Absence Of Methylation Research Articles

Effect of segmentation dimension on radiomics analysis for MGMT promoter methylation status in gliomas

Introduction and objective: We investigated the impact of 2D (2D_seg) and 3D (3D_seg) segmentation on the accuracy of prediction models in the radiomics analysis to determine the presence or absence of methylation in the O6-methylguanine DNA methyltransferase (MGMT) gene promoter region of gliomas. Materials and methods: Magnetic resonance imaging images of gliomas were obtained from the Cancer Imaging Archive for 50 methylated and 50 unmethylated cases respectively. For each case, 2D_seg and 3D_seg were performed, and 788 radiomics features, including wavelet transform, were obtained. Ten features were selected by LASSO regression. The coefficients of determination (R2) and root mean squared error (RMSE) were calculated by multiple regression analysis. Discriminant boundaries to discriminate methylation were created by linear discriminant analysis, and the sensitivity and specificity of each method were calculated. The discriminant accuracy of both methods was evaluated by receiver operating characteristics (ROC) analysis. Results: The R2 value and RMSE were 0.72/0.28 and 0.73/0.33 for 2D_seg and 3D_seg, respectively. Similarly, sensitivity and specificity were 82.5/67.5% and 85/62.5%, respectively. The area under the curve determined by ROC analysis was 0.80 and 0.79, respectively, i.e. slightly larger for 2D_seg. The p-value by the DeLong method was 0.73. Conclusions: In the radiomics analysis using 2D_seg and 3D_seg, no difference in discriminant accuracy was observed between them. Therefore, 2D segmentation should be chosen because it is easier to segment.

B-370 Clinical Utility of microRNA Quantitative Analysis Using Double Quencher Probes and Methylation Analysis by Methylation Sensitive Restriction Enzyme Polymerase Chain Reaction for Bladder Cancer

Abstract Background Histone modifications, DNA methylation, and microRNA (miRNA)-mediated gene silence are examples of epigenetic changes that regulate gene expression without causing sequence changes. The early phases of neoplastic transformation are characterized by the occurrence of these epigenetic changes, which are believed to be crucial for the development and progression of cancer. To increase the sensitivity and practicality of miRNA analysis, we developed a measuring technique using a ZEN double quencher into hydrolysis probe in reverse transcription quantitative polymerase chain reaction (RT-qPCR). Additionally, we developed a method for DNA methylation analysis employing methylation-sensitive restriction enzymes (MSRE) and methylation-insensitive restriction enzymes (MIRE) that can analyze a number of samples quickly. We examined the clinical usefulness of using these systems to understand the level of miRNA expression and methylation in bladder cancer. Methods In this study, 64 biopsied tissues from transurethral bladder tumor resection (TURBT) were used. For miRNA extraction, Cica geniusTM RNA Prep Kit (For Tissue) II (KANTO CHEMICAL CO.,INC.) and a solution that improves miRNA extraction efficiency were used. Using stem-loop RT primers, complementary DNA was created from the isolated miRNA solution. Quantitative analysis was performed by RT-qPCR using a ZEN double quencher probe and miR-21,30a, 200c, and U6 were used as target. Genomic DNA (gDNA) was extracted from the TURBT sample using High Pure PCR Template Preparation Kit (Roche). Restriction enzyme digestion was performed using HapII (Takara Bio) as MSRE and MspI (Takara Bio) as MIRE; the latter has the same recognition site as HapII but is not affected by the presence or absence of methylation. Real-time PCR was performed on the LightCycler 96 (Roche) using Universal ProbeLibrary probes (Roche) targeting ESM1, SFMBT2, and SEPTIN9. From the obtained Cp value, each methylation rate was calculated. The Fisher’s exact test was used to statistically analyze the relationship among miRNA, methylation data and other clinicopathological factors. Results For miR-30a, we observed a higher expression of miRNA was linked to more advanced T stage (P = 0.047). Additionally, ESM1 methylation exhibited a significant correlation with greater grade (P = 0.004) and T stage progress (P = 0.018). Data on miRNA abundance and methylation rate were combined in order to uncover a link with clinical data that was not apparent from either set of data separately. Samples with higher values for both miR-21 and ESM1 methylation rates were shown to have high grade (P = 0.007) and progress to the T stage (P = 0.006). Furthermore, samples with higher values for both miR-200c and SEPTIN9 methylation rates did not progress to the T stage (P = 0.013). Conclusion The combined miRNA and methylation data revealed relationships with clinical results that could not be discovered with either data separately, highlighting the clinical usefulness of the current study’s findings.

