MGMT is down-regulated independently of promoter DNA methylation in rats with all-trans retinoic acidinduced spina bifida aperta

O6-methylguanine DNA methyltransferase(MGMT), a DNA repair enzyme, has been reported in some congenital malformations, but it is less frequently reported in neural tube defects. This study investigated MGMT mRNA expression and methylation levels in the early embryo and in different embryonic stages, as well as the relationship between MGMT and neural tube defects. Spina bifida aperta was induced in rats by a single intragastric administration of all-trans retinoic acid on embryonic day(E) 10, whereas normal control rats received the same amount of olive oil on the same embryonic day. DNA damage was assessed by detecting γ-H2 A.X in spina bifida aperta rats. Real time-polymerase chain reaction was used to examine mRNA expression of MGMT in normal control and spina bifida aperta rats. In normal controls, the MGMT mRNA expression decreased with increasing embryonic days, and was remarkably reduced from E11 to E14, reaching a minimum at E18. In the spina bifida aperta model, γ-H2 A.X protein expression was increased, and mRNA expression of MGMT was markedly decreased on E14, E16, and E18. Bisulfite sequencing polymerase chain reaction for MGMT promoter methylation demonstrated that almost all CpG sites in the MGMT promoter remained unmethylated in both spina bifida aperta rats and normal controls, and there was no significant difference in methylation level between the two groups on either E14 or E18. Our results show that DNA damage occurs in spina bifida aperta rats. The mRNA expression of MGMT is downregulated, and this downregulation is independent of promoter DNA methylation.

Association of DNA methylation/demethylation with the functional outcome of stroke in a hyperinflammatory state

Inflammation is closely related to stroke prognosis, and high inflammation status leads to poor functional outcome in stroke. DNA methylation is involved in the pathogenesis and prognosis of stroke. However, the effect of DNA methylation on stroke at high levels of inflammation is unclear. In this study, we constructed a hyperinflammatory cerebral ischemia mouse model and investigated the effect of hypomethylation and hypermethylation on the functional outcome. We constructed a mouse model of transient middle cerebral artery occlusion and treated the mice with lipopolysaccharide to induce a hyperinflammatory state. To investigate the effect of DNA methylation on stroke, we used small molecule inhibitors to restrain the function of key DNA methylation and demethylation enzymes. 2,3,5-Triphenyltetrazolium chloride staining, neurological function scores, neurobehavioral tests, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot assay were used to evaluate the effects after stroke in mice. We assessed changes in the global methylation status by measuring DNA 5-mc and DNA 5-hmc levels in peripheral blood after the use of the inhibitor. In the group treated with the DNA methylation inhibitor, brain tissue 2,3,5-triphenyltetrazolium chloride staining showed an increase in infarct volume, which was accompanied by a decrease in neurological scores and worsening of neurobehavioral performance. The levels of inflammatory factors interleukin 6 and interleukin-1 beta in ischemic brain tissue and plasma were elevated, indicating increased inflammation. Related inflammatory pathway exploration showed significant overactivation of nuclear factor kappa B. These results suggested that inhibiting DNA methylation led to poor functional outcome in mice with high inflammation following stroke. Further, the effects were reversed by inhibition of DNA demethylation. Our findings suggest that DNA methylation regulates the inflammatory response in stroke and has an important role in the functional outcome of hyperinflammatory stroke.

Salsolinol as an RNA m~6A methylation inducer mediates dopaminergic neuronal death by regulating YAP1 and autophagy

