BACKGROUND: DNA methylation is a critical factor influencing plant growth, adaptability, and phenotypic plasticity. While extensively studied in model and crop species, it remains relatively unexplored in holm oak and other non-domesticated forest trees. This study conducts a comprehensive in-silico mining of DNA methyltransferase and demethylase genes within the holm oak genome to enhance our understanding of this essential process in these understudied species. The expression levels of these genes in adult and seedling leaves, as well as embryos, were analysed using quantitative real-time PCR (qRT-PCR). Global DNA methylation patterns were assessed through methylation-sensitive amplified polymorphism (MSAP) techniques. Furthermore, specific methylated genomic sequences were identified via MSAP sequencing (MSAP-Seq).
RESULTS: A total of 13 DNA methyltransferase and three demethylase genes were revealed in the holm oak genome. Expression levels of these genes varied significantly between organs and developmental stages. MSAP analyses revealed a predominance of epigenetic over genetic variation among organs and developmental stages, with significantly higher global DNA methylation levels observed in adult leaves. Embryos exhibited frequent demethylation events, while de novo methylation was prevalent in seedling leaves. Approximately 35% of the genomic sequences identified by MSAP-Seq were methylated, predominantly affecting nuclear genes and intergenic regions, as opposed to repetitive sequences and chloroplast genes. Methylation was found to be more pronounced in the exonic regions of nuclear genes compared to their promoter and intronic regions. The methylated genes were predominantly associated with crucial biological processes such as photosynthesis, ATP synthesis-coupled electron transport, and defence response.
CONCLUSIONS: This study opens a new research direction in analysing variability in holm oak by evaluating the epigenetic events and mechanisms based on DNA methylation. It sheds light on the enzymatic machinery governing DNA (de)methylation, and the changes in the expression levels of methylases and demethylases in different organs along the developmental stages. The expression level was correlated with the DNA methylation pattern observed, showing the prevalence of de novo methylation and demethylation events in seedlings and embryos, respectively. Several methylated genes involved in the regulation of transposable element silencing, lipid biosynthesis, growth and development, and response to biotic and abiotic stresses are highlighted. MSAP-seq integrated with whole genome bisulphite sequencing and advanced sequencing technologies, such as PacBio or Nanopore, will bring light on epigenetic mechanisms regulating the expression of specific genes and its correlation with the phenotypic variability and the differences in the response to environmental cues, especially those related to climate change.

Cytosine methylation contributes to the regulation of gene expression and normal hematopoiesis in mammals. It is catalyzed by the family of DNA methyltransferases that include DNMT1, DNMT3A, and DNMT3B. Peripheral T-cell lymphomas (PTCLs) represent aggressive mature T-cell malignancies exhibiting a broad spectrum of clinical features with poor prognosis and inadequately understood molecular pathobiology. To better understand the molecular landscape and identify candidate genes involved in disease maintenance, we profiled DNA methylation and gene expression of PTCLs. We found that the methylation patterns in PTCLs are deregulated and heterogeneous but share 767 hypo- and 567 hypermethylated differentially methylated regions (DMRs) along with 231 genes up- and 91 genes downregulated in all samples, suggesting a potential association with tumor development. We further identified 39 hypomethylated promoters associated with increased gene expression in the majority of PTCLs. This putative oncogenic signature included the TRIP13 (thyroid hormone receptor interactor 13) gene whose genetic and pharmacologic inactivation inhibited the proliferation of T-cell lines by inducing G2-M arrest and apoptosis. Our data thus show that human PTCLs have a significant number of recurrent methylation alterations that may affect the expression of genes critical for proliferation whose targeting might be beneficial in anti-lymphoma treatments.

Aberrant gene expression patterns in acute myeloid leukemia (AML) with balanced chromosomal translocations are often associated with dysregulation of epigenetic modifiers. The AML1/ETO (RUNX1/MTG8) fusion protein, caused by the translocation (8;21)(q22;q22), leads to the epigenetic repression of its target genes. We aimed in this work to identify critical epigenetic modifiers, on which AML1/ETO-positive AML cells depend on for proliferation and survival using shRNA library screens and global transcriptomics approaches. Using shRNA library screens, we identified 41 commonly depleted genes in two AML1/ETO-positive cell lines Kasumi-1 and SKNO-1. We validated, genetically and pharmacologically, DNMT1 and ATR using several AML1/ETO-positive and negative cell lines. We also demonstrated in vivo differentiation of myeloblasts after treatment with the DNMT1 inhibitor decitabine in a patient with an AML1/ETO-positive AML. Bioinformatic analysis of global transcriptomics after AML1/ETO induction in 9/14/18-U937 cells identified 973 differentially expressed genes (DEGs). Three genes (PARP2, PRKCD, and SMARCA4) were both downregulated after AML1/ETO induction, and identified in shRNA screens. In conclusion, using unbiased shRNA library screens and global transcriptomics, we have identified several driver epigenetic regulators for proliferation in AML1/ETO-positive AML. DNMT1 and ATR were validated and are susceptible to pharmacological inhibition by small molecules showing promising preclinical and clinical efficacy.

