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PubMed1999

Methylation-sensitive, single-strand conformation analysis (MS-SSCA): A rapid method to screen for and analyze methylation.

We have developed methylation-sensitive, single-strand conformation analysis (MS-SSCA) as a method of screening for methylation changes. Bisulfite modification converts cytosines to thymines, but methylated cytosines remain unchanged. This modification creates sequence differences between methylated and unmethylated samples, which can be resolved by SSCA. SSCA is 70-95% efficient at detecting single base changes in a fragment. As bisulfite modification of methylated DNA would typically involve several base changes in a fragment, the efficiency of detecting methylation using MS-SSCA could approach 100%. We applied this method to analyze the BRCA1 promoter CpG island in breast cancer samples. About 20% of sporadic breast cancers are hypermethylated at the BRCA1 promoter CpG island. MS-SSCA rapidly detected those tumors that had previously been shown to be methylated by Southern blotting. The variant bands detected by SSCA were analyzed by sequencing and shown to be methylated. MS-SSCA is a simple method for screening large numbers of samples for methylation and can accelerate genomic sequencing, as all bands can be isolated and sequenced directly.

BRCA1 Protein↗

PubMed1977

Genetics of ribosomal protein methylation in Escherichia coli. II. A mutant lacking a new type of methylated amino acid, N5-methylglutamine, in protein L3.

The ribosomes of an Escherichia coli mutant, designated prm-2, can be methylated in vitro by an enzymatic fraction from wild-type. This enzyme is inactive on the ribosomes from another mutant, prm-1, is reported previously to be methyl group-deficient in protein L11. In vitro methylation of prm-2 ribosomes resulted in the incorporation of about one methyl group per molecule of protein L3. After acid hydrolysis, all the methyl groups were found in a very basic compound which was identified as methylamine. This compound could have been generated by acid hydrolysis of N-methylated amide-groups from glutamine or asparagine. Therefore, chemically-synthesized N4-methyl-asparagine and N5-methylglutamine were chromatographed together with an enzymatic hydrolysate of methylated prm-2 proteins. In all the chromatogrphic systems studied the methylated amino acid was found in the same position as N5'-methylglutamine. These results indicate that mutant prm-2 lacks one residue of N5-methylglutamine present in ribosomal protein L3 of wild type E. coli.

Bacterial Proteins↗

PubMed2002

Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers.

AFLP analysis using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites has been used to analyse the methylation state of anonymous CCGG sequences in Arabidopsis thaliana. The technique was modified to improve the quality of fingerprints and to visualise larger numbers of scorable fragments. Sequencing of amplified fragments indicated that detection was generally associated with non-methylation of the cytosine to which the isoschizomer is sensitive. Comparison of EcoRI/ HpaII and EcoRI/ MspI patterns in different ecotypes revealed that 35-43% of CCGG sites were differentially digested by the isoschizomers. Interestingly, the pattern of digestion among different plants belonging to the same ecotype is highly conserved, with the rate of intra-ecotype methylation-sensitive polymorphisms being less than 1%. However, pairwise comparisons of methylation patterns between samples belonging to different ecotypes revealed differences in up to 34% of the methylation-sensitive polymorphisms. The lack of correlation between inter-ecotype similarity matrices based on methylation-insensitive or methylation-sensitive polymorphisms suggests that whatever the mechanisms regulating methylation may be, they are not related to nucleotide sequence variation.

Arabidopsis↗

PubMed1997

alpha-satellite DNA methylation in normal individuals and in ICF patients: heterogeneous methylation of constitutive heterochromatin in adult and fetal tissues.

The methylation profile of ten alpha-satellites was investigated in normal individuals and in ICF (Immunodeficiency, Centromeric instability, Facial abnormalities) patients. Two out of three ICF patients showed modified methylation of these sequences, reproducing a placental profile. CENP-B boxes, the binding sites of centromeric protein B, were always skewed toward nonmethylation. Unexpected results were observed in normal individuals: in somatic adult tissues the methylation pattern of alpha-satellite DNA varied between chromosomes, and in fetal tissues these satellites were homogeneously undermethylated. Detailed methylation analysis of CENP-B boxes revealed that unmethylated alpha-satellite units coexist with thoroughly methylated regions. These observations showed that the two major components of constitutive heterochromatin are differently methylated in normal somatic and fetal tissues, since classical satellites are consistently methylated. The definite changes in the methylation profile of heterochromatin in somatic chromosomes and the asynchronous timing of methylation of classical and alpha-satellites during development may reflect specific roles of highly repeated sequences in genomic organization.

