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PubMed1998

DNA methylation by wheat cytosine DNA methyltransferase: modulation by protease inhibitor E-64.

Cytosine DNA methyltransferase isolated from wheat seedlings and purified in the presence of metalloprotease and serine protease inhibitors has molecular mass and specific activity equal to about 85 kDa and 250 units/mg protein, respectively. Apparent K(m) for AdoMet and [I]50 for AdoHcy values are about 6 microM and 12 microM, respectively. The enzyme is active in wide pH range (pH 5.5-8.5) and is inhibited by NaCl. The enzyme rapidly loses its methyltransferase activity in the absence of substrates. Using the cysteine protease inhibitor E-64 it has been shown that rapid enzyme inactivation is caused by disappearance of essential enzyme SH-groups but is not due to proteolytic enzyme cleavage.

Cysteine Proteinase Inhibitors↗
PubMed1991

Patterns of DNA methylation--evolutionary vestiges of foreign DNA inactivation as a host defense mechanism. A proposal.

The proposals in this review are based on experimental work on the integration of foreign DNA in mammalian cells, on the establishment of specific de novo patterns of DNA methylation, and on the inhibition of transcription by the sequence-specific methylation of promoter sequences. It is suggested that eukaryotic cells have developed several mechanisms of defense against the uptake, integration, and continued expression of foreign DNA. In the course of evolution and continuing at present, cells have been exposed to foreign DNA, entire genomes or fragments of them. A particularly problematic organ system in that respect must be digestive tract in higher organisms. The defense mechanisms are thought to be the following: (i) degradation and/or excretion of foreign DNA; (ii) excision and loss of previously integrated DNA from the host genome; (iii) targeted inactivation of foreign genes by sequence-specific methylation. Genes whose products could be advantageous to the transformed cells can somehow be selectively excluded from this silencing mechanism. In part, the specificity of de novo methylation must reside in the DNA methyltransferase systems of the host cell. However, nucleotide sequence, structure, and chromatin arrangement in the foreign DNA could also play an important role. Since defense processes must have been activated many times in evolution, patterns of DNA methylation as they can be observed today, may represent vestiges of evolution, i.e. the sum total of selective de novo methylations, possibly demethylations, and mutations.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2026

CpGene: a web application for epigenetic signature identification from DNA methylation arrays.

MOTIVATION: DNA methylation (DNAme) is the best studied epigenetic mechanism that plays pivotal role in tissue differentiation and epigenetic disruption has been correlated to diverse disease types (e.g. cancer, metabolic disorders). While various DNAme array platforms have been discovered, data analysis remains a challenging task which often requires in-depth bioinformatic expertise. Here, we developed a user-friendly web-based application for data analysis and visualization that accommodates users ranging from early-career basic/translational researchers to experienced bioinformaticians. RESULTS: CpGene is a web application for analyzing DNA methylation array data. It supports Illumina 450K, EPIC, and EPICv2 methylation array platforms and processes .idat files with integrated preprocessing, normalization, and quality control. Biomarker discovery is available through either classic differential methylation point analysis or machine learning-based feature selection as well as gene enrichment analysis. Results are summarized with clear visualizations, to aid interpretation. By combining these functions in a unified interface, CpGene streamlines methylation analysis and helps identify CpG sites and genes with biological and clinical relevance. AVAILABILITY AND IMPLEMENTATION: CpGene is openly accessible as a web service through http://cpgene.duckdns.org:8001/ and it's source code is available on https://github.com/kostaslazaros/cpgenene.

DNA Methylation↗
PubMed2006

Assessing the effects of high methionine intake on DNA methylation.