Salsolinol(1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline,Sal)is a catechol isoquinoline that causes neurotoxicity and shares structural similarity with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine,an environmental toxin that causes Parkinson's disease.However,the mechanism by which Sal mediates dopaminergic neuronal death remains unclear.In this study,we found that Sal significantly enhanced the global level of N~6-methyladenosine(m~6A)RNA methylation in PC12 cells,mainly by inducing the downregulation of the expression of m~6A demethylases fat mass and obesity-associated protein(FTO)and alk B homolog 5(ALKBH5).RNA sequencing analysis showed that Sal downregulated the Hippo signaling pathway.The m~6A reader YTH domain-containing family protein 2(YTHDF2)promoted the degradation of m~6A-containing Yes-associated protein 1(YAP1)mRNA,which is a downstream key effector in the Hippo signaling pathway.Additionally,downregulation of YAP1 promoted autophagy,indicating that the mutual regulation between YAP1 and autophagy can lead to neurotoxicity.These findings reveal the role of Sal on m~6A RNA methylation and suggest that Sal may act as an RNA methylation inducer mediating dopaminergic neuronal death through YAP1 and autophagy.Our results provide greater insights into the neurotoxic effects of catechol isoquinolines compared with other studies and may be a reference for assessing the involvement of RNA methylation in the pathogenesis of Parkinson's disease.

Effects of histone acetylation and DNA methylation on p21^(WAF1)regulation

Cell cycle progression is regulated by interactions between cyclins and cyclin-dependent kinases (CDKs). p21(WAF1) is one of the CIP/KIP family which inhibits CDKs activity. Increased expression of p21(WAF1) may play an important role in the growth arrest induced in transformed cells. Although the stability of the p21( WAF1) mRNA could be altered by different signals, cell differentiation and numerous influencing factors. However, recent studies suggest that two known mechanisms of epigenesis, i.e.gene inactivation by methylation in promoter region and changes to an inactive chromatin by histone deacetylation, seem to be the best candidate mechanisms for inactivation of p21( WAF1). To date, almost no coding region p21(WAF1) mutations have been found in tumor cells, despite extensive screening of hundreds of various tumors. Hypermethylation of the p21(WAF1) promoter region may represent an alternative mechanism by which the p21(WAF1/CIP1) gene can be inactivated. The reduction of cellular DNMT protein levels also induces a corresponding rapid increase in the cell cycle regulator p21(WAF1) protein demonstrating a regulatory link between DNMT and p21(WAF1) which is independent of methylation of DNA. Both histone hyperacetylation and hypoacetylation appear to be important in the carcinoma process, and induction of the p21(WAF1) gene by histone hyperacetylation may be a mechanism by which dietary fiber prevents carcinogenesis. Here, we review the influence of histone acetylation and DNA methylation on p21(WAF1) transcription, and affection of pathways or factors associated such as p 53, E2A, Sp1 as well as several histone deacetylation inhibitors.

Unveiling DNA methylation in Alzheimer’s disease:a review of array-based human brain studies

The intricacies of Alzheimer’s disease pathogenesis are being increasingly illuminated by the exploration of epigenetic mechanisms,particularly DNA methylation.This review comprehensively surveys recent human-centered studies that investigate whole genome DNA methylation in Alzheimer’s disease neuropathology.The examination of various brain regions reveals distinctive DNA methylation patterns that associate with the Braak stage and Alzheimer’s disease progression.The entorhinal cortex emerges as a focal point due to its early histological alterations and subsequent impact on downstream regions like the hippocampus.Notably,ANK1 hypermethylation,a protein implicated in neurofibrillary tangle formation,was recurrently identified in the entorhinal cortex.Further,the middle temporal gyrus and prefrontal cortex were shown to exhibit significant hypermethylation of genes like HOXA3,RHBDF2,and MCF2L,potentially influencing neuroinflammatory processes.The complex role of BIN1 in late-onset Alzheimer’s disease is underscored by its association with altered methylation patterns.Despite the disparities across studies,these findings highlight the intricate interplay between epigenetic modifications and Alzheimer’s disease pathology.Future research efforts should address methodological variations,incorporate diverse cohorts,and consider environmental factors to unravel the nuanced epigenetic landscape underlying Alzheimer’s disease progression.

