Total Methylation Levels Research Articles

Differential Variability of Maize (Zea mays L.) Inbred Lines to Moisture-stress at Reproductive Stages and DNA Methylation Studies of Identified Contrasting Genotypes under Moisture-Stress Conditions

An investigation was undertaken to assess the effect of moisture stress during the reproductive initiation stage on the quality and quantity of pollen grains produced in eight inbred lines (UASBM22, UASBM13, UASBM09, UASBM11, UASBM06, UASBM14, UASBM02 and UASBM10) under greenhouse conditions in three blocks viz., well-watered, stress I (for 21 days from 28 days after sowing) and stress II (for 22 days from 32 days after sowing). Moisture stress significantly affected the number of pollen grains per anther and other plant growth-related traits. The moisture stress effect was not uniform across inbred lines. The inbred lines UASBM22, UASBM13, UASBM09 and UASBM11 recorded a significant reduction in the total number of pollen grains per anther and an increase in pollen sterility while in inbred lines, UASBM06, UASBM14, UASBM02 and UASBM10, the moisture stress effect was not significant. The changes in the DNA methylation pattern in leaves and immature anthers under moisture stress of the contrasting inbred lines (UASBM06 and UASBM13) were studied through methylation- sensitive random amplification polymorphism. An increase in total DNA methylation level in both leaves and anthers was observed in drought tolerant inbred line, UASBM06 under stress while the increase was only in the leaves of the susceptible inbred line UASBM13. Leaves and anthers of UASBM06 showed hypermethylation compared to UASBM13 in moisture-stress conditions. In maize, increased DNA methylation seems to be an important mechanism associated with drought responses which probably regulates the methylation-sensitive gene expression and acclimation responses in maize.

Targeted bisulfite sequencing of Scots pine adaptation-related genes

During environmental changes, epigenetic processes can enable adaptive responses faster than natural selection. In plants, very little is known about the role of DNA methylation during long-term adaptation. Scots pine is a widely distributed coniferous species which must adapt to different environmental conditions throughout its long lifespan. Thus, epigenetic modifications may contribute towards this direction. We provide bisulfite next-generation sequencing data from the putative promoters and exons of eight adaptation-related genes (A3IP2, CCA1, COL1, COL2, FTL2, MFT1, PHYO, and ZTL) in three Scots pine populations located in northern and southern parts of Finland. DNA methylation levels were studied in the two seed tissues: the maternal megagametophyte which contributes to embryo viability, and the biparental embryo which represents the next generation. In most genes, differentially methylated cytosines (DMCs) were in line with our previously demonstrated gene expression differences found in the same Scots pine populations. In addition, we found a strong correlation of total methylation levels between the embryo and megagametophyte tissues of a given individual tree, which indicates that DNA methylation can be inherited from the maternal parent. In conclusion, our results imply that DNA methylation differences may contribute to the adaptation of Scots pine populations in different climatic conditions.

Dietary selenomethionine reduced oxidative stress by resisting METTL3-mediated m6A methylation level of Nrf2 to ameliorate LPS-induced liver necroptosis in laying hens

Selenomethionine (SeMet) as the main form of daily dietary selenium, occupies essential roles in providing antioxidant and anti-inflammatory properties, which alleviates inflammatory liver damage. N6-methyladenosine (m6A) is one of the most prevalent and abundant internal transcriptional modifications that regulate gene expression. To investigate the protective mechanism of SeMet on liver injury and the regulatory effect of m6A methylation modification, we established the model by supplementing dietary SeMet, and LPS as stimulus in laying hens. LMH cells were intervened with SeMet (0.075 μM) and/or LPS (60 μg/mL). Subsequently, histopathology and ultrastructure of liver were observed. Western Blot, qRT-PCR, colorimetry, MeRIP-qPCR, fluorescent probe staining and AO/EB were used to detect total m6A methylation level, m6A methylation level of Nrf2, ROS, inflammatory and necroptosis factors. Studies showed that SeMet suppressed LPS-induced upregulation of total m6A methylation levels and METTL3 expression. Interestingly, SeMet reduced the m6A methylation level of Nrf2, activated antioxidant pathways and alleviated oxidative stress. LMH cells were transfected with 50 μm siMETTL3. SeMet/SiMETTL3 reversed the LPS-induced reduction in Nrf2 mRNA stability, slowed down its degradation rate. Moreover, LPS induced oxidative stress, led to necroptosis and activated NF-κB to promote the expression of inflammatory factors. SeMet/SiMETTL3 alleviated LPS-induced necroptosis and inflammation. Altogether, SeMet enhanced antioxidant and anti-inflammatory capacity by reducing METTL3-mediated m6A methylation levels of Nrf2, ultimately alleviating liver damage. Our findings provided new insights and therapeutic target for the practical application of dietary SeMet in the treatment and prevention of liver inflammation, and supplied a reference for comparative medicine.

