Adenine Methylation Research Articles

MRNA ADENOSINE METHYLASE promotes drought tolerance through N6-methyladenosine-dependent and independent impacts on mRNA regulation in Arabidopsis.

Among many mRNA modifications, adenine methylation at the N6 position (N6-methyladenosine, m6A) is known to affect mRNA biology extensively. The influence of m6A has yet to be assessed under drought, one of the most impactful abiotic stresses. We show that Arabidopsis thaliana (L.) Heynh. (Arabidopsis) plants lacking mRNA ADENOSINE METHYLASE (MTA) are drought-sensitive. Subsequently, we comprehensively assess the impacts of MTA-dependent m6A changes during drought on mRNA abundance, stability, and translation in Arabidopsis. During drought, there is a global trend toward hypermethylation of many protein-coding transcripts that does not occur in mta. We also observe complex regulation of m6A at a transcript-specific level, possibly reflecting compensation by other m6A components. Importantly, a subset of transcripts that are hypermethylated in an MTA-dependent manner exhibited reduced turnover and translation in mta, compared with wild-type (WT) plants, during drought. Additionally, MTA impacts transcript stability and translation independently of m6A. We also correlate drought-associated deposition of m6A with increased translation of modulators of drought response, such as RD29A, COR47, COR413, ALDH2B, ERD7, and ABF4 in WT, which is impaired in mta. m6A is dynamic during drought and, alongside MTA, promotes tolerance by regulating drought-responsive changes in transcript turnover and translation.

Adenosine Deaminase-Like Gene-Carried Lentivirus Toolkit for Identification of DNA N6-Methyladenine Origins.

Post-replicative DNA N6-methyladenine (pr6mdA) can form via bona fide methylase-catalyzed adenine methylation, playing a pivotal role in embryonic development and other biological processes. Surprisingly, pre-methylated adenine can be erroneously incorporated into DNA as misincorporated N6-methyladenine (i6mdA) via DNA polymerase-mediated replication. Despite pr6mdA and i6mdA sharing identical chemical structures, their biological functions diverge significantly, presenting a substantial challenge in distinguishing between the two. Here, for the first-time, it is exploited that the adenosine deaminase-like (Adal) protein and a corresponding activity-null mutant to construct an Adal lentivirus toolkit. With this newly designed toolkit, both pr6mdA and i6mdA can be identified and quantified simultaneously. The presence of 6mdA in the bone marrow cells of mice is shown, with its levels serving as indicators for growth with age, probably reflecting the cellular stress-caused changes in RNA decay, nucleotide pool sanitation, and transcription. Collectively, a powerful toolkit to advance understanding of both pr6mdA and i6mdA is demonstrated.

Direct transposition of native DNA for sensitive multimodal single-molecule sequencing

Concurrent readout of sequence and base modifications from long unamplified DNA templates by Pacific Biosciences of California (PacBio) single-molecule sequencing requires large amounts of input material. Here we adapt Tn5 transposition to introduce hairpin oligonucleotides and fragment (tagment) limiting quantities of DNA for generating PacBio-compatible circular molecules. We developed two methods that implement tagmentation and use 90–99% less input than current protocols: (1) single-molecule real-time sequencing by tagmentation (SMRT-Tag), which allows detection of genetic variation and CpG methylation; and (2) single-molecule adenine-methylated oligonucleosome sequencing assay by tagmentation (SAMOSA-Tag), which uses exogenous adenine methylation to add a third channel for probing chromatin accessibility. SMRT-Tag of 40 ng or more human DNA (approximately 7,000 cell equivalents) yielded data comparable to gold standard whole-genome and bisulfite sequencing. SAMOSA-Tag of 30,000–50,000 nuclei resolved single-fiber chromatin structure, CTCF binding and DNA methylation in patient-derived prostate cancer xenografts and uncovered metastasis-associated global epigenome disorganization. Tagmentation thus promises to enable sensitive, scalable and multimodal single-molecule genomics for diverse basic and clinical applications.

Characterization of winged helix domain fusion endonucleases as N6-methyladenine-dependent type IV restriction systems.

