The DNA methyltransferase-2 gene in the prawn Macrobrachium rosenbergii: characteristics and expression patterns during ovarian and embryonic development

DNA methylation plays important roles in eukaryotic development. However, studies of DNA methylation in Crustacea are very limited. In the present study, we isolated and identified a DNA methyltransferase 2 (Mar-Dnmt2) gene from the prawn Macrobrachium rosenbergii. The cDNA of Mar-Dnmt2 contained an 1,191-bp open reading frame that encoded a putative Dnmt2 protein of 396 amino acids with the ten motifs characteristic of DNA methyltransferases and exhibiting high identities with other known Dnmt2 homologs (30%-42%). By real-time PCR, an especially high level of Mar-Dnmt2 mRNA was found in the ovary, approximately 5-16 times higher than in other tissues. Moreover, Mar-Dnmt2 mRNA obviously decreased during embryonic development. On the other hand, DNA methylation was detected in this study; about 0.05% of cytosine residues were found to be methylated in the DNA of M. rosenbergii.     

DNA甲基转移酶

DNA甲基化在基因调节和动物发育中起着重要作用。负责DNA甲基化作用的酶尔为DNA甲基转移酶（Dnmts）。到目前为止,在哺乳动物细胞中已经鉴定了三种DNA甲基转移酶基因家族,即Dnmt1、Dnmt2和Dnmt3。鉴定和研究DNA甲基转移酶对阐明DNA甲基化机制起着关键的作用。     

The Methylation Cycle and its Possible Functions in Barley Endosperm Development

Barley endosperm development can be subdivided into the pre-storage, intermediate, storage and desiccation phase. Nothing is known about DNA methylation events involved in different endosperm-specific developmental programmes. A complete set of methylation cycle enzyme genes was identified and investigated by mRNA expression analysis. During the pre-storage phase, methionine synthase and S-adenosylmethionine (AdoMet) synthase genes are expressed at high levels, mainly to produce AdoMet, which might be used for methylation processes as indicated by high expression of methyltransferases HvMET1, HvCMT1 and HvDnmt3-1 as well as AdoHcy hydrolase genes. The methyltransferases, core histones and DNA-unwinding ATPases are co-expressed at the mRNA level. On the contrary, storage protein (prolamin) gene expression is repressed due to CpG methylation. Expression of genes responsible for starch biosynthesis is also developmentally regulated but not methylation-dependent. Thus, during pre-storage phase, activity of HvMET1 and HvCMT1 possibly maintains DNA replication and suppresses specific pathways of maturation. Besides, HvDnmt3-1 might be responsible for differentiation-specific de novo methylation. Expression of methyltransferases HvDnmt3-2 and HvCMT2 peaks during the onset of massive starch accumulation. The enzymes are likely responsible for DNA methylation involved in determining plastid division and amyloplast differentiation as concluded from the patterns of co-expressed genes. Levels of AdoMet decarboxylase mRNA, but not methyltransferase- and AdoHcy mRNA, increase at the beginning of desiccation together with methionine synthase and AdoMet synthase levels. This increase may be indicative for utilization of AdoMet in polyamine production protecting aleuron and embryo cell membranes during desiccation.     

Dnmt2 is the most evolutionary conserved and enigmatic cytosine DNA methyltransferase in eukaryotes