Genome-wide impact of cytosine methylation and DNA sequence context on UV-induced CPD formation.

Exposure to ultraviolet (UV) light is the primary etiological agent for skin cancers because UV damages cellular DNA. The most frequent form of UV damage is the cyclobutane pyrimidine dimer (CPD), which consists of covalent linkages between neighboring pyrimidine bases in DNA. In human cells, the 5' position of cytosine bases in CG dinucleotides is frequently methylated, and methylated cytosines in the TP53 tumor suppressor are often sites of mutation hotspots in skin cancers. It has been argued that this is because cytosine methylation promotes UV-induced CPD formation; however, the effects of cytosine methylation on CPD formation are controversial, with conflicting results from previous studies. Here, we use a genome-wide method known as CPD-seq to map UVB- and UVC-induced CPDs across the yeast genome in the presence or absence in vitro methylation by the CpG methyltransferase M.SssI. Our data indicate that cytosine methylation increases UVB-induced CPD formation nearly 2-fold relative to unmethylated DNA, but the magnitude of induction depends on the flanking sequence context. Sequence contexts with a 5' guanine base (e.g., GCCG and GTCG) show the strongest induction due to cytosine methylation, potentially because these sequence contexts are less efficient at forming CPD lesions in the absence of methylation. We show that cytosine methylation also modulates UVC-induced CPD formation, albeit to a lesser extent than UVB. These findings can potentially reconcile previous studies, and define the impact of cytosine methylation on UV damage across a eukaryotic genome.

Methylation Analysis of Rb, P16, MGMT, and hMLH-1in Mucoepidermoid Carcinomas. A Pilot Study

Mucoepidermoid carcinoma (MC) is the most common malignant epithelial neoplasm in the salivary glands. This neoplasm has varying proportions of mucous, epidermoid, intermediate, columnar, and clear cells. MCs have been associated with CRTC1-MAML2 genes; however, their pathogenesis is uncertain. Recently, epigenetic changes have been considered a possible aetiologic factor. To identify the methylation state of RB, P16, MGMT, and hMLH genes in the three severity grades of MC were used five MCs and one healthy minor salivary gland as a control group (CG) obtained from the Pathology and Oral Medicine Laboratory and analyzed using MS-PCR to compare the presence or absence of methylation in promotor regions. The Kruskal-Wallis test was performed, with p≤0.05 considered significant. CG was employed as the normalizer of methylation levels. All assays were performed in triplicate. The mean age of our population was 52.6±18.6 years old; the total population was female and included 2 low grade, 2 intermediate grade, and 1 high grade levels of severity. When comparing the methylation status of the three histopathological grades of MC against the control, statistically significant differences were observed in Rb-M, MGMT-M, and hMLH-1-NM for high-grade severity, with p values of 0.03, 0.05, and 0.04, respectively. Methylation is a possible mechanism for pathogenesis processing of high-grade MC. However, a larger sample population is necessary to validate this finding.