Winged helix (wH) domains, also termed winged helix-turn-helix (wHTH) domains, are widespread in all kingdoms of life and have diverse roles. In the context of DNA binding and DNA modification sensing, some eukaryotic wH domains are known as sensors of non-methylated CpG. In contrast, the prokaryotic wH domains in DpnI and HhiV4I act as sensors of adenine methylation in the 6mApT (N6-methyladenine, 6mA, or N6mA) context. DNA-binding modes and interactions with the probed dinucleotide are vastly different in the two cases. Here, we show that the role of the wH domain as a sensor of adenine methylation is widespread in prokaryotes. We present previously uncharacterized examples of PD-(D/E)XK-wH (FcyTI, Psp4BI), PUA-wH-HNH (HtuIII), wH-GIY-YIG (Ahi29725I, Apa233I), and PLD-wH (Aba4572I, CbaI) fusion endonucleases that sense adenine methylation in the Dam+ Gm6ATC sequence contexts. Representatives of the wH domain endonuclease fusion families with the exception of the PLD-wH family could be purified, and an in vitro preference for adenine methylation in the Dam context could be demonstrated. Like most other modification-dependent restriction endonucleases (MDREs, also called type IV restriction systems), the new fusion endonucleases except those in the PD-(D/E)XK-wH family cleave close to but outside the recognition sequence. Taken together, our data illustrate the widespread combinatorial use of prokaryotic wH domains as adenine methylation readers. Other potential 6mA sensors in modified DNA are also discussed.

Adenine Methylation Enhances the Conformational Flexibility of an RNA Hairpin Tetraloop.

The N6-methyladenosine modification is one of the most abundant post-transcriptional modifications in ribonucleic acid (RNA) molecules. Using molecular dynamics simulations and alchemical free-energy calculations, we studied the structural and energetic implications of incorporating this modification in an adenine mononucleotide and an RNA hairpin structure. At the mononucleotide level, we found that the syn configuration is more favorable than the anti configuration by 2.05 ± 0.15 kcal/mol. The unfavorable effect of methylation was due to the steric overlap between the methyl group and a nitrogen atom in the purine ring. We then probed the effect of methylation in an RNA hairpin structure containing an AUCG tetraloop, which is recognized by a "reader" protein (YTHDC1) to promote transcriptional silencing of long noncoding RNAs. While methylation had no significant conformational effect on the hairpin stem, the methylated tetraloop showed enhanced conformational flexibility compared to the unmethylated tetraloop. The increased flexibility was associated with the outward flipping of two bases (A6 and U7) which formed stacking interactions with each other and with the C8 and G9 bases in the tetraloop, leading to a conformation similar to that in the RNA/reader protein complex. Therefore, methylation-induced conformational flexibility likely facilitates RNA recognition by the reader protein.

Methylation Induces a Low‐energy Emissive State in N6‐methyladenine Containing Dinucleotides

AbstractMethylation of adenine at the N6 position is a crucial epigenetic modification that profoundly influences gene regulation and expression. Moreover, this modification intricately alters the excited state dynamics of adenine nucleobases. To explore the impact of N6‐methyladenine on the excited state dynamics within oligonucleotides, we conducted a comprehensive investigation of two dinucleotides containing N6‐methyladenosine, in conjunction with adenosine or guanosine. Using steady‐state and time‐resolved absorption and fluorescence spectroscopy techniques, we not only observed the customary monomer‐like and charge transfer emissive states, as reported in previous dinucleotides, but also identified an additional low‐energy emissive state. This unique state exhibits an extraordinary Stokes Shift exceeding 2.3 eV and has a relatively long lifetime of 4–5 ns. We propose that this state corresponds to a bonded exciplex state, governed by ground‐state geometries.

Epigenetics and the improvement of crop plants

Epigenetics is a term that refers to the changes in gene expression that are heritable and induced by DNA methylation, histones post-translational modifications, or sncRNA, not resulting from the DNA sequence rearrangements. Epigenetic modifications influence gene expression, and thus, the plasticity of plants' development and phenotype in response to external and internal factors. Until recently, the only known epigenetic modification of the DNA in eukaryotic organisms was 5-methylcytosine. The growing interest in epigenetics and the development of sensitive detection methods enabled the discovery of other modifications of the DNA nitrogenous bases, i.e., 4-methylcytosine and 6-methyladenine. However, whilst research on the 5mC distribution and role in eukaryotic organisms is widespread, analyses regarding 6mA are scarced. Nevertheless, there are indications of a potential epigenetic role of 6-methyladenine in eukaryotic genomes. Understanding epigenetic mechanisms, which are triggered in response to environmental changes, is crucial for agriculture. This review shows epigenetic mechanisms, with particular emphasis on adenine methylation in plants, as well as the role of epigenetic variation in epibreeding, affecting the improvement of agronomic traits.