Revolutionizing Non-Invasive Biomarker Discoveries: The Power of Methylation Screening Analysis in Cell-Free DNA Liquid Biopsy

Epigenetic changes of DNA, including methylation, have long been recognized as key indicators of various diseases, including aging, cancer, and neurological disorders. Biomarker discoveries based on distinct methylation patterns for both hypermethylation and hypomethylation lead the way in discovery of novel diagnosis and treatment targets. Many different approaches are present to detect the level of methylation in whole genome (whole genome bisulfite sequencing, microarray) as well as at specific loci (methylation specific PCR). Cell-free DNA (cf-DNA) found in body fluids like blood provides information about DNA methylation and serves as a less invasive approach for genetic screening. Cell-free DNA and methylation screening technologies, when combined, have the potential to transform the way we approach genetic screening and personalized therapy. These technologies can help enhance disease diagnostic accuracy and inform the development of targeted therapeutics by providing a non-invasive way for acquiring genomic information and identifying disease-associated methylation patterns. We highlight the clinical benefits of using cell-free DNA (cf-DNA) liquid biopsy analysis and available methylation screening technologies that have been crucial in identifying biomarkers for disease from patients using a non-invasive way. Powering such biomarker discoveries are various methods of cf-DNA methylation analysis such as Bisulfite Sequencing and most recently, Methylation-Specific Restriction Enzyme (MSRE-seq) Analysis, paving the way for novel epigenetic biomarker discoveries for more robust diagnosis such as early disease detection, prognosis, monitoring of disease progression and treatment response as well as discovery of novel drug targets.

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.

Reduced non-CpG methylation is a potential epigenetic target after spinal cord injury

DNA methylation is a critical epigenetic regulator in the occurrence and development of diseases and is closely related to various functional responses in relation to spinal cord injury.To investigate the role of DNA methylation in spinal cord injury,we constructed a library with reduced-representation bisulfite sequencing data obtained at various time points(day 0-42)after spinal cord injury in mice.Global DNA methylation levels,specifically non-CpG(CHG and CHH)methylation levels,decreased modestly following spinal cord injury.Stages post-spinal cord injury were classified as early(day 0-3),intermediate(day7-14),and late(day 28-42)based on similarity and hie rarchical cluste ring of global DNA methylation patterns.The non-CpG methylation level,which included CHG and CHH methylation levels,was markedly reduced despite accounting for a minor proportion of total methylation abundance.At multiple genomic sites,including the 5’untranslated regions,promoter,exon,intron,and 3’untranslated regions,the non-CpG methylation level was markedly decreased following spinal cord injury,whereas the CpG methylation level remained unchanged at these locations.Approximately one-half of the differentially methylated regions were located in intergenic areas;the other differentially methylated regions in both CpG and non-CpG regions were cluste red in intron regions,where the DNA methylation level was highest.The function of genes associated with differentially methylated regions in promoter regions was also investigated.From Gene Ontology analysis results,DNA methylation was implicated in a number of essential functional responses to spinal cord injury,including neuronal synaptic connection creation and axon regeneration.Notably,neither CpG methylation nor non-CpG methylation was implicated in the functional response of glial or inflammatory cells.In summary,our work elucidated the dynamic pattern of DNA methylation in the spinal co rd following injury and identified reduced nonCpG methylation as an epigenetic target after spinal cord injury in mice.

Identification of differentially expressed genes regulated by methylation in colon cancer based on bioinformatics analysis

BACKGROUND DNA methylation, acknowledged as a key modification in the field of epigenetics, regulates gene expression at the transcriptional level. Aberrant methylation in DNA regulatory regions could upregulate oncogenes and downregulate tumor suppressor genes without changing the sequences.However, studies of methylation in the control of gene expression are still inadequate. In the present research, we performed bioinformatics analysis to clarify the function of methylation and supply candidate methylation-related biomarkers and drivers for colon cancer.AIM To identify and analyze methylation-regulated differentially expressed genes(MeDEGs) in colon cancer by bioinformatics analysis.METHODS We downloaded RNA expression profiles, Illumina Human Methylation 450 K BeadChip data, and clinical data of colon cancer from The Cancer Genome Atlas project. MeDEGs were identified by analyzing the gene expression and methylation levels using the edgeR and limma package in R software. Gene ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were performed in the DAVID database and KEGG Orthology-Based Annotation System 3.0, respectively. We then conducted Kaplan–Meier survival analysis to explore the relationship between methylation and expression and prognosis. Gene set enrichment analysis(GSEA) and investigation of protein-protein interactions(PPI) were performed to clarify the function of prognosis-related genes.RESULTS A total of 5 up-regulated and 81 down-regulated genes were identified asMeDEGs. GO and KEGG pathway analyses indicated that MeDEGs were enriched in multiple cancer-related terms. Furthermore, Kaplan–Meier survival analysis showed that the prognosis was negatively associated with the methylation status of glial cell-derived neurotrophic factor(GDNF) and reelin(RELN). In PPI networks, GDNF and RELN interact with neural cell adhesion molecule 1. Besides, GDNF can interact with GDNF family receptor alpha(GFRA1), GFRA2, GFRA3, and RET. RELN can interact with RAFAH1 B1,disabled homolog 1, very low-density lipoprotein receptor, lipoprotein receptorrelated protein 8, and NMDA 2 B. Based on GSEA, hypermethylation of GDNF and RELN were both significantly associated with pathways including "RNA degradation," "ribosome," "mismatch repair," "cell cycle" and "base excision repair."CONCLUSION Aberrant DNA methylation plays an important role in colon cancer progression.MeDEGs that are associated with the overall survival of patients may be potential targets in tumor diagnosis and treatment.

