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DNA Methyltransferase (dna + methyltransferase)
Kinds of DNA Methyltransferase Selected AbstractsProtection of hematopoietic cells from O6 -alkylation damage by O6 -methylguanine DNA methyltransferase gene transfer: studies with different O6 -alkylating agents and retroviral backbonesEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2001Michael Jansen Abstract: Overexpression of O6 -methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O6 -alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O6 -alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8,12 µg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer. [source] Defective DNA methylation and CD70 overexpression in CD4+ T cells in MRL/lpr lupus-prone miceEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 5 2007Abstract We have determined that abnormal DNA methylation in T cells coincides with the development of autoimmunity, using a mouse model that exhibits an age-dependent lupus-like disease (MRL/lpr mice). Splenic CD4+ T cells were isolated from these mice at 5 and 16,wk of age (before and after autoimmunity is established) and the expression of DNA methyltransferase,1 (Dnmt1) and the methylation-sensitive gene Tnfsf7 (CD70) was measured. Bisulfite DNA sequencing was used to monitor the methylation status of the Tnfsf7 gene. We found that Dnmt1 steady-state mRNA levels were significantly lower in 16-wk-old MRL/lpr mice, which had established autoimmunity, compared to the 5-wk-old MRL/lpr mice. Furthermore, the expression of CD70 was higher in MRL/lpr mice at 16,wk. CD70 was overexpressed in MRL/lpr mice compared to age- and sex-matched MRL+/+ controls. Bisulfite DNA sequencing of the Tnfsf7 gene in MRL/lpr mice revealed that at 16,wk, CG pairs were hypomethylated compared to 5-wk-old mice, and that Tnfsf7 from MRL/lpr mice was hypomethylated at 16,wk relative to age-matched MRL+/+ controls. Our data indicate that decreased expression of Dnmt1 and the corresponding T cell DNA hypomethylation correlate with the development of age-dependent autoimmunity in MRL/lpr mice. [source] Regulation of transcription of the Dnmt1 gene by Sp1 and Sp3 zinc finger proteinsFEBS JOURNAL, Issue 12 2002Shotaro Kishikawa The Sp family is a family of transcription factors that bind to cis -elements in the promoter regions of various genes. Regulation of transcription by Sp proteins is based on interactions between a GC-rich binding site (GGGCGG) in DNA and C-terminal zinc finger motifs in the proteins. In this study, we characterized the GC-rich promoter of the gene for the DNA methyltransferase (Dnmt1) that is responsible for methylation of cytosine residues in mammals and plays a role in gene silencing. We found that a cis -element (nucleotides ,161 to ,147) was essential for the expression of the mouse gene for Dnmt1. DNA-binding assays indicated that transcription factors Sp1 and Sp3 bound to the same cis -element in this region in a dose-dependent manner. In Drosophila SL2 cells, which lack the Sp family of transcription factors, forced expression of Sp1 or Sp3 enhanced transcription from the Dnmt1 promoter. Stimulation by Sp1 and Sp3 were independent phenomena. Furthermore, cotransfection reporter assays with a p300-expression plasmid revealed the activation of the promoter of the Dnmt1 gene in the presence of Sp3. The transcriptional coactivator p300 interacted with Sp3 in vivo and in vitro. Our results indicate that expression of the Dnmt1 gene is controled by Sp1 and Sp3 and that p300 is involved in the activation by Sp3. [source] Advanced colorectal polyps with the molecular and morphological features of serrated polyps and adenomas: concept of a ,fusion' pathway to colorectal cancerHISTOPATHOLOGY, Issue 2 2006J R Jass Aim :,To establish and explain the pattern of molecular signatures across colorectal polyps. Methods and results :,Thirty-two sessile serrated adenomas (SSA), 10 mixed polyps (MP), 15 traditional serrated adenomas (SA), 49 hyperplastic polyps (HP) and 84 adenomas were assessed for mutation of KRAS and BRAF and aberrant expression of p53. The findings were correlated with loss of expression of O-6-methylguanine DNA methyltransferase (MGMT). KRAS mutation occurred more frequently (26.5%) than BRAF mutation (4.8%) in adenomas (P < 0.001) and particularly in adenomas with villous architecture (50%). Loss of expression of MGMT correlated with KRAS mutation in small tubular adenomas (P < 0.04). BRAF mutation was frequent in HPs (67%) and SSAs (81%), while KRAS mutation was infrequent (4% and 3%, respectively). Of MPs and SAs, 72% had either BRAF or KRAS mutation. Aberrant expression of p53 was uncommon overall, but occurred more frequently in MPs and SAs (12%) than adenomas (1%) (P < 0.04) and there was concordant loss of expression of MGMT. Conclusions :,Molecular alterations that are characteristic of the serrated pathway and adenoma,carcinoma sequence can co-occur in a minority of advanced colorectal polyps that then show morphological features of both pathways. These lesions account for only 2% of colorectal polyps, but may be relatively aggressive. [source] Hypermethylation