CCGG Methylation Research Articles

Early life social and ecological determinants of global DNA methylation in wild spotted hyenas.

Environmental factors early in life can have lasting influence on the development and phenotypes of animals, but the underlying molecular modifications remain poorly understood. We examined cross-sectional associations among early life socioecological factors and global DNA methylation in 293 wild spotted hyenas (Crocuta crocuta) in the Masai Mara National Reserve, Kenya, grouped according to three age classes (cub, subadult and adult). Explanatory variables of interest included annual maternal rank based on outcomes of dyadic agonistic interactions, litter size, wild ungulate prey density and anthropogenic disturbance in the year each hyena was born based on counts of illegal livestock in the Reserve. The dependent variable of interest was global DNA methylation, assessed via the LUminometric Methylation Assay, which provides a percentage methylation value calculated at CCGG sites across the genome. Among cubs, we observed approximately 2.75% higher CCGG methylation in offspring born to high- than low-ranking mothers. Among cubs and subadults, higher anthropogenic disturbance corresponded with greater %CCGG methylation. In both cubs and adults, we found an inverse association between prey density measured before a hyena was 3months old and %CCGG methylation. Our results suggest that maternal rank, anthropogenic disturbance and prey availability early in life are associated with later life global DNA methylation. Future studies are required to understand the extent to which these DNA methylation patterns relate to adult phenotypes and fitness outcomes.

Analysis of H3K27me3 expression and DNA methylation at CCGG sites in smoking and non-smoking patients with non-small cell lung cancer and their clinical significance.

Smoking frequently leads to epigenetic alterations, including DNA methylation and histone modifications. The effect that smoking has on the DNA methylation levels at CCGG sites, the expression of trimethylation of histone H3 at lysine 27 (H3K27me3) and enhancer of zeste homolog 2 (EZH2), and their interactions in patients with non-small cell lung cancer (NSCLC) were analyzed. There were a total of 42 patients with NSCLC, 22 with adenocarcinomas and 20 with squamous cell carcinomas enrolled in the present study. Expression of H3K27me3, EZH2 and proliferating cellular nuclear antigen (PCNA) were immunohistochemically detected. DNA methylation at CCGG sites was evaluated via histoendonuclease-linked detection of DNA methylation sites. The apoptotic index of cancerous tissues obtained from patients of different smoking statuses was evaluated via the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling method. The association with clinicopathological data was calculated relative to different smoking statuses. Compared with the non-smokers, smokers with NSCLC exhibited a significantly lower apoptotic index (P<0.05), and frequently had a lower level of DNA methylation at CCGG sites, lower H3K27me3 expression and a higher EZH2 expression (P<0.05). DNA methylation levels at CCGG sites were negatively correlated to the Brinkman index (P=0.017). Furthermore, there was a parallel association between the H3K27me3 and EZH2 expression levels in the majority of smokers, whereas in the majority of non-smokers, there was a diverging association (P=0.015). There was a diverging association between the PCNA and EZH2 expression levels in the majority of smokers; however, in the majority of non-smokers, there was a parallel association (P=0.048). In addition, the association between the CCGG methylation ratio and immunohistochemical expression of H3K27me3 was a parallel association in the majority of smokers, while in the majority of non-smokers there was a diverging association (P=0.049). Conclusively, patients with NSCLC and different smoking statuses exhibit different epigenetic characteristics. Additionally, DNA methylation levels at the CCGG sites may have the ability to determine associations between the expression levels of H3K27me3, EZH2 and PCNA.