The role of Herpes Virus promoter methylation in the realization of pathogenic potential in the example of Marek’s Disease

Marek’s disease virus is ubiquitous and can harm not only poultry, but also be oncogenic for humans. VBM and malignant tumors induced by them are a convenient and accessible natural model for studying herpesvirus-associated carcinogenesis. To date, according to our observations, there are additional risks of human infection with the Marek’s disease virus - the disease began to appear in broiler chickens 30 days and older, i.e. contact with poultry meat carries a risk of infection. In addition, COVID-19 disease may be accompanied by folic acid deficiency, i.e. a violation of the folate cycle in humans, which increases the risk of manifestation of diseases associated with DNA viruses, since a violation of the folate cycle can reduce the activity of DNA methylation, incl. viral DNA. Methylation is carried out enzymatically in the first minutes after DNA replication, i.e. postreplicatively. Since the DNA nucleotide sequence does not change, methylation is essentially an epigenetic event. We have studied the relationship between the methylation of promoters of the Marek’s disease virus and the copy number of the virus. The assessment of the presence or absence of methylation, as well as partial methylation, was carried out on the basis of identifying the difference between the threshold cycles dC(t). The presence of unmethylated sites included in the studied promoter sequence was detected on the basis of the ability of methylsensitive restrictases AciI and GlaI. A correlation was found between the concentration of genomic DNA of the Marek’s disease virus serotype 1 strain CVI 988 in cell culture and the presence of demethylated CpG islands in the composition of promoters located at position 9413-9865 bp. and 127943 - 128193 b.p. genomic DNA of the virus. The data obtained make it possible to explain the mechanism of the increase in the pathogenicity of herpesvirus infections under conditions of a decrease in the activity of viral DNA methylation in the body.

Disease-related blood-based differential methylation in cystic fibrosis and its representation in lung cancer revealed a regulatory locus in PKP3 in lung epithelial cells

ABSTRACT Cystic fibrosis (CF) is a monogenic disease, characterized by massive chronic lung inflammation. The observed variability in clinical phenotypes in monozygotic CF twins is likely associated with the extent of inflammation. This study sought to investigate inflammation-related aberrant DNA methylation in CF twins and to determine to what extent acquired methylation changes may be associated with lung cancer. Blood-based genome-wide DNA methylation analysis was performed to compare the DNA methylomes of monozygotic twins, from the European CF Twin and Sibling Study with various degrees of disease severity. Putatively inflammation-related and differentially methylated positions were selected from a large lung cancer case-control study and investigated in blood by targeted bisulphite next-generation-sequencing. An inflammation-related locus located in the Plakophilin-3 (PKP3) gene was functionally analysed regarding promoter and enhancer activity in presence and absence of methylation using luciferase reporter assays. We confirmed in a unique cohort that monozygotic twins, even if clinically discordant, have only minor differences in global DNA methylation patterns and blood cell composition. Further, we determined the most differentially methylated positions, a high proportion of which are blood cell-type-specific, whereas others may be acquired and thus have potential relevance in the context of inflammation as lung cancer risk factors. We identified a sequence in the gene body of PKP3 which is hypermethylated in blood from CF twins with severe phenotype and highly variably methylated in lung cancer patients and controls, independent of known clinical parameters, and showed that this region exhibits methylation-dependent promoter activity in lung epithelial cells.

Full methylation of H3K27 by PRC2 is dispensable for initial embryoid body formation but required to maintain differentiated cell identity.

Polycomb repressive complex 2 (PRC2) catalyzes methylation of histone H3 on lysine 27 and is required for normal development of complex eukaryotes. The nature of that requirement is not clear. H3K27me3 is associated with repressed genes, but the modification is not sufficient to induce repression and, in some instances, is not required. We blocked full methylation of H3K27 with both a small molecule inhibitor, GSK343, and by introducing a point mutation into EZH2, the catalytic subunit of PRC2, in the mouse CJ7 cell line. Cells with substantively decreased H3K27 methylation differentiate into embryoid bodies, which contrasts with EZH2 null cells. PRC2 targets had varied requirements for H3K27me3, with a subset that maintained normal levels of repression in the absence of methylation. The primary cellular phenotype of blocked H3K27 methylation was an inability of altered cells to maintain a differentiated state when challenged. This phenotype was determined by H3K27 methylation in embryonic stem cells through the first 4 days of differentiation. Full H3K27 methylation therefore was not necessary for formation of differentiated cell states during embryoid body formation but was required to maintain a stable differentiated state.

Staphylococcus ursi sp. nov., a new member of the 'Staphylococcus intermedius group' isolated from healthy black bears.

