Cooperative activity of DNA methyltransferases for maintenance of symmetrical and non-symmetrical cytosine methylation in Arabidopsis thaliana

Maintenance of cytosine methylation in plants is controlled by three DNA methyltransferases. MET1 maintains CG methylation, and DRM1/2 and CMT3 act redundantly to enforce non-CG methylation. RPS, a repetitive hypermethylated DNA fragment from Petunia hybrida, attracts DNA methylation when transferred into Petunia or other species. In Arabidopsis thaliana, which does not contain any RPS homologues, RPS transgenes are efficiently methylated in all sequence contexts. To test which DNA methylation pathways regulate RPS methylation, we examined maintenance of RPS methylation in various mutant backgrounds. Surprisingly, CG methylation was lost in a drm1/2/cmt3 mutant, and non-CG methylation was almost completely eliminated in a met1 mutant. An unusual cooperative activity of all three DNA methyltransferases is therefore required for maintenance of both CG and non-CG methylation in RPS. Other unusual features of RPS methylation are the independence of its non-CG methylation from the RNA-directed DNA methylation (RdDM) pathway and the exceptional maintenance of methylation at a CC(m)TGG site in some epigenetic mutants. This is indicative of activity of a methylation system in plants that may have evolved from the DCM methylation system that controls CC(m)WGG methylation in bacteria. Our data suggest that strict separation of CG and non-CG methylation pathways does not apply to all target regions, and that caution is required in generalizing methylation data obtained for individual genomic regions.     

Genomic distribution and inter-sample variation of non-CpG methylation across human cell types

DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG) dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types. We confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different pluripotent cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human cells using a large representative sample set.     

不同倍性西瓜基因组DNA甲基化水平与模式的MSAP分析

DNA甲基化是表观遗传修饰的主要方式之一,在基因表达调控中发挥重要作用。本研究以不同倍性（2x、3x、4x）西瓜为试材,采用基于DNA甲基化敏感酶的扩增多态性分析（Methylation-Sensitive Ampliftcation Polymorphism,MSAP）方法,在全基因组水平上探究西瓜同源多倍化过程中DNA序列中CCGG位点的甲基化水平及模式变化特征。研究中选用23对选扩引物,共检测到1883个基因位点。二倍体、三倍体、四倍体中检测到的位点数分别为647、655和581;其中发生甲基化的位点数分别为181、150和159。相应的扩增总甲基化率分别为28．0％、22．9％和27．4％：全甲基化位点数分别为121、80和82,相应的全甲基化率分别为18．7％、12．2％和14．1％。进一步对不同倍性西瓜DNA甲基化模式的变化特征进行分析,结果显示：四倍体西瓜与二倍体西瓜相比有超过半数的位点（54．4％）DNA甲基化模式发生了变化,其与三倍体西瓜相比也有近一半的位点（45．4％）DNA甲基化模式发生了变化,并且变化趋势都以四倍体西瓜甲基化程度升高为主：而三倍体西瓜与二倍体西瓜相比．虽然也有41．6％的位点DNA甲基化模式发生了改变,但变化趋势以三倍体西瓜甲基化程度降低略占优势：与之相似,三倍体西瓜与四倍体相比较。甲基化的变化趋势也是以三倍体西瓜甲基化程度降低为主。以上结果表明：不同倍性西瓜中DNA甲基化事件虽均有发生．但不论是从总甲基化率还是全甲基化率来看,DNA甲基化水平与倍性高低关系不大．三倍体西瓜表现出较为显著的低甲基化水平特征。DNA甲基化模式的分析也表明。与二倍体及四倍体西瓜相比．三倍体西瓜DNA甲基化模式的调整主要以去甲基化为主。显示出三倍体西瓜基因组独特的DNA甲基化特征。本研究为进一步从表观遗传学的角度探讨西瓜的三倍体优?     