of RAS effector related genes and DNA methyltransferase 1 expression in endometrial carcinogenesisINTERNATIONAL JOURNAL OF CANCER, Issue 2 2008Xiaoyun Liao Abstract Epigenetic aberration is known to be important in human carcinogenesis. Promoter methylation status of RAS effector related genes, RASSF1A, RASSF2A, hDAB2IP (m2a and m2b regions) and BLU, was evaluated in 76 endometrial carcinomas and their non-neoplastic endometrial tissue by methylation specific PCR. Hypermethylation of at least one of the 5 genes was detected in 73.7% of carcinomas. There were significant correlations between methylation of hDAB2IP and RASSF1A, RASSF2A (p = 0.042, p = 0.012, respectively). Significantly, more frequent RASSF1A hypermethylation was found in Type I endometrioid carcinomas than Type II carcinomas (p = 0.049). Among endometrioid cancers, significant association between RASSF1A hypermethylation and advanced stage, as well as between methylation of hDAB2IP at m2a region with deep myometrial invasion (p < 0.05) was observed. mRNA expression of RASSF1A, RASSF2A and BLU in endometrial cancer cell lines significantly increased after treatment with the demethylating agent 5-Aza-2,-deoxycytidine supporting the repressive effect of hypermethylation on their transcription. Immunohistochemical study of DNMT1 on eight normal endometrium, 16 hyperplastic endometrium without atypia, 40 atypical complex hyperplasia and 79 endometrial carcinomas showed progressive increase in DNMT1 immunoreactivity from normal endometrium to endometrial hyperplasia and endometrioid carcinomas (p = 0.001). Among carcinomas, distinctly higher DNMT1 expression was observed in Type I endometrioid carcinomas (p < 0.001). DNMT1 immunoreactivity correlated with RASSF1A and RASSF2A methylation (p < 0.05). The data suggested that hypermethylation of RAS related genes, particularly RASSF1A, was involved in endometrial carcinogenesis with possible divergent patterns in different histological types. DNMT1 protein overexpression might contribute to such aberrant DNA hypermethylation of specific tumor suppressor genes in endometrial cancers. © 2008 Wiley-Liss, Inc. [source] Regional DNA hypermethylation and DNA methyltransferase (DNMT) 1 protein overexpression in both renal tumors and corresponding nontumorous renal tissuesINTERNATIONAL JOURNAL OF CANCER, Issue 2 2006Eri Arai Abstract To evaluate the significance of altered DNA methylation during renal tumorigenesis, tumorous tissues (T) and corresponding nontumorous renal tissues (N) from 94 patients with renal tumors, and normal renal tissues (C) from 16 patients without renal tumors were investigated. DNA methylation status on CpG islands of the p16, human MutL homologue 1 (hMLH1), von-Hippel Lindau (VHL) and thrombospondin-1 (THBS-1) genes and the methylated in tumor (MINT) -1, -2, -12, -25 and -31 clones and DNA methyltransferase (DNMT) 1 expression were examined by bisulfite modification and immunohistochemistry, respectively. The average number of methylated CpG islands was significantly higher in N than in C, and was even higher in T. The average number of methylated CpG islands in N was significantly correlated with a higher histological grade of corresponding conventional renal cell carcinomas (RCCs). The average number of methylated CpG islands in RCCs was significantly correlated with macroscopic configuration with extranodular or multinodular growth, higher histological grade, infiltrating growth pattern and vascular involvement. The recurrence-free survival rate of patients with RCCs showing accumulation of DNA methylation was significantly lower than that of patients not showing this feature. The incidence of nuclear immunoreactivity for DNMT1 tended to be higher in proximal tubules from N than in those from C, and was significantly higher in RCCs. From the viewpoint of altered DNA methylation, N is at the precancerous stage, and N showing accumulation of DNA methylation may generate more malignant RCCs. Regional DNA hypermethylation may be associated with renal tumorigenesis from a precancerous condition to malignant progression and become a predictor of patient prognosis. © 2006 Wiley-Liss, Inc. [source] Frequent promoter hypermethylation and low expression of the MGMT gene in oligodendroglial tumorsINTERNATIONAL JOURNAL OF CANCER, Issue 3 2005Maria Möllemann Abstract Allelic losses on the chromosome arms 1p and 19q have been associated with favorable response to chemotherapy and good prognosis in anaplastic oligodendroglioma patients, but the molecular mechanisms responsible for this relationship are as yet unknown. The DNA repair enzyme O6 -methylguanine DNA methyltransferase (MGMT) may cause resistance to DNA-alkylating drugs commonly used in the treatment of anaplastic oligodendrogliomas and other malignant gliomas. We report on the analysis of 52 oligodendroglial tumors for MGMT promoter methylation, as well as mRNA and protein expression. Using sequencing of sodium bisulfite-modified DNA, we determined the methylation status of 25 CpG sites within the MGMT promoter. In 46 of 52 tumors (88%), we detected MGMT promoter hypermethylation as defined by methylation of