, GpCpT, GpCpC or GpCpG sites, where methylation is catalyzed by the DNA 5-cytosine 5'-GpC-3' methyltransferase M.CviPI. We tested a variety of sequences including various plasmid-based sites, a cloned disease-associated (CAG)83?(CTG)83 repeat and in vitro synthesized tracts of only (CAG)500?(CTG)500 or (CAG)800?(CTG)800. The repeat tracts are enriched for the preferred CpA and CpT motifs. We found that none of the tested enzymes can cleave their recognition sequences when they are 5'-GpC-3' methylated. A genomic site known to convert its non-CpG methylation levels upon C2C12 differentiation was confirmed through the use of these enzymes. These enzymes can be useful in rapidly and easily determining the most common non-CpG methylation status in various sequence contexts, as well as at expansions of (CAG)n?(CTG)n repeat tracts associated with diseases like myotonic dystrophy and Huntington disease.     

The effect of C(5) cytosine methylation at CpG sequences on mitomycin-DNA bonding profiles

Recent studies have documented that cytosine C(5) methylation of CpG sequences enhances mitomycin C (1) adduction. The reports differ on the extent and uniformity of 1 modification at the nucleotide level. We have determined the bonding profiles for mitomycin monoalkylation in two DNA restriction fragments where the CpG sequences were methylated. Three mitomycin substrates were used and two different enzymatic assays employed to monitor the extent of drug modification at the individual base sites. Drug DNA modification was accomplished with I and 10-decarbamoylmitomycin C (2) under reductive (Na2S2O4) condilions and with N-methyl-7-methoxyaziridinomitosene (3) under nonreductive conditions. The UvrABC incision assay permitted us to quantitate the sites of drug adduction, and the lambda-exonuclease stop assay provided a qualitative estimation of drug-DNA modification consistent with the UvrABC data. We learned that C(5) cytosine methylation (m5C) enhanced the extent of overall DNA modification. Using the UvrABC endonuclease assay, we found that modification by 1 increased 2.0 and 7.4 times for the two DNA restriction fragments. Analysis of the modification sites at the nucleotide sequence level revealed that guanine (G) was the only base modified and that the overall increased level of DNA adduction was due to enhanced modification of select m5CpG* (G* = mitomycin (mitosene) adduction sites) loci compared with CpG* sites: the largest differences reached two orders of magnitude. Significantly, not all CpG* sites underwent increased drug adduction upon C(5) cytosine methylation. The effect of C(5) cytosine methylation on the drug adduction profiles was less pronounced for G* sites located within dinucleotide sequences other than CpG*. We observed that DNA methylation often led to slightly diminished adduction levels at these sites. The different m5CpG* adduction patterns provided distinctive sequence-selective bonding profiles for 1-3. We have attributed the large differences in guanine reactivity to DNA structural factors created, in part, by C(5) cytosine methylation. The significance of these findings in cancer chemotherapy is briefly discussed.