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DNA Demethylation (dna + demethylation)
Selected AbstractsCytoplasmic localization of oocyte-specific variant of porcine DNA methyltransferase-1 during early developmentDEVELOPMENTAL DYNAMICS, Issue 7 2009Young Sun Jeong Abstract DNA methyltransferase-1 (Dnmt1) is involved in the maintenance of genomic methylation patterns. Rather than full-length Dnmt1, mouse oocytes have a truncated variant called Dnmt1o. Immunofluorescence data showed that Dnmt1o localized to the cytoplasm, but this has not been confirmed using more direct methods. The cytoplasmic localization of Dnmt1o has been assigned to the main cause of global DNA demethylation in early mouse embryos. We studied localization of Dnmt1o in mouse and pig embryos. We identified pig Dnmt1o protein and its transcript with unique 5,-end sequence. Physically separating mouse and pig 2-cell embryos into their nuclear and cytoplasmic components demonstrated that Dnmt1o of both species localized to the cytoplasm. Cloned pig embryos had Dnmt1o as the main form, with no indication of somatic Dnmt1. These findings indicate that Dnmt1o is cytoplasmic during early development; its presence in both pig and mouse embryos further suggests that Dnmt1o is conserved in mammals. Developmental Dynamics 238:1666,1673, 2009. © 2009 Wiley-Liss, Inc. [source] RNA expression microarray analysis in mouse prospermatogonia: Identification of candidate epigenetic modifiers,DEVELOPMENTAL DYNAMICS, Issue 4 2008Christophe Lefèvre Abstract The mammalian totipotent and pluripotent lineage exhibits genome-wide dynamics with respect to DNA methylation content. The first phase of global DNA demethylation and de novo remethylation occurs during preimplantation development and gastrulation, respectively, while the second phase occurs in primordial germ cells and primary oocytes/prospermatogonia, respectively. These dynamics are indicative of a comprehensive epigenetic resetting or reprogramming of the genome in preparation for major differentiation events. To gain further insight into the mechanisms driving DNA methylation dynamics and other types of epigenetic modification, we performed an RNA expression microarray analysis of fetal prospermatogonia at the stage when they are undergoing rapid de novo DNA remethylation. We have identified a number of highly or specifically expressed genes that could be important for determining epigenetic change in prospermatogonia. These data provide a useful resource in the discovery of molecular pathways involved in epigenetic reprogramming in the mammalian germ line. Developmental Dynamics 237:1082,1089, 2008. © 2008 Wiley-Liss, Inc. [source] Genome-wide and locus-specific DNA hypomethylation in G9a deficient mouse embryonic stem cellsGENES TO CELLS, Issue 1 2007Kohta Ikegami In the mammalian genome, numerous CpG-rich loci define tissue-dependent and differentially methylated regions (T-DMRs). Euchromatin from different cell types differs in terms of its tissue-specific DNA methylation profile as defined by these T-DMRs. G9a is a euchromatin-localized histone methyltransferase (HMT) and catalyzes methylation of histone H3 at lysines 9 and 27 (H3-K9 and -K27). To test whether HMT activity influences euchromatic cytosine methylation, we analyzed the DNA methylation status of approximately 2000 CpG-rich loci, which are predicted in silico, in G9a,/, embryonic stem cells by restriction landmark genomic scanning (RLGS). While the RLGS profile of wild-type cells contained about 1300 spots, 32 new spots indicating DNA demethylation were seen in the profile of G9a,/, cells. Virtual-image RLGS (Vi-RLGS) allowed us to identify the genomic source of ten of these spots. These were confirmed to be cytosine demethylated, not just at the Not I site detected by the RLGS but extending over several kilobase pairs in cis. Chromatin immunoprecipitation (ChIP) confirmed these loci to be targets of G9a, with decreased H3-K9 and/or -K27 dimethylation in the G9a,/, cells. These data indicate that G9a site-selectively contributes to DNA methylation. [source] DNA demethylation of vascular endothelial growth factor-C is associated with gene expression and its possible involvement of lymphangiogenesis in gastric cancerINTERNATIONAL JOURNAL OF CANCER, Issue 8 2007Shunji Matsumura Abstract Previous studies have indicated that lymphangiogenesis in solid tumors is associated with lymphatic metastasis. Overexpression of Vascular endothelial growth factor (VEGF)-C plays a major role in lymphangiogenesis in cancers. In the present study, DNA methylation and expression of the VEGF-C gene was investigated in gastric cancer (GC). Four GC cell lines (MKN-45, MKN-74, HSC-39 and HSC-43) showed no expression of VEGF-C, and the VEGF-C gene was found to be methylated in these cells. In contrast, 7 GC cell lines (MKN-1, MKN-7, MKN-28, TMK-1, KATO-III, SH101-P4 and HSC-44PE) expressed VEGF-C, and the VEGF-C gene was found to be unmethylated in these cell lines. In addition, expression of VEGF-C mRNA was retrieved by treatment with a demethylating agent, Aza-2,-deoxycytidine. In GC tissue samples, bisulfite DNA sequencing analysis revealed that VEGF-C was not methylated in 9 (29.0%) of 31 GC samples, whereas demethylation was not observed in corresponding non-neoplastic mucosa samples. Overexpression of VEGF-C mRNA was observed in 16 (51.6%) of 31 GC samples by quantitative reverse