Presence Of DNA Methylation Research Articles

MAP30 transgenic tobacco lines: from silencing to inducing.

DNA methylation is one of the most important epigenetic event that regulates gene expression. In addition to DNA methylation, transgene copy number may induce gene silencing. Therefore, the study of these cases is useful for understanding of gene silencing regulation. In this study, the methylation pattern of 35S promoter was investigated in the second generation of MAP30 transgenic tobacco lines. Therefore, the genomic DNA melting curve changes were investigated before and after bisulfite treatment by real time PCR. To determine the exact position of methylation, the samples were sequenced after bisulfite treatment. Observation of decrease in DNA melting curve of expressing line in comparison with silenced line confirmed the presence of DNA methylation in silenced line. In order to induce the MAP30 expression, the silenced line was treated using different concentrations of Azacytidine and green tea extracts. The results showed that all concentrations of green tea extracts for 6days and the concentrations of 3 and 10μM Azacytidine for 10 and 3days could induce the expression of MAP30 in silenced line respectively. Finally, the transgene copy number was estimated using real time PCR, as silenced line contained more than two copies while the lines expressing MAP30 contained only one or two copies. Finally, we found that the presence of DNA methylation and also multiple gene copy numbers in silenced line have been led to gene silencing. Moreover, the effect of green tea extract on DNA methylation showed incredible results for the first time.

LSD1 prevents aberrant heterochromatin formation in Neurospora crassa.

Heterochromatin is a specialized form of chromatin that restricts access to DNA and inhibits genetic processes, including transcription and recombination. In Neurospora crassa, constitutive heterochromatin is characterized by trimethylation of lysine 9 on histone H3, hypoacetylation of histones, and DNA methylation. We explored whether the conserved histone demethylase, lysine-specific demethylase 1 (LSD1), regulates heterochromatin in Neurospora, and if so, how. Though LSD1 is implicated in heterochromatin regulation, its function is inconsistent across different systems; orthologs of LSD1 have been shown to either promote or antagonize heterochromatin expansion by removing H3K4me or H3K9me respectively. We identify three members of the Neurospora LSD complex (LSDC): LSD1, PHF1, and BDP-1. Strains deficient for any of these proteins exhibit variable spreading of heterochromatin and establishment of new heterochromatin domains throughout the genome. Although establishment of H3K9me3 is typically independent of DNA methylation in Neurospora, instances of DNA methylation-dependent H3K9me3 have been found outside regions of canonical heterochromatin. Consistent with this, the hyper-H3K9me3 phenotype of Δlsd1 strains is dependent on the presence of DNA methylation, as well as HCHC-mediated histone deacetylation, suggesting that spreading is dependent on some feedback mechanism. Altogether, our results suggest LSD1 works in opposition to HCHC to maintain proper heterochromatin boundaries.

Cytosine methylation of rice mitochondrial DNA from grain and leaf tissues.

The rice leaf mitochondrial DNA is more methylated compared to the rice grain mitochondrial DNA. The old rice leaf mitochondrial DNA has also a higher methylation level than the young rice leaf mitochondrial DNA. The presence of DNA methylation in rice organelles has not been well characterized. We have previously shown that cytosine methylation of chloroplast DNA is different between leaf and grain, and varies between young and old leaves in rice. However, the variation in cytosine methylation of mitochondrial DNA is still poorly characterized. In this study, we have investigated cytosine methylation of mitochondrial DNA in the rice grain and leaf. Based on CpG, CHG, and CHH methylation analyses, the leaf mitochondrial DNA was found to be more methylated compared to the grain mitochondrial DNA. The methylation of the leaf mitochondrial DNA was also higher in old compared to young leaves. Differences in methylation were observed at different cytosine positions of the mitochondrial DNA between grain and leaf, although there were also positions with a similarlevel of high methylation in all the tissues examined. The differentially methylated cytosine positions in rice mitochondrial DNA were observed mostly in the intergenic region and in some mitochondrial-specific genes involved in ATP production, transcription, and translation. The functional importance of cytosine methylation in the life cycle of rice mitochondria is still to be determined.

DNA methylation analysis within the IL2RA gene promoter in youth with autoimmune thyroid disease.

