Cyanocobalamin prevents cardiomyopathy in type 1 diabetes by modulating oxidative stress and DNMT-SOCS1/3-IGF-1 signaling.

Patients with long-standing diabetes have a high risk for cardiac complications that is exacerbated by increased reactive oxygen species (ROS) production. We found that feeding cyanocobalamin (B12), a scavenger of superoxide, not only prevented but reversed signs of cardiomyopathy in type 1 diabetic Elmo1H/H Ins2Akita/+ mice. ROS reductions in plasma and hearts were comparable to those in mice treated with other antioxidants, N-acetyl-L-cysteine or tempol, but B12 produced better cardioprotective effects. Diabetes markedly decreased plasma insulin-like growth factor (IGF)-1 levels, while B12, but not N-acetyl-L-cysteine nor tempol, restored them. B12 activated hepatic IGF-1 production via normalization of S-adenosylmethionine levels, DNA methyltransferase (DNMT)-1/3a/3b mRNA, and DNA methylation of promoters for suppressor of cytokine signaling (SOCS)-1/3. Reductions of cardiac IGF-1 mRNA and phosphorylated IGF-1 receptors were also restored. Thus, B12 is a promising option for preventing diabetic cardiomyopathy via ROS reduction and IGF-1 retrieval through DNMT-SOCS1/3 signaling.

In Arabidopsis thaliana Cd differentially impacts on hormone genetic pathways in the methylation defective ddc mutant compared to wild type.

DNA methylation plays an important role in modulating plant growth plasticity in response to stress, but mechanisms involved in such control need further investigation. We used drm1 drm2 cmt3 mutant of Arabidopsis thaliana, defective in DNA methylation, to explore metabolic pathways downstream epigenetic modulation under cadmium (Cd) stress. To this aim, a transcriptomic analysis was performed on ddc and WT plants exposed to a long-lasting (21 d) Cd treatment (25/50 µM), focusing on hormone genetic pathways. Growth parameters and hormones amount were also estimated. Transcriptomic data and hormone quantification showed that, under prolonged Cd treatment, level and signalling of growth-sustaining hormones (auxins, CKs, GAs) were enhanced and/or maintained, while a decrease was detected for stress-related hormones (JA, ABA, SA), likely as a strategy to avoid the side effects of their long-lasting activation. Such picture was more effective in ddc than WT, already at 25 µM Cd, in line with its better growth performance. A tight relationship between methylation status and the modulation of hormone genetic pathways under Cd stress was assessed. We propose that the higher genome plasticity conferred to ddc by DNA hypomethylated status underlies its prompt response to modulate hormones genetic pathways and activity and assure a flexible growth.

Cytosine-5 RNA methylation links protein synthesis to cell metabolism.

Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.

DNA Modifications and Alzheimer's Disease.

Alzheimer's disease (AD) is a complex neurodegenerative disease, affecting millions of people worldwide. While a number of studies have focused on identifying genetic variants that contribute to the development and progression of late-onset AD, the majority of these only have a relatively small effect size. There are also a number of other risk factors, for example, age, gender, and other comorbidities; however, how these influence disease risk is not known. Therefore, in recent years, research has begun to investigate epigenetic mechanisms for a potential role in disease etiology. In this chapter, we discuss the current state of play for research into DNA modifications in AD, the most well studied being 5-methylcytosine (5-mC). We describe the earlier studies of candidate genes and global measures of DNA modifications in human AD samples, in addition to studies in mouse models of AD. We focus on recent epigenome-wide association studies (EWAS) in human AD, using microarray technology, examining a number of key study design issues pertinent to such studies. Finally, we discuss how new technological advances could further progress the research field.

Exercise and inflammation-related epigenetic modifications: focus on DNA methylation.

Epigenetics is the study of mitotically or meiotically heritable phenotypes that occur as a result of modifications to DNA, thereby regulating gene expression independently of changes in base sequence due to manipulation of the chromatin structure. These modifications occur through a variety of mechanisms, such as DNA methylation, post-translational histone modifications, and non-coding RNAs, and can cause transcriptional suppression or activation depending on the location within the gene. Environmental stimuli, such as diet and exercise, are thought to be able to regulate these mechanisms, with inflammation as a probable contributory factor. Research into these areas is still in its infancy however. This review will focus on DNA methylation in the context of inflammation (both pro- and anti-inflammatory processes) and exercise. The complexity and relative shortcomings of some existing techniques for studying epigenetics will be highlighted, and recommendations for future study approaches made.