Dnmt2 is the most strongly conserved cytosine DNA methyltransferase in eukaryotes. It has been found in all organisms possessing methyltransferases of the Dnmt1 and Dnmt3 families, whereas in many others Dnmt2 is the sole cytosine DNA methyltransferase. The Dnmt2 molecule contains all conserved motifs of cytosine DNA methyltransferases. It forms 3D complexes with DNA very similar to those of bacterial DNA methyltransferases and performs cytosine methylation by a catalytic mechanism common to all cytosine DNA methyltransferases. Catalytic activity of the purified Dnmt2 with DNA substrates is very low and could hardly be detected in direct biochemical assays. Dnmt2 is the sole cytosine DNA methyltransferase in Drosophila and other dipteran insects. Its overexpression as a transgene leads to DNA hypermethylation in all sequence contexts and to an extended life span. On the contrary, a null-mutation of the Dnmt2 gene leads to a diminished life span, though no evident anomalies in development are observed. Dnmt2 is also the sole cytosine DNA methyltransferase in several protists. Similar to Drosophila these protists have a very low level of DNA methylation. Some limited genome compartments, such as transposable sequences, are probably the methylation targets in these organisms. Dnmt2 does not participate in genome methylation in mammals, but seems to be an RNA methyltransferase modifying the 38th cytosine residue in anticodon loop of certain tRNAs. This modification enhances stability of tRNAs, especially in stressful conditions. Dnmt2 is the only enzyme known to perform RNA methylation by a catalytic mechanism characteristic of DNA methyltransferases. The Dnmt2 activity has been shown in mice to be necessary for paramutation establishment, though the precise mechanisms of its participation in this form of epigenetic heredity are unknown. It seems likely, that either of the two Dnmt2 activities could become a predominant one during the evolution of different species. The high level of the Dnmt2 evolutionary conservation proves its activity to have a significant adaptive value in natural environment.     

DNA methylation mediates the discriminatory power of associative long-term memory in honeybees

Memory is created by several interlinked processes in the brain, some of which require long-term gene regulation. Epigenetic mechanisms are likely candidates for regulating memory-related genes. Among these, DNA methylation is known to be a long lasting genomic mark and may be involved in the establishment of long-term memory. Here we demonstrate that DNA methyltransferases, which induce and maintain DNA methylation, are involved in a particular aspect of associative long-term memory formation in honeybees, but are not required for short-term memory formation. While long-term memory strength itself was not affected by blocking DNA methyltransferases, odor specificity of the memory (memory discriminatory power) was. Conversely, perceptual discriminatory power was normal. These results suggest that different genetic pathways are involved in mediating the strength and discriminatory power of associative odor memories and provide, to our knowledge, the first indication that DNA methyltransferases are involved in stimulus-specific associative long-term memory formation.     

TET家族DNA羟化酶与5-hmC在肿瘤中的作用机制的研究进展

DNA甲基化失调引起基因表达异常是表观遗传学的一个显著特点。目前已知,由DNA甲基转移酶（DNA methyltransferases,DMNTs）催化DNA甲基化,其酶基因突变或表达异常引起DNA甲基化水平的改变。近期研究发现了一种DNA去甲基化酶--TET（Ten-Eleventranslocation）家族DNA羟化酶,能通过多种途径催化5-甲基胞嘧啶（5．methylcytosine,5-mC）去甲基化,从而调控DNA基化的平衡。5-羟甲基胞嘧啶（5-hydroxymethylcytosine,5-hmC）作为DNA去甲基化多重步骤中重要的中间产物,其水平在肿瘤的发生和发展时期发生显著变化。该文从TET家族蛋白展开,介绍TET蛋白的结构、功能及作用机制以及多种人类肿瘤中丁E丁家族基因与5-hmC水平的相关性及其对肿瘤发生发展、诊断预后等临床意义的研究进展。     

Epigenetic cancer therapy: Proof of concept and remaining challenges

Over the past few years several drugs that target epigenetic modifications have shown clinical benefits, thus seemingly validating epigenetic cancer therapy. More recently, however, it has become clear that these drugs are either characterized by low specificity or that their target enzymes have low substrate specificity. As such, clinical proof-of-concept for epigenetic cancer therapies remains to be established. Human cancers are characterized by widespread changes in their genomic DNA methylation and histone modification patterns. Epigenetic cancer therapy aims to restore normal epigenetic modification patterns through the inhibition of epigenetic modifier enzymes. In this review, we provide an overview about the known functional roles of DNA methyltransferases, histone deacetylases, histone methyltransferases, and demethylases in cancer development. The available data identify several examples that warrant further consideration as drug targets. Future research should be directed toward targeted enzyme inhibition and toward exploring interactions between epigenetic pathways to maximize cancer specificity.     