Adolescent↗

PubMed1991

Stereochemical studies of the C-methylation of deoxycytidine catalyzed by HhaI methylase and the N-methylation of deoxyadenosine catalyzed by EcoRI methylase.

The steric course of methyl group transfer catalyzed by two DNA methylases, HhaI methylase, a DNA (cytosine-5)-methyltransferase, and EcoRI methylase, which methylates at N6 of adenosine, has been studied with (methyl-R)- and (methyl-S)-[methyl-2H1,3H]adenosylmethionine as the methyl donor, using as substrates poly-d(GC) (HhaI) and the dodecamer oligonucleotide duplex d(CGCGAATTCGCG) (EcoRI), respectively. The methylated nucleotides were degraded to convert the chiral methyl groups into acetic acid for configurational analysis. It was found that both enzymatic reactions proceed with inversion of configuration of the methyl group.

Base Sequence↗

PubMed1988

Synthesis and mass spectra of 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-ethyl-D-glucitol and the positional isomers of 4-O-acetyl-1,5-anhydro-di-O-ethyl-O- methyl-D-glucitol and 4-O-acetyl-1,5-anhydro-O-ethyl-di-O-methyl-D-glucitol.

Reductive cleavage of fully methylated, partially O-ethylated cellulose or fully ethylated, partially O-methylated cellulose and subsequent acetylation had previously been shown to produce 4-O-acetyl-1,5-anhydro-2,3,6-tri-O-methyl-, -6- O-ethyl-2,3-di-O-methyl-, -3-O-ethyl-2,6-di-O-methyl-, -2-O-ethyl-3,6-di-O- methyl-, -2,3-di-O-ethyl-6-O-methyl-, -2,6-di-O-ethyl-3-O-methyl-, -3,6-di-O- ethyl-2-O-methyl-, and -2,3,6-tri-O-ethyl-D-glucitol. Described herein is the independent synthesis of these derivatives, except for the first (which had been reported); and their 1H-n.m.r. spectra, chemical-ionization (NH3) mass spectra, and electron-impact mass spectra are tabulated.

Chemical Phenomena↗

PubMed1993

Different proportions of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor currents at the mossy fibre-granule cell synapse of developing rat cerebellum.

The mossy fibre-granule cell synapse undergoes major developmental changes during the second and third weeks after birth. We investigated synaptic transmission during postnatal days 10-22 by means of whole-cell patch-clamp recordings from granule cells in situ. Parasagittal slices were cut from rat cerebellar vermis, and excitatory postsynaptic currents were evoked in granule cells by mossy fibre stimulation with 1.2 mM Mg++ in the extracellular solution. In the majority of granule cells recorded at postnatal days 16-22, excitatory currents were characterized by a fast initial peak followed by a slower component, while in many of the cells recorded at more immature stages, the fast peak was virtually absent. Pharmacological and kinetic data indicated that the fast and slow components were mediated by non-N-methyl-D-aspartate and N-methyl-D-aspartate receptor activation, respectively. The magnitude of the non-N-methyl-D-aspartate current increased with developmental age, while the magnitude of the NMDA current did not change markedly. The age-dependent change of the non-N-methyl-D-aspartate currents could not be accounted for by changes in recording conditions or granule cell electrotonic properties. Furthermore, from postnatal day 11 to 16 the extent of Mg++ block on the N-methyl-D-aspartate receptor did not change, and could not explain the increasing non-N-methyl-D-aspartate/N-methyl-D-aspartate current ratio. We concluded therefore that the age-dependent increase of the non-N-methyl-D-aspartate current was the main cause of the different postsynaptic current waveforms observed at different ages. The developmental change in the proportion of N-methyl-D-aspartate and non-N-methyl-D-aspartate currents may be relevant to the processes regulating granule cell maturation and excitability.

2-Amino-5-phosphonovalerate↗

PubMed1996

N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor antagonism reduces Fos-like immunoreactivity in central trigeminal neurons after corneal stimulation in the rat.

