Internal Cytosine Research Articles

The potential association between Wolbachia infection and DNA methylation in Hylyphantes graminicola (Araneae: Linyphiidae)

DNA methylation plays an important role in biological regulation in vertebrates, while its presence is variable among insects. However, only a few studies have reported such methylation in spiders. Here, we detected DNA methylation levels and patterns in the small-sized spider Hylyphantes graminicola (Araneae: Linyphiidae) through methylation-sensitive amplified fragment length polymorphism (MSAP). Given the prevailing rate of Wolbachia infection in H. graminicola and the potential impact on host DNA methylation, four H. graminicola groups, i.e., Wolbachia-uninfected (W−) males, Wolbachia-infected (W+) males, W− females, and W+ females, were used for detection of DNA methylation. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of the DNA methyltransferase 1 (Dnmt1) gene in the four groups. Results demonstrated the existence of genomic DNA methylation in H. graminicola. The hemi- or fully methylated ratios at the internal cytosine (HMeCG or MeCG) was significantly higher than the hemi-methylated ratio at the external cytosine (HMeCCG) in both W− and W+ spiders. Compared to W− male and female spiders, hemi- or fully methylated ratios at the internal cytosine in W+ males and females were significantly lower, and Dnmt1 expression was significantly lower in W+ males and females than in W− males and females. These results suggest that a potential association may exist between Wolbachia infection and DNA methylation in H. graminicola.

The assessment of epigenetic diversity, differentiation, and structure in the 'Fuji' mutation line implicates roles of epigenetic modification in the occurrence of different mutant groups as well as spontaneous mutants.

The ‘Fuji’ line includes many varieties with a similar genetic background and consistent inducement factors with epigenetic occurrence, thus it may be considered an ideal candidate for epigenetic research. In this study, 91 bud mutations of ‘Fuji’ apple were used as the test materials. Using the genetic variation within ‘Fuji’ as the control, the characteristics of epigenetic variation at different levels in both varieties and mutant groups were examined. The results showed that: (1) the global genomic DNA methylation level of the 91 bud mutants of ‘Fuji’ ranged from 29.120%-45.084%, with an average of 35.910%. Internal cytosine methylation was the main DNA methylation pattern. Regarding the variation of methylation patterns of ‘Fuji’ mutants, the vast majority of loci maintained the original methylation pattern existed in ‘Fuji’. CHG methylation variation was the main type of variation; (2) the variation in methylation patterns between the mutant groups was greater than that of methylation levels. Among these patterns, the variation in CHG methylation patterns (including CHG hypermethylation and CHG demethylation) was expected to be dominant. The observed variation in methylation levels was more important in the Color mutant group; however, the variation in methylation patterns was more obvious in both the early maturation and Spur mutant groups. Moreover, the range of variation in the Early-maturation group was much wider than that in the Spur mutant group; (3) epigenetic diversity and genetic diversity were both low between the mutant groups. In the ‘Fuji’ mutant groups, there was few correlation between genetic and epigenetic variation, and epigenetic differentiation resulted in more loci with moderate or greater differentiation; (4) the purifying selection seemed to play a major role in the differentiation of different groups of ‘Fuji’ mutants (65.618%), but epigenetic diversity selection still occurred at nearly 35% of loci. Sixteen epigenetic outlier loci were detected.

Analysis of sulphur and chlorine induced DNA cytosine methylation alterations in fresh corn (Zea mays L. saccharata and rugosa) leaf tissues by methylation sensitive amplification polymorphism (MSAP) approach.

