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PubMed2005

Transgene-induced CCWGG methylation does not alter CG methylation patterning in human kidney cells.

Several reports suggest that C(m)CWGG methylation tends not to co-exist with (m)CG methylation in human cells. We have asked whether or not methylation at CCWGG sites can influence CG methylation. DNA from cells expressing an M.EcoRII-GFP fusion was actively methylated at CCWGG sites. CG methylation as measured by R.HpaII/R.MspI ratios was unchanged in cells expressing the transgene. Cloned representatives of C(m)CWGG methylated DNA often contained, or were adjacent to an ALU repeat, suggesting that M.EcoRII-GFP actively methylated gene-rich R-band DNA. The transgenic methyltransferase applied C(m)CWGG methylation to a representative human promoter that was heavily methylated at CG dinucleotides (the SERPINB5 promoter) and to a representative promoter that was essentially unmethylated at CG dinucleotides (the APC promoter). In each case, the CG methylation pattern remained in its original state, unchanged by the presence of neighboring C(m)CWGG sites. Q-PCR measurements showed that RNA expression from the APC gene was not significantly altered by the presence of C(m)CWGG in its promoter. Kinetic studies suggested that an adjacent C(m)CWGG methylation site influences neither the maintenance nor the de novo methylation activities of purified human Dnmt1. We conclude that C(m)CWGG methylation does not exert a significant effect on CG methylation in human kidney cells.

Base Sequence↗

PubMed1981

Methylation of membrane proteins in human erythrocytes. Identification and characterization of polypeptides methylated in lysed cells.

An in vitro system was developed for studying protein methylation reactions in human red blood cells. Packed erythrocytes were lysed by freeze-thawing in the presence of S-adenosyl[methyl-3H]methionine. Specific incorporation of base-labile methyl groups into the band 3 anion transport protein and the major sialoglycoprotein (glycophorin, periodic acid-Schiff reagent-1) was demonstrated by dodecyl sulfate gel electrophoresis at pH 2.4, selective extractions with Triton X-100 and lithium diiodosalicylate, and protease sensitivity. Two other unidentified intrinsic membrane proteins with Mr = 96,000 and 23,500 were also methylated. Little radioactivity was incorporated into membrane proteins when membranes were incubated with S-adenosyl-L-[methyl-3H]methionine in the absence of cytosol. No evidence was obtained for incorporation of methyl label into extrinsic proteins including bands 1, 2, 2.1, 4, 5, 6, or in zone 4.5. Proteolytic digestion of intact cells and isolated membranes revealed that one site of methylation on the band 3 polypeptide may be at the inner surface of the membrane near the junction of the cytoplasmic domain and the membrane domain. The rates of hydrolysis of the incorporated methyl groups were characterized over a range of pH values. These rates were compared to those of methyl esterified amino acids and peptides, including aspartic acid beta-methyl ester which has been isolated from proteolytic digests of methylated erythrocyte membranes (Janson, C. A., and Clarke, S. (1980) J. Biol. Chem. 225, 11640-11643). We find that the rates of base-catalyzed hydrolysis of beta-methyl esters of aspartic acid and gamma-methyl esters of glutamic acid are highly sensitive to the presence of substituents on the alpha-carboxyl and alpha-amino groups. The rate of hydrolysis of the membrane-incorporated methyl groups are consistent with those of internal aspartic acid and glutamic acid methyl ester residues.

Chymotrypsin↗

PubMed1989

7-Methyl bile acids: effects of chenodeoxycholic acid, cholic acid, and their 7 beta-methyl analogues on the formation of cholesterol gallstones in the prairie dog.

