Epigenetic signatures in in vitro-expanded equine chondrocytes induced by a histone deacetylase inhibitor.

DNA methylation plays pivotal roles in regulating gene expression and the secondary metabolism in plants. Salvia miltiorrhiza and Salvia bowleyana are traditional Chinese medicinal plants with roots enriched with tanshinone components. However, the regulatory mechanism of DNA methylation on tanshinone production remains elusive. Here, we analyzed 30-day-old hairy roots of S. miltiorrhiza and S. bowleyana using targeted high-performance liquid chromatography analysis and found significantly higher tanshinone content in S. miltiorrhiza. Whole-genome bisulfite sequencing revealed elevated DNA methylation levels in S. miltiorrhiza, potentially due to the upregulation of methylation-related genes, including DOMAINS REARRANGED METHYLTRANSFERASE 1 (DRM1), DECREASE IN DNA METHYLATION 1 (DDM1), CHROMOMETHYLASE 2 (CMT1), and CHROMOMETHYLASE 3 (CMT3), alongside the low expression of the demethylase gene REPRESSOR OF SILENCING 1 (ROS1) in S. miltiorrhiza. Additionally, four genes that are involved in tanshinone biosynthesis, including 1-DEOXY-D-XYLULOSE-5-PHOSPHATE REDUCTASE (DXS1), GERANYLGERANYL DIPHOSPHATE SYNTHASE (GGPPS2), 4-HYDROXY-3-METHYLBUT-2-ENYL PYROPHOSPHATE REDUCTASE (HDR2), and COPALYL PYROPHOSPHATE SYNTHASE (CPS3), showed lower methylation levels in the promoters of DXS1, GGPPS2, and CPS3 and a higher DNA methylation level in the gene body of HDR2 in S. miltiorrhiza, which may lead to their high expression and the accumulation of tanshinones. Consistently, overexpression of the SmCMT3 in S. miltiorrhiza significantly reduced the contents of cryptotanshinone, tanshinone I, and tanshinone IIA. Transcriptomic and methylome analyses confirmed that the expression levels of the tanshinone biosynthesis-related genes, including SmMK, SmCPS1, SmDXS2, and SmAACT1, were correlated with their promoter or gene body DNA methylation levels. Our findings reveal that DNA methylation critically regulates tanshinone biosynthesis in S. miltiorrhiza and S. bowleyana, offering valuable insights for breeding.

Puerarin alleviates cerebral ischemia/reperfusion (CIR)-induced neurocyte oxidative stress and apoptosis via DNA demethylation-mediated PI3K/Akt activation.

Fruit texture variation in peach (Prunus persica L. Batsch), especially softening accompanied by the melting phase, directly affects marketability and storage. Two tandem endopolygalacturonase (endoPG) genes at the F-M locus, PGF and PGM, determine the melting or nonmelting trait, which is associated with the freestone or clingstone trait. In this study, freestone melting flesh (FMF), clingstone melting flesh (CMF), and clingstone nonmelting flesh (CNMF) cultivars were sampled across various developmental and postharvest stages. PGF-PGM haplotypes were identified using molecular markers, and their contributions to the FMF/CMF/CNMF phenotypes were investigated. In FMF peach, the FMF phenotypes were related to elevated PGF expression with spatiotemporal differences between the flesh near the stone at early developmental stages and the whole flesh at the postharvest stage. PpNAC74 and PpNAC87, specifically expressed in the flesh near skin or stone, cooperatively activated PGF transcription in FMF. Two PGM alleles, PGMSer and PGMPhe, exhibited functional divergence with increased expression of the former dominantly conferring the CMF phenotype, and the recessive PGMPhe determining the CNMF phenotype. PpNAC1-PpNAC72-PpNAC100 synergistically activated PGMSer transcription during ethylene-induced melting in CMF. Transient overexpression or silencing of these PpNACs induced or repressed the expression of target endoPGs and accelerated or delayed softening, respectively. Whole-genome bisulfite sequencing suggested that DNA methylation mediates the expression of key softening-related genes and thus participates in the regulation of the flesh melting and freestone traits. Our findings elucidate the genetic and molecular regulatory network diversification that determines flesh texture and stone adhesion in peach fruit.

Epigenetic regulation of physiological resilience to ammonia nitrogen stress in the Pacific whiteleg shrimp Penaeus vannamei: Evidence from genome-wide DNA methylation dynamics.

