Interactions within the mammalian DNA methyltransferase family

Background  

In mammals, epigenetic information is established and maintained via the postreplicative methylation of cytosine residues by the DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is required for maintenance methylation whereas Dnmt3a and Dnmt3b are responsible for de novo methylation. Contrary to Dnmt3a or Dnmt3b, the isolated C-terminal region of Dnmt1 is catalytically inactive, despite the presence of the sequence motifs typical of active DNA methyltransferases. Deletion analysis has revealed that a large part of the N-terminal domain is required for enzymatic activity.     

Novel splice variants associated with one of the zebrafish <Emphasis Type="Italic">dnmt3</Emphasis>genes

Background  

DNA methylation and the methyltransferases are known to be important in vertebrate development and this may be particularly true for the Dnmt3 family of enzymes because they are thought to be the de novo methyltransferases. Mammals have three Dnmt3 genes; Dnmt3a, Dnmt3b, and Dnmt3L, two of which encode active enzymes and one of which produces an inactive but necessary cofactor. However, due to multiple promoter use and alternative splicing there are actually a number of dnmt3 isoforms present. Six different dnmt3 genes have recently been identified in zebrafish.     

Lack of involvement of known DNA methyltransferases in familial hydatidiform mole implies the involvement of other factors in establishment of imprinting in the human female germline

Background  

Differential methylation of the two alleles is a hallmark of imprinted genes. Correspondingly, loss of DNA methyltransferase function results in aberrant imprinting and abnormal post-fertilization development. In the mouse, mutations of the oocyte-specific isoform of the DNA methyltransferase Dnmt1 (Dnmt1o) and of the methyltransferase-like Dnmt3L gene result in specific failures of imprint establishment or maintenance, at multiple loci. We have previously shown in humans that an analogous inherited failure to establish imprinting at multiple loci in the female germline underlies a rare phenotype of recurrent hydatidiform mole.     

Increased Expression of DNA Methyltransferase 3a in Obese Adipose Tissue: Studies With Transgenic Mice

Epigenetic mechanisms are likely to be involved in the development of obesity. This study was designed to examine the role of a DNA methyltransferase (Dnmt3a), in obese adipose tissue. The gene expression of Dnmts was examined by quantitative real‐time PCR analysis. Transgenic mice overexpressing Dnmt3a in the adipose tissue driven by the aP2 promoter were created (Dnmt3a mice). DNA methylation of downregulated genes was examined using bisulfite DNA methylation analysis. Dnmt3a mice were fed a methyl‐supplemented or high‐fat diet, and subjected to body weight measurement and gene expression analysis of the adipose tissue. Expression of Dnmt3a was markedly upregulated in the adipose tissue of obese mice. The complementary DNA (cDNA) microarray analysis of Dnmt3a mice revealed a slight decrease in the gene expression of secreted frizzled‐related protein 1 (SFRP1) and marked increase in that of interferon responsive factor 9 (IRF9). In the SFRP1 promoter, DNA methylation was not markedly increased in Dnmt3a mice relative to wild‐type mice. In experiments with a high‐fat diet or methyl‐supplemented diet, body weight did not differ significantly with the genotypes. Gene expression levels of inflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and monocyte chemoattractant protein‐1 (MCP‐1) were higher in Dnmt3a mice than in wild‐type mice on a high‐fat diet. This study suggests that increased expression of Dnmt3a in the adipose tissue may contribute to obesity‐related inflammation. The data highlight the potential role of Dnmt3a in the adult tissue as well as in the developing embryo and cancer.     

Novel anticancer drug curaxin CBL0137 impairs DNA methylation by eukaryotic DNA methyltransferase Dnmt3a

Novel DNA intercalating anticancer drug curaxin CBL0137 significantly inhibited in vitro DNA methylation by eukaryotic DNA methyltransferase Dnmt3a catalytic domain (Dnmt3a-CD) at low micromolar concentrations (IC₅₀ 3–9 µM). CBL0137 reduced the binding affinity of Dnmt3a-CD to its DNA target, causing up to four-fold increase in the K_d of the enzyme/DNA complex. Binding of CBL0137 to Dnmt3a-CD was not observed. The observed decrease in methylation activity of Dnmt3a-CD in the presence of CBL0137 can be explained by curaxin’s ability to intercalate into DNA.     