Six Staphylococcus strains were isolated from healthy black bears (Ursus americanus) in the Great Smoky Mountains National Park, Tennessee, USA. Phylogenetic analysis based on complete genome, 16S rRNA, dnaJ, hsp60, rpoB and sodA genes, and MALDI-TOF-MS main spectral profiles revealed that the strains belonged to one species and showed the closest relatedness to members of the ‘ Staphylococcus intermedius group’ (SIG), which include Staphylococcus intermedius , Staphylococcus pseudintermedius, Staphylococcus delphini and Staphyloccoccus cornubiensis. The strains were positive in SIG-specific and negative in individual species-specific PCR assays for the nuc gene. The strains can be differentiated from the other SIG species by the absence of sucrose fermentation, from S. intermedius DSM 20373T, S. pseudintermedius CCUG 49543T and S. cornubiensis DSM 105366T by the absence of methyl β-d-glucopyranoside fermentation and from S. delphini DSM 20771T by fermentation of trehalose. DNA relatedness of the type strain MI 10-1553T with the type strains of S. delphini , S. pseudintermedius , S. intermedius and S. cornubiensis was ≤48.2 % by digital DNA–DNA hybridization and ≤92.3 % by average nucleotide identity calculations. Iso-C15:0, anteiso-C15 : 0 and anteiso-C17 : 0 were the most common fatty acids. Polar lipids consisted of phosphadidylglycerols, phospholipids, glycolipid, diphosphatidylglycerol and aminophospholipid. Cell-wall peptidoglycan was of type A3α l-Lys-Gly3 (Ser; similar to A11.2 and A11.3). The respiratory quinone belonged to menaquinone 7 (MK-7). The G+C content of MI 10-1553T was 39.3 mol%. The isolated strains represent a novel species of the genus Staphylococcus , for which we propose the name Staphylococcus ursi sp. nov. The type strain is MI 10-1553T (=ATCC TSD-55T=CCOS 1900T).

Regulation of interleukin 6 by a polymorphic CpG within the frontal cortex in Alzheimer’s disease

The cytokine interleukin 6 (IL-6) has been linked to the pathogenesis of Alzheimer’s disease (AD). This is the first study to investigate the genetic and epigenetic interactions in the control of IL-6 in human brain and its relation to AD neuropathology in prefrontal cortex tissues from AD and controls genotyped for the SNP −174 C/G rs1800795, a polymorphic CpG in which the G allele creates a CpG site. Within CC homozygotes there were significantly higher brain levels of IL-6 protein compared to G allele carriers. The C allele that resulted in an absence of methylation at a CpG was also associated with significant changes in methylation at neighboring CpGs. Furthermore, there were significant differences in methylation between CC and CG/GG at CpG sites in the AD and control groups. That DNA methylation was altered in the brains by the presence of rs1800795, which further correlated with protein levels suggests the presence of a polymorphic CpG and genetic-epigenetic interactions in the regulation of IL-6 in the prefrontal cortex within AD brains.

Mercury and methylmercury levels in soils associated with coal-fired power plants in central-northern Chile

Mercury pollution is a worldwide problem, and is associated with a number of natural and anthropogenic processes. The present work, conducted in Chile, a country that has traditionally depended heavily on fossil fuels for power generation, examines total mercury (THg) and monomethylmercury (MMHg) concentrations in soils across different sites exposed to coal fired power plant emissions. Samples from four selected (Renca, Laguna Verde, Las Ventanas, Huasco) and 1 control (Quintay) sites were analyzed using cold vapour and fluorescence spectroscopy (CV-AFS) for THg determination and chromatographic separation with atomic fluorescence detection (DI-GC-AFS) was followed for speciation analysis.From the sites analyzed, Renca and Las Ventanas showed high concentrations of total mercury, exhibiting ranges between 135 – 568 and 94–464 ng g−1 respectively, while Laguna Verde and Huasco exhibited lower values ranged 5–27 and 9–44 ng g−1 respectively. Conversely, analysis of MMHg concentrations showed that only Renca site possessed high values, ranging between 0.1 and 3.0 ng g−1, resulting in this site being considered contaminated. Conversely, other sites showed minimal values comparable to the control site (0.024 ± 0.003 ng g−1) in terms of MMHg concentrations. An analysis of the differences between MMHg and THg concentrations in contaminated sites, suggests an overall absence of methylation in soils of Las Ventanas, probably related to the very high levels of soil heavy metals, especially copper. Moreover, the influence of the composition and physicochemical properties of the different soils on the mobility of the species was assessed. Results obtained (as Log Kd) were 3.5 and 4.1 for Renca and Las Ventanas respectively, suggesting low mobility of mercury species in the environment for both sites.Finally, the data obtained allowed us to establish a first approximation of the differences in concentration and mobility of total and MMHg associated with coal fired power plants emission in central-northern Chile, an area previously understudied in a country heavily dependent on fossil-fuels.