more than 50% of the sequenced CpG sites. Real-time reverse transcription-PCR showed reduced MGMT mRNA levels relative to non-neoplastic brain tissue in the majority of tumors with hypermethylation. Similarly, immunohistochemical analysis showed either no or only small fractions of MGMT positive tumor cells. MGMT promoter hypermethylation was significantly more frequent and the percentage of methylated CpG sites in the investigated MGMT promoter fragment was significantly higher in tumors with loss of heterozygosity on chromosome arms 1p and 19q as compared to tumors without allelic losses on these chromosomes arms. Taken together, our data suggest that MGMT hypermethylation and low or absent expression are frequent in oligodendroglial tumors and likely contribute to the chemosensitivity of these tumors. [source] Single-molecule analysis of chromatin: Changing the view of genomes one molecule at a timeJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2008Santhi Pondugula Abstract Wrapping DNA into chromatin provides a wealth of regulatory mechanisms that ensure normal growth and development in eukaryotes. Our understanding of chromatin structure, including nucleosomes and non-histone protein,DNA interactions, has benefited immensely from nuclease and chemical digestion techniques. DNA-bound proteins, such as histones or site-specific factors, protect DNA against nuclease cleavage and generate large nucleosomal or small regulatory factor footprints. Chromatin subject to distinct modes of regulation often coincides with sites of nuclease hypersensitivity or nucleosome positioning. An inherent limitation of cleavage-based analyses has been the inability to reliably analyze regions of interest when levels of digestion depart from single-hit kinetics. Moreover, cleavage-based techniques provide views that are averaged over all the molecules in a sample population. Therefore, in cases of occupancy of multiple regulatory elements by factors, one cannot define whether the factors are bound to the same or different molecules in the population. The recent development of DNA methyltransferase-based, single-molecule MAP-IT technology overcomes limitations of ensemble approaches and has opened numerous new avenues in chromatin research. Here, we review the strengths, limitations, applications and future prospects of MAP-IT ranging from structural issues to mechanistic questions in eukaryotic chromatin regulation. J. Cell. Biochem. 105: 330,337, 2008. © 2008 Wiley-Liss, Inc. [source] Emerging Role of Epigenetics in the Actions of AlcoholALCOHOLISM, Issue 9 2008Shivendra D. Shukla This review deals with the recent developments on the epigenetic effects of ethanol. A large body of data have come from studies in liver and in neuronal systems and involve post-translational modifications in histones and methylations in DNA. Ethanol causes site selective acetylation, methylation, and phosphorylation in histone. With respect to methylations the methyl group donating system involving S-adenosyl methionine appears to play a central role. There is contrasting effect of acetylation versus methylation on the same site of histone, as it relates to the transcriptional activation. Epigenetic memory also appears to correlate with liver pathology and Mallory body formation. Experimental evidence supports transcriptional regulation of genes in the CNS by DNA methylations. These studies are contributing towards a better understanding of a novel epigenetic regulation of gene expression in the context of alcohol. The critical steps and the enzymes (e.g., histone acetyltransferase, histone deacetylase, DNA methyltransferase) responsible for the epigenetic modifications are prime targets for intense investigation. The emerging data are also beginning to offer novel insight towards defining the molecular actions of ethanol and may contribute to potential therapeutic targets at the nucleosomal level. These epigenetic studies have opened up a new avenue of investigation in the alcohol field. [source] Alterations of DNA methylation and clinicopathological diversity of human cancersPATHOLOGY INTERNATIONAL, Issue 9 2008Yae Kanai Alterations of DNA methylation can account for the histological heterogeneity, reflected in the stepwise progression and complex biological characteristics of human cancers, that genetic alterations alone cannot explain. Analysis of DNA methylation status in tissue samples can be an aid to understanding the molecular mechanisms of multistage carcinogenesis. Human cancer cells show a drastic change in DNA methylation status, that is, overall DNA hypomethylation and regional DNA hypermethylation, which results in chromosomal instability and silencing of tumor-suppressor genes. Overexpression of DNA methyltransferase (DNMT) 1 is not a secondary result of increased cell proliferative activity but may underline the CpG island methylator phenotype of cancers. Splicing alteration of DNMT3B may result in chromosomal instability through DNA hypomethylation of pericentromeric satellite regions. Alterations of DNA methylation are observed even in the precancerous stage frequently associated with chronic inflammation