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Results for “DNA Methylation Detection”

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PubMed2005

Methylated-CpG island recovery assay: a new technique for the rapid detection of methylated-CpG islands in cancer.

Hypermethylation of CpG islands is a phenomenon commonly observed during the development and progression of human tumors. Detection of methylated-CpG islands in easily accessible biological materials such as serum has the potential to be useful for the early diagnosis of cancer. Most currently used methods for detecting methylated-CpG islands are based on sodium bisulfite conversion of genomic DNA, followed by PCR reactions. Here we describe a method, methylated-CpG island recovery assay (MIRA) that does not depend on the use of sodium bisulfite but has similar sensitivity and specificity as bisulfite-based approaches. Methyl-CpG-binding domain proteins, such as methyl-CpG-binding domain protein-2 (MBD2), have the capacity to bind specifically to methylated DNA sequences. In the MIRA procedure, sonicated genomic DNA isolated from cells or tissue is incubated with a matrix containing glutathione-S-transferase-MBD2b in the presence of methyl-CpG-binding domain protein 3-like-1, a binding partner of MBD2 that increases the affinity of MBD2 for methylated DNA. Specifically bound DNA is eluted from the matrix and gene-specific PCR reactions are performed to detect CpG island methylation. Methylation can be detected using 1 ng of DNA or 3000 cells. MIRA is a specific and sensitive, but not laborious, technique that can be clinically useful in the detection and diagnosis of any DNA methylation-associated disease, including cancer.

Base Sequence↗
PubMed2000

DNA methylation and gastrointestinal malignancies: functional consequences and clinical implications.

Evidence now suggests that the transcriptional silencing of selected genes by DNA methylation plays a crucial role in the development and progression of human gastrointestinal malignancies. To date, genes involved in the regulation of the cell cycle, DNA repair, angiogenesis, and apoptosis have all been shown to be inactivated by the hypermethylation of their respective 5' CpG islands. The specific causes and effects of the changes in DNA methylation that occur in cancer remain unknown, however. Nevertheless, recently developed techniques for detecting changes in DNA methylation have dramatically increased the amount of information available on the patterns of methylation that occur as cancers progress. One key process involved in aberrant methylation is related to aging, and because it affects a large number of CpG islands, age-related methylation may be a primary cause of the increased incidence of cancer seen among older individuals. Other patterns of methylation are cancer-specific and are detected only in a subset of tumors exhibiting the CpG island methylator phenotype (CIMP). In this regard, the majority of sporadic colorectal cancers, exhibiting microsatellite instability (MSI) appear to be associated with CIMP, which leads to aberrant methylation of human MutL homologue (hMLH1) and the loss of its expression. We anticipate that as the various components of the molecular machinery involved in aberrant DNA methylation become better understood, they will prove to be useful targets, serving as the basis for the development of new methods for the diagnosis and treatment of gastrointestinal malignancies.

DNA↗
PubMed2002

Down-regulation of von Hippel-Lindau protein in N-nitroso compound-induced rat non-clear cell renal tumors.

Non-clear cell rat kidney tumors, inducible by N-nitroso compounds but lacking mutations in the von Hippel--Lindau (VHL) coding sequence, were examined for other VHL alterations. Neither mutations nor DNA methylation was detected in a putative promoter region. By immunohistochemistry, however, VHL protein level was evidently reduced in six of the eight eosinophilic renal epithelial tumors and in all the ten nephroblastomas. Immunoblotting of normal kidney detected two VHL proteins of 20 and 22kDa in a 16-day-old fetal rat but only 20kDa protein in an adult rat. This is the first demonstration of VHL alteration at the protein level.

Adenocarcinoma↗
PubMed1991

5-Azacytidine inhibits the induction of transient TK-deficient cells by 5-bromodeoxyuridine. A novel hypothesis for the facilitation of hypermethylation by 5-bromodeoxyuridine.