High methylation levels of PCDH10 predict poor prognosis in patients with pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies and is not a clinically homogeneous disease, but subsets of patients with distinct prognosis and response to therapy can be identified by genome-wide analyses. Mutations in major PDAC driver genes were associated with poor survival. By bioinformatics analysis, we identified protocadherins among the most frequently mutated genes in PDAC suggesting an important role of these genes in the biology of this tumor. Promoter methylation of protocadherins has been suggested as a prognostic marker in different tumors, but in PDAC this epigenetic modification has not been extensively studied. Thus, we evaluated whether promoter methylation of three frequently mutated protocadherins, PCDHAC2, PCDHGC5 and PCDH10 could be used as survival predictors in PDAC patients.MethodsDNA extracted from 23 PDACs and adjacent non-neoplastic pancreatic tissues were bisulfite treated. Combined Bisulfite Restriction Analysis (COBRA) coupled to denaturing high-performance liquid chromatography (dHPLC) detection and bisulfite genomic sequencing (BGS) were used to determine the presence of methylated CpG dinucleotides in the promoter amplicons analyzed.ResultsIn an exploratory analysis, two protocadherins showed the same pattern of CpG methylation in PDAC and adjacent non-neoplastic pancreatic tissues: lack of methylation for PCDHAC2, complete methylation for PCDHGC5. Conversely, the third protocadherin analyzed, PCDH10, showed a variable degree of CpG methylation in PDAC and absence of methylation in adjacent non-neoplastic pancreatic tissues. At Kaplan–Meier analysis, high levels of PCDH10 methylation defined according to the receiver operating characteristic (ROC) curve analysis were significantly associated with worse progression-free survival (PFS) rates (P = 0.008), but not with overall survival (OS). High levels of PCDH10 methylation were a prognostic factor influencing PFS (HR = 4.0: 95% CI, 1.3–12.3; P = 0.016), but not the OS.ConclusionsIn this study, we show for the first time that the methylation status of PCDH10 can predict prognosis in PDAC patients with a significant impact on the outcome in terms of progression-free survival. High levels of PCDH10 promoter methylation could be useful to identify patients at high risk of disease progression, contributing to a more accurate stratification of PDAC patients for personalized clinical management.

Methylation at the C-3′ in D-Ring of Strigolactone Analogs Reduces Biological Activity in Root Parasitic Plants and Rice

Strigolactones (SLs) regulate plant development and induce seed germination in obligate root parasitic weeds, e.g. Striga spp. Because organic synthesis of natural SLs is laborious, there is a large need for easy-to-synthesize and efficient analogs. Here, we investigated the effect of a structural modification of the D-ring, a conserved structural element in SLs. We synthesized and investigated the activity of two analogs, MP13 and MP26, which differ from previously published AR8 and AR36 only in the absence of methylation at C-3′. The de-methylated MP13 and MP26 were much more efficient in regulating plant development and inducing Striga seed germination, compared with AR8. Hydrolysis assays performed with purified Striga SL receptor and docking of AR8 and MP13 to the corresponding active site confirmed and explained the higher activity. Field trials performed in a naturally Striga-infested African farmer’s field unraveled MP13 as a promising candidate for combating Striga by inducing germination in host’s absence. Our findings demonstrate that methylation of the C-3′ in D-ring in SL analogs has a negative impact on their activity and identify MP13 and, particularly, MP26 as potent SL analogs with simple structures, which can be employed to control Striga, a major threat to global food security.

