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DNA methyltransferase inhibitors (DNMTis) reexpress hypermethylated genes in cancers and leukemias and also activate endogenous retroviruses (ERVs), leading to interferon (IFN) signaling, in a process known as viral mimicry. In the present study we show that in the subset of acute myeloid leukemias (AMLs) with mutations in

Non-coding transcription is an important determinant of heterochromatin formation. In Arabidopsis thaliana a specialized RNA polymerase V (Pol V) transcribes pervasively and produces long non-coding RNAs. These transcripts work with small interfering RNA to facilitate locus-specific establishment of RNA-directed DNA methylation (RdDM). Subsequent maintenance of RdDM is associated with elevated levels of Pol V transcription. However, the impact of DNA methylation on Pol V transcription remained unresolved. We found that DNA methylation strongly enhances Pol V transcription. The level of Pol V transcription is reduced in mutants defective in RdDM components working downstream of Pol V, indicating that RdDM is maintained by a mutual reinforcement of DNA methylation and Pol V transcription. Pol V transcription is affected only on loci that lose DNA methylation in all sequence contexts in a particular mutant, including mutants lacking maintenance DNA methyltransferases, which suggests that RdDM works in a complex crosstalk with other silencing pathways.

Background DNA methylation is a conserved and important epigenetic modification involved in the regulation of numerous biological processes, including plant development, secondary metabolism, and response to stresses. However, no information is available regarding the identification of cytosine-5 DNA methyltransferase (C5-MTase) and DNA demethylase (dMTase) genes in the orchid Dendrobium officinale. Results In this study, we performed a genome-wide analysis of DoC5-MTase and DodMTase gene families in D. officinale. Integrated analysis of conserved motifs, gene structures and phylogenetic analysis showed that eight DoC5-MTases were divided into four subfamilies (DoCMT, DoDNMT, DoDRM, DoMET) while three DodMTases were divided into two subfamilies (DoDML3, DoROS1). Multiple cis-acting elements, especially stress-responsive and hormone-responsive ones, were found in the promoter region of DoC5-MTase and DodMTase genes. Furthermore, we investigated the expression profiles of DoC5-MTase and DodMTase in 10 different tissues, as well as their transcript abundance under abiotic stresses (cold and drought) and at the seedling stage, in protocorm-like bodies, shoots, and plantlets. Interestingly, most DoC5-MTases were downregulated whereas DodMTases were upregulated by cold stress. At the seedling stage, DoC5-MTase expression decreased as growth proceeded, but DodMTase expression increased. Conclusions These results provide a basis for elucidating the role of DoC5-MTase and DodMTase in secondary metabolite production and responses to abiotic stresses in D. officinale.

Herein, a hand-drawing paper-based bipolar electrode (BPE) electrochemiluminescence (ECL) platform for M.SssI methyltransferase (M.SssI MTase) assay was proposed via employing high electrocatalytic Pt@CeO

DNA methylation plays important roles in various biological processes, and the alteration of DNA methyltransferase activity can induce the aberrant DNA methylation patterns. Despite the progress in methyltransferase activity assays, few methods enable the detection of both bacteria and human methyltransferases. Herein, we construct a universal and label-free chemiluminescent sensor for accurate quantification of both bacteria methyltransferases (e.g., M. SssI methyltransferase (M.SssI MTase)) and human methyltransferases (e.g., DNA (cytosine-5)-methyltransferase 1, (Dnmt1)) by integrating a dumbbell probe with BssHII endonuclease-mediated rolling circle amplification (RCA). We ingeniously design a structure-switchable dumbbell probe which integrates target-recognition, BssHII endonuclease-cleavage, RCA amplification and signal transduction in one probe for the detection of both M.SssI MTase and Dnmt1. Moreover, the introduction of two BssHII endonuclease recognition sites in a dumbbell probe can greatly reduce the false positivity resulting from the incomplete cleavage of dumbbell probe by BssHII, because once one of two recognition sites is identified by BssHII, the dumbbell probe can be completely digested by Exonuclease III (Exo III) and Exonuclease I (Exo I) to prevent the nonspecific RCA. This chemiluminescent sensor can accurately quantify M.SssI MTase in both 10% serum and various cell lysis buffers, and even sensitively detect Dnmt1 activity in MCF-7 cells. Furthermore, this chemiluminescent sensor can be used to screen the inhibitors of Dnmt1 and M.SssI MTase, with promising applications in disease diagnosis and drug discovery.

In plants and mammals, DNA methylation plays a critical role in transcriptional silencing by delineating heterochromatin from transcriptionally active euchromatin. A homeostatic balance between heterochromatin and euchromatin is essential to genomic stability. This is evident in many diseases and mutants for heterochromatin maintenance, which are characterized by global losses of DNA methylation coupled with localized ectopic gains of DNA methylation that alter transcription. Furthermore, we have shown that genome-wide methylation patterns in Arabidopsis thaliana are highly stable over generations, with the exception of rare epialleles. However, the extent to which natural variation in the robustness of targeting DNA methylation to heterochromatin exists, and the phenotypic consequences of such variation, remain to be fully explored. Here we describe the finding that heterochromatin and genic DNA methylation are highly variable among 725 A. thaliana accessions. We found that genic DNA methylation is inversely correlated with that in heterochromatin, suggesting that certain methylation pathway(s) may be redirected to genes upon the loss of heterochromatin. This redistribution likely involves a feedback loop involving the DNA methyltransferase, CHROMOMETHYLASE 3 (CMT3), H3K9me2, and histone turnover, as highly expressed, long genes with a high density of CMT3-preferred CWG sites are more likely to be methylated. Importantly, although the presence of CG methylation in genes alone may not affect transcription, genes containing CG methylation are more likely to become methylated at non-CG sites and silenced. These findings are consistent with the hypothesis that natural variation in DNA methylation homeostasis may underlie the evolution of epialleles that alter phenotypes.