This paper examines the mechanism by which 5-bromodeoxyuridine (BrdUrd) induces a high frequency of transient trifluorothymidine (F3TdR)-resistant variants in the TK6 human lymphoblast cell line (a TK +/- heterozygote). This phenomenon has previously been termed 'pseudomutation' (Liber et al., 1985). We now report that 5-azacytidine (5-AzaC), an inhibitor of DNA methylation, reverses BrdUrd-induced pseudomutation in a dose-dependent manner. The inhibition by 5-AzaC is highly specific and does not appear to involve nucleotide pool perturbations. 5-AzaC inhibits the pseudomutagenic effect (transient trifluorothymidine resistance in a thymidine kinase heterozygote), but not the stable mutagenic effect (stable 6-thioguanine resistance or trifluorothymidine resistance in a hypoxanthine-guanine phosphoribosyltransferase-proficient cell) induced by BrdUrd. 5-AzaC did not affect the induction nor expression of mutation induced by several other chemical mutagens at either the tk or hgprt loci. Inhibition of pseudomutation by 5-AzaC did not appear to be caused by a number of potential confounding factors. Although significant changes in the levels of DNA methylation were detected by HPLC analysis in BrdUrd-treated cells, the dose response for inhibition of pseudomutation by 5-AzaC was correlated with a significant decrease in 5-methylcytidine levels. These results and additional data in the literature have led us to postulate a novel mechanism in which the substitution of BrdUrd in a TpG dinucleotide(s) may serve as a substrate for non-heritable methylation and hence transiently inactivate tk gene expression.

Azacitidine↗
PubMed2004

Demethylating agent, 5-azacytidine, reverses differentiation of embryonic stem cells.

The de novo methylation activity is essential for embryonic development as well as embryonic stem (ES) cell differentiation, where the intensive and extensive DNA methylation was detected. In this study, we investigated the effects of a demethylating agent, 5-azacytidine (5-AzaC), on differentiated ES cells in order to study the possibility of reversing the differentiation process. We first induced differentiation of ES cells by forming embryoid bodies, and then the cells were treated with 5-AzaC. The cells showed some undifferentiated features such as stem cell-like morphology with unclear cell-to-cell boundary and proliferative responsiveness to LIF. Moreover, 5-AzaC increased the expressions of ES specific markers, SSEA-1, and alkaline phosphatase activity as well as ES specific genes, Oct4, Nanog, and Sox2. We also found that 5-AzaC demethylated the promoter region of H19 gene, a typical methylated gene during embryonic differentiation. These results indicate that 5-AzaC reverses differentiation state of ES cells through its DNA demethylating activity to differentiation related genes.

Alkaline Phosphatase↗
PubMed2005

Detection in fecal DNA of colon cancer-specific methylation of the nonexpressed vimentin gene.

BACKGROUND: Increased DNA methylation is an epigenetic alteration that is common in human cancers and is often associated with transcriptional silencing. Aberrantly methylated DNA has also been proposed as a potential tumor marker. However, genes such as vimentin, which are transcriptionally silent in normal epithelium, have not until now been considered as targets for cancer-associated aberrant methylation and for use as cancer markers. METHODS: We applied methylation-specific polymerase chain reaction to the vimentin gene, which is transcriptionally silent in normal colonocytes, and compared methylation of vimentin exon 1 in cancer tissues and in fecal DNA from colon cancer patients versus control samples from healthy subjects. RESULTS: Vimentin exon-1 sequences were unmethylated in 45 of 46 normal colon tissues. In contrast, vimentin exon-1 sequences were methylated in 83% (38 of 46) and 53% (57 of 107) of tumors from two independently collected groups of colon cancer patients. When evaluated as a marker for colon cancer detection in fecal DNA from another set of colon cancer patients, aberrant vimentin methylation was detected in fecal DNA from 43 of 94 patients, for a sensitivity of 46% (95% confidence interval [CI] = 35% to 56%). The sensitivity for detecting stage I and II cancers was 43% (26 of 60 case patients) (95% CI = 31% to 57%). Only 10% (20 of 198 case patients) of control fecal DNA samples from cancer-free individuals tested positive for vimentin methylation, for a specificity of 90% (95% CI = 85% to 94%). CONCLUSIONS: Aberrant methylation of exon-1 sequences within the nontranscribed vimentin gene is a novel molecular biomarker of colon cancer and can be successfully detected in fecal DNA to identify nearly half of individuals with colon cancer.

Adenocarcinoma↗
PubMed2002

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

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

Amino Acid Sequence↗
PubMed1983

Methylation of the T-DNA in Agrobacterium tumefaciens and in several crown gall tumors.

Methylation of the T-DNA in Agrobacterium tumefaciens and in four octopine-type (A6S/2, E9, 15955/1, 15955/01) and one nopaline-type (HT37#15) crown gall tumors was investigated using the isoschizomeric restriction endonucleases Msp I and Hpa II. T-DNA in the octopine-type Ti-plasmid pTiB6(806) was not methylated at the sequence 5'CCGG3' in Agrobacterium. With two possible exceptions, neither was the T-DNA of the nopaline-type Ti-plasmid pTiT37 methylated in the bacterium. In all tumor lines investigated, at least one copy of the T-DNA was not methylated. DNA methylation was not detected in the lines A6S/2, 15955/1, HT37#15, and the TL region of E9. DNA methylation of some copies of TR in the E9 tumor line, and possibly in the 15955/01 line, was detected. The methylation of some copies of TR in the E9 line may indicate that not all copies of TR are transcribed in this tumor.

