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At least 199 records · Page 11Linked to original sources
PubMed2005

Epigenetic regulation of lentiviral transgene vectors in a large animal model.

Transgenic animals are of outstanding relevance for genetic studies and the development of novel therapies for human diseases. A recent development is the generation of transgenic animals by lentiviral gene transfer. So far, studies on lentiviral transgenesis focused on first-generation (founder or F0) animals-most of which carry multiple integrants. Here, we analyze transgene expression and epigenetic regulation of individual integrants in lentiviral transgenic pigs after segregation to the F1 generation. Unexpectedly, one-third of lentiviral integrants exhibited low expression levels and were hypermethylated, as demonstrated by methylation-sensitive Southern blotting and bisulfite sequencing. Proviral methylation density correlated inversely with expression levels. In addition, treatment of isolated transgenic fibroblasts with the DNA methylase inhibitor 5-azacytidine induced a threefold increase in mean fluorescence intensity (MFI) from 8 to 26.1. Treatment with the histone deacetylase inhibitor trichostatin A enhanced MFI to only 11.1. Taken together, expression of lentiviral integrants in higher mammals is regulated by epigenetic modifications. In contrast to previous expectations, DNA methylation plays an important role in lentiviral expression.

Animals↗
PubMed2004

Dynamic regulation of estrogen receptor-beta expression by DNA methylation during prostate cancer development and metastasis.

Estrogen receptor (ER)-beta is thought to exert anti-proliferative effects in the normal prostate but supports prostate cancer (PCa) cell survival. We previously reported that the receptor's expression declined as PCa developed in the gland but reappeared in lymph node and bone metastases. To investigate whether hypermethylation was the underlying mechanism for these phenomena, we first identified two CpG islands (CGIs) encompassing 41 CpG dinucleotides, located separately in the untranslated exon 0N and the promoter region of ER-beta. Using immunostained, laser capture-microdissected samples from 56 clinical specimens, we demonstrated an inverse relationship exists between the extent of ER-beta CGI methylation and receptor expression in normal, hyperplastic, premalignant, and malignant foci of the prostate and in lymph node and bone metastases. Treatment of PCa cell lines (LNCaP and DU145), that express little ER-beta mRNA, with a demethylating agent increased levels of receptor expression thus corroborating our in vivo findings that methylation is involved in ER-beta silencing. Methylation centers in the promoter region and exon 0N were identified by hierarchical cluster analysis of bisulfite sequencing data obtained from 710 alleles. Methylation at these centers was insignificant in normal epithelium, reached 80 to 90% in grade 4/5 PCa, but declined to less than 20% in bone metastases. In addition, progressive methylation spreading from the exonic CGI to the promoter CGI, which correlated with loss of ER-beta expression, was detected in microdissected samples and in cell cultures. Using a new class of methylated oligonucleotides that mediate sequence-specific methylation in cellulo, we demonstrated that methylation of the promoter CGI, but not the exonic CGIs, led to transcriptional inactivation of ER-beta. Our results present the first evidence that epigenetic regulation of ER-beta is a reversible and tumor stage-specific process and that gene silencing via methylated oligonucleotides may have therapeutic potential in the treatment of advanced PCa.

Base Sequence↗
PubMed2002

Evidence for the role of promoter methylation in the regulation of MMP-9 gene expression.

Several studies have reported that elevated MMP-9 expression in lymphoma tissues correlated with tumor stage, grade, or prognosis. Because the DNA methylation pattern is critical for gene expression, detailed methylation analysis using genomic bisulfite sequencing was performed on a series of lymphoma cell lines. We found an inverse correlation between level of methylation of the MMP-9 promoter and the level of MMP-9 expression. Treating lymphoma cells with a DNA methylation inhibitor decreased MMP-9 promoter methylation and increased MMP-9 messenger RNA and protein secretion. This increased expression was potentiated by PMA, a known stimulus of MMP-9 in lymphoma cells. Finally, experiments using in vitro methylated MMP-9 promoter constructs confirmed the fact that DNA methylation exerts suppression on transcriptional activity. The results thus indicate that methylation may contribute to the transcriptional activity of the MMP-9 promoter.

Animals↗
PubMed2003

Promoter hypomethylation of a novel cancer/testis antigen gene CAGE is correlated with its aberrant expression and is seen in premalignant stage of gastric carcinoma.