Alpha-subunit of the interleukin-2 receptor (IL2RA) is involved in the regulation of T-cell function and has been related to autoimmune thyroid disease (AITD). Although the exact mechanisms are not fully understood, promoter methylation might account for differences in gene expression. The aim of this study was to investigate whether there are differences in the percentage of DNA methylation within the IL2RA gene promoter in young patients with AITD. In a cross-sectional design, the presence of DNA methylation in the IL2RA gene promoter was quantified, by real-time PCR and melting curve analysis, in modified genomic DNA isolated from blood samples of a total of 149 children and adolescents with AITD, including patients with Hashimoto thyroiditis (ΗΤ) (n=60), Graves' disease (GD) (n=9), concurrent diagnosis of HT and type 1 diabetes (T1DM+HT) (n=25), and healthy controls (n=55). The percentage of DNA methylation in the IL2RA gene promoter was significantly decreased in patients with GD (26.0±4.2%) but not in those with HT (36.3±1.4%) in comparison with controls (41.3±1.5%). The observed DNA hypomethylation in the IL2RA gene promoter in patients with GD might be related to its increased expression, thus contributing to the etiopathogenesis of GD in childhood and adolescence.

Multiple Myeloma Epigenetic Programming Prognostic of Outcome Converges with Loci Reprogrammed in Relapsed/Refractory Disease

The genetic and transcriptional program of multiple myeloma has identified novel markers of high-risk disease and mechanisms of pathogenesis. However, there remains a significant gap between prospective identification of high-risk patients and our ability to determine all patients that experience poor outcomes. Epigenetic alterations in myeloma have been less studied, but have significant potential as a translatable biomarker. To better understand how epigenetic programming may contribute to myeloma pathogenesis, we characterized the myeloma DNA methylome. DNA from CD138+ enriched myeloma specimens from the Multiple Myeloma Research Foundation (MMRF) CoMMpass study (NCT01454297) were obtained after receiving permission from the CoMMpass Tissue Use Committee and Emory IRB. Whole genome bisulfite sequencing (WGBS) was performed by adapter ligation (Kapa HyperPrep) using fully methylated sequencing adapters followed by bisulfite conversion (Qiagen Epitect), library amplification (Kapa Uracil+ HiFi polymerase), and sequencing (NovaSeq 6000 S4 150bp paired-end reads). Sequencing data were mapped with Bismark and CpG methylation calls were extracted using R. WGBS DNA methylation data was generated for 120 specimens, including 87 baseline, 24 paired relapse, and 9 paired peripheral blood specimens. This yielded ≥5x coverage at 21,393,650 CpGs in 90% of samples with an average coverage of 20x. Myeloma exhibited extensive genomic hypomethylation such that the median level was 42% (range 21-67%) as compared to 64% in plasma cells from healthy individuals. Principle component analysis indicated most variation corresponded with hypomethylation, which occurred in megabase domains devoid of gene expression. In contrast, DNA methylation was retained in the bodies of expressed genes. Principle components 2 and 3 separated samples with t(4;14) translocations from others. This may be due to overexpression of WHSC1 driving excessive histone 3 lysine 36 di-methylation (H3K36me2), which in turn impacts the function of PWWP-domain containing DNA methyltransferases (DNMT3A, DNMT3B). Given the 3-fold variability observed in average methylation, we sought to understand if this was indicative of outcome. Analysis of 87 baseline specimens identified 23,386 loci where DNA methylation (or lack thereof) was prognostic of outcome (FDR ≤0.01) (Figure 1a). These prognostic loci were clustered into contiguous regions often found in gene bodies and could be used to stratify patients by progression-free and overall survival (P-value <1e-7; Hazard ratio >8) (Figure 1b). Importantly, the prognostic value of these CpG were independent of t(4;14) status and ISS stage, indicating these high-risk patients would not have otherwise been identified. Furthermore, analysis of 24 relapse specimens from 22 patients indicated epigenetic remodeling occurred at these prognostic loci. Specifically, loci where the presence of DNA methylation was indicative of poor outcome gained DNA methylation in relapsed samples, and loci where lack of DNA methylation was indicative of poor outcome lost DNA methylation in relapse samples (Figure 1c). These relapse/refractory DNA methylation changes occurred in contiguous regions proximal to genes including PRKCE, MGMT, FHIT, WWOX, and HDAC9 (Figure 1d and data not shown). Cumulatively, these data identify myeloma epigenetic markers of outcome that undergo reprogramming in relapsed samples suggesting they may be indicative of therapeutic resistance. Disclosures Lonial: Genentech: Consultancy; Takeda: Consultancy, Research Funding; Amgen: Consultancy; BMS: Consultancy; Janssen: Consultancy, Research Funding; GSK: Consultancy; Karyopharm: Consultancy; Celgene Corporation: Consultancy, Research Funding. Boise:AstraZeneca: Honoraria, Research Funding; Genentech Inc.: Membership on an entity's Board of Directors or advisory committees.