DNA methylation,an epigenetic mechanism connecting folate to healthy embryonic development and aging

Experimental studies demonstrated that maternal exposure to certain environmental and dietary factors during early embryonic development can influence the phenotype of offspring as well as the risk of disease development at the later life. DNA methylation, an epigenetic phenomenon, has been suggested as a mechanism by which maternal nutrients affect the phenotype of their offspring in both honeybee and agouti mouse models. Phenotypic changes through DNA methylation can be linked to folate metabolism by the knowledge that folate, a coenzyme of one-carbon metabolism, is directly involved in methyl group transfer for DNA methylation. During the fetal period, organ-specific DNA methylation patterns are established through epigenetic reprogramming. However, established DNA methylation patterns are not immutable and can be modified during our lifetime by the environment. Aberrant changes in DNA methylation with diet may lead to the development of age-associated diseases including cancer. It is also known that the aging process by itself is accompanied by alterations in DNA methylation. Diminished activity of DNA methyltransferases (Dnmts) can be a potential mechanism for the decreased genomic DNA methylation during aging, along with reduced folate intake and altered folate metabolism. Progressive hypermethylation in promoter regions of certain genes is observed throughout aging, and repression of tumor suppressors induced by this epigenetic mechanism appears to be associated with cancer development. In this review, we address the effect of folate on early development and aging through an epigenetic mechanism, DNA methylation.     

Cytosine methyltransferases as tumor markers

Changes in DNA methylation patterns is a prominent characteristic of human tumors. Tumor cells display reduced levels of genomic DNA methylation and site-specific CpG island hypermethylation. Methylation of CpG dinucleotides is catalyzed by the enzyme family of DNA methyltransferases (DNMTs). In this review, the role of DNA methylation and DNMTs as key determinants of carcinogenesis is further elucidated. The chromatin modifying proteins that are known to interact with DNMTs are also described. Finally, the role of DNMTs as potential therapeutic targets is addressed.     

DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.

The establishment of DNA methylation patterns requires de novo methylation that occurs predominantly during early development and gametogenesis in mice. Here we demonstrate that two recently identified DNA methyltransferases, Dnmt3a and Dnmt3b, are essential for de novo methylation and for mouse development. Inactivation of both genes by gene targeting blocks de novo methylation in ES cells and early embryos, but it has no effect on maintenance of imprinted methylation patterns. Dnmt3a and Dnmt3b also exhibit nonoverlapping functions in development, with Dnmt3b specifically required for methylation of centromeric minor satellite repeats. Mutations of human DNMT3B are found in ICF syndrome, a developmental defect characterized by hypomethylation of pericentromeric repeats. Our results indicate that both Dnmt3a and Dnmt3b function as de novo methyltransferases that play important roles in normal development and disease.     

Expression of exogenous DNA methyltransferases: Application in molecular and cell biology

DNA methyltransferases might be used as powerful tools for studies in molecular and cell biology due to their ability to recognize and modify nitrogen bases in specific sequences of the genome. Methylation of the eukaryotic genome using exogenous DNA methyltransferases appears to be a promising approach for studies on chromatin structure. Currently, the development of new methods for targeted methylation of specific genetic loci using DNA methyltransferases fused with DNA-binding proteins is especially interesting. In the present review, expression of exogenous DNA methyltransferase for purposes of in vivo analysis of the functional chromatin structure along with investigation of the functional role of DNA methylation in cell processes are discussed, as well as future prospects for application of DNA methyltransferases in epigenetic therapy and in plant selection.     