Previously, we reported the identification and characterization of a novel cancer/testis antigen gene, CAGE(4), that was expressed in various histological types of tumors, but not in normal tissues, with the exception of the testis. To date, molecular mechanisms for the expression of CAGE have never been studied. In our expression analysis, we found that some cancer cell lines did not express CAGE. The expression of CAGE could be restored in these cell lines by treatment with 5(')-aza-2(')-deoxycytidine, suggesting that the expression of CAGE is mainly suppressed by hypermethylation. Bisulfite sequencing analysis of the 16 CpG sites of the CAGE promoter in various cancer cell lines and tissues revealed a close relationship between the methylation status of the CAGE promoter and the expression of CAGE. The transient transfection experiments displayed that the methylation of CpG sites inhibited the CAGE promoter activity in luciferase reporter assays. The methylation of the CpG sites inhibited the binding of transcription factors, shown by a mobility shift assay. A methylation-specific PCR analysis revealed that hypomethylation of the CAGE promoter was present at frequencies of more than 60% in breast, gastric, and lung cancers, and hepatocellular carcinomas, and at frequencies of less than 40% in prostate, uterine cervical, and laryngeal cancers. Promoter hypomethylation was found in chronic gastritis (19/55, 34.5%) and liver cirrhosis (13/22, 59%), but not in normal prostate, normal colon, or chronic hepatitis. These results suggest that the methylation status of the CpG sites of CAGE determines its expression, that the hypomethylation of CAGE precedes the development of gastric cancer and hepatocellular carcinoma, and that the high frequencies of hypomethylation of CAGE, in various cancers would be valuable as a cancer diagnostic marker.

Antigens, Nuclear↗
PubMed2001

Regulation of transcription of the glutathione S-transferase P1 gene by methylation of the minimal promoter in human leukemia cells.

To study the relationship between methylation and the transcriptional activity of the minimal promoter of the glutathione S-transferase GSTP1 gene encoding glutathione S-transferase P1-1, GSTP1 mRNA levels as well as basal promoter activity were compared in human leukemia cell lines. The K562 erythroleukemia cell line presented a strong GSTP1 promoter activity, as measured in transient transfection assays using a luciferase reporter plasmid, and correlated with a high mRNA whereas in Raji cells no mRNA was expressed. In order to establish a relationship between the expression and the methylation status, we used in vitro bisulfite sequencing which indicated that both methylated and unmethylated GSTP1 promoter alleles coexisted in K562 cells, whereas Raji lymphoma cells showed a nearly uniform hypermethylation of the promoter region. To determine the impact of methylation, we used in vitro SssI methylation of the minimal GSTP1 promoter, which led to the silencing of the promoter activity in transient transfection assays in expressing K562 as well as in non-expressing Raji cells. These data are in good agreement with previously obtained results and indicate that methylation of CpG sites of the basal promoter is an essential mechanism in the control of GSTP1 gene expression in human leukemia.

Base Sequence↗
PubMed2001

Influence of oocyte nuclei on demethylation of donor genome in cloned bovine embryos.

We recently demonstrated that satellite regions exhibit an aberrant DNA methylation in cloned bovine embryos. Here, we examined, using bisulfite-sequencing technology, whether the inefficient demethylation of cloned donor genomes could be rescued by the presence of oocytic nuclei. Both AciI digestion and sequencing analyses showed that satellite sequence was demethylated more efficiently in cloned tetraploid blastocysts than in diploid clones. When methyl-CpG density (the number of methyl-CpG sites per string) was scored, a significant decrease was observed in tetraploids (P<0.001). These results suggest that unknown mechanisms provided by oocytic nuclei could assist the demethylation of satellite sequences in tetraploid clones.

