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Abstract Background DNA methylation is an epigenetic modification that plays an important role in animal and plant development. Among the diverse types of DNA methylation modifications, methylation of cytosines catalyzed by DNA cytosine methyltransferases (DNMTs) is the most common. Recently, we characterized DNA methyltransferase genes including HlDnmt1 and HlDnmt from the Asian longhorned tick, Haemaphysalis longicornis. However, the dynamic expression and functions of these DNMTs at different developmental stages and feeding statuses of the important vector tick H. longicornis remain unknown. Results The expression levels of HlDnmt1 and HlDnmt were significantly different at the four developmental stages: eggs, larvae, nymphs, and adults, with the highest expression levels observed in the larval stage. HlDnmt1 and HlDnmt showed different expression trends in the midguts, ovary, Malpighian tubules, and salivary glands of engorged adults, with the highest expression of HlDnmt1 observed in the ovary and the lowest in the midguts; HlDnmt expression was the highest in the midguts and the lowest in the Malpighian tubules. After RNA interference, the relative expression of HlDnmt1 and HlDnmt in H. longicornis decreased significantly, resulting in a significant decrease in the biting rate of H. longicornis. RNA-seq revealed that the differentially expressed genes were mainly enriched in the biological processes of peptide biosynthesis and the cell components of ribosomes. Molecular functions were mainly concentrated on oxidoreductase activity, ribosome structure composition, serine-type endopeptidase activity, molecular function regulators, and endopeptidase inhibitor activity. KEGG enrichment analysis showed that the differentially expressed genes were mainly enriched in autophagy and lysosome pathways, amino sugar and nucleotide sugar metabolism, glyceride metabolism, ribosomes, and other pathways. Conclusions HlDnmt1 and HlDnmt played an important role during development and feeding of H. longicornis, and their functions were potentially associated with lysosome pathways. These results provide basic knowledge for understanding the epigenetic regulation of the development of the tick H. longicornis, which sheds light on control strategies for ticks and tick-borne diseases.

Abstract 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). Result 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. Conclusion 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.

Abstract Hepatocellular carcinoma (HCC) is a primary malignant tumour, ranking second in global mortality rates and posing significant health threats. Epigenetic alterations, particularly DNA methylation, have emerged as pivotal factors associated with HCC diagnosis, therapy, prognosis and malignant progression. However, a comprehensive analysis of the DNA methylation mechanism driving HCC progression and its potential as a therapeutic biomarker remains lacking. This review attempts to comprehensively summarise various aspects of DNA methylation, such as its mechanism, detection methods and biomarkers aiding in HCC diagnosis, treatment and prognostic assessment of HCC. It also explores the role of DNA methylation in regulating HCC's malignant progression and sorafenib resistance, alongside elaborating the therapeutic effects of DNA methyltransferase inhibitors on HCC. A detailed examination of these aspects underscores the significant research on DNA methylation in tumour cells to elucidate malignant progression mechanisms, identify diagnostic markers and develop new tumour‐specific inhibitors for HCC. Key points A comprehensive summary of various aspects of DNA methylation, such as its mechanism, detection methods and biomarkers aiding in diagnosis and treatment. The role of DNA methylation in regulating hepatocellular carcinoma's (HCC) malignant progression and sorafenib resistance, alongside elaborating therapeutic effects of DNA methyltransferase inhibitors. Deep research on DNA methylation is critical for discovering novel tumour‐specific inhibitors for HCC.

Abstract 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.

In cutaneous melanoma, epigenetic dysregulation is implicated in drug resistance and tumor immune escape. However, the epigenetic mechanisms that influence immune escape remain poorly understood. To elucidate how epigenetic dysregulation alters the expression of surface proteins that may be involved in drug targeting and immune escape, we performed a 3-dimensional surfaceome screen in primary melanoma cultures and identified the DNA-methyltransferase inhibitor decitabine as significantly upregulating the costimulatory molecule ICAM-1. By analyzing The Cancer Genome Atlas melanoma dataset, we further propose ICAM-1 upregulation on melanoma cells as a biomarker of a proinflammatory and antitumorigenic signature. Specifically, we showed that DNA-methyltransferase inhibitor administration upregulated the expression of the antigen-presenting machinery, HLA class I/II, as well as the secretion of the proinflammatory chemokines CXCL9 and CXCL10. Our in silico analysis on The Cancer Genome Atlas and ex vivo experiments on human primary melanoma samples revealed that increased ICAM-1 expression positively correlated with increased immunogenicity of human melanoma cells and correlated with increased immune cell infiltration. These findings suggest a therapeutic approach to modulate the immunogenic phenotype of melanoma cells, hence supporting the exploration of DNA-methyltransferase inhibitor as a potential inducer of infiltration in immunologically cold tumors.

Background: Acute myeloid leukemia (AML) is a heterogeneous hematopoietic malignancy whose prognosis is associated with several biomarkers. Decitabine, a deoxyribonucleic acid (DNA) methyltransferase (DNMT) inhibitor, combined with cytarabine, aclarubicin hydrochloride, and granulocyte colony-stimulating factor (DCAG), has been used in patients newly diagnosed with AML. This regimen has been especially used in older and fragile patients who are immunocompromised or have co-morbidities, as well as those with specific gene mutations. However, the integration of molecular risk stratification and treatment guidance for the DCAG regimen has not been well defined. Therefore, this study aimed to investigate the genetic mutations associated with AML and establish appropriate treatment strategies for patients newly diagnosed with AML. Methods: This study analyzed the clinical data and genetic mutations based on next-generation sequencing (NGS) in 124 newly diagnosed patients with AML who received the DCAG regimen at the People's Liberation Army (PLA) General Hospital from January 2008 to August 2020. Factors associated with the cumulative incidence of relapse (CIR) and leukemia-free survival (LFS) in patients newly diagnosed with AML were analyzed. Results: The most adverse prognosis of DCAG-treated patients was observed in those with FLT3-ITD, KIT, PTPN11, GATA2, or IDH1 mutations during univariable analysis, whereas PTPN11 mutation was solely significant in multivariable analysis, with an increased likelihood of CIR (P = 0.001) and reduced LFS duration (P = 0.077). Hyperleukocytosis was maintained as an independent risk factor for increased CIR risk (P = 0.044) and decreased LFS duration (P = 0.042) in multivariable analysis. In this study, we validated the risk classification of patients with AML receiving an epigenetic modifier-based induction regimen across a broad age range. Conclusion: NGS demonstrated a dismal overall outcome in patients with the rare PTPN11 mutations, indicating the need for new therapies that target this high-risk subtype of AML. These results offer a potential molecular stratification and treatment guidance for patients with AML.

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.

Introduction. Rearrangement of molecular pathways and activation of bypass signaling determine the progression of tumor cell resistance to various drugs. Study of the common features of resistant formation mechanisms is essential for breast and other cancer beneficial treatments.Materials and methods. The present work was performed on estrogen receptor α ERα-positive (ERα – estrogen receptor α) McF-7 breast cancer cells, established sublines resistant to the mTOR inhibitor rapamycin or antiestrogen tamoxifen, and ERα-negative MDA-MB-231 breast cancer cells. Methods used include MTT test, transient transfection, immunoblotting, real-time polymerase chain reaction and methylation analysis by bisulfite pyrosequencing.Results. We have shown that the resistance of breast cancer cells to targeted and hormonal drugs is associated with the suppression of DNA methyltransferase 3A (DNMT3A) and respective changes in DNA methylation; DNMT3A knockdown results in the partial resistance to both drugs demonstrating the pivotal role of DNMT3A suppression in the progression of cell resistance.Conclusion. Totally, the results obtained highlight the possible mechanism of tumor cell resistance to targeting/hormonal drugs based on the deregulation of DNMTs expression and demonstrate direct connection between DNMT3A suppression and resistance progression.