and/or persistent viral infection or with cigarette smoking. Precancerous conditions showing alterations of DNA methylation may generate more malignant cancers. Aberrant DNA methylation is significantly associated with aggressiveness of cancers and poorer outcome of cancer patients. Genome-wide analysis of DNA methylation status based on array-based technology may identify DNA methylation profiles that can be used as appropriate indicators for carcinogenetic risk estimation and prognostication. [source] A bicistronic SIN-lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell linesTHE JOURNAL OF GENE MEDICINE, Issue 9 2003Emmanuel Richard Abstract Background Erythropoietic protoporphyria (EPP) is an inherited disease characterised by a ferrochelatase (FECH) deficiency, the latest enzyme of the heme biosynthetic pathway, leading to the accumulation of toxic protoporphyrin in the liver, bone marrow and spleen. We have previously shown that a successful gene therapy of a murine model of the disease was possible with lentiviral vectors even in the absence of preselection of corrected cells, but lethal irradiation of the recipient was necessary to obtain an efficient bone marrow engraftment. To overcome a preconditioning regimen, a selective growth advantage has to be conferred to the corrected cells. Methods We have developed a novel bicistronic lentiviral vector that contains the human alkylating drug resistance mutant O6 -methylguanine DNA methyltransferase (MGMT G156A) and FECH cDNAs. We tested their capacity to protect hematopoietic cell lines efficiently from alkylating drug toxicity and correct enzymatic deficiency. Results EPP lymphoblastoid (LB) cell lines, K562 and cord-blood-derived CD34+ cells were transduced at a low multiplicity of infection (MOI) with the bicistronic constructs. Resistance to O6 -benzylguanine (BG)/N,N,-bis(2-chloroethyl)- N -nitrosourea (BCNU) was clearly shown in transduced cells, leading to the survival and expansion of provirus-containing cells. Corrected EPP LB cells were selectively amplified, leading to complete restoration of enzymatic activity and the absence of protoporphyrin accumulation. Conclusions This study demonstrates that a lentiviral vector including therapeutic and G156A MGMT genes followed by BG/BCNU exposure can lead to a full metabolic correction of deficient cells. This vector might form the basis of new EPP mouse gene therapy protocols without a preconditioning regimen followed by in vivo selection of corrected hematopoietic stem cells. Copyright © 2003 John Wiley & Sons, Ltd. [source] Prognostic significance of O6 -methylguanine DNA methyltransferase and p57 methylation in patients with diffuse large B-cell lymphomasAPMIS, Issue 2 2009SUN MI LEE To evaluate whether promoter methylation is related to responsiveness for chemotherapy or clinical outcome, we performed an association analysis between methylation and clinical outcomes. Patients with nodal diffuse large B-cell lymphomas (DLBCL) at a single institute (n=44) were studied for methylation of tumor-related genes, MGMT, p15INK4B, p16INK4A, p16INK4A, Mad2, TMS1/ASC, CASP8, and GSTP1. The clinical behavior of DLBCL after chemotherapy was followed up and analyzed. Hypermethylation of promoters of MGMT, p15INK4B, p16INK4A, p16INK4A, Mad2, and TMS1/ASC genes was observed in 52.3%, 31.8%, 54.5%, 47.7%, 50%, and 2.3% of the cases, respectively. Methylation of CASP8 and GSTP1 genes was not observed. Promoter methylation was not related to chemo-responsiveness, disease-free survival, and progress of disease after chemotherapy. However, in overall survival analyses, MGMT methylation (p<0.05) and responsiveness to chemotherapy (p<0.01) were significant prognostic factors in patients with DLBCL. In the low-risk group, patients with p57 methylation showed longer overall survival than patients without p57 methylation (p=0.02) and all patients with p57 methylation were alive during follow-up. Our results demonstrate that aberrant promoter methylation of MGMT and p57 is an additional biological marker for predicting increased overall survival in patients with DLBCL. [source] Association between enhanced type I collagen expression and epigenetic repression of the FLI1 gene in scleroderma fibroblastsARTHRITIS & RHEUMATISM, Issue 7 2006Youngqing Wang Objective Scleroderma (systemic sclerosis; SSc) is an autoimmune disease characterized by vasculopathy and widespread organ fibrosis. Altered fibroblast function, both in vivo and in vitro, is well documented and illustrated by augmented synthesis and deposition of extracellular matrix proteins. We undertook this study to investigate the possibility that epigenetic mechanisms mediate the emergence and persistence of the altered SSc fibroblast phenotype. Methods The effects of DNA methyltransferase and histone deacetylase inhibitors on collagen expression and the level of epigenetic mediators in fibroblasts were examined. The effects of transient transfection of SSc fibroblasts with FLI1 gene and normal cells with FLI1 antisense construct on collagen expression were determined. The methylation status of the FLI1 promoter was tested in cultured cells and in