DNA (cytosine) methylation mechanism is another way through which plants respond to various cues including soil fertility amendments and abiotic stresses, and the mechanism has been used to infer some physiological, biochemical or adaptation processes. Despite numerous studies on global DNA methylation profiling in various crop species, however, researches on fresh corn (Zea mays L. saccharata or rugosa) remain largely unreported. The study aimed at investigating sulphur and chlorine induced DNA methylation changes in the fresh corn leaves of field-grown plants at the milk stage. Methylation sensitive amplification polymorphism (MSAP) technique was used to profile sulphur (S) and chlorine (Cl) induced DNA methylation patterns, levels and polymorphism alterations at the CCGG sites in fresh corn leaves of TDN21, JKN2000 and JKN928 hybrid cultivars. Twelve primer pairs used effectively detected 325 MSAP bands, exhibiting differentially methylated sites in the genomic DNA of all the three cultivars, with control showing higher (48.9-56.3%) type I bands as compared to sulphur (34.8-44.9%) and chlorine (40.9-47.4%) treatment samples. Consequently, total methylation levels were greater in S and Cl treatment samples than control; accounting for 43.7-59.7, 51.1-65.2 and 46.8-55.1% of total sites in TDN21, JKN2000 and JKN928, respectively. Full methylation of the internal cytosine was greater than hemi-methylation. Further, demethylation polymorphic loci significantly exceeded methylation polymorphic loci, being greater in S than Cl and control samples in all cultivars. Sulphur and chlorine have a profound influence on DNA methylation patterns and levels at the milk stage, principally by increasing the demethylation loci in the internal cytosine of the fresh corn genome. We speculate that these methylation alterations play an integral role in photosynthates assimilation and physiochemical pathways regulating quality parameters in kernels, as well as abiotic stress responses in fresh corn.

Epigenetic DNA Methylation Mediating Octopus vulgaris Early Development: Effect of Essential Fatty Acids Enriched Diet.

The common octopus, Octopus vulgaris, is a good candidate for aquaculture but a sustainable production is still unviable due to an almost total mortality during the paralarvae stage. DNA methylation regulates gene expression in the eukaryotic genome, and has been shown to exhibit plasticity throughout O. vulgaris life cycle, changing profiles from paralarvae to adult stages. This pattern of methylation could be sensitive to small alterations in nutritional and environmental conditions during the species early development, thus impacting on its health, growth and survival. In this sense, a full understanding of the epigenetic mechanisms operating during O. vulgaris development would contribute to optimizing the culture conditions for this species. Paralarvae of O. vulgaris were cultured over 28 days post-hatching (dph) using two different Artemia sp. based diets: control and a long chain polyunsaturated fatty acids (LC-PUFA) enriched diet. The effect of the diets on the paralarvae DNA global methylation was analyzed by Methyl-Sensitive Amplification Polymorphism (MSAP) and global 5-methylcytosine enzyme-linked immunosorbent assay (ELISA) approaches. The analysis of different methylation states over the time revealed a global demethylation phenomena occurring along O. vulgaris early development being directly driven by the age of the paralarvae. A gradual decline in methylated loci (hemimethylated, internal cytosine methylated, and hypermethylated) parallel to a progressive gain in non-methylated (NMT) loci toward the later sampling points was verified regardless of the diet provided and demonstrate a pre-established and well-defined demethylation program during its early development, involving a 20% of the MSAP loci. In addition, a differential behavior between diets was also observed at 20 dph, with a LC-PUFA supplementation effect over the methylation profiles. The present results show significant differences on the paralarvae methylation profiles during its development and a diet effect on these changes. It is characterized by a process of demethylation of the genome at the paralarvae stage and the influence of diet to favor this methylation loss.

Cytosine methylation at CpCpG sites triggers accumulation of non-CpG methylation in gene bodies.

Methylation of cytosine is an epigenetic mark involved in the regulation of transcription, usually associated with transcriptional repression. In mammals, methylated cytosines are found predominantly in CpGs but in plants non-CpG methylation (in the CpHpG or CpHpH contexts, where H is A, C or T) is also present and is associated with the transcriptional silencing of transposable elements. In addition, CpG methylation is found in coding regions of active genes. In the absence of the demethylase of lysine 9 of histone 3 (IBM1), a subset of body-methylated genes acquires non-CpG methylation. This was shown to alter their expression and affect plant development. It is not clear why only certain body-methylated genes gain non-CpG methylation in the absence of IBM1 and others do not. Here we describe a link between CpG methylation and the establishment of methylation in the CpHpG context that explains the two classes of body-methylated genes. We provide evidence that external cytosines of CpCpG sites can only be methylated when internal cytosines are methylated. CpCpG sites methylated in both cytosines promote spreading of methylation in the CpHpG context in genes protected by IBM1. In contrast, CpCpG sites remain unmethylated in IBM1-independent genes and do not promote spread of CpHpG methylation.

