The G9a histone methyltransferase represses osteoclastogenesis and bone resorption by regulating NFATc1 function.

Epigenetic modifications affect cell differentiation via transcriptional regulation. G9a/EHMT2 is an important epigenetic modifier that catalyzes the methylation of histone 3 lysine 9 (H3K9) and interacts with various nuclear proteins. In this study, we investigated the role of G9a in osteoclast differentiation. When we deleted G9a by infection of Cre-expressing adenovirus into bone marrow macrophages (BMMs) from G9afl/fl (Ehmt2fl/fl) and induced osteoclastic differentiation by the addition of macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), the number of TRAP-positive multinucleated osteoclasts significantly increased compared with control. Furthermore, the mRNA expression of osteoclast markers, TRAP, and cathepsin K, and to a lesser extent, NFATc1, a critical transcription factor, increased in G9a KO cells. Infection of wild-type (WT) G9a-expressing adenovirus in G9a KO cells restored the number of TRAP-positive multinucleated cells. In G9a KO cells, increased nuclear accumulation of NFATc1 protein and decreased H3K9me2 accumulation were observed. Furthermore, ChIP experiments revealed that NFATc1 binding to its target, Ctsk promoter, was enhanced by G9a deletion. For in vivo experiments, we created G9a conditional knock-out (cKO) mice by crossing G9afl/fl mice with Rank Cre/+ (Tnfrsf11aCre/+) mice, in which G9a is deleted in osteoclast lineage cells. The trabecular bone volume was significantly reduced in female G9a cKO mice. The serum concentration of the C-terminal telopeptide of type I collagen (CTX), a bone-resorbing indicator, was higher in G9a cKO mice. In addition, osteoclasts differentiated from G9a cKO BMMs exhibited greater bone-resorbing activity. Our findings suggest that G9a plays a repressive role in osteoclastogenesis by modulating NFATc1 function.

Emerging methylation-based approaches in microbiome engineering.

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.

DNA hypomethylation in wood frog liver under anoxia and dehydration stresses.

Wood frogs are freeze-tolerant vertebrates that can endure weeks to months frozen during the winter without breathing and with as much as 65% of total body water frozen as extracellular ice. Underlying tolerances of anoxia and of cellular dehydration support whole body freezing. One pro-survival mechanism employed by these frogs is epigenetic modifications via DNA hypomethylation processes facilitating transcriptional repression or activation. These processes involve proteins such as DNA Methyltransferases (DNMTs), Methyl Binding Domain proteins (MBDs), Ten-Eleven Translocases (TETs), and Thymine Deglycosylase (TDG). The present study evaluates the responses of these proteins to dehydration and anoxia stresses in wood frog liver. DNMT relative protein expression was reduced in liver, but nuclear DNMT activity did not change significantly under anoxia stress. By contrast, liver DNMTs and nuclear DNMT activity were upregulated under dehydration stress. These stress-specific differences were speculated to arise from Post-Translational Modifications (PTMs). DNMT3A and DNMT3B showed increased relative protein expression during recovery from dehydration and anoxia. Further, MBD1 was elevated during both conditions suggesting transcriptional repression. TET proteins showed varying responses to anoxia likely due to the absence of oxygen, a main substrate required by TETs. Similarly, TDG, an enzyme that corrects DNA damage, was downregulated under anoxia potentially due to lower levels of reactive oxygen species that damage DNA, but levels returned to normal during reperfusion of oxygen. Our results indicate differential stress-specific responses that indicate the need for more research in the DNA hypomethylation mechanisms employed by the wood frog during stress.

Dysregulation of arginine methylation in tumorigenesis.

Protein methylation, similar to DNA methylation, primarily involves post-translational modification (PTM) targeting residues of nitrogen-containing side-chains and other residues. Protein arginine methylation, occurred on arginine residue, is mainly mediated by protein arginine methyltransferases (PRMTs), which are ubiquitously present in a multitude of organisms and are intricately involved in the regulation of numerous biological processes. Specifically, PRMTs are pivotal in the process of gene transcription regulation, and protein function modulation. Abnormal arginine methylation, particularly in histones, can induce dysregulation of gene expression, thereby leading to the development of cancer. The recent advancements in modification mediated by PRMTs and cancer research have had a profound impact on our understanding of the abnormal modification involved in carcinogenesis and progression. This review will provide a defined overview of these recent progression, with the aim of augmenting our knowledge on the role of PRMTs in progression and their potential application in cancer therapy.

