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PubMed2003

Transcriptional regulation of mouse delta-opioid receptor gene by CpG methylation: involvement of Sp3 and a methyl-CpG-binding protein, MBD2, in transcriptional repression of mouse delta-opioid receptor gene in Neuro2A cells.

Opioid receptors are expressed in a cell type-specific manner. Here we show that the mouse delta-opioid receptor (mDOR) gene is regulated by promoter region CpG methylation. The mDOR promoter containing a putative CpG island is highly methylated in Neuro2A cells, correlating with the repression of this gene in these cells. This is in contrast with the unmethylated state of the mDOR promoter in NS20Y cells, which express a high level of mDOR. Repression of mDOR transcription in Neuro2A cells could be partially relieved by chemically induced demethylation with 5-aza-2'-deoxycytidine. In addition, in vitro methylation of the luciferase reporter gene driven by the mDOR promoter resulted in an inhibition of transcription in NS20Y cells. Methyl-CpG-binding protein complex 1 (MeCP1) has been implicated in methylation-mediated transcriptional repression of several genes. Electrophoretic mobility shift assays showed that fully methylated, but not unmethylated, mDOR promoter fragment formed a MeCP1-like protein complex that contained methyl-CpG-binding domain protein 2 (MBD2) and Sp3. Furthermore, the expression level of Sp3 was decreased when Neuro2A cells were demethylated with 5-aza-2'-deoxycytidine, and increasing Sp3 levels in Schneider's Drosophila line 2 cells led to the repression of mDOR promoter activity when the promoter was methylated. These results demonstrate that Sp3 and MBD2 are involved in the transcriptional repression of mDOR in Neuro2A cells through binding to the methylated CpG sites in the promoter region and may play a role in the cell type-specific expression of mDOR.

Animals↗
PubMed2003

Loss of CpG methylation is strongly correlated with loss of histone H3 lysine 9 methylation at DMR-LIT1 in patients with Beckwith-Wiedemann syndrome.

To clarify the chromatin-based imprinting mechanism of the p57(KIP2)/LIT1 subdomain at chromosome 11p15.5 and the mouse ortholog at chromosome 7F5, we investigated the histone-modification status at a differentially CpG methylated region of Lit1/LIT1 (DMR-Lit1/LIT1), which is an imprinting control region for the subdomain and is demethylated in half of patients with Beckwith-Wiedemann syndrome (BWS). Chromatin-immunoprecipitation assays revealed that, in both species, DMR-Lit1/LIT1 with the CpG-methylated, maternally derived inactive allele showed histone H3 Lys9 methylation, whereas the CpG-unmethylated, paternally active allele was acetylated on histone H3/H4 and methylated on H3 Lys4. We have also investigated the relationship between CpG methylation and histone H3 Lys9 methylation at DMR-LIT1 in patients with BWS. In a normal individual and in patients with BWS with normal DMR-LIT1 methylation, histone H3 Lys9 methylation was detected on the maternal allele; however, it disappeared completely in the patients with the DMR-LIT1 imprinting defect. These findings suggest that the histone-modification status at DMR-Lit1/LIT1 plays an important role in imprinting control within the subdomain and that loss of histone H3 Lys9 methylation, together with CpG demethylation on the maternal allele, may lead to the BWS phenotype.

Base Sequence↗
PubMed1993

DNA methylation of the pepsinogen 1 gene during rat glandular stomach carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine or catechol.

The methylation patterns of the rat pepsinogen 1 (Pg1) gene in preneoplastic and neoplastic stomach lesions induced by genotoxic N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or the non-genotoxic carcinogen catechol were investigated. Male WKY/Ncrj rats were given MNNG in their drinking water (50 mg/l) for 30 weeks or 0.8% catechol throughout the experiment (60 weeks). MNNG induced Pg1 altered pyloric glands (PAPG), adenomatous hyperplasias and well-differentiated adenocarcinomas. Catechol also induced PAPG and adenomatous hyperplasias although cancers did not develop. Adenomatous hyperplasias and adenocarcinomas all consisted of gastric type cells resembling surface mucous cells or pyloric gland cells with little or no Pg1 expression. In MNNG-induced stomach cancers generally lacking Pg1, altered Pg1 gene methylation was observed with both CCGG and GCGC sites being methylated more than normal pyloric mucosa. MNNG or catechol-induced adenomatous hyperplasias also demonstrated essentially the same methylation changes in the CCGG, but not in the GCGC sites. In the mucosa containing PAPG in groups treated with MNNG or catechol the methylation patterns of the Pg1 gene were quite similar to those of normal pyloric mucosa, although the CCGG sites tended to demonstrate slightly increased methylation. The results suggest that the altered methylation of the Pg1 gene observed in stomach cancers is acquired early in the carcinogenic process and progressive methylation changes occur with tumor development.