Sensitive and accurate determination of DNA methyltransferase (DNA Mtase) activity is highly pursued for understanding fundamental biological processes related to DNA methylation, clinical disease diagnosis and drug discovery. Herein, we propose a new electrochemical immuno-DNA sensing platform for DNA Mtase activity assay and inhibitor screening. After homogeneous DNA methylation by CpG methyltransferase (M.SssI Mtase), the methylated DNA can be specifically recruited onto an electrode via its immunological binding with the immobilized anti-5-methylcytosine antibody. The recruited methylated DNA was simultaneously used as a substrate to facilitate successive template-free DNA extension and enzyme catalysis for the dual-step signal amplification of DNA Mtase activity. The developed immuno-DNA sensing strategy effectively integrates solution-phase DNA methylation, surface affinity binding recognition, and successive template-free DNA extension and enzyme catalysis-based signal amplification, rendering a highly specific, sensitive and accurate assay of DNA Mtase activity. A low detection limit of 0.039 U mL-1 could be achieved with a high selectivity. It was also applied for efficient evaluation of various inhibitors. Current affinity recognition of the immobilized antibody with methylated DNA switches the sensing platform into a DNA operation interface, facilitating the opportunity for combining various DNA-based signal amplification strategies to improve the detection performance. It would be used as a general strategy for the analysis of DNA Mtase activity, inhibitors and more analytes, and is anticipated to show potential for applications in disease diagnosis and drug discovery.

CpG island methylation plays an important role in diverse biological processes including the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. Due to the dependence of DNA methylation on DNA methyltransferase (MTase) activity, DNA MTases have become the potential targets in anticancer therapy. Herein we demonstrate for the first time the construction of a single quantum dot (QD) nanosensor with the capability of sensing methylcytosine sites for sensitive quantification of M.SssI CpG methyltransferase (M.SssI MTase). We design a biotin-/phosphate-modified double-stranded DNA (dsDNA) substrate with a 5'-G-C-G-mC-3'/3'-mC-G-mC-G-5' site for sensing M.SssI MTase. In the presence of M.SssI MTase, the methylation-responsive sequence of the dsDNA substrate is methylated and cleaved by GlaI endonuclease, producing two dsDNA fragments with a free 3'-OH terminus. In the presence of terminal deoxynucleotidyl transferase (TdT), multiple Cy5-dATPs can be sequentially added to the free 3'-OH terminus of dsDNA fragments to obtain biotin-/multiple Cy5-labeled dsDNAs. The resultant biotin-/multiple Cy5-labeled dsDNAs can assemble on the surface of the streptavidin-coated QD to obtain a QD-dsDNA-Cy5 nanostructure in which the fluorescence resonance energy transfer (FRET) from the QD to Cy5 can occur. The emission of Cy5 can be simply quantified by single-molecule detection. By the integration of sensing methylcytosine sites and enzymatic polymerization, the sensitivity of this nanosensor has been significantly enhanced. This nanosensor can detect as low as 2.1 × 10-7 U μL-1 M.SssI MTase with good selectivity against other cytosine MTases, and it can be further applied for the screening of MTase inhibitors and complex biological sample analysis, holding great potential in clinical diagnosis and drug discovery.

Little is known regarding the DNA methyltransferases (MTases) in hyperthermophilic archaea. In this study, we focus on an MTase from Aeropyrum pernix K1, a hyperthermophilic archaeon that is found in hydrothermal vents and whose optimum growth temperature is 90°C to 95°C. From genomic sequence analysis, A. pernix K1 has been predicted to have a restriction-modification system (R-M system). The restriction endonuclease from A. pernix K1 (known as ApeKI from New England BioLabs Inc. [catalog code R06435]) has been described previously, but the properties of the MTase from A. pernix K1 (M.ApeKI) have not yet been clarified. Thus, we demonstrated the properties of M.ApeKI. In this study, M.ApeKI was expressed in Escherichia coli strain JM109 and affinity purified using its His tag. The recognition sequence of M.ApeKI was determined by methylation activity and bisulfite sequencing (BS-seq). High-performance liquid chromatography (HPLC) was used to detect the position of the methyl group in methylated cytosine. As a result, it was clarified that M.ApeKI adds the methyl group at the C-5 position of the second cytosine in 5′-GCWGC-3′. Moreover, we also determined that the MTase optimum temperature was over 70°C and that it is strongly tolerant to high temperatures. M.ApeKI is the first highly thermostable DNA (cytosine-5)-methyltransferase to be evaluated by experimental evidence. IMPORTANCE In general, thermophilic bacteria with optimum growth temperatures over or equal to 60°C have been predicted to include only N4-methylcytosine or N6-methyladenine as methylated bases in their DNA, because 5-methylcytosine is susceptible to deamination by heat. However, from this study, A. pernix K1, with an optimum growth temperature at 95°C, was demonstrated to produce a DNA (cytosine-5)-methyltransferase. Thus, A. pernix K1 presumably has 5-methylcytosine in its DNA and may produce an original repair system for the expected C-to-T mutations. M.ApeKI was demonstrated to be tolerant to high temperatures; thus, we expect that M.ApeKI may be valuable for the development of a novel analysis system or epigenetic editing tool.

Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

Background Protein pulldown using Methyl-CpG binding domain (MBD) proteins followed by high-throughput sequencing is a common method to determine DNA methylation. Algorithms have been developed to estimate absolute methylation level from read coverage generated by affinity enrichment-based techniques, but the most accurate one for MBD-seq data requires additional data from an SssI-treated Control experiment. Results Using our previous characterizations of Methyl-CpG/MBD2 binding in the context of an MBD pulldown experiment, we build a model of expected MBD pulldown reads as drawn from SssI-treated DNA. We use the program BayMeth to evaluate the effectiveness of this model by substituting calculated SssI Control data for the observed SssI Control data. By comparing methylation predictions against those from an RRBS data set, we find that BayMeth run with our modeled SssI Control data performs better than BayMeth run with observed SssI Control data, on both 100 bp and 10 bp windows. Adapting the model to an external data set solely by changing the average fragment length, our calculated data still informs the BayMeth program to a similar level as observed data in predicting methylation state on a pulldown data set with matching WGBS estimates. Conclusion In both internal and external MBD pulldown data sets tested in this study, BayMeth used with our modeled pulldown coverage performs better than BayMeth run without the inclusion of any estimate of SssI Control pulldown, and is comparable to – and in some cases better than – using observed SssI Control data with the BayMeth program. Thus, our MBD pulldown alignment model can improve methylation predictions without the need to perform additional control experiments. Electronic supplementary material The online version of this article (10.1186/s12859-019-3011-2) contains supplementary material, which is available to authorized users.

