Bacterial epigenetics, particularly through DNA methylation, exerts significant influence over various biological processes such as DNA replication, uptake, and gene regulation in bacteria. In this review, we explore recent advances in characterizing bacterial epigenomes, accompanied by emerging strategies that harness bacterial epigenetics to elucidate and engineer diverse bacterial species with precision and effectiveness. Furthermore, we delve into the potential of epigenetic modifications to steer microbial functions and influence community dynamics, offering promising opportunities for understanding and modulating microbiomes. Additionally, we investigate the extensive diversity of DNA methyltransferases and emphasize their potential utility in the context of the human microbiome. In summary, this review highlights the potential of DNA methylation as a powerful toolkit for engineering microbiomes.

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

DNA methylation is one of induced changes under salinity stress causing reduction in the expression of several crucial genes required for normal plant\'s operation. Potential use of royal jelly (RJ), folic acid (FA) and 5-azacitidine (5-AZA) on two Egyptian faba bean varieties (Sakha-3 and Giza-716) grown under saline conditions was investigated. Salinity stress affects negatively on seeds germination (G %), mitotic index, membrane stability and induced a significant increase in chromosomal abnormalities (CAs). DNA methyltransferases genes (MT1 and MT2) were highly up-regulated (∼23 and 8 folds for MT1 and MT2 in shoots of Giza-716 stressed plants). On the other hand, down regulation of other studied stress related genes: superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), heat shock protein (HSP-17.9) and proline-rich protein (GPRP) were detected in stressed plants of both studied varieties. Treating plants with RJ and FA increase G%, chlorophyll content, improves membrane properties and reduces CAs compared to non-treated stressed plants. Exogenous application of 5-AZA, RJ and FA on salinity stressed plants was associated with a significant reduction in the transcription of MT1 and MT2 which was associated with significant up regulation in the expression of Cu/Zn-SOD, CAT, GR, GPRP and HSP-17.9 encoding genes. The Lowest expression of MT1 and MT2 were induced with 5-AZA treatment in both studied varieties. Exogenous application of the FA, RJ and 5-AZA modified the methylation state of stressed plants by regulation the expression of DNA methyltransferases, subsequently, modulated the expression of studied genes and could be proposed as a promising treatment to ameliorate hazardous effects of salt stress on different plants.

Nowadays, the explosion of knowledge in the field of epigenetics has revealed new pathways toward the treatment of multifactorial diseases, rendering the key players of the epigenetic machinery the focus of today\'s pharmaceutical landscape. Among epigenetic enzymes, DNA methyltransferases (DNMTs) are first studied as inhibition targets for cancer treatment. The increasing clinical interest in DNMTs has led to advanced experimental and computational strategies in the search for novel DNMT inhibitors. Considering the importance of epigenetic targets as a novel and promising pharmaceutical trend, the present study attempted to discover novel inhibitors of natural origin against DNMTs using a combination of structure and ligand-based computational approaches. Particularly, a pharmacophore-based virtual screening was performed, followed by molecular docking and molecular dynamics simulations in order to establish an accurate and robust selection methodology. Our screening protocol prioritized five natural-derived compounds, derivatives of coumarins, flavones, chalcones, benzoic acids, and phenazine, bearing completely diverse chemical scaffolds from FDA-approved \"Epi-drugs\". Their total DNMT inhibitory activity was evaluated, revealing promising results for the derived hits with an inhibitory activity ranging within 30-45% at 100 µM of the tested compounds.

