Epigenetic Control of a Transposon-Inactivated Gene in Neurospora Is Dependent on DNA Methylation

An unstable allele of the Neurospora am (GDH) gene resulting from integration of the retrotransposon Tad3-2 into 5'' noncoding sequences was found in previous work. We report that reversion to Am(+) depends on DNA methylation within and upstream of Tad. Levels of methylation were correlated with the proportion of Am(+) conidia, whether the cultures were derived from Am(-) or Am(+) isolates. Reversion to Am(+) did not occur when conidia were plated on 5-azacytidine, which reduces DNA methylation. The mutation dim-2, which appears to abolish DNA methylation, also prevented reversion to Am(+). The native am allele, in a strain that lacked Tad elements, was replaced with am::Tad3-2 or with a deletion derivative that prevents transposition of Tad. Transformants of both classes showed instability comparable with that of the original isolates, which contain multiple Tad elements. Deletion of the upstream enhancer-like sequences, URSamα and β, did not prevent the instability of am::Tad3-2. The results suggest that am expression is dependent on DNA methylation but not on proliferation or transposition of the Tad element and that the instability does not require the upstream sequences of am.     

Evidence that steroid 5alpha-reductase isozyme genes are differentially methylated in human lymphocytes

The synthesis of dihydrotestosterone (DHT) is catalyzed by steroid 5alpha-reductase isozymes 1 and 2, and this function determines the development of the male phenotype during embriogenesis and the growth of androgen sensitive tissues during puberty. The aim of this study was to determine the cytosine methylation status of 5alpha-reductase isozymes types 1 and 2 genes in normal and in 5alpha-reductase deficient men. Genomic DNA was obtained from lymphocytes of both normal subjects and patients with primary 5alpha-reductase deficiency due to point mutations in 5alpha-reductase 2 gene. Southern blot analysis of 5alpha-reductase types 1 and 2 genes from DNA samples digested with HpaII presented a different cytosine methylation pattern compared to that observed with its isoschizomer MspI, indicating that both genes are methylated in CCGG sequences. The analysis of 5alpha-reductase 1 gene from DNA samples digested with Sau3AI and its isoschizomer MboI which recognize methylation in GATC sequences showed an identical methylation pattern. In contrast, 5alpha-reductase 2 gene digested with Sau3AI presented a different methylation pattern to that of the samples digested with MboI, indicating that steroid 5alpha-reductase 2 gene possess methylated cytosines in GATC sequences. Analysis of exon 4 of 5alpha-reductase 2 gene after metabisulfite PCR showed that normal and deficient subjects present a different methylation pattern, being more methylated in patients with 5alpha-reductase 2 mutated gene. The overall results suggest that 5alpha-reductase genes 1 and 2 are differentially methylated in lymphocytes from normal and 5alpha-reductase deficient patients. Moreover, the extensive cytosine methylation pattern observed in exon 4 of 5alpha-reductase 2 gene in deficient patients, points out to an increased rate of mutations in this gene.     

Evidence for cytosine methylation of non-symmetrical sequences in transgenic Petunia hybrida.

A considerable proportion of cytosine residues in plants are methylated at carbon 5. According to a well-accepted rule, cytosine methylation is confined to symmetrical sequences such as CpG and CpNpG, which provide the signal for faithful transmission of symmetrical methylation patterns by maintenance methylase. Using a genomic sequencing technique, we have analysed cytosine methylation patterns within a hypermethylated and a hypomethylated state of a transgene in Petunia hybrida. Examination of a part of the transgene promoter revealed that in both states m5C residues located within non-symmetrical sequences could be detected. Non-symmetrical C residues in the two states were methylated at frequencies of 5.9 and 31.9%, respectively. Methylation appeared to be distributed heterogeneously, but some DNA regions were more intensively methylated than others. Our results show that at least in a transgene, a heterogeneous methylation pattern, which does not depend on symmetry of target sequences, can be established and conserved.     