The role of glutamate receptors in processing noxious sensory input from the cornea was assessed in barbiturate-anesthetized rats. Animals were treated with selective antagonists for N-methyl-D-aspartate or non-N-methyl-D-aspartate receptor subtypes prior to application of mustard oil to the corneal surface. Neural activation was estimated from the number of neurons that produced Fos, the protein product of the immediate early gene, c-fos, as detected by immunocytochemistry. Fos-positive neurons were found at two distinct regions of the spinal trigeminal nucleus: the subnucleus interpolaris/caudalis transition and the subnucleus caudalis/upper cervical cord transition. The number of Fos-positive neurons was reduced dose-dependently by the competitive N-methyl-D-aspartate receptor antagonist, 3-[(+/-)-2-carboxypiperazin-4-yl]-propyl-1-phosphonic acid (0.08-8 nmol, i.c.v.), or by the non-N-methyl-D-aspartate receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (2.5-250 nmol, i.c.v.). The greatest reduction in Fos-positive cells was seen at the subnucleus caudalis/upper cervical cord transition after blockade of either receptor subtype. Combined blockade of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors did not cause a further reduction in the number of Fos-positive neurons than was seen after the highest dose of either antagonist alone. Peripheral or central administration of the nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester, had no effect on the number of Fos-positive neurons after corneal stimulation. These results suggest that corneal input to neurons at the subnucleus caudalis/upper cervical cord transition, and to a lesser extent, at the subnucleus interpolaris/subnucleus caudalis transition depends on excitatory amino acid transmission. Both N-methyl-D-aspartate and non-N-methyl-D-aspartate glutamate receptor subtypes, but not the formation of nitric oxide, contribute to the processing of acute corneal stimuli by central trigeminal neurons.

Animals↗

PubMed1991

The effect of glutathione monoethyl ester on the potentiation of the acute toxicity of methyl parathion, methyl paraoxon or fenitrothion by diethyl maleate in the mouse.

Depletion of hepatic glutathione in the mouse by pretreatment with diethyl maleate (DEM) is known to potentiate the acute toxicities of many dimethyl-substituted organothiophosphate insecticides. However, certain studies have raised doubts regarding the participation of glutathione in the detoxification of methyl parathion in the mouse, and hence the putative mechanism of action of DEM-induced potentiation of this insecticide. The present study evaluates the hypothesis that DEM potentiates the acute toxicities of methyl parathion, methyl paraoxon, and fenitrothion by a mechanism other than glutathione depletion. One hour following pretreatment of mice with DEM (0.75 ml/kg i.p.), glutathione was markedly depleted and the acute toxicities of methyl parathion, methyl paraoxon and fenitrothion were potentiated. Administration of glutathione monoethyl ester (20 mmol/kg p.o.) to DEM-pretreated mice attenuated DEM-depletion of hepatic glutathione, or maintained glutathione at or above control levels. However, glutathione monoethyl ester did not alter the DEM-induced potentiation of the lethality of these insecticides. Furthermore, administration of glutathione monoethyl ester to naive mice increased hepatic glutathione levels, but did not affect the percentage of animals succumbing to a challenge dose of methyl parathion, methyl paraoxon, or fenitrothion. These data indicate that DEM potentiates the toxicity of methyl parathion, methyl paraoxon or fenitrothion by a mechanism unrelated to hepatic glutathione content.

Animals↗

PubMed2003

N-CoR mediates DNA methylation-dependent repression through a methyl CpG binding protein Kaiso.

The identification and characterization of molecular mechanisms utilized by cells to mediate transcriptional repression at methylated loci are fundamental to understanding the biological consequences of DNA methylation. Here we demonstrate that Kaiso, a methyl CpG binding protein belonging to the BTB/POZ family of transcription factors, is a component of the human N-CoR complex. In vitro, the Kaiso/N-CoR complex binds specific CpG-rich sequences in a methylation-dependent manner. In vivo, Kaiso targets the N-CoR complex to the MTA2 gene promoter in a methylation-dependent manner. Importantly, we demonstrate that Kaiso is required for transcriptional repression of the methylated MTA2 locus. Furthermore, this repression also requires a functional N-CoR deacetylase complex, which brings about histone hypoacetylation and methylation of H3 lysine 9 to the MTA2 locus. Thus, our data demonstrate a critical role for a methyl CpG binding protein in mediating DNA methylation-dependent repression and reveal the mechanism by which it represses transcription.

Animals↗

PubMed2004

Flavonoid methylation: a novel 4'-O-methyltransferase from Catharanthus roseus, and evidence that partially methylated flavanones are substrates of four different flavonoid dioxygenases.

