DNA cytosine methylation in plant development

Cytosine bases of the nuclear genome in higher plants are often extensively methylated.Cytosine methylation has been implicated in the silencing of both transposable elements (TEs) and endogenous genes,and loss of methylation may have severe functional consequences.The recent methylation profiling of the entire Arabidopsis genome has provided novel insights into the extent and pattern of cytosine methylation and its relationships with gene activity.In addition,the fresh studies also revealed the more dynami...     

DNA cytosine methylation and heat-induced deamination

The heat-induced conversion of 5-methylcytosine (m⁵C) residues to thymine residues and of cytosine to uracil residues in single-stranded DNA was studied. The calculated rates for deamination at 37°C and pH 7.4 were 9.5×10^–10 and 2.1×10^–10 sec^–1, respectively. N⁴-Methyldeoxycytidine, which is in the DNA of certain thermophilic bacteria, was more heat-resistant than was deoxycytidine and much more than was 5-methyldeoxycytidine. Thermophilic bacteria which contain N⁴-methylcytosine rather than m⁵C in their genomes may thereby largely avoid heat-induced mutation due to deamination, which is incurred by the many organisms that contain m⁵C in their DNA.     

Non-symmetrical cytosine methylation in tobacco pollen DNA

We have detected sequence-specific non-symmetrical cytosine methylation within a 140 bp region of the promoter for the tobacco auxin-binding protein gene T85 in pollen DNA. Direct sequencing of the population of bisulphite reaction products showed that, in this region, 10 out of a possible 49 cytosine residues were methylated at a high frequency in pollen whereas the corresponding region from somatic cells (leaf DNA) did not show a detectable level of methylation. The context of these sites was 1×m⁵CpTpC, 1×m⁵CpGpT, 1×m⁵CpCpT, 2×m⁵CpTpT, 2×m⁵CpGpG, and 3×m⁵CpApT of which only m⁵CpGpG and m⁵CpGpT fitted the consensus sequence for symmetrical methylation in plants.     

High-resolution analysis of cytosine methylation in ancient DNA

Epigenetic changes to gene expression can result in heritable phenotypic characteristics that are not encoded in the DNA itself, but rather by biochemical modifications to the DNA or associated chromatin proteins. Interposed between genes and environment, these epigenetic modifications can be influenced by environmental factors to affect phenotype for multiple generations. This raises the possibility that epigenetic states provide a substrate for natural selection, with the potential to participate in the rapid adaptation of species to changes in environment. Any direct test of this hypothesis would require the ability to measure epigenetic states over evolutionary timescales. Here we describe the first single-base resolution of cytosine methylation patterns in an ancient mammalian genome, by bisulphite allelic sequencing of loci from late Pleistocene Bison priscus remains. Retrotransposons and the differentially methylated regions of imprinted loci displayed methylation patterns identical to those derived from fresh bovine tissue, indicating that methylation patterns are preserved in the ancient DNA. Our findings establish the biochemical stability of methylated cytosines over extensive time frames, and provide the first direct evidence that cytosine methylation patterns are retained in DNA from ancient specimens. The ability to resolve cytosine methylation in ancient DNA provides a powerful means to study the role of epigenetics in evolution.     

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

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

Common methods for cytosine methylation analysis in DNA

The review considers the methods most commonly used to detect DNA methylation, their advantages, potential limitations, and selection for various purposes. A detailed protocol is described for bisulfite treatment, which is used as a preliminary step in the majority of DNA methylation assays.     

Conservation of Dcm-mediated cytosine DNA methylation in Escherichia coli

In Escherichia coli, cytosine DNA methylation is catalyzed by the DNA cytosine methyltransferase (Dcm) protein and occurs at the second cytosine in the sequence 5'CCWGG3'. Although the presence of cytosine DNA methylation was reported over 35?years ago, the biological role of 5-methylcytosine in E.?coli remains unclear. To gain insight into the role of cytosine DNA methylation in E.?coli, we (1) screened the 72 strains of the ECOR collection and 90 recently isolated environmental samples for the presence of the full-length dcm gene using the polymerase chain reaction; (2) examined the same strains for the presence of 5-methylcytosine at 5'CCWGG3' sites using a restriction enzyme isoschizomer digestion assay; and (3) quantified the levels of 5-methyl-2'-deoxycytidine in selected strains using liquid chromatography tandem mass spectrometry. Dcm-mediated cytosine DNA methylation is conserved in all 162 strains examined, and the level of 5-methylcytosine ranges from 0.86% to 1.30% of the cytosines. We also demonstrate that Dcm reduces the expression of ribosomal protein genes during stationary phase, and this may explain the highly conserved nature of this DNA modification pathway.     