Intra-Tumor Heterogeneity in Acute Myeloid Leukemia (AML): Results from a Real Life Cohort

Context. AML is an oligoclonal disease. Intratumoral genetic heterogeneity (ITH) is pervasive in cancers and has often been linked to poor outcome. Except in patients (pts) with complex karyotypes (Bochtler, J Clin Oncol 2013), the clinical relevance of ITH remains unknown in AML. We assessed ITH in a real-life AML cohort.Methods. Since Nov 2015, all newly diagnosed AML pts at our institution were analyzed by targeted sequencing with a 60-gene myeloid panel at an average depth of 815X. Variant Allele Frequencies (VAF) were adjusted for copy number variation to estimate Cancer Cell Fractions (CCFs) and rank mutations by decreasing CCFs. Mutations were segregated into different clones when distant by >10% CCF. Ancestral (as opposed to secondary) mutations are defined as those belonging to the first clone (Figure). We could next derive for each pt the total number of clones and the ITH index, defined as the ratio of secondary to ancestral mutations (according to Turajlic, Cell 2018).Results. We studied 201 consecutive AML pts (median age 67 years, secondary AML 24%). The median number of mutations was 4 (range 0-12). Clonal heterogeneity (>1 clone) was found in 71% of pts with a median of 2 clones per pt (range 1-5). 140 pts received intensive chemotherapy (IC), 50 hypomethylating agents (HMA) and 11 best supportive care (BSC) only.Signaling (FLT3, NRAS, KRAS, KIT, JAK2, CBL, CSF3R, BRAF, NF1, PTPN11, RIT1, CALR and MPL, 60%), DNA methylation (DNTM3A, TET2, IDH1/2, 54%), NPM1 (29%), spliceosome (U2AF1, SRSF2, SF3B1, ZRSR2, PRPF8, 27.9%) and tumor suppressors (PHF6, TP53, WT1, 27.4%) were the most frequently mutated pathways. ELN 2017 risk category was favorable, intermediate, adverse and not evaluable in 35%, 27%, 35% and 3% of pts respectively. Increasing ITH was associated with a higher number of clones (p=0.001) and of mutations (p<0.001). ITH was greater in pts with at least one mutation in histone modifiers (ASXL1, ASXL2, EZH2, KMT2A-PTD KMT2D, KDM6A, p=0.0004), cohesin (STAG2, RAD21, SMC3, SMC1A, CTCF, p=0.0018) or signaling (p=0.002) genes but was not affected by the presence of mutations in DNA methylation or spliceosome, tumor suppressor genes, transcription factors (RUNX1, CEBPA, ETV6, GATA2) or co-activators (BCOR, BCORL1, EP300, CREBBP), all p>0.05. ITH was not different among ELN 2017 risk categories (p=0.15). ITH was independent of age, WBC count and type of AML (secondary vs de novo), all p>0.2.There was no significant difference in the number of mutations (p=0.12), clones (p=0.58) or ITH indexes (p=0.38) according to IC/HMA/BSC treatment strategies. We next performed a preliminary analysis of the prognostic impact of ITH in the 140 pts treated with IC (median age 58y), with a median follow-up of only 12.5 months. 117 (84%) pts achieved CR and 32 (23%) were transplanted in first CR. Median event-free survival (EFS) was 18.7 vs 8.0 months in pts with adv and non-adv ELN risk, respectively (p=0.016). In univariate analyses, DNA methylation (p=0.04) and spliceosome (p=0.007) mutations were the two pathways predicting adverse outcome. The number of mutations (p=0.6) or of clones (p=0.8) had no impact on EFS. Pts with fewer secondary than ancestral mutations (ITH <1, ‘ITH low’ group, n=64 [46%]) had a median EFS of 13.6 months vs 19.8 months in the 76 (54%) pts with ITH index ≥1 (‘ITH high’ group, p=0.067). In a multivariate Cox model accounting for age, log(WBC), ELN risk, AML type (secondary vs de novo), presence of DNA methylation and spliceosome mutations and ITH, ITH-high was identified as a significant predictor of improved EFS (HR=0.54, p=0.03), independently of older age (HR=1.02, p=0.07), adv ELN risk (HR=2.4, p=0.005) and higher WBC count (HR=1.27, p=0.004). Censoring at transplant in first CR did not affect this result. In 14 pts who relapsed after a median of 12.9 months since diagnosis with paired NGS at relapse, the number of clones significantly decreased at relapse (p=0.046).Conclusion. Our results confirm that most AMLs are oligoclonal. Surprisingly, higher ITH is not related to older age or secondary AML. Our preliminary results suggest that lower ITH translates into shorter EFS in pts treated with IC. Longer follow-up and/or increased pt numbers are warranted to determine whether NGS-based assessments of clonal heterogeneity can improve AML risk stratification. [Display omitted] DisclosuresFenaux:Otsuka: Honoraria, Research Funding; Jazz: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding.