SETD3 protein is the actin-specific histidine N-methyltransferase.

Protein histidine methylation is a rare post-translational modification of unknown biochemical importance. In vertebrates, only a few methylhistidine-containing proteins have been reported, including β-actin as an essential example. The evolutionary conserved methylation of β-actin H73 is catalyzed by an as yet unknown histidine N-methyltransferase. We report here that the protein SETD3 is the actin-specific histidine N-methyltransferase. In vitro, recombinant rat and human SETD3 methylated β-actin at H73. Knocking-out SETD3 in both human HAP1 cells and in Drosophila melanogaster resulted in the absence of methylation at β-actin H73 in vivo, whereas β-actin from wildtype cells or flies was > 90% methylated. As a consequence, we show that Setd3-deficient HAP1 cells have less cellular F-actin and an increased glycolytic phenotype. In conclusion, by identifying SETD3 as the actin-specific histidine N-methyltransferase, our work pioneers new research into the possible role of this modification in health and disease and questions the substrate specificity of SET-domain-containing enzymes.

136 DNA Methylation Status and Transcript Profile of Genes Associated with Lipid Metabolism, Cell Survival, and Pluripotency in Bovine Blastocysts with Different Kinetics of Development

During in vitro production (IVP), blastocysts can be differentiated based on the kinetics of early cleavage. These groups present distinct patterns of global DNA methylation, an epigenetic characteristic generally responsible for suppression (presence of methylation) or activation (absence of methylation) of genes from different biological pathways. This work investigated the DNA methylation and mRNA levels of genes related to embryo development and viability. For this purpose, bovine embryos underwent IVP using conventional protocols. After 40 h of insemination, embryos were classified as FBL (fast cleavage: 4 cells or more) or SBL (slow cleavage: 2 or 3 cells), remaining in culture until blastocyst stage. Sexed semen was used to prevent differences due to sex, even without statistical differences in male:female ratio already reported. Blastocysts (40 per group) were analysed by EmbryoGENE Methylation DNA Array (Ispada et al. 2016 Proc. 49th SSR: 181) and later analysed through BioMark™HD (Fluidigm Corp., South San Francisco, CA, USA) for the transcripts profile. The PPIA gene was used as endogenous control for ΔCt calculation and submitted to Student’s t-test. Genome-wide DNA methylation analysis identified 47,713 methylated regions (7976 hypermethylated in FBL and 3608 hypermethylated in SBL). Fast embryos presented more hypermethylations distributed throughout the genome, such as introns, exons, promoter and repeat elements, whereas hypermethylation were more present in CpG islands in slow embryos. Differentially methylated regions were clustered by means of biological processes and the most affected pathways were related to lipid metabolism and cell differentiation and survival. Regarding the gene expression analysis, all results are presented in FBL in relation to SBL. Of genes involved in lipid metabolism, ACSL3, ELOVL6, PPARA, and FADS, previously identified as hypermethylated genes, were down-regulated, whereas PPARG and PTGS2 showed no statistical difference; SCD and FASN, although hypomethylated, were also down-regulated, and ACSL6, which did not differ in DNA methylation status, was down-regulated. Of genes involved in survival/death, BAX, HSPA1A, BID, NFE2L2, and GPX1 were hypermethylated; however, the first 2 were up-regulated, BID was down-regulated, and the last 2 were not statistically different. Although CASP9, TXNRD1, and FOXO3 were all hypomethylated, only CASP9 was up-regulated. Also, DDIT3 was down-regulated and NOS2 was up-regulated, although they did not differ in DNA methylation between groups. Of genes involved in cell differentiation, POU5F1 and SALL4 were both hypermethylated, but only the POU5F1 was down-regulated; NANOG, which did not differ in DNA methylation status between groups, was also down-regulated. In conclusion, although we did not find correlation in DNA methylation and RNA levels for all genes evaluated, the chosen pathways were indeed different between groups, which could lead to their potential suppression/activation and affect embryo viability. Also, this lower correlation may be a result of the influence of other epigenetic mechanisms differently activated between groups.

