DNA MTase Research Articles

Single-base resolution quantitative genome methylation analysis in the model bacterium Helicobacter pylori by enzymatic methyl sequencing (EM-Seq) reveals influence of strain, growth phase, and methyl homeostasis

BackgroundBacterial epigenetics is a rapidly expanding research field. DNA methylation by diverse bacterial methyltransferases (MTases) contributes to genomic integrity and replication, and many recent studies extended MTase function also to global transcript regulation and phenotypic variation. Helicobacter pylori is currently one of those bacterial species which possess the highest number and the most variably expressed set of DNA MTases. Next-generation sequencing technologies can directly detect DNA base methylation. However, they still have limitations in their quantitative and qualitative performance, in particular for cytosine methylation.ResultsAs a complementing approach, we used enzymatic methyl sequencing (EM-Seq), a technology recently established that has not yet been fully evaluated for bacteria. Thereby, we assessed quantitatively, at single-base resolution, whole genome cytosine methylation for all methylated cytosine motifs in two different H. pylori strains and isogenic MTase mutants. EM-Seq reliably detected both m5C and m4C methylation. We demonstrated that three different active cytosine MTases in H. pylori provide considerably different levels of average genome-wide single-base methylation, in contrast to isogenic mutants which completely lost specific motif methylation. We found that strain identity and changed environmental conditions, such as growth phase and interference with methyl donor homeostasis, significantly influenced quantitative global and local genome-wide methylation in H. pylori at specific motifs. We also identified significantly hyper- or hypo-methylated cytosines, partially linked to overlapping MTase target motifs. Notably, we revealed differentially methylated cytosines in genome-wide coding regions under conditions of methionine depletion, which can be linked to transcript regulation.ConclusionsThis study offers new knowledge on H. pylori global and local genome-wide methylation and establishes EM-Seq for quantitative single-site resolution analyses of bacterial cytosine methylation.

Expression of a unique M. tuberculosis DNA MTase Rv1509 in M. smegmatis alters the gene expression pattern and enhances virulence.

Mycobacterium tuberculosis (M. tb) genome encompasses 4,173 genes, about a quarter of which remain uncharacterized and hypothetical. Considering the current limitations associated with the diagnosis and treatment of tuberculosis, it is imperative to comprehend the pathomechanism of the disease and host-pathogen interactions to identify new drug targets for intervention strategies. Using in-silico comparative genome analysis, we identified one of the M. tb genes, Rv1509, as a signature protein exclusively present in M. tb. To explore the role of Rv1509, a likely methyl transferase, we constructed a knock-in Mycobacterium smegmatis (M. smegmatis) constitutively expressing Rv1509 (Ms_Rv1509). The Ms_Rv1509 led to differential expression of many transcriptional regulator genes as assessed by RNA-seq analysis. Further, in-vitro and in-vivo studies demonstrated an enhanced survival of Ms_Rv1509 inside the host macrophages. Ms_Rv1509 also promoted phagolysosomal escape inside macrophages to boost bacterial replication and dissemination. In-vivo infection studies revealed that Ms_Rv1509 survives better than BCG and causes pathological manifestations in the pancreas after intraperitoneal infection. Long-time survival of Ms_Rv1509 resulted in lymphocyte migration, increased T regulatory cells, giant cell formation, and likely granuloma formation in the pancreas, pointing toward the role of Rv1509 in M. tb pathogenesis.

Identification and virus-induced gene silencing (VIGS) analysis of methyltransferase affecting tomato (Solanum lycopersicum) fruit ripening.

Six methyltransferase genes affecting tomato fruit ripening were identified through genome-wide screening, VIGS assay, and expression pattern analysis. The data provide the basis for understanding new mechanisms of methyltransferases. Fruit ripening is a critical stage for the formation of edible quality and seed maturation, which is finely modulated by kinds of factors, including genetic regulators, hormones, external signals, etc. Methyltransferases (MTases), important genetic regulators, play vital roles in plant development through epigenetic regulation, post-translational modification, or other mechanisms. However, the regulatory functions of numerous MTases except DNA methylation in fruit ripening remain limited so far. Here, six MTases, which act on different types of substrates, were identified to affect tomato fruit ripening. First, 35 MTase genes with relatively high expression at breaker (Br) stage of tomato fruit were screened from the tomato MTase gene database encompassing 421 genes totally. Thereafter, six MTase genes were identified as potential regulators of fruit ripening via virus-induced gene silencing (VIGS), including four genes with a positive regulatory role and two genes with a negative regulatory role, respectively. The expression of these six MTase genes exhibited diverse patterns during the fruit ripening process, and responded to various external ripening-related factors, including ethylene, 1-methylcyclopropene (1-MCP), temperature, and light exposure. These results help to further elaborate the biological mechanisms of MTase genes in tomato fruit ripening and enrich the understanding of the regulatory mechanisms of fruit ripening involving MTases, despite of DNA MTases.