SSc and normal skin biopsy specimens. Results Increased levels of epigenetic mediators in SSc fibroblasts were noted. The addition of epigenetic inhibitors to cell cultures normalized collagen expression in SSc fibroblasts. The augmented collagen synthesis by SSc fibroblasts was linked to epigenetic repression of the collagen suppressor gene FLI1. Heavy methylation of the CpG islands in the FLI1 promoter region was demonstrated in SSc fibroblasts and skin biopsy specimens. Conclusion The results of this study indicate that epigenetic mechanisms may mediate the fibrotic manifestations of SSc. The signal transduction leading to the SSc fibrotic phenotype appears to converge on DNA methylation and histone deacetylation at the FLI1 gene. [source] Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of HP1352, a putative DNA methyltransferase in Helicobacter pyloriACTA CRYSTALLOGRAPHICA SECTION D, Issue 4 2003Katja Schirwitz The putative methyltransferase gene HP1352 from Helicobacter pylori strain 26695 was heterologously expressed in Escherichia coli. The 359-amino-acid gene product was purified and crystallized. The crystals belong to space group I212121 and show diffraction to at least 2.5,Å resolution. The unit-cell parameters are a = 69.6, b = 86.6, c = 140.0,Å. A greater than 90% complete native data set has been collected and structure determination using the molecular-replacement method is ongoing. [source] Epigenetic aberrations and therapeutic implications in gliomasCANCER SCIENCE, Issue 6 2010Atsushi Natsume Almost all cancer cells have multiple epigenetic abnormalities, which combine with genetic changes to affect many cellular processes, including cell proliferation and invasion, by silencing tumor-suppressor genes. In this review, we focus on the epigenetic mechanisms of DNA hypomethylation and CpG island hypermethylation in gliomas. Aberrant hypermethylation in promoter CpG islands has been recognized as a key mechanism involved in the silencing of cancer-associated genes and occurs at genes with diverse functions related to tumorigenesis and tumor progression. Such promoter hypermethylation can modulate the sensitivity of glioblastomas to drugs and radiotherapy. As an example, the methylation of the O6-methylguanine DNA methyltransferase (MGMT) promoter is a specific predictive biomarker of tumor responsiveness to chemotherapy with alkylating agents. Further, we reviewed reports on pyrosequencing , a simple technique for the accurate and quantitative analysis of DNA methylation. We believe that the quantification of MGMT methylation by pyrosequencing might enable the selection of patients who are most likely to benefit from chemotherapy. Finally, we also evaluated the potential of de novo NY-ESO-1, the most immunogenic cancer/testis antigen (CTA) discovered thus far, as an immunotherapy target. The use of potent epigenetics-based therapy for cancer cells might restore the abnormally regulated epigenomes to a more normal state through epigenetic reprogramming. Thus, epigenetic therapy may be a promising and potent treatment for human neoplasia. (Cancer Sci 2010) [source] Sequence-specific Methyltransferase- Induced Labeling of DNA (SMILing DNA)CHEMBIOCHEM, Issue 3 2004Goran Pljevalj Abstract A new concept for sequence-specific labeling of DNA by using chemically modified cofactors for DNA methyltransferases is presented. Replacement of the amino acid side chain of the natural cofactor S -adenosyl- L -methionine with an aziridine group leads to a cofactor suitable for DNA methyltransferase-catalyzed sequence-specific coupling with DNA. Sequence-specifically fluorescently labeled plasmid DNA was obtained by using the DNA methyltransferase from Thermus aquaticus (M.TaqI) as catalyst and attaching a fluorophore to the aziridine cofactor. First results suggest that all classes of DNA methyltransferases with different recognition sequences can be used. In addition, this novel method for DNA labeling should be applicable to a wide variety of reporter groups. [source] Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3bGENES TO CELLS, Issue 7 2006Akiko Tsumura DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1,/, or Dnmt3a,/,Dnmt3b,/, ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases. [source] De Novo DNA methylation independent establishment of maternal imprint on X chromosome in mouse oocytesGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 12 2008Hatsune Chiba Mouse blastocyst stage embryo stained for histone H3 lysine-27 trimethylation (red) and DNA (blue). H3K27me3 marks the inactive X chromosome. The study by Chiba et al. in this issue suggests that de novo DNA methyltransferases are dispensable for setting the imprint on the maternally-derived X chromsome in growing oocytes. See Chiba et al. in this issue. [source] Genetic evidence for a maternal effect locus controlling genomic imprinting and growthGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 4 2005Amanda R. Duselis Abstract Crosses between two species of deer mouse (Peromyscus) yield dramatic parent-of-origin effects. Female P. maniculatus (BW) crossed with male P. polionotus (PO) produce animals smaller