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PubMed1992

Purification, sequence, and cellular localization of a novel chromosomal protein that binds to methylated DNA.

Methylation of mammalian DNA can lead to repression of transcription and alteration of chromatin structure. Recent evidence suggests that both effects are the result of an interaction between the methylated sites and methyl-CpG-binding proteins (MeCPs). MeCP1 has previously been detected in crude nuclear extracts. Here we report the identification, purification, and cDNA cloning of a novel MeCP called MeCP2. Unlike MeCP1, the new protein is able to bind to DNA that contains a single methyl-CpG pair. By staining with an antibody, we show that the distribution of MeCP2 along the chromosomes parallels that of methyl-CpG. In mouse, for example, MeCP2 is concentrated in pericentromeric heterochromatin, which contains a large fraction (about 40%) of all genomic 5-methylcytosine.

Amino Acid Sequence↗

PubMed1981

[Limited accessibility of DNA methylation sites for bacterial methylases M. Eco RII and M.Eco dam in chromatin at different levels of organization].

The bacterial methylases M. Eco RII and M. Eco dam can methylate DNA in rat liver chromatin to form the 5-methylcytosine (m5C) and N6-methyladenine (m6A) residues, respectively. The CH3-accepting capacity of DNA in chromatin (mono- and dinucleosomes, mono- and dinucleomers) is 15 - 30 times less than that of free total DNA in rat liver. Such a low level of DNA methylation in chromatin in vitro suggests that the accessibility and recognition of methylation sites by DNA-methylases are decreased in comparison with free DNA both in the core-particle DNA and in the internucleosomal DNA. The degree of DNA methylation in chromatin particles depends on the ionic strength and Mg2+; when the former is decreased from 0.515 down to 0.176, the DNA methylation by both enzymes is increased 2-fold. An addition of Mg2+ (1 - 2 mM) decreases the CH3-accepting capacity of nucleomeric DNA, that of nucleosomal DNA remains unchanged. Thus, the accessibility of DNA for methylases is variable depending on the conformational changes of chromatin. The values of the m6A to m5C ratio for free and nucleosomal DNAs formed by methylation with a methylation of nucleomeric DNA, i. e. 1.01, 0.92 and 0.51, respectively. As Mg/4 concentration rises, the m6A/m5C ratio for nucleosomal and nucleomeric DNA is increased. It seems therefore that at different levels of organization and upon certain conformation changes the number and, probably, the nature of exposed DNA methylation sites in chromatin are different. Bacterial DNA-methylases can be used as an effective probe for a fine analysis of chromatin ultrastructure, in particular at its different functional states.

Animals↗

PubMed2006

A histone methylation-dependent DNA methylation pathway is uniquely impaired by deficiency in Arabidopsis S-adenosylhomocysteine hydrolase.

S-adenosylhomocysteine hydrolase (SAH) is a key enzyme in the maintenance of methylation homeostasis in eukaryotes because it is needed to metabolize the by-product of transmethylation reactions, S-adenosylhomocysteine (AdoHcy), which causes by-product inhibition of methyltransferases (MTase's). Complete loss of SAH function is lethal. Partial loss of SAH function causes pleiotropic effects including developmental abnormalities and reduced cytosine methylation. Here we describe a novel partial-function missense allele of the Arabidopsis SAH1 gene that causes loss of cytosine methylation specifically in non-CG contexts controlled by the CMT3 DNA MTase and transcriptional reactivation of a silenced reporter gene, without conferring developmental abnormalities. The CMT3 pathway depends on histone H3 lysine 9 methylation (H3 mK9) to guide DNA methylation. Our results suggest that this pathway is uniquely sensitive to SAH impairment because of its requirement for two transmethylation reactions that can both be inhibited by AdoHcy. Our results further suggest that gene silencing pathways involving an interplay between histone and DNA methylation in other eukaryotes can be selectively impaired by controlled SAH downregulation.

Adenosylhomocysteinase↗

PubMed1988

RglB facilitated cloning of highly methylated eukaryotic DNA: the human L1 transposon, plant DNA, and DNA methylated in vitro with human DNA methyltransferase.