Cytosine-to-uracil deamination by SssI DNA methyltransferase.

The prokaryotic DNA(cytosine-5)methyltransferase M.SssI shares the specificity of eukaryotic DNA methyltransferases (CG) and is an important model and experimental tool in the study of eukaryotic DNA methylation. Previously, M.SssI was shown to be able to catalyze deamination of the target cytosine to uracil if the methyl donor S-adenosyl-methionine (SAM) was missing from the reaction. To test whether this side-activity of the enzyme can be used to distinguish between unmethylated and C5-methylated cytosines in CG dinucleotides, we re-investigated, using a sensitive genetic reversion assay, the cytosine deaminase activity of M.SssI. Confirming previous results we showed that M.SssI can deaminate cytosine to uracil in a slow reaction in the absence of SAM and that the rate of this reaction can be increased by the SAM analogue 5'-amino-5'-deoxyadenosine. We could not detect M.SssI-catalyzed deamination of C5-methylcytosine ((m5)C). We found conditions where the rate of M.SssI mediated C-to-U deamination was at least 100-fold higher than the rate of (m5)C-to-T conversion. Although this difference in reactivities suggests that the enzyme could be used to identify C5-methylated cytosines in the epigenetically important CG dinucleotides, the rate of M.SssI mediated cytosine deamination is too low to become an enzymatic alternative to the bisulfite reaction. Amino acid replacements in the presumed SAM binding pocket of M.SssI (F17S and G19D) resulted in greatly reduced methyltransferase activity. The G19D variant showed cytosine deaminase activity in E. coli, at physiological SAM concentrations. Interestingly, the C-to-U deaminase activity was also detectable in an E. coli ung (+) host proficient in uracil excision repair.

Fidelity index determination of DNA methyltransferases.

DNA methylation is the most frequent form of epigenetic modification in the cell, which involves gene regulation in eukaryotes and protection against restriction enzymes in prokaryotes. Even though many methyltransferases exclusively modify their cognate sites, there have been reports of those that exhibit promiscuity. Previous experimental approaches used to characterize these methyltransferases do not provide the exact concentration at which off-target methylation occurs. Here, we present the first reported fidelity index (FI) for a number of DNA methyltransferases. We define the FI as the ratio of the highest amount of methyltransferase that exhibits no star activity (off-target effects) to the lowest amount that exhibits complete modification of the cognate site. Of the methyltransferases assayed, M.MspI and M.AluI exhibited the highest fidelity of ≥250 and ≥500, respectively, and do not show star activity even at very high concentrations. In contrast, M.HaeIII, M.EcoKDam and M.BamHI have the lowest fidelity of 4, 4 and 2, respectively, and exhibit star activity at concentrations close to complete methylation of the cognate site. The fidelity indexes provide vital information on the usage of methyltransferases and are especially important in applications where site specific methylation is required.

Epigenetics and the adaptive immune response.

Cells of the adaptive immune response undergo dynamic epigenetic changes as they develop and respond to immune challenge. Plasticity is a necessary prerequisite for the chromosomal dynamics of lineage specification, development, and the immune effector function of the mature cell types. The alterations in DNA methylation and histone modification that characterize activation may be integral to the generation of immunologic memory, thereby providing an advantage on secondary exposure to pathogens. While the immune system benefits from the dynamic nature of the epigenome, such benefit comes at a cost - increased likelihood of disease-causing mutation.

DNA methylation and methyl-CpG binding proteins: developmental requirements and function.

DNA methylation is a major epigenetic modification in the genomes of higher eukaryotes. In vertebrates, DNA methylation occurs predominantly on the CpG dinucleotide, and approximately 60% to 90% of these dinucleotides are modified. Distinct DNA methylation patterns, which can vary between different tissues and developmental stages, exist on specific loci. Sites of DNA methylation are occupied by various proteins, including methyl-CpG binding domain (MBD) proteins which recruit the enzymatic machinery to establish silent chromatin. Mutations in the MBD family member MeCP2 are the cause of Rett syndrome, a severe neurodevelopmental disorder, whereas other MBDs are known to bind sites of hypermethylation in human cancer cell lines. Here, we review the advances in our understanding of the function of DNA methylation, DNA methyltransferases, and methyl-CpG binding proteins in vertebrate embryonic development. MBDs function in transcriptional repression and long-range interactions in chromatin and also appear to play a role in genomic stability, neural signaling, and transcriptional activation. DNA methylation makes an essential and versatile epigenetic contribution to genome integrity and function.