DNA甲基化与肿瘤

表观遗传指不涉及DNA序列改变的,可随细胞分裂而遗传的基因组修饰作用;DNA甲基化是其中研究最多的基因表达调节机制。异常DNA甲基化可致肿瘤发生,它亦是肿瘤基因诊断和治疗的靶点。文章介绍DNA甲基化基本概念、作用效果及其可能机制;并讨论异常DNA甲基化与肿瘤的关联,包括肿瘤中DNA异常甲基化原因、异常甲基化致瘤机制及基因甲基化研究在肿瘤诊治中的应用等。     

The evidence for functional non-CpG methylation in mammalian cells

In mammalian genomes, the methylation of cytosine residues within CpG dinucleotides is crucial to normal development and cell differentiation. However, methylation of cytosines in the contexts of CpA, CpT, and CpC (non-CpG methylation) has been reported for decades, yet remains poorly understood. In recent years, whole genome bisulphite sequencing (WGBS) has confirmed significant levels of non-CpG methylation in specific tissues and cell types. Non-CpG methylation has several properties that distinguish it from CpG methylation. Here we review the literature describing non-CpG methylation in mammalian cells, describe the important characteristics that distinguish it from CpG methylation, and discuss its functional importance.     

The evidence for functional non-CpG methylation in mammalian cells

In mammalian genomes, the methylation of cytosine residues within CpG dinucleotides is crucial to normal development and cell differentiation. However, methylation of cytosines in the contexts of CpA, CpT, and CpC (non-CpG methylation) has been reported for decades, yet remains poorly understood. In recent years, whole genome bisulphite sequencing (WGBS) has confirmed significant levels of non-CpG methylation in specific tissues and cell types. Non-CpG methylation has several properties that distinguish it from CpG methylation. Here we review the literature describing non-CpG methylation in mammalian cells, describe the important characteristics that distinguish it from CpG methylation, and discuss its functional importance.     

Genetic and DNA Methylation Changes in Cotton (Gossypium) Genotypes and Tissues

In plants, epigenetic regulation is important in normal development and in modulating some agronomic traits. The potential contribution of DNA methylation mediated gene regulation to phenotypic diversity and development in cotton was investigated between cotton genotypes and various tissues. DNA methylation diversity, genetic diversity, and changes in methylation context were investigated using methylation-sensitive amplified polymorphism (MSAP) assays including a methylation insensitive enzyme (BsiSI), and the total DNA methylation level was measured by high-performance liquid chromatography (HPLC). DNA methylation diversity was greater than the genetic diversity in the selected cotton genotypes and significantly different levels of DNA methylation were identified between tissues, including fibre. The higher DNA methylation diversity (CHG methylation being more diverse than CG methylation) in cotton genotypes suggest epigenetic regulation may be important for cotton, and the change in DNA methylation between fibre and other tissues hints that some genes may be epigenetically regulated for fibre development. The novel approach using BsiSI allowed direct comparison between genetic and epigenetic diversity, and also measured CC methylation level that cannot be detected by conventional MSAP.     

Developmental changes in DNA methylation of pollen mother cells of David lily during meiotic prophase I

Epigenetic marks in the form of DNA methylation are involved in the development of germ cells and are important in the maintenance of fertility. However, the controlling system of the on-off switch for DNA methylation largely remains unclear. In this study, the extent of cytosine methylation during the meiotic prophase I in David lily is assessed using high pressure liquid chromatography to evaluate the DNA methylation rates. Comparing the degree of DNA methylation before, during, and after synizesis, both de novo methylation and demethylation occurred. Mainly the methylation level decreased by 21.3% (from 54.8 to 33.5%) during synizesis in the pollen mother cells. The developmental timing of genome-wide DNA methylation acquisition during pollen mother cell development is clarified in this paper. The relative amounts of 5-methyl-deoxycytidine of global methylation in leaf DNA in David lily were also higher than in other species reported.     

Delayed DNA methylation is an integral feature of DNA replication in mammalian cells