In vitro methylation of Bluescribe plasmid DNA (pBS) with human placental DNA methyltransferase to 6% 5-methylcytosine (mC) reduced transformation efficiencies in rglB+ host strains C600 and DS410 by almost 2 orders of magnitude. By contrast, the rglB- derivative of DS410 showed no reduction in transformation efficiency with methylation while the rglB- derivative of C600 was partially tolerant to methylation. Further, we show that the 1.8 kilobase (kb) and 1.2 kb KpnI fragments derived from the human L1 repeat have respectively 18.3% and 2.3% mC in vivo. Using these hyper- and hypo-methylated genomic segments ligated into the pBS plasmid, transformants with the highly methylated 1.8 kb L1 insert were recovered at 17 to 40 fold higher frequency with the rglB- host strains than with the rglB+ hosts. In addition, recombinant phage (lambda 2001) containing inserts of plant genomic DNA with 26.7% mC (from Petunia hybrida) when plated on rglB- hosts gave titres up to 222 times higher than on the rglB+ strains.

Cell Line↗

PubMed1977

Inhibition of DNA methylation by S-adenosylethionine with the production of methyl-deficient DNA in regenerating rat liver.

Ethionine, a liver carcinogen, was administered p.o. (300 mg/kg) to rats 17 hr after partial hepatectomy. At 6 hr after administration of the ethionine, hepatic S-adenosylethionine levels were 30- to 40-fold greater than the hepatic level of S-adenosylmethionine. A 10-fold ratio of S-adenosylethionine to S-adenosylmethionine still persited at 24 hr after ethionine administration. When given at 17 hr after partial hepatectomy, ethionine produced a 30% inhibition of DNA synthesis, measured by the incorporation of [methyl-3H]thymidine at 23 to 24 hr after partial hepatectomy (6 to 7 hr after ethionine administration). DNA synthesized during this interval was methyl deficient as judged by the reduced incorporation of radioactivity from L-[methyl-3H]methionine into 5-methylcytosine residues of DNA. In an assay for DNA methylation in vitro using whole nuclei, the methyl-deficient DNA was methylated by S-adenosylmethionine 8 times more than was control DNA; the DNA methylation was competitively inhibited by S-adenosylethionine. These data suggest that S-adenosylethionine, formed in vivo from ethionine, competitively inhibits the methylation of DNA in vivo by S-adenosylmethionine, resulting in the production of methyl-deficient DNA.

Animals↗

PubMed1999

Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation.

ATP-dependent nucleosome remodeling and core histone acetylation and deacetylation represent mechanisms to alter nucleosome structure. NuRD is a multisubunit complex containing nucleosome remodeling and histone deacetylase activities. The histone deacetylases HDAC1 and HDAC2 and the histone binding proteins RbAp48 and RbAp46 form a core complex shared between NuRD and Sin3-histone deacetylase complexes. The histone deacetylase activity of the core complex is severely compromised. A novel polypeptide highly related to the metastasis-associated protein 1, MTA2, and the methyl-CpG-binding domain-containing protein, MBD3, were found to be subunits of the NuRD complex. MTA2 modulates the enzymatic activity of the histone deacetylase core complex. MBD3 mediates the association of MTA2 with the core histone deacetylase complex. MBD3 does not directly bind methylated DNA but is highly related to MBD2, a polypeptide that binds to methylated DNA and has been reported to possess demethylase activity. MBD2 interacts with the NuRD complex and directs the complex to methylated DNA. NuRD may provide a means of gene silencing by DNA methylation.

Adenosine Triphosphatases↗

PubMed2003

Methylated DNA as a possible screening marker for neoplastic disease in several body fluids.

Early detection appears to be one of the most important approaches to reducing mortality caused by neoplasia. Changes in DNA methylation have been recognized as one of the most common molecular alterations in human tumors. Due to the ubiquity of DNA methylation changes and the possibility to detect methylated DNA in several body fluids, this specifically altered DNA may serve as a potential new screening marker for neoplastic disease.

Biomarkers, Tumor↗

PubMed1986

Effect of site-specific DNA methylation and mutagenesis on recognition by methylated DNA-binding protein from human placenta.