DNA methylation in sugarcane somaclonal variants assessed through methylation-sensitive amplified polymorphism

Micropropagation is an important tool for large-scale multiplication of plant superior genotypes. However, somaclonal variation is one of the drawbacks of this process. Changes in DNA methylation have been widely reported as one of the main causes of somaclonal variations in plants. In order to investigate the occurrence of changes in the methylation pattern of sugarcane somaclonal variants, the MSAP (methylation-sensitive amplified polymorphism) technique was applied to micro-propagated plantlets sampled at the third subculture phase. The mother plant, in vitro normal plantlets, and in vitro abnormal plantlets (somaclonal variants) of four sugarcane clones were screened against 16 MSAP selective primers for EcoRI/MspI and EcoRI/HpaII restriction enzymes. A total of 1005 and 1200 MSAP-derived markers with polymorphism percentages of 28.36 and 40.67 were obtained for EcoRI/HpaII and EcoRI/MspI restriction enzyme combinations, respectively. The genetic similarity between the mother plant and the somaclonal variants ranged from 0.877 to 0.911 (EcoRI/MspI) and from 0.928 to 0.955 (EcoRI/HpaII). Most of the MASPs among mother plant and micro-propagated plantlets were derived from EcoRI/MspI restriction enzymes suggesting alteration due to gain or loss of internal cytosine methylation. A higher rate of loss of methylation (hypomethylation) than gain of methylation (hypermethylation) was observed in the abnormal in vitro sugarcane plantlets. Although changes in the methylation pattern were also observed in the in vitro normal plantlets, they were lower than those observed for the in vitro abnormal plantlets. The MASP technique proved to be a promising tool to early assessment of genetic fidelity of micro-propagated sugarcane plants.

Epigenetic Silencing of Catalase Induces Accumulation of Reactive Oxygen Species in Chronic Lymphocytic Leukemia B Cells Leading to Activation of Axl: An Escape Strategy?