than either parent. PO females crossed with BW males yield lethal overgrowth that has been associated with loss-of-imprinting (LOI). Previously, we mapped two loci influencing fetal growth. These two loci, however, do not account for the LOI, nor for the dysmorphic phenotypes. Here we report that maternal genetic background strongly influences the LOI. Analyses of crosses wherein maternal genetic background is varied suggest that this effect is likely due to the action of a small number of loci. We have termed these putative loci Meil. Estimation of Meil loci number was confounded by skewed allelic ratios in the intercross line employed. We show that the Meil loci are not identical to any of the DNA methyltransferases shown to be involved in regulation of genomic imprinting. genesis 43:155,165, 2005. © 2005 Wiley-Liss, Inc. [source] Hypermethylation of gene promoters in hematological neoplasiaHEMATOLOGICAL ONCOLOGY, Issue 4 2002C. S. Chim Abstract Cancer cells are associated with global hypomethylation but with focal hypermethylation of specific gene promoters organized as CpG island. DNA methyltransferases, DNMT1 and 3 (3a and 3b), have been implicated in mediating maintenance and de novo methylation. Hypermethylation of gene promoters results in the inactivation of the corresponding genes, by preclusion of the formation of the transcription complex, due to the recruitment of MBP, MeCPs and histone deacetylase. This results in the deacetylation of histone and thus a compact chromatin complex unfavourable for the initiation of transcription. This methylation-associated gene silencing has been demonstrated in various genes including tumour suppressor genes (p15, p16, p73, VHL). Therefore, gene promoter hypermethylation collaborates with other mechanisms of gene inactivation such as deletion and intragenic mutations to fulfil Knudson's hypothesis. Hypermethylation may serve as a molecular disease marker for the detection of minimal residual disease. Emerging evidence suggests a possible prognostic value of gene promoter hypermethylation. Moreover, gene hypermethylation may also serve as a target for therapeutic invention by hypomethylating agents. Copyright © 2002 John Wiley & Sons, Ltd. [source] Alterations of DNA methylation and histone modifications contribute to gene silencing in hepatocellular carcinomasHEPATOLOGY RESEARCH, Issue 11 2007Yutaka Kondo Aim:, The aim of the present study was to examine DNA methylation and histone modification changes in hepatocellular carcinomas (HCC). Methods:, DNA methylation in the P16, RASSF1a, progesterone receptor (PGR) and estrogen receptor , (ER,) promoters was determined by quantitative bisulfite-pyrosequencing technique in HCC patients. Histone H3-lysine (K) 4, H3-K9 and H3-K27 modifications in all these four genes were examined by chromatin immunoprecipitation (ChIP) assay in HCC cell lines. Expression of two DNA methyltransferases (DNMT1 and DNMT3b) and three histone methyltransferases (SUV39H1, G9a and EZH2) in HCC patients was measured by real-time polymerase chain reaction. Results:, Aberrant DNA methylation was detected in all the HCC. Patients with DNA methylation in the RASSF1a, PGR andER, promoters in cancers also had substantial DNA methylation in their non-cancerous liver tissues, whereas DNA methylation in the P16 promoter was cancer specific. Epigenetic states in HCC cell lines showed that silencing of P16 and RASSF1a depended on DNA methylation and histone H3-K9 methylation. However, silencing of the PGR and ER, genes was more closely related to H3-K27 methylation rather than DNA methylation. Consistent with the alteration of histone status, higher expression of G9a and EZH2 was found in HCC than in non-cancerous liver tissues (P < 0.01). Conclusion:, These data suggest that multiple epigenetic silencing mechanisms are inappropriately active in HCC cells. [source] Conservation of DNA methylation in dipteran insectsINSECT MOLECULAR BIOLOGY, Issue 2 2004J. Marhold Abstract DNA methylation is a central mechanism of epigenetic regulation. Whereas vertebrate DNA methylation requires at least four different DNA methyltransferases, Drosophila melanogaster only utilizes a single, Dnmt2-like enzyme. This profound difference has raised the question of the evolutionary significance of the Drosophila methylation system. We have now identified Dnmt2-like open reading frames in the genome sequences of Drosophila pseudoobscura and Anopheles gambiae. These genes represent the only candidate DNA methyltransferases in their respective genomes. Consistent with a catalytic activity of Dnmt2 proteins, we could also demonstrate low but significant levels of DNA methylation in genomic DNA from these species. Lastly, we were also able to detect highly conserved Dnmt2-like open reading frames and concomitant DNA methylation in several additional Drosophila species, which suggests that Dnmt2-mediated DNA methylation has been conserved over a considerable evolutionary distance. [source] Nicotine induces the fragile histidine triad methylation in human esophageal squamous epithelial cellsINTERNATIONAL JOURNAL OF CANCER, Issue 5 2006Toshiya Soma Abstract The