Heat stress induces pathological and molecular responses in the gills of largemouth bass (Micropterus salmoides) revealed by histology, transcriptomics, and DNA methylomics.

Lemon (Citrus limon L.), an economically important Citrus species, produces high levels of citric acid. However, the regulatory mechanisms underlying citric acid accumulation in lemon fruit are poorly understood. In this study, we generated a haplotype-resolved genome for "Eureka", a widely cultivated commercial lemon cultivar. Based on the progenitor sequences, we elucidated the origin and identified some domesticated loci of the lemon haplomes, including those associated with citric acid metabolism. Comparative genomics analysis revealed that the gene families enriched in the pathways related to stress responses and soluble sugar biosynthesis were dramatically contracted in the lemon genome. Substantial allelic variations in sequences, gene expression and methylation levels were detected between the 2 haplotypes. Of note, transcript levels of vacuolar P-ATPases, PH5, encoding a proton pump involved in citric acid accumulation, were drastically higher in Eureka lemon relative to sweet lemon, which contains trace amount of citric acid in the fruit. In addition, whole-genome bisulfite sequencing revealed that the promoter of PH5 was highly methylated in sweet lemon but not in Eureka. Furthermore, demethylation of the PH5 promoter led to an increase in the citric acid content. Taken together, these findings demonstrate that low DNA methylation level in the promoter region of PH5 contributes to the abundant accumulation of citric acid in lemon fruit. Our study provides a valuable genetic resource for investigating the domestication mechanism in citrus and underpins genome-based genetic engineering to create either acid or acidless cultivars.

DNA methylation, metabolome, and transcriptome analysis reveal epigenomic differences between diploid Chinese cabbage seeds and tetraploid Chinese cabbage seeds.

ABSTRACTObesity‐related complications are often driven by chronic inflammation and oxidative stress, exacerbated by aberrant DNA methylation. Natural products with anti‐inflammatory and antioxidant properties may offer therapeutic potential. This study investigated the potential molecular mechanisms underlying the effects of Rosa roxburghii Tratt fermentation broth (RRTFB) on obesity through targeted methylation, while also examining its primary active components and assessing its potential therapeutic value. Male SD rats were fed a high‐fat diet (HFD) to induce obesity, with RRTFB administered as an intervention. Various methods, including reduced representation bisulfite sequencing analysis (RRBS), molecular docking, surface plasmon resonance (SPR), and other analytical methods were employed for the study. The results showed that, compared to the HFD‐fed rats, the RRTFB intervention groups (HFH and HFL) exhibited a significant reduction in MDA, IL‐6, TNF‐α and DNMT3a levels, along with increased SOD, GSH‐pX, and CAT activities in epididymal fat. RRBS revealed a significant number of differential methylation regions (DMRs) in genes related to fat metabolism, oxidative stress, and inflammation in HFD‐fed rats and HFH. Protein interaction analysis and subsequent validation experiments identified SIRT1 as a key regulator mediating the efficacy of RRTFB: RRTFB reduced SIRT1 promoter methylation and enhanced its expression. In 3T3‐L1 cells with Dnmt3a overexpression, SIRT1 levels were significantly reduced. ChIP‐qPCR further confirmed an enhanced binding of Dnmt3a to the SIRT1 promoter. Molecular docking and SPR confirmed that flavonoids, the active components of RRTFB, could directly bind to DNMT3a and modulate its activity. This study substantiates the potential of RRTFB as a phytochemo therapeutic strategy for combating obesity, highlighting its ability to mitigate obesity through DNMT3a/SIRT1‐mediated epigenetic regulation, with flavonoids identified as the primary bioactive components.