This review deals with the functional characteristics and biological roles of enzymes participating in DNA methylation and demethylation as key factors in epigenetic regulation of gene expression. The set of enzymes that carry out such processes in human cells is limited to representatives of two families, namely DNMT (DNA methyltransferases) and TET (DNA dioxygenases). The review presents detailed information known today about each functionally important member of these families and describes the catalytic activity and roles in the mammalian body while also providing examples of dysregulation of the expression and/or activity of these enzymes in conjunction with the development of some human disorders, including cancers, neurodegenerative diseases, and developmental pathologies. By combining the up-to-date information on the dysfunction of various enzymes that control the DNA “methylome” in the human body, we hope not only to draw attention to the importance of the maintenance of a required DNA methylation level (ensuring epigenetic regulation of gene expression and normal functioning of the entire body) but also to help identify new targets for directed control over the activity of the enzymes that implement the balance between processes of DNA methylation and demethylation.

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.

Objective To explore the regulative mechanism of hypoxia/reoxygen (H/R) for invasion ability of extravillous trophoblast (EVT). Methods Immortalized human EVT cell line HTR-8/SVneo in early pregnancy, primary EVT cells isolated from the villi of placenta in early pregnancy, and villi explants were cultured respectively. A simulation model of H/R environment in EVT cells invading the uterus was established under the conversion conditions of 1% O2 for 24 h to 20% O2 for another 24 h. Western blotting and immunofluorescence assay were used to detect the expression of key factors such as cell invasion, and Transwell chamber test was employed to observe cell migration and invasion. Results After H/R induction, the migration and invasion abilities of HTR-8/SVneo cells were weakened, and the epitaxial ability of cultured villi explants and the migration and invasion abilities of isolated primary EVT cells were also weakened. Under H/R conditions, the expression levels of MMP-2 and MMP-9, integrin-β1 and integrin-α5 were decreased in HTR-8/SVneo cells (P < 0.05), and the protein expression of DNA methyltransferase (DNMTs) was also down-regulated (P < 0.01). In the primary EVT cells under H/R condition, the expression levels of epithelial-mesenchymal transition (EMT) positive regulatory factors, Slug and Snails, were inhibited (P < 0.05), that of epithelial cell marker E-cadherin was increased (P < 0.01), protein levels of MMP-2 and MMP-9, integrin-β1 and integrin-α5 were decreased (P < 0.05), and cell invasion ability was reduced along with decreased DNMT1 and DNMT3A expression (P < 0.05). However, overexpression of DNMT1, DNMT3A and DNMT3B in HTR-8/SVneo cells promoted the up-regulation of interstitial markers N-cadherin, and MMP-2 and MMP-9 (P < 0.05). Conclusion H/R regulates the expression of EMT-related factors such as MMP-2/-9 through DNMTs, and thereby reduces the migration and invasion abilities of EVT cells.

DNA methyltransferase 1 (DNMT1) is the enzyme primarily responsible for propagation of the methylation pattern in cells. Mutations in DNMT1 have been linked to the development of adult-onset neurodegenerative disorders; these disease-associated mutations occur in the regulatory replication foci-targeting sequence (RFTS) domain of the protein. The RFTS domain is an endogenous inhibitor of DNMT1 activity that binds to the active site and prevents DNA binding. Here, we examine the impact of the disease-associated mutation A554V on normal RFTS-mediated inhibition of DNMT1. Wild-type and mutant proteins were expressed and purified to homogeneity for biochemical characterization. The mutation increased DNA binding affinity ~8-fold. In addition, the mutant enzyme exhibited increased DNA methylation activity. Circular dichroism (CD) spectroscopy revealed that the mutation does not significantly impact the secondary structure or relative thermal stability of the isolated RFTS domain. However, the mutation resulted in changes in the CD spectrum in the context of the larger protein; a decrease in relative thermal stability was also observed. Collectively, this evidence suggests that A554V disrupts normal RFTS-mediated autoinhibition of DNMT1, resulting in a hyperactive mutant enzyme. While the disease-associated mutation does not significantly impact the isolated RFTS domain, the mutation results in a weakening of the interdomain stabilizing interactions generating a more open, active conformation of DNMT1. Hyperactive mutant DNMT1 could be responsible for the increased DNA methylation observed in affected individuals.

Though DNMTs inhibitors were widely used in myelodysplastic syndrome and leukaemia, their application in solid tumours has been limited by low response rate and lack of optimal combination strategies. In gastric cancer (GC), the therapeutic implication of KRAS mutation or MEK/ERK activation for combinational use of DNMTs inhibitors with MEK/ERK inhibitors remains elusive. In this study, stable knockdown of DNMT1 expression by lentiviral transfection led to decreased sensitivity of GC cells to 5-Azacytidine. KRAS knockdown in KRAS mutant GC cells or the MEK/ERK activation by EGF stimulation in GC cells increased DNMT1 expression, while inhibition of MEK/ERK activity by Selumetinib led to decreased DNMT1 expression. 5-Azacytidine treatment, which led to dramatic decline of DNMTs protein levels and increased activity of MEK/ERK pathway, altered the activity of MEK/ERK inhibitor Selumetinib on GC cells. Both RAS-dependent gene expression signature and expression levels of multiple MEK/ERK-dependent genes were correlated with DNMT1 expression in TCGA stomach cancer samples. In conclusion, DNMT1 expression partially dictates 5-Azacytidine sensitivity and correlates with RAS/MEK/ERK activity in GC cells. Combining DNMTs inhibitor with MEK/ERK inhibitor might be a promising strategy for patients with GC.

Despite numerous studies on transcriptional level regulation by single genes in drug producing Actinomyces, the global regulation based on epigenetic modification is not well explored. N4-methylcytosine (m4C), an abundant epigenetic marker in Actinomycetes’ genome, but its regulatory mechanism remains unclear. In this study, we identify a m4C methyltransferase (SroLm3) in Streptomyces roseosporus L30 and multi-omics studies were performed and revealed SroLm3 as a global regulator of secondary metabolism. Notably, three BGCs in ΔsroLm3 strain exhibited decreased expression compared to wild type. In-frame deletion of sroLm3 in S.roseosporus L30 further revealed its role in enhancing daptomycin production. In summary, we characterized a m4C methyltransferase, revealed the function of m4C in secondary metabolism regulation and biosynthesis of red pigment, and mapped a series of novel regulators for daptomycin biosynthesis dominated by m4C methylation. Our research further indicated that m4C DNA methylation may contribute to a metabolic switch from primary to secondary metabolism in Actinomyces.