Reducing Artifactual EGFR T790M Mutations in DNA from Formalin-Fixed Paraffin-Embedded Tissue by Use of Thymine-DNA Glycosylase.

False-positive EGFR T790M mutations have been reported in formalin-fixed lung tumors, but the cause of the false positives has not been identified. The T790M mutation results from a C>T change at the cytosine of a CpG dinucleotide. The presence or absence of methylation at this cytosine has different consequences following deamination, resulting in a thymine or uracil, respectively, both of which however result in an artifactual change. Uracil-DNA glycosylase (UDG) can be used to eliminate DNA templates with uracil residues but is not active against artifactual thymines. We therefore investigated the use of thymine-DNA glycosylase (TDG) to reduce artifactual T790M mutations. Formalin-fixed normal lung tissues and lung squamous cell carcinomas were tested to measure the frequency of false-positive EGFR mutations by use of droplet digital PCR before and after treatment with either UDG or TDG. Methylation at the cytosine at EGFR T790 was assessed by pyrosequencing and by analysis of public databases. Artifactual EGFR T790M mutations were detected in all of the archival formalin-fixed normal lung and lung squamous cell carcinomas at mutant allele frequencies of 1% or lower. The cytosine at EGFR T790 showed high levels of methylation in all lung cancer samples and normal tissues. Pretreatment of the formalin-fixed DNA with either UDG or TDG reduced the false EGFR T790M mutations, but a greater reduction was seen with the TDG treatment. Both U:G and T:G lesions in formalin-fixed tissue are sources of false-positive EGFR T790M mutations. This is the first report of the use of TDG to reduce sequence artifacts in formalin-fixed DNA and is applicable to the accurate detection of mutations arising at methylated cytosines.

Methylation and the single neuronal cell

Neural Epigenomics The presence or absence of methylation on chromosomal DNA can drive or repress gene expression. Now, a comprehensive map of methylation variation in neuronal cell populations, including a between-species comparison, illustrates how epigenetic diversity plays important roles in neuronal development. Luo et al. examined how DNA methylation is both similar and different within neurons at the single-nucleus level in humans and mice. They identified 16 mouse and 21 human neuronal clusters, with greater complexity of excitatory neurons in deep brain layers than in superficial layers. Science , this issue p. [600][1] [1]: /lookup/doi/10.1126/science.aan3351

Defects in methylation of arginine 37 on CENP-A/Cse4 are compensated by the ubiquitin ligase complex Ubr2/Mub1.

The kinetochore, a supramolecular protein complex, provides the physical connection between chromatin and the microtubule and ensures correct chromosome segregation during mitosis. Centromeric regions are marked by the presence of the histone H3 variant CENP-A. Cse4, the CENP-A homologue from Saccharomyces cerevisiae, is methylated on arginine 37 in its N-terminus (R37), and the absence of methylation (cse4-R37A) causes synthetic genetic defects in combination with mutations or deletions in genes encoding components of the Ctf19/CCAN complex and with the CDEI binding protein Cbf1. Here, we report that the absence of the E3 ubiquitin ligase Ubr2 as well as its adaptor protein Mub1 suppresses the defects caused by the absence of Cse4-R37 methylation. Ubr2 is known to regulate the levels of the MIND complex component Dsn1 via ubiquitination and proteasome-mediated degradation. Accordingly, we found that overexpression of DSN1 also led to suppression of Cse4 methylation defects. Altogether, our data indicate that the absence of R37 methylation reduces the recruitment of kinetochore proteins to centromeric chromatin, and that this can be compensated for by stabilising the outer kinetochore protein Dsn1.

Blocked transcription through KvDMR1 results in absence of methylation and gene silencing resembling Beckwith-Wiedemann syndrome.