Cell Line↗
PubMed2005

Active RNA silencing at low temperature indicates distinct pathways for antisense-mediated gene-silencing in potato.

Previously, it was shown that low temperature (<or= 15 degrees C) inhibits RNA silencing-mediated defence by the control of siRNA generation. In contrast, we have found nine antisense potato lines out of 24 in which RNA silencing was not inhibited at low temperature. In these lines, the extent of endogenous repression varied in leaves and was found to be different in roots and tubers. In order to address the contribution of gene dosage and repetitive structure of the transgene loci to the temperature dependence/independence of antisense-mediated gene-silencing DNA gel blot analysis was performed. Interestingly, none of the studied features correlated with the observed silencing effect. In addition, the insertion of vector backbone sequences into the potato genome did not influence the temperature dependence. RNA-directed DNA methylation was detected in the majority of antisense lines, however, it was also independent of the type of RNA silencing. Thus, it is feasible that chromosomal flanking sequences or the chromatin structure surrounding the insertion determine which silencing pathway is activated.

Cold Temperature↗
PubMed2000

PCR amplification in bisulfite methylcytosine mapping in the GC-rich promoter region of amyloid precursor protein gene in autopsy human brain.

Alterations in DNA 5-methyldeoxycytidine pattern influence gene expression for certain mammalian genes in development, differentiation, carcinogenesis, and aging. Detection of DNA methylation at the promoter region, which generally represses transcription activity, is one important element in studying changes in molecular expression with aging and age-associated disorders. Bisulfite genomic sequencing is a useful method for mapping methylated cytosines. However, PCR amplification for bisulfite-treated DNA does not yield a sufficient amount of products that have a sufficient level of specificity, especially in the GC-rich sequences usually seen at the promoter regions of house keeping genes. We present a method for increasing the sensitivity and specificity of PCR amplification in bisulfite methylcytosine mapping in an extremely GC-rich promoter region of amyloid precursor protein (APP) gene from the cerebral cortex of human autopsy brain. The PCR used consists of two cycles using the lower primer alone to amplify the sense sequence, and then eight cycles at a theoretical annealing temperature (60 degrees C) and 30 cycles at a lower annealing temperature (50 degrees C) using both the upper and lower primers. The present method likely can also be applied to other GC-rich genomic sequences.

Aged↗
PubMed1989

Genomic footprinting with Taq polymerase.

The linear amplification of chemically sequenced DNA with DNA polymerase from Thermus aquaticus permits the direct detection of DNA methylation and protein-DNA interaction sites in vivo.

DNA-Directed DNA Polymerase↗
PubMed2026

Cell-free DNA methylation biomarkers for the early detection and tumor burden monitoring of gastric cancer.

Development of sensitive biomarkers is required to achieve early detection and tumor burden monitoring in gastric cancer (GC). We performed genome-wide methylation sequencing on 78 tissue and 241 plasma samples from 171 GC patients and 114 healthy controls from two independent clinical centers. Differentially methylated regions (DMRs) were screened using paired GC and normal tissues, and refined through cfDNA profiles with LASSO regression to construct a cfDNA-based biomarker, the GCML-score. The GCML-score, consisting of 13 DMRs, demonstrated excellent diagnostic performance (AUC: 0.95/0.99/0.95 overall and 0.96/0.99/0.82 in early GC for training/internal validation/external validation cohorts). In 12 patients receiving neoadjuvant chemotherapy, dynamic changes in GCML-score were consistent with radiological tumor burden, highlighting its monitoring potential. The GCML-score, derived from genome-wide cfDNA methylation profiling, provides a robust tool for early GC detection and real-time tumor burden monitoring, facilitating improved prognosis and personalized therapeutic strategies.

Journal Article↗
PubMed1991

Transformation of a partial nopaline synthase gene into tobacco suppresses the expression of a resident wild-type gene.

A portion of the nopaline synthase gene under the control of the cauliflower mosaic virus 35S promoter was used to transform a tobacco plant that had previously been transformed with a wild-type nopaline synthase (nos) gene. Unexpectedly, in all nine primary transformants tested the wild-type nos expression was virtually completely suppressed. In contrast, plants transformed with the control vector DNA, which differed only in the absence of the partial nos gene, did not show any inhibition of nos expression. Progeny plants were analyzed for the stability of the gene-silencing phenotype. All of the progeny that carried both the wild-type and partial nos genes had no detectable nopaline synthase activity. In addition, wild-type nos mRNA could not be detected in these plants. In most plants in which the wild-type gene was segregated away from the partial nos gene, wild-type levels of activity were detected. Although DNA methylation has been shown to be correlated with a decrease in promoter activity in plants, none of the progeny appeared to carry a methylated nos promoter. The underlying mechanism causing this gene suppression phenomenon is unclear at this time.