Chronic inflammation is strongly associated with an increased risk of developing colorectal cancer. DNA hypermethylation of CpG islands alters the expression of genes in cancer cells and plays an important role in carcinogenesis. Chronic inflammation is also associated with DNA methylation alterations and in a mouse model of inflammation-induced colon tumorigenesis, we previously demonstrated that inflammation-induced tumours have 203 unique regions with DNA hypermethylation compared to uninflamed epithelium. To determine if altering inflammation-induced DNA hypermethylation reduces tumorigenesis, we used the same mouse model and treated mice with the DNA methyltransferase (DNMT) inhibitor decitabine (DAC) throughout the tumorigenesis time frame. DAC treatment caused a significant reduction in colon tumorigenesis. The tumours that did form after DAC treatment had reduced inflammation-specific DNA hypermethylation and alteration of expression of associated candidate genes. When compared, inflammation-induced tumours from control (PBS-treated) mice were enriched for cell proliferation associated gene expression pathways whereas inflammation-induced tumours from DAC-treated mice were enriched for interferon gene signatures. To further understand the altered tumorigenesis, we derived tumoroids from the different tumour types. Interestingly, tumoroids derived from inflammation-induced tumours from control mice maintained many of the inflammation-induced DNA hypermethylation alterations and had higher levels of DNA hypermethylation at these regions than tumoroids from DAC-treated mice. Importantly, tumoroids derived from inflammation-induced tumours from the DAC-treated mice proliferated more slowly than those derived from the inflammation-induced tumours from control mice. These studies suggest that inhibition of inflammation-induced DNA hypermethylation may be an effective strategy to reduce inflammation-induced tumorigenesis.

In Norway, mine tailings waste can be deposited by coastal submarine dispersal. Mine tailings slurry includes fine particles, Exposure to mine tailings particles increases mortality in Atlantic cod larvae and results in increased expression of genes involved in epigenetic regulation.

We reconstituted two biochemical processes that may contribute to UV-induced mutagenesis in vitro and analysed the mutational profiles in the products. One process is translesion synthesis (TLS) by DNA polymerases (Pol) δ, η and ζ, which creates C>T transitions at pyrimidine dimers by incorporating two dAMPs opposite of the dimers. The other process involves spontaneous deamination of cytosine, producing uracil in pyrimidine dimers, followed by monomerization of the dimers by secondary UV irradiation, and DNA synthesis by Pol δ. The mutational spectrum resulting from deamination without translesion synthesis is similar to a mutational signature found in melanomas, suggesting that cytosine deamination encountered by the replicative polymerase has a prominent role in melanoma development. However, CC>TT dinucleotide substitution, which is also commonly observed in melanomas, was produced almost exclusively by TLS. We propose that both TLS-dependent and deamination-dependent mutational processes are likely involved in UV-induced melanoma development.

Research Question Is there any genetic association between oestrogen receptor alpha [ERα]-PvuII polymorphism and idiopathic male infertility? Design A total of 226 infertile and 213 fertile men participated in the present case-control study. ERα-PvuII genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP] method. Meta-analysis was also performed by pooling data collected from seven other eligible studies identified by searches of PubMed, Embase, Google Scholar, and Science Direct databases. Summary odds ratios were estimated by fixed- or random-effects models. The molecular effects of ERα-PvuII polymorphism were evaluated by bioinformatics tools. Results A significant protective association was reported between ERα-PvuII and male infertility in the homozygote model [OR=0.54, 95%CI=0.3–0.98, p=0.042]. Also, a similar association was observed in asthenozoospermia subgroup [OR=0.4, 95%CI=0.18–0.9, p=0.025]. Meta-analysis also revealed that the ER-PvuII polymorphism was significantly associated with the decreased risk of male infertility in the heterozygote co-dominant model [OR=0.80, 95%CI=0.64–0.99, p=0.042]. Moreover, similar protective results were reported in stratified analyses in Caucasian subgroup in the dominant genetic model [OR=0.66, 95%CI=0.45–0.96, p=0.029] and in the heterozygote co-dominant model [OR=0.62, 95%CI=0.41–0.93, p=0.021]. A significant association was also found in studies with sample size of less than 400 subjects in heterozygote co-dominant model [OR=0.69, 95%CI=0.50–0.95, p=0.023]. The bioinformatics data indicated that ER-PvuII polymorphism could significantly affect RNA structure of ERα [p=0.004]. Conclusion The ERα-PvuII polymorphism could be considered as a possible protective factor against male infertility.

DNA methylation carried out by different methyltransferase classes is a relevant epigenetic modification of DNA which plays a relevant role in the development of eukaryotic organisms. Accordingly, in Arabidopsis thaliana loss of DNA methylation due to combined mutations in genes encoding for DNA methyltransferases causes several developmental abnormalities. The present study describes novel growth disorders in the drm1 drm2 cmt3 triple mutant of Arabidopsis thaliana, defective both in maintenance and de novo DNA methylation, and highlights the correlation between DNA methylation and the auxin hormone pathway. By using an auxin responsive reporter gene, we discovered that auxin accumulation and distribution were affected in the mutant compared to the wild type, from embryo to adult plant stage. In addition, we demonstrated that the defective methylation status also affected the expression of genes that regulate auxin hormone pathways from synthesis to transport and signalling and a direct relationship between differentially expressed auxin-related genes and altered auxin accumulation and distribution in embryo, leaf and root was observed. Finally, we provided evidence of the direct and organ-specific modulation of auxin-related genes through the DNA methylation process.

Significance Site-specific modification of epigenetic marks such as DNA methylation to regulate gene expression is a unique approach to enhance economically important crop traits. This approach allows for the maintenance of the introduced modifications in the absence of the initial transgene inducer in subsequent generations and relies largely on methylation of cytosines in the CG-specific sequence context. We have developed a targeted DNA methylation tool based on a bacterial methyltransferase and the CRISPR-Cas9 platform to directly methylate cytosines at CG sites in Arabidopsis. These tools expand the currently available CRISPR-based targeted DNA methylation tools and provide an approach for the establishment of heritable targeted DNA methylation in plants., CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research.