Ultra-sensitive immunodetection of 5'methyl cytosine for DNA methylation analysis on oligonucleotide microarrays.

For the determination of methylation levels in genomic regulatory DNA sequences a high-sensitive assay for detecting 5'methyl-cytosines (5'mC) in non-bisulfite-treated DNA has been established. The system is designed for the application of immunofluorescence using a monoclonal antibody that specifically recognizes 5'mC in single-stranded DNA hybridized to oligonucleotide microarrays. For assay readout an ultra-sensitive fluorescence scanner with submicrometer resolution was used. To minimize autofluorescence 150-microm thin glass slides with an aldehyde-functionalized surface were developed. These methodological improvements allowed the detection of 5'mC in synthetic oligonucleotides hybridized to microarrays with atto molar analytical sensitivity. Using enzymatic fragmented genomic DNA from myeloid leukemia tumor cell lines differences in the methylation status of gene regulatory sequences for E-cadherin, p15/CDKN2b and p16/CDKN2a were demonstrated. Thus, this novel technique can potentially be used for DNA methylation analysis in various scientific fields.     

Four-stranded DNA structure and DNA base methylation in the mechanism of action of restriction endonucleases.

We examined the probability of short palindromec DNA sequences to occur as four-stranded structures held together in double-helical DNA by the additional hydrogen bonds postulated by McGavin (1971). The likeliness of the palindromes to be folded at their symmetry axes to allow the additional hydrogen bonding was considered using published physicochemical evidence and theoretical deductions. We deduced that both in vivo and in vitro the requirements may be met for duplex DNA folding which would approach palindrome complementary base bairs and thus allow the formation of the additional hydrogen bonds. However, we propose hydrogen bonding between guanine-cytosine base pairs to be different than that proposed by McGavin. Using CPK atom models we found that formation of the tertiary conformation already proposed by other authors and which we call the cage structure may be prevented or hindered by adenine, guanine or cytosine methylation. The available experimental data on recognition and cleavage site specificity of the Type 11 restriction endonucleases were confronted with the cage model as an alternative of the cruciform model and with the postulated effects of base methylation. The published data did not contradict the validity of the cage model and the role of base methylation in preventing the four-stranded palindrome structure. An applicability of the basic ideas of four-stranded DNA and base methylation effect to the mechanism of action of modification methylases and other restriction endonucleases was shortly discussed, but only tentative conclusions could be reached.     

Specific targeting of cytosine methylation to DNA sequences in vivo

Development of methods that will allow exogenous imposition of inheritable gene-specific methylation patterns has potential application in both therapeutics and in basic research. An ongoing approach is the use of targeted DNA methyltransferases, which consist of a fusion between gene-targeted zinc-finger proteins and prokaryotic DNA cytosine methyltransferases. These enzymes however have so far demonstrated significant and unacceptable levels of non-targeted methylation. We now report the development of second-generation targeted methyltransferase enzymes comprising enhanced zinc-finger arrays coupled to methyltransferase mutants that are functionally dominated by their zinc-finger component. Both in vitro plasmid methylation studies and a novel bacterial assay reveal a high degree of target-specific methylation by these enzymes. Furthermore, we demonstrate for the first time transient expression of targeted cytosine methyltransferase in mammalian cells resulting in the specific methylation of a chromosomal locus. Importantly, the resultant methylation pattern is inherited through successive cell divisions.     