Reduced Susceptibility of DNA Methyltransferase 1 Hypomorphic (Dnmt1(N/+)) Mice to Hepatic Steatosis upon Feeding Liquid Alcohol Diet

Background

Methylation at C-5 (5-mdC) of CpG base pairs, the most abundant epigenetic modification of DNA, is catalyzed by 3 essential DNA methyltransferases (Dnmt1, Dnmt3a and Dnmt3b). Aberrations in DNA methylation and Dnmts are linked to different diseases including cancer. However, their role in alcoholic liver disease (ALD) has not been elucidated.

Methodology/Principal Findings

Dnmt1 wild type (Dnmt1 ^+/+) and hypomorphic (Dnmt1 ^N/+) male mice that express reduced level of Dnmt1 were fed Lieber-DeCarli liquid diet containing ethanol for 6 weeks. Control mice were pair-fed calorie-matched alcohol-free liquid diet, and Dnmtase activity, 5-mdC content, gene expression profile and liver histopathology were evaluated. Ethanol feeding caused pronounced decrease in hepatic Dnmtase activity in Dnmt1 ^+/+ mice due to decrease in Dnmt1 and Dnmt3b protein levels and upregulation of miR-148 and miR-152 that target both Dnmt1 and Dnmt3b. Microarray and qPCR analysis showed that the genes involved in lipid, xenobiotic and glutathione metabolism, mitochondrial function and cell proliferation were dysregulated in the wild type mice fed alcohol. Surprisingly, Dnmt1 ^N/+ mice were less susceptible to alcoholic steatosis compared to Dnmt1 ^+/+ mice. Expression of several key genes involved in alcohol (Aldh3b1), lipid (Ppara, Lepr, Vldlr, Agpat9) and xenobiotic (Cyp39a1) metabolism, and oxidative stress (Mt-1, Fmo3) were significantly (P<0.05) altered in Dnmt1 ^N/+ mice relative to the wild type mice fed alcohol diet. However, CpG islands encompassing the promoter regions of Agpat9, Lepr, Mt1 and Ppara were methylation-free in both genotypes irrespective of the diet, suggesting that promoter methylation does not regulate their expression. Similarly, 5-mdC content of the liver genome, as measured by LC-MS/MS analysis, was not affected by alcohol diet in the wild type or hypomorphic mice.

Conclusions/Significance

Although feeding alcohol diet reduced Dnmtase activity, the loss of one copy of Dnmt1 protected mice from alcoholic hepatosteatosis by dysregulating genes involved in lipid metabolism and oxidative stress.     

5-Aza-2′-deoxycytidine Leads to Reduced Embryo Implantation and Reduced Expression of DNA Methyltransferases and Essential Endometrial Genes

Background

The DNA demethylating agent 5-aza-2′-deoxycytidine (5-aza-CdR) incorporates into DNA and decreases DNA methylation, sparking interest in its use as a potential therapeutic agent. We aimed to determine the effects of maternal 5-aza-CdR treatment on embryo implantation in the mouse and to evaluate whether these effects are associated with decreased levels of DNA methyltransferases (Dnmts) and three genes (estrogen receptor α [Esr1], progesterone receptor [Pgr], and homeobox A10 [Hoxa10]) that are vital for control of endometrial changes during implantation.

Methods and Principal Findings

Mice treated with 5-aza-CdR had a dose-dependent decrease in number of implantation sites, with defected endometrial decidualization and stromal cell proliferation. Western blot analysis on pseudo-pregnant day 3 (PD3) showed that 0.1 mg/kg 5-aza-CdR significantly repressed Dnmt3a protein level, and 0.5 mg/kg 5-aza-CdR significantly repressed Dnmt1, Dnmt3a, and Dnmt3b protein levels in the endometrium. On PD5, mice showed significantly decreased Dnmt3a protein level with 0.1 mg/kg 5-aza-CdR, and significantly decreased Dnmt1 and Dnmt3a with 0.5 mg/kg 5-aza-CdR. Immunohistochemical staining showed that 5-aza-CdR repressed DNMT expression in a cell type–specific fashion within the uterus, including decreased expression of Dnmt1 in luminal and/or glandular epithelium and of Dnmt3a and Dnmt3b in stroma. Furthermore, the 5′ flanking regions of the Esr1, Pgr, and Hoxa10 were hypomethylated on PD5. Interestingly, the higher (0.5 mg/kg) dose of 5-aza-CdR decreased protein expression of Esr1, Pgr, and Hoxa10 in the endometrium on PD5 in both methylation-dependent and methylation-independent manners.