Abstract Programmed cell death protein 1(PD-1) is a type of immune-inhibitory checkpoint protein, which delivers inhibitory signals to cytotoxic T cells by binding to the programmed death ligand-1 (PD-L1) displayed on the surface of cancer cells. Antibodies blocking PD-1/PD-L1 interaction have been extensively used in treatment of human malignancies and have achieved promising outcomes in recent years. However, gradual development of resistance to PD-1/PD-L1 blockade has decreased the effectiveness of this immunotherapy in cancer patients. The underlying epigenetic mechanisms need to be elucidated for application of novel strategies overcoming this immunotherapy resistance. Epigenetic aberrations contribute to cancerogenesis by promoting different hallmarks of cancer. Moreover, these alterations may lead to therapy resistance, thereby leading to poor prognosis. Recently, the epigenetic regulatory drugs have been shown to decrease the resistance to PD-1/PD-L1 inhibitors in certain cancer patients. Inhibitors of the non-coding RNAs, DNA methyltransferases, and histone deacetylases combined with PD-1/PD-L1 inhibitors have shown considerable therapeutic efficacy against carcinomas as well as blood cancers. Importantly, DNA methylation-mediated epigenetic silencing can inhibit antigen processing and presentation, which promotes cancerogenesis and aggravates resistance to PD-1/PD-L1 blockade immunotherapy. These observations altogether suggest that the combination of the epigenetic regulatory drugs with PD-1/PD-L1 inhibitors may present potential solution to the resistance caused by monotherapy of PD-1/PD-L1 immunotherapy.

Abstract Background DNA methyltransferases (MTases) are enzymes that induce methylation, one of the representative epigenetic modifications of DNA, and are also useful tools for analyzing epigenomes. However, regarding DNA cytosine 5-methylation, MTases identified so far have drawbacks in that their recognition sequences overlap with those for intrinsic DNA methylation in mammalian cells and/or that the recognition sequence is too long for fine epigenetic mapping. To identify MTases with short recognition sequences that never overlap with the CG dinucleotide, we systematically investigated the 25 candidate enzymes identified using a database search, which showed high similarity to known cytosine 5-MTases recognizing short sequences. Results We identified MTases with six new recognition sequences, including TCTG, CC, CNG, TCG, GCY, and GGCA. Because the recognition sequence never overlapped with the CG dinucleotide, MTases recognizing the CC dinucleotide were promising. Conclusions In the current study, we established a procedure for producing active CC-methylating MTases and applied it to nucleosome occupancy and methylome sequencing to prove the usefulness of the enzyme for fine epigenetic mapping. MTases that never overlap with CG dinucleotides would allow us to profile multiple epigenomes simultaneously.

Abstract DNA methylation plays a crucial role in the survival of bacteriophages (phages), yet the understanding of their genome methylation remains limited. In this study, DNA methylation patterns are analyzed in 8848 metagenome‐assembled high‐quality phages from 104 fecal samples using single‐molecule real‐time sequencing. The results demonstrate that 97.60% of gut phages exhibit methylation, with certain factors correlating with methylation densities. Phages with higher methylation densities appear to have potential viability advantages. Strikingly, more than one‐third of the phages possess their own DNA methyltransferases (MTases). Increased MTase copies are associated with higher genome methylation densities, specific methylation motifs, and elevated prevalence of certain phage groups. Notably, the majority of these MTases share close homology with those encoded by gut bacteria, suggesting their exchange during phage–bacterium interactions. Furthermore, these MTases can be employed to accurately predict phage–host relationships. Overall, the findings indicate the widespread utilization of DNA methylation by gut DNA phages as an evasion mechanism against host defense systems, with a substantial contribution from phage‐encoded MTases.

Colorectal cancer is a complex disease resulting from the interaction of genetics, epigenetics, and environmental factors. DNA methylation is frequently found in tumor suppressor genes to promote cancer development. Several factors are associated with changes in the DNA methylation pattern, and recently, the gastrointestinal microbiota could be associated with this epigenetic change. The predominant phyla in gut microbiota are Firmicutes and Bacteroidetes; however, an enrichment of Bacteroides fragilis, Fusobacterium nucleatum, and Streptococcus bovis, among others, has been reported in colorectal cancer, although the composition could be influenced by several factors, including diet, age, sex, and cancer stage. Fusobacterium nucleatum, a gram-negative anaerobic bacillus, is mainly associated with colorectal cancer patients positive for the CpG island methylator phenotype, although hypermethylation in genes such as MLH1, CDKN2A, MTSS1, RBM38, PKD1, PTPRT, and EYA4 has also been described. Moreover, Hungatella hathewayi, a gram-positive, rod-shaped bacterium, is related to hypermethylation in SOX11, THBD, SFRP2, GATA5, ESR1, EYA4, CDX2, and APC genes. The underlying epigenetic mechanism is unclear, although it could be implicated in the regulation of DNA methyltransferases, enzymes that catalyze the transfer of a methyl group on cytosine of CpG sites. Since DNA methylation is a reversible event, changes in gut microbiota could modulate the gene expression through DNA methylation and improve the colorectal cancer prognosis.

DNA methylation results in a variety of human diseases and the DNA methylation process is mediated by DNA methyltransferases, which have therefore become potential targets for disease treatment. In this study, a turn-off nanogold biological probe system was successfully created for determining the activity of DNA methyltransferases (M.SssI MTase). A dumbbell-shaped DNA probe with a site-recognizable region of M. SssI MTase and a fluorescent signal probe based on a DNA-templated gold nanocluster (DNA-AuNC) probe combined for the quantitative detection of M. SssI MTase. This dumbbell-shaped DNA probe was methylated by M. SssI MTase, and the dumbbell-shaped DNA probe with a methyl group was recognized by an endonuclease (GlaI) and cleaved into hairpin DNA. The dGTP was added to the 3′-OH terminus of hairpin DNA fragments in the presence of terminal deoxynucleotidyl transferase (TdT), and the hairpin DNA was extended with a G-rich sequence that can be used as an inactivation probe. When the inactivation probe was combined with the signal probe, the fluorescent signal disappeared due to the photoinduced electron transfer effect. Methyltransferase activity was then detected based on the turn-off principle of the fluorescence signal from the DNA-AuNCs. The bioprobe enabled sensitive detection of M. SssI MTase with a detection limit of 0.178 U mL−1 and good specificity. The bioprobe demonstrated good detection efficiency in both human serum and cell lysates, and its unique fluorescence turn-off mechanism provided good resistance to interference, thus increasing its potential application in complex biological samples. Moreover, it is suitable for screening and assessing the inhibitory activity of M. SssI MTase inhibitors, and therefore has significant potential for disease diagnosis and drug discovery.

Aims: New methods of DNA recombination that capture the principal advantages of the BioBrick standard (ease of design) and Golden Gate assembly (decreased labor) are demonstrated here. Methods & materials: Both methods employ DNA methyltransferase expression vectors, available from Addgene, that protect selected sites on different plasmids from particular Type II restriction endonucleases. No other reagents are required. Results: The 4R/2M discontinuous DNA assembly is more efficient (produces more desired recombinant plasmids) and as specific (produces few undesired recombination products) as conventional subcloning. The 5RM continuous DNA assembly is approximately as efficient and specific as conventional Golden Gate assembly, even though in vivo methylation of one plasmid is incomplete. Conclusion: Both methylase-assisted methods streamline BioBrick assembly workflows without complicating the design of synthetic parts.

Epigenetics modulates expression levels of various important genes in both prokaryotes and eukaryotes. These epigenetic traits are heritable without any change in genetic DNA sequences. DNA methylation is a universal mechanism of epigenetic regulation in all kingdoms of life. In bacteria, DNA methylation is the main form of epigenetic regulation and plays important roles in affecting clinically relevant phenotypes, such as virulence, host colonization, sporulation, biofilm formation et al. In this review, we survey bacterial epigenomic studies and focus on the recent developments in the structure, function, and mechanism of several highly conserved bacterial DNA methylases. These methyltransferases are relatively common in bacteria and participate in the regulation of gene expression and chromosomal DNA replication and repair control. Recent advances in sequencing techniques capable of detecting methylation signals have enabled the characterization of genome-wide epigenetic regulation. With their involvement in critical cellular processes, these highly conserved DNA methyltransferases may emerge as promising targets for developing novel epigenetic inhibitors for biomedical applications.