Catharanthus roseus (Madagascar periwinkle) flavonoids have a simple methylation pattern. Characteristic are B-ring 5' and 3' methylations and a methylation in the position 7 of the A-ring. The first two can be explained by a previously identified unusual O-methyltransferase (CrOMT2) that performs two sequential methylations. We used a homology based RT-PCR strategy to search for cDNAs encoding the enzyme for the A-ring 7 position. Full-length cDNAs for three proteins were characterized (CrOMT5, CrOMT6, CrOMT7). The deduced polypeptides shared 59-66% identity among each other, with CrOMT2, and with CrOMT4 (a previously characterized protein of unknown function). The five proteins formed a cluster separate from all other OMTs in a relationship tree. Analysis of the genes showed that all C. roseus OMTs had a single intron in a conserved position, and a survey of OMT genes in other plants revealed that this intron was highly conserved in evolution. The three cDNAs were cloned for expression of His-tagged recombinant proteins. CrOMT5 was insoluble, but CrOMT6 and CrOMT7 could be purified by affinity chromatography. CrOMT7 was inactive with all compounds tested. The only substrates found for CrOMT6 were 3'-O-methyl-eriodictyol (homoeriodictyol) and the corresponding flavones and flavonols. The mass spectrometric analysis showed that the enzyme was not the expected 7OMT, but a B-ring 4'OMT. OMTs with this specificity had not been described before, and 3',4'-dimethylated flavonoids had not been found so far in C. roseus, but they are well-known from other plants. The identification of this enzyme activity raised the question whether methylation could be a part of the mechanisms channeling flavonoid biosynthesis. We investigated four purified recombinant 2-oxoglutarate-dependent flavonoid dioxygenases: flavanone 3beta-hydroxylase, flavone synthase, flavonol synthase, and anthocyanidin synthase. 3'-O-Methyl-eriodictyol was a substrate for all four enzymes. The activities were only slightly lower than with the standard substrate naringenin, and in some cases much higher than with eriodictyol. Methylation in the A-ring, however, strongly reduced or abolished the activities with all four enzymes. The results suggested that B-ring 3' methylation is no hindrance for flavonoid dioxygenases. These results characterized a new type of flavonoid O-methyltransferase, and also provided new insights into the catalytic capacities of key dioxygenases in flavonoid biosynthesis.

Amino Acid Sequence↗

PubMed1996

The role of phosphatidylethanolamine methylation in the secretion of very low density lipoproteins by cultured rat hepatocytes: rapid inhibition of phosphatidylethanolamine methylation by bezafibrate increases the density of apolipoprotein B48-containing lipoproteins.

The role of phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine (PE) methylation pathway in the secretion of very low density lipoproteins (VLDL) by cultured rat hepatocytes has been investigated by determining the effects of inhibitors. We have shown that bezafibrate and clofibric acid, known hypolipidemic agents, are potent inhibitors of PE methylation (see accompanying paper by Nishimaki-Mogami et al. (1996) Biochim. Biophys. Acta 1304). In hepatocytes incubated with ethanolamine, which maintained cellular PE levels and PE methylation activity, bezafibrate (200 microM) decreased the secretion of triacylglycerol (TG), PC, apolipoproteins B48, and E in VLDL by 50-75%. In contrast, bezafibrate at this concentration had marginal effect on VLDL secretion (83-115% of control) by hepatocytes that had been cultured in the absence of ethanolamine. In these cells PE levels and PE methylation activity had decreased by approx. 40%. VLDL secretion was decreased at concentrations similar to those required to inhibit PE methylation, and was accompanied by an increase in cellular TG levels. The same ethanolamine-dependent effects were produced by clofibric acid and also by 3-deazaadenosine (DZA), an inhibitor of cellular methylation reactions. These results indicate that PC synthesis via the PE methylation pathway plays a significant role in VLDL secretion in rat hepatocytes if the pathway is maintained at levels comparable to those in vivo. The reductions of PE methylation by bezafibrate and DZA did not affect the total amount of apolipoprotein B48 secreted into the medium. The decrease in apolipoprotein B48 in VLDL caused by bezafibrate was accompanied by an increase in apolipoprotein B48 in the HDL density range. In contrast, the amount of apolipoprotein B100 in VLDL and density determined by sequential flotation were unaffected. These findings indicate that rapid reduction of PC synthesis via PE methylation does not affect the secretion of apolipoprotein B48- or B100-containing lipoprotein particles, but does impair the lipidation of particles containing apolipoprotein B48, but not apolipoprotein B100.