The purpose of this study was to compare the effects of the naturally occurring bile acids (chenodeoxycholic acid and cholic acid) with their 7-methyl analogues (3 alpha,7 alpha-dihydroxy-7 beta-methyl-5 beta-cholanoic acid and 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid) on gallstone formation and prevention and cholesterol metabolism in the prairie dog. Sixty animals were fed a semipurified diet, containing 0.4% cholesterol, with one of the following acids (0.1%): chenodeoxycholic, cholic, 3 alpha,7 alpha-dihydroxy-7 beta-methyl-5 beta-cholanoic, or 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid. This concentration of dietary bile acids amounts to a dose of 27-30 mg/kg.day. After 8 wk, 89% of control animals had gallstones and 94% had cholesterol crystals. Chenodeoxycholic and 3 alpha,7 alpha-dihydroxy-7 beta-methyl-5 beta-cholanoic acids decreased the incidence of gallstones to 50%. Cholic acid and 3 alpha,7 alpha,12 alpha-tri-hydroxy-7 beta-methyl-5 beta-cholanoic acid did not prevent gallstone formation. The liver cholesterol level was decreased by chenodeoxycholic acid, whereas cholic and 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acids increased serum and liver cholesterol. Each administered bile acid became the predominant biliary bile acid and 7-methyl analogues did not increase secondary bile acids. Fecal analysis of radioactive metabolites using 14C-labeled 7-methyl analogues showed that these compounds are resistant to bacterial 7-dehydroxylation. It was concluded that 3 alpha,7 alpha-dihydroxy-7 beta-methyl-5 beta-cholanoic acid inhibited gallstone formation as effectively as chenodeoxycholic acid, whereas both cholic and 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acids were not effective. The effects of 7-methyl analogues on the parameters of cholesterol metabolism that we studied were similar to those of their parent compounds, chenodeoxycholic and cholic acids. Thus, 3 alpha,7 alpha-dihydroxy-7 beta-methyl-5 beta-cholanoic acid but not 3 alpha,7 alpha,12 alpha-trihydroxy-7 beta-methyl-5 beta-cholanoic acid offers promise in cholelitholytic therapy for the prevention and possibly dissolution of cholesterol gallstones.

Animals↗

PubMed2005

DNA methylation profiling identifies CG methylation clusters in Arabidopsis genes.

Cytosine DNA methylation in vertebrates is widespread, but methylation in plants is found almost exclusively at transposable elements and repetitive DNA. Within regions of methylation, methylcytosines are typically found in CG, CNG, and asymmetric contexts. CG sites are maintained by a plant homolog of mammalian Dnmt1 acting on hemi-methylated DNA after replication. Methylation of CNG and asymmetric sites appears to be maintained at each cell cycle by other mechanisms. We report a new type of DNA methylation in Arabidopsis, dense CG methylation clusters found at scattered sites throughout the genome. These clusters lack non-CG methylation and are preferentially found in genes, although they are relatively deficient toward the 5' end. CG methylation clusters are present in lines derived from different accessions and in mutants that eliminate de novo methylation, indicating that CG methylation clusters are stably maintained at specific sites. Because 5-methylcytosine is mutagenic, the appearance of CG methylation clusters over evolutionary time predicts a genome-wide deficiency of CG dinucleotides and an excess of C(A/T)G trinucleotides within transcribed regions. This is exactly what we find, implying that CG methylation clusters have contributed profoundly to plant gene evolution. We suggest that CG methylation clusters silence cryptic promoters that arise sporadically within transcription units.

Arabidopsis↗

PubMed2002

Epstein-barr virus-positive gastric carcinoma demonstrates frequent aberrant methylation of multiple genes and constitutes CpG island methylator phenotype-positive gastric carcinoma.

CpG island methylation is an important mechanism for inactivating the genes involved in tumorigenesis. Gastric carcinoma (GC) is one of the tumors that exhibits a high frequency of aberrant CpG island methylation. There have been many reports suggesting a close link between Epstein-Barr virus (EBV) and the development of GC. However, little is known about the oncogenic mechanism of EBV in gastric carcinogenesis. Twenty-one cases of EBV-positive GC and 56 cases of EBV-negative GC were examined for aberrant DNA methylation of the CpG islands of 19 genes or loci and the differences in the methylation frequency between EBV-positive and -negative GCs were investigated to determine a role of aberrant methylation in EBV-related gastric carcinogenesis. The average number of methylated genes or loci was higher in EBV-positive GCs than in EBV-negative GCs (13.4 versus 7.8, respectively, P < 0.001). EBV-positive GCs showed methylation in at least 10 CpG islands (52.6% of the tested genes), whereas 62.5% of EBV-negative GCs showed methylation in <10 CpG islands. THBS1, APC, p16, 14-3-3 sigma, MINT1, and MINT25 were methylated at a frequency >90% in EBV-positive GCs. The methylation frequency difference in the respective CpG islands between EBV-positive and -negative GCs was statistically significant (P < 0.05). Among these genes or loci, the methylation frequency of p16 in the EBV-positive GCs was more than three times higher than in the EBV-negative GCs. The PTEN, RASSF1A, GSTP1, MGMT, and MINT2 were methylated in EBV-positive GCs at a frequency of more than three times that of the EBV-negative GCs. These results demonstrate a relationship between EBV and aberrant methylation in GC and suggest that aberrant methylation may be an important mechanism of EBV-related gastric carcinogenesis.

