Добірка наукової літератури з теми "N-4 Cytosine Methylation"

ABSTRACT 5-Methylcytosine in chromosomal DNA represents a potential source of frequent spontaneous mutation for hyperthermophiles. To determine the relevance of this threat for the archaeon Sulfolobus acidocaldarius, the mode of GGCC methylation by its restriction-modification system, SuaI, was investigated. Distinct isoschizomers of the SuaI endonuclease were used to probe the methylation state of GGCC in native S. acidocaldarius DNA. In addition, the methylation sensitivity of the SuaI endonuclease was determined with synthetic oligonucleotide substrates and modified natural DNAs. The results show that the SuaI system uses N 4 methylation to block cleavage of its recognition site, thereby avoiding the creation of G · T mismatches by spontaneous deamination at extremely high temperature.

Background: Maternal hyperglycemia is a well-recognized risk factor for fetal congenital heart disease. However, the underlying cellular and molecular mechanisms are not well characterized. We hypothesize that maternal hyperglycemia leading to congenital heart are linked to abnormal DNA methylation and mRNA expression at cardiac specific loci. Methods: Hyperglycemia was induced in normal 8-week old CD-1 female mice with a one-time intraperitoneal injection of 150 mg/kg of streptozotocin (STZ) 2 weeks prior to mating. Histological analysis of fetal cardiac morphology was evaluated for malformations on embryonic day (E) 16.5 of control pups and pups exposed to maternal hyperglycemia. We used a massively-parallel sequencing-based methylation sensitive restriction based assay to examine genome-wide cytosine methylation levels at >1.65 million loci in neonatal hearts on post-natal (P) day 0. Functional validation was performed with real time quantitative polymerase chain reaction (RT-qPCR). Results: Cardiac structural defects occurred in 28% of the pups (n=12/45) of hyperglycemic dams versus 7% (n=4/61) of controls. Notable phenotypes were hypoplastic left or right ventricle, double outlet right ventricle, ventricular septal defect, and left ventricular outflow tract obstruction. A 10-fold increase in DNA methylation of gene promoter regions was seen in many cardiac important genes in the experimental versus control P0 neonates and have corresponding decreases in gene expression in 21/32 genes functionally validated. Conclusion: Maternal hyperglycemia alters DNA methylation and mRNA expression of some cardiac genes during heart development. Quantitative, genome-wide assessment of cytosine methylation can be used as a discovery platform to gain insight into the mechanisms of hyperglycemia-induced cardiac anomalies.

ABSTRACT BslI is a thermostable type II restriction endonuclease with interrupted recognition sequence CCNNNNN/NNGG (/, cleavage position). The BslI restriction-modification system fromBacillus species was cloned and expressed inEscherichia coli. The system is encoded by three genes: the 2,739-bp BslI methylase gene (bslIM), thebslIRα gene, and the bslIRβ gene. The α and β subunits of BslI can be expressed independently inE. coli in the absence of BslI methylase (M.BslI) protection. BslI endonuclease activity can be reconstituted in vitro by mixing the two subunits together. Gel filtration chromatography and native polyacrylamide gel electrophoresis indicated that BslI forms heterodimers (αβ), heterotetramers (α2β2), and possibly oligomers in solution. Two β subunits can be cross-linked by a chemical cross-linking agent, indicating formation of heterotetramerBslI complex (α2β2). In DNA mobility shift assays, neither subunit alone can bind DNA. DNA mobility shift activity was detected after mixing the two subunits together. Because of the symmetric recognition sequence of the BslI endonuclease, we propose that its active form is α2β2. M.BslI contains nine conserved motifs of N-4 cytosine DNA methylases within the β group of aminomethyltransferase. Synthetic duplex deoxyoligonucleotides containing cytosine hemimethylated or fully methylated at N-4 inBslI sites in the first or second cytosine are resistant toBslI digestion. C-5 methylation of the second cytosine on both strands within the recognition sequence also renders the site refractory to BslI digestion. Two putative zinc fingers are found in the α subunit of BslI endonuclease.

Estrogen receptor α (ERα) has an important role in mammary carcinogenesis, prognosis, and treatment. In human and canine mammary cancer, the most aggressive tumors show loss of ERα expression, which in human breast cancer has been attributed to methylation of the cytosine followed by guanine (CpG) island within the estrogen receptor α gene ( ESR1) promoter. This study aimed to investigate the role of ESR1 CpG island (CGI) methylation in ERα expression in canine mammary tumors. Twenty-one canine mammary samples were sorted into three groups: malignant tumor (n = 9), benign tumor (n = 8), and normal gland (n = 4). Immunohistochemical analysis and reverse-transcription quantitative real-time PCR were performed to assess ERα expression and ESR1 mRNA levels. The methylation status was determined using sodium-bisulfite-treated DNA sequencing. All normal mammary glands and benign tumors showed high ERα expression (score range, 5–8). Six of the nine malignant tumors did not show ERα expression (score 0), two had score 2, and one had score 4. Lower ERα ( P < .005) and ESR1 mRNA levels ( P < .005) were found in malignant mammary tumors than in the other two groups. Canine ESR1 has an intragenic and non-promoter-associated CGI, different from humans. No significant variation in methylation percentage was observed among the groups, suggesting that ESR1 is not regulated by DNA methylation, unlike that in humans. This difference should be considered in further research using ERα as a biomarker for mammary tumors in canine studies on ERα-targeting therapy.