Animals↗
PubMed2001

KG-1 and KG-1a model the p15 CpG island methylation observed in acute myeloid leukemia patients.

p15 and p16 are tumor suppressor genes that have 5' CpG islands and both are subject to hypermethylation associated with their transcriptional inactivation in hematological malignancies. In this study, we used sodium bisulfite sequencing to obtain a complete map of the 5-methylcytosine status of 80 CpGs covering approximately 900 bp in the 5' p15 CpG island, and 53 CpGs covering approximately 700 bp in the 5' p16 CpG island in the hematopoietic cell lines HL60, KG-1, and KG-1a, two normal human bone marrow samples (NBM), and eight cytosine arabinoside (ara-C)-resistant adult acute myeloid leukemia (AML) patients. We found methylation of the p15 CpG island in 75% of the AML cases studied spread throughout the 5' region analyzed but only minimal methylation of p15 in NBM. Further, the p16 CpG island was not aberrantly methylated in NBM or the eight AML patients studied. Two distinct modes of p15 methylation in AML were identified, variegated and complete. Interestingly, KG-1 and KG-1a model the methylation of p15 observed in AML, where KG-1 methylation is variegated and KG-1a methylation is complete. Both KG-1 and KG-1a had no detectable p15 mRNA or protein. These results demonstrate that rather than continuous increases in p15 methylation, surprisingly two punctuated modes of aberrant p15 methylation, variegated and complete, were observed in vitro and in vivo. Thus aberrant methylation of tumor suppressor genes is not a binary switch but in the case of p15 occurs in two independent and stable states.

Bone Marrow↗
PubMed1997

Tissue-specific methylation occurs in the essential promoter element of the tyrosine hydroxylase gene.

Expression of tyrosine hydroxylase (TH) is regulated in a tissue-specific manner by multiple mechanisms. In catecholaminergic cells, the expression of TH-mRNA is up-regulated by forskolin (FK) and is suppressed by retinoic acid (RA). We have previously provided evidence that, in N-18 cells, the expression of TH-mRNA is suppressed by DNA methylation of the TH gene itself. In the present study, using a catecholaminergic cell line, N1E-115, we performed deletional and mutational analyses on the 5'-flanking region of the mouse TH gene. The results indicate that a cAMP response element (CRE) mediates constitutive transcription of the TH gene, as well as responsiveness to FK and RA. Using bisulfite sequencing methods, we analyzed the methylation status of the TH gene 5'-flanking region in various cell lines and rat tissues. We found that three cytosine residues in the domain surrounding the CRE of the TH gene promoter were specifically methylated in N-18 cells and TH non-expressing rat tissues. In contrast, these cytosines were undermethylated in TH expressing cell lines and tissues. The inverse correlation between the frequency of cytosine methylation at these specific sites and the levels of TH expression supports a role for DNA methylation in the regulation of tissue-specific gene expression.

Animals↗
PubMed2002

Genome-wide profiling of DNA methylation reveals transposon targets of CHROMOMETHYLASE3.

DNA methylation has been implicated in a variety of epigenetic processes, and abnormal methylation patterns have been seen in tumors. Analysis of methylation patterns has traditionally been conducted either by using Southern analysis after cleavage with methyl-sensitive restriction endonucleases or by bisulfite sequencing. However, neither method is practical for analyzing more than a few genes. Here, we describe a simple technique for genome-wide mapping of DNA methylation patterns. Fragmentation by a methyl-sensitive restriction endonuclease is followed by size fractionation and hybridization to microarrays. We demonstrate the utility of this method by characterizing methylation patterns in Arabidopsis methylation mutants. This analysis reveals that CHROMOMETHYLASE3 (CMT3), which was previously shown to maintain CpXpG methylation, preferentially methylates transposons, even when they are present as single copies within the genome. Methylation profiling has potential applications in disease research and diagnostic screening.

Arabidopsis↗
PubMed2005

Promoter methylation and differential expression of pi-class glutathione S-transferase in endometrial carcinoma.