Background: A challenge for novel therapeutic strategies will be the fine tuning of intracellular reactive oxygen species (ROS) signaling in order to effectively deprive cells from ROS-induced tumor promoting events and subsequent tipping the balance to ROS-induced apoptotic signaling. ROS plays a critical role in regulation of the pro-survival receptor tyrosine kinase (RTK) signaling pathways in human cancers. Studies have identified mitochondrial metabolism as the key source for abundant ROS in chronic lymphocytic leukemia (CLL). Unlike in other malignant cells, increased oxidative phosphorylation but not increased aerobic glycolysis has been found in CLL B-cells. While we have detected constitutively phosphorylated/active RTKs like Axl in CLL B-cells, the mechanism of activation remains largely undefined. Here we report that (i) elevated ROS activates the Axl signaling pathway in CLL B-cells and, (ii) mechanism of increased ROS accumulation in the leukemic B-cells.Methods: CLL B- and normal B-cells were purified from peripheral blood of previously untreated CLL patients and normal healthy individuals, respectively using a RossetteSep B-Cell enrichment kit. CLL B-cells were exposed to H2O2 for 5 min and cell lysates were analyzed for activation of RTKs including Axl by immunoprecipitation/Western blots. We also examined expression status of SIRT3, acetylated-superoxide dismutase (SOD)2 and catalase in CLL B-cells by Western blots. Catalase mRNA levels in CLL B-/normal B-cells were determined by qRT-PCR. In some experiments, genomic DNA was isolated from CLL B-/normal B-cells for catalase promoter methylation studies. Finally, accumulation of O2- and H2O2 in CLL B- and normal B-cells were measured by flow cytometry after treating the cells withdihydroethidium (DHE) and dichlorohydrofluorescein diacetate (DCFDA), respectively.Results: We found that enforced induction of ROS significantly increases tyrosine phosphorylation levels on multiple cellular proteins in CLL B-cells (Fig. 1). Further analysis finds that increased ROS activates Axl and its downstream targets AKT/Erk1/2 MAPK and the fibroblast growth factor receptor (FGFR) which we recently defined as a downstream target of Axl; while ROS accumulation did not show any significant effect on other RTKs, cMET or IGFR1, in CLL B-cells. Interestingly, the histone deacetylase SIRT3 which activates mitochondrial SOD2 via deacetylation, we found, was overexpressed in CLL B-cells as compared to normal B-cells indicating more efficient conversion of O2- into H2O2 in the leukemic B-cells (Fig. 2). However expression of catalase, which converts H2O2 into O2 and H2O, was reduced significantly in CLL B-cells as compared to normal B-cells both at mRNA and protein levels. To delineate the mechanism of reduced expression of catalase in CLL B-cells we studied the human catalase promoter region for potential methylation sites using the method of HpaII restriction enzyme digestion where HpaII is unable to cut DNA (CCGG site) when the internal cytosine is methylated. Initial findings from this approach suggest that while the catalase gene promoter in CLL B-cells may contain methylated cytosines, the catalase gene in normal B-cells may not confer methylation in the promoter region. Next, genomic DNA from CLL B-cells (n=10) and normal B-cells (n=6) was subjected to bisulfite treatment, followed by PCR amplification and sequencing. This approach revealed no significant promoter methylation of the catalase gene in normal B-cells, while moderate to heavily methylated promoter of the catalase gene was detected in CLL B-cells from majority of CLL patients (9 of 10). Further studies are in progress to examine catalase promoter methylation status in a larger cohort of CLL patients and its association with H2O2 accumulation, RTK phosphorylation and risk-factors. Indeed, flow cytometric analysis finds lower levels of O2- but accumulation of H2O2 in CLL B-cells as compared to normal B-cells. Taken together, these findings suggest that although SOD2 remains highly active, increased accumulation of H2O2 may occur in CLL B-cells due to epigenetic silencing of the catalase gene.Conclusion: These observations may explain, at least in part, the expression of constitutively active Axl signaling pathway in CLL B-cells that plays a role in CLL B cell survival and may account for the prolonged survival and/or apoptotic resistance of the leukemic B-cells. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Analysis of cytosine methylation status in potato by methylation-sensitive amplified polymorphisms under low-temperature stress

China is the leading producer of potatoes (Solanum tuberosum) in the world. However, low-temperature stress can severely restrict the yield and quality of potato crops in that country. We used methylation-sensitive amplified polymorphisms (MSAPs) to analyze how such stress might alter the status of cytosine methylation in potato cultivar ‘Atlantic’. Over 2400 loci were identified and assessed for DNA methylation in chilling-treated and control plants. The level of cytosine methylation increased from 37.71% to 56.51% after 48 h of treatment but then decreased to 42.14% after 60 h of chilling. Stress-induced changes in the banding patterns of DNA methylation primarily resulted from full methylation of CG (internal cytosine of 5’-CCGG-3’) on double DNA strands. We sequenced 78 polymorphic DNA fragments and blasted them against NCBI. Expression patterns of 4 genes were then assessed via semi-quantitative RT-PCR. The expression patterns of genes related to chilling stress appeared to be regulated by the methylation or demethylation status in potato.

Salt tolerance and alterations in cytosine methylation in the interspecific hybrids of Fraxinus velutina and Fraxinus mandshurica

For cross-pollination trees, the optimal breeding method is hybridization. Tree heterosis is commonly present and is the main research focus in tree crossbreeding. Salt stress and interspecific hybridization may lead to DNA methylation changes. The study crossed Fraxinus mandshurica (female parent) with Fraxinus velutina (male parent) to obtain interspecific F1 hybrid progenies that could obtain the good characters of parents. The results showed that growth and survival rate of the interspecific hybrid progenies (F1 hybrids of F. mandshurica × F. velutina) were significantly higher than those of intraspecific open pollinated plants from parental F. mandshurica and F. velutina. Salt tolerance and cytosine methylation in interspecific F1 hybrids and the intraspecific open pollinated plants from parents were examined. Membrane permeability, ROS and antioxidant activity, malondialdehyde, and photosynthesis were measured after salt treatment and genomic methylation was analyzed using a methylation-sensitive amplified polymorphism protocol. F1 hybrids exhibited heterosis for growth in normal as well as high salt conditions. DNA methylation in the F1 hybrids was lower than the intraspecific open pollinated plants from parents. Salt treatments changed DNA methylation patterns in F1 hybrids. Genomic DNA of the intraspecific open pollinated plants from parents had internal cytosine methylation (average of 13.22 %), whereas F1 hybrid seedlings had external cytosine methylation (average of 7.34 %). Such changes in DNA methylation patterns in F1 hybrids suggest a connection between salt tolerance and epigenetic mechanisms in plants. We observed that alteration of DNA methylation was closely correlated with the adaptation to the salt stress and provided epigenetic mechanisms of salt tolerance in the interspecific hybridization of trees.