Exposure of children to brominated flame retardants and heavy metals in Morocco: Urine and blood levels in association with global cytosine and adenine methylation

Persistent pollutants, namely brominated flame retardants (BFRs) and heavy metals, are compounds that are added to a wide range of products and materials for preventing ignition, increasing the functionality of materials or improving their performance, e.g. electric conductivity. The exposure of children might consequently be inferred, through indoor dust and hand-to-mouth or toy-chewing behaviors. The current study is aimed at assessing the exposure of Moroccan children to BFRs and heavy metal elements, and evaluating their associations with global DNA methylation. First, parents responded to a questionnaire pertaining to children’s lifestyle, then blood and urine samples were collected from (n = 93) children aged between 5 and 11 years for biomonitoring and DNA methylation analysis. BFRs were detected in 54.84% of samples with a median concentration of 0.01 nmol/mL (range: 0.004–0.051 nmol/mL) while metal elements were detected in more than 90% of samples. BFRs showed no variations with global DNA methylation, unlike metal elements, which revealed significant associations with global DNA methylation markers, namely 5-mC, 5-hmC and N⁶-mA levels. Moroccan children may be exposed to flame retardants and heavy metals through several routes. Further research is required to assess the exposure and the health impacts of environmental pollutants and ultimately protect the Moroccan population by the prevention of adverse health effects.

Adenine methylation is very scarce in the Drosophila genome and not erased by the ten-eleven translocation dioxygenase.

N6-methyladenine (6mA) DNA modification has recently been described in metazoans, including in Drosophila, for which the erasure of this epigenetic mark has been ascribed to the ten-eleven translocation (TET) enzyme. Here, we re-evaluated 6mA presence and TET impact on the Drosophila genome. Using axenic or conventional breeding conditions, we found traces of 6mA by LC-MS/MS and no significant increase in 6mA levels in the absence of TET, suggesting that this modification is present at very low levels in the Drosophila genome but not regulated by TET. Consistent with this latter hypothesis, further molecular and genetic analyses showed that TET does not demethylate 6mA but acts essentially in an enzymatic-independent manner. Our results call for further caution concerning the role and regulation of 6mA DNA modification in metazoans and underline the importance of TET non-enzymatic activity for fly development.

Adenine methylation is very scarce in the Drosophila genome and not erased by the ten-eleven translocation dioxygenase

N6-methyladenine (6mA) DNA modification has recently been described in metazoans, including in Drosophila, for which the erasure of this epigenetic mark has been ascribed to the ten-eleven translocation (TET) enzyme. Here, we re-evaluated 6mA presence and TET impact on the Drosophila genome. Using axenic or conventional breeding conditions, we found traces of 6mA by LC-MS/MS and no significant increase in 6mA levels in the absence of TET, suggesting that this modification is present at very low levels in the Drosophila genome but not regulated by TET. Consistent with this latter hypothesis, further molecular and genetic analyses showed that TET does not demethylate 6mA but acts essentially in an enzymatic-independent manner. Our results call for further caution concerning the role and regulation of 6mA DNA modification in metazoans and underline the importance of TET non-enzymatic activity for fly development.

Distinct epigenetic signatures of classical and hypervirulent Klebsiella pneumoniae.