The diagnostic role of DNA methylation in sporadic endometrial cancer: a systematic review and meta-analysis.

BackgroundAlthough increasing numbers of methylated genes have been identified as biomarkers for endometrial cancer, the results have been inconsistent. We therefore carried out a systematic review and meta-analysis to evaluate the diagnostic accuracy of methylated genes as markers for sporadic endometrial cancer.ResultsA total of 22 studies including 1930 participants (sporadic endometrial cancer patients and normal individuals) met our eligibility criteria. The pooled sensitivity and specificity were 0.93 (95% confidence interval: 0.91−0.94) and 0.48 (95% confidence interval: 0.46–0.50), respectively. The area under the summary receiver operating characteristic curve was 0.8834. The presence of DNA methylation was significantly associated with lymph node metastasis of endometrial cancer (pooled odds ratio: 0.28, 95% confidence interval: 0.15–0.52, p < 0.001).Materials and MethodsWe searched the relevant literature systematically using the PubMed and Web of Science databases up to April 2017. Diagnostic accuracy variables were pooled and analyzed using Meta-DiSc software. Sensitivity analysis and publication bias were evaluated using Review Manager.ConclusionsThis meta-analysis suggests that the detection of DNA methylation is associated with lymph node metastasis, with high sensitivity but relatively low specificity for the diagnosis of sporadic endometrial cancer.

Methylation specific targeting of a chromatin remodeling complex from sponges to humans

DNA cytosine methylation and methyl-cytosine binding domain (MBD) containing proteins are found throughout all vertebrate species studied to date. However, both the presence of DNA methylation and pattern of methylation varies among invertebrate species. Invertebrates generally have only a single MBD protein, MBD2/3, that does not always contain appropriate residues for selectively binding methylated DNA. Therefore, we sought to determine whether sponges, one of the most ancient extant metazoan lineages, possess an MBD2/3 capable of recognizing methylated DNA and recruiting the associated nucleosome remodeling and deacetylase (NuRD) complex. We find that Ephydatia muelleri has genes for each of the NuRD core components including an EmMBD2/3 that selectively binds methylated DNA. NMR analyses reveal a remarkably conserved binding mode, showing almost identical chemical shift changes between binding to methylated and unmethylated CpG dinucleotides. In addition, we find that EmMBD2/3 and EmGATAD2A/B proteins form a coiled-coil interaction known to be critical for the formation of NuRD. Finally, we show that knockdown of EmMBD2/3 expression disrupts normal cellular architecture and development of E. muelleri. These data support a model in which the MBD2/3 methylation-dependent functional role emerged with the earliest multicellular organisms and has been maintained to varying degrees across animal evolution.

Clinical Utility of Molecular Profiling in Higher Risk MDS Treated with Azacitidine: Can We Tailor Therapy Accordingly?