We previously identified M.ApeKI from Aeropyum pernix K1 as a highly thermostable DNA (cytosine-5)-methyltransferase. M.ApeKI uses the type II restriction-modification system (R-M system), among the best-studied R-M systems. Although endonucleases generally utilize Mg (II) as a cofactor, several reports have shown that MTases exhibit different reactions in the presence of metal ions. This study aim was to evaluate the enzymatic properties of DNA (cytosine-5)-methyltransferase M.ApeKI from archaea in the presence of metal ions. We evaluated the influence of metal ions on the catalytic activity and DNA binding of M.ApeKI. The catalytic activity was inhibited by Cu (II), Mg (II), Mn (II), and Zn (II), each at 5 mM. DNA binding was more strongly inhibited by 5 mM Cu (II) and 10 mM Zn (II). To our knowledge, this is the first report showing that DNA binding of typeII MTase is inhibited by metal ions.

The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic biological chassis. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here, we report imitating methylation patterns rapidly in TXTL (IMPRINT), a generalized, rapid, and scalable approach based on cell-free transcription-translation (TXTL) to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from a bacterium\'s restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the bacterium\'s DNA methylation pattern, circumventing restriction and enhancing transformation. With IMPRINT, we efficiently multiplex methylation by diverse DNA methyltransferases and enhance plasmid transformation in gram-negative and gram-positive bacteria. We also develop a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to screen a ribosome-binding site library in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling the use of sophisticated genetic manipulation tools across the bacterial world.

Epigenetic mechanisms, such as DNA methylation, have been proposed to mediate plastic responses in insects. The pea aphid (Acyrthosiphon pisum), like the majority of extant aphids, displays cyclical parthenogenesis - the ability of mothers to switch the reproductive mode of their offspring from reproducing parthenogenetically to sexually in response to environmental cues. The pea aphid genome encodes two paralogs of the de novo DNA methyltransferase gene, dnmt3a and dnmt3x. Here we show, using phylogenetic analysis, that this gene duplication event occurred at least 150 million years ago, likely after the divergence of the lineage leading to the Aphidomorpha (phylloxerans, adelgids and true aphids) from that leading to the scale insects (Coccomorpha) and that the two paralogs are maintained in the genomes of all aphids examined. We also show that the mRNA of both dnmt3 paralogs is maternally expressed in the viviparous aphid ovary. During development both paralogs are expressed in the germ cells of embryos beginning at stage 5 and persisting throughout development. Treatment with 5-azactyidine, a chemical that generally inhibits the DNA methylation machinery, leads to defects of oocytes and early-stage embryos and causes a proportion of later stage embryos to be born dead or die soon after birth. These phenotypes suggest a role for DNA methyltransferases in reproduction, consistent with that seen in other insects. Taking the vast evolutionary history of the dnmt3 paralogs, and the localisation of their mRNAs in the ovary, we suggest there is a role for dnmt3a and/or dnmt3x in early development, and a role for DNA methylation machinery in reproduction and development of the viviparous pea aphid.

DNA methylation is an epigenetic mechanism that introduces a methyl group at the C5 position of cytosine. This reaction is catalyzed by DNA methyltransferases (DNMTs) and is essential for the regulation of gene transcription. The DNMT1 and DNMT3A or -3B family proteins are known targets for the inhibition of DNA hypermethylation in cancer cells. A selective non-nucleoside DNMT3A inhibitor was developed that mimics S-adenosyl-l-methionine and deoxycytidine; however, the mechanism of selectivity is unclear because the inhibitor-protein complex structure determination is absent. Therefore, we performed docking and molecular dynamics simulations to predict the structure of the complex formed by the association between DNMT3A and the selective inhibitor. Our simulations, binding free energy decomposition analysis, structural isoform comparison, and residue scanning showed that Arg688 of DNMT3A is involved in the interaction with this inhibitor, as evidenced by its significant contribution to the binding free energy. The presence of Asn1192 at the corresponding residues in DNMT1 results in a loss of affinity for the inhibitor, suggesting that the interactions mediated by Arg688 in DNMT3A are essential for selectivity. Our findings can be applied in the design of DNMT-selective inhibitors and methylation-specific drug optimization procedures.