Research Progress on the Role of Epigenetic Methylation Modification in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) stands as the prevailing form of primary liver cancer, characterized by a poor prognosis and high mortality rate. A pivotal factor in HCC tumorigenesis is epigenetics, specifically the regulation of gene expression through methylation. This process relies significantly on the action of proteins that modify methylation, including methyltransferases, their associated binding proteins, and demethylases. These proteins are crucial regulators, orchestrating the methylation process by regulating enzymes and their corresponding binding proteins. This orchestration facilitates the reading, binding, detection, and catalysis of gene methylation sites. Methylation ences the development, prolisignificantly influferation, invasion, and prognosis of HCC. Furthermore, methylation modification and its regulatory mechanisms activate distinct biological characteristics in HCC cancer stem cells, such as inducing cancer-like differentiation of stem cells. They also influence the tumor microenvironment (TME) in HCC, modulate immune responses, affect chemotherapy resistance in HCC patients, and contribute to HCC progression through signaling pathway feedback. Given the essential role of methylation in genetic information, it holds promise as a potential tool for the early detection of HCC and as a target to improve drug resistance and promote apoptosis in HCC cells.

5-Fluorouracil dose escalation generated desensitized colorectal cancer cells with reduced expression of protein methyltransferases and no epithelial-to-mesenchymal transition potential.

Background: Colorectal cancer (CRC) is one of the most frequently diagnosed cancers. In many cases, the poor prognosis of advanced CRC is associated with resistance to treatment with chemotherapeutic drugs such as 5-Fluorouracil (5-FU). The epithelial-to-mesenchymal transition (EMT) and dysregulation in protein methylation are two mechanisms associated with chemoresistance in many cancers. This study looked into the effect of 5-FU dose escalation on EMT and protein methylation in CRC. Materials and Methods: HCT-116, Caco-2, and DLD-1 CRC cell lines were exposed to dose escalation treatment of 5-FU. The motility and invasive potentials of the cells before and after treatment with 5-FU were investigated through wound healing and invasion assays. This was followed by a Western blot which analyzed the protein expressions of the epithelial marker E-cadherin, mesenchymal marker vimentin, and the EMT transcription factor (EMT-TF), the snail family transcriptional repressor 1 (Snail) in the parental and desensitized cells. Western blotting was also conducted to study the protein expressions of the protein methyltransferases (PMTs), Euchromatic histone lysine methyltransferase 2 (EHMT2/G9A), protein arginine methyltransferase (PRMT5), and SET domain containing 7/9 (SETD7/9) along with the global lysine and arginine methylation profiles. Results: The dose escalation method generated 5-FU desensitized CRC cells with distinct morphological features and increased tolerance to high doses of 5-FU. The 5-FU desensitized cells experienced a decrease in migration and invasion when compared to the parental cells. This was reflected in the observed reduction in E-cadherin, vimentin, and Snail in the desensitized cell lines. Additionally, the protein expressions of EHMT2/G9A, PRMT5, and SETD7/9 also decreased in the desensitized cells and global protein lysine and arginine methylation became dysregulated with 5-FU treatment. Conclusion: This study showed that continuous, dose-escalation treatment of 5-FU in CRC cells generated 5-FU desensitized cancer cells that seemed to be less aggressive than parental cells.

Exogenous application of 5-azacitidin, royal jelly and folic acid regulate plant redox state, expression level of DNA methyltransferases and alleviate adverse effects of salinity stress on Vicia faba L. plants.

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.

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

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.

Mice with NOP2/sun RNA methyltransferase 5 deficiency die before reaching puberty due to fatal kidney damage.