Conclusions

The effects of 5-aza-CdR on embryo implantation in mice were associated with altered expression of endometrial Dnmts and genes controlling endometrial changes, suggesting that altered gene methylation, and not cytotoxicity alone, contributes to implantation defects induced by 5-aza-CdR.     

Diurnal expression of Dnmt3b mRNA in mouse liver is regulated by feeding and hepatic clockwork

DNA methyltransferase 3B (DNMT3B) is critically involved in de novo DNA methylation and genomic stability, while the regulatory mechanism in liver is largely unknown. We previously reported that diurnal variation occurs in the mRNA expression of Dnmt3b in adult mouse liver. The aim of this study was to determine the mechanism underlying the diurnal expression pattern. The highest level and the lowest level of Dnmt3b mRNA expression were confirmed to occur at dawn and in the afternoon, respectively, and the expression pattern of Dnmt3b closely coincided with that of Bmal1. Since the diurnal pattern of Dnmt3b mRNA expression developed at weaning and scheduled feeding to separate the feeding cycle from the light/dark cycle led to a phase-shift in the expression, it could be assumed that feeding plays a critical role as an entrainment signal. In liver-specific Bmal1 knockout (L-Bmal1 KO) mice, L-Bmal1 deficiency resulted in significantly higher levels of Dnmt3b at all measured time points, and the time when the expression was the lowest in wild-type mice was shifted to earlier. Investigation of global DNA methylation revealed a temporal decrease of 5-methyl-cytosine percentage in the genome of wild-type mice in late afternoon. By contrast, no such decrease in 5-methyl-cytosine percentage was detected in L-Bmal1 KO mice, suggesting that altered Dnmt3b expression affects the DNA methylation state. Taken together, the results suggest that the feeding and hepatic clockwork generated by the clock genes, including Bmal1, regulate the diurnal variation in Dnmt3b mRNA expression and the consequent dynamic changes in global DNA methylation.     

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.     

Np95 interacts with de novo DNA methyltransferases,Dnmt3a and Dnmt3b,and mediates epigenetic silencing of the viral CMV promoter in embryonic stem cells

Recent studies have indicated that nuclear protein of 95 kDa (Np95) is essential for maintaining genomic methylation by recruiting DNA methyltransferase (Dnmt) 1 to hemi‐methylated sites. Here, we show that Np95 interacts more strongly with regulatory domains of the de novo methyltransferases Dnmt3a and Dnmt3b. To investigate possible functions, we developed an epigenetic silencing assay using fluorescent reporters in embryonic stem cells (ESCs). Interestingly, silencing of the cytomegalovirus promoter in ESCs preceded DNA methylation and was strictly dependent on the presence of either Np95, histone H3 methyltransferase G9a or Dnmt3a and Dnmt3b. Our results indicate a regulatory role for Np95, Dnmt3a and Dnmt3b in mediating epigenetic silencing through histone modification followed by DNA methylation.     

Diet and cell size both affect queen-worker differentiation through DNA methylation in honey bees (Apis mellifera, Apidae)

Background

Young larvae of the honey bee (Apis mellifera) are totipotent; they can become either queens (reproductives) or workers (largely sterile helpers). DNA methylation has been shown to play an important role in this differentiation. In this study, we examine the contributions of diet and cell size to caste differentiation.

Methodology/Principal Findings

We measured the activity and gene expression of one key enzyme involved in methylation, Dnmt3; the rates of methylation in the gene dynactin p62; as well as morphological characteristics of adult bees developed either from larvae fed with worker jelly or royal jelly; and larvae raised in either queen or worker cells. We show that both diet type and cell size contributed to the queen-worker differentiation, and that the two factors affected different methylation sites inside the same gene dynactin p62.

Conclusions/Significance

We confirm previous findings that Dnmt3 plays a critical role in honey bee caste differentiation. Further, we show for the first time that cell size also plays a role in influencing larval development when diet is kept the same.     