5-甲基胞嘧啶修饰对沉默抗生素基因的激活

对真核生物的表观遗传学研究表明,5-甲基胞嘧啶修饰参与了多种重要生理功能。虽然在原核生物中也存在5-甲基胞嘧啶修饰,但其具体功能尚未确定。大肠杆菌编码的Dcm甲基转移酶负责DNA的5-甲基胞嘧啶修饰[1],有研究报道显示,Dcm与细菌的限制修饰系统相关[2];也有研究报道dcm基因能影响大肠杆菌中核糖体基因的表达,从而影响初级代谢和次级代谢[3]。本期介绍了高婕、贺新义等发表的论文"大肠杆菌甲基转移酶dcm基因的表达对变铅青链霉菌的多效性影响"[4],作者巧妙地利用变铅青链霉菌的DNA无甲基化修饰这一特点,将大肠杆菌dcm基因导入变铅青链霉菌,研究了5-甲基胞嘧啶修饰在变铅青链霉菌中的功能。结果发现,DNA的5-甲基胞嘧啶修饰不仅可影响变铅青链霉菌的形态和生理分化,而且还能激活放线紫红素沉默基因的表达。论文作者以变铅青链霉菌为材料,拓展了对原核生物DNA5-甲基胞嘧啶修饰的生理功能的认识。以此为基础的深入研究,不仅有助于揭示5-甲基胞嘧啶修饰在原核生物中的功能,而且有可能为沉默抗生素基因的表达或抗生素产量的提高提供一个新的途径。     

Effects of 5 cytosine methylation on the B-Z transition in DNA restriction fragments and recombinant plasmids

Alternating (dC-dG)n regions in DNA restriction fragments and recombinant plasmids were methylated at the 5 position of the cytosine residues by the HhaI methylase. Methylation lowers the concentration of NaCl or MgCl2 necessary to cause the B-Z conformational transition in these sequences. Ionic strengths higher than physiological conditions are required to form the Z conformation when the methylated (dC-dG)n tract is contiguous with regions that do not form Z structures, in contrast to the results with the DNA polymer poly(m5dC-dG) . poly(m5dC-dG). In supercoiled plasmids containing (dC-dG)n sequences, methylation reduces the number of negative supercoils necessary to stabilize the Z conformation. Calculations of the observed free energy contributions of the B-Z junction and cytosine methylation suggest that two junctions offset the favorable effect of methylation on the Z conformation in (dC-dG)n sequences (about 29 base-pairs in length). Studies with individual methylated topoisomers demonstrate that increasing Na+ concentration up to approximately 0.2 M inhibits the formation of the Z conformation in the (m5dC-dG)n region of supercoiled plasmids. The results suggest that methylation may serve as a triggering mechanism for Z DNA formation in supercoiled DNAs.     

Presence and role of cytosine methylation in DNA viruses of animals

Nucleotide composition varies greatly among DNA viruses of animals, yet the evolutionary pressures and biological mechanisms driving these patterns are unclear. One of the most striking discrepancies lies in the frequency of CpG (the dinucleotide CG, linked by a phosphate group), which is underrepresented in most small DNA viruses (those with genomes below 10 kb) but not in larger DNA viruses. Cytosine methylation might be partially responsible, but research on this topic has focused on a few virus groups. For several viruses that integrate their genome into the host genome, the methylation status during this stage has been studied extensively, and the relationship between methylation and viral-induced tumor formation has been examined carefully. However, for actively replicating viruses—particularly small DNA viruses—the methylation status of CpG motifs is rarely known and the effects on the viral life cycle are obscure. In vertebrate host genomes, most cytosines at CpG sites are methylated, which in vertebrates acts to regulate gene expression and facilitates the recognition of unmethylated, potentially pathogen-associated DNA. Here we briefly introduce cytosine methylation before reviewing what is currently known about CpG methylation in DNA viruses.     