Adenocarcinoma↗
PubMed2004

High throughput screening of methylation status of genes in prostate cancer using an oligonucleotide methylation array.

Recent work using high-throughput microarray technology has discovered altered expression of a large number of genes in prostate cancer. Many of these alterations may be the consequence of changes in methylation status in the CpG islands of promoter or exon 1 regions of these genes. In order to determine the methylation status of a large number of genes and ESTs we combined the principle of match/mismatch hybridization with the technique of whole genome labeling to develop a highly specific oligonucleotide-based methylation microarray. Using this array, we analyzed the methylation status of 105 genes and ESTs in three prostate cancer cell lines. Between 32 and 47% of these genes and ESTs were methylated in these cell lines. By correlating the methylation status of this array with the results of Affymetrix expression arrays of three prostate cancer cell lines, we determined that methylation of genes played a significant role (37%) in down-regulating the expression of certain genes in prostate cancer. We also tested this array on a number of primary prostate tissue samples. Our results indicated that a subset of genes in this microarray (25/105) were methylated in all prostate cancer samples but not in normal prostate, suggesting the potential significance of alterations in the methylation status of certain genes in the development of prostate cancer.

Acetylation↗
PubMed2002

Maternal methyl supplements in mice affect epigenetic variation and DNA methylation of offspring.

This study was designed to determine if maternal dietary methyl supplements increase DNA methylation and methylation-dependent epigenetic phenotypes in mammalian offspring. Female mice of two strains were fed two levels of dietary methyl supplement or control diet prior to and during pregnancy. Offspring of these mice vary in phenotype, which is epigenetically determined and affects health and 2-y survival. Phenotype and DNA methylation of a long terminal repeat (LTR) controlling expression of the agouti gene were assayed in the resulting offspring. Methyl supplements increase the level of DNA methylation in the agouti LTR and change the phenotype of offspring in the healthy, longer-lived direction. This shows that methyl supplements have strong effects on DNA methylation and phenotype and are likely to affect long-term health. Optimum dietary supplements for the health and longevity of offspring should be intensively investigated. This should lead to public policy guidance that teaches optimal, rather than minimal, dose levels of maternal supplements.

Animals↗
PubMed1974

Enzymatic methylations: III. Cadaverine-induced conformational changes of E. coli tRNA fMet as evidenced by the availability of a specific adenosine and a specific cytidine residue for methylation.