transcription-polymerase chain reaction. Of the 9 GC cases with VEGF-C demethylation, 8 (88.9%) overexpressed VEGF-C. In contrast, of the 22 GC cases without VEGF-C demethylation, 8 (36.4%) overexpressed VEGF-C (p = 0.0155). Furthermore, lymphatic vessel density determined by immunostaining of podoplanin in GC tissues was associated with overexpression of VEGF-C (p < 0.0001). These results suggest that demethylation and activation of the VEGF-C gene is likely involved in lymphangiogenesis in GC. © 2007 Wiley-Liss, Inc. [source] Mechanisms of DNA breaks induction in vivo by 5-azacytidine: paths of micronucleus induction by azaCJOURNAL OF APPLIED TOXICOLOGY, Issue 3 2008P. Morales-Ramírez Abstract The aim of the present study was to correlate the time-response curves of micronucleated polychromatic erythrocyte (MN-PCE) induction by 5-azacytidine (azaC) with the possible processes involved in DNA break production; this is based on the results previously published by other authors. The MN-PCE induction at two different doses of azaC was determined by sampling blood from the tails of mice before the acute treatment and over nine periods of 8 h each afterwards. Both doses caused two peaks of MN-PCE induction, one at 32 h and another at 48 h, approximately; a shoulder was detected that remained high from 56 h up to the end of the study (72 h). These results suggest that azaC induced DNA breaks and subsequently MN (micronucleus) by three different mechanisms, and in agreement with data in the literature, these could be successively the following: (i) during excision of the large adduct comprising the DNA methyl transferase covalently linked to DNA; (ii) failure of recombination repair or mismatch repair; and (iii) persistent chromosome fragility in G-C rich sites due to DNA demethylation and chromatin decondensation. Copyright © 2007 John Wiley & Sons, Ltd. [source] Maternal chromatin remodeling during maturation and after fertilization in mouse oocytesMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 2 2004Marcella Spinaci Abstract Immunofluorescence staining with antibodies against acetylated histone H4 and 5-methylcytosine was carried out to investigate female chromatin remodeling throughout oocyte maturation and chromatin rearrangement involving both male and female genomes after fertilization. Oocyte cytoplasm remodels female chromatin in preparation of the fertilizing event and the subsequent chromatin rearrangement. Histone H4 are in fact progressively deacetylated whereas demethylating enzymes do not seem to be active over this period. The acetylase/deacetylase balance seems to be cell cycle dependent as female chromatin is deacetylated during maturation and reacetylated at telophase II stage both after fertilization and activation. On the contrary, DNA demethylation seems to be strictly selective. It is in fact confined to the remodeling of paternal genome after fertilization of mature oocytes as the ooplasm is not effective in demethylating either paternal chromatin in germinal vesicle breakdown (GVBD) fertilized oocytes or maternal genome of partenogenetically activated oocytes. Surprisingly, we induced maternal chromatin demethylation after fertilization by treating oocytes with a combination of a methyltransferase inhibitor, 5-azacytidine (5-AzaC), and a reversible and specific inhibitor of histone deacetylase, trichostatin A (TSA). This treatment likely induces a hyperacetylation of histones (thus favoring the access to demethylating enzymes by opening female chromatin structure) associated with a block of reparative methylation by inhibiting methytransferases. This manipulation of chromatin remodeling may have applications regarding the biological significance of aberrant DNA methylation. Mol. Reprod. Dev. 69: 215,221, 2004. © 2004 Wiley-Liss, Inc. [source] Flowering and dwarfism induced by DNA demethylation in Pharbitis nilPHYSIOLOGIA PLANTARUM, Issue 1 2010Yuiko Iwase Flowering and dwarfism induced by 5-azacytidine and zebularine, which both cause DNA demethylation, were studied in a short-day (SD) plant Pharbitis nil (synonym Ipomoea nil), var. Violet whose photoinduced flowering state does not last for a long period of time. The DNA demethylating reagents induced flowering under non-inductive long-day (LD) conditions. The flower-inducing effect of 5-azacytidine did not last for a long period of time, and the plants reverted to vegetative growth. The progeny of the plants that were induced to flower by DNA demethylation did not flower under the non-inductive photoperiodic conditions. These results suggest that the flowering-related genes were activated by DNA demethylation and then remethylated again in the progeny. The DNA demethylation also induced dwarfism. The dwarfism did not last for a long period of time, was not heritable and was overcome by gibberellin A3 but not by t -zeatin or kinetin. The change in the genome-wide methylation state was examined by methylation-sensitive amplified fragment length polymorphism (MS-AFLP) analysis. The analysis detected many more polymorphic fragments between the DNA samples isolated from the cotyledons treated with SD than from the cotyledons under LD conditions, indicating that the DNA methylation state was altered by photoperiodic conditions. Seven LD-specific fragments were extracted from the gel of the MS-AFLP and were sequenced. One of these fragments was highly homologous with the genes encoding