Patients with long-standing diabetes have a high risk for cardiac complications that is exacerbated by increased reactive oxygen species (ROS) production. We found that feeding cyanocobalamin (B12), a scavenger of superoxide, not only prevented but reversed signs of cardiomyopathy in type 1 diabetic Elmo1H/H Ins2Akita/+ mice. ROS reductions in plasma and hearts were comparable to those in mice treated with other antioxidants, N-acetyl-L-cysteine or tempol, but B12 produced better cardioprotective effects. Diabetes markedly decreased plasma insulin-like growth factor (IGF)-1 levels, while B12, but not N-acetyl-L-cysteine nor tempol, restored them. B12 activated hepatic IGF-1 production via normalization of S-adenosylmethionine levels, DNA methyltransferase (DNMT)-1/3a/3b mRNA, and DNA methylation of promoters for suppressor of cytokine signaling (SOCS)-1/3. Reductions of cardiac IGF-1 mRNA and phosphorylated IGF-1 receptors were also restored. Thus, B12 is a promising option for preventing diabetic cardiomyopathy via ROS reduction and IGF-1 retrieval through DNMT-SOCS1/3 signaling., Kakoki et al report a role for cyanocobalamin (B12) in preventing cardiomyopathy in type 1 diabetic mice, by reducing levels of reactive oxygen species and restoring IGF-1 levels via DNMT-SOCS1/3 signalling. The authors therefore propose B12 as a promising option to prevent diabetic cardiomyopathy through its cardioprotective effects.

CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research.

DNA methylation has evolved to silence mutagenic transposable elements (TEs) while typically avoiding the targeting of endogenous genes. Mechanisms that prevent DNA methyltransferases from ectopically methylating genes are expected to be of prime importance during periods of dynamic cell cycle activities including plant embryogenesis. However, virtually nothing is known regarding how DNA methyltransferase activities are precisely regulated during embryogenesis to prevent the induction of potentially deleterious and mitotically stable genic epimutations. Here, we report that microRNA-mediated repression of CHROMOMETHYLASE 3 (CMT3) and the chromatin features that CMT3 prefers help prevent ectopic methylation of thousands of genes during embryogenesis that can persist for weeks afterwards. Our results are also consistent with CMT3-induced ectopic methylation of promoters or bodies of genes undergoing transcriptional activation reducing their expression. Therefore, the repression of CMT3 prevents epigenetic collateral damage on endogenous genes. We also provide a model that may help reconcile conflicting viewpoints regarding the functions of gene-body methylation that occurs in nearly all flowering plants.

DNA methylation plays crucial roles in transposon silencing and genome integrity. CHROMOMETHYLASE3 (CMT3) is a plant-specific DNA methyltransferase responsible for catalyzing DNA methylation at the CHG (H = A, T, C) context. Here, we identified a positive role of CMT3 in heat-induced activation of retrotransposon ONSEN. We found that the full transcription of ONSEN under heat stress requires CMT3. Interestingly, loss-of-function CMT3 mutation led to increased CHH methylation at ONSEN. The CHH methylation is mediated by CMT2, as evidenced by greatly reduced CHH methylation in cmt2 and cmt2 cmt3 mutants coupled with increased ONSEN transcription. Furthermore, we found more CMT2 binding at ONSEN chromatin in cmt3 compared to wild-type accompanied with an ectopic accumulation of H3K9me2 under heat stress, suggesting a collaborative role of H3K9me2 and CHH methylation in preventing heat-induced ONSEN activation. In summary, this study identifies a non-canonical role of CMT3 in preventing transposon silencing and provides new insights into how DNA methyltransferases regulate transcription under stress conditions., Author summary DNA methylation is generally known to silence transposon and maintain genome integrity. Environmental stress has been reported to release the transcriptional silencing of some transposable elements. DNA methylation is involved in the transcriptional restriction of heat-induced Copia-type retrotransposon ONSEN in Arabidopsis when subjected to heat stress. Here, we identified a non-canonical and positive role of the DNA methyltransferase CMT3 in ONSEN reactivation under heat stress. We showed that CMT3 prevents CMT2-mediated CHH methylation and H3K9me2 accumulation under heat at ONSEN chromatin to modulate ONSEN transcription. Our work revealed the molecular mechanism of CMT3 in heat-induced ONSEN activation and sheds new light on the survival mechanism of certain transposons in the host genome under stress conditions.

This phase 2 study was designed to compare systemic decitabine exposure, demethylation activity, and safety in the first 2 cycles with cedazuridine 100 mg/decitabine 35 mg vs standard decitabine 20 mg/m2 IV. Adults with International Prognostic Scoring System intermediate-1/2- or high-risk myelodysplastic syndromes (MDS) or chronic myelomonocytic leukemia (CMML) were randomized 1:1 to receive oral cedazuridine/decitabine or IV decitabine in cycle 1, followed by crossover to the other treatment in cycle 2. All patients received oral cedazuridine/decitabine in subsequent cycles. Cedazuridine and decitabine were given initially as separate capsules in a dose-confirmation stage and then as a single fixed-dose combination (FDC) tablet. Primary end points: mean decitabine systemic exposure (geometric least-squares mean [LSM]) of oral/IV 5-day area under curve from time 0 to last measurable concentration (AUClast), percentage long interspersed nuclear element 1 (LINE-1) DNA demethylation for oral cedazuridine/decitabine vs IV decitabine, and clinical response. Eighty patients were randomized and treated. Oral/IV ratios of geometric LSM 5-day AUClast (80% confidence interval) were 93.5% (82.1-106.5) and 97.6% (80.5-118.3) for the dose-confirmation and FDC stages, respectively. Differences in mean %LINE-1 demethylation between oral and IV were ≤1%. Clinical responses were observed in 48 patients (60%), including 17 (21%) with complete response. The most common grade ≥3 adverse events regardless of causality were neutropenia (46%), thrombocytopenia (38%), and febrile neutropenia (29%). Oral cedazuridine/decitabine (100/35 mg) produced similar systemic decitabine exposure, DNA demethylation, and safety vs decitabine 20 mg/m2 IV in the first 2 cycles, with similar efficacy. This study is registered at www.clinicaltrials.gov as #NCT02103478.