Cloning, purification and characterization of translationally fused protein DNA methyltransferase M•HhaI-EGFP

Design of the transcriptionally-fused protein M•HhaI-EGFP, composed of bacterial DNA-methyltransferase M•HhaI and enhanced green fluorescent protein (GFP) is described. The mentioned M•HhaI-EGFP was expressed in Escherichia coli ER1821 and purified by affinity chromatography on Ni-NTA agarose. According to expectations M•HhaI-EGFP fused protein retained significant features of corresponding original proteins: the ability to transfer methyl group to the C5 carbon atom of internal cytosine in CGCG site and absorption–emission spectral characteristics. The created transcriptionally-fused protein M•HhaI-EGFP could be used in various experiments in molecular biology.

Drought resistance and DNA methylation of interspecific hybrids between Fraxinus mandshurica and Fraxinus americana

The advantages of F1 hybrid progenies of Fraxinus mandshurica and Fraxinus americana were analyzed under drought conditions in the field and greenhouse. For cross-pollination trees, the optimal breeding method is hybridization. Tree heterosis is commonly present and is the main research focus in tree crossbreeding. In this study, Fraxinus mandshurica was used as female and Fraxinus americana was used as male parents to generate F1 hybrid generation. The adaptability and overwintering survival of F1 hybrids were evaluated under natural conditions. The advantages of F1 generation were analyzed under drought conditions in the field and greenhouse. The results showed that hybrid F1 generation (IH-10 and IH-15) had significantly higher growth and survival rate than the intraspecific progenies from either F. mandshurica or F. americana (MA-13 and FA-43, respectively) in the field. After 12 days of drought stress, growth, chlorophyll a and b, superoxide dismutase and peroxidase of IH-10 and IH-15 were higher than those of MA-13 and FA-43, suggesting an advantage of drought resistance for the hybrid progenies. Relationship of drought resistance and DNA methylation of interspecific hybrids was analyzed. Without drought treatment, methylation degree in the hybrid progenies (IH-10 and IH-15) was lower than that in the paternal or maternal progenies (MA-13 and FA-43). Drought stress enhanced the methylation rate in both hybrid and parental progenies, with higher increases in MA-13 and FA-43 than those in IH-10 and IH-15. Methylation in MA-13 and FA-43 mainly occurred in the full methylation of the internal cytosine, while in IH-10, methylation occurred in both full methylation of the internal cytosine and hemi-methylation of the external cytosine. These results showed that DNA methylation pattern was reconstructed in the hybrid progenies. Further analysis indicated that methylated DNA fragments were associated with growth and cell metabolism. Southern blot and transcriptional analysis showed that DNA methylation in FmaHSP70, FmaCO, FmaWRKY2 and FmaEF1A altered their gene expression. We observed that alteration of DNA methylation is closely correlated with the adaptation to the drought stress and provided epigenetic mechanisms of drought resistance in the interspecific hybridization of trees.