Methylation of DNA occurs at cytosines within CpG (cytosine-guanine) dinucleotides and is 1 of several epigenetic mechanisms that serve to establish and maintain tissue-specific patterns of gene expression. The methyl groups transferred in mammalian DNA methylation reactions are ultimately derived from methionine. High dietary methionine intake might therefore be expected to increase DNA methylation. Because of the circular nature of the methionine cycle, however, methionine excess may actually impair DNA methylation by inhibiting remethylation of homocysteine. Although little is known regarding the effect of dietary methionine supplementation on mammalian DNA methylation, the available data suggest that methionine supplementation can induce hypermethylation of DNA in specific genomic regions. Because locus-specific DNA hypomethylation is implicated in the etiology of various cancers and developmental syndromes, clinical trials of "promethylation" dietary supplements are already under way. However, aberrant hypermethylation of DNA could be deleterious. It is therefore important to determine whether dietary supplementation with methionine can effectively support therapeutic maintenance of DNA methylation without causing excessive and potentially adverse locus-specific hypermethylation. In the viable yellow agouti (Avy) mouse, maternal diet affects the coat color distribution of offspring by perturbing the establishment of methylation at the Avy metastable epiallele. Hence, the Avy mouse can be employed as a sensitive epigenetic biosensor to assess the effects of dietary methionine supplementation on locus-specific DNA methylation. Recent developments in epigenomic approaches that survey locus-specific DNA methylation on a genome-wide scale offer broader opportunities to assess the effects of high methionine intake on mammalian epigenomes.

Animals↗
PubMed1984

Reversal of DNA methylation with 5-azacytidine alters chromosome replication patterns in human lymphocyte and fibroblast cultures.

Prior studies demonstrated that developmental or induced methylation of DNA can inactivate associated gene loci. Such DNA methylation can be reversed and specific genes reactivated by treatment with 5-azacytidine (5- azaC ). The present cytogenetic studies using replication banding methods show that 5- azaC treatment also results in an increase or decrease in replication staining at one or more band locations in human lymphocyte and fibroblast chromosomes. New replication band locations are not formed. These changes in replication staining, which reflect changes in timing of replication, are different between these two tissues. However, in both tissues, the delayed onset of replication in the heterocyclic, inactive X is shortened by 5- azaC . A correlation is thus suggested between the induced temporal change to earlier DNA replication, and induced hypomethylation and gene activation. The temporal effect on chromosome replication in 5- azaC -treated cells depends on the portion of the S-period studied. Toward the beginning of S, early-replication patterns are increased in both lymphocytes and fibroblasts. Toward the end of S, late-replication patterns are increased only in lymphocytes, suggesting a differential effect of 5- azaC in: (1) early-vs. late-S, and (2) lymphocytes vs. fibroblasts. Generally, 5- azaC has its greatest effect on the inactive chromosome regions that are typically late-replicating prior to 5- azaC treatment. These observed changes in replication band staining suggest that DNA methylation may modify regional groups of genes in concert.

Azacitidine↗
PubMed1983

Effect of several inhibitors of enzymatic DNA methylation on the in vivo methylation of different classes of DNA sequences in a cultured human cell line.

We have previously demonstrated that, while most enzymatic formation of 5-methylcytosine in the DNA of mammalian cells occurs very shortly after strand synthesis, there is also a minor fraction of methylation which occurs in some DNA sequences up to at least several hours after strand synthesis. Using a human cell line, we have examined the effects on these two classes of enzymatic DNA methylation of several compounds which have been reported to be inhibitors of methylation reactions. We have found that cycloleucine, ethionine, and 5'-deoxy-5'-methylthioadenosine (MTA) are all effective as inhibitors of enzymatic DNA methylation, but that there is no differential effect between the delayed and non-delayed methylation reactions. Tubericidin (7-deaza-adenosine) plus homocysteine inhibited delayed DNA methylation much more than non-delayed methylation (by up to 4 times). By contrast, 5-azacytidine produced a higher level of inhibition of DNA methylation at sites in the DNA in which the methylation occurred very shortly after strand synthesis. Also 5-azacytidine was by far the most potent inhibitor of DNA methylation of the compounds tested. S-Adenosyl-homocysteine and caffeine were found to have no effect on DNA methylation. These results are discussed in relation to the number and specificity of DNA methylases in these cells and to the cellular functions of those DNA sequences in which methylation is delayed for some hours after strand synthesis.

5-Methylcytosine↗
PubMed2002

An overview of the analysis of DNA methylation in mammalian genomes.