CpG Islands↗

PubMed2000

Regulation of N-methyl-D-aspartate receptor expression and N-methyl-D-aspartate-induced cellular response during chronic hypoxia in differentiated rat PC12 cells.

The purpose of the present study was to examine the effect of chronic hypoxia on N-methyl-D-aspartate-mediated cellular responses in differentiated PC12 cells. PC12 cells were differentiated by treatment with nerve growth factor. Patch-clamp analysis in differentiated PC12 cells showed that extracellularly applied N-methyl-D-aspartate induced an inward current that was abolished by the presence of the N-methyl-D-aspartate receptor antagonist MK-801. Results from Ca(2+) imaging experiments showed that N-methyl-D-aspartate induced an elevation in intracellular free Ca(2+) which was also abolished by MK-801. We also examined the effect of hypoxia on the N-methyl-D-aspartate-induced current in nerve growth factor-treated cells. We found that the N-methyl-D-aspartate-induced inward current and the N-methyl-D-aspartate-induced elevation in intracellular free Ca(2+) were markedly attenuated by chronic hypoxia. We next examined the possibility that the reduced N-methyl-D-aspartate responsiveness was due to down-regulation of N-methyl-D-aspartate receptor levels. Northern blot and immunoblot analyses showed that both messenger RNA and protein levels for N-methyl-D-aspartate receptor subunit 1 were markedly decreased during hypoxia. However, the messenger RNA for N-methyl-D-aspartate receptor subunit 2C was increased, whereas the protein level for subunit 2C did not change. Our results indicate that differentiated PC12 cells express functional N-methyl-D-aspartate receptors and that chronic exposure to hypoxia attenuates the N-methyl-D-aspartate-induced Ca(2+) accumulation in these cells via down-regulation of N-methyl-D-aspartate receptor subunit 1. This mechanism may play an important role in protecting PC12 cells against hypoxic stress.

Animals↗

PubMed1992

Tissue distribution of enzymic methylation of glutathione S-transferase and its effects on catalytic activity. Methylation of glutathione S-transferase 11-11 inhibits conjugating activity towards 1-chloro-2,4-dinitrobenzene.