Chemical Expansion of the Methyltransferase Reaction: Tools for DNA Labeling and Epigenome Analysis.

DNA is the genetic matter of life composed of four major nucleotides which can be further furnished with biologically important covalent modifications. Among the variety of enzymes involved in DNA metabolism, AdoMet-dependent methyltransferases (MTases) combine the recognition of specific sequences and covalent methylation of a target nucleotide. The naturally transferred methyl groups play important roles in biological signaling, but they are poor physical reporters and largely resistant to chemical derivatization. Therefore, an obvious strategy to unlock the practical utility of the methyltransferase reactions is to enable the transfer of "prederivatized" (extended) versions of the methyl group.However, previous enzymatic studies of extended AdoMet analogs indicated that the transalkylation reactions are drastically impaired as the size of the carbon chain increases. In collaborative efforts, we proposed that, akin to enhanced SN2 reactivity of allylic and propargylic systems, addition of a π orbital next to the transferable carbon atom might confer the needed activation of the reaction. Indeed, we found that MTase-catalyzed transalkylations of DNA with cofactors containing a double or a triple C-C bond in the β position occurred in a robust and sequence-specific manner. Altogether, this breakthrough approach named mTAG (methyltransferase-directed transfer of activated groups) has proven instrumental for targeted labeling of DNA and other types of biomolecules (using appropriate MTases) including RNA and proteins.Our further work focused on the propargylic cofactors and their reactions with DNA cytosine-5 MTases, a class of MTases common for both prokaryotes and eukaryotes. Here, we learned that the 4-X-but-2-yn-1-yl (X = polar group) cofactors suffered from a rapid loss of activity in aqueous buffers due to susceptibility of the triple bond to hydration. This problem was remedied by synthetically increasing the separation between X and the triple bond from one to three carbon units (6-X-hex-2-ynyl cofactors). To further optimize the transfer of the bulkier groups, we performed structure-guided engineering of the MTase cofactor pocket. Alanine replacements of two conserved residues conferred substantial improvements of the transalkylation activity with M.HhaI and three other engineered bacterial C5-MTases. Of particular interest were CpG-specific DNA MTases (M.SssI), which proved valuable tools for studies of mammalian methylomes and chemical probing of DNA function.Inspired by the successful repurposing of bacterial enzymes, we turned to more complex mammalian C5-MTases (Dnmt1, Dnmt3A, and Dnmt3B) and asked if they could ultimately lead to mTAG labeling inside mammalian cells. Our efforts to engineer mouse Dnmt1 produced a variant (Dnmt1*) that enabled efficient Dnmt1-directed deposition of 6-azide-hexynyl groups on DNA in vitro. CRISPR-Cas9 editing of the corresponding codons in the genomic Dnmt1 alleles established endogenous expression of Dnmt1* in mouse embryonic stem cells. To circumvent the poor cellular uptake of AdoMet and its analogs, we elaborated their efficient internalization by electroporation, which has finally enabled selective catalysis-dependent azide tagging of natural Dnmt1 targets in live mammalian cells. The deposited chemical groups were then exploited as "click" handles for reading adjoining sequences and precise genomic mapping of the methylation sites. These findings offer unprecedented inroads into studies of DNA methylation in a wide range of eukaryotic model systems.

A Survey of Archaeal Restriction-Modification Systems.

