Differentially Methylated Genes Research Articles

DNA Methylation Profiling of Ovarian Tissue of Climbing Perch (Anabas testudienus) in Response to Monocrotophos Exposure.

Epigenetic modifications like DNA methylation can alter an organism's phenotype without changing its DNA sequence. Exposure to environmental toxicants has the potential to change the resilience of aquatic species. However, little information is available on the dynamics of DNA methylation in fish gonadal tissues in response to organophosphates. In the present work, reduced-representation bisulfite sequencing was performed to identify DNA methylation patterns in the ovarian tissues of Anabas testudienus exposed to organophosphates, specifically monocrotophos (MCP). Through sequencing, an average of 41,087 methylated cytosine sites were identified and distributed in different parts of genes, i.e., in transcription start sites (TSS), promoters, exons, etc. A total of 1058 and 1329 differentially methylated regions (DMRs) were detected as hyper-methylated and hypo-methylated in ovarian tissues, respectively. Utilizing whole-genome data of the climbing perch, the DMRs, and their associated overlapping genes revealed a total of 22 genes within exons, 45 genes at transcription start sites (TSS), and 218 genes in intergenic regions. Through gene ontology analysis, a total of 16 GO terms particularly involved in ovarian follicular development, response to oxidative stress, oocyte maturation, and multicellular organismal response to stress associated with reproductive biology were identified. After functional enrichment analysis, relevant DMGs such as steroid hormone biosynthesis (Cyp19a, 11-beta-HSD, 17-beta-HSD), hormone receptors (ar, esrrga), steroid metabolism (StAR), progesterone-mediated oocyte maturation (igf1ar, pgr), associated with ovarian development in climbing perch showed significant differential methylation patterns. The differentially methylated genes (DMGs) were subjected to analysis using real-time PCR, which demonstrated altered gene expression levels. This study revealed a molecular-level alteration in genes associated with ovarian development in response to chemical exposure. This work provides evidence for understanding the relationship between DNA methylation and gene regulation in response to chemicals that affect the reproductive fitness of aquatic animals.

Genome-wide DNA methylation mediates the resistance to vibriosis in Cynoglossus semilaevis

Chinese tongue sole (Cynoglossus semilaevis) is an economically important marine fish in China. However, vibriosis has caused huge mortality and economic losses in its culturing industry. To reveal the effect of DNA methylation on the resistance to vibriosis in tongue sole, we conducted RNA sequencing and whole genome bisulfite sequencing (WGBS), and compared the gene expressions and DNA methylation patterns between the resistant and susceptible families. We identified a total of 741 significantly differentially expressed genes (DEGs) in kidney and 17460 differentially methylated genes (DMGs), which were both enriched in immune-related pathways, such as “cAMP signaling pathway” and “NOD-like receptor signaling pathway”. Through the correlation analysis of DEGs and DMGs, we identified two important immune pathways, including “complement and coagulation cascades”, and “cytokine-cytokine receptor interaction”, which played important roles in regulating the inflammation level and immune homeostasis. For example, the expression of proinflammatory cytokine il17c was down-regulated under the regulation of DNA methylation; in addition, the expression of protease-activated receptor 3 (par3) was up-regulated, which could induce the up-expressionof il8. These results demonstrated that the regulation of DNA methylation on the genes involved in immune responses might contribute to the resistance to vibriosis in tongue sole, and provided a basis for the control of diseases in fish aquaculture.

Whole-genome bisulfite sequencing reveals the function of DNA methylation in the allotransplantation immunity of pearl oysters.