ribosomal proteins. [source] Overexpression of the growth arrest and DNA damage,induced 45, gene contributes to autoimmunity by promoting DNA demethylation in lupus T cellsARTHRITIS & RHEUMATISM, Issue 5 2010Yaping Li Objective Demethylation of CD11a and CD70 regulatory regions in CD4+ T cells contributes to the development of autoreactivity and overstimulation of autoantibodies. Because growth arrest and DNA damage,induced 45, (GADD45,) reduces epigenetic silencing of genes by removing methylation marks, this study examined whether the gadd45A gene could contribute to autoimmunity by promoting DNA demethylation in T cells from patients with systemic lupus erythematosus (SLE). Methods Levels of GADD45,, CD11a, and CD70 messenger RNA (mRNA) and protein were detected by real-time reverse transcription,polymerase chain reaction and Western blotting or flow cytometry. Global DNA methylation was evaluated using Methylamp global DNA methylation quantification kits. Detection of CD4+ T cell proliferation and autologous B cell IgG antibodies was performed using commercially available kits. CD11a and CD70 promoter methylation was determined with bisulfite sequencing. Results Elevated gadd45A mRNA expression and global DNA hypomethylation were observed in CD4+ T cells from SLE patients. The levels of gadd45A mRNA were inversely proportional to the levels of DNA methylation. Positive correlations were found between gadd45A and CD11a/CD70 mRNA levels. Expression of gadd45A mRNA was increased in CD4+ T cells following ultraviolet B irradiation, and this was accompanied by increased levels of CD11a and CD70 mRNA. Moreover, increased expression of gadd45A, CD11a, and CD70 mRNA was accompanied by increased autoreactivity and excessive B cell stimulation in gadd45A -transfected CD4+ T cells. CD11a promoter methylation was also significantly reduced in transfected cells. Transfection of gadd45A small interfering RNA inhibited the autoreactivity of SLE CD4+ T cells and led to significant increases in the methylation levels of the CD11a and CD70 promoter regions. Conclusion These findings indicate that gadd45A may contribute to lupus-like autoimmunity by promoting DNA demethylation in SLE CD4+ T cells. [source] DNA demethylation at specific CpG sites in the IL1B promoter in response to inflammatory cytokines in human articular chondrocytesARTHRITIS & RHEUMATISM, Issue 11 2009Ko Hashimoto Objective To determine whether changes in the DNA methylation status in the promoter region of the gene encoding interleukin-1, (IL-1,) account for expression of IL1B messenger RNA (mRNA) after long-term treatment of human articular chondrocytes with inflammatory cytokines. Methods IL-1,, tumor necrosis factor , (TNF,) plus oncostatin M (OSM), or 5-azadeoxycytidine (5-aza-dC) was added twice weekly for 4,5 weeks to primary cultures of normal human articular chondrocytes derived from the femoral head cartilage of patients with a fracture of the femoral neck. Expression of MMP13, IL1B, TNFA, and DNMT1 was determined by SYBR Green,based quantitative reverse transcription,polymerase chain reaction (RT-PCR) analysis of genomic DNA and total RNA extracted from the same sample before and after culture. Bisulfite modification was used to identify which CpG sites in the IL1B promoter showed differential methylation between IL1B -expressing and IL1B -nonexpressing cells. The percentages of cells that were methylated at that critical CpG site (,299 bp) were quantified by a method that depended on methylation-sensitive restriction enzymes and real-time RT-PCR. Secretion of IL-1, into the culture media was assessed by enzyme-linked immunosorbent assay. Results Healthy chondrocytes did not express IL1B mRNA, but the levels were increased 5-fold by treatment with 5-aza-dC and were increased 100,1,000-fold by treatment with TNF,/OSM. The percentage CpG methylation was decreased by 5-aza-dC treatment but was reduced considerably more by IL-1, and was almost abolished by TNF,/OSM. The mRNA was translated into protein in cytokine-treated chondrocytes. Conclusion These novel findings indicate that inflammatory cytokines can change the DNA methylation status at key CpG sites, resulting in long-term induction of IL1B in human articular chondrocytes. [source] AID in reprogramming: Quick and efficientBIOESSAYS, Issue 5 2010Identification of a key enzyme called AID, its activity in DNA demethylation, may help to overcome a pivotal epigenetic barrier in reprogramming somatic cells toward pluripotency Abstract Current methods of reprogramming differentiated cells into induced pluripotent stem cells remain slow and inefficient. In a recent report published online in Nature, Bhutani et al.1 developed a cell fusion strategy, achieving quick and efficient reprogramming toward pluripotency. Using this assay, they identified an immune system protein called activation-induced cytidine deaminase, or AID, which unexpectedly is actually able to "aid" in reprogramming due to its involvement in DNA demethylation that is required for induction of the two key pluripotency genes, Oct4 and Nanog. More recently, Popp et al.2 also reported online in Nature that AID is important for complete cell reprogramming in mammals. Together, these findings provide new insights into how cells are reprogrammed, identify the specific role of AID in cell fate reversal, and advance the field of regenerative medicine. [source] | |||||||||||||