DNA methylation in plants.

DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of siRNA-DNA sequence identity; therefore, it is mainly associated with CNG and non-symmetrical methylations (rare in animals) in coding and promoter regions of silenced genes. Cytoplasmic viral RNA can affect methylation of homologous nuclear sequences and it maybe one of the feedback mechanisms between the cytoplasm and the nucleus to control gene expression.

Chromatin and siRNA pathways cooperate to maintain DNA methylation of small transposable elements in Arabidopsis.

BACKGROUND: DNA methylation occurs at preferred sites in eukaryotes. In Arabidopsis, DNA cytosine methylation is maintained by three subfamilies of methyltransferases with distinct substrate specificities and different modes of action. Targeting of cytosine methylation at selected loci has been found to sometimes involve histone H3 methylation and small interfering (si)RNAs. However, the relationship between different cytosine methylation pathways and their preferred targets is not known. RESULTS: We used a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related siRNA silencing component (AGO4) in methylating target loci. We found that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that correspond to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants. CONCLUSION: We conclude that the targets of both DNA methylation and histone H3K9 methylation pathways are transposable elements genome-wide, irrespective of element type and position. Our findings also suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism alone. Thus, parallel pathways would be needed to maintain silencing of transposable elements.

Low-frequency normal mode in DNA HhaI methyltransferase and motions of residues involved in the base flipping.

The results of normal-mode analyses are in accord with the proposal that a low-frequency motion of the HhaI methyltransferase enzyme is responsible for base flipping in bound DNA. The vectors of the low-frequency normal mode of residues Ser-85 and Ile-86 point directly to the phosphate and ribose moieties of the DNA backbone near the target base in position to rotate the dihedral angles and flip the base out of the DNA duplex. The vector of residue Gln-237 on the major groove is in the proper orientation to assist base separation. Our results favor the major groove pathway and the protein active process in base flipping.

Role of the arabidopsis DRM methyltransferases in de novo DNA methylation and gene silencing.

Proper DNA methylation patterning requires the complementary processes of de novo methylation (the initial methylation of unmethylated DNA sequences) and maintenance methylation (the faithful replication of preexisting methylation). Arabidopsis has two types of methyltransferases with demonstrated maintenance activity: MET1, which maintains CpG methylation and is homologous to mammalian DNMT1, and CHROMOMETHYLASE 3 (CMT3), which maintains CpNpG (N = A, T, C, or G) methylation and is unique to the plant kingdom. Here we describe loss-of-function mutations in the Arabidopsis DOMAINS REARRANGED METHYLASE (DRM) genes and provide evidence that they encode de novo methyltransferases. drm1 drm2 double mutants retained preexisting CpG methylation at the endogenous FWA locus but blocked de novo CpG methylation that is normally associated with FWA transgene silencing. Furthermore, drm1 drm2 double mutants blocked de novo CpNpG and asymmetric methylation and gene silencing of the endogenous SUPERMAN (SUP) gene, which is normally triggered by an inverted SUP repeat. However, drm1 drm2 double mutants did not show reactivation of previously established SUPERMAN epigenetic silenced alleles. Thus, drm mutants prevent the establishment but not the maintenance of gene silencing at FWA and SUP, suggesting that the DRMs encode the major de novo methylation enzymes affecting these genes.

A cytosine methyltransferase homologue is essential for repeat-induced point mutation in Neurospora crassa.

During sexual development, Neurospora crassa inactivates genes in duplicated DNA segments by a hypermutation process, repeat-induced point mutation (RIP). RIP introduces C:G to T:A transition mutations and creates targets for subsequent DNA methylation in vegetative tissue. The mechanism of RIP and its relationship to DNA methylation are not fully understood. Mutations in DIM-2, a DNA methyltransferase (DMT) responsible for all known cytosine methylation in Neurospora, does not prevent RIP. We used RIP to disrupt a second putative DMT gene in the Neurospora genome and tested mutants for defects in DNA methylation and RIP. No effect on DNA methylation was detected in the tissues that could be assayed, but the mutants showed recessive defects in RIP. Duplications of the am and mtr genes were completely stable in crosses homozygous for the mutated potential DMT gene, which we call rid (RIP defective). The same duplications were inactivated normally in heterozygous crosses. Disruption of the rid gene did not noticeably affect fertility, growth, or development. In contrast, crosses homozygous for a mutation in a related gene in Ascobolus immersus, masc1, reportedly fail to develop and heterozygous crosses reduce methylation induced premeiotically [Malagnac, F., Wendel, B., Goyon, C., Faugeron, G., Zickler, D., et al. (1997) Cell 91, 281-290]. We isolated homologues of rid from Neurospora tetrasperma and Neurospora intermedia to identify conserved regions. Homologues possess all motifs characteristic of eukaryotic DMTs and have large distinctive C- and N-terminal domains.