DNA methylation at position C5 of the pyrimidine ring of cytosine in mammalian genomes has received a great deal of research interest due to its importance in many biological phenomena. It is associated with events such as epigenetic gene silencing and the maintenance of genome integrity. Aberrant DNA methylation, particularly that of chromosomal regions called CpG islands, is an important step in carcinogenesis. In order to elucidate methylation profiling of complex genomes, various methods have been developed. Many of these methods are based on the differential reactivity of cytosine and 5-methylcytosine to various chemicals. The combined use of these chemical reactions and other preexisting methods has enabled the discrimination of cytosine and 5-methylcytosine in complex genomes. The use of proteins that preferentially bind to methylated DNA has also successfully been used to discriminate between methylated and unmethylated sites. The chemical and structural dissection of the in vivo processes of enzymatic methylation and the binding of methyl-CpG binding proteins provides evidence for the complex mechanisms that nature has acquired. In this review we summarize the methods available for the discrimination between cytosine and 5-methylcytosine in complex genomes.

Animals↗
PubMed2005

Atypical RNA polymerase subunits required for RNA-directed DNA methylation.

RNA-directed DNA methylation, one of several RNA interference-mediated pathways in the nucleus, has been documented in plants and in human cells. Despite progress in identifying the DNA methyltransferases, histone-modifying enzymes and RNA interference proteins needed for RNA-directed DNA methylation, the mechanism remains incompletely understood. We screened for mutants defective in RNA-directed DNA methylation and silencing of a transgene promoter in Arabidopsis thaliana and identified three drd complementation groups. DRD1 is a SNF2-like protein required for RNA-directed de novo methylation. We report here that DRD2 and DRD3 correspond to the second-largest subunit and largest subunit, respectively, of a fourth class of DNA-dependent RNA polymerase (polymerase IV) that is unique to plants. DRD3 is a functionally diversified homolog of NRPD1a or SDE4, identified in a separate screen for mutants defective in post-transcriptional gene silencing. The identical DNA methylation patterns observed in all three drd mutants suggest that DRD proteins cooperate to create a substrate for RNA-directed de novo methylation.

Arabidopsis↗
PubMed2006

RNAi, DRD1, and histone methylation actively target developmentally important non-CG DNA methylation in arabidopsis.

Cytosine DNA methylation protects eukaryotic genomes by silencing transposons and harmful DNAs, but also regulates gene expression during normal development. Loss of CG methylation in the Arabidopsis thaliana met1 and ddm1 mutants causes varied and stochastic developmental defects that are often inherited independently of the original met1 or ddm1 mutation. Loss of non-CG methylation in plants with combined mutations in the DRM and CMT3 genes also causes a suite of developmental defects. We show here that the pleiotropic developmental defects of drm1 drm2 cmt3 triple mutant plants are fully recessive, and unlike phenotypes caused by met1 and ddm1, are not inherited independently of the drm and cmt3 mutations. Developmental phenotypes are also reversed when drm1 drm2 cmt3 plants are transformed with DRM2 or CMT3, implying that non-CG DNA methylation is efficiently re-established by sequence-specific signals. We provide evidence that these signals include RNA silencing though the 24-nucleotide short interfering RNA (siRNA) pathway as well as histone H3K9 methylation, both of which converge on the putative chromatin-remodeling protein DRD1. These signals act in at least three partially intersecting pathways that control the locus-specific patterning of non-CG methylation by the DRM2 and CMT3 methyltransferases. Our results suggest that non-CG DNA methylation that is inherited via a network of persistent targeting signals has been co-opted to regulate developmentally important genes.

Arabidopsis↗
PubMed1999

DNA methylation and chromatin modification.

DNA methylation and chromatin modification are two global mechanisms that regulate gene expression. Recent studies provide insight into the mechanism of transcriptional silencing by a methyl-CpG binding protein, MeCP2. MeCP2 is shown to interact with the Sin3/histone deacetylase co-repressor complex. Thus, this interaction can provide a mechanistic explanation for the long-known relationship between DNA methylation and chromatin structure. Moreover, several studies have shown that inhibition of histone deacetylases by specific inhibitors can reactivate endogenous genes or reporter constructs previously silenced by DNA methylation. Taken together, the data strongly suggest that DNA methylation can pattern chromatin modification.