Variations of DNA methylation and genes expression for brown fiber during its development process

Brown cotton is a kind of naturally colored cotton which accumulates brown pigment in fiber. In the experiment, the variations of DNA methylation and genes expression were studied during the development processes from 5 DPA to 25 DPA for brown fiber. By using the methylation-sensitive amplified polymorphism technique, we choose 66 pairs of selective-amplification primers to assess the status and levels of cytosine methylation. The hemimethylation of the external cytosine and the full methylation of the internal cytosine were scored. As a result, with the development of fiber from 5 DPA to 25 DPA, the ratios of the external hemimethylation and internal full methylation were gradually increased. In detail, at the stages of 5, 10, 15 and 25 DPA, the percentages of external hemimethylation were 8.29, 8.81, 9.77 and 10.09 %, the ratios of internal full methylation were 17.91, 19.20, 20.02 and 20.78 %, respectively. The development of brown fiber triggered the increase of methylated loci in the whole genome. For further analysis, we used cDNA-AFLP protocol to identify the specially expressed genes for further analysis. Totally, 30 polymorphic transcription-derived fragments (TDFs) were isolated and sequenced. By homologous alignment analysis, 19 TDFs were found similar with genes from cotton. Among them, the homologous genes of TDF5, TDF6, TDF10, TDF12, TDF17, TDF22, TDF23 and TDF25 were all from fiber. Furthermore, 13 TDFs were found to be homologous with reported functional genes.

The role of DNA methylation on Octopus vulgaris development and their perspectives

DNA methylation is a common regulator of gene expression and development in mammalian and other vertebrate genomes. DNA methylation has been studied so far in a few bivalve mollusk species, finding a wide spectrum of levels. We focused our study in the common octopus, Octopus vulgaris, an important organism for neuroscience, physiology and ethology research as well as for human consumption. We aim to confirm the existence of DNA methylation in O. vulgaris and ultimately, if methylation plays a role in gene regulation during octopus development. We used a genome-wide approach, methylation-sensitive amplified polymorphism (MSAP), firstly in four different tissues from the same specimens from adult benthonic individuals to test whether gene expression is regulated by methylation. Secondly, we tested the hypothesis that methylation underlies development by assessing MSAP patters from paralarvae to adult developmental stages. Our data indicate that octopus genome is widely methylated since clear differences can be observed, and the methylation pattern changes with the development. The statistical analyses showed significant differences in methylation pattern between paralarvae, where higher internal cytosine methylation is observed, and the three other post-hatching stages. This suggests an important role of cytosine methylation during the first step of development, when major morphological changes take place. However, methylation seems to have little effect on gene expression during the benthonic phase, since no significant effect was revealed in the analyses of molecular variance (AMOVA) performed. Our observations highlight the importance of epigenetic mechanisms in the first developmental steps of the common octopus and opens new perspectives to overcome high mortality rate during paralarvae growth. Thus, better understanding the molecular regulation patterns could lead to new approaches that increase the efficiency of husbandry of this emergent species for aquaculture.

DNA methylation alterations of upland cotton (Gossypium hirsutum) in response to cold stress

DNA methylation plays an important role in regulating gene expression in plants. In the experiment, we studied effects of cold on DNA methylation variation in upland cotton. Using the methylation-sensitive amplified polymorphism procedure, we chose 66 pairs of selective amplification primers to assess the status and levels of cytosine methylation. The hemimethylation of the external cytosine and the full methylation of the internal cytosine were scored. As a result, cold triggered the demethylation of hemimethylated or internally full methylated cytosine. With the prolongation of cold treatment, the demethylation loci increased and the methylation loci decreased. Nevertheless, this change could be reverted when cotton was subsequently recovered under normal temperature. In addition, 29 polymorphic bands that appeared in the electrophoretogram were sequenced. By homologous alignment analysis, most of these 29 fragments were identified as genes or DNA clones involved in abiotic stress response. The variation in methylation loci existed at both coding and non-coding regions. Furthermore, the expression of the abiotic stress-related genes, GhCLSD (Seq21), GhARK (Seq22), GhARM (Seq15, Seq18, Seq19 and Seq21) and GhTPS (Seq8), were tested. The results revealed that cold treatment induced down-regulation of GhCLSD, GhARK and GhARM, but up-regulated the expression of GhTPS. These changes were in accordance with the alteration of DNA methylation. Thus, cold may affect the gene expression via changing the methylation status in the cytosine nucleotide.