In the pearl culture industry, a major challenge is the overactive immunological response in pearl oysters resulting from allotransplantation, leading to shell-bead rejection and death. To better understand the molecular mechanisms of postoperative recovery and the regulatory role of DNA methylation in gene expression, we analyzed the changes in DNA methylation levels after allotransplantation in pearl oyster Pinctada fucata martensii, and elucidated the regulatory function of DNA methylation in promoter activity of nicotinic acetylcholine receptor (nAChR) gene. We constructed nine DNA methylomes at different time points after allotransplantation and used bisulfite genomic sequencing PCR technology (BSP) to verify the methylation status in the promoter of nAChR. We performed Dual luciferase assays to determine the effect of the dense methylation region in the promoter on transcriptional activity and used DNA pull-down and mass spectrometry analysis to assess the capability of transcription factor binding with the dense methylation region. The DNA methylomes reveal that CG-type methylation is predominant, with a trend opposite to non-CG-type methylation. Promoters, particularly CpG island-rich regions, were less frequently methylated than gene function elements. We identified 5,679 to 7,945 differentially methylated genes (DMGs) in the gene body, and 2,146 to 3,385 DMGs in the promoter at each time point compared to the pre-grafting group. Gene ontology and pathway enrichment analyses showed that these DMGs were mainly associated with "cellular process", "Membrane", "Epstein-Barr virus infection", "Notch signaling pathway", "Fanconi anemia pathway", and "Nucleotide excision repair". Our study also found that the DNA methylation patterns of the promoter region of nAChR gene were consistent with the DNA methylomics data. We further demonstrated that the dense methylation region in the promoter of nAChR affects transcriptional activity, and that the methylation status in the promoter modulates the binding of different transcription factors, particularly transcriptional repressors. These findings enhance our understanding of the immune response and regulation mechanism induced by DNA methylation in pearl oysters after allotransplantation.

Circulating epigenomic biomarkers correspond with kidney disease susceptibility in high-risk populations with type 2 diabetes mellitus

AimsTo investigate epigenomic indices of diabetic kidney disease (DKD) susceptibility among high-risk populations with type 2 diabetes mellitus. MethodsKDIGO (Kidney Disease: Improving Global Outcomes) clinical guidelines were used to classify people living with or without DKD. Differential gene methylation of DKD was then assessed in a discovery Aboriginal Diabetes Study cohort (PROPHECY, 89 people) and an external independent study from Thailand (THEPTARIN, 128 people). Corresponding mRNA levels were also measured and linked to levels of albuminuria and eGFR. ResultsIncreased DKD risk was associated with reduced methylation and elevated gene expression in the PROPHECY discovery cohort of Aboriginal Australians and these findings were externally validated in the THEPTARIN diabetes registry of Thai people living with type 2 diabetes mellitus. ConclusionsNovel epigenomic scores can improve diagnostic performance over clinical modelling using albuminuria and GFR alone and can distinguish DKD susceptibility.

Identification and analysis of RNA-5-methylcytosine-related key genes in osteoarthritis

Background5-methylcytosine (m5C) modification is widely associated with many biological and pathological processes. However, knowledge of m5C modification in osteoarthritis (OA) remains lacking. Thus, our study aimed to identify common m5C features in OA.ResultsIn the present study, we identified 1395 differentially methylated genes (DMGs) and 1673 differentially expressed genes (DEGs) using methylated RNA immunoprecipitation next-generation sequencing (MeRIP-seq) and RNA-sequencing. A co-expression analysis of DMGs and DEGs showed that the expression of 133 genes was significantly affected by m5C methylation. A protein–protein interaction network of the 133 genes was constructed using the STRING database, and the cytoHubba plug-in of Cytoscape was used to hub genes were screen out 11 hub genes, including MMP14, VTN, COL15A1, COL6A2, SPARC, COL5A1, COL6A3, COL6A1, COL8A2, ADAMTS2 and COL7A1. The Pathway enrichment analysis by the ClueGO and CluePedia plugins in Cytoscape showed that the hub genes were significantly enriched in collagen degradation and extracellular matrix degradation.ConclusionsOur study indicated that m5C modification might play an important role in OA pathogenesis, and the present study provides worthwhile insight into identifying m5C-related therapeutic targets in OA.

Analysis of Methylome, Transcriptome, and Lipid Metabolites to Understand the Molecular Abnormalities in Polycystic Ovary Syndrome.