A partially purified tRNA methylase fraction from rat liver, containing m(2)G- m(1)A- and m(5)C-methylase, was used to study the influence of Mg(++) and of the biogenic polyamine cadaverine on the enzymatic methylation of E.coli tRNA(fMet)in vitro. In presence of 1 or 10 mM Mg(++), guanosine no. 27 was methylated to m(2)G. In 1 mM Mg(++) plus 30 mM cadaverine, guanosine in position 27 and adenosine in position 59 were methylated. In presence of 30 mM cadaverine alone tRNA(fMet) accepted three methyl groups: in addition to guanosine no. 27 and adenosine no. 59 cytidine no. 49 was methylated. In order to correlate tRNA(fMet) tertiary structure changes with the methylation patterns, differentiated melting curves of tRNA(fMet) were measured under the methylation conditions. It was shown that the thermodynamic stability of tRNA(fMet) tertiary structure is different in presence of Mg(++), or Mg(++) plus cadaverine, or cadaverine alone. From the differentiated melting curves and from the methylation experiments one can conclude that at 37 degrees in the presence of Mg(++) tRNA(fMet) has a compact structure with the extra loop and the TpsiC-loop protected by tertiary structure interactions. In Mg(++) plus cadaverine, the TpsiC-loop is available, while the extra loop is yet engaged in teritary structure (G-15: C-49) interactions. In cadaverine alone, the TpsiC-loop and the extra loop are free; hence under these conditions the open tRNA(fMet) clover leaf may be the substrate for methylation. In general, cadaverine destabilizes tRNA tertiary structure in the presence of Mg(++), and stabilizes tRNA(fMet) tertiary structure in the absence of Mg(++). This may be explained by a competition of cadaverine with Mg(++) for specific binding sites on the tRNA. On the basis of these experiments a possible role of biogenic polyamines in vivo may be discussed: as essential components of procaryotic and eucaryotic ribosomes they may together with ribosomal factors facilitate tRNA-ribosome binding during protein biosynthesis by opening the tRNA tertiary structure, thus making the tRNA's TpsiC-loop available for interaction with the complementary sequence of the ribosomal 5S RNA.

Adenosine↗
PubMed1982

Studies on the biological role of DNA methylation: V. The pattern of E.coli DNA methylation.

The distribution of the methylatable sites GATC and CCATGG was studied by analyzing the molecular average size of restriction fragments of E. coli DNA. Both sites were found to be randomly distributed, reflecting a random pattern of methylation. The methylation pattern of specific sequences such as the origin of replication and rRNA genes has been studied in wild type E. coli and a methylation deficient (dam- dcm-) mutant. These sequences were found to be methylated in wild type cells and unmethylated in the mutant indicating that there is no effect of the state of methylation of these sequences on their expression. Analysis of the state of methylation of GATC sites in newly replicating DNA using the restriction enzyme Dpn I (cleaves only when both strands are methylated) revealed no detectable hemimethylated DNA suggesting that methylation occurs at the replication fork. Taking together the results presented here and previously published data (5), we arrive at the conclusion that the most likely function of E. coli DNA methylations is probably in preventing nuclease activity.

Base Composition↗
PubMed2006

Rapid and sensitive detection of CpG-methylation using methyl-binding (MB)-PCR.

Methylation of CpG islands is associated with transcriptional repression and, in cancer, leads to the abnormal silencing of tumor suppressor genes. We have developed a novel technique for detecting CpG-methylated DNA termed methyl-binding (MB)-PCR. This technique utilizes a recombinant protein with high affinity for CpG-methylated DNA that is coated onto the walls of a PCR vessel and selectively captures methylated DNA fragments from a mixture of genomic DNA. The retention and, hence, the degree of methylation of a specific DNA fragment (e.g. a CpG island promoter of a specific gene) is detected in the same tube by gene-specific PCR. MB-PCR does not require bisulfite treatment or methylation-sensitive restriction and provides a quick, simple and extremely sensitive technique allowing the detection of methylated DNA, in particular in tumor tissue or tumor cells from limited samples. Using this novel approach, we determined the methylation status of several established and candidate tumor suppressor genes and identified the ICSBP gene, encoding the myeloid and B-cell-specific transcription factor interferon consensus sequence-binding protein, as a target for aberrant hypermethylation in acute myeloid leukemia.

Acute Disease↗
PubMed2002

Enzymatic regional methylation assay: a novel method to quantify regional CpG methylation density.

We have developed a novel quantitative method for rapidly assessing the CpG methylation density of a DNA region in mammalian cells. After bisulfite modification of genomic DNA, the region of interest is PCR amplified with primers containing two dam sites (GATC). The purified PCR products are then incubated with 14C-labeled S-adenosyl-L-methionine (SAM) and dam methyltransferase as an internal control to standardize DNA quantity. This is followed by an incubation with 3H-labeled SAM and SssI methyltransferase for methylation quantification. By use of standard mixtures of cell line DNA with a defined methylation status in every assay, the ratio (3H/14C signal) of each sample can be converted into percentage values to assess the methylation density of the amplified sequence. This methylation-sensitive technique, termed ERMA (Enzymatic Regional Methylation Assay) provides several advantages over existing methods used for methylation analysis as it determines an exact measurement of the methylation density of the region studied. We demonstrate a use of this technique in determining the methylation density of the promoter region of the tumor suppressor gene p15INK4B and changes that occur after treatment with demethylating agents.