Advances in DNA methylation and its role in cytoplasmic male sterility in higher plants

The impact of epigenetic modifications like DNA methylation on plant phenotypes has expanded the possibilities for crop development.DNA methylation plays a part in the regulation of both the chromatin structure and gene expression,and the enzyme involved,DNA methyltransferase,executes the methylation process within the plant genome.By regulating crucial biological pathways,epigenetic changes actively contribute to the creation of the phenotype.Therefore,epigenome editing may assist in overcoming some of the drawbacks of genome editing,which can have minor off-target consequences and merely facilitate the loss of a gene’s function.These drawbacks include gene knockout,which can have such off-target effects.This review provides examples of several molecular characteristics of DNA methylation,as well as some plant physiological processes that are impacted by these epigenetic changes in the plants.We also discuss how DNA alterations might be used to improve crops and meet the demands of sustainable and environmentally-friendly farming.

DNA Methylation of KLRC1 and KLRC3 in Autoimmune Thyroiditis:Perspective of Different Water Iodine Exposure

Objective This study aimed to identify differentially methylated genes(DMGs) associated with natural killer cells in patients with autoimmune thyroiditis(AIT), focusing on the influence of varying water iodine exposure levels.Methods Participants were divided into categories based on median water iodine(MWI)concentrations: iodine-fortified areas(IFA, MWI < 10 μg/L), iodine-adequate areas(IAA, 40 ≤ MWI ≤ 100μg/L), and iodine-excessive areas(IEA, MWI > 300 μg/L). A total of 176 matched AIT cases and controls were recruited and divided into 89, 40, and 47 pairs for IFA, IAA, and IEA, respectively. DMGs were identified using 850K Bead Chip analysis for 10/10 paired samples. Validation of DNA methylation and m RNA expression levels of the DMGs was conducted using Methyl Target^(TM) and QRT-PCR for 176/176paired samples.Results KLRC1, KLRC3, and SH2D1B were identified as significant DMGs. Validation revealed that KLRC1 was hypomethylated and highly expressed, whereas KLRC3 was hypermethylated and highly expressed in individuals with AIT. Furthermore, KLRC1 was hypomethylated and highly expressed in both IFA and IEA.Conclusion The DNA methylation status of KLRC1 and KLRC3 may play crucial roles in AIT pathogenesis. Additionally, DNA methylation of KLRC1 seems to be influenced by different iodine concentrations in water.

