Assessing the DNA methylation status of single cells with the comet assay

The comet assay (single cell gel electrophoresis) is a cost-effective, sensitive, and simple technique that is traditionally used for analyzing and quantifying DNA damage in individual cells. The aim of this study was to determine whether the comet assay could be modified to detect changes in the levels of DNA methylation in single cells. We used the difference in methylation sensitivity of the isoschizomeric restriction endonucleases HpaII and MspI to demonstrate the feasibility of the comet assay to measure the global DNA methylation level of individual cells. The results were verified with the well-established cytosine extension assay. We were able to show variations in DNA methylation after treatment of cultured cells with 5-azacytidine and succinylacetone, an accumulating metabolite in human tyrosinemia type I.     

Sequence specificity of cytosine methylation in the DNA of the Chinese hamster ovary (CHO-K1) cell line

We have determined the DNA renaturation kinetics for those DNA sequences of the Chinese hamster ovary (CHO-K1) cells in which enzymatic cytosine methylation occurred immediately after strand synthesis and for those in which methylation was delayed after strand synthesis. DNA sequences showing immediate or delayed methylation were found to be distributed throughout all repetition classes of the DNA of these cells, with a slight concentration of immediate methylation in moderately repetitive sequences and with delayed methylation being slightly over-represented in the highly repetitive fraction. However, DNA sequences showing both classes of methylation were represented equally in unique DNA sequences. We interpret these data to mean that the methylase acting near the replication forks (the ‘immediate’ methylase) is a relatively inefficient enzyme, missing some 20% of hemimethylated sites produced by DNA replication in these cells. We suggest that the methylase performing maintenance methylation at sites remote from the replication forks (the ‘delayed’ methylase) is simply a back-up enzyme for the first and that it has no true sequence specificity. The implications of this for the function(s) of DNA methylation in mammalian cells are discussed.     

CLOCK,PER2 and BMAL1 DNA Methylation: Association with Obesity and Metabolic Syndrome Characteristics and Monounsaturated Fat Intake

The circadian clock system instructs 24-h rhythmicity on gene expression in essentially all cells, including adipocytes, and epigenetic mechanisms may participate in this regulation. The aim of this research was to investigate the influence of obesity and metabolic syndrome (MetS) features in clock gene methylation and the involvement of these epigenetic modifications in the outcomes. Sixty normal-weight, overweight and obese women followed a 16-weeks weight reduction program. DNA methylation levels at different CpG sites of CLOCK, BMAL1 and PER2 genes were analyzed by Sequenom's MassARRAY in white blood cells obtained before the treatment. Statistical differences between normal-weight and overweight?+?obese subjects were found in the methylation status of different CpG sites of CLOCK (CpGs 1, 5-6, 8 and 11-14) and, with lower statistical significance, in BMAL1 (CpGs 6-7, 8, 15 and 16-17). The methylation pattern of different CpG sites of the three genes showed significant associations with anthropometric parameters such as body mass index and adiposity, and with a MetS score. Moreover, the baseline methylation levels of CLOCK CpG 1 and PER2 CpGs 2-3 and 25 correlated with the magnitude of weight loss. Interestingly, the percentage of methylation of CLOCK CpGs 1 and 8 showed associations with the intake of monounsaturated and polyunsaturated fatty acids. This study demonstrates for the first time an association between methylation status of CpG sites located in clock genes (CLOCK, BMAL1 and PER2) with obesity, MetS and weight loss. Moreover, the methylation status of different CpG sites in CLOCK and PER2 could be used as biomarkers of weight-loss success, particularly CLOCK CPGs 5-6. (Author correspondence: garaulet@um.es)     

The evolution of CpNpG methylation in plants

Summary CpNpG and CpG methylation was surveyed in a range of vascular and nonvascular plants to determine firstly when CpNpG methylation evolved and secondly whether the two methylation systems found in higher plants were likely to be under common or separate control. Although both systems exist in a wide range of vascular plant taxa, the nonvascular plant taxa appear to contain only CpNpG methylation and this in only very limited amounts. The data suggest that both systems may have evolved at the same time and that speciation involved loss of one or the other methylation system or the evolution of differentiation stage-specific control systems.     