Glutathione S-transferases (GSTs) were isolated from rat liver, lung, heart, kidney, testis and brain by coupled affinity chromatography and subunits were resolved by reverse-phase h.p.l.c. The reverse-phase h.p.l.c. technique was improved from our previously published work [Johnson, Neal, Collins & Siegel (1990) Biochem. J. 270, 483-489] by changing from a C4 to a C18 wide-pore reverse-phase column; this resulted in baseline or near-baseline resolution of all GST subunits. There were significant tissue-dependent differences in the expression of GST subunits and the level of GST subunits present was quantitatively determined for each of the tissues. The extent of methylation of GSTs in vitro and distribution of GST methyltransferase (GST-MT) was determined in cytosolic fractions from each of these tissues. Purified GST isoenzymes were methylated with partially purified liver GST-MT. Methylation of Mu class subunits 3 and 4, the preferred substrates of methylation in liver, was substoichiometric in all tissues. The extent of methylation of subunit 3 ranged from 0.13% to 0.94% and subunit 4 from 0.03% to 0.60%. Methylation of Alpha class subunits was either not detectable or 5-10-fold less than that of Mu class subunits 3 and 4. Pi class subunit 7 was methylated to a greater extent than the Alpha class subunits but less than Mu class isoenzymes. A notable exception to this low level of methylation was GST 11-11, found mainly in testis and brain. Methylation of subunit 11 reached 21.9% (219 pmol of methyl group/nmol of subunit 11) when this isoenzyme was incubated with partially purified liver GST methyltransferase. Methylation of GST 11-11 was found to inhibit the conjugating activity of this isoenzyme towards 1-chloro-2,4-dinitrobenzene; the degree of inhibition of conjugating activity correlated with the extent of methylation of GST 11-11. GST-MT activity toward GST subunits 3, 4 and 11 was present in kidney and liver, detectable in lung and heart, but absent from brain and testis. Anion-exchange chromatography of GST-MTs from liver and kidney suggested the presence of four different forms of GST-MT (I-IV) and indicated that GST-MT isoenzymes III and IV were present at significantly lower concentrations in kidney than liver. The present paper shows that methylation is an enzyme-catalysed reaction that differs in substrate-specificity with respect to different GST isoenzymes, that expression of GST-MT is tissue-dependent and multiple forms of the enzyme are present in liver and kidney, and that methylation inhibits GST activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Analysis of Variance↗

PubMed1997

Mapping promoter regions that are hypersensitive to methylation-mediated inhibition of transcription: application of the methylation cassette assay to the Epstein-Barr virus major latency promoter.

Methylation-associated transcriptional repression is recognized in many settings and may play a role in normal differentiation and in tumorigenesis. Both sequence-specific and nonspecific mechanisms have been elaborated. Recently, we have presented evidence that methylation-associated inhibition of the Epstein-Barr virus (EBV) major latency promoter (BamHI C promoter or Cp) in Burkitt's lymphoma and Hodgkin's disease may play an important role in the pathogenesis of these tumors by protecting them from CD8+ cytotoxic T-cell immunosurveillance. The mechanism of transcriptional repression may relate to specific inhibition of the binding of a cellular transcription factor by methylation. To dissect the viral promoter with regard to transcriptional sensitivity to methylation, we have devised an assay that allows the methylation of discrete regions of reporter plasmids. During the course of the assay, methylation patterns appeared to be stable; there was no evidence of either spread or reversal of the imposed methylation pattern. Application of the assay to the 3.8-kb region upstream of the major EBV latency promoter with natural Cp reporter plasmids showed that sensitivity to methylation is not homogeneously distributed but is concentrated in two discrete regions. The first of these methylation-hypersensitive regions (MHRI) is the previously identified EBNA-2 response element, which includes the methylation-sensitive CBF2 binding site. The second (MHRII) is a sequence further downstream whose potential role in methylation-mediated transcriptional repression had been previously unsuspected. In chimeric enhancer/promoter plasmids, methylation of this downstream region was sufficient to virtually abolish simian virus 40 enhancer-driven transcription. Further dissection indicated that methylation of the EBNA-2 response element (MHRI) was sufficient to abolish EBNA-2-mediated Cp activity while methylation of a region including the EBNA-2 response element and downstream sequence (MHRI and MHRII) was sufficient to abolish all Cp-mediated reporter activity, including that driven by the EBNA-1-dependent enhancer in the origin of plasmid replication, oriP.

Binding Sites↗

PubMed2006

A genome-wide screen for promoter methylation in lung cancer identifies novel methylation markers for multiple malignancies.

BACKGROUND: Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The "rules" governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets. METHODS AND FINDINGS: In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5' CpG islands, are induced from undetectable levels by 5-aza-2'-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors. CONCLUSIONS: By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.

Adenocarcinoma↗

PubMed2003

Frequent HOXA11 and THBS2 promoter methylation, and a methylator phenotype in endometrial adenocarcinoma.