Acetylation↗
PubMed2001

Programming the transcriptional state of replicating methylated dna.

CpG methylation is maintained in daughter chromatids by the action of DNA methyltransferase at the replication fork. An opportunity exists for transcription factors at replication forks to bind their cognate sequences and thereby prevent remethylation by DNA methyltransferase. To test this hypothesis, we injected a linearized, methylated, and partially single-stranded reporter plasmid into the nuclei of Xenopus oocytes and followed changes in the transcriptional activity after DNA replication. We find that dependent on Gal4-VP16, the action of DNA methyltransferase, and replication-coupled chromatin assembly DNA replication provides a window of time in which regulatory factors can activate or repress gene activity. Demethylation in the promoter region near the GAL4 binding sites of the newly synthesized DNA did not occur even though the Gal4 binding sites were occupied and transcription was activated. We conclude that "passive" demethylation at the replication fork is not simply dependent on the presence of DNA binding transcriptional activators.

Animals↗
PubMedDate not supplied

Comparative effects of L-methionine, S-adenosyl-L-methionine and 5'-methylthioadenosine on the growth of preneoplastic lesions and DNA methylation in rat liver during the early stages of hepatocarcinogenesis.

Male Wistar rats, initiated with diethylnitrosamine (DENA), were subjected to a selection treatment, according to the "resistant hepatocyte" model, followed or not followed by phenobarbital (PB). Rats received, for 3 weeks after selection, 4 i.m. doses (96 mmol/kg) of L-methionine, S-adenosyl-L-methionine (SAM), or 5'-methylthioadenosine (MTA), a SAM catabolite formed during polyamine synthesis or by spontaneous splitting of SAM at physiologic temperature and pH. They were then killed. In some rats, SAM and MTA treatments were started 20 weeks after initiation. The animals were killed 3 weeks later and persistent (neoplastic) nodules (PN) were collected. Some rat groups received 1/2 and 1/4 of the above SAM and MTA doses, or 1/8 of the above MTA dose. SAM and MTA, but not methionine, caused a dose-dependent decrease in number and surface area of gamma-glutamyltranspeptidase (GGT)-positive foci, and in labeling index (LI) of focal cells, coupled with remodeling. SAM and MTA liver contents, SAM/S-adenosylhomocysteine (SAH) ratio and overall methylation of liver DNA were low during the development of GGT-positive foci. SAM, but not methionine, caused a dose-dependent recovery of SAM content and DNA methylation, and a partial reconstitution of liver MTA pool. Exogenous MTA only induced the reconstitution of MTA pool, without affecting SAM level and DNA methylation. Recovery of SAM and MTA pool and DNA methylation was found in the rats subjected to SAM plus MTA, indicating the absence of inhibition of DNA methyltransferases in vivo by MTA. MTA also inhibited liver reparative growth in partially hepatectomized rats, without modifying SAM content and DNA methylation of regenerating liver (RL). A high activity of ornithine decarboxylase (ODC) was found in the liver, during the development of preneoplastic foci, and in PN. This activity was inhibited by SAM and MTA treatments. Although MTA was more effective than SAM, the decrease in ODC activity was coupled with a larger fall in DNA synthesis in SAM-treated than in MTA-treated rats. Thus the antipromotion effect of SAM could not merely depend on its (spontaneous) transformation into MTA. Although MTA production may play a role in the SAM antipromotion effect, other mechanisms could be involved. A role of DNA methylation in the inhibition of growth by SAM is suggested. MTA is a potential chemopreventive agent for liver carcinogenesis.

Adenosine↗
PubMed2005

Cell and stage of transformation-specific effects of folate deficiency on methionine cycle intermediates and DNA methylation in an in vitro model.