Cytosine methylation levels in the genome ofStellaria longipes

Environment-induced alteration of DNA methylation levels was investigated inStellaria longipes (Caryophyllaceae). Total cytosine methylation levels were measured using HPLC in 6 genets representing two ecotypes (alpine and prairie) grown in short day photoperiod and cold temperature (SDC) and long day photoperiod and warm temperature (LDW) conditions. The levels of methylated cytosine were 16.54-22.20% among the three genets from the alpine and 12.62–24.70% in the three prairie genets when they were grown in SDC conditions. After the plants were moved to the LDW conditions, all of the three genets from the alpine showed decreasing levels of DNA methylation up to 6 days of growing in LDW. When the plants continued to grow in LDW for 10 days the average methylation level in the prairie genotypes showed no significant change. Cytosine methylation level was also detected inHpall andSau3AI restriction sites using the coupled restriction enzyme digestion and random amplification (CRED-RA) procedure, in which 15 random primers were used. Fifty per cent of the amplified bands with either or both of these two restriction sites were identified as being methylated in an alpine genotype (1C) and approximately 66% were found to be methylated in a prairie genotype (7C). It was observed that the change in growing conditions from SDC to LDW induced a decrease of methylation levels inHpall sites.     

Methylated cytosine at Dcm (CC T A GG) sites in Escherichia coli: Possible function and evolutionary implications

The frequency and distribution of methylated cytosine (5-MeC) at CC _T ^A GG (Dcm sites) in 49 E. coli DNA loci (207,530 bp) were determined. Principal observations of this analysis were: (1) Dcm frequency was higher than expected from random occurrence but lower than calculated with Markov chain analysis; (2) CCTGG sites were found more frequently in coding than in noncoding regions, while the opposite was true for CCAGG sites; (3) Dcm site distribution does not exhibit any identifiably regular pattern on the chromosome; (4) Dcm sites at oriC are probably not important for accurate initiation of DNA replication; (5) 5-MeC in codons was more frequently found in first than in second and third positions; (6) there are probably few genes in which the mutation rate is determined mainly by DNA methylation. It is proposed that the function of Dcm methylase is to protect chromosomal DNA from restriction-enzyme EcoRII. The Dcm methylation contribution to determine frequency of oligonucleotides, mutation rate, and recombination level, and thus evolution of the E. coli genome, could be interpreted as a consequence of the acquisition of this methylation.Correspondence to: M.C. Gómez-Eichelmann     

Parental 5-methylcytosine methylation patterns are stable upon inter-species hybridization of Xiphophorus (Teleostei: Poeciliidae) fish

Cytosine methylation appears to be established as an important DNA base modification involved in regulation of gene expression but is poorly understood from an evolutionary viewpoint. Xiphophorus progeny from inter-species crosses and backcrosses that are utilized in contemporary tumor induction studies were analyzed for cytosine methylation pattern inheritance using Southern blot analyses. Methylation patterns at CCGG sequences of 411 independent chromosomes in three distinct inter-species crosses were analyzed. In every case the non-recurrent parental methylation pattern remained unaltered for each of the genes studied, once introduced into the recurrent parental genetic background. Through F₁ inter-species hybridization and succeeding meiosises leading to first generation (BC₁) and second generation (BC₂) backcross hybrid progeny, we demonstrate that parental species methylation patterns are stable.     

Measurement of DNA methylation using stable isotope dilution and gas chromatography-mass spectrometry

A simple, highly selective, and sensitive method has been developed to quantify methylation of DNA extracted from human peripheral blood mononuclear cells. Assay has been performed at nucleobases level. Cytosine and 5-methylcytosine DNA content has been detected by gas chromatography-mass spectrometry using [2-(13)C]cytosine and [2-(13)C]5-methylcytosine as internal standards. The methylation level has been calculated as 5-methylcytosine/total cytosine ratio. The working range selected on calibration curve, obtained by evaluation of standards and matrix-added standards measurements, is suitable for 5 microg DNA analysis. In this range, healthy human DNA methylation percentage is within 5-6%.     

PAGE separation of hemi-methylated or unmethylated oligonucleotide substrates to distinguish between maintenance and de novo DNA methyltranferase activity

DNA methyltransferase (DNMT) enzymes catalyze the addition of a methyl group to cytosine residues in DNA. Appropriate cytosine methylation of CpG dinucleotides is required for normal mammalian development and homeostasis, and quantitative methods are necessary to assess DNMT activity in various cell extracts. The method described in this report utilizes incorporation of S-[methyl-(3)H]-adenosyl-L-methionine into hemi-methylated or unmethylated oligonucleotides to distinguish between maintenance and de novo DNMT activity, respectively. However, unlike previously described methods, this protocol uses native polyacrylamide gel electrophoresis to detect the incorporation of radioactivity into substrate oligonucleotides. This approach distinguishes between incorporation of radioactivity into target substrate oligonucleotides and incorporation into non-specific cellular DNA that often contaminates nuclear extracts, and permits the reproducible quantitation and comparison of de novo and maintenance DNMT activities in various cell lines. Electrophoretic separation of the methylated substrates is a cost-effective, specific, and reproducible approach to quantitate DNMT activities in nuclear extracts.     