PURPOSE: This study was designed to determine whether there is a methylator phenotype in stage I and II endometrioid endometrial adenocarcinoma, and if so, whether methylation correlates with recurrence. EXPERIMENTAL DESIGN: Bisulfite-converted DNAs from 24 stage I and II primary cancers (12 recurrent and 12 nonrecurrent), and 5 endometrial cancer cell lines were analyzed for methylation in the promoter regions of seven genes. A methylation index (MeI) was calculated for each tumor. Frequent HOXA11 and THBS2 methylation prompted analysis of case-matched bloods and 25 additional nonrecurrent primary cancers. Statistical analysis included Fisher's exact and Student t tests. RESULTS: Rates of methylation in the initial tumor series were as follows: HOXA11, 70.8%; THBS2, 62.5%; MLH1, 33.3%; CTNNB1, 16.7%; VDR, 4.2%; CDKN2A, 4.2%; and THBS1, 0%. There was no difference in the MeI of recurrent and nonrecurrent cases. However, cell lines had higher mean MeI. High rates of HOXA11 and THBS2 methylation were confirmed in the additional nonrecurrent tumors. None of the 24 case-matched bloods had HOXA11 methylation, whereas three blood DNAs showed THBS2 methylation. There was a statistically significant difference in the rate of HOXA11 methylation in recurrent and nonrecurrent tumors (P = 0.0167). CONCLUSIONS: Endometrial adenocarcinomas have a methylator phenotype. No correlation between MeI and clinicopathologic variables in early stage tumors was observed. High rates of methylation were found in the HOXA11 and THBS2 promoter regions. HOXA11 promoter methylation was significantly more frequent in recurrent than nonrecurrent cases. HOXA11 methylation in early stage endometrial cancer is associated with poor outcome.

Adenocarcinoma↗

PubMed1997

Low frequency of p16/CDKN2A methylation in sporadic melanoma: comparative approaches for methylation analysis of primary tumors.

Methylation of the 5' CpG island of the p16 tumor suppressor gene represents one possible mechanism for inactivation of this cell cycle regulatory gene that is also a melanoma predisposition locus. We have investigated the potential contribution of somatic silencing of the p16 gene by DNA methylation in 30 cases of sporadic cutaneous melanoma. The methylation status of the 5' CpG island of p16 was initially determined by Southern analysis and then reevaluated (in a blinded manner) using methylation-specific PCR, methylation-sensitive single nucleotide primer extension, and bisulfite genomic sequencing. All methodologies yielded concordant results, and significant levels of methylation were observed in 3 of the 30 (10%) melanoma DNAs analyzed. Of the three tumors found to be methylated, two were also positive for LOH on 9p21 (where the p16 gene resides), implying that both p16 alleles were inactivated, one via deletion and the other via methylation-associated transcriptional silencing. The association between methylation and transcriptional silencing of p16 was also further supported by inducing p16 expression with a DNA demethylating agent (5-aza-2'-deoxycytidine) in a melanoma cell line known to harbor a methylated p16 allele. Although methylation-associated gene silencing does not represent a common mechanism for p16 inactivation in sporadic melanoma, our findings provide support that PCR-based techniques, such as methylation-specific PCR and methylation-sensitive single nucleotide primer extension, can be reliably used for the accurate detection and quantitation of aberrant levels of DNA methylation in tumor specimens.

Chromosome Deletion↗

PubMed2004

Differentiation between peptides containing acetylated or tri-methylated lysines by mass spectrometry: an application for determining lysine 9 acetylation and methylation of histone H3.

Histone acetylation and methylation play a critical role in transcription and gene regulation. Identification of sites of lysine acetylation and methylation in histones or other proteins by mass spectrometry (MS) is of increasing interest. In this paper, we report the use of MS to differentiate between peptides containing acetylated or tri-methylated lysines. High accuracy matrix-assisted laser desorption/ionization-time of flight MS gives better than five parts per million measurement accuracy, which is sufficient to verify acetylation and/or methylation. Electrospray ionization tandem mass spectrometry was used to assign modification sites and to differentiate acetylation from methylation. Typically, an immonium ion at m/z 98 corresponds to a mono-methylated lysine and an immonium ion at m/z 126 corresponds to an acetylated lysine. The neutral loss ion (MH(+)-59) is unique for a tri-methylated lysine. For a peptide with two or more modification sites of acetylation or tri-methylation or one site containing partial acetylation and tri-methylation, the a(2)-, b(2)-type ion is the characteristic index for an acetylated lysine whereas the b(2)-59 ion is indicative of a tri-methylated lysine in the N-terminus. The y-type ions and y-59 ions are characteristic of an acetylated lysine and a tri-methylated lysine at the C-terminus, respectively. We demonstrated that a lysine in a peptide modified by methylation or acetylation can be differentiated by MS using our method. Even if more then one lysine is present in a peptide and different modifications of this amino acid occur, they can be distinguished. This method was successful for the determination of the acetylation and methylation status of lysine 9 of histone H3 in chicken erythrocytes and human HeLa cell lines.