Cell Cycle Proteins↗
PubMed1987

Enzymatic protein carboxyl methylation in rat posterior pituitary: neurophysins in rapid-turnover pool determine methyl accepting capacity.

In vitro stimulation of intact rat posterior pituitary by either veratridine or K+ depolarization results in the concomitant release of neurophysins and in a decrease (70-80%) in their carboxyl methylation as measured either with L-[methyl-3H]methionine in the intact lobes after stimulation or in their homogenates with [methyl-3H]S-adenosyl-L-methionine and purified protein carboxyl methyltransferase. A similar reduction in neurophysin methylation (60%) was observed when the arrival of newly synthesized neurophysins at the posterior pituitary was blocked by colchicine. Experimental data indicate that the reduction in neurophysin content of the lobes after 12 h of colchicine treatment (less than 7%) or after in vitro stimulation (about 10%) cannot account for the marked reduction in neurophysin methylation. The results suggest that the granule pool characterized by rapid turnover of neurophysins probably represents the major source of methyl acceptor proteins in the lobe. In spite of the marked reduction in neurophysin methyl accepting capacity observed after stimulation, there was no parallel increase in methyl accepting capacity of the released neurophysins. We propose that a neurophysin subfraction that might be associated with the membrane of releasable granules participates in the methylation reaction in situ.

Animals↗
PubMed1987

Fixation of the unmethylated or the 5'-CCGG-3' methylated adenovirus late E2A promoter-cat gene construct in the genome of hamster cells: gene expression and stability of methylation patterns.

The late E2A promoter of adenovirus type 2 (Ad2) DNA can be inactivated by in vitro methylation of three 5'-CCGG-3' sequences at positions +23, +5, and -215 relative to the cap site in this promoter. This inactivation has been documented in transient expression experiments both in Xenopus laevis oocytes and in mammalian cells (K.-D. Langner, L. Vardimon, D. Renz, and W. Doerfler, Proc. Natl. Acad. Sci. USA 81:2950-2954, 1984; K.-D. Langner, U. Weyer, and W. Doerfler, Proc. Natl. Acad. Sci. USA 83:1598-1602, 1986). In the present study, in vitro-methylated or unmethylated promoter-gene assemblies were permanently fixed by integration in the hamster genome. In individually established cell lines, the degree of promoter methylation was correlated to gene activity. The pAd2E2AL-CAT construct, in which the late E2A promoter controls expression of the procaryotic chloramphenicol acetyltransferase (cat) gene, was fixed in BHK21 hamster cells by cotransfection with and selection for the pSV2-neo construct (P. J. Southern and P. Berg, J. Mol. Appl. Genet. 1:327-341, 1982) in which the early simian virus 40 promoter controls the gene for neomycin phosphotransferase. The pAd2E2AL-CAT construct was transfected in the unmethylated or in the 5'-CCGG-3' methylated form. The pSV2-neo plasmid was cotransfected in the unmethylated form. The stability of in vitro-imposed methylation patterns and cat gene expression were followed and correlated in a number of established cell lines which contained the constructs integrated in a non-rearranged configuration. The foreign DNA did not persist in the episomal state but was integrated, frequently in multiple tandems of the plasmid DNA. Among 19 cell lines established after transfecting the unmethylated pAd2E2AL-CAT construct, the late E2A promoter remained unmethylated (examined in 10 cell lines), and the cat gene was expressed in 18 cell lines. On the other hand, among 14 cell lines which were generated by transfection with the methylated construct, 7 cell lines did not express the cat gene, and the three 5'-CCGG-3' sequences in the late E2A promoter remained almost completely methylated. In five cell lines, the E2A promoter sequences were partly demethylated and the cat gene was expressed at low levels. Last, in two cell lines, demethylations were found to be extensive and strong cat expression was observed. It remained a question of considerable interest what factors determined the stability of methylation patterns that had been preimposed by in vitro methylation on specific sequences in a promoter, after this promoter was fixed by integration in the mammalian genome.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetyltransferases↗
PubMed2006

Control of the DNA methylation system component MBD2 by protein arginine methylation.