TCERG1L hypermethylation is a risk factor of diabetic retinopathy in Chinese children with type 1 diabetes

●AIM:To identify the differential methylation sites(DMS)and their according genes associated with diabetic retinopathy(DR)development in type 1 diabetes(T1DM)children.●METHODS:This study consists of two surveys.A total of 40 T1DM children was included in the first survey.Because no participant has DR,retina thinning was used as a surrogate indicator for DR.The lowest 25%participants with the thinnest macular retinal thickness were included into the case group,and the others were controls.The DNA methylation status was assessed by the Illumina methylation 850K array BeadChip assay,and compared between the case and control groups.Four DMS with a potential role in diabetes were identified.The second survey included 27 T1DM children,among which four had DR.The methylation patterns of the four DMS identified by 850K were compared between participants with and without DR by pyrosequencing.●RESULTS:In the first survey,the 850K array revealed 751 sites significantly and differentially methylated in the case group comparing with the controls(|Δβ|>0.1 and Adj.P<0.05),and 328 of these were identified with a significance of Adj.P<0.01.Among these,319 CpG sites were hypermethylated and 432 were hypomethylated in the case group relative to the controls.Pyrosequencing revealed that the transcription elongation regulator 1 like(TCERG1L,cg07684215)gene was hypermethylated in the four T1DM children with DR(P=0.018),which was consistent with the result from the first survey.The methylation status of the other three DMS(cg26389052,cg25192647,and cg05413694)showed no difference(all P>0.05)between participants with and without DR.●CONCLUSION:The hypermethylation of the TCERG1L gene is a risk factor for DR development in Chinese children with T1DM.

DNA hypomethylation promotes learning and memory recovery in a rat model of cerebral ischemia/reperfusion injury

Cerebral ischemia/reperfusion injury impairs learning and memory in patients.Studies have shown that synaptic function is involved in the formation and development of memory,and that DNA methylation plays a key role in the regulation of learning and memory.To investigate the role of DNA hypomethylation in cerebral ischemia/reperfusion injury,in this study,we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery and then treated the rats with intraperitoneal 5-aza-2′-deoxycytidine,an inhibitor of DNA methylation.Our results showed that 5-aza-2′-deoxycytidine markedly improved the neurological function,and cognitive,social and spatial memory abilities,and dose-dependently increased the synaptic density and the expression of SYP and SHANK2 proteins in the hippocampus in a dose-dependent manner in rats with cerebral ischemia/reperfusion injury.The effects of 5-aza-2′-deoxycytidine were closely related to its reduction of genomic DNA methylation and DNA methylation at specific sites of the Syp and Shank2 genes in rats with cerebral ischemia/reperfusion injury.These findings suggest that inhibition of DNA methylation by 5-aza-2′-deoxycytidine promotes the recovery of learning and memory impairment in a rat model of cerebral ischemia/reperfusion injury.These results provide theoretical evidence for stroke treatment using epigenetic methods.

Advances in microfluidic-based DNA methylation analysis

DNA methylation has been extensively investigated in recent years,not least because of its known relationship with various diseases.Progress in analytical methods can greatly increase the relevance of DNA methylation studies to both clinical medicine and scientific research.Microflu-idic chips are excellent carriers for molecular analysis,and their use can provide improvements from multiple aspects.On-chip molecular analysis has received extensive attention owing to its advantages of portability,high throughput,low cost,and high efficiency.In recent years,the use of novel microfluidic chips for DNA methylation analysis has been widely reported and has shown obvious superiority to conventional methods.In this review,wefirst focus on DNA methylation and its applications.Then,we discuss advanced microfluidic-based methods for DNA methylation analysis and describe the great progress that has been made in recent years.Finally,we summarize the advantages that microfluidic technology brings to DNA methylation analysis and describe several challenges and perspectives for on-chip DNA methylation analysis.This review should help researchers improve their understanding and make progress in developing microfluidic-based methods for DNA methylation analysis.

Methylation status of c-fms oncogene in HCC and its relationship with clinical pathology

INTRODUCTIONThe mechanism that DNA hypomethylation leads toactivation of oncogene and occurrence of malignantneoplasm is being increasingly recognized byresearchers. Normal DNA methylation playsimportant role in stabilizing the phenotype of cell.DNA methylation status reduction and/or patternalteration are related to activation and abnormallyhigh expression of some oncogenes and cellularmalignancy[1-6]. c-fms oncogene encodes for colonystimulating factor 1 receptor (CSF-1R)[7], c-fms/CSF-1R was highly expressed in hepatocellularcarcinoma (HCC) tissue, but the mechanismremained obscure[8,9].