Acetylation↗

PubMed2002

Methylation patterns of the human beta-glucuronidase gene locus: boundaries of methylation and general implications for frequent point mutations at CpG dinucleotides.

Methylation of CpG islands spanning promoter regions is associated with control of gene expression, although it is unclear what mechanisms define the boundaries between methylated and unmethylated regions in the genome. Methylation of genomic DNA in mammals also affects the frequency of inherited diseases by predisposing them to CpG mutations. To gain insight into these issues, we investigated patterns of cytosine methylation on almost the entire beta-glucuronidase gene (GUSB) from normal leukocyte DNAs by bisulfite genomic sequencing. We mapped the boundaries of methylation that flank the 5'- and 3'-ends of the CpG island region, and correlated methylation status with transitional mutations at CpG sites. GenBank sequence analyses showed that the CpG island of human GUSB is juxtaposed with multiple Alu repeats and also includes multiple Sp1 sites upstream and downstream of the transcription start, which has been suggested to prevent CpG islands from becoming methylated. We show that cytosine methylation is extensive across the entire gene except for CpG sites in the proximal promoter region, exon 1, and part of intron 1; the unmethylated CpG island is embedded between densely methylated flanking regions containing multiple Alu repeats; a sharp boundary separates the methylated and unmethylated regions of the 5'-flank of the CpG island, but a gradual change in methylation density over 1.0 kb is observed in the 3'-flank of the CpG island; boundaries of the 5'-end and 3'-end of the CpG island contain multiple Sp1 sites in addition to Alu repeats; methylation in both strands is symmetrical except at the boundary regions between methylated and unmethylated regions; and nonmethylation of exon 1 correlates with the absence of transitional mutations at CpG sites in exon 1.

3' Flanking Region↗

PubMed1999

Patterns of cytosine methylation in an elite rice hybrid and its parental lines, detected by a methylation-sensitive amplification polymorphism technique.

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we assessed the extent and pattern of cytosine methylation in the rice genome, using the technique of methylation-sensitive amplified polymorphism (MSAP), which is a modification of the amplified fragment length polymorphism (AFLP) method that makes use of the differential sensitivity of a pair of isoschizomers to cytosine methylation. The tissues assayed included seedlings and flag leaves of an elite rice hybrid, Shanyou 63, and the parental lines Zhenshan 97 and Minghui 63. In all, 1076 fragments, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified using 16 pairs of selective primers. A total of 195 sites were found to be methylated at cytosines in one or both parents, and the two parents showed approximately the same overall degree of methylation (16.3%), as revealed by the incidence of differential digestion by the isoschizomers. Four classes of patterns were identified in a comparative assay of cytosine methylation in the parents and hybrid; increased methylation was detected in the hybrid compared to the parents at some of the recognition sites, while decreased methylation in the hybrid was detected at other sites. A small proportion of the sites was found to be differentially methylated in seedlings and flag leaves; DNA from young seedlings was methylated to a greater extent than that from flag leaves. Almost all of the methylation patterns detected by MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrate that the MSAP technique is highly efficient for large-scale detection of cytosine methylation in the rice genome. We believe that the technique can be adapted for use in other plant species.

Base Sequence↗

PubMed1997

Dual mode of N-methyl-D-aspartate-induced neuronal death in hippocampal slice cultures in relation to N-methyl-D-aspartate receptor properties.