DNA methylation is vital for proper chromatin structure and function in mammalian cells. Genetic removal of the enzymes that catalyze DNA methylation results in defective imprinting, transposon silencing, X chromosome dosage compensation, and genome stability. This epigenetic modification is interpreted by methyl-DNA binding domain (MBD) proteins. MBD proteins respond to methylated DNA by recruiting histone deacetylases (HDAC) and other transcription repression factors to the chromatin. The MBD2 protein is dispensable for animal viability, but it is implicated in the genesis of colon tumors. Here we report that the MBD2 protein is controlled by arginine methylation. We identify the protein arginine methyltransferase enzymes that catalyze this modification and show that arginine methylation inhibits the function of MBD2. Arginine methylation of MBD2 reduces MBD2-methyl-DNA complex formation, reduces MBD2-HDAC repression complex formation, and impairs the transcription repression function of MBD2 in cells. Our report provides a molecular description of a potential regulatory mechanism for an MBD protein family member. It is the first to demonstrate that protein arginine methyltransferases participate in the DNA methylation system of chromatin control.

Amino Acid Sequence↗
PubMed1999

Methylation-mediated transcriptional silencing in euchromatin by methyl-CpG binding protein MBD1 isoforms.

DNA methylation of promoter-associated CpG islands is involved in the transcriptional repression of vertebrate genes. To investigate the mechanisms underlying gene inactivation by DNA methylation, we characterized a human MBD1 protein, one of the components of MeCP1, which possesses a methyl-CpG binding domain (MBD) and cysteine-rich (CXXC) domains. Four novel MBD1 isoforms (MBD1v1, MBD1v2, MBD1v3, and MBD1v4) were identified by the reverse transcription-PCR method. We found that these transcripts were alternatively spliced in the region of CXXC domains and the C terminus. Green fluorescent protein-fused MBD1 was localized to multiple foci on the human genome, mostly in the euchromatin regions, and particularly concentrated in the pericentromeric region of chromosome 1. Both the MBD sequence and genome methylation were required for proper localization of the MBD1 protein. We further investigated whether MBD1 isoforms are responsible for transcriptional repression of human genes. A bacterially expressed MBD1 protein bound preferentially to methylated DNA fragments containing CpG islands from the tumor suppressor genes p16, VHL, and E-cadherin and from an imprinted SNRPN gene. All MBD1 isoforms inhibited promoter activities of these genes via methylation. Interestingly, MBD1 isoforms v1 and v2 containing three CXXC domains also suppressed unmethylated promoter activities in mammalian cells. These effects were further manifested in Drosophila melanogaster cells, which lack genome methylation. Sp1-activated transcription of methylated p16 and SNRPN promoters was inhibited by all of the MBD1 isoforms, whereas the isoforms v1 and v2 reduced Sp1-activated transcription from unmethylated promoters as well. These findings suggested that the MBD1 isoforms have different roles in methylation-mediated transcriptional silencing in euchromatin.

Alternative Splicing↗
PubMed2000

Genomic targeting of methylated DNA: influence of methylation on transcription, replication, chromatin structure, and histone acetylation.

We have developed a strategy to introduce in vitro-methylated DNA into defined chromosomal locations. Using this system, we examined the effects of methylation on transcription, chromatin structure, histone acetylation, and replication timing by targeting methylated and unmethylated constructs to marked genomic sites. At two sites, which support stable expression from an unmethylated enhancer-reporter construct, introduction of an in vitro-methylated but otherwise identical construct results in specific changes in transgene conformation and activity, including loss of the promoter DNase I-hypersensitive site, localized hypoacetylation of histones H3 and H4 within the reporter gene, and a block to transcriptional initiation. Insertion of methylated constructs does not alter the early replication timing of the loci and does not result in de novo methylation of flanking genomic sequences. Methylation at the promoter and gene is stable over time, as is the repression of transcription. Surprisingly, sequences within the enhancer are demethylated, the hypersensitive site forms, and the enhancer is hyperacetylated. Nevertheless, the enhancer is unable to activate the methylated and hypoacetylated reporter. Our findings suggest that CpG methylation represses transcription by interfering with RNA polymerase initiation via a mechanism that involves localized histone deacetylation. This repression is dominant over a remodeled enhancer but neither results in nor requires region-wide changes in DNA replication or chromatin structure.