Heterosis, or hybrid vigor, refers to the superior phenotypes of a hybrid compared with their parents and is widely exploited in agriculture. Interspecific hybrids within the Oryza genus demonstrate significant potential for the systematic improvement of rice varieties. Nevertheless, the mechanistic basis underlying heterosis in interspecific Oryza hybrids remains poorly understood. Here, we systematically performed phenotypic characterization, whole-genome bisulfite sequencing, RNA sequencing, and small RNA profiling using Oryza sativa L. ssp. japonica cv. Nipponbare (NIP), Oryza rufipogon Griff. acc. CWR, and their resulting F1 hybrid (named as NC). NIP and CWR showed distinct phenotypic and molecular differences. The interspecific hybrid, NC, exhibited significant yield heterosis. In the hybrid, most epigenetic and transcriptional features displayed additive inheritance patterns relative to parental lines. Analysis revealed that domestication-selected genes maintained relatively low DNA methylation coupled with high expression levels in both hybrid and parental lines. Additionally, we identified that non-additive miRNAs were potentially involved in regulating fertility, cell growth, and cell division processes in the hybrid. A significant negative correlation was observed between DNA methylation level and gene expression. Functional enrichment analysis revealed that hybrid-MPV DEGs were significantly associated with flowering time regulation, carbohydrate metabolism, photosynthesis, protein phosphorylation, seed development, and defense responses. Through weighted gene co-expression network analysis, we identified 102 functional gene modules, six of which were significantly associated with yield-related heterosis. Collectively, our results provide a multi-omics framework for understanding interspecific hybridization between elite cultivars and wild rice relatives, highlighting CWR as an untapped genetic reservoir for rice improvement.

cfDNAFE: Comprehensively extracting multi-omics features of cell-free DNA for noninvasive diagnosis.

Comparative genome analysis and global methylation patterns for epigenetic study in the brackish water flea Diaphanosoma celebensis.

Characterization of methylation profile in biofluid cell-free DNA and identification of differentially methylated genes for phenotypic representations in Parkinson's disease.

Sex hormones are important factors in maintaining brain function and acting as brain protectors. Recent research suggests that neuronal damage in brain aging may be linked to the methylation of the estrogen receptor α (ERα). However, the mechanism of Zuogui Pills (ZGW) in brain-aging ERα DNA methylation and neuronal repair remains unknown. D-galactose-induced ovary removal mice were used as a model of aging. Changes in estrous cycle were detected in mice by vaginal cell smear. Animal behavior tests, including the Morris water maze (MWM) and new object recognition (NOR) test, were conducted. Hematoxylin-eosin (HE) and Nissl-staining were carried out to assess hippocampal neurogenesis. Enzyme-linked immunosorbent assay (ELISA) was performed for 5- methylcytosine methylation levels, and immunohistochemistry (IHC) and western blotting (WB) experiments were performed to assess ERα/DNA methyltransferase 1 (DNMT1) expression after ZGW treatment. Finally, bisulfite sequencing PCR (BSP) analysis was performed to identify methylated differentially expressed estrogen receptor 1 (ESR1) gene in D-gal-induced senescent neurons before and after ZGW treatment. We found that ERα methylation was involved in the delayed brain ageing process of ZGW. Mechanistically, ZGW can improve the learning and memory ability of brain-aging mice, reduce the expression of 5-methylcytosine (5-mc) in serum, increase the amount of ERα, inhibit the expression of DNMT1, and significantly reduce methylated expression of the ESRI gene. Our data suggested that ZGW slowed down D-gal-induced brain aging in mice, and these results showed that ZGW is beneficial for aging. It may be used for neuronal protection in aging.

White lupin exhibits remarkable adaptability to phosphorus (P)-deficient soil through the development of cluster roots (CR), thereby enhancing P use sufficiency. Despite its crucial role, the underlying mechanism governing CR formation remains elusive. Here, we reveal an elevated DNA methylation level through whole-genome bisulfite sequencing in CR in response to P deficiency, particularly in gene and flanking regions, suggesting a responsive epigenetic mechanism. To further investigate the potential involvement of epigenetic remodelling, we treated lupin plants with the DNA methyltransferase (DNMT) inhibitor 5-azacytidine, which led to a disruption of total DNMT activity and impaired CR formation under phosphorus-deficient conditions. Integrated analysis of methylome and RNA-Seq highlights the methylation of CAFFEIC ACID O-METHYLTRANSFERASE 1 (COMT1), a key enzyme in melatonin synthesis, as pivotal for promoting CR formation in white lupin. Functional validation through overexpression or gene silencing of LaCOMT1 in transgenic lupin roots confirms the positive impact of LaCOMT1 on CR formation. Furthermore, melatonin application directly increases CR numbers, indicating the role of methylation-activated LaCOMT1 in promoting CR formation via melatonin synthesis. Those findings provide insights into the epigenomic landscape of white lupin, establishing a direct genetic link between epigenetic mechanisms and P-deficiency-induced CR formation.