Endosperm-specific hypomethylation,and meiotic inheritance and variation of DNA methylation level and pattern in sorghum (<Emphasis Type="Italic">Sorghum bicolor</Emphasis> L.) inter-strain hybrids

Understanding dynamics and inheritance of DNA methylation represents important facets for elucidating epigenetic paradigms in plant development and evolution. Using four sets of sorghum (Sorghum bicolor L.) inter-strain hybrids and their inbred parents, the developmental stability and inheritance of cytosine methylation in two tissues, leaf and endosperm, by MSAP analysis were investigated. It was found that in all lines (inbred and hybrid) studied, endosperm exhibited a markedly reduced level of full methylation of the external cytosine or both cytosines at the CCGG sites relative to leaf, which caused a variable reduction in the estimated total methylation level in endosperm by 6.89–19.69% (11.47% on average). For both tissues, a great majority of cytosine methylation profiles transmitted to F1 hybrids, however, from 1.69 to 3.22% of the profiles showed altered patterns in hybrids. Both inherited and altered methylation profiles can be divided into distinct groups, and their frequencies are variable among the cross-combinations, and between the two tissues. The variations in methylation level and pattern detected in the hybrids were not caused by parental heterozygosity, and they could be either non-random or stochastic among hybrid individuals. Homology analysis of isolated bands that showed endosperm-specific hypomethylation or variation in hybrids indicated that diverse sequences were involved, including known-function cellular genes and mobile elements. RT-PCR analysis of six genes representing endosperm-specific hypomethylation in MSAP profiles indicated that all showed higher expression in endosperm than in leaf, suggesting involvement of methylation state in regulating tissue-specific or tissue-biased expression in sorghum. Analysis on leaf-RNA from 5-azacytidine-treated plants further corroborated this possibility. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tissue-specific differences in cytosine methylation and their association with differential gene expression in sorghum

It has been well established that DNA cytosine methylation plays essential regulatory roles in imprinting gene expression in endosperm, and hence normal embryonic development, in the model plant Arabidopsis (Arabidopsis thaliana). Nonetheless, the developmental role of this epigenetic marker in cereal crops remains largely unexplored. Here, we report for sorghum (Sorghum bicolor) differences in relative cytosine methylation levels and patterns at 5'-CCGG sites in seven tissues (endosperm, embryo, leaf, root, young inflorescence, anther, and ovary), and characterize a set of tissue-specific differentially methylated regions (TDMRs). We found that the most enriched TDMRs in sorghum are specific for the endosperm and are generated concomitantly but imbalanced by decrease versus increase in cytosine methylation at multiple 5'-CCGG sites across the genome. This leads to more extensive demethylation in the endosperm than in other tissues, where TDMRs are mainly tissue nonspecific rather than specific to a particular tissue. Accordingly, relative to endosperm, the other six tissues showed grossly similar levels though distinct patterns of cytosine methylation, presumably as a result of a similar extent of concomitant decrease versus increase in cytosine methylation that occurred at variable genomic loci. All four tested TDMRs were validated by bisulfite genomic sequencing. Diverse sequences were found to underlie the TDMRs, including those encoding various known-function or predicted proteins, transposable elements, and those bearing homology to putative imprinted genes in maize (Zea mays). We further found that the expression pattern of at least some genic TDMRs was correlated with its tissue-specific methylation state, implicating a developmental role of DNA methylation in regulating tissue-specific or -preferential gene expression in sorghum.     

DNA methylation as an epigenetic regulator of neural 5-lipoxygenase expression: evidence in human NT2 and NT2-N cells

Increased expression of 5-lipoxygenase is associated with various neuropathologies and may be related to epigenetic gene regulation. DNA methylation in promoter regions is typically associated with gene silencing. We found that human NT2 cells, which differentiate into neuron-like NT2-N cells, express 5-lipoxygenase and we investigated the relationship between 5-lipoxygenase expression and the methylation state of the 5-lipoxygenase core promoter. We used the demethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor valproate to alter DNA methylation and to induce histone modifications. 5-Lipoxygenase expression and DNA methylation were assayed with RT-PCR and bisulfite genomic sequencing, respectively. Neuronal differentiation of proliferating NT2 precursors decreased 5-lipoxygenase expression. 5-Aza-2'-deoxycytidine increased 5-lipoxygenase mRNA levels only in proliferating cells, whereas valproate increased 5-lipoxygenase mRNA levels in a cell cycle-independent manner. In both precursors and differentiated cells, CpG dinucleotides of the promoter were poorly methylated. In precursors, both 5-aza-2'-deoxycytidine and valproate further reduced the number of methylated CpGs. Moreover, we found evidence for cytosine methylation in CpWpG (W=adenine or thymine) and other asymmetrical sequences; CpWpG methylation was reduced by valproate in NT2-N but not in NT2 cells. This is the first report demonstrating that the dynamics of DNA methylation relates to neural 5-lipoxygenase gene expression.     