▪Background Hypomethylating agent (HMA) therapy represents the standard of care for patients with higher risk myelodysplastic syndromes although only 50% of patients respond to treatment. Recent evidence from molecular profiling through next-generation sequencing (NGS) in myeloid diseases has been conflicting as to the value of somatic mutations as a biomarker for response to HMA. TET2 mutations, predominantly in the absence of ASXL1 mutations (or variant allele frequency (VAF) < 10%), have been shown to predict HMA response in MDS and chronic myelomonocytic leukemia (CMML) (Itzykson et al., 2011; Bejar et al., 2014 (80% decitabine)) while DNMT3A mutations predict response to frontline HMA treatment in acute myeloid leukemia (AML) (Coombs et al., 2016). Therefore, our goal was to identify molecular predictors of response and outcomes to azacitidine in myeloid malignancies.Patients and Methods Genetically profiled higher-risk MDS, CMML and oligoblastic AML (20-30% blasts) cases were retrospectively identified from the Moffitt Cancer Center MDS database. We evaluated gene mutations associated with DNA methylation (TET2, DNMT3A, IDH1, IDH2, and WT1) and up to 19 additional genes. NGS was performed prior to the initiation of HMA in all patients. The lower limit of VAF detection was set at 5% and the minimum depth of coverage at each position was 500X. Clinical variables and outcomes of MDS patients were characterized at the time of sample procurement. Fisher's exact and t-tests were used for comparative analyses. Kaplan-Meier curves were used to estimate overall survival and analyzed from the date of mutation identification. Multivariate Cox regression models were created to adjust for clinical characteristics.Results From May 2013 to February 2016, a total of 77 patients with NGS for somatic mutations prior to HMA therapy were identified with a median age of 70 years and male predominance (66%). Of the cohort, 97% of patients (n=75) were treated with azacitidine with 17% of patients (n=13) proceeding to allogeneic hematopoietic stem cell transplant (AHSCT). A total of 86% of patients (n=66) had at least one pathogenic mutation. Mutations in DNA methylation occurred in 43% of patients (n=33) while TET2 mutation without clonal ASXL1 mutations occurred in 13% of patients (n=10). At a median follow up 17 months, the median OS of the entire cohort was 12.5 months. Patients with a DNA methylation mutation had a median OS that was not reached (NR) vs a median OS of 11.5 months in wildtype (WT) patients (HR 0.38, 95% CI 0.21 to 0.75; P = 0.005), which remained significant when censoring patients at time of AHSCT (P = 0.002). TP53 mutant (MT) patients (n=14, 18% of cohort) had a median OS of 7.9 months vs 15.4 months in WT patients (HR 3.69, 95% CI 3.04 to 28.8; P = 0.0001). The presence of DNA methylation or TP53 mutation in comparison to wildtype patients significantly stratified prognosis in azacitidine treated patients (Figure 1A, P = 0.0001). In multivariable analysis incorporating age and revised international prognostic scoring system (IPSS-R) category, DNA methylation (HR 0.45, 95% CI 0.21 to 0.96; P = 0.04) and TP53 (HR 2.34, 95% CI 1.04 to 5.26; P = 0.04) mutation status remained predictive for survival. Notably, response rates in DNA methylation mutant patients were similar to WT patients (40% versus 42%) with no difference in treatment duration. However, the overall response rate of TET2 MT/ASXL1 WT patients was 70% versus 36% in the rest of the cohort (P =0.08) and 0% in TET2 MT/ASXL1 MT patients (0/6, P =0.01) with a significantly longer duration of treatment in the TET2 MT/ASXL1 WT cohort (median number of cycles 7.5 versus 4; P = 0.002). Additionally, TET2 MT/ASXL1 WT patients had longer survival (median OS NR vs 12.2 months; P = 0.047). When censoring for transplant, the impact of TET2 MT/ASXL1 WT genoptype was significantly strengthened with 70% of patients alive past 15 months (median OS NR vs 9.9 months; HR 0.24, 95% CI 0.19 to 0.75; P = 0.007; Figure 1B).Conclusion In patients with myeloid malignancies, molecular profiling via NGS can predict outcomes to azacitidine therapy. Patients with mutations of DNA methylation have improved OS whereas OS is poor in TP53 MT patients. Most importantly, TET2 MT/ASXL1 WT identifies a genotypic subgroup with particularly good outcomes when treated with HMA without AHSCT and potentially challenges the early timing of AHSCT for higher risk patients and this molecular profile. [Display omitted] DisclosuresPadron:Novartis: Honoraria; Incyte: Research Funding; CTI: Honoraria, Research Funding; KALOBIOS: Research Funding. Vaupel:Genoptix, a Novartis Company: Employment. Hall:Genoptix, a Novartis Company: Employment. Komrokji:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Speakers Bureau; Boehringer-Ingelheim: Research Funding; Incyte: Consultancy.

Hormone stimulation of androgen receptor mediates dynamic changes in DNA methylation patterns at regulatory elements.

DNA methylation is an epigenetic modification that contributes to stable gene silencing by interfering with the ability of transcriptional regulators to bind to DNA. Recent findings have revealed that hormone stimulation of certain nuclear receptors induces rapid, dynamic changes in DNA methylation patterns alongside transcriptional responses at a subset of target loci, over time. However, the ability of androgen receptor (AR) to dynamically regulate gene transcription is relatively under-studied and its role in the regulation of DNA methylation patterns remains to be elucidated. Here we demonstrate in normal prostate cells that hormone stimulated AR activity results in dynamic changes in the transcription rate and DNA methylation patterns at the AR target genes, TIPARP and SGK1. Time-resolved chromatin immunoprecipitation experiments on the SGK1 locus reveals dynamic recruitment of AR and RNA Polymerase II, as well as the recruitment of proteins involved in the DNA demethylation process, TET1 and TDG. Furthermore, the presence of DNA methylation at dynamic regions inhibits protein binding and transcriptional activity of SGK1. These findings establish AR activity as a contributing factor to the dynamic regulation of DNA methylation patterns at target genes in prostate biology and infer further complexity involved in nuclear receptor mediation of transcriptional regulation.