Gardening the genome: DNA methylation in Arabidopsis thaliana

DNA methylation has two essential roles in plants and animals - defending the genome against transposons and regulating gene expression. Recent experiments in Arabidopsis thaliana have begun to address crucial questions about how DNA methylation is established and maintained. One cardinal insight has been the discovery that DNA methylation can be guided by small RNAs produced through RNA-interference pathways. Plants and mammals use a similar suite of DNA methyltransferases to propagate DNA methylation, but plants have also developed a glycosylase-based mechanism for removing DNA methylation, and there are hints that similar processes function in other organisms.     

Mammalian DNA methyltransferases show different subnuclear distributions

In mammalian cells, DNA methylation patterns are precisely maintained after DNA replication with defined changes occurring during development. The major DNA methyltransferase (Dnmt1) is associated with nuclear replication sites during S-phase, which is consistent with a role in maintenance methylation. The subcellular distribution of the recently discovered de novo DNA methyltransferases, Dnmt3a and Dnmt3b, was investigated by immunofluorescence and by epitope tagging. We now show that both Dnmt3a and Dnmt3b are distributed throughout the nucleoplasm but are not associated with nuclear DNA replication sites during S-phase. These results suggest that de novo methylation by Dnmt3a and Dnmt3b occurs independently of the replication process and might involve an alternative mechanism for accessing the target DNA. The different subcellular distribution of mammalian DNA methyltransferases might thus contribute to the regulation of DNA methylation.     

Arabidopsis Trithorax histone methyltransferases are redundant in regulating development and DNA methylation

Although the Trithorax histone methyltransferases ATX1–5 are known to regulate development and stress responses by catalyzing histone H3K4 methylation in Arabidopsis thaliana, it is unknown whether and how these histone methyltransferases affect DNA methylation. Here, we found that the redundant ATX1–5 proteins are not only required for plant development and viability but also for the regulation of DNA methylation. The expression and H3K4me3 levels of both RNA-directed DNA methylation (RdDM) genes (NRPE1, DCL3, IDN2, and IDP2) and active DNA demethylation genes (ROS1, DML2, and DML3) were downregulated in the atx1/2/4/5 mutant. Consistent with the facts that the active DNA demethylation pathway mediates DNA demethylationmainly at CG and CHG sites, and that the RdDM pathway mediates DNA methylation mainly at CHH sites, whole-genome DNA methylation analyses showed that hyper-CG and CHG DMRs in atx1/2/4/5 significantly overlapped with those in the DNA demethylation pathway mutant ros1 dml2 dml3 (rdd), and that hypo-CHH DMRs in atx1/2/4/5 significantly overlapped with those in the RdDM mutant nrpe1, suggesting that the ATX paralogues function redundantly to regulate DNA methylation by promoting H3K4me3 levels and expression levels of both RdDM genes and active DNA demethylation genes. Given that the ATX proteins function as catalytic subunits of COMPASS histone methyltransferase complexes, we also demonstrated that the COMPASS complex components function as a whole to regulate DNA methylation. This study reveals a previously uncharacterized mechanism underlying the regulation of DNA methylation.     

Plant DNA methyltransferases

DNA methylation is an important modification of DNA that plays a role in genome management and in regulating gene expression during development. Methylation is carried out by DNA methyltransferases which catalyse the transfer of a methyl group to bases within the DNA helix. Plants have at least three classes of cytosine methyltransferase which differ in protein structure and function. The METI family, homologues of the mouse Dnmt1 methyltransferase, most likely function as maintenance methyltransferases, but may also play a role in de novo methylation. The chromomethylases, which are unique to plants, may preferentially methylate DNA in heterochromatin; the remaining class, with similarity to Dnmt3 methyltransferases of mammals, are putative de novo methyltransferases. The various classes of methyltransferase may show differential activity on cytosines in different sequence contexts. Chromomethylases may preferentially methylate cytosines in CpNpG sequences while the Arabidopsis METI methyltransferase shows a preference for cytosines in CpG sequences. Additional proteins, for example DDM1, a member of the SNF2/SWI2 family of chromatin remodelling proteins, are also required for methylation of plant DNA.