Flowering plants and mammals contain imprinted genes that are primarily expressed in the endosperm and placenta in a parent-of-origin manner. In this study, we show that early activation of the geminivirus genes C2 and C3 in Arabidopsis (Arabidopsis thaliana) plants, encoding a viral suppressor of RNA interference and a replication enhancer protein, respectively, is correlated with the transient vegetative expression of VARIANT IN METHYLATION5 (VIM5), an endosperm imprinted gene that is conserved in diverse plant species. VIM5 is a ubiquitin E3 ligase that directly targets the DNA methyltransferases MET1 and CMT3 for degradation by the ubiquitin-26S proteasome proteolytic pathway. Infection with Beet severe curly top virus induced VIM5 expression in rosette leaf tissues, possibly via the expression of the viral replication initiator protein, leading to the early activation of C2 and C3 coupled with reduced symmetric methylation in the C2-3 promoter and the onset of disease symptoms. These findings demonstrate how this small DNA virus recruits a host imprinted gene for the epigenetic activation of viral gene transcription. Our findings reveal a distinct strategy used by plant pathogens to exploit the host machinery in order to inhibit methylation-mediated defense responses when establishing infection.

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Kr&uuml, ppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%&ndash, 70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGF&beta, 1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGF&beta, 1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGF&beta, 1-induced and uninduced PLOD2 expression.

Herein, a novel label-free photoelectrochemical (PEC) sensing platform with near-zero background noise was developed for M.SssI CpG methyltransferase (M.SssI MTase) activity assay based on a new Schottky junction of Bi

DNA methylation plays an important role in modulating plant growth plasticity in response to stress, but mechanisms involved in such control need further investigation. We used drm1 drm2 cmt3 mutant of Arabidopsis thaliana, defective in DNA methylation, to explore metabolic pathways downstream epigenetic modulation under cadmium (Cd) stress. To this aim, a transcriptomic analysis was performed on ddc and WT plants exposed to a long-lasting (21 d) Cd treatment (25/50 µM), focusing on hormone genetic pathways. Growth parameters and hormones amount were also estimated. Transcriptomic data and hormone quantification showed that, under prolonged Cd treatment, level and signalling of growth-sustaining hormones (auxins, CKs, GAs) were enhanced and/or maintained, while a decrease was detected for stress-related hormones (JA, ABA, SA), likely as a strategy to avoid the side effects of their long-lasting activation. Such picture was more effective in ddc than WT, already at 25 µM Cd, in line with its better growth performance. A tight relationship between methylation status and the modulation of hormone genetic pathways under Cd stress was assessed. We propose that the higher genome plasticity conferred to ddc by DNA hypomethylated status underlies its prompt response to modulate hormones genetic pathways and activity and assure a flexible growth.

Cancer therapies that target epigenetic repressors can mediate their effects by activating retroelements within the human genome. Retroelement transcripts can form double-stranded RNA (dsRNA) that activates the MDA5 pattern recognition receptor

In this article, a new concept for general pathogenesis has been proposed. Advances in molecular genetics have led to the realization that essential concepts in the framework of molecular biology are still missing. Clinical medicine is plagued by similar shortcomings: The questioning of current paradigms could open new vistas and invite challenging approaches. This article presents an unconventional idea. Foreign DNA which is regularly ingested with the essential food supply is not completely degraded. Small quantities of fragmented DNA rather persist transiently in the gastro-intestinal tract of mice and can be traced to various organ systems, except for cells in the germ line. Foreign DNA entering and persisting in mammalian cells can stochastically lead to genome-wide alterations of transcriptional and CpG DNA methylation profiles. In the course of food-ingested DNA invading somatic cells, completely new cell types can be generated which might be involved in the causation of common ailments. Projects emanating from this perception merit critical analysis and rigorous pursuit.

Overwhelming evidence suggests that in addition to the genetic changes of DNA mutations, epigenetic changes of DNA methylation and histone modifications play important role in regulation of gene expression. DNA methylation is the most frequent epigenetic alteration observed in mammalian genomes, and generally it is negatively correlated with gene expression. Various methods are available for the detection of DNA methylation changes. Although the recent high-throughput methods for DNA methylation analysis have various advantages, they require high levels of technical expertise, costly equipment, and reagents. Because of these reasons, many of the global DNA methylation analysis methods are mainly performed at core facility, and laboratories with limited resources and expertise are not able to use these methods. Methylation-Sensitive-Random Amplified Polymorphic DNA-Polymerase Chain Reaction (MS-RAPD-PCR) is a restriction enzyme digestion and PCR-based method for the analysis of DNA methylation changes. This method is cost-effective, requires simple and basic instrumentation, and therefore can easily be performed in any laboratory with basic setup having a regular DNA thermal cycler and DNA gel electrophoresis system. Additional advantages of this method over other methods for DNA methylation analysis are that it requires very less amount of DNA and can screen DNA methylation changes globally at genome-wide level with high sensitivity. This method has been successfully used to detect changes in DNA methylation either occurring naturally or induced by various toxicants and environmental factors. A detail experimental protocol for MS-RAPD-PCR is described in this chapter.