Methylated DNA-binding protein (MDBP) from human placenta is the first protein shown to bind specifically to certain DNA sequences only when they are methylated at cytosine residues. Among the sites recognized by MDBP is pB site 1, a pBR322-derived sequence which has a high affinity for MDBP when methylated at all CpG positions. We have substituted pB site 1 with 5-methyl-cytosine (m5C) residues at one to three of its CpG dinucleotides on one strand by the use of m5C-containing oligonucleotides. MDBP binds best when all three CpG dinucleotides in the region 5'-ATCGTCACGGCGAT-3' are methylated. Even more binding is obtained when both strands are methylated. Alteration of various residues in this binding site by oligonucleotide-directed mutagenesis decreased the binding. However, two mutations which increased the dyad symmetry of part of the binding site yielded ligands with a higher affinity for MDBP.

Base Sequence↗

PubMed2000

MBD2-MBD3 complex binds to hemi-methylated DNA and forms a complex containing DNMT1 at the replication foci in late S phase.

BACKGROUND: In vertebrates and plants, DNA methylation is one of the major mechanisms regulating gene expression. Recently, a family of methyl-CpG-binding proteins has been identified, and some members, such as MeCP2 and MBD2, were shown to mediate gene repression by recruiting histone deacetylase complexes to methylated genes. However, the function of another member of this family, MBD3, remained elusive. RESULTS: It was shown that MBD2 and MBD3 form homo- and hetero-dimers (or multimers) in vitro and in vivo. Significantly, the MBD2-MBD3 complex showed an affinity to hemi-methylated DNAs, a property that has never been reported with any member of the family proteins. MBD2 and MBD3 were co-localized with DNMT1 at replication foci in 293 cell nuclei at late S phase. Moreover, by a co-immunoprecipitation experiment, DNMT1 was shown to form a complex with MBD2 and MBD3. Finally, the abundance of MBD3 was highest in the late S phase when the DNMT1 is also most abundant, whereas the MBD2 level was largely constant throughout the cell cycle. CONCLUSIONS: The results suggest that MBD3 may play an important role in the S phase. We hypothesize that the MBD2-MBD3 complex recognizes hemi-methylated DNA concurrent with DNA replication and recruits histone deacetylase complexes, as well as DNMT1, to establish and/or maintain the transcriptionally repressed chromatin.

Chromatin↗

PubMed1999

Evaluation of methylated DNA binding protein-1 in mouse liver.

DNA methylation (DNA-5-methylcytosine [MeC]) plays a key role in regulation of gene expression. Highly methylated genes tend to be silenced whereas hypomethylated genes have an increased potential for expression. One way in which methylation may lead to inhibition of transcription is by permitting the binding of methylated DNA-binding proteins, which then block access of transcription factors to DNA. A particular methylated DNA-binding protein, MDBP-1, has been identified in nuclear extracts prepared from various human and rat tissues, and binds in a sequence-specific manner to DNA containing methylated cytosines (P. C. Supakar et al., 1988, Nucleic Acids Res. 16, 8029-8044). In the current study, an electrophoretic mobility shift assay (EMSA) was employed to assess the presence of MDBP-1 in protein isolated from mouse liver and to examine characteristics of its binding to DNA. The ligand used in our EMSA was a 32P-end-labeled, double-stranded, symmetrically methylated oligonucleotide containing the protein's binding site, 5'-CTAGATMGT-CAMGGMGAT-3' (M denotes 5-methyl-cytosine). Utilizing protein extracted from B6C3F1 mouse liver, we observed a complex that is competed by non-labeled, double-stranded, fully methylated ligand but not by an excess of a 26-mer, double-stranded oligonucleotide containing the binding site for AP-1. No complex was formed when a nonmethylated, double-stranded ligand, or our single-stranded ligands (methylated or unmethylated) were used as EMSA ligands. Complex formation was observed utilizing double-stranded, hemimethylated ligands; however, the affinity of MDBP-1 for these was one-tenth the affinity of MDBP-1 for fully methylated, double-stranded ligand. Additionally, protein isolated from C57BL/6 and C3H/He mouse liver was found to bind specifically to fully methylated ligand and hemimethylated ligands for MDBP-1, with similar characteristics as the binding of B6C3F1 mouse liver protein. These results indicate that MDBP-1 is present in mouse liver, and that the degree of methylation determines the strength of binding of MDBP-1 to DNA.