BACKGROUND: NOP2/Sun RNA methyltransferase 5 (NSUN5) is an RNA methyltransferase that has a broad distribution and plays critical roles in various biological processes. However, our knowledge of the biological functions of NSUN5 in mammals is very limited. Therefore, in this study, we investigate the role of NSUN5 in mice. METHODS: In the present research, we built a mouse model (Nsun5-/-) using the CRISPR/Cas9 system to investigated the specific role of NSUN5. RESULTS: We observed that Nsun5-/- mice had a reduced body weight compared to wild-type mice. Additionally, their survival rate gradually decreased to 20% after postnatal day (PD) 21. Further examination revealed the Nsun5-/- mice had multiple organ damage, with the most severe damage occurring in the kidneys. Moreover, we observed glycogen deposition and fibrosis, along with a notable shorting of the primary foot processes of glomeruli in Nsun5-/- kidneys. Furthermore, we found that the kidneys of Nsun5-/- mice showed increased expression of the apoptotic signal Caspase-3 and accumulated stronger DNA damage at PD 21. CONCLUSIONS: In our study, we found that mice lacking NSUN5 died before puberty due to kidney fatal damage caused by DNA damage and cell apoptosis. These results suggest that NSUN5 plays a vital role in preventing the accumulation of DNA damage and cell apoptosis in the kidney.

Different expression patterns of DNA methyltransferases during horse testis development.

DNA methyltransferases (DNMTs) are important epigenetic modification during spermatogenesis. To further evaluate the pattern of DNMTs in horse testes during development, we investigated the expression and localization of DNMT1, DNMT3a and DNMT3b at different time points. The qRT-PCR results showed that DNMT1 expression was maintained in testes tissue from 6-month-old (0.5y) to 2-year-old (2y) of age and decreased after 3-year-old (3y) (P < 0.01). The expression levels of DNMT3a and DNMT3b peaked in testes tissue at 3y (P < 0.01). At 4-year-old (4y), the expression of DNMT3a and DNMT3b was decreased and became similar to that at 0.5y. Immunofluorescence of DNMT1, DNMT3a and DNMT3b on testis samples confirmed the differential expression and localization of these three DNA methylation transferases during horse development. Further molecular biological studies are needed to understand the implications of the expression patterns of these DNMTs in horse testes.

Unraveling the relationship between histone methylation and nonalcoholic fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) poses a significant health challenge in modern societies due to shifts in lifestyle and dietary habits. Its complexity stems from genetic predisposition, environmental influences, and metabolic factors. Epigenetic processes govern various cellular functions such as transcription, chromatin structure, and cell division. In NAFLD, these epigenetic tendencies, especially the process of histone methylation, are intricately intertwined with fat accumulation in the liver. Histone methylation is regulated by different enzymes like methyltransferases and demethylases and influences the expression of genes related to adipogenesis. While early-stage NAFLD is reversible, its progression to severe stages becomes almost irreversible. Therefore, early detection and intervention in NAFLD are crucial, and understanding the precise role of histone methylation in the early stages of NAFLD could be vital in halting or potentially reversing the progression of this disease.

Relationship between arginine methylation and vascular calcification.

In patients on maintenance hemodialysis (MHD), vascular calcification (VC) is an independent predictor of cardiovascular disease (CVD), which is the primary cause of death in chronic kidney disease (CKD). The main component of VC in CKD is the vascular smooth muscle cells (VSMCs). VC is an ordered, dynamic activity. Under the stresses of oxidative stress and calcium-­phosphorus imbalance, VSMCs undergo osteogenic phenotypic transdifferentiation, which promotes the formation of VC. In addition to traditional epigenetics like RNA and DNA control, post-translational modifications have been discovered to be involved in the regulation of VC in recent years. It has been reported that the process of osteoblast differentiation is impacted by catalytic histone or non-histone arginine methylation. Its function in the osteogenic process is comparable to that of VC. Thus, we propose that arginine methylation regulates VC via many signaling pathways, including as NF-B, WNT, AKT/PI3K, TGF-/BMP/SMAD, and IL-6/STAT3. It might also regulate the VC-related calcification regulatory factors, oxidative stress, and endoplasmic reticulum stress. Consequently, we propose that arginine methylation regulates the calcification of the arteries and outline the regulatory mechanisms involved.

Insights into E.†coli Cyclopropane Fatty Acid Synthase (CFAS) Towards Enantioselective Carbene Free Biocatalytic Cyclopropanation.