Orientador: Pablo Agustin Vargas Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba Resumo: O ameloblastoma é um tumor odontogênico epitelial benigno clinicamente agressivo com alterações epigenéticas pouco conhecidas. Nesse tumor, a expressão das proteínas MSH2 e MSH6 da via antimutagênica do Sistema de Reparo Mismatch se encontram reduzidas, sugerindo a ocorrência de alterações epigenéticas. Os objetivos desse trabalho são investigar o perfil imunohistoquímica dos marcadores epigenéticos envolvidos com a metilação do DNA e com a acetilação de histonas em ameloblastomas e carcinomas ameloblásticos, além de reportar o perfil de metilação dos genes MSH2, MSH3 e MSH6 (MutS) nos ameloblastomas. Foram realizadas reações imunohistoquímicas para os marcadores DNMT1, DNMT3A, DNMT3B e H3K9ac em 10 germes dentais, 38 ameloblastomas convencionais e seis carcinomas ameloblásticos, buscando determinar a comparação das expressões entre os grupos. Em seguida, 59 ameloblastomas convencionais organizados em um bloco de tissue microarray contendo informações clínicas, patológicas, genéticas e relato de recidiva, foram submetidos às mesmas reações para correlacionar com as variáveis qualitativas. Foram observadas a superexpressão de DNMT3B nos ameloblastomas em relação aos germes dentais (p = 0,0001), assim como aumentos nas expressões de DNMT1 (p = 0,0175), DNMT3A (p = 0,0113) e H3K9ac (p = 0,0004) nos carcinomas ameloblásticos em relação aos ameloblastomas. Adicionalmente, os casos de ameloblastomas que apresentavam a mutação BRAFV600E (p = 0,0114), envolvimento maxilar (p = 0,0072), e rompimento de corticais vestíbulo-palatina (p = 0,0066) possuíam um aumento na expressão de DNMT1, enquanto que casos com recidivas (p = 0,0477) possuíam aumento na expressão de DNMT3B. Para determinar o perfil de metilação dos genes MutS, 10 folículos dentários e 10 ameloblastomas convencionais frescos foram submetidos a análises quantitativas de PCR em tempo real (qPCR) assay, utilizando a técnica de restrição enzimática. Foram observados que os ameloblastomas apresentam baixo percentual dos genes MutS metilados, sem apresentar diferença significativa com os folículos dentários (p < 0,05). Esses resultados sugerem que novos perfis de genes metilados estejam presentes nos ameloblastomas, favorecendo o seu comportamento clínico mais agressivo e a sua progressão para carcinomas ameloblásticos. Contudo, a baixa expressão de MSH2 e MSH6 reportada na literatura não parece estar associada com alterações nos perfis de metilações dos seus respectivos genes, sugerindo que outros mecanismos possam estar presentes Abstract: Ameloblastoma is a benign epithelial odontogenic tumour clinically aggressive with epigenetics alterations fewer know. In this tumour, the MSH2 and MSH6 proteins in the Mismatch repair system antimutagenic pathway are low-expressed, suggesting the occurrence of epigenetic changes. This study aims to investigate the immunohistochemical profile of the epigenetic markers involved with the DNA methylation and histones acetylation in ameloblastomas and ameloblastic carcinomas, besides to reporting the MSH2, MSH3 and MSH6 (MutS) genes methylation profile in ameloblastomas. Immunohistochemical reactions were performed for DNMT1, DNMT3A, DNMT3B and H3K9ac markers in 10 dental germs, 38 conventional ameloblastomas and six ameloblastic carcinomas, seeking to determine the comparison of expressions between groups. Then, 59 conventional ameloblastomas organized in a tissue microarray block containing clinical, pathological, genetic information and a report of recurrence, underwent the same reactions to correlate with qualitative variables. Overexpression of DNMT3B in ameloblastomas was observed in relation to dental germs (p = 0.0001), as well as increases in the expressions of DNMT1 (p = 0.0175), DNMT3A (p = 0.0113) and H3K9ac (p = 0.0004) in ameloblastic carcinomas in relation to ameloblastomas. Additionally, ameloblastomas cases that presented the BRAFV600E mutation (p = 0.0114), maxillary involvement (p = 0.0072), and the vestibular-palatine cortical bone ruptured (p = 0.0066) had an increase in DNMT1 expression, while cases with recurrences (p = 0.0477) showed DNMT3B expression increased. Ten dental follicles and 10 conventional ameloblastomas fresh samples were selected to determine the MutS genes methylation profile. The samples were subjected to quantitative analyzes of real-time PCR (qPCR) assay, using the enzymatic restriction technique. It was observed that ameloblastomas have a low percentage of methylated MutS genes, without significant difference with dental follicles (p

DNA methyltransferase (MTase) has a crucial role in many biological processes, its abnormal expression level has been regarded as a predictive cancer biomarker. Herein, a sensitive fluorescence method based on thermosensitive poly (N-isopr-opylacrylamide) was developed to assay of M.SssI activity. When the M.SssI was introduced, dsDNA was methylated at palindromic sequence 5′-CmCGG-3′ and became resistant to cleavage by the endonuclease HpaII. Therefore, a biotin modified ssDNA and a FAM modified ssDNA were designed including the recognized sites for both methyltransferase M.SssI and endonuclease HpaII. By SA-biotin intereaction, the DNA was conjugated to thermosensitive poly (N-isopropylacrylamide) modified by SA, the methylated substrate fluorescence was increased with the concentration of M.SssI increasing. The proposed method has a low detection limit of 0.18 U/mL. This simple method can be a useful tool to apply in diagnosis and biomedical research, which was successfully investigated in the serum sample.

DNA methyltransferase (MTase) plays an important role in many biological processes and has been recognized as a predictive cancer biomarker far before other signs of malignancy and a therapeutic target in cancer treatment. Thus simple and sensitive determination of DNA MTase activity is urgently required. The commercially available glucometer is considered as the most successful point-of-care (POC) sensor up to date, and researchers extend its application in monitoring different types of targets rather than only glucose. Here, we developed a simple strategy for the sensitive detection of the DNA MTase (using M.SssI as an example) activity by using a glucometer as the signal transducer. A S1/S2 hybrid probe was designed including a specific recognition sequence for both DNA MTase and restriction endonuclease, and a complementary sequence for biotin-S3. Firstly, the S1/S2 hybrid probe was self-assembled on the gold electrode and methylated by M.SssI MTase to form the methylated dsDNA. Then, HpaII endonuclease specifically cleaved the residue of the unmethylated dsDNA. Subsequently, biotin-S3 hybridized with the overhang sequence of the methylated dsDNA. Finally, the biotin tag was successively combined with streptavidin (STV) and biotin-invertase. The invertase efficiently catalyzed the hydrolysis of sucrose to generate abundant glucose, which led to an amplified response of glucometer. This strategy could detect DNA MTase activity as low as 0.3 U mL-1 with good selectivity against other two cytosine MTases (HaeIII MTase and AluI MTase), and be successfully applied for screening the DNA MTase inhibitors (5-azacytidine and 5-aza-2'-deoxycytidine), implying our proposed method holds great promising application in early cancer diagnosis and therapeutics.