Folate is an essential co-factor in the remethylation of homocysteine to methionine, thereby ensuring the supply of S-adenosylmethionine, the methyl group donor for most biological methylations, including that of DNA. Aberrant patterns and dysregulation of DNA methylation are consistent events in carcinogenesis and hence, DNA methylation is considered to be mechanistically related to the development of cancer. Folate deficiency appears to increase the risk of several malignancies, and aberrant DNA methylation has been considered to be a leading mechanism by which folate deficiency enhances carcinogenesis. Although diets deficient in methyl group donors (choline, folate, methionine and vitamin B12) have been consistently observed to induce DNA hypomethylation, the effect of an isolated folate deficiency on DNA methylation remains highly controversial and unresolved. Whether or not isolated folate deficiency can modulate DNA methylation is an important issue because it would establish a mechanistic link between folate deficiency and cancer. We examined the effects of isolated folate deficiency on methionine cycle intermediates, genomic and site-specific DNA methylation and DNA methyltransferase in an in vitro model of folate deficiency, using untransformed NIH/3T3 and CHO-K1 cells, and human HCT116 and Caco-2 colon cancer cells. Our data demonstrate that the effect of folate deficiency on the methionine cycle pathway and DNA methylation in these cells is highly complex and appears to depend on the cell type and stage of transformation, and may be gene and site-specific. The direction of changes of methionine cycle intermediates in response to folate deficiency is not uniformly consistent with the known biochemical effect of folate on the methionine cycle pathway. Moreover, the effect of folate deficiency on DNA methylation appears to be mediated by both methionine cycle intermediate-dependent and independent pathways.

Animals↗
PubMed1994

The domains of a type I DNA methyltransferase. Interactions and role in recognition of DNA methylation.

The DNA methyltransferases of type I restriction-modification systems are trimeric enzymes composed of one DNA specificity (S) subunit and two modification (M) subunits. The S subunit contains two large regions, each of which recognizes one part of the split, asymmetrical DNA target sequence. Each M subunit contains an amino acid motif for binding the methyl group donor and cofactor, S-adenosyl methionine. The EcoKI methyltransferase has a strong preference for methylating a hemimethylated DNA target rather than an unmodified target. We have used partial proteolytic digestion of EcoKI methyltransferase to generate polypeptide domains that we have identified by amino acid sequencing. The S subunit was cut into two large, folded domains each containing one DNA binding region. Binding of DNA partially protected the S subunit from digestion. The M subunit was also cut into two large domains joined together by a short flexible loop, and a C-terminal tail region. The short loop contained part of the S-adenosyl methionine binding motif, and cofactor binding protected the loop and the two large domains from proteolysis. The C-terminal domain of M remained associated with the N-terminal domain of the S subunit even after the rest of the protein had been digested. The conformation of the tail region of the M subunit was sensitive to the methylation state of DNA in ternary complexes also containing S-adenosyl methionine, and could differentiate between unmethylated and hemimethylated DNA substrates.

Amino Acid Sequence↗
PubMed2004

DNA methylation and breast cancer.

DNA methylation and chromatin structure patterns are tightly linked components of the epigenome, which regulate gene expression programming. Two contradictory changes in DNA methylation patterns are observed in breast cancer; regional hypermethylation of specific genes and global hypomethylation. It is proposed here that independent mechanisms are responsible for these alterations in DNA methylation patterns and that these alterations deregulate two different processes in breast cancer. Regional hypermethylation is brought about by specific regional changes in chromatin structure, whereas global demethylation is caused by a general increase in demethylation activity. Hypermethylation silences growth regulatory genes resulting in uncontrolled growth whereas hypomethylation leads to activation of genes required for metastasis. DNA methylation inhibitors activate silenced tumor suppressor genes resulting in arrest of tumor growth and are now being tested as candidate anticancer drugs. Demethylation inhibitors are proposed here to be potential novel candidate antimetastatic agents, which would bring about methylation and silencing of metastatic genes. Future therapeutic application of either methylation or demethylation inhibitors in cancer therapy would require understanding of the relative role of these processes in the evolution of cancer.

Animals↗
PubMed2000

DNA methylation, nuclear structure, gene expression and cancer.