Animals↗

PubMed1993

Patterns of frog virus 3 DNA methylation and DNA methyltransferase activity in nuclei of infected cells.

The iridovirus frog virus 3 (FV3) can replicate in culture in fat head minnow (FHM) fish cells or in BHK-21 hamster cells. Viral DNA replication commences about 3 h after infection of FHM cells with FV3. Between 3 and 6 h postinfection (p.i.), a portion of the intranuclear FV3 DNA is partly unmethylated. At later times, p.i., all of the viral DNA in the nuclear and cytoplasmic compartments is methylated at the 5'-CCGG-3' sequences. Cytoplasmic FV3 DNA has not been found unmethylated. We have cloned viral DNA fragments from methylated virion DNA. By using the genomic sequencing technique, it has been demonstrated for segments of the FV3 DNA replicated both in FHM fish and BHK21 hamster cells that in a stretch encompassing a total of 350 bp, all of the analyzed 5'-CG-3' dinucleotides are methylated. The modified nucleotide 5-methyldeoxycytidine is present exclusively in the 5'-CG-3' dinucleotide combination. In the cloned FV3 DNA fragment p21A, an open reading frame has been located. The 5' region of this presumptive viral gene is also methylated in all 5'-CG-3' positions. DNA methyltransferase activity has been detected in the nuclei of FV3-infected FHM cells at 4, 11, and 20 h p.i. In the cytoplasmic fraction, comparable activity has not been observed. These data are consistent with the interpretation that FV3 DNA is newly synthesized and de novo methylated in the nuclei of infected FHM cells and subsequently exported into the cytoplasm for viral assembly.

Amino Acid Sequence↗

PubMed1996

The design of agents to control DNA methylation adducts. Enhanced major groove methylation of DNA by an N-methyl-N-nitrosourea functionalized phenyl neutral red intercalator.

An N-methyl-N-nitrosourea (MNU) moiety [CH3N(N=O)C(=O)NH-] linked to the C4'-position of the 5-substituted phenyl ring of phenyl neutral red (PNR), 2-methyl-3-amino-5-[p-[[2-[(N-nitroso-N-methylcarbamoyl)amino]ethy l] carbamoyl]phenyl]-7-(dimethylamino)phenazenium chloride (MNU-PNR), has been synthesized as an approach to design a molecule that will deliver alkylating agents with some preference to guanine (Gua) in the major groove of DNA. The PNR nucleus was chosen because previous studies suggested the following: (1) PNR binds with a slight preference for G/C rich sequences; and (2) PNR intercalates into DNA from the major groove with the 5-phenyl ring pointing out into the major groove (Müller, W., Bünemann, H., and Dattagupta, N. (1975) Eur. J. Biochem. 54, 279-291). It is demonstrated that MNU-PNR yields 2.6 and 6.0 times more N7-methylguanine (7-MeGua) than MNU at low salt (10 mM Tris buffer) and high salt (10 mM Tris buffer + 200 mM NaCl), respectively. It is also shown that the ratio of 7-MeGua (a major groove adduct) to N3-methyladenine (a minor groove adduct) is approximately 5 times higher for MNU-PNR than for MNU. The yield of the 7-MeGua adduct is decreased by the coaddition of a nonmethylating analogue of MNU-PNR or NaCl, but increased in the presence of the minor groove intercalator, ethidium bromide. Using a 32P-end-labeled restriction fragment, the enhanced methylation by MNU-PNR at 7-Gua is confirmed, and it is demonstrated that the sequence-dependent formation of 7-MeGua from MNU-PNR is the same as that seen with MNU. UV, circular dichrosism, and viscosity studies are consistent with MNU-PNR binding to DNA via an intercalation-based process.

Adenine↗

PubMed2001

Targeting silence: the use of site-specific recombination to introduce in vitro methylated DNA into the genome.