Pearl millet (Pennisetum glaucum) is a major staple food in arid and semi-arid regions of sub-Saharan Africa, India, and South Asia. However, how epigenetic mechanisms regulate tissue-specific gene expression in this crop remains poorly understood. In this study, we profiled multiple epigenetic features in the young panicles and roots of pearl millet using RNA-seq, ATAC-seq, whole-genome bisulfite sequencing, and ChIP-seq (H3K4me3 and H3K36me3). We identified thousands of genes that were differentially expressed between these two tissues. Root-specific genes were enriched for plant hormone signaling, oxidative phosphorylation, and stress responses. Analysis of chromatin accessibility revealed that root-specific accessible chromatin regions (ACRs) were enriched in binding motifs for stress-responsive transcription factors (e.g., NAC, WRKY), whereas ACRs in young panicles were enriched in motifs for developmental regulators (e.g., AP2/ERF). DNA methylation profiling revealed 25,141 tissue-specific differentially methylated regions, with CHH methylation—rather than CG or CHG methylation—showing the strongest tissue specificity. Promoters of root-specific genes had higher levels of CHH methylation compared to those of young panicle–specific genes, suggesting that the roles of CHH methylation in regulating transcription might be tissue dependent. Notably, promoter-associated H3K4me3 marked panicle-specific genes, whereas root-specific expression was primarily linked to chromatin accessibility, suggesting a transcription factor–mediated regulatory mechanism. Together, our findings highlight the distinct epigenetic frameworks governing tissue-specific gene expression in pearl millet and provide valuable insights for advancing the genetic improvement of this crop.Supplementary InformationThe online version contains supplementary material available at 10.1007/s42994-025-00243-2.

The incidence of urothelial carcinoma (UC) ranks second among all urological cancers, accounting for over 90% of malignant tumors in bladder. Patients diagnosed with UC experience a lower quality of life due to rapid progression of the disease. Early and non-invasive detection is curial for diagnosing UC and improving patient outcomes. This study aimed to develop and validate a DNA methylation assay for the early detection and monitoring for UC, with a focus on its diagnostic and prognostic implications. The DNA methylation assay with real-time methylation specific polymerase chain reaction (RT-MSP) technique measures the methylation level of SOX1-OT and HIST1H4F in urine samples. A cohort comprising 436 patients diagnosed with UC or other urologic disease as well as 79 healthy patients was retrospectively utilized to evaluate this assay. Furthermore, UC patients who underwent surgery are included to assess its ability to detect recurrence. The DNA methylation assay demonstrated significantly increased methylation level in tumor tissues and a high positive rate in urine samples from UC patients. The methylation profile effectively distinguishes UC from other non-UC urologic disease, exhibiting a high sensitivity (85.2%) and specificity (90.0%) for UC diagnosis. Furthermore, the assay showed promising ability in differentiating UC patients based on tumor grade, malignancy potential, and disease stage. Additionally, the DNA methylation assay demonstrated a superior ability to detect UC recurrence with a high area under curve (AUC) of 0.979. This research proposes a novel DNA methylation assay with urine as sample for detection, representing a cost-efficient and non-invasive method for diagnosing and monitoring UC disease.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-14646-0.

Acute myeloid leukemia (AML) is a highly heterogeneous disease, and molecular events such as DNMT3A gene mutations are associated with poor prognosis in AML patients. Consequently, there is an urgent need for a novel therapeutic approach for AML. DNMT3A mRNA and protein expression were confirmed inDNMT3A-mutant AML cellsvia RT-qPCR and Western blotting. Cell proliferation and apoptosis were assessed via CCK-8 and Annexin V/PI staining, respectively. Flow cytometry was used to analyze surface antigens and CD44v6 CAR-T-cell transfection efficiency. CD44v6-directed CAR plasmids were constructed, and lentiviruses were packaged. Methylation-specific PCR was used to evaluate differences in promoter methylation, whereas ELISA was used to measure cytokine secretion. In this study, we found that the DNMT3A-mutant group presented significantly increased expression of CD44v6 on the cell surface. Methylation of the CD44 promoter region was lower in the mutant group than in the control group. CD44v6 CAR-T cells exhibited specific cytotoxicity against DNMT3A-mutant AML cells. Furthermore, pretreatment with low concentrations of decitabine significantly enhanced the killing effect of CD44v6 CAR-T cells on DNMT3A-mutant AML cells (P < 0.05). Additionally, decitabine treatment upregulated the expression of CD44v6 on the surface of DNMT3A-mutant AML cells (P < 0.05). CD44v6 is a promising CAR-T-cell therapy target in AML patients with DNMT3A mutations. Notably, treatment with decitabine resulted in increased CD44v6 expression on the cell surface of DNMT3A-mutant AML cells. This increase in CD44v6 expression facilitates improved recognition and targeting by CD44v6 CAR-T cells.