In the majority of sites of methylation in the DNA of mammalian cells, the symmetry of methylation is restored within a few minutes of the passage of a replication fork. However, it has been shown that daughter strand methylation in immortalised cell lines is delayed in a substantial minority of sites for up to several hours after replication. We report here the results of two new approaches to the determination of the functional significance of delayed DNA methylation in mammalian cells. Firstly, we demonstrate that normal, nontransformed cells (human peripheral lymphocytes in short-term primary culture) have comparable proportions of delayed DNA methylation to many immortalised cell lines, showing that delayed DNA methylation is not just a secondary consequence of abnormally high methionine requirements commonly observed in transformed cells and that delayed DNA methylation would be unlikely not to occur in vivo. Secondly, we have used 5-aza-2'-deoxycytidine (5azadCyd) to derive subclones of cells from the Chinese hamster ovary cell line which have stably hypomethylated DNA. In three of these subclones which had lost on average one fourth of the methylation sites from their genomes, the proportion of daughter strand methylation which was delayed after replication was reduced by less than 10%. If delayed DNA methylation were site-specific, this implies that of the order of twice the number of "immediate" methylation sites than delayed methylation sites had been lost from the genomes of these hypomethylated subclones. Thus, delayed DNA methylation is an integral part of the process whereby replicating mammalian cells maintain the pattern of methylation in their genomes. These observations are discussed in relation to the significance of delayed DNA methylation for the accurate maintenance of methylation patterns in the genome and the consequent implications for the possible role of methylated deoxycytidines in mammalian gene control.     

Genetic Analysis of the Cardiac Methylome at Single Nucleotide Resolution in a Model of Human Cardiovascular Disease

Epigenetic marks such as cytosine methylation are important determinants of cellular and whole-body phenotypes. However, the extent of, and reasons for inter-individual differences in cytosine methylation, and their association with phenotypic variation are poorly characterised. Here we present the first genome-wide study of cytosine methylation at single-nucleotide resolution in an animal model of human disease. We used whole-genome bisulfite sequencing in the spontaneously hypertensive rat (SHR), a model of cardiovascular disease, and the Brown Norway (BN) control strain, to define the genetic architecture of cytosine methylation in the mammalian heart and to test for association between methylation and pathophysiological phenotypes. Analysis of 10.6 million CpG dinucleotides identified 77,088 CpGs that were differentially methylated between the strains. In F1 hybrids we found 38,152 CpGs showing allele-specific methylation and 145 regions with parent-of-origin effects on methylation. Cis-linkage explained almost 60% of inter-strain variation in methylation at a subset of loci tested for linkage in a panel of recombinant inbred (RI) strains. Methylation analysis in isolated cardiomyocytes showed that in the majority of cases methylation differences in cardiomyocytes and non-cardiomyocytes were strain-dependent, confirming a strong genetic component for cytosine methylation. We observed preferential nucleotide usage associated with increased and decreased methylation that is remarkably conserved across species, suggesting a common mechanism for germline control of inter-individual variation in CpG methylation. In the RI strain panel, we found significant correlation of CpG methylation and levels of serum chromogranin B (CgB), a proposed biomarker of heart failure, which is evidence for a link between germline DNA sequence variation, CpG methylation differences and pathophysiological phenotypes in the SHR strain. Together, these results will stimulate further investigation of the molecular basis of locally regulated variation in CpG methylation and provide a starting point for understanding the relationship between the genetic control of CpG methylation and disease phenotypes.     

A SNF2-like protein facilitates dynamic control of DNA methylation

DRD1 is a SNF2-like protein previously identified in a screen for mutants defective in RNA-directed DNA methylation of a seed promoter in Arabidopsis. Although the initial study established a role for DRD1 in RNA-directed DNA methylation, it did not address whether DRD1 is needed for de novo or maintenance methylation, or whether it is required for methylation of other target sequences. We show here that DRD1 is essential for RNA-directed de novo methylation and acts on different target promoters. In addition, an unanticipated role for DRD1 in erasure of CG methylation was shown when investigating maintenance methylation after segregating away the silencing trigger. DRD1 is unique among known SNF2-like proteins in facilitating not only de novo methylation of target sequences in response to RNA signals, but also loss of methylation when the silencing inducer is withdrawn. The opposing roles of DRD1 could contribute to the dynamic regulation of DNA methylation.     