BackgroundSmad7 is protective in a mouse model of rheumatoid arthritis. Here we investigated whether Smad7-expressing CD4+ T cells and the methylation of Smad7 gene in CD4+ T cells contribute to the disease activity of RA in patients.MethodsPeripheral CD4+ T cells were collected from 35 healthy controls and 57 RA patients. Smad7 expression by CD4+ T cells were determined and correlated with the clinical parameters of RA including RA score and serum levels of IL-6, CRP, ESR, DAS28-CRP, DAS28-ESR, Swollen joints and Tender joints. Bisulfite sequencing (BSP-seq) was used to determine the DNA methylation in Smad7 promoter (-1000 to +2000) region in CD4+ T cells. In addition, a DNA methylation inhibitor, 5-Azacytidine (5-AzaC), was added to CD4+ T cells to examine the possible role of Smad7 methylation in CD4+ T cell differentiation and functional activity.ResultsCompared to the heath controls, Smad7 expression was significantly decreased in CD4+ T cells from RA patients and inversely correlated with the RA activity score and serum levels of IL-6 and CRP. Importantly, loss of Smad7 in CD4+ T cell was associated with the alteration of Th17/Treg balance by increasing Th17 over the Treg population. BSP-seq detected that DNA hypermethylation occurred in the Smad7 promoter region of CD4+ T cells obtained from RA patients. Mechanistically, we found that the DNA hypermethylation in the Smad7 promoter of CD4+ T cells was associated with decreased Smad7 expression in RA patients. This was associated with overreactive DNA methyltransferase (DMNT1) and downregulation of the methyl-CpG binding domain proteins (MBD4). Inhibition of DNA methylation by treating CD4+ T cells from RA patients with 5-AzaC significantly increased Smad7 mRNA expression along with the increased MBD4 but reduced DNMT1 expression, which was associated with the rebalance in the Th17/Treg response.ConclusionDNA hypermethylation at the Smad7 promoter regions may cause a loss of Smad7 in CD4+ T cells of RA patients, which may contribute to the RA activity by disrupting the Th17/Treg balance.

Glioma‐initiating cells (GICs), a major source of glioblastoma recurrence, are characterized by the expression of neural stem cell markers and the ability to grow by forming nonadherent spheres under serum‐free conditions. Bone morphogenetic proteins (BMPs), members of the transforming growth factor‐β family, induce differentiation of GICs and suppress their tumorigenicity. However, the mechanisms underlying the BMP‐induced loss of GIC stemness have not been fully elucidated. Here, we show that paired related homeobox 1 (PRRX1) induced by BMPs decreases the CD133‐positive GIC population and inhibits tumorigenic activity of GICs in vivo. Of the two splice isoforms of PRRX1, the longer isoform, pmx‐1b, but not the shorter isoform, pmx‐1a, induces GIC differentiation. Upon BMP stimulation, pmx‐1b interacts with the DNA methyltransferase DNMT3A and induces promoter methylation of the PROM1 gene encoding CD133. Silencing DNMT3A maintains PROM1 expression and increases the CD133‐positive GIC population. Thus, pmx‐1b promotes loss of stem cell‐like properties of GICs through region‐specific epigenetic regulation of CD133 expression by recruiting DNMT3A, which is associated with decreased tumorigenicity of GICs.

DNA methylation mediated by DNA methyltransferase is an important epigenetic process that regulates gene expression in mammals, which plays a key role in silencing certain genes, such as tumor suppressor genes, in cancer, and it has become a promising therapeutic target for cancer treatment. Similar to other epigenetic targets, DNA methyltransferase can also be modulated by chemical agents. Four agents have already been approved to treat hematological cancers. In order to promote the development of a DNA methyltransferase inhibitor as an anti-tumor agent, in the current review, we discuss the relationship between DNA methylation and tumor, the anti-tumor mechanism, the research progress and pharmacological properties of DNA methyltransferase inhibitors, and the future research trend of DNA methyltransferase inhibitors.

Abstract Background DNA methylation plays a crucial role in multiple cellular processes such as gene regulation, chromatin stability, and genetic imprinting. In mammals, DNA methylation is achieved by DNA methyltransferases (DNMTs). A number of studies have associated alterations in DNMT activity to tumorigenesis; however, the exact role of DNMTs in shaping the genome in triple negative breast cancer (TNBC) is still being unraveled. Methods In the current study, we employed two DNMT inhibitors (Decitabine and 5-Azacytidine), two TNBC models (MDA-MB-231 and BT-549) and whole transcriptome RNA-Seq and characterized the transcriptional alterations associated with DNMT inhibition. Colony forming unit (CFU), flow cytometry, and fluorescent microscopy were used to assess cell proliferation, cell cycle distribution, and cell death, respectively. Ingenuity pathway analysis (IPA) was used for network and pathway analyses. Results Remarkably, DNMT inhibition induced the expression of genes involved in endoplasmic reticulum response to stress, response to unfolder protein, as well as cobalamin metabolic processes. In contrast, suppression of cellular processes related to cell cycle and mitosis were hallmarks of DNMT inhibition. Concordantly, DNMT inhibition led to significant inhibition of TNBC cell proliferation, G2-M cell cycle arrest and induction of cell death. Mechanistically, DNMT inhibition activated TP53, NUPR1, and NFkB (complex) networks, while RARA, RABL6, ESR1, FOXM1, and ERBB2 networks were suppressed. Our data also identified the long noncoding RNA (lncRNA) transcriptional portrait associated with DNMT inhibition and identified 25 commonly upregulated and 60 commonly downregulated lncRNAs in response to Decitabine and 5-Azacytidinec treatment in both TNBC models. TPT1-AS1 was the most highly induced (6.3 FC), while MALAT1 was the most highly suppressed (− 7.0 FC) lncRNA in response to DNMT inhibition. Conclusions Taken together, our data provides a comprehensive view of transcriptome alterations in the coding and noncoding transcriptome in TNBC in response to DNMT inhibition.

Background: DNA methyltransferase (DNMT) enzymes, encoded by DNMT1, DNMT3A andDNMT3B genes, play a major role in the development of cancers through aberrant promotermethylation. Due to little information about the biological and clinical significance of expressionchanges of these genes in Laryngeal Squamous Cell carcinoma (LSCC), the current study wasdesigned to evaluate the contribution of DNMTs expression as potential diagnostic biomarkersin progression of LSCC. Methods: DNMT1, DNMT3A and DNMT3B expressions in tumoral and normal tissues fromthirty-three LSCC patients were evaluated by relative comparative real-time PCR, prior toany therapeutic intervention. Relationship between genes expression and clinicopathologicalfeatures were also analyzed. Results: The mRNA expression levels of all three DNMTs (DNMT1, DNMT3A and DNMT3B)were significantly elevated in LSCC tumor specimens compared to that of non-tumor tissues(P

Background : It is well established that obesity is a major health risk in diabetes and associated diseases. Epigenetic changes, specially DNA methylation, play an important role in regulation of adipokines. The objective of the present study was to evaluate the DNA methylation status at the promoter region of the leptin gene in obese individuals and its association with metabolic risk factors. Methods : The study included obese (n=100) and non-obese (n=75) individuals aged 25-45 years, and measured their physical, biochemical parameters (glucose, insulin, and lipid profiles) and leptin, DNA methyltransferase 1 (DNMT1), and DNA methyltransferase 3 beta (DNMT3b) mRNA expressions with real-time reverse transcription-polymerase chain reaction (qRT-PCR). DNA methylation of the leptin gene at the promoter region was analyzed by methyl-specific qPCR . Results : The study found that the DNA methylation level at the promoter area of the leptin gene was negatively associated with weight in obese subjects. Furthermore, study findings showed that the DNA methylation level was negatively associated with fasting insulin, glucose, homeostatic model assessment for insulin resistance, and total cholesterol. There was also a higher expression of DNMT1 and DNMT-3b in obese subjects as compared with non-obese subjects. Conclusion : The leptin epigenetic profile may be associated with obesity and its associated metabolic risk factors.