When compared with bacteria, relatively little is known about the restriction-modification (RM) systems of archaea, particularly those in taxa outside of the haloarchaea. To improve our understanding of archaeal RM systems, we surveyed REBASE, the restriction enzyme database, to catalog what is known about the genes and activities present in the 519 completely sequenced archaeal genomes currently deposited there. For 49 (9.4%) of these genomes, we also have methylome data from Single-Molecule Real-Time (SMRT) sequencing that reveal the target recognition sites of the active m6A and m4C DNA methyltransferases (MTases). The gene-finding pipeline employed by REBASE is trained primarily on bacterial examples and so will look for similar genes in archaea. Nonetheless, the organizational structure and protein sequence of RM systems from archaea are highly similar to those of bacteria, with both groups acquiring systems from a shared genetic pool through horizontal gene transfer. As in bacteria, we observe numerous examples of "persistent" DNA MTases conserved within archaeal taxa at different levels. We experimentally validated two homologous members of one of the largest "persistent" MTase groups, revealing that methylation of C(m5C)WGG sites may play a key epigenetic role in Crenarchaea. Throughout the archaea, genes encoding m6A, m4C, and m5C DNA MTases, respectively, occur in approximately the ratio 4:2:1.

A programmable DNA nanodevice for colorimetric detection of DNA methyltransferase activity using functionalized hemin/G-quadruplex DNAzyme

The measurement of DNA methyltransferase (MTase) activity and screening of DNA MTase inhibitors holds significant importance for the diagnosis and therapy of methylation-related illness. Herein, we developed a colorimetric biosensor (PER-FHGD nanodevice) to detect DNA MTase activity by integrating the primer exchange reaction (PER) amplification and functionalized hemin/G-quadruplex DNAzyme (FHGD). By replacing the native hemin cofactor into the functionalized cofactor mimics, FHGD has exhibited significantly improved catalytic efficiency, thereby enhancing the detection performance of the FHGD-based system. The proposed PER-FHGD system is capable of detecting Dam MTase with excellent sensitivity, exhibiting a limit of detection (LOD) as low as 0.3 U/mL. Additionally, this assay demonstrates remarkable selectivity and ability for Dam MTase inhibitors screening. Furthermore, using this assay, we successfully detect the Dam MTase activity both in serum and in E. coli cell extracts. Importantly, this system has the potential to serve as a universal strategy for FHGD-based diagnosis in point-of-care (POC) tests, by simply altering the recognition sequence of the substrate for other analytes.

Characterisation of Type II DNA Methyltransferases of Metamycoplasma hominis.

Bacterial virulence, persistence and defence are affected by epigenetic modifications, including DNA methylation. Solitary DNA methyltransferases modulate a variety of cellular processes and influence bacterial virulence; as part of a restriction-modification (RM) system, they act as a primitive immune system in methylating the own DNA, while unmethylated foreign DNA is restricted. We identified a large family of type II DNA methyltransferases in Metamycoplasma hominis, comprising six solitary methyltransferases and four RM systems. Motif-specific 5mC and 6mA methylations were identified with a tailored Tombo analysis on Nanopore reads. Selected motifs with methylation scores >0.5 fit with the gene presence of DAM1 and DAM2, DCM2, DCM3, and DCM6, but not for DCM1, whose activity was strain-dependent. The activity of DCM1 for CmCWGG and of both DAM1 and DAM2 for GmATC was proven in methylation-sensitive restriction and finally for recombinant rDCM1 and rDAM2 against a dam-, dcm-negative background. A hitherto unknown dcm8/dam3 gene fusion containing a (TA) repeat region of varying length was characterized within a single strain, suggesting the expression of DCM8/DAM3 phase variants. The combination of genetic, bioinformatics, and enzymatic approaches enabled the detection of a huge family of type II DNA MTases in M. hominis, whose involvement in virulence and defence can now be characterized in future work.

Synthesis of S-Adenosyl-L-Methionine Analogs with Extended Transferable Groups for Methyltransferase-Directed Labeling of DNA and RNA.