This study aimed to identify differentially methylated genes (DMGs) and differentially expressed genes (DEGs) to investigate new biomarkers for the diagnosis and treatment of polycystic ovary syndrome (PCOS). To explore the potential biomarkers of PCOS diagnosis and treatment, we performed methyl-binding domain sequencing (MBD-seq) and RNA sequencing (RNA-seq) on ovarian granulosa cells (GCs) from PCOS patients and healthy controls. MBD-seq was also performed on the ovarian tissue of constructed prenatally androgenized (PNA) mice. Differential methylation and expression analysis were implemented to identify DMGs and DEGs, respectively. The identified gene was further verified by real-time quantitative PCR (RT-qPCR) and methylation-specific PCR (MSP) in clinical samples. Furthermore, ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) was carried out on PCOS patients and healthy controls to identify differential lipid metabolites. Compared to the control group, 13,526 DMGs related to the promoter region and 2429 DEGs were found. The function analysis of DMGs and DEGs showed that they were mainly enriched in glycerophospholipid, ovarian steroidogenesis, and other lipid metabolic pathways. Moreover, 5753 genes in DMGs related to the promoter region were screened in the constructed PNA mice. Integrating the DMGs data from PCOS patients and PNA mice, we identified the following 8 genes: CDC42EP4, ERMN, EZR, PIK3R1, ARHGEF18, NECTIN2, TSC2, and TACSTD2. RT-qPCR and MSP verification results showed that the methylation and expression of TACSTD2 were consistent with sequencing data. Additionally, 15 differential lipid metabolites were shown in the serum of PCOS patients. The differential lipids were involved in glycerophospholipid and glycerolipid metabolism. Using integration of methylome and lipid metabolites profiling we identified 8 potential epigenetic markers and 15 potential lipid metabolite markers for PCOS. Our results suggest that aberrant DNA methylation and lipid metabolite disorders may provide novel insights into the diagnosis and etiology of PCOS.

Identification of Potential Abnormal Methylation-Modified Genes in Coronary Artery Ectasia.

Coronary artery ectasia (CAE) is an easily recognized abnormality of coronary artery anatomy and morphology. However, its pathogenesis remains unclear. This study aimed to identify abnormal methylation-modified genes in patients with CAE, which could provide a research basis for CAE. Peripheral venous blood samples from patients with CAE were collected for RNA sequencing to identify differentially expressed genes (DEGs), followed by functional enrichment. Then, the DNA methylation profile of CAE was downloaded from GSE87016 (HumanMethylation450 BeadChip data, involving 11 cases and 12 normal controls) to identify differentially methylated genes (DMGs). Finally, after taking interaction genes between DEGs and DMGs, abnormal methylation-modified genes were identified, followed by protein-protein interaction analysis and expression validation using reverse transcriptase polymerase chain reaction. A total of 152 DEGs and 4318 DMGs were obtained from RNA sequencing and the GSE87016 dataset, respectively. After taking interaction genes, 9 down-regulated DEGs due to hypermethylation and 11 up-regulated DEGs due to hypomethylation were identified in CAE. A total of 10 core abnormal methylation-modified genes were identified, including six down-regulated DEGs due to hypermethylation (netrin G1, ADAM metallopeptidase domain 12, immunoglobulin superfamily member 10, sarcoglycan dela, Dickkopf WNT signaling pathway inhibitor 3, and GATA binding protein 6), and four up-regulated DEGs due to hypomethylation (adrenomedullin, ubiquitin specific peptidase 18, lymphocyte antigen 6 family member E, and MX dynamin-like GTPase 1). Some signaling pathways were identified in patients with CAE, including cell adhesion molecule, O-glycan biosynthesis, and the renin-angiotensin system. Abnormal methylation-modified DEGs involved in signaling pathways may be involved in CAE development.

Prenatal transportation stress does not impact resting skeletal muscle mitochondrial function or antioxidant activity in Brahman calves

In cattle, prenatal transportation stress has been associated with differential methylation of genes related to metabolism, but the effects of prenatal transportation stress on skeletal muscle mitochondria and oxidative stress have not been investigated. We tested the hypothesis that prenatally stressed calves would exhibit increased skeletal muscle mitochondrial function resulting in greater oxidative stress than calves from non-stressed dams. Serum and longissimus thoracis muscle samples were collected from yearling Brahman calves whose mothers were stressed by transportation at five time points during gestation [i.e., prenatally stressed (PNS); eight bulls and six heifers] and control calves (CON; four bulls and six heifers). Serum was evaluated for concentration of the stress hormone, cortisol and for a marker of muscle perturbation, creatine kinase activity. Muscle samples were analyzed for concentration of a by-product of lipid peroxidation, malondialdehyde, and activity of the antioxidants, superoxide dismutase and glutathione peroxidase. Additionally, muscle mitochondrial volume density and function were estimated by citrate synthase and cytochrome c oxidase activities, respectively. Data were analyzed using mixed linear models with sex, treatment, and the sex × treatment interaction as fixed effects. No investigated variable differed between CON and PNS calves (p ≥ 0.3). These data suggest that prenatal transportation stress does not have an impact on skeletal muscle mitochondrial metabolism or markers of stress or muscle damage in Brahman yearling calves at rest. However, previously reported negative impacts of prenatal stress on inflammatory responses suggest that PNS calves may be differentially equipped to handle an acute stressor. Future research should investigate the energetic and inflammatory implications of acute stressors in animals subjected to prenatal stress.