Aberrant DNA Methylation of CDH1, p16 and DAPK in Childhood Acute Lymphoblastic Leukemia

Results: CDH1 was methylated in 26 (60.4%) patients, p16 in two (4.6%) patients and DAPK in six (13.9%) patients at the time of diagnosis. Twenty nine (67.4%) patients had methylation of at least one gene. None of the healthy BM donors showed methylation of the above genes. Age was the only factor which showed significant association with the presence of DNA methylation (P=0.03). None of the other clinicopathological factors showed association with initial methylation status. At the time of morphologic CR, all patients who had aberrant DNA methylation at the time of diagnosis had no detectable residual methylation.

Sensitivity and specificity of immunoprecipitation of DNA containing 5-Methylcytosine

BackgroundAttempts to enrich or identify DNA with cytosine methylation have been commonly carried out using anti-5-methylcytosine or anti-MBD2 (methyl-CpG binding domain protein 2) antibody in immunoprecipitation (IP) assays. However, a careful and systematic control experiment to examine the sensitivity and specificity of this approach has not been reported. It is of critical importance to understand the potential pitfalls of this approach and to avoid potential misinterpretation of findings.FindingsWe found that increased concentration of antibody used in the assay increased the amount of overall DNA captured as expected. The increased number of methylated cytosines in/on the DNA fragment also increased the amount of DNA captured by the antibody. Importantly, the antibody can bind to some fully unmethylated DNA fragments, even when fully methylated DNA is present in the same experiment.ConclusionThe sensitivity of anti-5-methylcytosine antibody and anti-MBD2 antibody/MBD2 binding varies with the number of methylated cytosines on the DNA target. The specificity of these antibodies can also vary for different DNA target sequences. DNA fragments with fewer CpG sites may not bind to these antibodies even when all are methylated while DNA fragments with more CpG sites may bind to the antibodies when only some of these sites are methylated. More importantly, binding of DNA to these antibodies does not always indicate the presence of DNA methylation. It is clear that false positive and false negative findings can be easily reached even though it does not nullify these convenient and simple methods completely. Great caution should be taken for the interpretation of IP results using these antibodies and rigorous confirmation by sodium bisulfite sequencing is essential.

DNA Methylation Detection and Inhibitor Screening Based on the Discrimination of the Aggregation of Long and Short DNA on a Negatively Charged Indium Tin Oxide Microelectrode

On the basis of the different diffusivity and existence of electrostatic repulsion between long and short DNA on the negatively changed indium tin oxide (ITO) microelectrode, a simple but sensitive immobilization free solution-phase electrochemical method for DNA methylation detection and inhibitor screening has been developed. Electroactive substance (methylene blue) tagged at the penultimate base T close to the 3'-terminal first, in the absence of DNA methylation, methylene blue-labeled electroactive fragments cannot be generated by Dpn I and results in a weak electrochemical response being detected on the ITO electrode. On the contrary, a remarkable electrochemical response can be achieved by the cleavage in the presence of DNA methylation since methylene blue-labeled electroactive fragments can be generated and aggregate on the ITO electrode. The proposed system does not need complex operation procedures such as bisulfite treatment, PCR amplification, and electrode immobilization. Six ITO microelectrodes had been assembled on the same microchip, which can achieve the parallel detection of the same sample and improve the experimental efficiency of drug screening. The system was used to conveniently and specifically monitor the change of the DNA methylation level with high sensitivity and selectivity. The proposed system has the potential application to screen the drugs as inhibitors on the activity of methyltransferase in the clinic.

5-methyl-cytosine and 5-hydroxy-methyl-cytosine in the genome of Biomphalaria glabrata, a snail intermediate host of Schistosoma mansoni