S-Adenosyl-L-methionine (AdoMet) is a ubiquitous methyl donor for a variety of biological methylation reactions catalyzed by methyltransferases (MTases). AdoMet analogs with extended propargylic chains replacing the sulfonium-bound methyl group can serve as surrogate cofactors for many DNA and RNA MTases, enabling covalent derivatization and subsequent labeling of their cognate target sites in DNA or RNA. Although AdoMet analogs with saturated aliphatic chains are less popular than propargylic ones, they can be useful for dedicated studies that require certain chemical derivatization. Here we describe synthetic procedures for the preparation of two AdoMet analogs, one with a transferable 6-azidohex-2-ynyl group (carrying an activating C≡C triple bond and a terminal azide functionality), and the other one with a transferable ethyl-2,2,2-d3 group (an isotope-labeled aliphatic moiety). Our synthetic approach is based on direct chemoselective alkylation of S-adenosyl-L-homocysteine at sulfur with a corresponding nosylate or triflate, respectively, under acidic conditions. We also describe synthetic routes to 6-azidohex-2-yn-1-ol and conversion of the alcohols to corresponding nosylate and triflate alkylators. Using these protocols, the synthetic AdoMet analogs can be prepared within 1 to 2 weeks. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 6-azidohex-2-yn-1-ol Basic Protocol 2: Synthesis of 4-nitrobenzenesulfonate Basic Protocol 3: Synthesis of trifluoromethanesulfonates Basic Protocol 4: S-Alkylation of AdoHcy with sulfonates Basic Protocol 5: Purification and characterization of AdoMet analogs.

HpaII-assisted and linear amplification-enhanced isothermal exponential amplification fluorescent strategy for rapid and sensitive detection of DNA methyltransferase activity.

The detection of methyltransferase (MTase) activity is of great significance in methylation-related disease diagnosis and drug screening. Herein, a HpaII-assisted and linear amplification-enhanced exponential amplification strategy is proposed for sensitive and label-free detection of M.SssI MTase activity. The P1 probe contains self-complementary sequence 5'-CTAGCCGGCTAG-3' at 3'-terminal. After denaturation and annealing, P1 probes hybridize with itself to generate P1 duplexes. M.SssI MTase induces methylation of cytosine at 5'-CG-3' in P1 duplexes, and thus, HpaII fails to cleave at 5'-CCGG-3' due to methylation sensitivity, leaving P1 duplex intact. Then, these intact P1 duplexes are extended along 3'-terminal through Vent (exo-) DNA polymerase to generate dsDNA, which is recognized and nicked at the recognition sites by Nt.BstNBI, releasing two copies of primer X. Primer X hybridizes with X' at the amplification template T1 (X'-Y'-X') and then serves as primers to trigger the exponential amplification reaction (EXPAR). The point of inflection (POI) values of real-time fluorescence curves is linearly correlated with the logarithm of M.SssI MTase concentration in the range of 0.125 [Formula: see text] 8 U mL-1 with a low detection limit of 0.034 U mL-1. In the absence of M.SssI, P1 duplexes are cut by HpaII and separated into ssDNA under the executed temperature of EXPAR and thus unable to trigger the amplification. The strategy provides good selectivity against other types of MTases and protein and is able to detect M.SssI activity in human serum. Furthermore, the analytical method has the generality and can be extended to the analysis of other types of DNA MTases.

DNA Methylation and RNA-DNA Hybrids Regulate the Single-Molecule Localization of a DNA Methyltransferase on the Bacterial Nucleoid.