Potential Involvement of DNA Methylation in Hybrid Sterility in Hermaphroditic Argopecten Scallops.

DNA methylation is an important epigenetic modification factor in regulating fertility. Corresponding process remains poorly investigated in hermaphroditic scallops. The interspecific F1 hybrids between the hermaphroditic bay scallops (Argopecten irradians) and Peruvian scallops (Argopecten purpuratus) exhibited significant heterosis in yield, but sterility in hybrids obstructs the utilization of the genetic resources. However, the determination mechanism of hybrid sterility in the hermaphroditic Argopecten scallops is still unclear. In this study, the effect of DNA methylation in the hybrid sterility of hermaphroditic Argopecten scallops was explored. The results showed that the mean methylation level was higher in sterile hybrids than fertile hybrids, especially on chromosome 11 of the paternal parent. A total of 61,062 differentially methylated regions (DMRs) were identified, containing 3619 differentially methylated genes (DMGs) and 1165 differentially methylated promoters that are located in the DMRs of CG sequence context. The hyper-methylated genes were enriched into five KEGG pathways, including ubiquitin-mediated proteolysis, ECM-receptor interaction, non-homologous end-joining, notch signaling, and the mismatch repair pathways. The DMGs might induce hybrid sterility by inhibition of oogenesis and egg maturation, induction of apoptosis, increased ROS, and insufficient ATP supply. Our results would enrich the determination mechanism of hybrid sterility and provide new insights into the utilization of the genetic resources of the interspecific hybrids.

Regulatory role of N6-methyladenosine in intramuscular fat deposition in chicken

Intramuscular fat (IMF) has a pivotal influence on meat quality, with its deposition being a multifaceted physiological interaction of several regulatory factors. N6-methyladenosine (m6A), the preeminent epigenetic alteration among eukaryotic RNA modifications, holds a crucial role in moderating post-transcriptional gene expression. However, there is a dearth of comprehensive understanding regarding the functional machinery of m6A modification in the context of IMF deposition in poultry. Our current study entails an analysis of the disparities in IMF within the breast and leg of 180-day-old Jingyuan chickens. We implemented methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to delve into the distribution of m6A and its putative regulatory frameworks on IMF deposition in chickens. The findings demonstrated a markedly higher IMF content in leg relative to breast (P < 0.01). Furthermore, the expression of METTL14, WTAP, FTO, and ALKBH5 was significantly diminished in comparison to that of breast (P < 0.01). The m6A peaks in the breast and leg primarily populated 3'untranslated regions (3'UTR) and coding sequence (CDS) regions. The leg, when juxtaposed with the breast, manifested 176 differentially methylated genes (DMGs), including 151 hyper-methylated DMGs and 25 hypo-methylated DMGs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed a pronounced enrichment of DMGs in the biosynthesis of amino acids, peroxisome, Fatty acid biosynthesis, fatty acid elongation, and cell adhesion molecules (CAMs) pathways. Key DMGs, namely ECH1, BCAT1, and CYP1B1 were implicated in the regulation of muscle lipid anabolism. Our study offers substantial insight and forms a robust foundation for further exploration of the functional mechanisms of m6A modification in modulating IMF deposition. This holds profound theoretical importance for improving and leveraging meat quality in indigenous chicken breeds.

Whole-Genome Methylation Sequencing of Large Yellow Croaker (Larimichthys crocea) Liver Under Hypoxia and Acidification Stress.