Expression patterns of dnmt3aa,dnmt3ab,and dnmt4 during development and fin regeneration in zebrafish

Epigenetic modifications such as DNA methylation and chromatin modifications are critical for regulation of spatiotemporal gene expression during development. In mammals, the de novo-type DNA methyltransferases (Dnmts), Dnmt3a and Dnmt3b, are responsible for the creation of DNA methylation patterns during development. In addition to developmental processes, we recently showed that DNA methylation levels are dynamically changed during zebrafish fin regeneration, suggesting that the de novo-type Dnmts might play roles in the regulation of gene expression during regeneration processes. Here, we showed the detailed expression profiles of three zebrafish dnmt genes (dnmt3aa, dnmt3ab, and dnmt4), which were identified as the orthologues of mammalian dnmt3a and dnmt3b, during embryonic and larval development, as well as fin regeneration processes. dnmt3aa and dnmt3ab are expressed in the brain, pharyngeal arches, pectoral fin buds, intestine, and swim bladder; the specific expression of dnmt3aa is observed in the pronephric duct during larval development. dnmt4 expression is observed in the zona limitans intrathalamica, midbrain–hindbrain boundary, ciliary marginal zone, pharyngeal arches, auditory capsule, pectoral fin buds, intestine, pancreas, liver, and hematopoietic cells in the aorta–gonad–mesonephros and caudal hematopoietic tissue from 48 to 72 h post-fertilization. Furthermore, during fin regeneration, strong dnmt3aa expression, and faint dnmt3ab and dnmt4 expression are detected in blastema cells at 72 h post-amputation. Taken together, our results suggest that zebrafish Dnmt3aa, Dnmt3ab, and Dnmt4 may play roles in the formation of various organs, such as the brain, kidney, digestive organs, and/or hematopoietic cells, as well as in the differentiation of blastema cells.     

Detection of DNA Methylation by Dnmt3a Methyltransferase using Methyl-Dependent Restriction Endonucleases

DNA methylation at cytosine residues in CpG sites by DNA methyltransferases (MTases) is associated with various cell processes. Eukaryotic MTase Dnmt3a is the key enzyme that establishes the de novo methylation pattern. A new in vitro assay for DNA methylation by murine MTase Dnmt3a was developed using methyl-dependent restriction endonucleases (MD-REs), which specifically cleave methylated DNA. The Dnmt3a catalytic domain (Dnmt3a-CD) was used together with KroI and PcsI MD-REs. The assay consists in consecutive methylation and cleavage of fluorescently labeled DNA substrates, then the reaction products are visualized in polyacrylamide gel to determine the DNA methylation efficiency. Each MD-RE was tested with various substrates, including partly methylated ones. PcsI was identified as an optimal MDRE. PcsI recognizes two methylated CpG sites located 7 bp apart, the distance roughly corresponding to the distance between the active centers of the Dnmt3a-CD tetramer. An optimal substrate was designed to contain two methylated cytosine residues and two target cytosines in the orientation suitable for methylation by Dnmt3a-CD. The assay is reliable, simple, and inexpensive and, unlike conventional methods, does not require radioactive compounds. The assay may be used to assess the effectiveness of Dnmt3a inhibitors as potential therapeutic agents and to investigate the features of the Dnmt3a-CD function.     

Inhibition of murine DNA methyltransferase Dnmt3a by DNA duplexes containing pyrimidine-2(1<Emphasis Type="Italic">H</Emphasis>)-one

Here we studied the inhibition of the catalytic domain of Dnmt3a methyltransferase (Dnmt3a-CD) by DNA duplexes containing the mechanism-based inhibitor pyrimidine-2(1H)-one (P) instead of the target cytosine. It has been shown that conjugates of Dnmt3a-CD with P-DNA (DNA containing pyrimidine-2(1H)-one) are not stable to heating at 65°C in 0.1% SDS. The yield of covalent intermediate increases in the presence of the regulatory factor Dnmt3L. The importance of the DNA minor groove for covalent intermediate formation during the methylation reaction catalyzed by Dnmt3a-CD has been revealed. P-DNA was shown to inhibit Dnmt3a-CD; the IC₅₀ is 830 nM. The competitive mechanism of inhibition of Dnmt3a-CD by P-DNA has been elucidated. It is suggested that therapeutic effect of zebularine could be achieved by inhibition of not only Dnmt1 but also Dnmt3a.