Analyses on expression of trimethylation of histone H3 at lysine 27 and DNA methylation at CCGG sites in smoking and nonsmoking non-small cell lung cancer patients and their clinical significance.

e23179 Background: Smoking usually leads to epigenetic alterations like DNA methylation and histone modifications. We analyzed the effect that smoking had on DNA methylation level at CCGG sites and expression of trimethylation of histone H3 lysine 27 (H3K27me3), enhancer of zeste homolog 2 (EZH2) and their interactions in non-small cell lung cancer (NSCLC) patients. Methods: 42 NSCLC patients, 22 adenocarcinomas and 20 squamous cell carcinomas were enrolled. Expression of H3K27me3, EZH2 and PCNA were immunohistochemically detected. Apoptotic index in NSCLC with different smoking status was evaluated via TUNEL method. DNA methylation at CCGG sites was evaluated by HELMET method. Their correlation with clinicopathological data were calculated in relation to different smoking status. Results: Compared to the nonsmokers, smoker NSCLC patients were found more often of lower level of DNA methylation at CCGG sites, lower H3K27me3 expression and higher EZH2 expression, and lower apoptotic cell index ( P&lt; 0.05). DNA methylation level at CCGG sites negatively correlated to the Binkman Index( P= 0.017). Further, trend of H3K27me3 and EZH2 expression was in the same direction in most smokers, while in nonsmokers, their expression trend was mostly in the opposite direction ( P= 0.015). Trend of PCNA and EZH2 expression was in the opposite direction in most smokers, while in nonsmokers, their expression trend was mostly in the same direction ( P= 0.048). In addition, trend of CCGG methylation ratio and immunohistochemical expression of H3K27me3 was in the same direction in most smokers, while in nonsmokers, the trend was mostly in the opposite direction ( P= 0.049). Conclusions: NSCLC patients of different smoking status exhibit completely different epigenetic characteristics, and DNA methylation at CCGG sites would probably determine the expression trend of H3K27me3, EZH2 and PCNA.

DNA damage, repair monitoring and epigenetic DNA methylation changes in seedlings of Chernobyl soybeans

This pilot study was carried out to assess the effect of radio-contaminated Chernobyl environment on plant genome integrity 27 years after the accident. For this purpose, nuclei were isolated from root tips of the soybean seedlings harvested from plants grown in the Chernobyl area for seven generations. Neutral, neutral-alkaline, and methylation-sensitive comet assays were performed to evaluate the induction and repair of primary DNA damage and the epigenetic contribution to stress adaptation mechanisms. An increased level of single and double strand breaks in the radio-contaminated Chernobyl seedlings at the stage of primary root development was detected in comparison to the controls. However, the kinetics of the recovery of DNA breaks of radio-contaminated Chernobyl samples revealed that lesions were efficiently repaired at the stage of cotyledon. Methylation-sensitive comet assay revealed comparable levels in the CCGG methylation pattern between control and radio-contaminated samples with a slight increase of approximately 10% in the latter ones. The obtained preliminary data allow us to speculate about the onset of mechanisms providing an adaptation potential to the accumulated internal irradiation after the Chernobyl accident.Despite the limitations of this study, we showed that comet assay is a sensitive and flexible technique which can be efficiently used for genotoxic screening of plant specimens in natural and human-made radio-contaminated areas, as well as for safety monitoring of agricultural products.

RDNA genetic imbalance and nucleolar chromatin restructuring is induced by distant hybridization between Raphanus sativus and Brassica alboglabra.

The expression of rDNA in hybrids inherited from only one progenitor refers to nucleolar dominance. The molecular basis for choosing which genes to silence remains unclear. We report genetic imbalance induced by distant hybridization correlates with formation of rDNA genes (NORs) in the hybrids between Raphanus sativus L. and Brassica alboglabra Bailey. Moreover, increased CCGG methylation of rDNA in F1 hybrids is concomitant with Raphanus-derived rDNA gene silencing and rDNA transcriptional inactivity revealed by nucleolar configuration restriction. Newly formed rDNA gene locus occurred through chromosomal in F1 hybrids via chromosomal imbalance. NORs are gained de novo, lost, and/or transposed in the new genome. Inhibition of methyltransferases leads to changes in nucleolar architecture, implicating a key role of methylation in control of nucleolar dominance and vital nucleolar configuration transition. Our findings suggest that gene imbalance and methylation-related chromatin restructuring is important for rDNA gene silencing that may be crucial for synthesis of specific proteins.