Emergence and spread of the hypervirulent pathotype of Klebsiella pneumoniae have significantly increased infection rates in community as well as healthcare settings. There is an increasing interest to identify discriminating features between classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp) to facilitate our understanding of the rapid emergence and dissemination of the hypervirulent pathotype. Here, we sought to identify unique epigenetic signatures of hvKp pathotype that differ from its classical counterpart using single-base resolution methylome analysis of native DNA sequencing on the Oxford Nanopore Technologies platform. The overall global adenine methylation in GATC motifs (i.e., Dam methylation motif) and cytosine methylation in CCWGG motifs (i.e., Dcm methylation motif) were significantly higher in hvKp isolates compared to that in cKp isolates, irrespective of their position in chromosomes or putative extra-chromosomal genetic elements. Notably, we observed significant enrichment of hypermethylated GATC and CCWGG motifs in the virulome of hvKp compared to hvKp genes not directly associated with virulence. We also observed increased methylation of GATC and CCWGG motifs in the capsule synthesis locus of hvKp isolates compared to cKp isolates. Furthermore, we identified several differentially methylated genes (DMGs) between the two pathotypes; interestingly, these DMGs include metal ion transporters, multidrug efflux pumps, transcriptional regulators of stress response, and genes associated with biofilm formation. Our results highlight hypermethylation of GATC and CCWGG motifs as unique epigenetic signatures of hvKp isolates.IMPORTANCEHypervirulent Klebsiella pneumoniae (hvKp) is a more virulent and rapidly evolving hypermucoviscous pathotype of classical K. pneumoniae (cKp). The hypervirulent pathotype is a major public health concern and is associated with high infection rates in community as well as hospital settings. With the recent emergence of multidrug-resistant hvKp, it has become imperative to investigate non-classical mechanisms such as epigenetics in addition to canonical biochemical and genetic mechanisms that delineate and differentiate the hypervirulent pathotype from its classical counterpart. Here, we identify genome-wide differences in adenine and cytosine methylation marks at well-characterized motifs between the two pathotypes. Overall, significantly higher levels of methylation were observed across chromosomal DNA and extrachromosomal elements in hvKp compared to cKp. Among hvKp isolates, the genes associated with virulence are particularly enriched for methylation marks. Our findings shed light on how epigenetic signatures may help distinguish the pathogenic potential of bacteria.

Integrative DNA methylome and transcriptome analysis reveals DNA adenine methylation is involved in Salmonella enterica Typhimurium response to oxidative stress.

Salmonella could survive and replicate in macrophages, where it encounters multiple stresses. Deficiency of a DNA adenine methyltransferase impairs the survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) in hydrogen peroxide. To investigate whether DNA methylation is involved in the expression of oxidative stress-responsive genes, we combined RNA-seq and single-molecule real-time sequencing to integrate transcriptome and methylome analysis. Here, we show that (i) the entire amount of m6A GATC remains stable during oxidative stress; (ii) no significant association is observed between DNA methylation and transcription level in most genes; (iii) coincidence of level change between transcription and m6A GATC in the regulatory regions is identified in 49 genes under oxidative stress. Some of them are known to contribute to bacterial defenses against oxidative stress through reducing H2O2 levels, directing aberrant protein product degradation and inducing outer membrane protein expression. Specifically, the transcription level of ahpCF is negatively correlated to the m6A GATC level in its regulatory region. The transcription of smpB is elevated along with the decrease in m6A level at position 2,879,954 on the minus strand. In contrast, mRNA levels of STM14_2773 and ycfR are positively correlated with m6A GATC content in their regulatory regions. Highly stable DNA methylome and coupled change of m6A GATC with gene expression in specific positions suggest that DNA methylation homeostasis at genome-wide and plasticity in specific regions are crucial for bacterial response to oxidative stress. These findings provide new insights into the epigenetic regulatory mechanisms of gene expression in S. Typhimurium in the host microenvironment.IMPORTANCEThe intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) comes across a wide variety of stresses from entry to dissemination, such as reactive oxygen species. To adapt itself to oxidative stress, Salmonella must adopt various and complex strategies. In this study, we revealed that DNA adenine methyltransferase was essential for S. Typhimurium to survive in hydrogen peroxide. We then screened out oxidative stress-responsive genes that were potentially regulated by DNA methylation in S. Typhimurium. Our results show that the DNA methylome is highly stable throughout the genome, and the coupled change of m6A GATC with gene expression is identified in only a few positions, which suggests the complexity of the DNA methylation and gene expression regulation networks. The results may shed light on our understanding of m6A-mediated gene expression regulation in bacteria.

Differential adenine methylation analysis reveals increased variability in 6mA in the absence of methyl-directed mismatch repair.

Methylation greatly influences the bacterial genome by guiding DNA repair and regulating pathogenic and stress-response phenotypes. But, the rate of epigenetic changes and their consequences on molecular phenotypes are underexplored. Through a detailed characterization of genome-wide adenine methylation in a commonly used laboratory strain of Escherichia coli, we reveal that mismatch repair deficient populations experience an increase in epimutations resulting in a genome-wide reduction of 6mA methylation in a manner consistent with genetic drift. Our findings highlight how methylation patterns evolve and the constraints on epigenetic evolution due to post-replicative DNA repair, contributing to a deeper understanding of bacterial genome evolution and how epimutations may introduce semi-permanent variation that can influence adaptation.