Abstract In patients with prostate cancer (PCa), there is a high rate of overdiagnosis and frequent overtreatment. Therefore, there is an urgent need for more accurate prediction of biochemical recurrence (BCR). DNA methylation regulation patterns play crucial roles in tumorigenicity, progression, and treatment efficacy in PCa. However, the global relationship between epigenetic alterations, changes in mRNA levels, and pathologic phenotypes of PCa remain largely undefined. Here, we conducted a systematic analysis to identify global coexpression and comethylation modules in PCa. We identified coregulated methylation and expression modules and the relationships between epigenetic modifications, tumor progression, and the corresponding immune microenvironment in PCa. Our results show that DNA methyltransferases (DNMTs) are strongly associated with pathologic phenotypes and immune infiltration patterns in PCa. We built a two‐factor predictive model using the expression features of DNMT3B and DNMT1. The model was used to predict the BCR status of patients with PCa and achieved area under the receiver operating characteristic curve values of 0.70 and 0.88 in the training and independent testing datasets, respectively.

The intestine is inhabited by a large number of commensal bacteria that are immunologically non-self, potentially causing inflammation. However, in a healthy intestine, inflammation is strictly controlled at low levels to maintain homeostasis. We previously reported that the gut microbiota induce DNA methylation of the gene encoding Toll-like receptor (TLR) 4, a pattern recognition receptor that recognizes lipopolysaccharides of gram-negative bacteria, in colonic epithelial cells, suggesting its role in controlling intestinal inflammation. However, there remains a question of how gut microbiota cause methylation of only specific genes including TLR4, despite the fact that DNA methyltransferase (DNMT) is common to all genes targeted for methylation. Here, we identified RBM14 as an adaptor molecule that recruits DNMT to the TLR4 gene. RBM14 was shown to bind DNMT3 and be expressed at significantly higher levels in an intestinal epithelial cell (IEC) line with hypermethylated TLR4 gene than in an IEC line with hypomethylated TLR4 gene. In addition, RBM14 interacted with DNA regions of the TLR4 gene, and knockdown of RBM14 suppressed DNA methylation of the TLR4 gene in IECs. Furthermore, RBM14 expression was higher in colonic epithelial cells of conventional mice than in those of germ-free mice. Collectively, these results indicate that the gut microbiota induce methylation of the TLR4 gene in colonic epithelial cells by upregulating RBM14, which can recruit DNMT3 to the gene. The regulation of adaptor molecules such as RBM14, which bind to specific target genes and recruit DNMT, can explain, at least in part, how gut microbiota contribute to the maintenance of intestinal homeostasis through epigenetic control of specific gene expression in IECs.

ABSTRACT Opportunistic 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.

Background: Low birth weight (LBW) is a risk factor associated with endometriosis. Our study aimed to analyze the risk of endometriosis in women with a LBW history and the relationships of progesterone receptor B (PR-B) gene promoter methylation, DNA methyltransferase-1 (DNMT1) expression, PR-B expression, and vascular endothelial growth factors (VEGF) with endometriosis. Methods: This study was conducted in two stages, a retrospective case-control design and a cross-sectional design, with 52 cases of endometriosis and 30 controls, which were further subdivided into LBW and non-LBW groups, at Hasan Sadikin General Hospital and its hospital networks from October 2017 to August 2021. Menstrual blood was taken from subjects and analyzed using pyrosequencing techniques to assess DNA methylation, while q-RT PCR was used to assess gene expression. Results: There were significant differences in PR-B methylation, DNMT1 expression, PR-B expression, and VEGF expression (p < 0.001) between the case and control groups. There was a significant negative correlation between PR-B methylation and PR-B expression (r = −0.558; p = 0.047). Based on a multiple logistic analysis, the most dominant factor affecting endometriosis incidence is PR-B (OR 10.40, 95% CI 3.24–33.4, R2 = 45.8). We found that patients with a low birth weight history had a 1.41-times-higher risk of developing endometriosis (95% CI 0.57–3.49, p = 0.113), although the relationship was not statistically significant. Conclusion: Endometriosis is associated with PR-B gene promoter hypermethylation, decreased PR-B expression, and increased DNMT1 and VEGF expression. The methylation of PR-B is the most dominant factor affecting endometriosis incidence.

Abstract Background Environmental factors, such as oxidative stress, have the potential to modify the epigenetic landscape of cells. We have previously shown that DNA methyltransferase (DNMT) activity can be inhibited by sublethal doses of hydrogen peroxide (H2O2). However, site-specific changes in DNA methylation and the reversibility of any changes have not been explored. Using bead chip array technology, differential methylation was assessed in Jurkat T-lymphoma cells following exposure to H2O2. Results Sublethal H2O2 exposure was associated with an initial genome-wide decrease in DNA methylation in replicating cells, which was largely corrected 72 h later. However, some alterations were conserved through subsequent cycles of cell division. Significant changes to the variability of DNA methylation were also observed both globally and at the site-specific level. Conclusions This research indicates that increased exposure to H2O2 can result in long-term alterations to DNA methylation patterns, providing a mechanism for environmental factors to have prolonged impact on gene expression.

Methylation of cytosine DNA residues is the most studied and stable of all epigenetic modifications. Methylation of eukaryotic DNA is carried out by DNA methyltransferases. DNA methylation is an active mechanism for controlling gene transcription and is usually associated with prolonged silencing of DNA. The effect of peptides AEDG and AEDL on the growth of calluses of tobacco (Nicotiana tabacum) at low concentrations (10−7 M) is similar to the effect of phytohormones, has a regulatory character, and is possibly epigenetic in nature. Peptides increase the expression of DNA methyltransferase genes. One of the possible mechanisms of regulation of DNA methyltransferase genes by AEDG and AEDL is their ability to bind to free DNA regions at certain CNG sites, which are also methylation sites of plant cytosine methyltransferases. The AEDG peptide preferably binds to the CAG site and the AEDL peptide to the CTG site. By binding to the same sites as DNA methyltransferase, peptides block methylation sites, thereby reducing the level of DNA methylation. The specific binding of peptides to different sites that we discovered can be of great importance in gene regulation, since peptides with different structures can block different DNA regions for methylation of certain genes, thereby activating or silencing their expression.

Abstract 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.