Pi-class glutathione S-transferase (GSTP1), located on chromosome 11q13, codes for a phase II metabolic enzyme that detoxifies reactive electrophilic intermediates. The protein also interacts with steroid hormones in the human body. The role of GSTP1 in endometrial carcinoma has not been reported. In this study, we aimed at determining the expression of GSTP1 in relation to the epigenetic and genetic changes of the gene in endometrial carcinoma. The GSTP1 protein and mRNA expression was assessed by immunohistochemistry on tissue microarray and quantitative real-time reverse transcriptase-polymerase chain reaction, respectively. Its methylation status was studied by methylation-specific polymerase chain reaction and bisulfite sequencing. Possible mutations in coding region of GSTP1 were assessed by cDNA sequencing. Ninety-seven cases of endometrial carcinoma with available tissue blocks and clinical data were studied. Our results showed that 68.0% (66 of 97) of the cases showed reduced protein expression while 64% (16 of 25) showed reduced mRNA expression; 30.9% (30 of 97) of the cases demonstrated methylated alleles in at least one of the six methylation-specific polymerase chain reaction reactions. The methylation status significantly correlated with reduced protein expression (P = 0.008) and reduced mRNA expression (P = 0.003). Methylation at non-CpG sites including CpCpG trinucleotides and CpT dinucleotides were also observed. cDNA sequencing did not reveal genetic alterations in coding region of the gene. The extent of myometrial invasion was found to be significantly correlated with both the methylation status (P = 0.009) and the protein expression (P = 0.036) of the GSTP1 gene. We postulated that hypermethylation of the GSTP1 gene promoter region may act as a dynamic regulation mechanism contributing to reduced GSTP1 expression, which is associated with myometrial invasion potential of the endometrial carcinoma.

Adult↗
PubMed2004

Enzymatic regional methylation assay for determination of CpG methylation density.

DNA methylation is a critical mechanism for epigenetic gene regulation. In mammalian cells, CpG methylation density often influences the transcription level of related genes. Here, we report a short and accurate method to determine the CpG methylation density of any DNA region by sequentially applying bisulfite PCR and SssI (CpG) methylase. We introduced simple and efficient binding and washing steps that greatly improve the previous methodology and considerably enhance the accuracy of the assay. The accuracy of this method allows the detection of differences at the level of a single CpG methylation site when homogeneous PCR product was used as substrate. We validated our method by bisulfite sequencing multiple clones of samples with variable levels of CpG methylation. We envision that for its accuracy, simplicity, low-cost, flexibility, minimum instrumentation requirements, and high-throughput potential our method could greatly benefit research on DNA methylation.

Base Sequence↗
PubMed2024

Triphenyl Phosphate Alters Methyltransferase Expression and Induces Genome-Wide Aberrant DNA Methylation in Zebrafish Larvae.

Emerging environmental contaminants, organophosphate flame retardants (OPFRs), pose significant threats to ecosystems and human health. Despite numerous studies reporting the toxic effects of OPFRs, research on their epigenetic alterations remains limited. In this study, we investigated the effects of exposure to 2-ethylhexyl diphenyl phosphate (EHDPP), tricresyl phosphate (TMPP), and triphenyl phosphate (TPHP) on DNA methylation patterns during zebrafish embryonic development. We assessed general toxicity and morphological changes, measured global DNA methylation and hydroxymethylation levels, and evaluated DNA methyltransferase (DNMT) enzyme activity, as well as mRNA expression of DNMTs and ten-eleven translocation (TET) methylcytosine dioxygenase genes. Additionally, we analyzed genome-wide methylation patterns in zebrafish larvae using reduced-representation bisulfite sequencing. Our morphological assessment revealed no general toxicity, but a statistically significant yet subtle decrease in body length following exposure to TMPP and EHDPP, along with a reduction in head height after TPHP exposure, was observed. Eye diameter and head width were unaffected by any of the OPFRs. There were no significant changes in global DNA methylation levels in any exposure group, and TMPP showed no clear effect on DNMT expression. However, EHDPP significantly decreased only DNMT1 expression, while TPHP exposure reduced the expression of several DNMT orthologues and TETs in zebrafish larvae, leading to genome-wide aberrant DNA methylation. Differential methylation occurred primarily in introns (43%) and intergenic regions (37%), with 9% and 10% occurring in exons and promoter regions, respectively. Pathway enrichment analysis of differentially methylated region-associated genes indicated that TPHP exposure enhanced several biological and molecular functions corresponding to metabolism and neurological development. KEGG enrichment analysis further revealed TPHP-mediated potential effects on several signaling pathways including TGF&#x3b2;, cytokine, and insulin signaling. This study identifies specific changes in DNA methylation in zebrafish larvae after TPHP exposure and brings novel insights into the epigenetic mode of action of TPHP.

Animals↗
PubMed2026

Seeing and Feeling DNA Methylation: Single-Molecule Biophysics Meets Machine Learning.