The DNA methyltransferase (DNMT) family comprises a conserved set of DNA-modifying enzymes that have a central role in epigenetic gene regulation. Recent studies have shown that the functions of the canonical DNMT enzymes - DNMT1, DNMT3A and DNMT3B - go beyond their traditional roles of establishing and maintaining DNA methylation patterns. This Review analyses how molecular interactions and changes in gene copy numbers modulate the activity of DNMTs in diverse gene regulatory functions, including transcriptional silencing, transcriptional activation and post-transcriptional regulation by DNMT2-dependent tRNA methylation. This mechanistic diversity enables the DNMT family to function as a versatile toolkit for epigenetic regulation.

DNA methylation is a common modification occurring in all living organisms. 5-methylcytosine, which is produced in a reaction catalysed by C5-methyltransferases, can spontaneously undergo deamination to thymine, leading to the formation of T:G mismatches and C→T transitions. In Escherichia coli K-12, such mismatches are corrected by the Very Short Patch (VSP) repair system, with Vsr endonuclease as the key enzyme. Neisseria meningitidis possesses genes that encode DNA methyltransferases, including C5-methyltransferases. We report on the mutagenic potential of the meningococcal C5-methyltransferases M.NmeDI and M.NmeAI resulting from deamination of 5-methylcytosine. N. meningitidis strains also possess genes encoding potential Vsr endonucleases. Phylogenetic analysis of meningococcal Vsr endonucleases indicates that they belong to two phylogenetically distinct groups (type I or type II Vsr endonucleases). N. meningitidis serogroup C (FAM18) is a representative of meningococcal strains that carry two Vsr endonuclease genes (V.Nme18IIP and V.Nme18VIP). The V.Nme18VIP (type II) endonuclease cut DNA containing T:G mismatches in all tested nucleotide contexts. V.Nme18IIP (type I) is not active in vitro, but the change of Tyr69 to His69 in the amino acid sequence of the protein restores its endonucleolytic activity. The presence of tyrosine in position 69 is a characteristic feature of type I meningococcal Vsr proteins, while type II Vsr endonucleases possess His69. In addition to the T:G mismatches, V.Nme18VIP and V.Nme18IIPY69H recognize and digest DNA with T:T or U:G mispairs. Thus, for the first time, we demonstrate that the VSP repair system may have a wider significance and broader substrate specificity than DNA lesions that only result from 5-methylcytosine deamination.

Background We present a new methylation-sensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics such as demethylation, de novo methylation, and preservation of methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied the technique to analyze aluminum (Al)-tolerant and non-tolerant control and Al-stressed inbred triticale lines. The approach is based on detailed analysis of events affecting HpaII and MspI restriction sites in control and stressed samples, and takes advantage of molecular marker profiles generated by EcoRI/HpaII and EcoRI/MspI MSAP platforms. Methods Five Al-tolerant and five non-tolerant triticale lines were exposed to aluminum stress using the physiologicaltest. Total genomic DNA was isolated from root tips of all tolerant and non-tolerant lines before and after Al stress following metAFLP and MSAP approaches. Based on codes reflecting events affecting cytosines within a given restriction site recognized by HpaII and MspI in control and stressed samples demethylation (DM), de novo methylation (DNM), preservation of methylated sites (MSP), and preservation of nonmethylatedsites (NMSP) were evaluated. MSAP profiles were used for Agglomerative hierarchicalclustering (AHC) based on Squared Euclidean distance and Ward’s Agglomeration method whereas MSAP characteristics for ANOVA. Results Relative quantitative MSAP analysis revealed that both Al-tolerant and non-tolerant triticale lines subjected to Al stress underwent demethylation, with demethylation of CG predominating over CHG. The rate of de novo methylation in the CG context was ~3-fold lower than demethylation, whereas de novo methylation of CHG was observed only in Al-tolerant lines. Conclusions Our relative quantitative MSAP approach, based on methylation events affecting cytosines within HpaII–MspI recognition sequences, was capable of quantifying de novo methylation, demethylation, methylation, and non-methylated status in control and stressed Al-tolerant and non-tolerant triticale inbred lines. The method could also be used to analyze methylation events affecting CG and CHG contexts, which were differentially methylated under Al stress. We cannot exclude that the methylation changes revealed among lines as well as between Al-tolerant and non-tolerant groups of lines were due to some experimental errors or that the number of lines was too small for ANOVA to prove the influence of Al stress. Nevertheless, we suspect that Al tolerance in triticale could be partly regulated by epigenetic factors acting at the level of DNA methylation. This method provides a valuable tool for studies of abiotic stresses in plants.

The ability to target epigenetic marks like DNA methylation to specific loci is important in both basic research and in crop plant engineering. However, heritability of targeted DNA methylation, how it impacts gene expression, and which epigenetic features are required for proper establishment are mostly unknown. Here, we show that targeting the CG-specific methyltransferase M.SssI with an artificial zinc finger protein can establish heritable CG methylation and silencing of a targeted locus in Arabidopsis. In addition, we observe highly heritable widespread ectopic CG methylation mainly over euchromatic regions. This hypermethylation shows little effect on transcription while it triggers a mild but significant reduction in the accumulation of H2A.Z and H3K27me3. Moreover, ectopic methylation occurs preferentially at less open chromatin that lacks positive histone marks. These results outline general principles of the heritability and interaction of CG methylation with other epigenomic features that should help guide future efforts to engineer epigenomes., The ability to target DNA methylation to specific loci is important for both basic and applied research. Here, the authors fuse CG-specific methyltransferase SssI with an artificial zinc finger protein for DNA methylation targeting and show the chromatin features favorable for efficient gain of methylation.