Cyclopropane fatty acid synthases (CFAS) are a class of S-adenosylmethionine (SAM) dependent methyltransferase enzymes able to catalyse the cyclopropanation of unsaturated phospholipids. Since CFAS enzymes employ SAM as a methylene source to cyclopropanate alkene substrates, they have the potential to be mild and more sustainable biocatalysts for cyclopropanation transformations than current carbene-based approaches. This work describes the characterisation of E. coli CFAS (ecCFAS) and its exploitation in the stereoselective biocatalytic synthesis of cyclopropyl lipids. ecCFAS was found to convert phosphatidylglycerol (PG) to methyl dihydrosterculate 1 with up to 58 % conversion and 73 % ee and the absolute configuration (9S,10R) was established. Substrate tolerance of ecCFAS was found to be correlated with the electronic properties of phospholipid headgroups and for the first time ecCFAS was found to catalyse cyclopropanation of both phospholipid chains to form dicyclopropanated products. In addition, mutagenesis and in silico experiments were carried out to identify the enzyme residues with key roles in catalysis and to provide structural insights into the lipid substrate preference of ecCFAS. Finally, the biocatalytic synthesis of methyl dihydrosterculate 1 and its deuterated analogue was also accomplished combining recombinant ecCFAS with the SAM regenerating AtHMT enzyme in the presence of CH3I and CD3I respectively.

CRISPR-Cas9 Gene Editing: Curing Genetic Diseases by Inherited Epigenetic Modifications.

Introduction CRISPR-Cas9 gene editing, leveraging bacterial defense mechanisms, offers precise DNA modifications, holding promise in curing genetic diseases. This review critically assesses its potential, analyzing evidence on therapeutic applications, challenges, and future prospects. Examining diverse genetic disorders, it evaluates efficacy, safety, and limitations, emphasizing the need for a thorough understanding among medical professionals and researchers. Acknowledging its transformative impact, a systematic review is crucial for informed decision-making, responsible utilization, and guiding future research to unlock CRISPR-Cas9's full potential in realizing the cure for genetic diseases. Methods A comprehensive literature search across PubMed, Scopus, and the Web of Science identified studies applying CRISPR-Cas9 gene editing for genetic diseases, following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Inclusion criteria covered in vitro and in vivo models targeting various genetic diseases with reported outcomes on disease modification or potential cure. Quality assessment revealed a generally moderate to high risk of bias. Heterogeneity prevented quantitative meta-analysis, prompting a narrative synthesis of findings. Discussion CRISPR-Cas9 enables precise gene editing, correcting disease-causing mutations and offering hope for previously incurable genetic conditions. Leveraging inherited epigenetic modifications, it not only fixes mutations but also restores normal gene function and controls gene expression. The transformative potential of CRISPR-Cas9 holds promise for personalized treatments, improving therapeutic outcomes, but ethical considerations and safety concerns must be rigorously addressed to ensure responsible and safe application, especially in germline editing with potential long-term implications.

EZH2 as a major histone methyltransferase in PDGF-BB-activated orbital fibroblast in the pathogenesis of Graves' ophthalmopathy.

Graves' ophthalmopathy (GO) is an extra-thyroidal complication of Graves' disease which can lead to vision loss in severe cases. Currently, treatments of GO are not sufficiently effective, so novel therapeutic strategies are needed. As platelet-derived growth factor (PDGF)-BB induces several effector mechanisms in GO orbital fibroblasts including cytokine production and myofibroblast activation, this study aims to investigate the roles of histone lysine methyltransferases (HKMTs) in PDGF-BB-activated GO orbital fibroblasts by screening with HKMTs inhibitors library. From the total of twelve selective HKMT inhibitors in the library, EZH2, G9a and DOT1L inhibitors, DZNeP, BIX01294 and Pinometostat, respectively, prevented PDGF-BB-induced proliferation and hyaluronan production by GO orbital fibroblasts. However, only EZH2 inhibitor, DZNeP, significantly blocked pro-inflammatory cytokine production. For the HKMTs expression in GO orbital fibroblasts, PDGF-BB significantly and time-dependently induced EZH2, G9a and DOT1L mRNA expression. To confirm the role of EZH2 in PDGF-BB-induced orbital fibroblast activation, EZH2 silencing experiments revealed suppression of PDGF-BB-induced collagen type I and α-SMA expression along with decreasing histone H3 lysine 27 trimethylation (H3K27me3) level. In a more clinically relevant model than orbital fibroblast culture experiments, DZNeP treated GO orbital tissues significantly reduced pro-inflammatory cytokine production while slightly reduced ACTA2 mRNA expression. Our data is the first to demonstrate that among all HKMTs EZH2 dominantly involved in the expression of myofibroblast markers in PDGF-BB-activated orbital fibroblast from GO presumably via H3K27me3. Thus, EZH2 may represent a novel therapeutics target for GO.