DNA methylation in poultry:a review

As an important epigenetic modification,DNA methylation is involved in many biological processes such as animal cell differentiation,embryonic development,genomic imprinting and sex chromosome inactivation.As DNA methylation sequencing becomes more sophisticated,it becomes possible to use it to solve more zoological problems.This paper reviews the characteristics of DNA methylation,with emphasis on the research and application of DNA methylation in poultry.

Effects of maternal methyl donor intake during pregnancy on ileum methylation and function in an intrauterine growth restriction pig model

Background Intrauterine growth retardation(IUGR)affects intestinal growth,morphology,and function,which leads to poor growth performance and high mortality.The present study explored whether maternal dietary methyl donor(MET)supplementation alleviates IUGR and enhances offspring’s growth performance by improving intestinal growth,function,and DNA methylation of the ileum in a porcine IUGR model.Methods Forty multiparous sows were allocated to the control or MET diet groups from mating until delivery.After farrowing,8 pairs of IUGR and normal birth weight piglets from 8 litters were selected for sampling before suckling colostrum.Results The results showed that maternal MET supplementation tended to decrease the IUGR incidence and increased the average weaning weight of piglets.Moreover,maternal MET supplementation significantly reduced the plasma concentrations of isoleucine,cysteine,urea,and total amino acids in sows and newborn pig-lets.It also increased lactase and sucrase activity in the jejunum of newborn piglets.MET addition resulted in lower ileal methionine synthase activity and increased betaine homocysteine S-methyltransferase activity in the ileum of newborn piglets.DNA methylation analysis of the ileum showed that MET supplementation increased the methyla-tion level of DNA CpG sites in the ileum of newborn piglets.Down-regulated differentially methylated genes were enriched in folic acid binding,insulin receptor signaling pathway,and endothelial cell proliferation.In contrast,up-regulated methylated genes were enriched in growth hormone receptor signaling pathway and nitric oxide biosyn-thetic process.Conclusions Maternal MET supplementation can reduce the incidence of IUGR and increase the weaning litter weight of piglets,which may be associated with better intestinal function and methylation status.

Methylome and transcriptome data integration reveals potential roles of DNA methylation and candidate biomarkers of cow Streptococcus uberis subclinical mastitis

Background:Mastitis caused by different pathogens including Streptococcus uberis(S.uberis)is responsible for huge economic losses to the dairy industry.In order to investigate the potential genetic and epigenetic regulatory mecha‑nisms of subclinical mastitis due to S.uberis,the DNA methylome(whole genome DNA methylation sequencing)and transcriptome(RNA sequencing)of milk somatic cells from cows with naturally occurring S.uberis subclinical mastitis and healthy control cows(n=3/group)were studied.Results:Globally,the DNA methylation levels of CpG sites were low in the promoters and first exons but high in inner exons and introns.The DNA methylation levels at the promoter,first exon and first intron regions were nega‑tively correlated with the expression level of genes at a whole‑genome‑wide scale.In general,DNA methylation level was lower in S.uberis‑positive group(SUG)than in the control group(CTG).A total of 174,342 differentially methylated cytosines(DMCs)(FDR<0.05)were identified between SUG and CTG,including 132,237,7412 and 34,693 DMCs in the context of CpG,CHG and CHH(H=A or T or C),respectively.Besides,101,612 methylation haplotype blocks(MHBs)were identified,including 451 MHBs that were significantly different(dMHB)between the two groups.A total of 2130 differentially expressed(DE)genes(1378 with up‑regulated and 752 with down‑regulated expression)were found in SUG.Integration of methylome and transcriptome data with MethGET program revealed 1623 genes with signifi‑cant changes in their methylation levels and/or gene expression changes(MetGDE genes,MethGET P‑value<0.001).Functional enrichment of genes harboring≥15 DMCs,DE genes and MetGDE genes suggest significant involvement of DNA methylation changes in the regulation of the host immune response to S.uberis infection,especially cytokine activities.Furthermore,discriminant correlation analysis with DIABLO method identified 26 candidate biomarkers,including 6 DE genes,15 CpG‑DMCs and 5 dMHBs that discriminated between SUG and CTG.Conclusion:The integration of methylome and transcriptome of milk somatic cells suggests the possible involve‑ment of DNA methylation changes in the regulation of the host immune response to subclinical mastitis due to S.uberis.The presented genetic and epigenetic biomarkers could contribute to the design of management strategies of subclinical mastitis and breeding for mastitis resistance.