Journal Article↗
PubMed2026

MethylModes: computationally efficient detection of multimodal distributions in DNA methylation data.

SUMMARY: MethylModes is an R package and Shiny application to identify multimodal distributions in human DNA methylation at individual CpG sites. Multimodal distributions, which can be the result of nearby genetic variation, environmental exposures, or assay artifacts, are susceptible to confounding and important to identify for methylation analysis. MethylModes is easily incorporated into existing quality control pipelines of array-based DNA methylation data. The underlying algorithm uses kernel smoothing of probe-level data to locate the number and location of peaks. The algorithm can be parallelized across probes for efficient implementation at genome-scale. We provide a case study implementation of MethylModes in the Health and Retirement Study as well as the Airwave Health Monitoring Study. AVAILABILITY AND IMPLEMENTATION: MethylModes is available on GitHub at https://github.com/lutiffan/methylModes as an R package wrapping an R Shiny application. We include a toy dataset to validate installation. The codebase is also published on Zenodo at https://doi.org/10.5281/zenodo.17448517.

DNA Methylation↗
PubMed2006

Transcriptional silencing of multiple genes in trophozoites of Entamoeba histolytica.

In a previous work we described the transcriptional silencing of the amoebapore A (AP-A) gene (Ehap-a) of Entamoeba histolytica strain HM-1:IMSS. The silencing occurred following transfection with a plasmid containing a 5' upstream region (473 bp) of Ehap-a that included a truncated segment (140 bp) of a short interspersed nuclear element (SINE1). Silencing remained in effect even after removal of the plasmid (clone G3). Neither short interfering RNA nor methylated DNA were detected, but the chromatin domain of Ehap-a in the gene-silenced trophozoites was modified. Two other similar genes (Ehap-b and one encoding a Saposin-like protein, SAPLIP 1) also became silenced. In the present work we demonstrate the silencing of a second gene of choice, one that encodes the light subunit of the Gal/GalNAc inhibitable lectin (Ehlgl1) and the other, the cysteine proteinase 5 (EhCP-5). This silencing occurred in G3 trophozoites transfected with a plasmid in which the 473 bp 5' upstream Ehap-a fragment was directly ligated to the second gene. Transcriptional silencing occurred in both the transgene and the chromosomal gene. SINE1 sequences were essential, as was a direct connection between the Ehap-a upstream region and the beginning of the open reading frame of the second gene. Gene silencing did not occur in strain HM-1:IMSS with any of these plasmid constructs. The trophozoites with two silenced genes were virulence-attenuated as were those of clone G3. In addition, trophozoites not expressing Lgl1 and AP-A proteins had a significantly reduced ability to cap the Gal/GalNAc-lectin to the uroid region when incubated with antibodies against the heavy (170 kDa) subunit of the lectin. Lysates of trophozoites lacking cysteine proteinase 5 and AP-A proteins had 30% less cysteine proteinase activity than those of HM-1:IMSS strain or the G3 clone. Silencing of other genes in G3 amoebae could provide a model to study their various functions. In addition, double gene-silenced, virulence-attenuated trophozoites may be an important tool in vaccine development.

Animals↗
PubMed2004

Methylation assay by nucleotide incorporation: a quantitative assay for regional CpG methylation density.

Although the aberrant methylation in CpG islands is of great interest as a causative role in human malignancies, it has been very difficult to accurately determine methylation density. Here we report a novel microplate-based quantitative methylation assay, designated MANIC, for a region containing a number of CpG sites based on incorporation of hapten-labeled dCTP at cytosine sites where the methylated cytosines have not been converted to uracil by the bisulfite treatment. Validation using control DNAs revealed that the method was sensitive enough to detect < 1.25% methylated DNA and that calibration curve was linear. With this approach, we determined relative methylation density of O6-methylguanine-DNA methyltransferase gene promoter containing 12 CpG sites among the 12 colorectal cancers and corresponding normal mucosal tissues. Consequently, MANIC showed a high concordance with results by a quantitative method, bisulfite PCR single-stranded conformational polymorphism (BiPS). MANIC is a technique that avoids cumbersome procedures such as electrophoresis or the use of radiolabeling and is applicable to any sequence regardless of the total number of CpG sites or heterogeneity in methylation status.

Chromosome Mapping↗