Tissue culture-induced alteration in cytosine methylation in new rice recombinant inbred lines

Zizania DNA introgression could induce a large number of genetic and epigenetic changes of the new rice recombinant inbred lines genome. In this present study, we employed inter-simple sequence repeat (ISSR) to further study the genetic and epigenetic changes that are induced by tissue culture. Changes induced by tissue culture are mostly epigenetic. One kind of methylation alterations is that the unmethylated CCGG sequence was transformed into external hemimethylated cytosines. This type change, however, did not transmit to progenies of the plant. Another kind of methylation alterations is tissue culture-induced occurrence of de novo methylation at the external cytosines of the CCGG sites that are originally with hyper-methylated internal cytosines. Interestingly, many of these methylated external cytosines became demethylated again upon plant regeneration, implying that the hypomethylated state of these loci is required for successful plant regeneration. Because the changed methylation patterns are highly similar among calli subcultured for different intervals, and because apparent concordance exists between randomly selected individual regenerants, we are tempted to deduce that the DNA methylation changes did not occur randomly, rather, the methylation events likely to have hot spots, and/or occur non-randomly. Sequence analysis indicates that the loci underwent methylation alterations are predominantly genic, sharing significant homology to known-function genes or expressed sequence tag (EST) sequences. This finding implies that the epigenetic changes induced by tissue culture could potentially affect the expression of the relevant genes.

Detection of Epigenetic Variations in the Protoplast-Derived Germlings of Ulva reticulata Using Methylation Sensitive Amplification Polymorphism (MSAP)

Regeneration of protoplasts into de novo plants was reported for a large number of seaweed species. The regeneration of protoplasts into different morphotypes as a result of epigenetic variations was discussed for the first time in this study. The loci assessed for methylation modifications in normal filamentous thalli showed a frequency of 32.43% as unmethylated DNA, 24.32% as a hemimethylated, and 20.27% as a methylation of internal cytosine at both the strands. The corresponding methylation values for disk-type thalli were 27.02%, 32.43%, and 14.86%, respectively. The hypermethylation condition was apparent in the disk-type thalli with methylation ratio of 72.97% compared to that of normal filamentous thalli with 67.56%. The frequency of methylation polymorphic sites among the two morphotypes was 53%. The present study reveals the distinct expression of cytosine methylation and is thus correlated to differential morphogenesis of plants regenerated from cultured cells. The number of protoplasts regenerating into filamentous thalli declined with increasing temperature from 15°C, 20°C, 25°C, and 30°C. The disk-type variant had higher thermal stability at 30°C over normal filamentous thalli. Further, this variant could maintain itself for more than a year in the laboratory indicating its suitability for in vitro germplasm maintenance and propagation.Electronic supplementary materialThe online version of this article (doi:10.1007/s10126-012-9434-7) contains supplementary material, which is available to authorized users.