Large yellow croaker (Larimichthys crocea) is an important aquaculture species in China. This study analysed whole-genome methylation differences in liver tissues of young fish under different hypoxic and acidification conditions. Differentially methylated regions (DMRs) and differentially methylated genes (DMGs) were identified. Gene ontology (GO) and Kyoto encyclopaedia of genes and genomes (KEGG) enrichment analyses of DMGs were conducted to explore the mechanism of coping with hypoxic acidification. The main methylation type was CG, accounting for > 70% of total methylation, significantly higher than CHG and CHH methylation types. GO enrichment analysis of DMGs revealed strong enrichment of nervous system development, cell periphery, plasma membrane, cell junction organisation, cell junction, signalling receptor activity, molecular sensor activity, cell-linked tissue junction organisation, cell-cell adhesion and nervous system development. KEGG enrichment analysis of DMR-related genes identified cell adhesion molecules, cortisol synthesis and secretion and aldosterone synthesis and secretion as the three key pathways regulating the physiological responses to hypoxia and acidification. Long-term hypoxic and acidification stress affected the immune system, nervous system and stress responses of large yellow croaker. Whole-genome sequencing analysis of exposed tissues was used to investigate changes that occur in L. crocea in response to hypoxic and acidic conditions at the DNA methylation level. The findings contribute to our comprehensive understanding of functional methylation in large yellow croaker and will support future research on the response mechanisms of this species under different environmental pressures.

Osteoarthritis related epigenetic variations in miRNA expression and DNA methylation

Osteoarthritis (OA) is chronic arthritis characterized by articular cartilage degradation. However, a comprehensive regulatory network for OA-related microRNAs and DNA methylation modifications has yet to be established. Thus, we aimed to identify epigenetic changes in microRNAs and DNA methylation and establish the regulatory network between miRNAs and DNA methylation. The mRNA, miRNA, and DNA methylation expression profiles of healthy or osteoarthritis articular cartilage samples were downloaded from Gene Expression Omnibus (GEO) database, including GSE169077, GSE175961, and GSE162484. The differentially expressed genes (DEGs), differentially expressed miRNAs (DEMs), and differentially methylated genes (DMGs) were analyzed by the online tool GEO2R. DAVID and STRING databases were applied for functional enrichment analysis and protein-protein interaction (PPI) network. Potential therapeutic compounds for the treatment of OA were identified by Connectivity map (CMap) analysis. A total of 1424 up-regulated DEGs, 1558 down-regulated DEGs, 5 DEMs with high expression, 6 DEMs with low expression, 1436 hypermethylated genes, and 455 hypomethylated genes were selected. A total of 136 up-regulated and 65 downregulated genes were identified by overlapping DEGs and DEMs predicted target genes which were enriched in apoptosis and circadian rhythm. A total of 39 hypomethylated and 117 hypermethylated genes were obtained by overlapping DEGs and DMGs, which were associated with ECM receptor interactions and cellular metabolic processes, cell connectivity, and transcription. Moreover, The PPI network showed COL5A1, COL6A1, LAMA4, T3GAL6A, and TP53 were the most connective proteins. After overlapping of DEGs, DMGs and DEMs predicted targeted genes, 4 up-regulated genes and 11 down-regulated genes were enriched in the Axon guidance pathway. The top ten genes ranked by PPI network connectivity degree in the up-regulated and downregulated overlapping genes of DEGs and DMGs were further analyzed by the CMap database, and nine chemicals were predicted as potential drugs for the treatment of OA. In conclusion, TP53, COL5A1, COL6A1, LAMA4, and ST3GAL6 may play important roles in OA genesis and development.

The identification of hub-methylated differentially expressed genes in osteoarthritis patients is based on epigenomic and transcriptomic data.

Osteoarthritis (OA) refers to a commonly seen degenerative joint disorder and a major global public health burden. According to the existing literature, osteoarthritis is related to epigenetic changes, which are important for diagnosing and treating the disease early. Through early targeted treatment, costly treatments and poor prognosis caused by advanced osteoarthritis can be avoided. This study combined gene differential expression analysis and weighted gene co-expression network analysis (WGCNA) of the transcriptome with epigenome microarray data to discover the hub gene of OA. We obtained 2 microarray datasets (GSE114007, GSE73626) in Gene Expression Omnibus (GEO). The R software was utilized for identifying differentially expressed genes (DEGs) and differentially methylated genes (DMGs). By using WGCNA to analyze the relationships between modules and phenotypes, it was discovered that the blue module (MEBlue) has the strongest phenotypic connection with OA (cor = 0.92, p = 4e-16). The hub genes for OA, also known as the hub methylated differentially expressed genes, were identified by matching the MEblue module to differentially methylated differentially expressed genes. Furthermore, this study used Gene set variation analysis (GSVA) to identify specific signal pathways associated with hub genes. qRT-PCR and western blotting assays were used to confirm the expression levels of the hub genes in OA patients and healthy controls. Three hub genes were discovered: HTRA1, P2RY6, and RCAN1. GSVA analysis showed that high HTRA1 expression was mainly enriched in epithelial-mesenchymal transition and apical junction; high expression of P2RY6 was mainly enriched in the peroxisome, coagulation, and epithelial-mesenchymal transition; and high expression of RCAN1 was mainly enriched in epithelial-mesenchymal-transition, TGF-β-signaling, and glycolysis. The results of the RT-qPCR and WB assay were consistent with the findings. The three genes tested may cause articular cartilage degeneration by inducing chondrocyte hypertrophy, regulating extracellular matrix accumulation, and improving macrophage pro-inflammatory response, resulting in the onset and progression of osteoarthritis. They can provide new ideas for targeted treatment of osteoarthritis.