A comparative analysis of developmental profiles for DNA methylation in 5-azacytidine-induced early-flowering flax lines and their control

Earlier experiments demonstrated that DNA from young plants of 5-azacytidine-induced flax (Linum usitatissimum) lines that flower earlier-than-normal is hypomethylated relative to DNA from their control lines and detected differences in methylation level between plants sampled at different ages, which suggested that the methylation level in flax changes during development. To investigate this possibility, and its potential impact on the difference in methylation level between early-flowering and control lines, developmental profiles were established for the cytosine methylation levels in DNA from post-germination seedlings and from the shoot tips of main stems and the cotyledons sampled throughout vegetative phase. The methylation profiles for two early-flowering lines and their control lines were compared. The methylation profiles were then compared to profiles for DNA content, tissue weight and chlorophyll content (green tissues); these additional parameters provided information on tissue status in terms of cell division, tissue expansion and/or photosynthetic maturity. With one exception, methylation levels were either static or increased with plant age and/or tissue maturity; the highest methylation levels were seen in senescent cotyledons. Although DNA from immature plants or tissues of the early-flowering lines was usually hypomethylated, the hypomethylation was not always apparent in tissues from older plants.     

Premeiotic disruption of the Neurospora crassa malate synthase gene by native and divergent DNAs

Summary Repeat-induced point mutation (RIP) has been used to generate new mutations in the previously uncharacterised gene for malate synthase in Neurospora crassa. Molecular clones carrying the am (NADP-glutamate dehydrogenase) gene and the malate synthase gene from either N. crassa or Aspergillus nidulans have been introduced into Neurospora as ectopic duplicate copies by transformation, selecting for the am function in a deletion host. A number of meiotic progeny derived from these transformants were unable to use acetate as sole carbon source, yielded no detectable malate synthase activity and demonstrated extensive cytosine methylation of their duplicated sequences. The new locus has been designated acu-9 and has been assigned to linkage group VII.     

A DNA repair process in Escherichia coli corrects U:G and T:G mismatches to C:G at sites of cytosine methylation

Escherichia coli contains a base mismatch correction system called VSP repair that is known to correct T:G mismatches to C:G when they occur in certain sequence contexts. The preferred sequence context for this process is the site for methylation by the E. coli DNA cytosine methylase (Dcm). For this reason, VSP repair is thought to counteract potential mutagenic effects of deamination of 5-methylcytosine to thymine. We have developed a genetic reversion assay that quantitates the frequency of C to T mutations at Dcm sites and the removal of such mutations by DNA repair processes. Using this assay, we have studied the repair of U: G mismatches in DNA to C: G and have found that VSP repair is capable of correcting these mismatches. Although VSP repair substantially affects the reversion frequency, it may not be as efficient at correcting U: G mismatches as the uracil DNA glycosylase-mediated repair process.     

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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AM946602–AM946620 and FM872474–FM872476.     

Epigenetic responses to drought stress in rice (Oryza sativa L.)

Cytosine methylation polymorphism plays a key role in gene regulation, mainly in expression of genes in crop plants. The differential expression of cytosine methylation over drought stress response was analyzed in rice using drought susceptible but agronomically superior lines IR 20 and CO 43, and drought tolerant genotypes PL and PMK 3 and their F₁ hybrids. The parents and hybrids were subjected to two moisture regimes viz., one under drought condition and another under control condition. The cytosine methylation polymorphism in genomic DNA was quantified under both the conditions at the reproductive stage of the plant using the Methylation Sensitive Amplified Polymorphism (MSAP) technique devised by Xiong et al. (261:439–446, 1999). The results depicted that under drought condition, hyper-methylation was predominant in the drought susceptible genotypes while drought tolerant genotypes presented hypo-methylation behavior. While imposing drought, spikelet sterility per cent was positively correlated to percentage of methylation whereas, panicle length, number of seed per panicle, panicle weight, 100 seed weight, and yield/plant were negatively correlated indicating the role of epigenetic regulation in yield attributing traits in response to drought. Thus, methylation can be considered as an important epigenetic regulatory mechanism in rice plants to adapt drought situation. From this study, we speculate that the hyper- methylation may be an indicator of drought susceptibility and the hypo-methylation for drought tolerance and this methylation polymorphism can be effectively used in drought screening program.