Persistence of cytosine methylation of DNA following fertilisation in the mouse

Normal development of the mammalian embryo requires epigenetic reprogramming of the genome. The level of cytosine methylation of CpG-rich (5meC) regions of the genome is a major epigenetic regulator and active global demethylation of 5meC throughout the genome is reported to occur within the first cell-cycle following fertilization. An enzyme or mechanism capable of catalysing such rapid global demethylation has not been identified. The mouse is a widely used model for studying developmental epigenetics. We have reassessed the evidence for this phenomenon of genome-wide demethylation following fertilisation in the mouse. We found when using conventional methods of immunolocalization that 5meC showed a progressive acid-resistant antigenic masking during zygotic maturation which gave the appearance of demethylation. Changing the unmasking strategy by also performing tryptic digestion revealed a persistence of a methylated state. Analysis of methyl binding domain 1 protein (MBD1) binding confirmed that the genome remained methylated following fertilisation. The maintenance of this methylated state over the first several cell-cycles required the actions of DNA methyltransferase activity. The study shows that any 5meC remodelling that occurs during early development is not explained by a global active loss of 5meC staining during the cleavage stage of development and global loss of methylation following fertilization is not a major component of epigenetic reprogramming in the mouse zygote.     

Hunting for the 5th base: Techniques for analyzing DNA methylation

Background

In the recent past large progress has been made in the analysis of the epigenome, the entirety of epigenetic modifications, and its meaning for the implementation of the genetic code. Besides histone modifications and miRNA expression, DNA methylation is one of the key players in the field of epigenetics, involved in numerous regulatory processes.

Methods

In the present review we focus on methods for the analysis of DNA methylation patterns and present an overview about techniques and basic principles available for this purpose.

Results and general significance

We here discuss advantages and disadvantages of various methods and their feasibility for specific tasks of DNA methylation analysis.     

Murine DNA cytosine C(5)-methyltransferase: in vitro studies of de novo methylation spreading

The preference of murine DNA (cytosine-5)-methyltransferase (Dnmt1) for single stranded DNA substrates is increased up to 50-fold by the presence of a proximal 5-methyl cytosine (5(me)C). This modulation is distance-dependent and is due to an enhanced binding affinity and minor changes in catalytic efficiency. No modulation was observed with double stranded DNA. Modulation requires that the 5(me)C moiety be attached to the DNA strand containing the CpG methylation target. Our results support a model in which 5(me)C binding by the enzyme occurs to at least one site outside the region involved in CpG recognition. No modulation in response to 5(me)C is observed with the bacterial enzyme M.SssI, which lacks the large N-terminal regulatory domain found in Dnmt1. We suggest that this allosteric modulation involves the N-terminal domain of Dnmt1.     

The CHH motif in sugar beet satellite DNA: a modulator for cytosine methylation

Methylation of DNA is important for the epigenetic silencing of repetitive DNA in plant genomes. Knowledge about the cytosine methylation status of satellite DNAs, a major class of repetitive DNA, is scarce. One reason for this is that arrays of tandemly arranged sequences are usually collapsed in next‐generation sequencing assemblies. We applied strategies to overcome this limitation and quantified the level of cytosine methylation and its pattern in three satellite families of sugar beet (Beta vulgaris) which differ in their abundance, chromosomal localization and monomer size. We visualized methylation levels along pachytene chromosomes with respect to small satellite loci at maximum resolution using chromosome‐wide fluorescent in situ hybridization complemented with immunostaining and super‐resolution microscopy. Only reduced methylation of many satellite arrays was obtained. To investigate methylation at the nucleotide level we performed bisulfite sequencing of 1569 satellite sequences. We found that the level of methylation of cytosine strongly depends on the sequence context: cytosines in the CHH motif show lower methylation (44–52%), while CG and CHG motifs are more strongly methylated. This affects the overall methylation of satellite sequences because CHH occurs frequently while CG and CHG are rare or even absent in the satellite arrays investigated. Evidently, CHH is the major target for modulation of the cytosine methylation level of adjacent monomers within individual arrays and contributes to their epigenetic function. This strongly indicates that asymmetric cytosine methylation plays a role in the epigenetic modification of satellite repeats in plant genomes.     

Toxoplasma gondii and Cryptosporidium parvum lack detectable DNA cytosine methylation

Epigenetic factors play a role in the expression of virulence traits in Apicomplexa. Apicomplexan genomes encode putative DNA cytosine methylation enzymes. To assess the presence of cytosine methylation of Toxoplasma gondii and Cryptosporidium parvum DNA, we used mass spectrometry analysis and confirmed that these organisms lack detectable methylcytosine in their DNA.