Animals↗

PubMed1996

Size-related properties of vestibular afferent fibers in the frog: differential synaptic activation of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors.

Vestibular afferent fibers exhibit a specific, cell size-related uptake of aspartate and glycine [Straka H. et al. (1995) Neuroscience 70, 685-696]. A similar, size-related coexistence of glycine and glutamate had been reported earlier for these fibers [Reichenberger I. and Dieringer N. (1994) J. comp. Neurol. 349, 603-614]. Taken together, these results suggest a size-related co-release of both amino acids and the activation of different glutamate receptors in second order vestibular neurons. To test this hypothesis we stimulated the VIIIth nerve and recorded the responses of central vestibular neurons in the isolated brainstem of frogs before and during the application of the N-methyl-D-aspartate antagonists (7-chlorokynurenic acid and D-(-)-2-amino-5-phosphonovaleric acid). The presence of either one of these antagonists provoked a dose-dependent and Mg(2+)-sensitive partial block of the monosynaptic responses recorded extra- or intracellularly. This implies that afferent-evoked responses in central vestibular neurons are composed of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptor-mediated components. In most of the intracellularly recorded neurons (21 out of 24) the relative amplitude of the N-methyl-D-aspartate receptor-mediated component decreased with an increase in stimulus intensity. Since electric stimulation recruits thick afferents at a lower current intensity than thin afferent fibers, our results imply a co-activation of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors by thick vestibular afferents. At a given stimulus intensity the amplitude of the N-methyl-D-aspartate receptor-mediated component differed between neurons. The results of this study extend the list of known anatomical, histochemical and physiological properties that distinguish thick from thinner vestibular afferent fibers. In spite of this detailed knowledge, however, the physiological role of thick vestibular afferents is so far unclear. The novel concept of a size-related co-activation of N-methyl-D-aspartate and non-N-methyl-D-aspartate receptors by vestibular afferent fibers establishes the basis for more specific physiological hypotheses.

2-Amino-5-phosphonovalerate↗

PubMed1996

Asparagine 229 mutants of thymidylate synthase catalyze the methylation of 3-methyl-2'-deoxyuridine 5'-monophosphate.

The conserved Asn 229 of thymidylate synthase (TS) forms a cyclic hydrogen bond network with the 3-NH and 4-O of the nucleotide substrate 2'-deoxyuridine 5'-monophosphate (dUMP). Asn 229 is not essential for substrate binding or catalysis [Liu, l., & Santi, D. B. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 8604-8608] but is a major determinant in substrate specificity [Liu, l., & Santi, D. V. (1993) Biochemistry 32, 9263-9267]. 3-Methyl-dUMP (3-MedUMP) is neither a substrate nor an inhibitor of wild type TS but is converted to 3-methyl 2'-deoxythymidine 5'-monophosphate by many TS Asn 229 mutants. Some of the Asn 229 mutants (N229C, -I, -M, -A, and -V) have kcat values for 3-MedUMP methylation which are up to about 20% of that for wild type TS-catalyzed methylation of dUMP, and some mutants (N229C and -A) catalyze methylation of 3-MedUMP more efficiently than that of dUMP. Mutants with hydrophobic side chains tended to be more active in catalysis of methylation of 3-MedUMP than those with hydrophilic side chains. The ability of 3-MedUMP to serve as a substrate for Asn 229 mutants shows that the active form of dUMP involves the neutral pyrimidine base and that ionization of the 3-NH group does not occur in the course of catalysis. In contrast to the negligible binding of 3-MedUMP to wild type TS, both 3-MedUMP and dUMP showed similar Km values with the Asn 229 mutants, suggesting similar binding affinities to the mutants. The X-ray crystal structure of the TS N229C--3-MedUMP complex showed that the side chain of Cys 229 was rotated away from the pyrimidine ring to allow placement of a water molecule and the 3-methyl group of 3-MedUMP in the active site. Our results suggest that the inability of 3-MedUMP to undergo methylation by wild type TS is due to its inability to bind to the enzyme, which in turn is simply a result of steric interference of the 3-methyl group with the side chain of Asn 229.

Amino Acid Sequence↗

PubMed2007

Methylation analysis of the intergenic differentially methylated region of DLK1-GTL2 in human.