Development of a N6-methyladenosine-directed single quantum dot-based biosensor for sensitive detection of METTL3/14 complex activity in breast cancer tissues

The METTL3/14 complex is an important RNA N6-Methyladenosine (m6A) methyltransferase in organisms, and the abnormal METTL3/14 complex activity is associated with the pathogenesis and various cancers. Sensitive detection of METTL3/14 complex is essential to tumor pathogenesis study, cancer diagnosis, and anti-cancer drug discovery. However, traditional methods for METTL3/14 complex assay suffer from poor specificity, costly antibodies, unstable RNA substrates, and low sensitivity. Herein, we construct a single quantum dot (QD)-based förster resonance energy transfer (FRET) biosensor for sensitive detection of METTL3/14 complex activity. In the presence of METTL3/14 complex, it catalyzes the methylation of adenine in the substrate probe, leading to the formation of m6A that protects the substrate probes from MazF-mediated cleavage. The hybridization of methylated DNA substrate with biotinylated capture probe initiates polymerization reaction to obtain a biotinylated double-stranded DNA (dsDNA) with the incorporation of numerous Cy5 fluorophores. Subsequently, the Cy5-incorporated dsDNA can self-assembly onto the 605QD surface to form the 605QD-dsDNA-Cy5 nanostructure, causing FRET between 605QD donor and Cy5 acceptor. This biosensor has excellent sensitivity with a limit of detection (LOD) of 3.11 × 10−17 M, and it can measure the METTL3/14 complex activity in a single cell. Moreover, this biosensor can be used to evaluate the METTL3/14 complex kinetic parameters and screen potential inhibitors. Furthermore, it can differentiate the METTL3/14 complex expression in healthy human tissues and breast cancer patient tissues, providing a powerful tool for cancer pathogenesis study, clinical diagnosis, prognosis monitoring, and drug discovery.

A DNA adenine demethylase impairs PRC2-mediated repression of genes marked by a specific chromatin signature

BackgroundThe Fe (II)- and α-ketoglutarate-dependent AlkB family dioxygenases are implicated in nucleotide demethylation. AlkB homolog1 (ALKBH1) is shown to demethylate DNA adenine methylation (6mA) preferentially from single-stranded or unpaired DNA, while its demethylase activity and function in the chromatin context are unclear.ResultsHere, we find that loss-of-function of the rice ALKBH1 gene leads to increased 6mA in the R-loop regions of the genome but has a limited effect on the overall 6mA level. However, in the context of mixed tissues, rather than on individual loci, the ALKBH1 mutation or overexpression mainly affects the expression of genes with a specific combination of chromatin modifications in the body region marked with H3K4me3 and H3K27me3 but depleted of DNA CG methylation. In the similar context of mixed tissues, further analysis reveals that the ALKBH1 protein preferentially binds to genes marked by the chromatin signature and has a function to maintain a high H3K4me3/H3K27me3 ratio by impairing the binding of Polycomb repressive complex 2 (PRC2) to the targets, which is required for both the basal and stress-induced expression of the genes.ConclusionOur findings unravel a function of ALKBH1 to control the balance between the antagonistic histone methylations for gene activity and provide insight into the regulatory mechanism of PRC2-mediated H3K27me3 deposition within the gene body region.

To Me, or not to Me: Effects of mRNA adenine methylation on biotic stress responses.

To Me, or not to Me: Effects of mRNA adenine methylation on biotic stress responses.

Adenine DNA methylation is involved in regulating ethanol and osmotic stress responses in Lacticaseibacillus paracasei Zhang