Oral diseases are among the most common diseases worldwide and are associated with systemic illnesses, and the rising occurrence of oral diseases significantly impacts the quality of life for many individuals. It is crucial to detect and treat these conditions early to prevent them from advancing. DNA methylation is a fundamental epigenetic process that contributes to a variety of diseases including various oral diseases. Taking advantage of its reversibility, DNA methylation becomes a viable therapeutic target by regulating various cellular processes. Understanding the potential role of this DNA alteration in oral diseases can provide significant advances and more opportunities for diagnosis and therapy. This article will review the biology of DNA methylation, and then mainly discuss the key findings on DNA methylation in oral cancer, periodontitis, endodontic disease, oral mucosal disease, and clefts of the lip and/or palate in the background of studies on global DNA methylation and gene-specific DNA methylation.

Methylation of proteins and nucleic acids plays a fundamental role in epigenetic regulation, and discovery of methyltransferase (MT) inhibitors is an area of intense activity. Because of the diversity of MTs and their products, assay methods that detect S-adenosylhomocysteine (SAH) - the invariant product of S-adenosylmethionine (SAM)-dependent methylation reactions - offer some advantages over methods that detect specific methylation events. However, direct, homogenous detection of SAH requires a reagent capable of discriminating between SAH and SAM, which differ by a single methyl group. Moreover, MTs are slow enzymes and many have submicromolar affinities for SAM; these properties translate to a need for detection of SAH at low nanomolar concentrations in the presence of excess SAM. To meet these needs, we leveraged the exquisite molecular recognition properties of a naturally occurring SAH-sensing RNA aptamer, or riboswitch. By splitting the riboswitch into two fragments, such that SAH binding induces assembly of a trimeric complex, we engineered sensors that transduce binding of SAH into positive fluorescence polarization (FP) and time resolved Förster resonance energy transfer (TR-FRET) signals. The split riboswitch configuration, called the AptaFluor™ SAH Methyltransferase Assay, allows robust detection of SAH (Z\' > 0.7) at concentrations below 10 nM, with overnight signal stability in the presence of typical MT assay components. The AptaFluor assay tolerates diverse MT substrates, including histones, nucleosomes, DNA and RNA, and we demonstrated its utility as a robust, enzymatic assay method for several methyltransferases with SAM Km values < 1 µM. The assay was validated for HTS by performing a pilot screen of 1,280 compounds against the SARS-CoV-2 RNA capping enzyme, nsp14. By enabling direct, homogenous detection of SAH at low nanomolar concentrations, the AptaFluor assay provides a universal platform for screening and profiling MTs at physiologically relevant SAM concentrations.

BACKGROUND: Myelodysplastic syndrome (MDS) is a group of clonal hematopoietic stem cell disorders. DNA hypermethylation is considered to be the key mechanism of pathogenesis for MDS. Studies have demonstrated that DNA methylation can be regulated by the co-effect between long non-coding RNAs (lncRNAs) and DNA methyltransferases (DNMTs). The aim of this study was to identify DNMTs-associated differentially expressed (DE) lncRNAs, which may be a novel diagnostic and therapeutic target for MDS.
METHODS: Two gene expression profile datasets (GSE4619 and GSE19429) were downloaded from the Gene Expression Omnibus (GEO) database. Systematic bioinformatics analysis was conducted. Then we verified the expression of PRKCQ-AS1 in MDS patients and features in SKM-1 cells.
RESULTS: Bioinformatics analysis revealed that the DNMT-associated DE-lncRNA PRKCQ-AS1 was functionally related to DNA methylation. The target genes of PRKCQ-AS1 associated with DNA methylation are mainly methionine synthetase (MTR) and ten-eleven-translocation 1 (TET1). Moreover, the high expression of PRKCQ-AS1 was verified in real MDS cases. Further cellular analysis in SKM-1 cells revealed that overexpressed PRKCQ-AS1 promoted methylation levels of long interspersed nuclear element 1 (LINE-1) and cell proliferation, and apparently elevated both mRNA and protein levels of MTR and TET1, while knockdown of PRKCQ-AS1 showed opposite trend in SKM-1 cells.
CONCLUSIONS: DNMT-associated DE-lncRNA PRKCQ-AS1 may affects DNA methylation levels by regulating MTR and TET1.