BACKGROUND: Resistance to targeted therapies represents a significant hurdle to successfully treating hepatocellular carcinoma (HCC). While epigenetic abnormalities are critical determinants of HCC relapse and therapeutic resistance, the underlying mechanisms are poorly understood. We aimed to address whether and how dysregulated epigenetic regulators have regulatory and functional communications in establishing and maintaining drug resistance.
METHODS: HCC-resistant cells were characterized by CCK-8, IncuCyte Live-Cell analysis, flow cytometry and wound-healing assays. Target expression was assessed by qPCR and Western blotting. Global and promoter DNA methylation was measured by dotblotting, methylated-DNA immunoprecipitation and enzymatic digestion. Protein interaction and promoter binding of DNMT3a-TET2 were investigated by co-immunoprecipitation, ChIP-qPCR. The regulatory and functional roles of DNMT3a and TET2 were studied by lentivirus infection and puromycin selection. The association of DNMT and TET expression with drug response and survival of HCC patients was assessed by public datasets, spearman correlation coefficients and online tools.
RESULTS: We identified the coordination of DNMT3a and TET2 as an actionable mechanism of drug resistance in HCC. The faster growth and migration of resistant HCC cells were attributed to DNMT3a and TET2 upregulation followed by increased 5mC and 5hmC production. HCC patients with higher DNMT3a and TET2 had a shorter survival time with a less favorable response to sorafenib therapy than those with lower expression. Cancer stem cell-like cells (CSCs) displayed DNMT3a and TET2 overexpression, which were insensitive to sorafenib. Either genetic or pharmacological suppression of DNMT3a or/and TET2 impaired resistant cell growth and oncosphere formation, and restored sorafenib sensitivity. Mechanistically, DNMT3a did not establish a regulatory circuit with TET2, but formed a complex with TET2 and HDAC2. This complex bound the promoters of oncogenes (i.e., CDK1, CCNA2, RASEF), and upregulated them without involving promoter DNA methylation. In contrast, DNMT3a-TET2 crosstalk silences tumor suppressors (i.e., P15, SOCS2) through a corepressor complex with HDAC2 along with increased promoter DNA methylation.
CONCLUSIONS: We demonstrate that DNMT3a and TET2 act coordinately to regulate HCC cell fate in DNA methylation-dependent and -independent manners, representing strong predictors for drug resistance and poor prognosis, and thus are promising therapeutic targets for refractory HCC.

BACKGROUND: Despite recent advances in epithelial ovarian carcinoma (EOC) treatment, its recurrence and mortality rates have not improved significantly. DNA hypermethylation has generally been associated with an ominous prognosis and chemotherapy resistance, but the role of DNA methyltransferases (DNMTs) in EOC remains to be investigated.
METHODS: In the current study, we systematically retrieved gene expression data from patients with EOC and studied the immunohistochemical expression of DNMTs in 108 primary and 26 relapsed tumors.
RESULTS: Our results showed that the DNMT1, DNMT3A, DNMT3B and DNMT3L RNA levels were higher and the DNMT2 level was lower in tumors compared to non-neoplastic tissue, and DNMT3A and DNMT2 expression decreased from Stage-II to Stage-IV carcinomas. The proteomic data also suggested that the DNMT1 and DNMT3A levels were increased in the tumors. Similarly, the DNMT1, DNMT3A and DNMT3L protein levels were overexpressed and DNMT2 expression was reduced in high-grade carcinomas compared to non-neoplastic tissue and low-grade tumors. Moreover, DNMT1 and DNMT3L were increased in relapsed tumors compared to their primaries. The DNMT3A, DNMT1 and DNMT3B mRNA levels were correlated with overall survival.
CONCLUSIONS: Our study demonstrates that DNMT1 and DNMT3L are upregulated in primary high-grade EOC and further increase in relapses, whereas DNMT3A is upregulated only in the earlier stages of cancer progression. DNMT2 downregulation highlights the presumed tumor-suppressor activity of this gene in ovarian carcinoma.

OBJECTIVE: Schizophrenia is a serious mental disorder with complex clinical manifestations, while its pathophysiological mechanism is not fully understood. Accumulated evidence suggested the alteration in epigenetic pathway was associated with clinical features and brain dysfunctions in schizophrenia. DNA methyltransferases (DNMTs), a key enzyme for DNA methylation, are related to the development of schizophrenia, whereas the current research evidence is not sufficient. The aim of study was to explore the effects of gene polymorphisms of DNMTs on the susceptibility and symptoms of schizophrenia.
METHODS: The study was case-control study that designed and employed the Diagnostic and Statistical Manual of Mental Disorders-Fifth Edition (DSM-5) as the diagnostic standard. 134 hospitalized patients with schizophrenia in the Third People\'s Hospital of Zhongshan City from January 2018 to April 2020 (Case group) as well as 64 healthy controls (Control group) from the same region were involved. Single nucleotide polymorphisms (SNPs) of DNMT1 genes (r s2114724 and rs 2228611) and DNMT3B genes (rs 2424932, rs 1569686, rs 6119954 and rs 2424908) were determined with massARRAY. Linkage disequilibrium analysis and haplotype analysis were performed, and genotype and allele frequencies were compared. The Hardy-Weinberg equilibrium was tested by the Chi-square test in SPSS software (version 20.0, SPSS Inc., USA). The severity of clinical symptoms was assessed by the Positive and Negative Syndrome Scale (PANSS). The correlation between DNMT1 genes (rs 2114724 and rs 2228611) and DNMT3B genes (rs2424932, rs1569686, rs6119954 and rs2424908) and clinical features was analyzed.
RESULTS: There were no significant differences in genotype, allele frequency and haplotype of DNMT1 genes (rs 2114724 and rs 2228611) and DNMT3B genes (rs 2424932, rs 1569686, rs 6119954 and rs 2424908) between the case and healthy control group. There were significant differences in the PANSS total positive symptom scores, P3 (hallucinatory behavior), P6 (suspicious/persecution), G7 (motor retardation), and G15 (preoccupation) in patients with different DNMT1 gene rs 2114724 and rs 2228611 genotypes. The linkage disequilibrium analysis of gene polymorphic loci revealed that rs 2114724-rs 2228611 was complete linkage disequilibrium, and rs 1569686-rs 2424908, rs 2424932-rs 1569696 and rs 2424932-rs 2424908 were strongly linkage disequilibrium.
CONCLUSIONS: The polymorphisms alteration in genetic pathway may be associated with development of specific clinical features in schizophrenia.