AbstractAs an important feature of epigenetics, DNA methylation is a critical way to regulate gene expression and thus bacterial growth and metabolism. Lacticaseibacillus paracasei Zhang is a probiotic strain originated from koumiss. This study hypothesized that adenine methylation was involved in bacterial regulation of physiological adaptations when grown under environmental stress. Thus, this study compared the growth of an adenine methyltransferase‐inactivated mutant (ΔpglX) and the wild‐type L. paracasei Zhang when cultured under ethanol and osmotic stress. Transcriptomic and proteomic analyses were implemented to identify their differentially expressed genes/proteins (DEGs/DEPs) when grown under the stressed environments. The membrane fatty acid composition was also determined. Comparing with the wild‐type, the viable counts of the mutant under ethanol and osmotic stress increased by 2.6 times and decreased by half, respectively (ethanol stress: mutant, 4.30 ± 0.0038 × 108 CFU/mL; wild‐type, 1.64 ± 0.010 × 108 CFU/mL; osmotic stress: mutant, 2.21 ± 0.029 × 108 CFU/mL; wild‐type, 4.30 ± 0.13 × 108 CFU/mL). Common to both conditions, many identified DEGs and DEPs were involved in carbohydrate metabolism and fatty acid biosynthesis, generally, with more carbohydrate metabolism pathways but fewer fatty acid biosynthesis pathways, accompanied by increases in the membrane fatty acids. Our observations suggested that L. paracasei Zhang maximizes its energy metabolism by enhancing carbohydrate metabolism and improves its membrane strength via increasing the fatty acid content under environmental stress. This is the first report analyzing the epigenetic‐level stress responsive mechanism of L. paracasei Zhang, providing insights into methylation‐based regulation of bacterial adaptive responses.

TROP2 translation mediated by dual m6A/m7G RNA modifications promotes bladder cancer development

RNA modifications, including adenine methylation (m6A) of mRNA and guanine methylation (m7G) of tRNA, are crucial for the biological function of RNA. However, the mechanism underlying the translation of specific genes synergistically mediated by dual m6A/m7G RNA modifications in bladder cancer (BCa) remains unclear. We demonstrated that m6A methyltransferase METTL3-mediated programmable m6A modification of oncogene trophoblast cell surface protein 2 (TROP2) mRNA promoted its translation during malignant transformation of bladder epithelial cells. m7G methyltransferase METTL1 enhanced TROP2 translation by mediating m7G modification of certain tRNAs. TROP2 protein inhibition decreased the proliferation and invasion of BCa cells in vitro and in vivo. Moreover, synergistical knockout of METTL3/METTL1 inhibited BCa cell proliferation, migration, and invasion; however, TROP2 overexpression partially abrogated its effect. Furthermore, TROP2 expression was significantly positively correlated with the expression levels of METTL3 and METTL1 in BCa patients. Overall, our results revealed that METTL3/METTL1-mediated dual m6A/m7G RNA modifications enhanced TROP2 translation and promoted BCa development, indicating a novel RNA epigenetic mechanism in BCa.

Roles of adenine methylation in the physiology of Lacticaseibacillus paracasei

Lacticaseibacillus paracasei is an economically important bacterial species, used in the food industry and as a probiotic. Here, we investigate the roles of N6-methyladenine (6mA) modification in L. paracasei using multi-omics and high-throughput chromosome conformation capture (Hi-C) analyses. The distribution of 6mA-modified sites varies across the genomes of 28 strains, and appears to be enriched near genes involved in carbohydrate metabolism. A pglX mutant, defective in 6mA modification, shows transcriptomic alterations but only modest changes in growth and genomic spatial organization.

Global DNA Adenine Methylation in Caenorhabditis elegans after Multigenerational Exposure to Silver Nanoparticles and Silver Nitrate.

Multigenerational and transgenerational reproductive toxicity in a model nematode Caenorhabditis elegans has been shown previously after exposure to silver nanoparticles (Ag-NPs) and silver ions (AgNO3). However, there is a limited understanding on the transfer mechanism of the increased reproductive sensitivity to subsequent generations. This study examines changes in DNA methylation at epigenetic mark N6-methyl-2'-deoxyadenosine (6mdA) after multigenerational exposure of C. elegans to pristine and transformed-via-sulfidation Ag-NPs and AgNO3. Levels of 6mdA were measured as 6mdA/dA ratios prior to C. elegans exposure (F0) after two generations of exposure (F2) and two generations of rescue (F4) using high-performance liquid chromatography with tandem mass spectrometry (LC-MS/MS). Although both AgNO3 and Ag-NPs induced multigenerational reproductive toxicity, only AgNO3 exposure caused a significant increase in global 6mdA levels after exposures (F2). However, after two generations of rescue (F4), the 6mdA levels in AgNO3 treatment returned to F0 levels, suggesting other epigenetic modifications may be also involved. No significant changes in global DNA methylation levels were observed after exposure to pristine and sulfidized sAg-NPs. This study demonstrates the involvement of an epigenetic mark in AgNO3 reproductive toxicity and suggests that AgNO3 and Ag-NPs may have different toxicity mechanisms.