Hippocampal slices prepared from neonatal rats were cultured for several weeks, and excitotoxicity induced in CA1 pyramidal neurons by N-methyl-D-aspartate was evaluated at different culture stages. CA1 neurons cultured for one week exhibited cell death predominantly within 1-3 h after a 15-min N-methyl-D-aspartate insult (early death), whereas those cultured for three weeks showed cell death mainly a few hours to 24 h after the insult (delayed death). CA1 neurons cultured for two weeks were in a transitional state, expressing only weak early and delayed forms of cell death in response to N-methyl-D-aspartate. The N-methyl-D-aspartate-induced early cell death observed in the one-week group depended on external Cl- but did not require external Ca2+; rather, early cell death was enhanced in Ca2+-free solution. This early cell death was accompanied by cell swelling, but cell swelling alone produced by osmotic changes failed to induce cell death. There was no evidence that CA1 neurons in the one-week group were more responsive to N-methyl-D-aspartate than those in the two other groups. Delayed cell death examined in the three-week group depended on external Ca2+ but was independent of Cl-. The density of N-methyl-D-aspartate-induced whole-cell currents recorded from CA1 neurons in Mg2+-free solution remained unchanged during three weeks of culture. However, the N-methyl-D-aspartate receptor channel in the three-week group was more resistant to Mg2+ block than that in the one- or two-week group. The incidence of N-methyl-D-aspartate-induced delayed cell death was higher in the three-week group than in the two-week group in normal solution but not in Mg2+-free solution. Thus, Mg2+ block-resistant properties of the N-methyl-D-aspartate receptor channel acquired during prolonged culture may account for the high incidence of N-methyl-D-aspartate-induced delayed cell death in the three-week group. However, the N-methyl-D-aspartate receptor subunits expressed in the CA1 subfield did not show any feature specific to the three-week group. These results show that two mechanistically distinct modes of N-methyl-D-aspartate-induced neuronal death are manifested differentially at different culture stages, depending on the intrinsic neuron properties (i.e. early cell death) and on the properties or the responsiveness of the N-methyl-D-aspartate receptor (i.e. delayed cell death).

Animals↗

PubMed2002

De novo methylation of MMLV provirus in embryonic stem cells: CpG versus non-CpG methylation.

DNA methyltransferases, Dnmt3a and Dnmt3b, are required for de novo methylation in embryonic stem (ES) cells and postimplantation embryos. However, the mechanism of de novo methylation is largely unknown. In this study, we have analyzed the sequence specificity of Dnmt3a and Dnmt3b during de novo methylation of murine Moloney leukemia virus provirus DNA in virus-infected ES cells. Provirus DNA from infected wild-type (J1), Dnmt1-/- (c/c), and Dnmt3a3b-/- (3a3b-/-) ES cells were analyzed using the bisulfite sequencing method. We demonstrate that Dnmt3 enzymes methylate predominantly CpG sites in vivo and confirm that Dnmt3 enzymes, but not Dnmt1, are responsible for de novo methylation. However, the sequence context and CpG density do not appear to influence de novo methylation, though strand bias is detectable. Interestingly, non-CpG methylation is detected as a component of de novo methylation. CpA methylation was detected at approximately 1.4% of all sites in J1 and approximately 1.0% in c/c, but only approximately 0.2% in 3a3b-/-. Few methylated CpT or CpC sites were detected. Similar results from nearest neighbor analysis of global endogenous methylation levels indicated a correlation between Dnmt3a and Dnmt3b presence and CpA methylation. These results demonstrate that the Dnmt3 enzymes methylate predominantly CpG sites and at a low frequency CpA sites with no apparent sequence preferences.

5-Methylcytosine↗

PubMed2001

Methylation in the p53 promoter is a supplementary route to breast carcinogenesis: correlation between CpG methylation in the p53 promoter and the mutation of the p53 gene in the progression from ductal carcinoma in situ to invasive ductal carcinoma.