Although there is growing interest in the possibility that alterations in histone methylation may play a role in carcinogenesis, it has not been explored adequately in humans. Similarly, there are no reports of associations between this and a similar epigenetic event, DNA methylation. Using immunohistochemical staining, we compared the methylation of DNA and histones in histopathologically normal oral epithelium, dysplastic oral lesions, and squamous cell cancers (SCCs) from subjects with squamous cell cancer (n= 48) with those of normal oral epithelium from subjects without oral cancer (n= 93) who were matched on age and race. Monoclonal antibodies specific for 5 methyl cytosine (5-mc), lysine 4 of histone H3(H3-Lys4), and lysine 9 of histone H3(H3-Lys9) were used in this study. The percentages of cells positive and a weighted average of the immunostaining intensity scores were calculated for each of these tissues, and Spearman correlation analyses were employed to study associations between DNA and histone methylation. Correlations between DNA and histone methylation, H3-Lys4 and H3-Lys9 were positive and statistically significant in all tissue types; they were strongest in normal oral epithelium from non-cancer subjects (n= 0.63,p< 0.001 andr= 0.62,p< 0.001 respectively). Similarly, the positive correlations between H3-Lys4 and H3-Lys9 were statistically significant in all tissue types and strongest in normal oral epithelium from non-cancer subjects (r= 0.77,p< 0.001). Patterns of DNA and histone methylation are similar in tissues across the spectrum of oral carcinogenesis, and there is a significant positive association between these two epigenetic mechanisms.

Abstract Abstract 4629 Recently, mutations in DNA methyltransferase 3A (DNMT3A) have been found in patients with acute myeloid leukemia (AML) and have been confirmed to be connected with adverse clinical outcome. As DNMT3A plays direct role in the process of DNA methylation by adding methyl group to the cytosine residue of CpG dinucleotides, the question is obvious: What is the impact of DNMT3A mutations on DNA methylation levels? We examined 79 AML patients at diagnosis for the presence of aberrant DNA methylation of 12 tumor suppressor genes (TSG) (CDKN2B, CALCA, CDH1, ESR1, SOCS1, MYOD1, DAPK1, TIMP3, ICAM1, TERT, CTNNA1, EGR1) by methylation specific real-time PCR (MethyLight) and for mutations in the gene DNMT3A by direct sequencing. Next we studied methylation status of 24 HOX genes from all four clusters A-D using methylation-restriction endonucleases followed by RQ-PCR arrays in 10 AML samples compared to 4 healthy donor samples. Sequencing of cDNA between amino acids 300 and 930 revealed that 32 of 79 AML patients had DNMT3A mutation. The reason for higher DNMT3A mutation incidence in our patients’ cohort consists in preferential selection of AML patients with a higher percentage of probability of DNMT3A mutation (it means normal karyotype and mutations in NPM1, FLT3 and/or IDH1/2 genes). MethyLight assessment of 12 TSG showed subsequent frequencies of hypermethylation: CDKN2B (47%), CALCA (43%), CDH1 (22%), SOCS1 (24%), MYOD1 (18%), ESR1 (14%). The remaining 6 genes were weakly methylated in less than 10 % AML patients at diagnosis and were therefore excluded from further analysis. DNA methylation arrays revealed a set of differentially methylated HOX genes (n=12): 11 HOX genes were hypermethylated (HOXA4, HOXA6, HOXB13, HOXB3, HOXB4, HOXB7, HOXB8, HOXC8, HOXD10, HOXD11, HOXD3) and HOXA5 was hypomethylated compared to healthy donor samples. Comparing overall cumulative DNA methylation levels and numbers of simultaneously hypermethylated genes to mutational status of DNMT3A gene, we did observe lower levels of DNA methylation (P<0.0001) as well as displaying lower numbers of concurrently hypermethylated genes (P<0.0001) in patients with DNMT3a mutations. We observed the same trend also in DNA methylation levels of HOX genes when compared mutant (n=4) versus wild-type (n=6) DNMT3A patients. These results clearly show that numbers of simultaneously hypermethylated genes and DNA methylation levels of chosen tumor suppressor genes (TSG) as well as HOX genes differs between AML patients with wild type and mutant DNMT3A. This study is part of the COST Action BM0801 (EuGESMA) and is supported by NS10632-3/2009, OC10042 and IHBT00023736. Disclosures: No relevant conflicts of interest to declare.