Rice melatonin deficiency causes premature leaf senescence via DNA methylation regulation

In a study of DNA methylation changes in melatonin-deficient rice mutants,mutant plants showed premature leaf senescence during grain-filling and reduced grain yield.Melatonin deficiency led to transcriptional reprogramming,especially of genes involved in chlorophyll and carbon metabolism,redox regulation,and transcriptional regulation,during dark-induced leaf senescence.Hypomethylation of mCG and mCHG in the melatonin-deficient rice mutants was associated with the expression change of both protein-coding genes and transposable element-related genes.Changes in gene expression and DNA methylation in the melatonin-deficient mutants were compensated by exogenous application of melatonin.A decreased S-adenosyl-L-methionine level may have contributed to the DNA methylation variations in rice mutants of melatonin deficiency under dark conditions.

N6-methyladenosine methylation regulates the tumor microenvironment of Epstein-Barr virus-associated gastric cancer

BACKGROUND N6-methyladenosine(m6A)methylation modification exists in Epstein-Barr virus(EBV)primary infection,latency,and lytic reactivation.It also modifies EBV latent genes and lytic genes.EBV-associated gastric cancer(EBVaGC)is a distinctive molecular subtype of GC.We hypothesized EBV and m6A methylation regulators interact with each other in EBVaGC to differentiate it from other types of GC.AIM To investigate the mechanisms of m6A methylation regulators in EBVaGC to determine the differentiating factors from other types of GC.METHODS First,The Cancer Gene Atlas and Gene Expression Omnibus databases were used to analyze the expression pattern of m6A methylation regulators between EBVaGC and EBV-negative GC(EBVnGC).Second,we identified Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)functional enrichment of m6A-related differentially expressed genes.We quantified the relative abundance of immune cells and inflammatory factors in the tumor microenvironment(TME).Finally,cell counting kit-8 cell proliferation test,transwell test,and flow cytometry were used to verify the effect of insulin-like growth factor binding protein 1(IGFBP1)in EBVaGC cell lines.RESULTS m6A methylation regulators were involved in the occurrence and development of EBVaGC.Compared with EBVnGC,the expression levels of m6A methylation regulators Wilms tumor 1-associated protein,RNA binding motif protein 15B,CBL proto-oncogene like 1,leucine rich pentatricopeptide repeat containing,heterogeneous nuclear ribonucleoprotein A2B1,IGFBP1,and insulin-like growth factor 2 binding protein 1 were significantly downregulated in EBVaGC(P<0.05).The overall survival rate of EBVaGC patients with a lower expression level of IGFBP1 was significantly higher(P=0.046).GO and KEGG functional enrichment analyses showed that the immunity pathways were significantly activated and rich in immune cell infiltration in EBVaGC.Compared with EBVnGC,the infiltration of activated CD4+T cells,activated CD8+T cells,monocytes,activated dendritic cells,and plasmacytoid dendritic cells were significantly upregulated in EBVaGC(P<0.001).In EBVaGC,the expression level of proinflammatory factors interleukin(IL)-17,IL-21,and interferon-γ and immunosuppressive factor IL-10 were significantly increased(P<0.05).In vitro experiments demonstrated that the expression level of IGFBP1 was significantly lower in an EBVaGC cell line(SNU719)than in an EBVnGC cell line(AGS)(P<0.05).IGFBP1 overexpression significantly attenuated proliferation and migration and promoted the apoptosis levels in SNU719.Interfering IGFBP1 significantly promoted proliferation and migration and attenuated the apoptosis levels in AGS.CONCLUSION m6A regulators could remodel the TME of EBVaGC,which is classified as an immune-inflamed phenotype and referred to as a“hot”tumor.Among these regulators,we demonstrated that IGFBP1 affected proliferation,migration,and apoptosis.