Genome-wide DNA methylation and transcriptome analyses reveal important epigenetic genes associated with valgus-varus deformity in broilers

ABSTRACT 1. Valgus-varus deformity (VVD) is a common leg bone problem in broilers that causes serious economic losses to the breeding industry. The genetic aetiology of VVD is not clear, which restricts the genetic control of VVD. 2. In this study, knee cartilage of 35-day-old VVD and normal broilers was sequenced by whole-genome bisulphite sequencing (WGBS). The unique whole-genome DNA methylation profile of VVD broilers was described, and the methylation data and transcription data were used for joint analysis. 3. The mean methylation level of the VVD group was greater than that in the normal group. A total of 4315 differentially methylated regions (DMRs) were detected from methylation data, with the highest DMR density on chromosomes 25, 27, 31 and 33. DMRs were mainly located in introns, which accounted for more than 60%, followed by promoter and exon regions. 4. A total of 2326 differentially methylated genes (DMGs) were identified from DMRs, including 1159 genes with upregulated DMRs, 936 genes with downregulated DMRs, and 231 genes with two types of DMRs. 5. The ESPL1 gene may be an important epigenetic gene of VVD. The methylation of particular CpG17, CpG18 and CpG19 sites in the promoter region of the ESPL1 gene may hinder the binding of transcription factors and promoters and increase the expression of ESPL1.

Exogenous glutathione can alleviate chromium toxicity in kenaf by activating antioxidant system and regulating DNA methylation

Glutathione (GSH) participates in plant response to heavy metals (HMs) stress, however, the epigenetic regulating mechanisms of GSH in HMs detoxification remains unclear. In this study, to reveal the potential epigenetic regulating mechanisms, kenaf seedlings were treated with/without GSH under chromium (Cr) stress. A comprehensive physiological, genome-wide DNA methylation and gene functional analysis were performed. Results showed that external GSH obviously recovered Cr-induced growth inhibition, significantly decreased H2O2, O2.- and MDA accumulation, increased the activities of antioxidant enzymes (SOD, CAT, GR and APX) in kenaf exposed to Cr. In addition, the expression level of the main DNA methyltransferase (MET1, CMT3 and DRM1) and demethylase (ROS1, DEM, DML2, DML3 and DDM1) genes were investigated by qRT-PCR. The result indicated that Cr stress decreased DNA methyltransferase genes expression while increased demethylase genes expression; however, apply exogenous GSH led to the recovery trend. These indicating exogenous GSH alleviation Cr stress on kenaf seedlings by increasing DNA methylation level. At the same time, the MethylRAD-seq genome-wide DNA methylation analysis showed the DNA methylation level was significantly increased after GSH treatment compared with Cr treatment alone. The differentially methylated genes (DMGs) were uniquely enriched in DNA repair, flavin adenine dinucleotide binding and oxidoreductase activity. Furthermore, a ROS homeostasis-associated DMG, HcTrx, was selected for further functional analysis. Results showed that the knock-down of HcTrx kenaf seedlings displayed yellow-green phenotype and impaired antioxidant enzyme activity; in contrast, the overexpression lines of HcTrx improved chlorophyll levels and enhanced Cr tolerance in Arabidopsis. Taken together, our results illustrate the novel role of GSH-mediated Cr detoxification in kenaf by modulating the DNA methylation, and thus further affect the activation of antioxidant defense systems. The present characterized Cr tolerant gene resource could be further used for kenaf Cr tolerant breeding via genetic improvement.