DNA methylation and adaptive response in forest tree species

Progressive increase of temperatures as well as longer seasonal drought periods revealed by climate studies correspond to fast environmental changes that forest species face with their actual genetic background. Natural selective processes cannot develop an adaptive response within this time frame. Thus the capability of forest tree species to adapt to the new environments will depend on their genetic background, but also rely on their phenotypic plasticity. Several reports have shown the involvement of epigenetic modifiers as the basis of the phenotypic plasticity, and in particular to the adaptation to abiotic stresses. DNA methylation (methylation of cytosine residues)is one the most important epigenetic modification in eukaryotes. Itis involved in specific biological processes such as gene transcription regulation, gene silencing, mobile element control or genome imprinting.Therefore, there is a great interest in analyzing cytosine methylation levels and distribution within the genome. In order to analyze methylation-sensitive anonymous CCGG restriction sites we used MSAP technique (Methylation-Sensitive Amplified Polymorphism), an AFLP-based technique for the analysis of cytosine methylation. The technique is based on the use of isoschizomers that show differential cleavage sensitivity to cytosine methylation. HpaII and MspI are isoschizomers that are frequently used to detect cytosine methylation. HpaII cannot cleave if one or both cytosines are fully methylated (in both strands), whereas MspI cleaves C5mCGG but not 5mCCGG sequences). For each sample, MSAP analysis is performed using both EcoRI/HpaII and EcoRI/MspI digested samples. Comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) “Methylation-insensitive polymorphisms” that are associated with genetic variability and will show common EcoRI/HpaII and EcoRI/MspI profiles among samples; and (2) “Methylation-sensitive polymorphisms” that are associated with epigenetic variability and detected as amplified fragments differing in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns of the same sample. Thus, full methylation of the internal cytosine at the assayed CCGG sites will be associated with fragments detected in EcoRI/MspI pattern which are absent or less intense in EcoRI/HpaII profile. Hemi-methylation of the external cytosine will be associated with fragments observed in EcoRI/HpaII pattern which are absent in EcoRI/MspI profile. We have optimized DNA methylation analysis for two forest tree species, including both angiosperm (Fagus sp.) and gymnosperm (Pinus sp.) species. The set-up of the MSAP technology has allowed study intra-specific variability of DNA methylation in Pinus pinea L. a specie which is characterized by a low genetic variability and high level of phenotypic plasticity. Representative populations spanning the whole distribution of this specie within Spanish geography were chosen for this study, including coastal and interior populations. MSAP patterns from vegetative propagated trees were compared among and within populations. Percentage of methylation-sensitive polymorphisms was calculated and selective markers were identified. To test a possible role of DNA methylation in the plasticity of Fagus sylvatica L. in response to water deficit we analyzed two populations of beech from Spain and Sweden which differ in the response to water stress. Patterns of DNA methylation clearly differed between populations. Although the rate of DNA methylation was similar between irrigated and non-irrigated trees in each polpulation after AMOVA, some specific MSAPs associated with water stress response were identified.

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Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes

Cytosine residues in the vertebrate genome are enzymatically modified to 5-methylcytosine, which participates in transcriptional repression of genes during development and disease progression. 5-Methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. Analysis of 5-methylcytosine and 5-hydroxymethylcytosine is confounded, as these modifications are indistinguishable by traditional sequencing methods even when supplemented by bisulfite conversion. Here we demonstrate a simple enzymatic approach that involves cloning, identification, and quantification of 5-hydroxymethylcytosine in various CCGG loci within murine and human genomes. 5-Hydroxymethylcytosine was prevalent in human and murine brain and heart genomic DNAs at several regions. The cultured cell lines NIH3T3 and HeLa both displayed very low or undetectable amounts of 5-hydroxymethylcytosine at the examined loci. Interestingly, 5-hydroxymethylcytosine levels in mouse embryonic stem cell DNA first increased then slowly decreased upon differentiation to embryoid bodies, whereas 5-methylcytosine levels increased gradually over time. Finally, using a quantitative PCR approach, we established that a portion of VANGL1 and EGFR gene body methylation in human tissue DNA samples is indeed hydroxymethylation.

Is the cytosine DNA methyltransferase gene MET1 regulated by DNA methylation in Arabidopsis thaliana plants?

The methylation patterns of the MET1 gene in organs of Arabidopsis thaliana were studied by Southern blot hybridization of DNA samples hydrolyzed with differentially methylation-sensitive restriction endonucleases. A highly methylated on internal cytosine residue CCGG site was found 1.5 kb upstream of the gene, whereas CCGG sites located in more proximal parts of the 5'-flanking region and the gene itself are essentially unmethylated. This methylation pattern was observed in different organs of plants belonging to two different ecotypes as well as in different transgenic plant lines. The methylation level ofa CCGG site in exon 3 (2.1 kb from the gene's 5'-end) occurred to be variable between different transgenic plant lines and two ecotypes studied. Transcription levels of the MET1 gene vary slightly in organs of wild-type plants without any obvious correlation with its methylation. The transgenic antisense MET1 constructs expressed in plant genome do affect both MET1 methylation and its transcription but again without any obvious correlation. The comparative investigation of transcription levels of different genes of cytosine DNA methyltransferase family MET (MET1, MET2a, MET2b, MET3) and their methylation patterns shows that there may exist some mechanisms defending the most actively transcribed gene MET1 of this family from methylation mediated silencing. In contrast to DRM2 gene we could not find any adenine methylated GATC sites in the MET1 gene.