C5 cytosine methylation at CpG sites enhances sequence selectivity of mitomycin C-DNA bonding

We have established that UvrABC nuclease is equally efficient in cutting mitomycin C (MC)-DNA monoadducts formed at different sequences and that the degree of UvrABC cutting represents the extent of drug-DNA bonding. Using this method we determined the effect of C5 cytosine methylation on the DNA monoalkylation by MC and the related analogues N-methyl-7-methoxyaziridinomitosene (MS-NMA) and 10-decarbamoylmitomycin C (DC-MC). We have found that C5 cytosine methylation at CpG sites greatly enhances MC and MS-NMA DNA adduct formation at those sites while reducing adduct formation at non-CpG sequences. In contrast, although DC-MC DNA bonding at CpG sites is greatly enhanced by CpG methylation, its bonding at non-CpG sequences is not appreciably affected. These cumulative results suggest that C5 cytosine methylation at CpG sites enhances sequence selectivity of drug-DNA bonding. We propose that the methylation pattern and status (hypo- or hypermethylation) of genomic DNA may determine the cells' susceptibility to MC and its analogues, and these effects may, in turn, play a crucial role in the antitumor activities of the drugs.     

Differences in the methylation patterns of the VaSTS1 and VaSTS10 genes of Vitis amurensis Rupr.

Resveratrol is a plant-derived phenol but the mechanism that regulates its biosynthesis remains unidentified. Stilbene synthase (STS) catalyzes resveratrol formation in vivo and we have proposed that inducers of resveratrol production affect STS expression through an unidentified epigenetic mechanism. To investigate the role of DNA methylation in resveratrol biosynthesis, we treated both rolB transgenic and empty vector control Vitis amurensis cell cultures with the DNA demethylation agent, 5-azacytidine. Treated cells had increased resveratrol production through activation of VaSTS10 expression. The lowest levels of cytosine methylation were at the 5′- and 3′-ends of the VaSTS1 protein-coding sequence. Cytosine methylation decreased mostly at the 5′- and 3′-ends of VaSTS10 after azaC treatment with an intriguing regularity in the number of cytosine nucleotides within the 5′- and 3′- ends of the protein-coding sequences. Thus, cytosine methylation is crucial for the regulation of the resveratrol biosynthetic pathway.     

Direct electrochemical detection of DNA methylation for retinoblastoma and CpG fragments using a nanocarbon film

We describe the direct electrochemical detection of DNA methylation in relatively long sequences by using a nanocarbon film electrode. The film was formed by employing the electron cyclotron resonance sputtering method and had a nanocrystalline sp² and sp³ mixed bond structure. Our methylation detection technique measures the differences between the oxidation currents of both 5-methylcytosine and cytosine without a bisulfite reaction or labeling. This was possible because this film electrode has a wide potential window while maintaining the high electrode activity needed to quantitatively detect both bases by direct oxidation. By optimizing the electrode surface conditions using electrochemical pretreatment, we used this film to quantitatively detect single cytosine methylation regardless of the methylation position in the sequence including retinoblastoma gene fragments (∼24mers). This was probably due to the high stability of this film electrode, which we achieved by controlling the surface hydrophilicity to suppress the fouling, and by maintaining electrode activity against all the bases. The pH optimization of the oligonucleotide measurements was also useful for distinguishing both bases separately. Under the optimized conditions, this film electrode allowed us to realize the quantitative detection of DNA methylation ratios solely by measuring methylated 5′-cytosine-phosphoguanosine (CpG) repetition oligonucleotides (60mers) with different methylation ratios.     

Analysis of DNA methylation in different maize tissues

DNA methylation plays an important role in gene expression regulation during biological development and tissue differentiation in plants. This study adopted methylation-sensitive Amplified fragment length polymorphism （AFLP） to compare the levels of DNA cytosine methylation at CCGG sites in tassel, bracteal leaf, and ear leaf from maize inbred lines, 18 White and 18 Red, respectively, and also examined specific methylation patterns of the three tissues. Significant differences in cytosine methylation level among the three tissues and the same changing tendency in two inbred lines were detected. Both MSAP （methylation sensitive amplification polymorphism） ratio and full methylation level were the highest in bracteal leaf, and the lowest in tassel. Meanwhile, different methylation levels were observed in the same tissue from the inbred lines, 18 White and 18 Red. Full methylation of internal cytosine was the dominant type in the maize genome. The differential methylation patterns in the three tissues were observed. In addition, sequencing of nine differentially methylated fragments and the subsequent blast search revealed that the cytosine methylated 5 ＇ -CCGG-3 ＇ sequences were distributed in repeating sequences, in the coding and noncoding regions. Southern hybridization was used to verify the methylation polymorphism. These results clearly demonstrated the power of the MSAP technique for large-scale DNA methylation detection in the maize genome, and the complexity of DNA methylation change during plant growth and development. The different methylation levels may be related to specific gene expression in various tissues.     