The use of cryopreserved semen for insemination of mares facilitates breeding management but often results in reduced conception rates. This has been mainly attributed to changes in sperm membrane function caused by the freezing-thawing procedure. However, semen processing may also contribute to epigenetic changes in spermatozoa. In the present study, we therefore addressed changes in sperm DNA-methylation induced by cryopreservation of stallion semen. We hypothesised that the cryoprotectant may influence the DNA-methylation level of frozen-thawed semen. For this purpose, semen was collected from fertile Shetland pony stallions. Global DNA-methylation was assessed by ELISA (5-mC DNA ELISA Kit, Zymo Research, Irvine, CA, USA) with a monoclonal antibody sensitive and specific for 5-methylcytosin after DNA extraction and denaturation (100 ng of DNA per sample). The level of 5-methylcytosin in DNA is reported as the amount of methylated cytosine relative to the cytosine genomic content (%). Statistical analysis was done with the SPSS Statistics 21 software. Values are means ± standard error of the mean. In Experiment 1, 1.5 mL of raw semen (n = 6 stallions, 1 ejaculate each) was shock-frozen at –196°C for 15 min and thawed at 38°C for 60 s. Semen motility and membrane integrity were completely absent, while DNA-methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In Experiment 2, 3 ejaculates per stallion (n = 6) were included. Semen quality and DNA-methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (Minitube, Tiefenbach, Germany; cryoprotectant: 5% glycerol) or BotuCrio (Nidacon, Mölndal, Sweden; cryoprotectants: 1% glycerol and 4% methylformamid) extender. Semen was frozen in 0.5-mL straws in a computer-controlled rate freezer (IceCube 14 M; Sylab, Purkersdorf, Austria, cooling rates: 20°C to 5°C: 0.3°C min–1, 5°C to 25°C: 10°C min–1, –25°C to –140°C: 25°C min–1). Semen motility, morphology, and membrane integrity were significantly reduced (e.g. total motility before freezing: 88.8 ± 1.4%) by cryopreservation but not influenced by the extender used (e.g. total motility: Ghent 69.5 ± 2.0, BotuCrio 68.4 ± 2.2%; P < 0.001 v. nonfrozen semen). Cryopreservation significantly (P < 0.01) increased the level of DNA-methylation (before freezing: 0.6 ± 0.1%, Ghent 6.4 ± 3.7, BotuCrio 4.4 ± 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA-methylation was not correlated with semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA-methylation level in stallion semen. In the present study, this effect occurred irrespective of the cryoprotectant but was not seen after shock-freezing in the absence of cryoprotectants. The reduced fertility of mares after insemination with frozen-thawed semen may at least in part be explained by methylation of sperm DNA, which occurs in response to the cryopreservation procedure.

Methylation of a base in a specific DNA sequence protects the DNA from nucleolytic cleavage by restriction enzymes recognizing the same sequence. The MboII restriction–modification (R–M) system of Moraxella bovis ATCC 10900 consists of a restriction endonuclease gene and two methyltransferase genes. The enzymes encoded by this system recognize an asymmetrical sequence 5′-GAAGA-3′/3′-CTTCT-5′. M1.MboII modifies the last adenine in the recognition sequence 5′-GAAGA-3′ to N 6-methyladenine. A second methylase, M2.MboII, was cloned and purified to electrophoretic homogeneity using a four-step chromatographic procedure. It was demonstrated that M2.MboII modifies the internal cytosine in the recognition sequence 3′-CTTCT-5′, yielding N 4-methylcytosine, and moreover is able to methylate single-stranded DNA. The protein exists in solution as a monomer of molecular mass 30 000±1000 Da under denaturing conditions. Divalent cations (Ca2+, Mg2+, Mn2+ and Zn2+) inhibit M2.MboII methylation activity. It was found that the isomethylomer M2.NcuI from Neisseria cuniculi ATCC 14688 behaves in the same manner. Functional analysis showed that the complete MboII R–M system, consisting of two methyltransferases genes and the mboIIR gene, is the most stable and the least harmful to bacterial cells.

Cytosine (C) in DNA is often modified to 5-methylcytosine (m 5 C) to execute important cellular functions. Despite the significance of m 5 C for epigenetic regulation in mammals, damage to m 5 C has received little attention. For instance, almost no studies exist on erroneous methylation of m 5 C by alkylating agents to doubly or triply methylated bases. Owing to chemical evidence, and because many prokaryotes express methyltransferases able to convert m 5 C into N 4 ,5-dimethylcytosine (m N 4,5 C) in DNA, m N 4,5 C is probably present in vivo . We screened a series of glycosylases from prokaryotic to human and found significant DNA incision activity of the Escherichia coli Nei and Fpg proteins at m N 4,5 C residues in vitro . The activity of Nei was highest opposite cognate guanine followed by adenine, thymine (T) and C. Fpg-complemented Nei by exhibiting the highest activity opposite C followed by lower activity opposite T. To our knowledge, this is the first description of a repair enzyme activity at a further methylated m 5 C in DNA, as well as the first alkylated base allocated as a Nei or Fpg substrate. Based on our observed high sensitivity to nuclease S1 digestion, we suggest that m N 4,5 C occurs as a disturbing lesion in DNA and that Nei may serve as a major DNA glycosylase in E. coli to initiate its repair. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.