Bacterial DNA methyltransferases (MTases) function in restriction modification systems, cell cycle control, and the regulation of gene expression. DnmA is a recently described DNA MTase that forms N6-methyladenosine at nonpalindromic 5'-GACGAG-3' sites in Bacillus subtilis, yet how DnmA activity is regulated is unknown. To address DnmA regulation, we tested substrate binding in vitro and found that DnmA binds poorly to methylated DNA and to an RNA-DNA hybrid with the DNA recognition sequence. Further, DnmA variants with amino acid substitutions that disrupt cognate sequence recognition or catalysis also bind poorly to DNA. Using superresolution fluorescence microscopy and single-molecule tracking of DnmA-PAmCherry, we characterized the subcellular DnmA diffusion and detected its preferential localization to the replisome region and the nucleoid. Under conditions where the chromosome is highly methylated, upon RNA-DNA hybrid accumulation, or with a DnmA variant with severely limited DNA binding activity, DnmA is excluded from the nucleoid, demonstrating that prior methylation or accumulation of RNA-DNA hybrids regulates the association of DnmA with the chromosome in vivo. Furthermore, despite the high percentage of methylated recognition sites and the proximity to putative endonuclease genes conserved across bacterial species, we find that DnmA fails to protect B. subtilis against phage predation, suggesting that DnmA is functionally an orphan MTase involved in regulating gene expression. Our work explores the regulation of a bacterial DNA MTase and identifies prior methylation and RNA-DNA hybrids as regulators of MTase localization. These MTase regulatory features could be common across biology. IMPORTANCE DNA methyltransferases (MTases) influence gene expression, cell cycle control, and host defense through DNA modification. Predicted MTases are pervasive across bacterial genomes, but the vast majority remain uncharacterized. Here, we show that in the soil microorganism Bacillus subtilis, the DNA MTase dnmA and neighboring genes are remnants of a phage defense system that no longer protects against phage predation. This result suggests that portions of the bacterial methylome may originate from inactive restriction modification systems that have maintained methylation activity. Analysis of DnmA movement in vivo shows that active DnmA localizes in the nucleoid, suggesting that DnmA can search for recognition sequences throughout the nucleoid region with some preference for the replisome. Our results further show that prior DNA methylation and RNA-DNA hybrids regulate DnmA dynamics and nucleoid localization, providing new insight into how DNA methylation is coordinated within the cellular environment.

A fluorescent biosensor based on exponential amplification reaction-initiated CRISPR/Cas12a (EIC) strategy for ultrasensitive DNA methyltransferase detection

DNA methyltransferase (DNA MTase) catalyzes the process of DNA methylation, and the aberrant DNA MTase activity is closely associated with cancer incidence and progression. Inspired by the exponential amplification reaction (EXPAR) characteristics, we developed an EXPAR-initiated CRISPR/Cas12a (EIC) strategy for sensitively detecting DNA MTase activity. A hairpin probe (HP) was designed with a palindromic sequence in the stem as substrate and NH2-modified 3’ end to prevent nonspecific amplification. HP could be methylated by DNA adenine methyltransferase (Dam MTase) and then digested by DpnI to generate an oligonucleotide that can serve as an EXPAR primer. With the assistance of Nt.BstNBI nicking enzyme and Vent(exo-) polymerase, this primer bound to template and induced EXPAR. Interestingly, the product of Cycle 1 in EXPAR can function as primer to initiate Cycle 2. Both EXPAR products can further activate the collateral cleavage of CRISPR/Cas12a-crRNA, resulting in the fragmentation of fluorescence reporters and fluorescence recovery. Due to the highly efficient amplification (about 5 times signal-to-noise of SDA) and the robust trans-cleavage of CRISPR/Cas12a, the EIC system owned an extreme limit of detection (LOD) of 2 × 10−4 U/mL and a broad detection range from 2 × 10−4 to 10 U/mL for Dam MTase. In addition, this method has succeeded in inhibitor screening and evaluation, showing magnificent promise in drug discovery and cancer therapy.

DNA Methyltransferase Regulates Nitric Oxide Homeostasis and Virulence in a Chronically Adapted Pseudomonas aeruginosa Strain.