Prostate cancer (PCa) is a heterogeneous tumor that commonly occurs among males worldwide. This study explored the potential role that long non-coding RNA MCM3AP antisense RNA 1 (MCM3AP-AS1) plays in PCa progression, and investigated its mechanism. MCM3AP-AS1 and neuropeptide Y receptor Y1 (NPY1R) expression was determined in PCa cells. The regulatory role of MCM3AP-AS1 in PCa cells was defined using scratch test, Transwell assay, 5-ethynyl-2′-deoxyuridine (EdU) assay, and flow cytometry. Methylation-specific PCR (MSP) was used to test the methylation level of NPY1R. Subsequently, the interaction among MCM3AP-AS1, DNA methyltransferase (DNMT)1/DNMT3 (A/B), and NPY1R was investigated using RNA immunoprecipitation, RNA pull-down, and chromatin immunoprecipitation. Finally, we observed xenograft tumor in nude mice. MCM3AP-AS1 was highly, whereas NPY1R was poorly, expressed in PCa. Lentivirus-mediated overexpression of MCM3AP-AS1 promoted proliferation, invasion, and migration while suppressing apoptosis of PCa cells, whereas opposite trends were detected after inhibition of the mitogen-activated protein kinase (MAPK) pathway. MCM3AP-AS1 promoted methylation of NPY1R promoter via recruitment of DNMT1/DNMT3 (A/B), thereby downregulating NPY1R expression to activate the MAPK pathway. Furthermore, overexpressed MCM3AP-AS1 was observed to facilitate PCa development in vivo, which could be reversed by overexpressed NPY1R. Altogether, MCM3AP-AS1 silencing inhibits PCa progression by disrupting methylation of the NPY1R promoter to inactivate the MAPK pathway.

Abstract Background Atherosclerosis (AS), which characterized with the accumulation of lipids on the vessel wall, is the pathological basis of many cardiovascular diseases (CVD) and seriously threatens human health. Resveratrol (RES) has been reported to be benefit for AS treatment. This research aimed to observe the effects of RES on AS induced by high-fat diet (HFD) and LPS in ApoE−/− mice and investigate the underlying mechanism. Methods ApoE−/− mice were fed with HFD companied with LPS to induce AS and RES was administrated for 20 weeks. Splenic CD4+ T cells were cultured and treated with anti-CD3/CD28 together with LPS, and RES was added. Serum lipids and the atherosclerotic areas of aortas were detected. The activation of CD4+ T cells were investigated both in vivo and in vitro and the expression of DNA methyltransferases (Dnmt) in CD4+ T cells were measured. Results In vivo, administration of RES prevented HFD and LPS induced dysfunction of serum lipids including TC (total cholesterol), TG (triglyceride), LDL-C (low density lipoprotein cholesterol) and HDL-C (high density lipoprotein cholesterol), ameliorated the thickened coronary artery wall and decreased the areas of atherosclerotic lesion on aortas. Besides, RES decreased the number of CD4+ T cells in peripheral blood, decreased the expression of CD25 and CD44, but not affected the expression of L-selectin (CD62L). In vitro, RES decreased the expression of Ki67, CD25 and CD44 in CD4+ T cells. Moreover, RES increased the secretion of IL-2, IL-10 and TGF-β1, decreased IL-6. In addition, RES decreased both the mRNA and protein level of Dnmt1 and Dnmt3b in CD4+ T cells. Conclusion These results indicated that RES ameliorated AS induced by HFD companied with LPS in ApoE−/− mice, inhibited the proliferation and activation of CD4+ T cells and regulated the expression of Dnmt1 and Dnmt3b.

The tumor suppressor gene, p53, is inactivated in the human hepatocellular carcinoma cells line, Hep3B. Berberine has been reported to inhibit the proliferation of cancer cells. This study examined whether apoptosis was induced in berberine-treated Hep3B cells and observed the association between apoptosis and the expression of p53 and DNA methyltransferase (DNMT). The cell viability was measured using an MTT assay. Apoptosis of Hep3B was measured using annexin V flow cytometry. Berberine-treated cells were examined for their DNMT enzymatic activity, mRNA expression, and protein synthesis. The p53 levels were examined by Western blot analysis. The berberine treatment resulted in increased Hep3B cell death and apoptosis in a time- and dose-dependent manner. The DNMT3b activity, mRNA expression, and protein levels all decreased after the berberine treatment. In contrast, the p53 protein levels increased with a concomitant decrease in DNMT3b. No change in the expression of ERK was observed, but the P-ERK levels decreased in a dose dependent manner. These results indicate that a treatment of Hep3B cells with berberine can reduce the expression of DNMT3b, leading to an increase in the tumor suppressant gene p53 and an increase in cell apoptosis. This shows that berberine can effectively suppress the proliferation of liver cancer cells.

Abstract Background Degeneration of ovarian function is an obvious feature of female aging. In addition, studies have shown that autophagy decreases with age, and DNA methylation is a hallmark epigenetic pattern during aging. However, it is not clear whether the expression and DNA methylation of autophagy genes are involved in the declines in ovarian function that occur during aging. Results Three groups of rats were used: 6-month-old (6 M) rats, 12-month-old (12 M) rats and 24-month-old (24 M) rats. Serum E2 levels and the mRNA and protein expression levels of Atg5, Atg12, Atg16L, Beclin1 and Lc3B were significantly decreased in aged rats. In addition, the methylation levels of the Atg5 gene were significantly increased in aged rats. The expression of the Dnmt1 and Dnmt2 genes decreased with aging; however, the expression of the Dnmt3A and Dnmt3B genes gradually increased with aging. Conclusions Decreased autophagic activity was involved in the declines in ovarian function in aging rats. Upregulation of the DNA methyltransferases Dnmt3A and Dnmt3B may have led to methylation of the autophagy genes Atg5 and Lc3B to ultimately cause the observed decreases in autophagic activity.

DNA methyltransferase I plays the central role in maintenance of CpG DNA methylation patterns across the genome and alteration of CpG methylation patterns is a frequent and significant occurrence across many cancers. Cancer cells carrying hypomorphic alleles of Dnmt1 have become important tools for understanding Dnmt1 function and CpG methylation. In this study, we analyse colorectal cancer cells with a homozygous deletion of exons 3 to 5 of Dnmt1, resulting in reduced Dnmt1 activity. Although this cell model has been widely used to study the epigenome, the effects of the Dnmt1 hypomorph on cell signalling pathways and the wider proteome are largely unknown. In this study, we perform the first quantitative proteomic analysis of this important cell model and identify multiple signalling pathways and processes that are significantly dysregulated in the hypomorph cells. In Dnmt1 hypomorph cells, we observed a clear and unexpected signature of increased Epithelial-to-Mesenchymal transition (EMT) markers as well as reduced expression and sub-cellular re-localization of Beta-Catenin. Expression of wild-type Dnmt1 in hypomorph cells or knock-down of wild-type Dnmt1 did not recapitulate or rescue the observed protein profiles in Dnmt1 hypomorph cells suggesting that hypomorphic Dnmt1 causes changes not solely attributable to Dnmt1 protein levels. In summary, we present the first comprehensive proteomic analysis of the widely studied Dnmt1 hypomorph colorectal cancer cells and identify redistribution of Dnmt1 and its interaction partner Beta-Catenin.