fragile histidine triad (FHIT) gene has been proposed to have an important role in very early carcinogenesis. Methylation of the FHIT gene is associated with transcriptional inactivation in esophageal squamous cell carcinoma, and FHIT inactivation has been linked to smoking-related carcinogenesis. In this study, we confirmed methylation of the FHIT gene in human esophageal squamous epithelial cells (HEECs) and examined whether nicotine induced alteration of FHIT. Methylation status in the promoter region of the FHIT gene and p16INK4A gene was determined by methylation-specific PCR in HEECs exposed to nicotine under various conditions. Methylation status of the FHIT gene was confirmed by DNA-sequencing analysis. Protein expression of Fhit and the DNA methyltransferases (DNMTs) DNMT1 and DNMT3a were assessed by immunoblot analysis. In the absence of nicotine, methylation of the FHIT gene and attenuation of Fhit protein were not detected in HEECs. Nicotine induced the methylation of FHIT gene and attenuated Fhit protein in association with increased expression of DNMT3a. Reexpression of Fhit protein in HEECs was found after cessation of moderate- to long-term exposure to nicotine. Our results show that nicotine induces methylation of the FHIT gene followed by loss of Fhit protein expression in HEECs. Continuous smoking may thus increase the risk of esophageal cancer. © 2006 Wiley-Liss, Inc. [source] Transcriptional and post-transcriptional control of DNA methyltransferase 3B is regulated by phosphatidylinositol 3 kinase/Akt pathway in human hepatocellular carcinoma cell linesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 1 2010Chuanzhong Mei Abstract DNA methyltransferases (DNMTs) are essential for maintenance of aberrant methylation in cancer cells and play important roles in the development of cancers. Unregulated activation of PI3K/Akt pathway is a prominent feature of many human cancers including human hepatocellular carcinoma (HCC). In present study, we found that DNMT3B mRNA and protein levels were decreased in a dose- and time-dependent manner in HCC cell lines with LY294002 treatment. However, we detected that LY294002 treatment did not induce increase of the degradation of DNMT3B protein using protein decay assay. Moreover we found that Akt induced alteration of the expression of DNMT3B in cells transfected with myristylated variants of Akt2 or cells transfected with small interfering RNA respectively. Based on DNMT3B promoter dual-luciferase reporter assay, we found PI3K pathway regulates DNMT3B expression at transcriptional level. And DNMT3B mRNA decay analysis suggested that down-regulation of DNMT3B by LY294002 is also post-transcriptional control. Furthermore, we demonstrated that LY294002 down-regulated HuR expression in a time-dependent manner in BEL-7404. In summary, we have, for the first time, demonstrate that PI3K/Akt pathway regulates the expression of DNMT3B at transcriptional and post-transcriptional levels, which is particularly important to understand the effects of PI3K/Akt and DNMT3B on hepatocarcinogenesis. J. Cell. Biochem. 111: 158,167, 2010. © 2010 Wiley-Liss, Inc. [source] Effect of Alcohol Consumption on CpG Methylation in the Differentially Methylated Regions of H19 and IG-DMR in Male Gametes,Implications for Fetal Alcohol Spectrum DisordersALCOHOLISM, Issue 9 2009Lillian A. Ouko Background:, Exposure to alcohol in utero is the main attributable cause of fetal alcohol spectrum disorders (FASD) which in its most severe form is characterized by irreversible behavioral and cognitive disability. Paternal preconception drinking is not considered to be a significant risk factor, even though animal studies have demonstrated that chronic paternal alcohol consumption has a detrimental effect on the physical and mental development of offspring even in the absence of in utero alcohol exposure. It has been documented that alcohol can reduce the levels and activity of DNA methyltransferases resulting in DNA hypomethylation and that reduced methyltransferase activity can cause activation of normally silenced genes. The aim of this study was to establish a link between alcohol use in men and hypomethylation of paternally imprinted loci in sperm DNA in genomic regions critical for embryonic development, thus providing a mechanism for paternal effects in the aetiology of FASD. Methods:, Sperm DNA from male volunteers was bisulfite treated and the methylation patterns of 2 differentially methylated regions (DMRs), H19 and IG-DMR, analyzed following sequencing of individual clones. The methylation patterns were correlated with the alcohol consumption levels of the volunteer males. Results:, There was a pattern of increased demethylation with alcohol consumption at the 2 imprinted loci with a significant difference observed at the IG-DMR between the nondrinking and heavy alcohol consuming groups. Greater inter-individual variation in average methylation was observed at the H19 DMR and individual clones were more extensively demethylated than those of the IG-DMR. CpG site #4 in the IG-DMR was preferentially demethylated among all individuals and along with the H19 DMR CpG site #7 located within the CTCF binding site 6 showed significant demethylation in the alcohol consuming groups compared with the control group. Conclusion:, This study demonstrates a correlation between chronic alcohol use and demethylation of normally hypermethylated imprinted regions in sperm DNA. We hypothesize that, should these epigenetic changes in imprinted genes be transmitted through fertilization, they would alter the critical gene expression dosages required for normal prenatal development resulting in offspring with features of FASD. [source] Progressive up-regulation of genes encoding DNA methyltransferases in the colorectal adenoma-carcinoma sequenceMOLECULAR CARCINOGENESIS, Issue 9 2007Wolfgang M. Schmidt Abstract Epigenetic silencing is a prominent feature of cancer. Here, we investigated the expression of DNA demethylase and three DNA methyltransferases during colorectal tumorigenesis comparing the genes encoding DNA methyltransferases 1 (DNMT1), 3A, and 3B (DNMT3A and DNMT3B) with methyl-CpG binding domain protein 2 (MBD2), recently described as the only active DNA demethylase. Total RNA isolated from normal colonic mucosa (n,=,24), benign adenomas (n,=,18), and malignant colorectal carcinomas (n,=,32) was analyzed by reverse transcriptase-PCR with subsequent quantification by capillary gel electrophoresis. In contrast to MBD2, expression of DNMT1 and DNMT3A increased in parallel to the degree of dysplasia, with significant overexpression in the malignant lesion when compared with mucosa or with benign lesions (DNMT1). Pairwise comparisons between tumors and matched, adjacent healthy mucosa tissue (n,=,13) revealed that expression of all three genes encoding DNA methyltransferases increased by two- to three-fold. Our data suggest a relevant role of the DNA methyltransferases during colorectal tumorigenesis. This increase is not counterbalanced by enhanced expression of the demethylating component MBD2. As a consequence, epigenetic regulation in the adenoma-carcinoma sequence may be driven by increased methylating activity rather than suppressed demethylation. © 2007 Wiley-Liss, Inc. [source] Elevated mRNA levels of DNA methyltransferase-1 as an independent prognostic factor in primary nonsmall cell lung cancerCANCER, Issue 5 2006Hojoong Kim MD Abstract BACKGROUND. Despite many reports about the involvement of DNA methyltransferases (DNMTs) in human cancers, including nonsmall cell lung cancer (NSCLC), the clinicopathologic significance of DNMTs in primary NSCLC remains to be elucidated. METHODS. The relation between the mRNA levels of DNMTs (1 and 3b) and the promoter methylation of the p16, RAR,2, H-cadherin, GSTP1, RIZ, and FHIT genes and the clinicopathologic features in 102 fresh-frozen tissues and paraffin blocks were retrospectively studied. The mRNA levels of the DNMTs were assessed via semiquantitative reverse-transcription polymerase chain reaction (RT-PCR), and the methylation status of the CpG islands were determined by methylation-specific PCR. RESULTS. The mRNA levels of DNMT1 and DNMT3b were elevated in 53% and 58% of 102 NSCLCs, respectively. Hypermethylation of p16, RAR,2, H-cadherin, GSTP1, RIZ, and FHIT occurred in 37%, 38%, 34%, 18%, 9%, and 31% of patients, respectively. Univariate analysis showed that elevated DNMT mRNA levels were not significantly associated with the hypermethylation of 6 genes. However, the elevated mRNA levels of DNMT1 were determined to be significantly associated with the hypermethylation of the p16 promoter (odds ratio [OR] = 2.70, 95% confidence interval [95% CI], 1.02,7.15; P = .02), after controlling for age, gender, pack-years smoked, histology, and pathologic stage. The hazard of failure in cases with elevated mRNA levels of DNMT1 was 3.51 (95% CI, 1.18,12.76; P = .02) times higher than that in those without. The elevated mRNA levels of DNMT3b were not ultimately associated with patient prognosis. CONCLUSIONS. Elevated mRNA expression of DNMT1 may be an independent prognostic factor in NSCLC and CpG island hypermethylation in NSCLC may be maintained by a complex interaction of several factors rather than by a simple transcriptional up-regulation of DNMT1. Cancer 2006. © 2006 American Cancer Society. [source] Sequence-specific Methyltransferase- Induced Labeling of DNA (SMILing DNA)CHEMBIOCHEM, Issue 3 2004Goran Pljevalj Abstract A new concept for sequence-specific labeling of DNA by using chemically modified cofactors for DNA methyltransferases is presented. Replacement of the amino acid side chain of the natural cofactor S -adenosyl- L -methionine with an aziridine group leads to a cofactor suitable for DNA methyltransferase-catalyzed sequence-specific coupling with DNA. Sequence-specifically fluorescently labeled plasmid DNA was obtained by using the DNA methyltransferase from Thermus aquaticus (M.TaqI) as catalyst and attaching a fluorophore to the aziridine cofactor. First results suggest that all classes of DNA methyltransferases with different recognition sequences can be used. In addition, this novel method for DNA labeling should be applicable to a wide variety of reporter groups. [source] | |||||||||||||||||||||||||||||||