Role of epigenetic DNA alterations in the pathogenesis of systemic lupus erythematosus

Epigenetic alternations in genomic DNA encompass cytosine methylation in cytosine and guanine (CpG) dinucleotide islands, which are usually extended in the promoter and first exon of genes. The DNA methylation is carried out by DNA methyltransferases (DNMT) and it serves as an epigenetic method of gene expression modulation. The epigenetic alternations in genomic DNA have been implicated in the development of malignant and autoimmune diseases. The epigenetic aberration in regulatory DNA sequences may also be responsible for the emergence of changes in the immune system in patients with systemic lupus erythematosus (SLE). The agents 5-azacytidine (azacitidine) and 5-aza-2'-deoxycytidine (decitabine) belong to inhibitors of methyltransferase. These compounds affect the methylation level of promoter sequences and cause phenotypic changes in peripheral blood mononuclear cells (PBMC), which are similar to those observed in PBMC of SLE patients. The lack of methylcytosine in CpG dinucleotides may be responsible for the antigenic properties of microbial DNA. The presence of low-apoptotic methylated DNA fragments has been identified in plasma of SLE patients. These DNA fragments exhibit antigenic properties and may elicit the humoral response responsible for the flare of SLE. The low methylation of CpG residues in the regulatory sequences may also contribute to the elevated expression of human endogenous retroviruses (HERVs) in PBMC of SLE patients. The HERV components exhibit a profound similarity with nuclear antigens and may be responsible for the enhancement of the production of anti-antinuclear antibodies (ANA). Recent advances in the investigation of epigenetic DNA changes have formed the basis of improved understanding of etiopathogenesis of SLE, which may thereby facilitate improvement in therapeutic principles of this disease.     

Cytosine Methylation Associated with Repeat-Induced Point Mutation Causes Epigenetic Gene Silencing in Neurospora Crassa

Repeated DNA sequences are frequently mutated during the sexual cycle in Neurospora crassa by a process named repeat-induced point mutation (RIP). RIP is often associated with methylation of cytosine residues in and around the mutated sequences. Here we demonstrate that this methylation can silence a gene located in nearby, unique sequences. A large proportion of strains that had undergone RIP of a linked duplication flanking a single-copy transgene, hph (hygromycin B phosphotransferase), showed partial silencing of hph. These strains were all heavily methylated throughout the single-copy hph sequences and the flanking sequences. Silencing was alleviated by preventing methylation, either by 5-azacytidine (5AC) treatment or by introduction of a mutation (eth-1) known to reduce intracellular levels of S-adenosylmethionine. Silenced strains exhibited spontaneous reactivation of hph at frequencies of 10(-4) to 0.5. Reactivated strains, as well as cells that were treated with 5AC, gave rise to cultures that were hypomethylated and partially hygromycin resistant, indicating that some of the original methylation was propagated by a maintenance mechanism. Gene expression levels were found to be variable within a population of clonally related cells, and this variation was correlated with epigenetically propagated differences in methylation patterns.     

Analysis of Cytosine Methylation Pattern in Response to Water Deficit in Pea Root Tips

Abstract: The correlation between environmental stress and DNA methylation has been studied by following the methylation status of cytosine residues in the DNA of pea root tips exposed to water deficit. DNA methylation was evaluated by two complementary approaches: (i) immunolabelling by means of a monoclonal antibody against 5-methylcytosine; (ii) MSAP (Methylation-Sensitive Amplified Polymorphism) to verify if methylation and de-methylation in response to water deficit may be related to specific DNA sequences. Immunolabelling showed that water stress induces cytosine hypermethylation in the pea genome. Regarding the CCGG target sequence, an increase in methylation specifically in the second cytosine (about 40 % of total site investigated) was revealed by MSAP analyses. In addition, MSAP band profile detected in three independent repetitions was highly reproducible suggesting that, at least for the CCGG target sequence, methylation was addressed to specific DNA sequences.