Imprinting is a non-Mendelian form of inheritance where epigenetic modifications control mono-allelic expression depending on the parental origin. Methylation of CpG-dinucleotides at differentially methylated regions (DMRs) is one of the best-studied mechanisms directing expression to one specific parental allele. We studied the methylation patterns of the intergenic (IG)-DMR of DLK1 and GTL2. The methylation marks of the IG-DMR were analysed in human gametes, preimplantation embryos, amniocytes and blood of babies born after intracytoplasmic sperm injection (ICSI) and blood from adults using a bisulphite sequencing technique. In oocytes, the IG-DMR was mainly unmethylated while in sperm cells a generally methylated pattern was detected. This germ-line specific methylation mark was maintained in the preimplantation embryos until the second cleavage stage. Afterwards in the preimplantation embryos, intermediate methylation patterns (26-74% methylation) occurred, which may point to relaxation of the imprints. Intermediate patterns were also present in amniocytes, blood from ICSI babies and adults. We hypothesise that in the early cleavage stage embryo a strict differential methylation pattern is needed for the correct imprint establishment of surrounding imprinted genes. Once correct imprinting of the involved gene(s) is acquired, a more relaxed state of the IG-region is allowed.

Calcium-Binding Proteins↗

PubMed1979

Attractants and repellents influence methylation and demethylation of methyl-accepting chemotaxis proteins in an extract of Escherichia coli.

During bacterial chemotaxis, attractants and repellents alter the methylation levels of the methyl-accepting chemotaxis proteins (MCPs). These methylation levels represent a balance between two enzymatic processes: methylation and demethylation. In vivo experiments previously have shown that chemoeffectors influence the demethylation process; effects on the methylation system have not been reported. Here we show that in a cell-free extract of Escherichia coli both methylation and demethylation of the MCPs are affected by attractants and repellents. Attractants enhance methylation and inhibit demethylation. Repellents inhibit methylation and stimulate demethylation. The cell-free system provides an opportunity for further study of the mechanisms by which attractants and repellents influence the levels of methylation of the MCPs.

Bacterial Proteins↗

PubMed1999

Isolation of DNA fragments associated with methylated CpG islands in human adenocarcinomas of the lung using a methylated DNA binding column and denaturing gradient gel electrophoresis.

We have constructed a library of DNA fragments heavily methylated in human adenocarcinomas of the lung to permit the comprehensive isolation of methylated CpG islands in cancer. Heavily methylated genomic DNA fragments from tumors of nine male patients were enriched using a methylated DNA binding column and used for construction of the library. From this library, DNA fragments having properties of CpG islands were isolated on the basis of their reduced rate of strand dissociation during denaturing gradient gel electrophoresis. Approximately 1,000 clones, corresponding to 0.3% of the library were analyzed, and nine DNA fragments were identified as being associated with CpG islands that were methylated in tumor DNA. One CpG island was methylated specifically in tumor DNA, whereas the remaining eight CpG islands were methylated both in normal and tumor DNA derived from the same patients. Our results suggest that the number of CpG islands methylated specifically in tumors is not large. The library, which contains DNA fragments from methylated CpG islands comprehensively, is expected to be valuable when elucidating epigenetic processes involved in carcinogenesis.

Adenocarcinoma↗

PubMed1999

Tandem B1 elements located in a mouse methylation center provide a target for de novo DNA methylation.

A cis-acting methylation center that signals de novo DNA methylation is located upstream of the mouse Aprt gene. In the current study, two approaches were taken to determine if tandem B1 repetitive elements found at the 3' end of the methylation center contribute to the methylation signal. First, bisulfite genomic sequencing demonstrated that CpG sites within the B1 elements were methylated at relative levels of 43% in embryonal stem cells deficient for the maintenance DNA methyltransferase when compared with wild type embryonal stem cells. Second, the ability of the B1 elements to signal de novo methylation upon stable transfection into mouse embryonal carcinoma cells was examined. This approach demonstrated that the B1 elements were methylated de novo to a high level in the embryonal carcinoma cells and that the B1 elements acted synergistically. The results from these experiments provide strong evidence that the tandem B1 repetitive elements provide a significant fraction of the methylation center signal. By extension, they also support the hypothesis that one role for DNA methylation in mammals is to protect the genome from expression and transposition of parasitic elements.

Animals↗