BackgroundBiomphalaria glabrata is the mollusc intermediate host for Schistosoma mansoni, a digenean flatworm parasite that causes human intestinal schistosomiasis. An estimated 200 million people in 74 countries suffer from schistosomiasis, in terms of morbidity this is the most severe tropical disease after malaria. Epigenetic information informs on the status of gene activity that is heritable, for which changes are reversible and that is not based on the DNA sequence. Epigenetic mechanisms generate variability that provides a source for potentially heritable phenotypic variation and therefore could be involved in the adaptation to environmental constraint. Phenotypic variations are particularly important in host-parasite interactions in which both selective pressure and rate of evolution are high. In this context, epigenetic changes are expected to be major drivers of phenotypic plasticity and co-adaptation between host and parasite. Consequently, with characterization of the genomes of invertebrates that are parasite vectors or intermediate hosts, it is also essential to understand how the epigenetic machinery functions to better decipher the interplay between host and parasite.MethodsThe CpGo/e ratios were used as a proxy to investigate the occurrence of CpG methylation in B. glabrata coding regions. The presence of DNA methylation in B. glabrata was also confirmed by several experimental approaches: restriction enzymatic digestion with isoschizomers, bisulfite conversion based techniques and LC-MS/MS analysis.ResultsIn this work, we report that DNA methylation, which is one of the carriers of epigenetic information, occurs in B. glabrata; approximately 2% of cytosine nucleotides are methylated. We describe the methylation machinery of B. glabrata. Methylation occurs predominantly at CpG sites, present at high ratios in coding regions of genes associated with housekeeping functions. We also demonstrate by bisulfite treatment that methylation occurs in multiple copies of Nimbus, a transposable element.ConclusionsThis study details DNA methylation for the first time, one of the carriers of epigenetic information in B. glabrata. The general characteristics of DNA methylation that we observed in the B. glabrata genome conform to what epigenetic studies have reported from other invertebrate species.

Association of DNA promoter hypermethylation of decoy receptor 1 (DCR1) with poor response to irinotecan in metastatic colorectal cancer.

3552 Background: Heterogeneity in the biology of colorectal cancer (CRC) is associated with variable responses to standard chemotherapy. We aimed to identify DNA hypermethylated genes as predictive biomarkers for irinotecan treatment of patients with metastatic CRC. Methods: The presence of DNA methylation for a selected panel of 22 genes was assessed by methylation specific PCR (MSP) on primary tumors of 185 patients with metastatic CRC treated with first-line capecitabine (CAP, n=90) or a combination of capecitabine and irinotecan (CAPIRI, n=95) in the phase 3 CAIRO trial. Methylation status of each gene was correlated to progression free survival (PFS) by treatment regimen. Genes for which methylation status associated with response to irinotecan, were validated in 166 patients treated with first-line CAP (n=78) or CAPIRI (n=88). Results: Decoy Receptor 1 (DCR1) was identified as a novel hypermethylated gene in CRC. In CAPIRI treated patients, DCR1 methylation was correlated to a shorter PFS compared to patients with unmethylated DCR1 (hazard ratio [HR]=0.4 (95%CI =0.3-0.7), p = 0.0009). In patients with methylated DCR1 PFS did not improve with CAPIRI treatment, compared to treatment with CAP (discovery set: HR=0.8 (95%CI=0.5-1.3, p=0.4); validation set: HR=1.1 (95%CI 0.7-1.7, p=0.6)), in contrast to patients with unmethylated DCR1 (discovery set: HR=2.5 (95%CI 1.7-3.3, p=0.00004); validation set: HR=1.7 (95%CI 1.1-2.0, p=0.004)). Conclusions: CRC patients with methylated DCR1 did not benefit from adding irinotecan to capecitabine therapy, indicating that DCR1 methylation status may guide selecting metastatic CRC patients for irinotecan-based therapy.

Genome-wide association between DNA methylation and alternative splicing in an invertebrate

BackgroundGene bodies are the most evolutionarily conserved targets of DNA methylation in eukaryotes. However, the regulatory functions of gene body DNA methylation remain largely unknown. DNA methylation in insects appears to be primarily confined to exons. Two recent studies in Apis mellifera (honeybee) and Nasonia vitripennis (jewel wasp) analyzed transcription and DNA methylation data for one gene in each species to demonstrate that exon-specific DNA methylation may be associated with alternative splicing events. In this study we investigated the relationship between DNA methylation, alternative splicing, and cross-species gene conservation on a genome-wide scale using genome-wide transcription and DNA methylation data.ResultsWe generated RNA deep sequencing data (RNA-seq) to measure genome-wide mRNA expression at the exon- and gene-level. We produced a de novo transcriptome from this RNA-seq data and computationally predicted splice variants for the honeybee genome. We found that exons that are included in transcription are higher methylated than exons that are skipped during transcription. We detected enrichment for alternative splicing among methylated genes compared to unmethylated genes using fisher’s exact test. We performed a statistical analysis to reveal that the presence of DNA methylation or alternative splicing are both factors associated with a longer gene length and a greater number of exons in genes. In concordance with this observation, a conservation analysis using BLAST revealed that each of these factors is also associated with higher cross-species gene conservation.ConclusionsThis study constitutes the first genome-wide analysis exhibiting a positive relationship between exon-level DNA methylation and mRNA expression in the honeybee. Our finding that methylated genes are enriched for alternative splicing suggests that, in invertebrates, exon-level DNA methylation may play a role in the construction of splice variants by positively influencing exon inclusion during transcription. The results from our cross-species homology analysis suggest that DNA methylation and alternative splicing are genetic mechanisms whose utilization could contribute to a longer gene length and a slower rate of gene evolution.