DNA methylation has emerged as an important component of transcriptional regulation. However, our understanding of how DNA methylation influences transcription, chromatin structure, replication timing, and imprinting has been limited by the lack of experimental systems that permit control of the methylation state of genes in a chromosomal context. Here, we describe a novel technique that allows for efficient introduction of methylated and unmethylated DNA into defined sites in the mammalian genome. This protocol utilizes bacterial CpG methyltransferases to methylate the DNA of interest in vitro, followed by site-specific targeting using Cre recombinase. Long-term maintenance of the methylation state in vivo allows analysis of the biological consequences of methylation by direct comparison of the methylated and unmethylated state in the same genomic position.

Animals↗

PubMed2001

Solution structure of the methyl-CpG binding domain of human MBD1 in complex with methylated DNA.

In vertebrates, the biological consequences of DNA methylation are often mediated by protein factors containing conserved methyl-CpG binding domains (MBDs). Mutations in the MBD protein MeCP2 cause the neurodevelopmental disease Rett syndrome. We report here the solution structure of the MBD of the human methylation-dependent transcriptional regulator MBD1 bound to methylated DNA. DNA binding causes a loop in MBD1 to fold into a major and novel DNA binding interface. Recognition of the methyl groups and CG sequence at the methylation site is due to five highly conserved residues that form a hydrophobic patch. The structure indicates how MBD may access nucleosomal DNA without encountering steric interference from core histones, and provides a basis to interpret mutations linked to Rett syndrome in MeCP2.

Acetylation↗

PubMed1998

Allelic losses and DNA methylation at DNA mismatch repair loci in sporadic colorectal cancer.

Normal and tumor DNA samples of 35 patients with sporadic colorectal carcinoma were analyzed for microsatellite alterations at 12 markers linked to mismatch repair loci: hMLH1, hMSH2, hMSH3, hMSH6, hPMS1 and hPMS2. Remarkably, no correlation was observed between the replication error phenotype (RER+) and allelic losses at these loci. Hemizygous deletions, seen in 6/35 (17%) informative cases at hMLH1, 4/27 (15%) at hMSH2/hMSH6 and 6/34 (18%) at hMSH3, were rarely found in RER+ tumors. Since mismatch repair protein components act in molecular complexes of defined stoichiometry we propose that hemizygous deletion of the corresponding loci may be involved in colorectal tumorigenesis through defects in cellular functions other than replication error correction. The analysis of the methylation status of the promoter region of hMLH1 revealed that methylation might be an important mechanism of this locus inactivation in RER+ sporadic colorectal cancer.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.

Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10,000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.

Animals↗

PubMed1980

Studies on the biological role of dna methylation; IV. Mode of methylation of DNA in E. coli cells.

Two pairs of restriction enzyme isoschizomers were used to study in vivo methylation of E. coli and extrachromosomal DNA. By use of the restriction enzymes MboI (which cleaves only the unmethylated GATC sequence) and its isoschizomer Sau3A (indifferent to methylated adenine at this sequence), we found that all the GATC sites in E. coli and in extrachromosomal DNAs are symmetrically methylated on both strands. The calculated number of GATC sites in E. coli DNA can account for all its m6Ade residues. Foreign DNA, like mouse mtDNA, which is not methylated at GATC sites became fully methylated at these sequences when introduced by transfection into E. coli cells. This experiment provides the first evidence for the operation of a de novo methylation mechanism for E. coli methylases not involved in restriction modification. When the two restriction enzyme isoschizomers, EcoRII and ApyI, were used to analyze the methylation pattern of CCTAGG sequences in E. coli C and phi X174 DNA, it was found that all these sites are methylated. The number of CCTAGG sites in E. coli C DNA does not account for all m5Cyt residues.

Base Sequence↗

PubMed1976

Studies on the biological role of DNA methylation. II. Role of phiX174 DNA methylation in the process of viral progeny DNA synthesis.

In vivo inhibition of bacteriophage phiX174 DNA methylation by nicotinamide resulted in the accumulation of replicative intermediates with multiple-genome length single-stranded "tails". These abnormal replicative intermediates could not be chased into viral single-stranded circular DNA. The effect of nicotinamide on phage maturation and accumulation of abnormal replicative intermediates could be reversed by washing out the inhibitor. The results suggest that the single methyl group present in the viral DNA serves as a recognition site for a specific endonuclease, probably the gene A protein product, that is responsible for the excision of the single-stranded one-genome long viral DNA, before final maturation of the virus occurs.

Coliphages↗