Differential Methylation during Maize Leaf Growth Targets Developmentally Regulated Genes

DNA methylation is an important and widespread epigenetic modification in plant genomes, mediated by DNA methyltransferases (DMTs). DNA methylation is known to play a role in genome protection, regulation of gene expression, and splicing and was previously associated with major developmental reprogramming in plants, such as vernalization and transition to flowering. Here, we show that DNA methylation also controls the growth processes of cell division and cell expansion within a growing organ. The maize (Zea mays) leaf offers a great tool to study growth processes, as the cells progressively move through the spatial gradient encompassing the division zone, transition zone, elongation zone, and mature zone. Opposite to de novo DMTs, the maintenance DMTs were transcriptionally regulated throughout the growth zone of the maize leaf, concomitant with differential CCGG methylation levels in the four zones. Surprisingly, the majority of differentially methylated sequences mapped on or close to gene bodies and not to repeat-rich loci. Moreover, especially the 5' and 3' regions of genes, which show overall low methylation levels, underwent differential methylation in a developmental context. Genes involved in processes such as chromatin remodeling, cell cycle progression, and growth regulation, were differentially methylated. The presence of differential methylation located upstream of the gene anticorrelated with transcript expression, while gene body differential methylation was unrelated to the expression level. These data indicate that DNA methylation is correlated with the decision to exit mitotic cell division and to enter cell expansion, which adds a new epigenetic level to the regulation of growth processes.

Variation in Genomic Methylation in Natural Populations of Chinese White Poplar

BackgroundIt is thought that methylcytosine can be inherited through meiosis and mitosis, and that epigenetic variation may be under genetic control or correlation may be caused by neutral drift. However, DNA methylation also varies with tissue, developmental stage, and environmental factors. Eliminating these factors, we analyzed the levels and patterns, diversity and structure of genomic methylcytosine in the xylem of nine natural populations of Chinese white poplar.Principal FindingsOn average, the relative total methylation and non-methylation levels were approximately 26.567% and 42.708% (P<0.001), respectively. Also, the relative CNG methylation level was higher than the relative CG methylation level. The relative methylation/non-methylation levels were significantly different among the nine natural populations. Epigenetic diversity ranged from 0.811 (Gansu) to 1.211 (Shaanxi), and the coefficients of epigenetic differentiation (GST = 0.159) were assessed by Shannon’s diversity index. Co-inertia analysis indicated that methylation-sensitive polymorphism (MSP) and genomic methylation pattern (CG-CNG) profiles gave similar distributions. Using a between-group eigen analysis, we found that the Hebei and Shanxi populations were independent of each other, but the Henan population intersected with the other populations, to some degree.ConclusionsGenome methylation in Populus tomentosa presented tissue-specific characteristics and the relative 5′-CCGG methylation level was higher in xylem than in leaves. Meanwhile, the genome methylation in the xylem shows great epigenetic variation and could be fixed and inherited though mitosis. Compared to genetic structure, data suggest that epigenetic and genetic variation do not completely match.

Methylation-sensitive amplification polymorphism (MSAP) protocol to assess CpG and CpNpG methylation in the banana genome

Introduction. The technique presented provides molecular markers for DNA methylation studies in the Musa sp. genome. This technique can be used for studying DNA methylation in natural populations, in mapping populations, and also in plant organs or during plant development. The principle, key advantages, starting plant material, time requi- red and expected results are presented. Materials and methods. This part describes the required materials, and the protocols for producing MSAP autoradiography with HpaII / MspI (CCGG methylation) and for EcoRII / PspGI (CCWGG methylation). It mentions the main pro- blems which can occur. Results. The protocol makes it possible to detect CCWGG and CCGG methylated sites. France / Musa sp. / methods / methylation / DNA / marqueur genetique