BackgroundFibrillin-1 (FBN1) methylation risk from control to colorectal cancer (CRC), the variation regularities of FBN1 methylation, and DNA methyltransferase (DNMT) catalyzed with FBN1 methylation had not been reported yet; these were all studied in this paper.MethodsFBN1 methylation roles were investigated with big data and meta-analysis.ResultsThe 6 independent studies were searched including 702 tissue and 448 feces. FBN1 methylation frequencies of CRC, adenoma or polyp, and control in tissue were 79.1%, 69.4%, and 2.7%, respectively; those in feces were 74.6%, 50.7%, and 10.8%, respectively. FBN1 methylation of control samples was used as a standard reference; this study showed that ORs (95% CI) of FBN1 methylation in CRC and control tissues were 124.79 (62.86–248.35); those in feces were detected to be 30.87 (16.48–57.85). FBN1 methylation risk in tissue was higher than that in feces; there was a quadratic equation between the methylation rate of tissue and that of feces. There was another quadratic curve in the variation process of FBN1 methylation; this curve reflected the overall metabolism regularity of DNMT.ConclusionsThe transcriptional inactivation of FBN1 gene might start from normal colonic epithelium; the quadratic curve of FBN1 methylation catalyzed by DNMT can gradually produce powerful strength, accelerate expansion, and eventually lead to CRC. The overall metabolism regularity of DNMT maintains the changing process of FBN1 methylation; it has the changing feature of the same quadratic curve. FBN1 methylation is a promising biomarker. FBN1 methylation risk size in feces reflects that in tissue in non-invasive detection.

The rearrangement of molecular pathways and the activation of bypass signaling determine the progression of tumor cell resistance to various drugs that specifically block target signaling proteins. The present work was performed on the MCF-7 breast cancer cells and established sublines, resistant to mTOR inhibitor rapamycin or antiestrogen tamoxifen, developed under prolonged cell treatment with rapamycin or tamoxifen, respectively. We have shown that both resistant sublines demonstrate the cross-resistance to rapamycin and tamoxifen and are characterized with the common signaling changes, namely—blocking of the estrogen receptor α (ERα) transcriptional activity and constitutive activation of Akt signaling. Analysis of the epigenetic machinery revealed the drastic suppression of the level of DNA methyltransferase 3A (DNMT3A) in both the resistant sublines that were correlated with the demethylation of the LINE-1 repeats. Knockdown of the DNMT3A via siRNA results in the progression of partial resistance of MCF-7 cells to both tamoxifen and rapamycin, supporting the important role of DNA methylation in the formation of the resistant phenotype. Totally, the results obtained highlight the possible mechanism of the tumor cell resistance to targeting/hormonal drugs based on the rearrangement of DNA methylation profile and activation of the bypass signaling pathways.

Growing evidence suggests that epigenetic mechanisms, such as DNA methylation and demethylation, and histone modifications, are involved in the development of alcohol and drug addiction. However, studies of alcohol use disorder (AUD) that are focused on epigenetic DNA modifications and gene expression changes remain conflicting. Our aim was to study the effect of repeated ethanol consumption on epigenetic regulatory enzymes such as DNA methyltransferase and demethylase enzymes and whether those changes affected dynorphin/kappa-opioid receptor system in the Nucleus Accumbens (NAc). Two groups of male alcohol-preferring Alko Alcohol (AA) rats, rats which are selectively bred for high voluntary alcohol consumption and one group of male Wistar rats were used. The first group of AA rats had access to alcohol (10% ethanol solution) for 90 min on Mondays, Wednesdays and Fridays over a period of 3 weeks to establish a stable baseline of ethanol intake (AA-ethanol). The second group of AA rats (AA-water) and the Wistar rats (Wistar-water) were provided with water. Using qPCR, we found that voluntary alcohol drinking increased Dnmt1, −3a, and −3b mRNA levels and did not affect Tet family transcripts in the AA-ethanol group when compared with AA- and Wistar-water rats. DNMT and TET enzymatic activity measurements showed similar results to qPCR, where DNMT activity was increased in AA-ethanol group compared with AA-water and Wistar-water groups, with no statistically significant difference between groups in TET enzyme activity. In line with previous data, we found an increased percentage of global DNA methylation and hydroxymethylation in the AA-ethanol group compared with control rats. Finally, we investigated changes of selected candidate genes from dynorphin/kappa-opioid receptor system (Pdyn, Kor) and Dnmt3a genes that might be important in AUD-related behaviour. Our gene expression and promoter methylation analysis revealed a significant increase in the mRNA levels of Pdyn, Kor, and Dnmt3a in the AA-ethanol group, however, these changes can only be partially associate with the aberrant DNA methylation in promoter areas of the selected candidate genes. Thus, our findings suggest that the aberrant DNA methylation is rather one of the several mechanisms involved in gene expression regulation in AA rat model.

Genomic analysis has demonstrated that many fungi possess essential gene clusters for the production of previously unobserved secondary metabolites; however, these genes are normally reduced or silenced under most conditions. These cryptic biosynthetic gene clusters have become treasures of new bioactive secondary metabolites. The induction of these biosynthetic gene clusters under stress or special conditions can improve the titers of known compounds or the production of novel compounds. Among the inducing strategies, chemical-epigenetic regulation is considered a powerful approach, and it uses small-molecule epigenetic modifiers, which mainly act as the inhibitors of DNA methyltransferase, histone deacetylase, and histone acetyltransferase, to promote changes in the structure of DNA, histones, and proteasomes and to further activate cryptic biosynthetic gene clusters for the production of a wide variety of bioactive secondary metabolites. These epigenetic modifiers mainly include 5-azacytidine, suberoylanilide hydroxamic acid, suberoyl bishydroxamic acid, sodium butyrate, and nicotinamide. This review gives an overview on the method of chemical epigenetic modifiers to trigger silent or low-expressed biosynthetic pathways to yield bioactive natural products through external cues of fungi, mainly based on the research progress in the period from 2007 to 2022. The production of about 540 fungal secondary metabolites was found to be induced or enhanced by chemical epigenetic modifiers. Some of them exhibited significant biological activities such as cytotoxic, antimicrobial, anti-inflammatory, and antioxidant activity.

Synucleinopathies are a group of neurodegenerative diseases with common pathological lesions associated with the excessive accumulation and abnormal intracellular deposition of toxic species of α-synuclein. The shared clinical features are chronic progressive decline of motor, cognitive, and behavioral functions. These disorders include Parkinson’s disease, dementia with Lewy body, and multiple system atrophy. Vigorous research in the mechanisms of pathology of these illnesses is currently under way to find disease-modifying treatment and molecular markers for early diagnosis. α-Synuclein is a prone-to-aggregate, small amyloidogenic protein with multiple roles in synaptic vesicle trafficking, neurotransmitter release, and intracellular signaling events. Its expression is controlled by several mechanisms, one of which is epigenetic regulation. When transmitted to the nucleus, α-synuclein binds to DNA and histones and participates in epigenetic regulatory functions controlling specific gene transcription. Here, we discuss the various aspects of α-synuclein involvement in epigenetic regulation in health and diseases.

ABSTRACT 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.

DNA methylation, mediated by DNA methyltransferases (Dnmts), is a typical epigenetic process that plays an important role in affecting organism acclimatization and adaptation to environmental changes. However, information about Dnmts and their associations with the cold tolerance of ticks remains meager. Hence, in the present study, the Dnmts in important vector ticks Dermacentor silvarum and Haemaphysalis longicornis were cloned and identified, and their functions in cold response were further explored. Results showed that the length of DsDnmt and DsDnmt1 in D. silvarum, and HlDnmt1 and HlDnmt in H. longicornis were 1,284, 549, 1,500, and 1,613 bp, respectively. Bioinformatics in protein analysis revealed that they were all unstable hydrophilic proteins and were mainly characterized with Dcm (DNA cytosine methyltransferase domain), Dnmt1-RFD (DNA methyltransferase replication foci domain), zf-CXXC (zinc finger-CXXC domain), and BAH (Bromo adjacent homology domain). The relative expression of these Dnmts was reduced after cold treatment for 3 days (P < 0.05), and increased with the extension of treatment. Western blot revealed that Dnmt1 decreased first and then increased significantly (P < 0.05) in both tick species, whereas other Dnmts fluctuated at varying degrees. RNA interference significantly silenced the genes Dnmts (P < 0.01), and mortality increased significantly (P < 0.05), when exposed to sub-lethal temperature, underscoring the important roles of Dnmts during the cold response of D. silvarum and H. longicornis. The above results lay the foundation for further understanding of the epigenetic regulation of DNA methylation in cold acclimatization and adaptation of ticks.