Protein Arginine Methyltransferases in Pancreatic Ductal Adenocarcinoma: New Molecular Targets for Therapy.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignant disease with a low 5-year overall survival rate. It is the third-leading cause of cancer-related deaths in the United States. The lack of robust therapeutics, absence of effective biomarkers for early detection, and aggressive nature of the tumor contribute to the high mortality rate of PDAC. Notably, the outcomes of recent immunotherapy and targeted therapy against PDAC remain unsatisfactory, indicating the need for novel therapeutic strategies. One of the newly described molecular features of PDAC is the altered expression of protein arginine methyltransferases (PRMTs). PRMTs are a group of enzymes known to methylate arginine residues in both histone and non-histone proteins, thereby mediating cellular homeostasis in biological systems. Some of the PRMT enzymes are known to be overexpressed in PDAC that promotes tumor progression and chemo-resistance via regulating gene transcription, cellular metabolic processes, RNA metabolism, and epithelial mesenchymal transition (EMT). Small-molecule inhibitors of PRMTs are currently under clinical trials and can potentially become a new generation of anti-cancer drugs. This review aims to provide an overview of the current understanding of PRMTs in PDAC, focusing on their pathological roles and their potential as new therapeutic targets.

A Computational Approach for the Discovery of Novel DNA Methyltransferase Inhibitors.

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.

Methylated chalcones are required for rhizobial nod gene induction in the Medicago truncatula rhizosphere.

Legume nodulation requires the detection of flavonoids in the rhizosphere by rhizobia to activate their production of Nod factor countersignals. Here we investigated the flavonoids involved in nodulation of Medicago truncatula. We biochemically characterized five flavonoid-O-methyltransferases (OMTs) and a lux-based nod gene reporter was used to investigate the response of Sinorhizobium medicae NodD1 to various flavonoids. We found that chalcone-OMT 1 (ChOMT1) and ChOMT3, but not OMT2, 4, and 5, were able to produce 4,4'-dihydroxy-2'-methoxychalcone (DHMC). The bioreporter responded most strongly to DHMC, while isoflavones important for nodulation of soybean (Glycine max) showed no activity. Mutant analysis revealed that loss of ChOMT1 strongly reduced DHMC levels. Furthermore, chomt1 and omt2 showed strongly reduced bioreporter luminescence in their rhizospheres. In addition, loss of both ChOMT1 and ChOMT3 reduced nodulation, and this phenotype was strengthened by the further loss of OMT2. We conclude that: the loss of ChOMT1 greatly reduces root DHMC levels; ChOMT1 or OMT2 are important for nod gene activation in the rhizosphere; and ChOMT1/3 and OMT2 promote nodulation. Our findings suggest a degree of exclusivity in the flavonoids used for nodulation in M. truncatula compared to soybean, supporting a role for flavonoids in rhizobial host range.

Rectifying METTL4-Mediated N6-Methyladenine Excess in Mitochondrial DNA Alleviates Heart Failure.