DNA methylation at 5-methylcytosine (5mC) is crucial for embryonic development and cellular function, while aberrant patterns strongly drive disease onset and progression. Its reversible nature offers substantial therapeutic potential, emphasizing the need for precise, context-specific genome wide 5mC mapping. Conventional techniques such as bisulfite sequencing and ensemble biosensor assays are hindered by DNA degradation, amplification bias, high cost, and inability to resolve single-molecule structural and mechanical effects of methylation. This review examines advances in single-molecule biophysical methods (nanopore sensing, smFRET, optical/magnetic tweezers, and AFM) that provide direct, label-free/minimally invasive 5mC detection, along with quantitative insights into DNA conformation, mechanics, and protein-DNA interactions. These techniques complement traditional methylome mapping by linking genomic localization to molecular mechanisms. Emerging machine-learning approaches are revolutionizing analysis, particularly in nanopore sensing, while promising applications in smFRET, tweezers, and AFM address throughput and reproducibility challenges. Their convergence promises scalable, high-resolution epigenetic profiling, advancing precision epigenomics toward clinical application.

DNA Methylation↗
PubMed2005

Expression of aquaporin-5 in and fluid secretion from immortalized human salivary gland ductal cells by treatment with 5-aza-2'-deoxycytidine: a possibility for improvement of xerostomia in patients with Sjögren's syndrome.

The aim of the present study was to investigate the possibility that ductal cells, which preferentially survive and/or proliferate in Sjögren's syndrome (SS) salivary glands of patients with SS, could acquire the functional expression of membrane water channel aquaporin-5 (AQP5). Thus, in this study, we demonstrate that an immortalized normal human salivary gland ductal cell (NS-SV-DC) line, lacking the expression of AQP5, acquires AQP5 gene expression in response to treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR), a DNA demethylating agent. Confocal microscopic analysis revealed the localization of AQP5 expression mainly at the apical and lateral sides of the plasma membrane. The expressed AQP5 protein was functionally active because AQP5 expression resulted in a significant increase in the osmotically directed net fluid rate across monolayers of NS-SV-DC cells. By the analysis of bisulfite sequencing of CpG islands in the AQP5 promoter, hypermethylation within the consensus Sp1-binding sites was commonly observed in parental cell clones, whereas demethylation at the CGs, one in the second consensus Sp1 element and the other outside of the third consensus Sp1 element in the AQP5 promoter, was detected in NS-SV-DC cells after treatment with 5-Aza-CdR. By analyzing the luciferase activity of transfected AQP5 promoter vectors, it became evident that demethylation at the CGs cooperatively functions between these two sites to induce AQP5 expression. Our data, therefore, suggest that treatment of ductal cells with 5-Aza-CdR could result in the expression of the AQP5 gene, thereby leading to increased fluid secretion from ductal cells in SS salivary glands.

Aquaporin 5↗
PubMed2005

Inactivation of RASSF1A tumor suppressor gene by aberrant promoter hypermethylation in human pituitary adenomas.

Aberrant promoter methylation and resultant silencing of several tumor suppressor genes play an important role in the pathogenesis of many tumor types. The human Ras association domain family 1A gene (RASSF1A), recently cloned from the lung tumor locus at 3p21.3, was shown to be frequently inactivated by hypermethylation of its promoter region in a number of malignancies. We have investigated the expression and epigenetic changes of this novel universal tumor suppressor gene in pituitary adenomas and correlated the data with clinicopathologic findings. Fresh frozen normal pituitary tissues and 52 primary pituitary adenomas including all major types were examined. Methylation-specific polymerase chain reaction (MSP), combined bisulfite restriction analysis (COBRA), bisulfite sequencing and semiquantitative reverse transcription-polymerase chain reaction were used to analyze DNA promoter methylation status and the mRNA expression of RASSF1A, respectively. High levels of RASSF1A transcript and no methylation of the RASSF1A promoter were found in normal pituitary tissues. RASSF1A promoter methylation was detected in 20 of 52 (38%) adenomas including all major types of pituitary adenomas. However, a lower frequency of methylation of the RASSF1A promoter was found in gonadotroph cell adenomas (15%) compared with growth hormone cell, prolactin cell, or adrenocorticotropic hormone cell adenomas (54, 46 and 50%, respectively). Methylation frequency was higher in the most aggressive adenomas (87% in grade IV tumors, P=0.0163). In addition, methylation of the RASSF1A promoter potentially correlated with higher labeling index of the proliferation marker Ki-67 (P=0.1475). Loss or significant reduction of RASSF1A messenger RNA transcripts was identified in 18 of 20 (90%) adenomas with hypermethylation of RASSF1A (P<0.0001). Our data suggest that promoter hypermethylation of RASSF1A and resultant alterations of RASSF1A expression may play a critical role in pituitary tumorigenesis and may be involved in tumor progression.