BackgroundDNA methylation is an epigenetic mechanism involved in gene regulation and cellular differentiation. Accurate and comprehensive assessment of DNA methylation patterns is thus essential for understanding their role in various biological processes and disease mechanisms. Bisulfite sequencing has long been the default method for analyzing methylation marks due to its single-base resolution, but the associated DNA degradation poses a concern. Although several methods have been proposed to circumvent this issue, there is no clear consensus on which method might be better suited for specific study designs.ResultsWe conducted a comparative evaluation of four DNA methylation detection approaches: whole-genome bisulfite sequencing (WGBS), Illumina methylation microarray (EPIC), enzymatic methyl-sequencing (EM-seq) and third-generation sequencing by Oxford Nanopore Technologies (ONT). DNA methylation profiles were assessed across three human genome samples derived from tissue, cell line, and whole blood. We systematically compared these methods in terms of resolution, genomic coverage, methylation calling accuracy, cost, time, and practical implementation. EM-seq showed the highest concordance with WGBS, indicating strong reliability due to their similar sequencing chemistry. ONT sequencing, while showing lower agreement with WGBS and EM-seq, captured certain loci uniquely and enabled methylation detection in challenging genomic regions. Despite a substantial overlap in CpG detection among methods, each method identified unique CpG sites, emphasizing their complementary nature.ConclusionsOur findings underscore the strengths and limitations of current DNA methylation detection methods. EM-seq and ONT emerge as robust alternatives to WGBS and EPIC, offering unique advantages: EM-seq delivers consistent and uniform coverage, while ONT excels in long-range methylation profiling and access to challenging genomic regions. These insights provide practical guidance for method selection based on specific experimental goals.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13072-025-00616-3.

BackgroundEpitranscriptomic data indicate that aberrant tRNA modifications in malignant diseases can promote tumor growth by facilitating oncogene translation. NSUN2, a 5-methylcytosine (m5C) methyltransferase of tRNA, is elevated in an array of solid cancers, including triple-negative breast cancer (TNBC). However, it remains unclear how NSUN2 drives aggressive behavior and if NSUN2 could be an effective therapeutic target for TNBC.MethodsFunctional experiments, including RNA interference, lentivirus transduction, and in vivo xenograft models, were conducted to evaluate the role of NSUN2 in TNBC cell proliferation, metastasis, and chemoresistance. Ribosome sequencing (Ribo-seq), tRNA m5C bisulfite sequencing, and codon usage bias analysis were employed to explore the translational mechanisms underlying NSUN2-mediated tRNA modifications. Glycolysis assays and molecular docking were used to investigate metabolic reprogramming and protein interactions.ResultsNSUN2 was significantly upregulated in TNBC and correlated with poor patient prognosis. Mechanistically, NSUN2 mediates m5C modification of tRNAVal−CAC, enhancing the codon-frequency-dependent translation of key glycolysis-related genes, including ALDH3A2, ALDH7A1, HK1, and PFKM. Depletion of NSUN2 disrupted tRNAVal−CAC m5C modification, impairing the translation of these metabolic enzymes and suppressing glycolysis, which ultimately inhibited TNBC cell proliferation, migration, and invasion both in vitro and in vivo. Furthermore, NSUN2 overexpression conferred resistance to docetaxel, while its inhibition sensitized TNBC cells to docetaxel treatment. Clinically, elevated expression levels of NSUN2 and glycolysis-related genes were observed in docetaxel-resistant TNBC tissues, further supporting the role of NSUN2 in chemoresistance.ConclusionsThis study identifies NSUN2 as a critical regulator of TNBC progression through tRNAVal−CAC m5C modification and codon-biased translation of glycolysis-related mRNAs. Our findings reveal a novel NSUN2–tRNAVal−CAC axis that orchestrates metabolic reprogramming and translational control in TNBC, offering a promising prognostic biomarker and therapeutic target.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s11658-025-00781-z.