ABSTRACTOpportunistic pathogens such as Pseudomonas aeruginosa adapt their genomes rapidly during chronic infections. Understanding their epigenetic regulation may provide biomarkers for diagnosis and reveal novel regulatory mechanisms. We performed single-molecule real-time sequencing (SMRT-seq) to characterize the methylome of a chronically adapted P. aeruginosa clinical strain, TBCF10839. Two N6-methyladenine (6mA) methylation recognition motifs (RCCANNNNNNNTGAR and TRGANNNNNNTGC [modification sites are in bold]) were identified and predicted as new type I methylation sites using REBASE analysis. We confirmed that the motif TRGANNNNNNTGC was methylated by the methyltransferase (MTase) M.PaeTBCFII, according to methylation sensitivity assays in vivo and vitro. Transcriptomic analysis showed that a ΔpaeTBCFIIM knockout mutant significantly downregulated nitric oxide reductase (NOR) regulation and expression of coding genes such as nosR and norB, which contain methylated motifs in their promoters or coding regions. The ΔpaeTBCFIIM strain exhibited reduced intercellular survival capacity in NO-producing RAW264.7 macrophages and attenuated virulence in a Galleria mellonella infection model; the complemented strain recovered these defective phenotypes. Further phylogenetic analysis demonstrated that homologs of M.PaeTBCFII occur frequently in P. aeruginosa as well as other bacterial species. Our work therefore provided new insights into the relationship between DNA methylation, NO detoxification, and bacterial virulence, laying a foundation for further exploring the molecular mechanism of DNA methyltransferase in regulating the pathogenicity of P. aeruginosa.IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen which causes acute and chronic infections that are difficult to treat. Comparative genomic analysis has showed broad genome diversity among P. aeruginosa clinical strains and revealed their different regulatory traits compared to the laboratory strains. While current investigation of the epigenetics of P. aeruginosa is still lacking, understanding epigenetic regulation may provide biomarkers for diagnosis and facilitate development of novel therapies. Denitrification capability is critical for microbial versatility in response to different environmental stress conditions, including the bacterial infection process, where nitric oxide (NO) can be generated by phagocytic cells. The denitrification regulation mechanisms have been studied intensively at genetic and biochemical levels. However, there is very little evidence about the epigenetic regulation of bacterial denitrification mechanism. P. aeruginosa TBCF10839 is a chronically host-adapted strain isolated from a cystic fibrosis (CF) patient with special antiphagocytosis characteristics. Here, we investigated the regulatory effect of an orphan DNA MTase, M.PaeTBCFII, in P. aeruginosa TBCF10839. We demonstrated that the DNA MTase regulates the transcription of denitrification genes represented by NOR and affects antiphagocytic ability in bacteria. In silico analysis suggested that DNA methylation modification may enhance gene expression by affecting the binding of transacting factors such as DNR and RpoN. Our findings not only deepen the understanding of the role of DNA MTase in transcriptional regulation in P. aeruginosa but also provide a theoretical foundation for the in-depth study of the molecular mechanism of the epigenetic regulation on denitrification, virulence, and host-pathogen interaction.

Methylation-powered engineering of a dual-color light-up RNA nanosensor for label-free and ultrasensitive sensing of multiple DNA methyltransferases

DNA methylation is one important epigenetic modification that involves in regulating multiple cellular functions. DNA methyltransferases (MTases) maintain methylation patterns in genomic DNA, and their aberrant activities may lead to multiple diseases including cancers. However, the efficient sensing of multiple DNA MTases is limited by the diversity of catalytic substrates and the rareness of methylation-dependent restriction endonucleases. Herein, inspired by the intrinsic superiorities of light-up RNA aptamers and unique properties of T7-based transcription amplification, we construct a novel methylation-powered engineering of a dual-color light-up RNA nanosensor for label-free and ultrasensitive sensing of multiple DNA MTases. Taking advantage of ultrahigh specificity of MDREs toward methylated DNAs, high efficiency of bicyclic cascade amplifications and high signal-to-noise ratio of light-up RNA aptamer-fluorophore pairs, this nanosensor exhibits good specificity and high sensitivity with a detection limit of 3.77 × 10−3 U/mL for M.SssI and 7.21 × 10−5 U/mL for Dam. Furthermore, it can screen potential inhibitors and quantify M.SssI and Dam MTases activities in E. coli cells and human serum samples. Notably, this assay can be completed homogeneously within 2 h in a single tube in a label-free manner, providing a powerful platform for sensing multiple DNA MTases in biomedical research and clinical therapeutics.

Simple Detection of DNA Methyltransferase with an Integrated Padlock Probe.

DNA methyltransferases (MTases) can be regarded as biomarkers, as demonstrated by many studies on genetic diseases. Many researchers have developed biosensors to detect the activity of DNA MTases, and nucleic acid amplification, which need other probe assistance, is often used to improve the sensitivity of DNA MTases. However, there is no integrated probe that incorporates substrates and template and primer for detecting DNA MTases activity. Herein, we first designed a padlock probe (PP) to detect DNA MTases, which combines target detection with rolling circle amplification (RCA) without purification or other probe assistance. As the substrate of MTase, the PP was methylated and defended against HpaII, lambda exonuclease, and ExoI cleavage, as well as digestion, by adding MTase and the undestroyed PP started RCA. Thus, the fluorescent signal was capable of being rapidly detected after adding SYBRTM Gold to the RCA products. This method has a detection limit of approximately 0.0404 U/mL, and the linear range was 0.5–110 U/mL for M.SssI. Moreover, complex biological environment assays present prospects for possible application in intricacy environments. In addition, the designed detection system can also screen drugs or inhibitors for MTases.