Acetylation↗
PubMed2005

Methylation-associated silencing of the nuclear receptor 1I2 gene in advanced-type neuroblastomas, identified by bacterial artificial chromosome array-based methylated CpG island amplification.

To identify genes whose expression patterns are altered by methylation of DNA, we established a method for scanning human genomes for methylated DNA sequences, namely bacterial artificial chromosome array-based methylated CpG island amplification (BAMCA). In the course of a program using BAMCA to screen neuroblastoma cell lines for aberrant DNA methylation compared with stage I primary neuroblastoma tumors, we identified CpG methylation-dependent silencing of the nuclear receptor 1I2 (NR1I2) gene. NR1I2 was methylated in a subset of neuroblastoma cell lines and also in advanced-stage primary tumors with amplification of MYCN. Its methylation status was inversely associated with gene expression. Treatment with the demethylating agent 5-aza-2'-deoxycytidine restored NR1I2 transcription in neuroblastoma cell lines lacking endogenous expression of this gene. A CpG island located around exon 3 of NR1I2 showed promoter activity, and its methylation status was clearly and inversely correlated with NR1I2 expression status. The gene product, NR1I2, has a known function in regulating response to xenobiotic agents but it also suppressed growth of neuroblastoma cells in our experiments. We identified some possible transcriptional targets of NR1I2 by expression array analysis. The high prevalence of NR1I2 silencing by methylation in aggressive neuroblastomas, together with the growth-suppressive activity of NR1I2, suggests that this molecule could serve as a diagnostic marker to predict prognosis for neuroblastomas.

Cell Line, Tumor↗
PubMed2006

Examination of a CpG island methylator phenotype and implications of methylation profiles in solid tumors.

The CpG island methylator phenotype (CIMP), thoroughly described in colorectal cancer and to a lesser extent in other solid tumors, is important in understanding epigenetics in carcinogenesis and may be clinically useful for classification of neoplastic disease. Therefore, we investigated whether this putative phenotype exists in exposure-related solid tumors, where somatic gene alterations and enhanced clonal growth are selected for by carcinogens, and examined the ability of methylation profiles to classify malignant disease. We studied promoter hypermethylation of 16 tumor suppressor genes and 3 MINT loci (acknowledged classifiers of CIMP) in 344 bladder cancers, 346 head and neck squamous cell carcinomas (HNSCC), 146 non-small-cell lung cancer (NSCLC), and 71 malignant pleural mesotheliomas (MPM). We employed rigorous statistical methods to examine the distribution of promoter methylation and the usefulness of these profiles for disease classification. In bladder cancer, HNSCC, and NSCLC, there was a significant correlation (P < 0.0001) between methylation of the three MINT loci and methylation index, although the distribution of methylated loci varied significantly across these disease. Although there was a significant (P < 0.001) association between gene methylation profile and disease, rates of misclassification of each disease by their methylation profile ranged from 28% to 32%, depending on the classification scheme used. These data suggest that a form of CIMP exists in these solid tumors, although its etiology remains elusive. Whereas the gene profiles of hypermethylation among examined loci could not unequivocally distinguish disease type, the existence of CIMP and the relative preponderance of hypermethylation in these cancers suggest that methylation analysis may be clinically useful as a targeted screening tool.

Carcinoma, Non-Small-Cell Lung↗
PubMed2004

Methylation profiles of thirty four promoter-CpG islands and concordant methylation behaviours of sixteen genes that may contribute to carcinogenesis of astrocytoma.