Role and mechanism of action of C. PvuII, a regulatory protein conserved among restriction-modification systems.

The PvuII restriction-modification system is a type II system, which means that its restriction endonuclease and modification methyltransferase are independently active proteins. The PvuII system is carried on a plasmid, and its movement into a new host cell is expected to be followed initially by expression of the methyltransferase gene alone so that the new host's DNA is protected before endonuclease activity appears. Previous studies have identified a regulatory gene (pvuIIC) between the divergently oriented genes for the restriction endonuclease (pvuIIR) and modification methyltransferase (pvuIIM), with pvuIIC in the same orientation as and partially overlapping pvuIIR. The product of pvuIIC, C. PvuII, was found to act in trans and to be required for expression of pvuIIR. In this study we demonstrate that premature expression of pvuIIC prevents establishment of the PvuII genes, consistent with the model that requiring C. PvuII for pvuIIR expression provides a timing delay essential for protection of the new host's DNA. We find that the opposing pvuIIC and pvuIIM transcripts overlap by over 60 nucleotides at their 5' ends, raising the possibility that their hybridization might play a regulatory role. We furthermore characterize the action of C. PvuII, demonstrating that it is a sequence-specific DNA-binding protein that binds to the pvuIIC promoter and stimulates transcription of both pvuIIC and pvuIIR into a polycistronic mRNA. The apparent location of C. PvuII binding, overlapping the -10 promoter hexamer and the pvuIICR transcriptional starting points, is highly unusual for transcriptional activators.

Expression of prokaryotic HhaI DNA methyltransferase is transforming and lethal to NIH 3T3 cells.

In neoplastic cells, levels of DNA methyltransferase activity are often increased, and evidence is accruing to suggest an important role for this event in tumorigenesis. To evaluate this possibility further, and to investigate the contribution of increasing de novo, as opposed to maintenance, DNA methylation in mammalian cells, we expressed the bacterial HhaI methyltransferase in cultured murine fibroblasts. This enzyme is a pure de novo DNA methyltransferase that methylates the internal C in the sequence GCGC. We find that both constitutive and induced expression of the wild-type HhaI results, primarily, in lethality to the cells. However, surviving cell clones that express low levels of M. HhaI demonstrate increased tumorigenicity as assessed by soft agar cloning efficiency (8.6% for sense HhaI-transduced PA 317 cells versus 0.4% for antisense controls; 1.7% for sense HhaI-transfected NIH 3T3 cells versus 0% for a mutant HhaI control) and tumorigenicity in nude mouse heterotransplants (75% for sense HhaI-transduced PA 317 cells versus 18.5% for antisense controls). DNA isolated from the clonogenic sense HhaI clones, versus clones expressing the mutant HhaI gene, has no increase in overall CpG methylation but an average of 27% (range, 16.7-38.9) increase in methylcytosine content at GCGC sites. These findings suggest that eukaryotic cells tolerate a narrow window of increase de novo DNA methylating capacity, above which cell death occurs and within cell transformation results. Our results further emphasize the potential role of increased DNA methyltransferase activity in the evolution of cancer.

Ethanol consumption inhibits fetal DNA methylation in mice: implications for the fetal alcohol syndrome.

Acute ethanol administration (3 g/kg twice a day) to pregnant mice, from the 9th thru the 11th day of gestation, resulted in hypomethylation of fetal deoxyribonucleic acid (DNA). Nuclei isolated from the fetuses of the ethanol-treated mice had lower levels of methylase activity relative to controls even in the presence of excess S-adenosylmethionine, which serves as the methyl donor for the enzyme DNA methyltransferase. Acetaldehyde, at concentrations as low as 3 to 10 microM, inhibited DNA methyltransferase activity in vitro. Since DNA methylation is thought to play an important role in the regulation of gene expression during embryogenesis, ethanol-associated alterations in fetal DNA methylation may contribute to the developmental abnormalities seen in the fetal alcohol syndrome.