Uncovering the role of DNA methyltransferases in grapevine – Plasmopara viticola interaction: From genome-wide characterization to global methylation patterns

Epigenetic regulation has recently gained prominence in the field of plant-pathogen interactions, providing a deeper understanding of the molecular mechanisms associated with plant infection. In grapevine interaction with pathogens, epigenetic regulation still remains a black box. In this work, we characterized grapevine DNA methyltransferase gene family and identified nine DNA methyltransferases genes across eight grapevine chromosomes coding for 17 proteins. We also assessed the modulation of global cytosine methylation and gene expression levels of these genes with the aim of establishing a connection between DNA methylation and grapevine resistance towards downy mildew. Our results revealed that, in the incompatible interaction, an early hypomethylation, coupled with downregulation of DNMT and CMT genes occurs very early after pathogen inoculation. Additionally, the compatible interaction is characterized by a hypermethylation at 6hpi. A temporal delay is evident between the shifts in DNA methyltransferases gene expression in both compatible and incompatible interactions which in turn may be reflected in the global methylation percentage. Overall, we present the first evidence of an epigenetic regulation role in grapevine defense against P. viticola.

Matrine inhibits advanced glycation end products-induced macrophage M1 polarization by reducing DNMT3a/b-mediated DNA methylation of GPX1 promoter

Advanced glycation end products (AGEs) are characterized diabetic metabolites inducing macrophage M1 polarization which is crucial in diabetes-exacerbated atherosclerosis. Matrine was proved anti-atherosclerotic. The current study was aimed to investigate the inhibitory effects of matrine on AGEs- induced macrophage M1 polarization and underlying molecular mechanisms. Primary mouse macrophages were exposed to AGEs. Receptor for AGEs (RAGE) and toll-like receptor 4 (TLR4) were over-expressed by vectors. Matrine was used to treat these cells. Inducible nitric oxide synthase (iNOS) expression and pro-inflammatory cytokine production were used to evaluate macrophage M1 polarization. Oxidative stress was assessed by intracellular reactive oxygen species (ROS) generation, total antioxidant capacity (TAC) and malondialdehyde (MDA) contents. Relative mRNA expression level was determined by real-time PCR. Western blotting was used to evaluate protein and protein phosphorylation levels. Bisulfite sequencing PCR (BSP) was used to evaluate DNA methylation. Matrine reduced AGEs exposure-elevated expressions of DNA methyltransferase (DNA MTase, DNMT)3a and DNMT3b in macrophages which were not affected by RAGE or TLR4 over expressions. DNA methylation rate of GPX1 promoter was reduced from 97.22% to 66.67% in AGEs- exposed macrophages treated by matrine. GPX1 expression was up-regulated by matrine, which further suppressed AGEs/RAGE-mediated oxidative stress. Thus, the activation of down-stream TLR4/STAT1 signaling pathway was inhibited by matrine treatment which eventually suppressed AGEs- induced macrophage M1 polarization. However, these effects of matrine were impaired by RAGE and TLR4 overexpression. Results from this study suggested that matrine inhibited AGEs- induced macrophage M1 polarization by suppressing RAGE-induced oxidative stress-mediated TLR4/STAT1 signaling pathway. Matrine exerted anti-oxidant effects via increasing GPX1 expression by inhibiting DNMT3a/b-induced GPX1 promoter DNA methylation.