Aberrant methylation in the CpG sites located in the promoter region of several tumor suppressor genes has been reported in various types of cancers. However, the methylation status of the p53 promoter has not been clearly determined and no information is available on its role in breast cancer. The aim of the study was to determine the presence and timing of the methylation of CpG sites in the p53 promoter, in the progression from ductal carcinoma in situ to invasive cancer. We also explored the correlation between the CpG methylation of the p53 promoter and p53 mutation during the progression of breast cancer. The corresponding lesions of both the invasive and noninvasive types were microdissected in paraffin-embedded tissue of 26 breast carcinomas. Bisulfite-modified DNA sequencing for methylation status in the p53 promoter was carried out, and double-strand DNA sequencing was performed in the promoter region and exons 4 to 9 of the p53 gene. CpG site methylation in the p53 promoter was detected in three cases (11.5%). Two noninvasive and three invasive lesions harbored CpG methylation in the p53 promoter. Methylations in more than one site were observed in three lesions, all of which contained methylation in two sites. The methylated CpG sites were located near the AP1 and YY-1 binding sites and at the YY-1 binding site. The p53 mutation was not found in the lesions where methylation in p53 promoter region was evident. In 16 cases (61.5%), neither methylation nor p53 mutation was detected. We conclude that the methylation in the p53 promoter region is found in the breast cancer irrespective of the status of invasion, and that the hypermethylation in the p53 promoter region is an alternative pathway to tumorigenesis where there is no p53 gene mutation.

Breast Neoplasms↗

PubMed2006

CpG island promoter methylation and silencing of 14-3-3sigma gene expression in LNCaP and Tramp-C1 prostate cancer cell lines is associated with methyl-CpG-binding protein MBD2.

14-3-3sigma proteins regulate numerous cellular processes that are important to cancer development. One of its biological roles involves G2 cell-cycle arrest following DNA damage. It has also been reported that the loss of 14-3-3sigma expression via CpG methylation may contribute to malignant transformation by impairing the G2 cell-cycle checkpoint function, thereby allowing an accumulation of genetic defects. However, how the CpG methylation-dependent silencing mechanism works in relation to promoter methylation associated with methyl-CpG-binding proteins (MeCPs) is still unclear. To better understand the mechanism, we first examined the methylation status of the 14-3-3sigma promoter-associated CpG islands and 14-3-3sigma gene expression in a subset of prostate cancer cell lines using methylation-specific PCR (MSP), an HhaI-based DNA methylation assay, and reverse transcription-PCR (RT-PCR). We found that the 14-3-3sigma expression is lost in LNCaP and Tramp-C1 prostate cancer cell lines and that this expression is restored after treatment with epigenetic silencing modifiers 5-aza-2'-deoxycytidine (5-aza) and trichostatin A (TSA). These results imply transcriptional silencing via promoter-associated CpG methylation. Chromatin immunoprecipitation analysis revealed that methyl-CpG-binding protein 2 (MBD2) is associated preferentially to the methylated CpG island in the 14-3-3sigma promoter in LNCaP and Tramp-C1 cells but not in 14-3-3sigma-expressing PC3 and DU145 cells, which contain an unmethylated CpG island in the 14-3-3sigma promoter region. The 14-3-3sigma gene silencing because of CpG methylation correlates with binding of MBD2. In addition, the activation of 14-3-3sigma gene expression by a combination of 5-aza and TSA also involves the release of the MBD2 from the 14-3-3sigma promoter-methylated CpG island in LNCaP and Tramp-C1 cells. Furthermore, MBD2 knockdown by siRNA stimulated 14-3-3sigma expression in LNCaP cells. We also investigated whether the loss of 14-3-3sigma expression in LNCaP and Tramp-C1 cells affects cell proliferation by MTT assays. Interestingly, we observed that 14-3-3sigma-inactivated LNCaP and Tramp-C1 cells had markedly decreased cell proliferation and protein expression of proliferation cell nuclear antigen (PCNA) after restoration of 14-3-3sigma expression with 5-aza and TSA treatment. On the other hand, the same treatment did not significantly affect 14-3-3sigma-active PC3 and DU145 cells, which normally express 14-3-3sigma. Finally, 14-3-3sigma knockdown by siRNA resulted in increased proliferation in PC3 and DU145 cells. These findings suggest that the transcriptional silencing of the 14-3-3sigma gene is caused by promoter CpG island methylation associated with MBD2, and that this may play an important role in prostate cancer progression during the invasive and metastatic stages of the disease.

14-3-3 Proteins↗