The role of DNA methylation on gene expression in the vertebrae of ancestrally benzo[a]pyrene exposed F1 and F3 male medaka

ABSTRACT Benzo[a]pyrene (BaP) is ubiquitously present in the aquatic environment and has been identified as a bone toxicant. Previous studies have demonstrated that ancestral BaP exposure can cause transgenerational bone deformities in fish. Transgenerational effects are thought to be caused by heritable epigenetic changes, such as DNA methylation, histone modification, and non-coding RNAs. To investigate the role of DNA methylation in BaP-induced transgenerational skeletal deformities and the related transcriptomic changes in deformed vertebrae, we examined the vertebrae of male F1 and F3 medaka fish using high-throughput RNA sequencing (RNA-seq) and whole-genome bisulphite sequencing (WGBS). The histological results revealed that osteoblast numbers at the vertebral bone decreased in the BaP-derived F1 and F3 adult males in comparison with the control group. Differentially methylated genes (DMGs) associated with osteoblastogenesis (F1 and F3), chondrogenesis (F1 and F3), and osteoclastogenesis (F3) were identified. However, RNA-seq data did not support the role of DNA methylation in the regulation of genes involved in skeletogenesis since there was very little correlation between the level of differential methylation and gene expression profiles related to skeletogenesis. Although DNA methylation plays a major role in the epigenetic regulation of gene expression, the dysregulation of vertebral gene expression patterns observed in the current study is most likely to be mediated by histone modification and miRNAs. Notably, RNA-seq and WGBS data indicated that genes related to nervous system development are more sensitive to ancestral BaP exposure, indicating a more complex transgenerational phenotype in response to ancestral BaP exposure.

Analysis of core mutation and TET2/ASXL1 mutations DNA methylation profile in myelodysplastic syndrome

ABSTRACTObjectivesThe study aims to analyze genetic mutation and clinical characteristics and study their correlation with survival prognosis of patients with myelodysplastic syndromes (MDS). Moreover, the differential DNA methylation profiles between TET2 mutated (Mut)/ASXL1 wild-type (WT) and TET2-Mut/ASXL1-Mut MDS samples were investigated to explore the mechanism of MDS patients with TET2/ASXL1 mutations.MethodsThe clinical data of 195 patients diagnosed with MDS were selected and statistically analyzed. The DNA methylation sequencing data set was obtained from the GEO and bioinformatics analyzed.ResultsOf the 195 MDS patients, 42 (21.5%) carried TET2 mutations. 81% of TET2-Mut patients could detect comutated genes. The most commonly comutated gene in MDS patients with TET2-Mut was ASXL1, which had a tendency towards poorer prognosis (P = 0.08). GO analysis showed that highly methylated differentially methylated genes (DMGs) was mainly enriched in biological processes such as cell surface receptor signal pathway and cell secretion. Hypomethylated DMGs was mainly enriched in cell differentiation and cell development. KEGG analysis showed that hypermethylated DMGs was mainly enriched in Ras signal pathway and MAPK signal pathway. Hypomethylated DMGs was mainly enriched in extracellular matrix receptor interaction and focal adhesion. PPI network analysis identified 10 hub genes of hypermethylated and hypomethylated DMGs that may be associated with patients with TET2-Mut/ASXL1-Mut respectively.ConclusionsOur results illustrate the interrelationships between genetic mutations and clinical phenotypes and disease outcomes, with substantial potential for clinical application. Differentially methylated hub genes might represent potential biomarkers and provide novel insights and possible targets for MDS with double TET2/ASXL1 mutations.

Comprehensive analysis of differences in N6-methyladenosine RNA methylomes in Helicobacter pylori infection.