A cascade amplification strategy for the detection of DNA methyltransferase activity by elemental labeling inductively coupled plasma mass spectrometry

A cascade amplification strategy combining strand displacement amplification (SDA) and multicomponent nucleic acid enzyme (MNAzyme) was proposed for detecting DNA MTase activity by AuNPs labeling inductively coupled plasma mass spectrometry (ICP-MS). With the absence of Dam MTase, the hairpin strand containing the recognition site of nicking endonuclease performed SDA reaction and generated abundant short strands. These strands hybridized with partzyme A to block the progress of the MNAzyme reaction. In the presence of Dam MTase, the action of methylation-sensitive nicking endonuclease was blocked and SDA process was inhibited, leading to activation of subsequent MNAzyme reaction. The completely assembled MNAzyme cleaved the substrates on the magnetic beads and the released AuNPs were detected by ICP-MS. The method shows an ultra-high sensitivity with a limit of detection of 1.51 × 10−4 U mL−1, around 1–2 orders of magnitude lower than most reported methods. It was applied to the screening of inhibitors of Dam MTase as well as Dam MTase analysis in serum samples. Recoveries of 94.6–104% were obtained for Dam MTase in spiked serum samples, demonstrating good potential of the method for highly sensitive detecting DNA MTase activity in complex biological samples.

An Isothermal Autocatalytic Hybridization Reaction Circuit for Sensitive Detection of DNA Methyltransferase and Inhibitors Assay.

Abnormal DNA methylation contributes to the annoying tumorigenesis and the elevated expression of methylation-related methyltransferase (MTase) is associated with many diseases. Hence DNA MTase could serve as a promising biomarker for cancer-specific diagnosis as well as a potential therapeutic target. Herein, we developed an isothermal autocatalytic hybridization reaction (AHR) circuit for the sensitive detection of MTase and its inhibitors by integrating the catalytic hairpin assembly (CHA) converter with the hybridization chain reaction (HCR) amplifier. The initiator-mediated HCR amplifier could generate amplified fluorescent readout, as well as numerous newly activated triggers for motivating the CHA converter. The CHA converter is designed to expose the identical sequence of HCR initiators that reversely powered the HCR amplifier. Thus, the trace amount of target could produce exponentially amplified fluorescent readout by the autocatalytic feedback cycle between HCR and CHA systems. Then an auxiliary hairpin was introduced to mediate the assay of Dam MTase via the well-established AHR circuit. The Dam MTase-catalyzed methylation of auxiliary hairpin leads to its subsequent efficient cleavage by DpnI endonuclease, thus resulting in the release of HCR initiators to initiate the AHR circuit. The programmable nature of the auxiliary hairpin allows its easy adaption into other MTase assay by simply changing the recognition site. This proposed AHR circuit permits a sensitive, robust, and versatile analysis of MTase with the limit of detection (LOD) of 0.011 U/mL. Lastly, the AHR circuit could be utilized for MTase analysis in real complex samples and for evaluating the cell-cycle-dependent expression of MTase. This developed MTase-sensing strategy holds promising potential for biomedical analysis and clinical diagnosis.

Bisubstrate Strategies to Target Methyltransferases

AbstractBisubstrate strategies address the lack of specificity encountered with S‐adenosyl‐L‐methionine (SAM) analogues targeting methyltransferases (MTases). Specifically, a bisubstrate inhibitor mimics the transition state complex associating a substrate and the SAM methyl group donor to a particular methyltransferase. These potential inhibitors contain both parts of the substrate and the methyl group donor. In this review, bisubstrate inhibitors of several MTases including DNA, RNA, protein, catechol or nicotinamide MTases are discussed in order to evaluate the interest of these promising molecules for the development of selective therapeutics against cancers, neuropsychiatric disorders and viral infections.

Ultrasensitive detection of DNA methyltransferase activity: a novel dual-amplification fluorescence technique.

DNA methyltransferase (MTase) is an important regulatory enzyme in various biological processes. However, current methods for investigating MTase activity are still limited in terms of sensitivity and/or generality. Herein, we proposed a dual amplification fluorescence strategy for the ultrasensitive detection of DNA adenine methylation methyltransferase (Dam MTase) activity based on strand displacement amplification (SDA) coupled with rolling circle amplification (RCA). In this study, the hairpin probe could not be cleaved by Nt.AlwI nicking endonuclease (Nt.AlwI) in the presence of Dam MTase, and the subsequent SDA-RCA reaction was blocked, resulting in a weak fluorescence signal. Moreover, the blocking effect was more pronounced at a higher concentration of Dam MTase. This assay provides a very low detection limit (down to 0.0067 U ml-1), as well as good selectivity against other types of MTases (e.g., CpG methyltransferase (M.SssI MTase)). In addition, the analytical mode improves the generality and can be extended to the detection of other types of DNA MTases.