Adenoma↗
PubMed2006

DNA methylation-dependent silencing of CST6 in human breast cancer cell lines.

Cystatin M (CST6) is a candidate breast cancer tumor suppressor that is expressed in normal and premalignant breast epithelium, but not in metastatic breast cancer cell lines. CST6 is subject to epigenetic silencing in MCF-7 breast cancer cells related to methylation of the CpG island that encompasses the CST6 proximal promoter region and exon 1. In the current study, CST6 CpG island methylation and expression status was examined in a panel of breast cancer cell lines. Seven of 12 (58%) cell lines lack detectable expression of CST6 and treatment of these cells with 5-aza-2'-deoxycytidine resulted in a significant increase in CST6 expression, suggesting that the loss of expression may be related to methylation-dependent epigenetic silencing. Bisulfite sequencing of CST6 in a subset of breast cancer cell lines revealed CpG island hypermethylation in CST6-negative cells, and an absence of CpG island methylation in cells that express CST6. The extent of regional methylation was strongly associated with the lack of expression of CST6 among these cell lines. In particular, hypermethylation of the proximal promoter was significantly associated with CST6 gene silencing, and methylation of a number of individual CpGs was found to be statistically correlated with extinction of gene expression. These results establish a strong link between CST6 promoter hypermethylation and loss of CST6 expression in breast cancer cell lines, and suggest that methylation-dependent epigenetic silencing of CST6 may represent an important mechanism for loss of CST6 during breast carcinogenesis in vivo.

Antimetabolites, Antineoplastic↗
PubMed2000

Epigenetic regulation of androgen receptor gene expression in human prostate cancers.

Epigenetic mechanisms including DNA methylation and histone deacetylation are thought to play important roles in gene transcriptional inactivation. Heterogenous expression of androgen receptor (AR), which appears to be related to variable responses to endocrine therapy in prostate cancer (PCa) may also be due to epigenetic factors. The methylation status of the 5' CpG island of the AR in 3 prostate cancer cell lines and 10 primary and 14 hormone-refractory PCa samples was determined using the bisulfite PCR methods. In DU145, CpG-rich regions of the AR were hypermethylated. By an immunohistochemical analysis, only one PCa sample had no AR expression, the others being heterogenous. Bisulfite sequencing and methylation-specific PCR analysis showed aberrant methylation of AR 5'-regulatory region in 20% of 10 primary and 28% of 14 hormone-refractory PCa samples. To clarify the effect of epigenetic regulation on AR expression, we treated three prostate cancer cell lines with a demethylating agent, 5-aza-2'-deoxycytidine (azaC), and a histone deacetylase inhibitor, Trichostatin A (TSA). In DU145, re-expression of AR mRNA was detected after treatment with azaC and/or TSA. Our results suggest that epigenetic regulations including CpG methylation and histone acetylation may play important roles in the regulation of the AR.

Antimetabolites, Antineoplastic↗
PubMed2006

Heritable germline epimutation of MSH2 in a family with hereditary nonpolyposis colorectal cancer.

Epimutations in the germline, such as methylation of the MLH1 gene, may contribute to hereditary cancer syndrome in human, but their transmission to offspring has never been documented. Here we report a family with inheritance, in three successive generations, of germline allele-specific and mosaic hypermethylation of the MSH2 gene, without evidence of DNA mismatch repair gene mutation. Three siblings carrying the germline methylation developed early-onset colorectal or endometrial cancers, all with microsatellite instability and MSH2 protein loss. Clonal bisulfite sequencing and pyrosequencing showed different methylation levels in different somatic tissues, with the highest level recorded in rectal mucosa and colon cancer tissue, and the lowest in blood leukocytes. This mosaic state of germline methylation with different tissue distribution could act as the first hit and provide a mechanism for genetic disease inheritance that may deviate from the mendelian pattern and be overlooked in conventional leukocyte-based genetic diagnosis strategy.

Adult↗