DNA methylation, chromatin structure, transcription, and cancer have traditionally been studied as separate phenomena. Recent data provide now direct physical and functional links between these processes revealing a complex network of interactions and mutual dependences. Methylated DNA is bound by methyl-CpG binding protein (MeCP) complexes that include histone deacetylases (HDACs). This recruitment of HDACs is suggested to promote local chromatin condensation and thereby repress gene expression. Most recently, also complexes of DNA methyltransferase (Dnmt1) with transcriptional repressors, DMAP1 and pRB, have been described providing a direct link to transcriptional regulation and tumor suppression. Inactivation of the DNA methyltransferase genes (Dnmt1, 3a, and 3b) was found to be lethal in mice and several human diseases (ICF and Rett syndrome) turned out to be linked to DNA methylation. In particular, global hypomethylation has been found in tumor samples together with cancer-type-specific, local hypermethylation. Taken together, these lines of evidence clearly underscore the central role of DNA methylation in the regulation of gene expression and chromatin structure during normal development and diseases like cancer. J. Cell. Biochem. Suppl. 35:78-83, 2000.

Animals↗
PubMed2005

The essential role of histone H3 Lys9 di-methylation and MeCP2 binding in MGMT silencing with poor DNA methylation of the promoter CpG island.

Silencing of the O (6)-methylguanine-DNA methyltransferase (MGMT) gene, a key to DNA repair, is involved in carcinogenesis. Recent studies have focused on DNA hypermethylation of the promoter CpG island. However, cases showing silencing with DNA hypomethylation certainly exist, and the mechanism involved is not elucidated. To clarify this mechanism, we examined the dynamics of DNA methylation, histone acetylation, histone methylation, and binding of methyl-CpG binding proteins at the MGMT promoter region using four MGMT negative cell lines with various extents of DNA methylation. Histone H3K9 di-methylation (H3me2K9), not tri-methylation, and MeCP2 binding were commonly seen in all MGMT negative cell lines regardless of DNA methylation status. 5Aza-dC, but not TSA, restored gene expression, accompanied by a decrease in H3me2K9 and MeCP2 binding. In SaOS2 cells with the most hypomethylated CpG island, 5Aza-dC decreased H3me2K9 and MeCP2 binding with no effect on DNA methylation or histone acetylation. H3me2K9 and DNA methylation were restricted to in and around the island, indicating that epigenetic modification at the promoter CpG island is critical. We conclude that H3me2K9 and MeCP2 binding are common and more essential for MGMT silencing than DNA hypermethylation or histone deacetylation. The epigenetic mechanism leading to silent heterochromatin at the promoter CpG island may be the same in different types of cancer irrespective of the extent of DNA methylation.

Azacitidine↗
PubMed2003

Progressive alterations in global and GC-rich DNA methylation during tumorigenesis.

DNA methylation plays a key role in the regulation of gene expression, and failure to maintain normal patterns of methylation often contributes to carcinogenesis. We have characterized progressive methylation changes during the promotion stage of carcinogenesis using a SENCAR mouse skin initiation/promotion tumorigenesis model. Mice were initiated with a dermal application of 75 microg dimethylbenz[a]anthracene (DMBA) and promoted with 9, 18, 27, and 36 mg cigarette smoke condensate (CSC) thrice weekly for time periods up to 29 weeks, when a large increase in tumor number was produced by the highest three doses. Global and GC-specific methylation were assessed using SssI methylase and arbitrarily primed PCR, respectively. Changes in GC-specific methylation were dose- and time-dependent. CSC doses required to detect these changes were 27 mg at 6 weeks and 18 mg at 9 weeks. This effect appears to be reversible; changes in GC-specific methylation were less marked after 9 weeks promotion with 27 mg CSC followed by 6 weeks of recovery in comparison to 9 and 15 weeks promotion with 27 mg CSC and no recovery period. Both tumor and non-tumor tissue promoted with 27 mg CSC for 29 weeks exhibited changes in GC-specific methylation that were more pronounced in tumors. Tumor tissue was globally hypomethylated, whereas non-tumor tissue did not exhibit changes in global methylation. In conclusion, as expected for a mechanism underlying tumor promotion, CSC alters methylation in a threshold-exhibiting, reversible, progressive fashion during promotion. Progressive alterations in global and GC-rich methylation appear to be mechanistically important during tumor promotion.

9,10-Dimethyl-1,2-benzanthracene↗