Background: Helicobacter pylori (H.pylori) infection is an important factor in the occurrence of human gastric diseases, but its pathogenic mechanism is not clear. N6-methyladenosine (m6A) is the most prevalent reversible methylation modification in mammalian RNA and it plays a crucial role in controlling many biological processes. However, there are no studies reported that whether H. pylori infection impacts the m6A methylation of stomach. In this study, we measured the overall level changes of m6A methylation of RNA under H. pylori infection through in vitro and in vivo experiment. Methods: The total quantity of m6A was quantified in gastric tissues of clinical patients and C57 mice with H. pylori infection, as well as acute infection model [H. pylori and GES-1 cells were cocultured for 48h at a multiplicity of infection (MOI) from of 10:1 to 50:1]. Furthermore, we performed m6A methylation sequencing and RNA-sequencing on the cell model and RNA-sequencing on animal model. Results: Quantitative detection of RNA methylation showed that H. pylori infection group had higher m6A modification level. M6A methylation sequencing identified 2,107 significantly changed m6A methylation peaks, including 1,565 upregulated peaks and 542 downregulated peaks. A total of 2,487 mRNA was upregulated and 1,029 mRNA was downregulated. According to the comprehensive analysis of MeRIP-seq and RNA-seq, we identified 200 hypermethylation and upregulation, 129 hypermethylation but downregulation, 19 hypomethylation and downregulation and 106 hypomethylation but upregulation genes. The GO and KEGG pathway analysis of these differential methylation and regulatory genes revealed a wide range of biological functions. Moreover, combining with mice RNA-seq results, qRT- PCR showed that m6A regulators, METTL3, WTAP, FTO and ALKBH5, has significant difference; Two key genes, PTPN14 and ADAMTS1, had significant difference by qRT- PCR. Conclusion: These findings provide a basis for further investigation of the role of m6A methylation modification in H. pylori-associated gastritis.

Hesperidin inhibits methylation and autophagy in LPS and high glucose-induced human villous trophoblasts

IntroductionGestational diabetes mellitus (GDM) is the first occurrence of diabetes due to abnormal maternal sugar metabolism after pregnancy, which may lead to adverse pregnancy outcomes. Hesperidin is known to decrease in the cord blood of GDM with obesity, but its role is unknown. This study aims to explore the potential function of hesperidin in GDM with obesity to develop new therapeutic ideas. MethodsPeripheral blood and placental tissues from GDM and GDM with obesity patients were collected to isolate human villous trophoblasts and detection. Bioinformatics was used to analyze the differential methylation genes between GDM and GDM with obesity. Immunofluorescence was applied for the detection of CK7 expression. Cells vitality was detected by CCK8 and transwell. Molecular docking was applied to predict the binding of hesperidin and ATG7 protein. Inflammation and m6A levels was analyzed by ELISA. ATG7, LC3, TLR4 and P62 proteins was analyzed by Western blot. ResultsThe methylation of ATG7 gene was up-regulated in GDM with obesity compared with GDM. The m6A and autophagy proteins levels in GDM with obesity were higher than that in GDM. LPS with 2.5–25 mM glucose induced the increase of autophagy proteins, inflammation and m6A levels in human villous trophoblasts. Hesperidin formed hydrogen bonds and hydrophobic interactions with ATG7 proteins. Hesperidin (0.25 μM) inhibited the autophagy proteins and m6A level in LPS and 25 mM glucose-induced human villous trophoblasts. DiscussionGDM with obesity followed the increase of autophagy proteins and m6A levels. Hesperidin inhibited the autophagy proteins and m6A level in LPS and glucose-induced human villous trophoblasts.

Folic Acid Supplementation Promotes Hypomethylation in Both the Inflamed Colonic Mucosa and Colitis-Associated Dysplasia.

The purpose of this study was to assess the effect of folic acid (FA) supplementation on colitis-associated colorectal cancer (CRC) using the azoxymethane/dextran sulfate sodium (AOM/DSS) model. Mice were fed a chow containing 2 mg/kg FA at baseline and randomized after the first DSS treatment to receive 0, 2, or 8 mg/kg FA chow for 16 weeks. Colon tissue was collected for histopathological evaluation, genome-wide methylation analyses (Digital Restriction Enzyme Assay of Methylation), and gene expression profiling (RNA-Seq). A dose-dependent increase in the multiplicity of colonic dysplasias was observed, with the multiplicity of total and polypoid dysplasias higher (64% and 225%, respectively) in the 8 mg FA vs. the 0 mg FA group (p < 0.001). Polypoid dysplasias were hypomethylated, as compared to the non-neoplastic colonic mucosa (p < 0.05), irrespective of FA treatment. The colonic mucosa of the 8 mg FA group was markedly hypomethylated as compared to the 0 mg FA group. Differential methylation of genes involved in Wnt/β-catenin and MAPK signaling resulted in corresponding alterations in gene expression within the colonic mucosa. High-dose FA created an altered epigenetic field effect within the non-neoplastic colonic mucosa. The observed decrease in site-specific DNA methylation altered oncogenic pathways and promoted colitis-associated CRC.