Role of CpG context and content in evolutionary signatures of brain DNA methylation

DNA methylation is essential in brain function and behavior; therefore, understanding the role of DNA methylation in brain-based disorders begins with the study of DNA methylation profiles in normal brain. Determining the patterns and scale of methylation conservation and alteration in an evolutionary context enables the design of focused but effective methylation studies of disease states. We applied an enzymatic-based approach, Methylation Mapping Analysis by Paired-end Sequencing (Methyl-MAPS), which utilizes second-generation sequencing technology to provide an unbiased representation of genome-wide DNA methylation profiles of human and mouse brains. In this large-scale study, we assayed CpG methylation in cerebral cortex of neurologically and psychiatrically normal human postmortem specimens, as well as mouse forebrain specimens. Cross-species human-mouse DNA methylation conservation analysis shows that DNA methylation is not correlated with sequence conservation. Instead, greater DNA methylation conservation is correlated with increasing CpG density. In addition to CpG density, these data show that genomic context is a critical factor in DNA methylation conservation and alteration signatures throughout mammalian brain evolution. We identify key genomic features that can be targeted for identification of epigenetic loci that may be developmentally and evolutionarily conserved and wherein aberrations in DNA methylation patterns can confer risk for disease.     

Controlling DNA methylation: many roads to one modification

Genetic, biochemical and cytological studies on DNA methylation in several eukaryotic organisms have resulted in leaps of understanding in the past three years. Discoveries of mechanistic links between DNA methylation and histone methylation, and between these processes and RNA interference (RNAi) machineries have reinvigorated the field. The details of the connections between DNA methylation, histone modifications and RNA silencing remain to be elucidated, but it is already clear that no single pathway accounts for all DNA methylation found in eukaryotes. Rather, different taxa use one or more of several general mechanisms to control methylation. Despite recent progress, classic questions remain, including: What are the signals for DNA methylation? Are "de novo" and "maintenance" methylation truly separate processes? How is DNA methylation regulated?     

Role of CpG context and content in evolutionary signatures of brain DNA methylation

DNA methylation is essential in brain function and behavior; therefore, understanding the role of DNA methylation in brain-based disorders begins with the study of DNA methylation profiles in normal brain. Determining the patterns and scale of methylation conservation and alteration in an evolutionary context enables the design of focused but effective methylation studies of disease states. We applied an enzymatic-based approach, Methylation Mapping Analysis by Paired-end Sequencing (Methyl-MAPS), which utilizes second-generation sequencing technology to provide an unbiased representation of genome-wide DNA methylation profiles of human and mouse brains. In this large-scale study, we assayed CpG methylation in cerebral cortex of neurologically and psychiatrically normal human postmortem specimens, as well as mouse forebrain specimens. Cross-species human-mouse DNA methylation conservation analysis shows that DNA methylation is not correlated with sequence conservation. Instead, greater DNA methylation conservation is correlated with increasing CpG density. In addition to CpG density, these data show that genomic context is a critical factor in DNA methylation conservation and alteration signatures throughout mammalian brain evolution. We identify key genomic features that can be targeted for identification of epigenetic loci that may be developmentally and evolutionarily conserved and wherein aberrations in DNA methylation patterns can confer risk for disease.     

Comprehensive DNA methylation analysis of human peripheral blood leukocytes and lymphoblastoid cell lines

DNA methylation is involved in development and in human diseases. Genomic DNA derived from lymphoblastoid cell lines (LCLs) is commonly used to study DNA methylation. There are potential confounding factors regarding the use of LCL-derived DNA, however, such as Epstein-Barr (EB) viral infection and artifacts induced during cell culture. Recently, several groups compared the DNA methylation status of peripheral blood leukocytes (PBLs) and LCLs and concluded that the DNA methylation profiles between them might be consistent. To confirm and extend theses results, we performed a comprehensive DNA methylation analysis using both PBLs and LCLs derived from the same individuals. Using the luminometric methylation assay, we revealed that the global DNA methylation level was different between PBLs and LCLs. Furthermore, the direction of change was not consistent. Comparisons of genome-wide DNA methylation patterns of promoter regions revealed that methylation profiles were largely conserved between PBLs and LCLs. A preliminary analysis in a small number of samples suggested that the methylation status of an LCL may be better correlated with PBLs from the same individual than with LCLs from other individuals. Expectedly, DNA methylation in promoter regions overlapping with CpG islands was associated with gene silencing in both PBLs and LCLs. With regard to methylation differences, we found that hypermethylation was more predominant than hypomethylation in LCLs compared with PBLs. These findings suggest that LCLs should be used for DNA methylation studies with caution as the methylation patterns of promoter regions in LCLs are not always the same as those in PBLs.