Control of genic DNA methylation in Arabidopsis

Detailed features of genomic DNA methylation have been revealed by recent genome-wide analyses on several model organisms. An unexpected feature conserved among plants and some animals is the presence of DNA methylation within transcribed genes. For understanding the controlling mechanisms of the enigmatic genic methylation, genetic and genomic approaches using Arabidopsis may be effective.

Suppression of microRNA accumulation via RNA interference in Arabidopsis thaliana

MicroRNAs (miRNAs) are key regulatory molecules in plants. These small RNAs are processed in the nucleus from longer precursor transcripts that form distinct secondary structures. The miRNAs target specific messenger RNAs (mRNAs) and consequently down-regulate gene expression. The importance of these regulatory molecules is wide-ranging, however, few loss-of-function mutants have been identified in miRNA genes and understanding the biology of miRNA-target pairings has largely depended upon creating alterations in the sequences of the target genes. Here we demonstrate using Arabidopsis thaliana, that it is possible to use RNA interference (RNAi) to suppress accumulation of miRNAs. Significantly reduced accumulation of miR163 and miR171a was achieved using hairpin RNAi constructs that were designed to target both the primary miRNA transcripts and their promoters. The presence of DNA methylation in the targeted promoter regions suggests that inhibition of transcription of the miRNA precursors is responsible. Reduction of miRNA accumulation resulted in an increase in accumulation of the mRNA targets of these miRNAs. This demonstrates that knock-down of miRNA expression can be achieved, thereby providing a straightforward approach for disrupting miRNA-target pairings and studying miRNA functions.

Quantitative DNA-methylation in Daphnia magna and effects of multigeneration Zn exposure

Little research on the epigenetic phenomenon of DNA methylation has been performed with invertebrates. However, a few studies have shown that effects of transient chemical exposure can be transferred to non-exposed generations through epigenetic inheritance. If this is a wide-spread phenomenon it may have major consequences for current ecological risk assessments. The presence of DNA methylation in Daphnia magna has only very recently been demonstrated, although not quantified. In the present study, the overall content of 5-methyl-2′-deoxycytidine (5mdC) in the DNA of D. magna was determined. Additionally, the effect of transgenerational Zn exposure on the 5mdC content was assessed. An optimized LC-MS-MS method proved to be very suitable for measuring overall levels of 5mdC in relatively small samples of D. magna DNA. The average [5mdC]/[dG] ratio ranged from 0.13% to 0.81%. A slight but significant decrease in DNA methylation was found in the offspring (F1) of Zn exposed daphnids in the parental generation. However, this effect did not propagate into the next generation (F2). The presence of DNA methylation in D. magna and the finding that different exposure histories can entail different levels of methylation, open the way for further investigations on the implications of epigenetics in aquatic toxicology.

Occurrence of DNA methylation in Daphnia magna and influence of multigeneration Cd exposure

Most of the research on the epigenetic phenomenon of DNA methylation has been performed with vertebrates and plants. Knowledge on DNA methylation in Daphnia magna, a key test organism in aquatic toxicology, is completely lacking. Through epigenetic inheritance, effects of transient chemical exposure could be transferred to non-exposed generations, which could have a major impact on ecological risk assessment procedures. In this study, we determined if CpG methylation occurs in D. magna and if this can be influenced by exposure to toxic substances. Homologs of human DNA methyltransferases DNMT1, DNMT2 and DNMT3A were found in the partially available D. magna genome. Using an optimized “Amplification of Intermethylated Sites (AIMS)” technique, two methylated fragments were discovered in D. magna DNA. No homology was found for these sequences. The methylation and the D. magna origin of the fragments were confirmed with Southern analysis. This optimized AIMS technique was then applied to DNA of D. magna which were exposed to 180 µg/L Cd for two generations. Exposure resulted in a significant decrease in reproduction. The same methylated fragments with the same band intensity were observed in DNA of both non-exposed and exposed daphnids. As such, it could not be demonstrated that Cd exposure altered DNA methylation. However, the presence of DNA methylation in D. magna shows that potentially epigenetic effects may occur in this species.