BACKGROUND: Myocardial mitochondrial dysfunction underpins the pathogenesis of heart failure (HF), yet therapeutic options to restore myocardial mitochondrial function are scarce. Epigenetic modifications of mitochondrial DNA (mtDNA), such as methylation, play a pivotal role in modulating mitochondrial homeostasis. However, their involvement in HF remains unclear. METHODS: Experimental HF models were established through continuous angiotensin II and phenylephrine (AngII/PE) infusion or prolonged myocardial ischemia/reperfusion injury. The landscape of N6-methyladenine (6mA) methylation within failing cardiomyocyte mtDNA was characterized using high-resolution mass spectrometry and methylated DNA immunoprecipitation sequencing. A tamoxifen-inducible cardiomyocyte-specific Mettl4 knockout mouse model and adeno-associated virus vectors designed for cardiomyocyte-targeted manipulation of METTL4 (methyltransferase-like protein 4) expression were used to ascertain the role of mtDNA 6mA and its methyltransferase METTL4 in HF. RESULTS: METTL4 was predominantly localized within adult cardiomyocyte mitochondria. 6mA modifications were significantly more abundant in mtDNA than in nuclear DNA. Postnatal cardiomyocyte maturation presented with a reduction in 6mA levels within mtDNA, coinciding with a decrease in METTL4 expression. However, an increase in both mtDNA 6mA level and METTL4 expression was observed in failing adult cardiomyocytes, suggesting a shift toward a neonatal-like state. METTL4 preferentially targeted mtDNA promoter regions, which resulted in interference with transcription initiation complex assembly, mtDNA transcriptional stalling, and ultimately mitochondrial dysfunction. Amplifying cardiomyocyte mtDNA 6mA through METTL4 overexpression led to spontaneous mitochondrial dysfunction and HF phenotypes. The transcription factor p53 was identified as a direct regulator of METTL4 transcription in response to HF-provoking stress, thereby revealing a stress-responsive mechanism that controls METTL4 expression and mtDNA 6mA. Cardiomyocyte-specific deletion of the Mettl4 gene eliminated mtDNA 6mA excess, preserved mitochondrial function, and mitigated the development of HF upon continuous infusion of AngII/PE. In addition, specific silencing of METTL4 in cardiomyocytes restored mitochondrial function and offered therapeutic relief in mice with preexisting HF, irrespective of whether the condition was induced by AngII/PE infusion or myocardial ischemia/reperfusion injury. CONCLUSIONS: Our findings identify a pivotal role of cardiomyocyte mtDNA 6mA and the corresponding methyltransferase, METTL4, in the pathogenesis of mitochondrial dysfunction and HF. Targeted suppression of METTL4 to rectify mtDNA 6mA excess emerges as a promising strategy for developing mitochondria-focused HF interventions.

Salt Tolerance Assessment in Triticum Aestivum and Triticum Durum.

BACKGROUND: Salt stress is a multicomponent phenomenon; it includes many processes that directly or indirectly affect the plant. Attempts have been made to comprehensively consider the processes of salt stress in plants Triticum aestivum (variety Orenburgskaya 22) and Triticum durum (variety Zolotaya). METHODS: The study used methods of light and fluorescence microscopy, methods of immunofluorodetection, expression of DNA methyltransferase genes, genes of the ion transporter and superoxide dismutase families, as well as biochemical determination of total antioxidant activity using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent. RESULTS: According to morphometric indicators, the Orenburgskaya 22 variety showed greater tolerance to the action of 150 mM NaCl than the Zolotaya variety. The level of expression of genes of the HKT ion transporter family in the Orenburgskaya 22 variety is higher than in the Zolotaya variety. It was found that the expression of the DNA methyltransferase gene DRM2.1, which post-translationally methylates cytosine residues, is 22.3 times higher in Zolotaya compared to Orenburg 22 when exposed to salt. The accumulation of toxic ions is accompanied by an increase in reactive oxygen species (ROS) and increased damage to root tissue, especially in the Zolotaya variety. Using fluorescence microscopy using the Carboxy-H2DFF marker in the Orenburgskaya 22 variety at high NaCl concentrations, the highest fluorescence intensity was determined in the cap zone; in the Zolotaya variety-in the zones of the cap and root meristem. Excess ROS is more successfully removed in the Orenburgskaya 22 variety, which has a higher level of antioxidant activity (AOA), as well as the level of expression of the Cu/ZnSOD and MnSOD superoxide dismutase genes. Using programmed cell death (PCD) markers based on the release of cytochrome c from mitochondria into the cytoplasm, DNA breakage and the release of phosphatidylserine from mitochondria, the degree of damage to root cells was assessed in both wheat varieties. It has been proven that wheat cell death occurs through the mitochondrial pathway. It was noted that the salt-sensitive variety Zolotaya had a significant number of necrotic cells. CONCLUSION: Based on the data obtained, it was concluded that the Orenburgskaya 22 variety exhibits greater resistance to salinity than the Zolotaya variety. These data may be of practical importance for enhancing protective mechanisms under abiotic stress.