Background Zinc-finger protein 471 (ZNF471) is a member of the Krüppel-associated box domain zinc finger protein (KRAB-ZFP) family. ZNF471 is methylated in squamous cell carcinomas of tongue, stomach and esophageal. However, its role in breast carcinogenesis remains elusive. Here, we studied its expression, functions, and molecular mechanisms in breast cancer. Methods We examined ZNF471 expression by RT-PCR and qPCR. Methylation-specific PCR determined its promoter methylation. Its biological functions and related molecular mechanisms were assessed by CCK-8, clonogenicity, wound healing, Transwell, nude mice tumorigenicity, flow cytometry, BrdU-ELISA, immunohistochemistry and Western blot assays. Results ZNF471 was significantly downregulated in breast cell lines and tissues due to its promoter CpG methylation, compared with normal mammary epithelial cells and paired surgical-margin tissues. Ectopic expression of ZNF471 substantially inhibited breast tumor cell growth in vitro and in vivo, arrested cell cycle at S phase, and promoted cell apoptosis, as well as suppressed metastasis. Further knockdown of ZNF471 verified its tumor-suppressive effects. We also found that ZNF471 exerted its tumor-suppressive functions through suppressing epithelial-mesenchymal transition, tumor cell stemness and AKT and Wnt/β-catenin signaling. Conclusions ZNF471 functions as a tumor suppressor that was epigenetically inactivated in breast cancer. Its inhibition of AKT and Wnt/β-catenin signaling pathways is one of the mechanisms underlying its anti-cancer effects.

The enhancer/promoter of the vitellogenin II gene (VTG) has been extensively studied as a model system of vertebrate transcriptional control. While deletion mutagenesis and in vivo footprinting identified the transcription factor (TF) binding sites governing its tissue specificity, DNase hypersensitivity and DNA methylation studies revealed the epigenetic changes accompanying its hormone-dependent activation. Moreover, upon induction with estrogen (E(2)), the region flanking the estrogen-responsive element (ERE) was reported to undergo active DNA demethylation. We now show that although the VTG ERE is methylated in embryonic chicken liver and in LMH/2A hepatocytes, its induction by E(2) was not accompanied by extensive demethylation. In contrast, E(2) failed to activate a VTG enhancer/promoter-controlled luciferase reporter gene methylated by SssI. Surprisingly, this inducibility difference could be traced not to the ERE but rather to a single CpG in an E-box (CACGTG) sequence upstream of the VTG TATA box, which is unmethylated in vivo but methylated by SssI. We demonstrate that this E-box binds the upstream stimulating factor USF1/2. Selective methylation of the CpG within this binding site with an E-box-specific DNA methyltransferase, Eco72IM, was sufficient to attenuate USF1/2 binding in vitro and abolish the hormone-induced transcription of the VTG gene in the reporter system.

DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. In this study we present Homology Assisted Repair Dependent Epigenetic eNgineering (HARDEN), a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways. This method allows for stable targeted DNA methylation through the process of homology directed repair (HDR) via an in vitro methylated exogenous repair template. We demonstrate that HARDEN can be applied to the neurodegenerative disease genes C9orf72 and APP, and methylation can be induced via HDR with both single and double stranded methylated repair templates. HARDEN allows for higher targeted DNA methylation levels than a dCas9-DNMT3a fusion protein construct at C9orf72, and genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HARDEN, whereas the dCas9-DNMT3a fusion construct causes global off-target methylation. HARDEN is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that recapitulates DNA methylation patterns seen in patients, demonstrating that DNA methylation of the 5′ regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.

Aberrant DNA methylation by DNA methyltransferases (MTase) is related to the initiation and progression of many diseases. Thus, site-specific identification of DNA methylation and detection of MTase activity are very important to diagnose and treat methylation-related diseases. Herein, a single-molecule counting based ultrasensitive assay was developed for facile and direct detection of MTase activity and inhibitor screening without the assistance of restriction endonuclease. A double-strand DNA (dsDNA) was designed with the recognition site of M. SssI MTase and assembled on the coverslip surface. After the dsDNA was methylated by M. SssI, the biotin conjugated anti-5-methylcytosine antibody (5mC Ab) would specifically bind the CpG methylation site, and subsequently, the streptavidin-labeled quantum dots (QS585) bind the biotins. By taking and counting the image spots of fluorescently labeled methylated dsDNA molecules, the single-molecule imaging of methylated dsDNA molecules was recorded to quantify the DNA MTase activity. The spot number shows a linear relation with the logarithm of M. SssI concentration in the concentration range of 0.001-1 U/mL. Compared with most of the state of the art methods, the proposed assay displays a lower detection limit of 0.0005 U/mL and can detect the DNA MTase more directly. Moreover, it can selectively detect M. SssI in more complex samples. In addition, it is further demonstrated that the protocol could be successfully applied to evaluate the inhibition efficiency of M. SssI inhibitors. This assay is anticipated to provide a new approach for clinical diagnosis of methylation-related diseases and screening of new anticancer drugs.

Pelargonidin, a well-known natural anthocyanidin found in berries strawberries, blueberries, red radishes and other natural foods, has been found to possess health beneficial effects including anti-cancer effect. Herein, we investigated the effect of pelargonidin on cellular transformation in mouse skin epidermal JB6 (JB6 P+) cells induced by tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Pelargonidin treatment significantly decreased colony formation and suppressed cell viability of JB6 P+ cells. Pelargonidin also induced the anti-oxidant response element (ARE)-luciferase activation in HepG2-C8 cells overexpressing the ARE-luciferase reporter. Knockdown of nuclear factor E2-related factor 2 (Nrf2) in shNrf2 JB6 P+ cells enhanced TPA-induced colony formation and attenuated pelargonidin's blocking effect. Pelargonidin reduced the protein levels of genes encoding methyltransferases (DNMTs) and histone deacetylases (HDACs). Importantly, pelargonidin decreased the DNA methylation in the Nrf2 promoter region of JB6 P+ cells and increased Nrf2 downstream target genes expression, such as NAD(P)H/quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1), involved in cellular protection. In summary, our results showed that pelargonidin blocks TPA-induced cell transformation. The possible molecular mechanisms of its potential anti-cancer effects against neoplastic transformation may be attributed to its activation of Nrf2-ARE signaling pathway and its cytoprotective effect.