Abstract Background Endothelial cell (EC) dysfunctions, including turnover enrichment, gap junction disruption, inflammation, and oxidation, play vital roles in the initiation of vascular disorders and atherosclerosis. Hemodynamic forces, i.e., atherprotective pulsatile (PS) and pro-atherogenic oscillatory shear stress (OS), can activate mechanotransduction to modulate EC function and dysfunction. This review summarizes current studies aiming to elucidate the roles of epigenetic factors, i.e., histone deacetylases (HDACs), non-coding RNAs, and DNA methyltransferases (DNMTs), in mechanotransduction to modulate hemodynamics-regulated EC function and dysfunction. Main body of the abstract OS enhances the expression and nuclear accumulation of class I and class II HDACs to induce EC dysfunction, i.e., proliferation, oxidation, and inflammation, whereas PS induces phosphorylation-dependent nuclear export of class II HDACs to inhibit EC dysfunction. PS induces overexpression of the class III HDAC Sirt1 to enhance nitric oxide (NO) production and prevent EC dysfunction. In addition, hemodynamic forces modulate the expression and acetylation of transcription factors, i.e., retinoic acid receptor α and krüppel-like factor-2, to transcriptionally regulate the expression of microRNAs (miRs). OS-modulated miRs, which stimulate proliferative, pro-inflammatory, and oxidative signaling, promote EC dysfunction, whereas PS-regulated miRs, which induce anti-proliferative, anti-inflammatory, and anti-oxidative signaling, inhibit EC dysfunction. PS also modulates the expression of long non-coding RNAs to influence EC function. i.e., turnover, aligmant, and migration. On the other hand, OS enhances the expression of DNMT-1 and -3a to induce EC dysfunction, i.e., proliferation, inflammation, and NO repression. Conclusion Overall, epigenetic factors play vital roles in modulating hemodynamic-directed EC dysfunction and vascular disorders, i.e., atherosclerosis. Understanding the detailed mechanisms through which epigenetic factors regulate hemodynamics-directed EC dysfunction and vascular disorders can help us to elucidate the pathogenic mechanisms of atherosclerosis and develop potential therapeutic strategies for atherosclerosis treatment.

Abstract Background The presence and activity of CRISPR-Cas defense systems is a hallmark of many prokaryotic microorganisms. Here, the distribution of sequences related to the highly iterated palindrome 1 (HIP1) element and the DNA methylation of CGATCG motifs embedded within HIP1 as a vital part of the CRISPR1 repeat sequence was analyzed in the cyanobacterium Synechocystis sp. PCC 6803. Previously suggested functions of HIP1 include organization of chromosomal structure, DNA recombination or gene regulation, all of which could be relevant in CRISPR-Cas functionality. Results The CRISPR1 repeat-spacer array contains more than 50 CGATCG elements that are double-methylated (5mCG6mATCG) by the enzymes M.Ssp6803I and M.Ssp6803III. Hence, more than 200 possible methylation events cluster over a stretch of 3600 bp of double-stranded DNA. Bisulfite sequencing showed that these motifs were highly methylated at the m5CGATCG positions whereas specific motifs within the CRISPR1 cas genes were hypomethylated suggesting a lowered accessibility for the DNA methylase to these regions. Assays for conjugation and CRISPR1-mediated DNA interference revealed a 50% drop in conjugation efficiency in the mutant lacking the 5mC methylation of CGATCG motifs, while the highly efficient DNA interference activity was not affected by the lack of m5CGATCG DNA-methylation, nor was the capability to differentiate between self and non-self targets based on the protospacer adjacent motifs (PAMs) GTA and GTC versus the non-PAM AGC. A third DNA methylation mediated by M.Ssp6803II modifies the first cytosine in the motif GGCC yielding GGm4CC. We found a remarkable absence of GGCC motifs and hence the corresponding methylation over an 11 kb stretch encompassing all the cas genes involved in interference and crRNA maturation but not adaptation of the CRISPR1 system. Conclusions The lack of GGCC tetranucleotides along the CRISPR1 interference and maturation genes supports the reported hybrid character of subtype I-D CRISPR-Cas systems. We report tight and very high 5mC methylation of the CRISPR1 repeat sequences. Nevertheless, cells lacking the 5mC methylation activity were unaffected in their CRISPR1-mediated interference response but the efficiency of conjugation was reduced by 50%. These results point to an unknown role of m5CGATCG DNA-methylation marks in conjugation and DNA transformation.

Xanthomonas euvesicatoria (Xe) is a gram-negative phytopathogenic bacterium that causes bacterial spot disease in tomato/pepper leading to economic losses in plantations. DNA methyltransferases (MTases) are critical for the survival of prokaryotes; however, their functions in phytopathogenic bacteria remain unclear. In this study, we characterized the functions of two putative DNA MTases, XvDMT1 and XvDMT2, in Xe by generating XvDMT1- and XvDMT2-overexpressing strains, Xe(XvDMT1) and Xe(XvDMT2), respectively. Virulence of Xe(XvDMT2), but not Xe(XvDMT1), on tomato was dramatically reduced. To postulate the biological processes involving XvDMTs, we performed a label-free shotgun comparative proteomic analysis, and results suggest that XvDMT1 and XvDMT2 have distinct roles in Xe. We further characterized the functions of XvDMTs using diverse phenotypic assays. Notably, both Xe(XvDMT1) and Xe(XvDMT2) showed growth retardation in the presence of sucrose and fructose as the sole carbon source, with Xe(XvDMT2) being the most severely affected. In addition, biofilm formation and production of exopolysaccharides were declined in Xe(XvDMT2), but not Xe(XvDMT1). Xe(XvDMT2) was more tolerant to EtOH than Xe(XvDMT1), which had enhanced tolerance to sorbitol but decreased tolerance to polymyxin B. Using single-molecule real-time sequencing and methylation-sensitive restriction enzymes, we successfully predicted putative motifs methylated by XvDMT1 and XvDMT2, which are previously uncharacterized 6mA and 5mC DNA MTases, respectively. This study provided new insights into the biological functions of DNA MTases in prokaryotic organisms.

In this study, a sensitive method for detecting DNA methyltransferase (MTase) activity was developed by combining the effective fluorescence resonance energy transfer (FRET) of cationic conjugated polymers and exonuclease (Exo) III–mediated signal amplification. DNA adenine MTase targets the GATC sequence within a substrate and converts the adenine in this sequence into N6-methyladenine. In the method developed in this study, the methylated substrate is cleaved using Dpn I, whereby a single-stranded oligodeoxynucleotide (oligo) is released. Afterward, the oligo is hybridized to the 3ʹ protruding end of the F-DNA probe to form a double-stranded DNA, which is then digested by Exo III. Subsequently, due to weak electrostatic interactions, only a weak FRET signal is observed. The introduction of the Exo-III–mediated target-recycling reaction improved the sensitivity for detecting MTase. This detection method was found to be sensitive for MTase detection, with the lowest detection limit of 0.045 U/mL, and was also suitable for MTase-inhibitor screening, whereby such inhibitors can be identified for disease treatment.