The maternally methylated KvDMR1 ICR regulates imprinted expression of a cluster of maternally expressed genes on human chromosome 11p15.5. Disruption of imprinting leads to Beckwith-Wiedemann syndrome (BWS), an overgrowth and cancer predisposition condition. In the majority of individuals with BWS, maternal-specific methylation at KvDMR1 is absent and genes under its control are repressed. We analyzed a mouse model carrying a poly(A) truncation cassette inserted to prevent RNA transcripts from elongation through KvDMR1. Maternal inheritance of this mutation resulted in absence of DNA methylation at KvDMR1, which led to biallelic expression of Kcnq1ot1 and suppression of maternally expressed genes. This study provides further evidence that transcription is required for establishment of methylation at maternal gametic DMRs. More importantly, this mouse model recapitulates the molecular phenotypic characteristics of the most common form of BWS, including loss of methylation at KvDMR1 and biallelic repression of Cdkn1c, suggesting that deficiency of maternal transcription through KvDMR1 may be an underlying cause of some BWS cases.

Un-methylation of the survivin gene has no effect on immunohistochemical expression of survivin protein in lung cancer patients with squamous cell carcinoma.

Survivin is a member of the inhibitor of apoptosis (IAP) family. The function of the survivin protein is to inhibit caspase activation, thereby leading to negative regulation of apoptosis or programmed cell death. This has been shown by the disruption of survivin induction pathways leading to an increased apoptosis and decreased tumour growth. These data suggest that survivin may provide a new target for cancer treatment, which would distinguish transformed cells from normal cells. In the present study, we aimed to investigate exon 1 of the survivin gene by means of methylation-specific PCR and evaluate its impact on survivin protein expression following DNA isolation and bisulphite modification in paraffin-embedded normal and tumour tissues of lung cancer patients with squamous cell carcinoma. We used 41 squamous cancer tissues with methylation in exon 1 of the survivin gene and non-methylation in corresponding tumours. However, the immunohistochemistry staining of these samples demonstrated an increased survivin protein compared to normal tissue. While there is almost no other study to date on this subject matter, we believe that the absence of methylation in exon 1 of the survivin gene may not affect disease prognosis as it has no effect on expression, and possible promoter methylation or transcription factors (Tab. 1, Fig. 4, Ref. 15).

Metabolic Disposition of Luteolin Is Mediated by the Interplay of UDP-Glucuronosyltransferases and Catechol-O-Methyltransferases in Rats.

Luteolin partially exerts its biologic effects via its metabolites catalyzed by UDP-glucuronosyltransferases (UGTs) and catechol-O-methyltransferases (COMTs). However, the interplay of UGTs and COMTs in mediating luteolin disposition has not been well clarified. In this study, we investigated the glucuronidation and methylation pathways of luteolin mediated by the interplay of UGTs and COMTs in vivo and in vitro. A total of nine luteolin metabolites was detected in rat plasma and bile by liquid chromatography-tandem mass spectrometry, namely, three glucuronides, two methylated metabolites, and four methylated glucuronides. Luteolin-3'-glucuronide (Lut-3'-G) exhibited the highest systemic exposure among these metabolites. Kinetics studies in rat liver S9 fractions suggested two pathways, as follows: 1) Luteolin was glucuronidated to luteolin-7-glucuronide, luteolin-4'-glucuronide, and Lut-3'-G by UGTs, and then Lut-7-G was methylated to chrysoeriol-7-glucuronide and diosmetin-7-glucuronide by COMTs. 2) Alternatively, luteolin was methylated to chrysoeriol and diosmetin by COMTs, and then chrysoeriol and diosmetin were glucuronidated by UGTs to their respective glucuronides. The methylation rate of luteolin was significantly increased by the absence of glucuronidation, whereas the glucuronidation rate was increased by the absence of methylation, but to a lesser extent. In conclusion, two pathways mediated by the interplay of UGTs and COMTs are probably involved in the metabolic disposition of luteolin. The glucuronidation and methylation of luteolin compensate for each other, although glucuronidation is the predominant pathway.