Adrenocortical carcinoma is rare and aggressive endocrine cancer of the adrenal gland. Within adrenocortical carcinoma, a recently described subtype characterized by a CpG island methylator phenotype (CIMP) has been associated with an especially poor prognosis. However, the drivers of CIMP remain unknown. Furthermore, the functional relation between CIMP and poor clinical outcomes of patients with adrenocortical carcinoma stays elusive.
Here, we show that CIMP in adrenocortical carcinoma is linked to the increased expression of DNA methyltransferases DNMT1 and DNMT3A driven by a gain of gene copy number and cell hyperproliferation. Importantly, we demonstrate that CIMP contributes to tumor aggressiveness by favoring tumor immune escape. This effect could be at least partially reversed by treatment with the demethylating agent 5-azacytidine.
In sum, our findings suggest that co-treatment with demethylating agents might enhance the efficacy of immunotherapy and could represent a novel therapeutic approach for patients with high CIMP adrenocortical carcinoma.

The pineal gland-derived indoleamine hormone, melatonin, regulates multiple cellular processes, ranging from chronobiology, proliferation, apoptosis, and oxidative damage to pigmentation, immune regulation, and mitochondrial metabolism. While melatonin is best known as a master regulator of the circadian rhythm, previous studies also have revealed connections between circadian cycle disruption and genomic instability, including epigenetic changes in the pattern of DNA methylation. For example, melatonin secretion is associated with differential circadian gene methylation in night shift workers and the regulation of genomic methylation during embryonic development, and there is accumulating evidence that melatonin can modify DNA methylation. Since the latter one impacts cancer initiation, and also, non-malignant diseases development, and that targeting DNA methylation has become a novel intervention target in clinical therapy, this review discusses the potential role of melatonin as an under-investigated candidate epigenetic regulator, namely by modulating DNA methylation via changes in mRNA and the protein expression of DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) proteins. Furthermore, since melatonin may impact changes in the DNA methylation pattern, the authors of the review suggest its possible use in combination therapy with epigenetic drugs as a new anticancer strategy.

DNA methylation is a critical epigenetic regulator in the occurrence and development of diseases and is closely related to various functional responses in relation to spinal cord injury. To investigate the role of DNA methylation in spinal cord injury, we constructed a library with reduced-representation bisulfite sequencing data obtained at various time points (day 0-42) after spinal cord injury in mice. Global DNA methylation levels, specifically non-CpG (CHG and CHH) methylation levels, decreased modestly following spinal cord injury. Stages post-spinal cord injury were classified as early (day 0-3), intermediate (day 7-14), and late (day 28-42) based on similarity and hierarchical clustering of global DNA methylation patterns. The non-CpG methylation level, which included CHG and CHH methylation levels, was markedly reduced despite accounting for a minor proportion of total methylation abundance. At multiple genomic sites, including the 5\' untranslated regions, promoter, exon, intron, and 3\' untranslated regions, the non-CpG methylation level was markedly decreased following spinal cord injury, whereas the CpG methylation level remained unchanged at these locations. Approximately one-half of the differentially methylated regions were located in intergenic areas; the other differentially methylated regions in both CpG and non-CpG regions were clustered in intron regions, where the DNA methylation level was highest. The function of genes associated with differentially methylated regions in promoter regions was also investigated. From Gene Ontology analysis results, DNA methylation was implicated in a number of essential functional responses to spinal cord injury, including neuronal synaptic connection creation and axon regeneration. Notably, neither CpG methylation nor non-CpG methylation was implicated in the functional response of glial or inflammatory cells. In summary, our work elucidated the dynamic pattern of DNA methylation in the spinal cord following injury and identified reduced non-CpG methylation as an epigenetic target after spinal cord injury in mice.