BACKGROUND: Astrocytoma is a common aggressive intracranial tumor and presents a formidable challenge in the clinic. Association of altered DNA methylation patterns of the promoter CpG islands with the expression profile of cancer-related genes, has been found in many human tumors. Therefore, DNA methylation status as such may serve as an epigenetic biomarker for both diagnosis and prognosis of human tumors, including astrocytoma. METHODS: We used the methylation specific PCR in conjunction with sequencing verification to establish the methylation profile of the promoter CpG island of thirty four genes in astrocytoma tissues from fifty three patients (The WHO grading: I: 14, II: 15, III: 12 and IV: 12 cases, respectively). In addition, compatible tissues (normal tissues distant from lesion) from three non-astrocytoma patients were included as the control. RESULTS: Seventeen genes (ABL, APC, APAF1, BRCA1, CSPG2, DAPK1, hMLH1, LKB1, PTEN, p14ARF, p15INK4b, p27KIP1, p57KIP2, RASSF1C, RB1, SURVIVIN, and VHL) displayed a uniformly unmethylated pattern in all the astrocytoma and non-astrocytoma tissues examined. However, the MAGEA1 gene that was inactivated and hypermethylated in non-astrocytoma tissues, was partially demethylated in 24.5% of the astrocytoma tissues (co-existence of the hypermethylated and demethylated alleles). Of the astrocytoma associated hypermethylated genes, the methylation pattern of the CDH13, cyclin a1, DBCCR1, EPO, MYOD1, and p16INK4a genes changed in no more than 5.66% (3/53) of astrocytoma tissues compared to non-astrocytoma controls, while the RASSF1A, p73, AR, MGMT, CDH1, OCT6, MT1A, WT1, and IRF7 genes were more frequently hypermethylated in 69.8%, 47.2%, 41.5%, 35.8%, 32%, 30.2%, 30.2%, 30.2% and 26.4% of astrocytoma tissues, respectively. Demethylation mediated inducible expression of the CDH13, MAGEA1, MGMT, p73 and RASSF1A genes was established in an astrocytoma cell line (U251), demonstrating that expression of these genes is likely regulated by DNA methylation. AR gene hypermethylation was found exclusively in female patients (22/27, 81%, 0/26, 0%, P < 0.001), while the IRF7 gene hypermethylation preferentially occurred in the male counterparts (11/26, 42.3% to 3/27, 11%, P < 0.05). Applying the mathematic method "the Discovery of Association Rules", we have identified groups consisting of up to three genes that more likely display the altered methylation patterns in concert in astrocytoma. CONCLUSIONS: Of the thirty four genes examined, sixteen genes exhibited astrocytoma associated changes in the methylation profile. In addition to the possible pathological significance, the established concordant methylation profiles of the subsets consisting of two to three target genes may provide useful clues to the development of the useful prognostic as well as diagnostic assays for astrocytoma.

Adolescent↗
PubMed2026

Characterization of DNA methylation in PBMCs and donor-matched iPSCs shows age-related methylation is reset during stem cell reprogramming.

DNA methylation is an important epigenetic mechanism that helps define and maintain cellular functions. It is influenced by many factors, including environmental exposures, genotype, cell type, sex, and aging. Since age is the primary risk factor for developing neurodegenerative diseases, it is important to determine if age-related DNA methylation is retained when cells are reprogrammed to an induced Pluripotent Stem Cell (iPSC) state. Here, we selected peripheral blood mononuclear cells (PBMCs; n&#x2009;=&#x2009;99) from a cohort of diverse and healthy individuals enrolled in the Genetic and Epigenetic Signatures of Translational Aging Laboratory Testing (GESTALT) study to reprogram to iPSCs. After reprogramming, the resulting iPSCs were evaluated for DNA methylation signatures to determine if they reflect the confounding factors of aging and environmental effects. Data from genome-wide DNA methylation arrays in both cell types showed that age-related methylation measured by epigenetic clocks is largely reset to an early methylation age after reprogramming of PBMCs to iPSCs. We further examined the epigenetic age of each cell type using an Epigenome-wide Association Study (EWAS) and identified a set of methylation Quantitative Trait Loci in each cell type. Our results show that age-related DNA methylation is largely reset in iPSCs, and each cell type has a unique set of methylation sites that are modified by population-level genetic variation.

DNA Methylation↗