Using SD-AFLP and MSAP to assess CCGG methylation in the banana genome

Two amplified fragment length polymorphism (AFLP)-derived techniques were used to assess methylation at CCGG sites in the banana genome. Assessment of these techniques revealed that, while amplification steps are very reproducible, the ligation step is more subject to variability. Overall, these techniques produced an error rate of 0.2% per analysed band. Statistical approach highlights the fact that sample duplication is necessary to produce reliable results. This study involved 18 primer pairs and found that in the banana genome, roughly 80% of CCGG sites are unmethylated, 5% are methylated at the internal cytosine, and 15% are methylated at the external or both cytosines.

Stable transgene expression and random gene silencing in wheat

Wheat genes for pathogenesis-related (PR-)proteins, chitinase and beta-1,3-glucanase, under the control of maize ubiquitin promoter-intron were used for transforming the spring wheat 'Bobwhite', using a biolistic approach. Twenty of the 24 primary transgenic lines expressing the PR-protein genes in the T0 generation were silenced in either the T1 or T2 generations. Two apparently genetically identical regenerants arising from a single callus co-bombarded with chitinase and beta-1,3-glucanase transgene combinations, but differing in the expression of the transgenes were selected for further characterization. In one homozygous line, transgene silencing was observed in the T3 plants, while the other line homozygous for the transgene loci stably expressed and inherited the transgenes to at least the T4 generation. Southern blot analyses of genomic DNA from the two lines using the isoschizomeric methylation-sensitive enzymes, MspI and HpaII, revealed a higher degree of methylation of CCGG sequences in the line with the silenced transgene locus. Analysis by reverse transcriptase-polymerase chain reaction, Northern blotting and Western blotting detected stable expression of the transgenes in the line with a lesser extent of methylation, whereas the line with a higher level of CCGG methylation had no transgene expression by the T3 generation. The germination of seeds from the silenced plants in the presence of a cytidine analogue, 5-azacytidine (azaC), did not lead to a reversion of this phenotype.

Bacillus subtilis phage SPR codes for a DNA methyltransferase with triple sequence specificity.

SPR, a temperate Bacillus subtilis phage, codes for a DNA methyltransferase that can methylate the sequences GGCC (or GGCC) and CCGG at the cytosines indicated. We show here that it can also methylate the sequence CC(A/T)GG and protect it from cleavage with EcoRII and ApyI. This methylation can be seen in vivo as well as in vitro with purified SPR methyltransferase. SPR19 and SPR83 are two mutant phages, defective in GGCC or CCGG methylation, respectively. These mutants have not lost their ability to methylate CC(A/T)GG sites. Mutation SPR26 has lost the ability to methylate all three sites. Thus the SPR methyltransferase codes for three genetically distinguishable methylation abilities.

Analysis of the variations in proviral cytosine methylation that accompany transformation and morphological reversion in a line of Rous sarcoma virus-infected Rat-1 cells.

Cells of the A11 lineage of Rat-1 contain a single complete Rous sarcoma provirus. Variation in the activity of this provirus accompanies fluctuations in the lineage between normal and transformed phenotypes. Increased proviral cytosine methylation of the doublet CpG in the tetranucleotide CCGG correlates with transcriptional inactivity and this pattern of cytosine hypermethylation is stable, even when the cells are transformed by another virus. However, transformation can also be induced by 5-azacytidine (but not by other mutagens) and in these transformants reduced proviral cytosine methylation is accompanied by increased proviral transcription. Differences in CCGG methylation between normal and transformed cells are found mainly in the 3' half of the provirus; sites near and within the src gene are heavily methylated only when the provirus is transcriptionally inactive. On the other hand, both transformed and normal A11 derivatives show little, if any, cytosine methylation of CCGG sequences in and flanking the 5' portion of the provirus.