The m6A methylation and expression profiles of mouse neural stem cells after hypoxia/reoxygenation

BackgroundIschemia–reperfusion injury to the central nervous system often causes severe complications. The activation of endogenous neural stem cells (NSCs) is considered a promising therapeutic strategy for nerve repair. However, the specific biological processes and molecular mechanisms of NSC activation remain unclear, and the role of N6-methyladenosine (m6A) methylation modification in this process has not been explored.MethodsNSCs were subjected to hypoxia/reoxygenation (H/R) to simulate ischemia–reperfusion in vivo. m6A RNA methylation quantitative kit was used to measure the total RNA m6A methylation level. Quantitative real-time PCR was used to detect methyltransferase and demethylase mRNA expression levels. Methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were conducted for NSCs in control and H/R groups, and the sequencing results were analyzed using bioinformatics. Finally, the migration ability of NSCs was identified by wound healing assays, and the proliferative capacity of NSCs was assessed using the cell counting kit-8, EdU assays and cell spheroidization assays.ResultsOverall of m6A modification level and Mettl14 mRNA expression increased in NSCs after H/R treatment. The m6A methylation and expression profiles of mRNAs in NSCs after H/R are described for the first time. Through the joint analysis of MeRIP-seq and RNA-seq results, we verified the proliferation of NSCs after H/R, which was regulated by m6A methylation modification. Seven hub genes were identified to play key roles in the regulatory process. Knockdown of Mettl14 significantly inhibited the proliferation of NSCs. In addition, separate analysis of the MeRIP-seq results suggested that m6A methylation regulates cell migration and differentiation in ways other than affecting mRNA expression. Subsequent experiments confirmed the migration ability of NSCs was suppressed by knockdown of Mettl14.ConclusionThe biological behaviors of NSCs after H/R are closely related to m6A methylation of mRNAs, and Mettl14 was confirmed to be involved in cell proliferation and migration.

Brain-derived neurotrophic factor (BDNF): an effect biomarker of neurodevelopment in human biomonitoring programs.

The present narrative review summarizes recent findings focusing on the role of brain-derived neurotrophic factor (BDNF) as a biomarker of effect for neurodevelopmental alterations during adolescence, based on health effects of exposure to environmental chemical pollutants. To this end, information was gathered from the PubMed database and the results obtained in the European project Human Biomonitoring for Europe (HBM4EU), in which BDNF was measured at two levels of biological organization: total BDNF protein (serum) and BDNF gene DNA methylation (whole blood) levels. The obtained information is organized as follows. First, human biomonitoring, biomarkers of effect and the current state of the art on neurodevelopmental alterations in the population are presented. Second, BDNF secretion and mechanisms of action are briefly explained. Third, previous studies using BDNF as an effect biomarker were consulted in PubMed database and summarized. Finally, the impact of bisphenol A (BPA), metals, and non-persistent pesticide metabolites on BDNF secretion patterns and its mediation role with behavioral outcomes are addressed and discussed. These findings were obtained from three pilot studies conducted in HBM4EU project. Published findings suggested that exposure to some chemical pollutants such as fine particle matter (PM), PFAS, heavy metals, bisphenols, and non-persistent pesticides may alter circulating BDNF levels in healthy population. Therefore, BDNF could be used as a valuable effect biomarker to investigate developmental neurotoxicity of some chemical pollutants.

P10.27.A THE INFLUENCE OF DEXAMETHASONE ON TOTAL DNA METHYLATION LEVEL IN GLIOBLASTOMA CELL LINES

Abstract BACKGROUND Dexamethasone (DEX) is a first-line antiedematic drug for glioblastoma (GBM) patients. There is also some evidence it also inhibits GBM cell proliferation and migration, thus impacting patients’ outcomes. However, other studies show that the use of DEX is associated with poor treatment outcomes in GBM patients. The exact mechanism of DEX action is unclear, and no definitive conclusion has yet been reached on its benefits in neurooncological treatment. Temozolomide (TMZ) is a first-line chemotherapeutic in GBM. Epigenetics offers a connection between genetic and environmental factors that influence the development of the disease. The best-characterized epigenetic mark is 5-methylcytosine (m5C) in DNA. The aim of that project is to show the effects of DEX administration on the total DNA methylation level. MATERIAL AND METHODS Using the nucleotide post-labeling method, we analyzed the total amount of m5C in DNA of GBM (T98G, U118, U138), cancer (HeLa) and normal (HaCaT) cell lines treated with DEX, and a combination of DEX and with TMZ. RESULTS We adjusted the DEX doses to the ones achieved in the central nervous system during treatment. We observed dose-dependent increase in total DNA methylation in all cell lines. However, the exposition of GBM cells to the combination of DEX and TMZ caused an adverse synergistic effect resulting in DNA demethylation in high doses of both drugs. In lower concentrations of both drugs DEX kept the increased DNA methylation and attenuated the demethylating effect of TMZ. CONCLUSION Total DNA methylation changes in glioma cell lines under DEX treatment suggest the new mechanism of that drug action and promote clinical implications for adjusting DEX and TMZ therapy in GBM patients. Our results show the potential and possible obstacles of the combined therapy of TMZ with DEX. Our experiments show that combined therapy with both drugs leads to total DNA hypomethylation in high doses of both drugs. Therefore the conclusion would be to adjust DEX administration during TMZ chemotherapy.

Genome-wide methylation profile of mitochondrial DNA across bovine preimplantation development

ABSTRACT This study characterized variations in the methylation profile of mitochondrial DNA (mtDNA) during initial bovine embryo development and correlated the presence of methylation with mtDNA transcription. Bovine oocytes were obtained from abattoir ovaries and submitted to in vitro culture procedures. Oocytes and embryos were collected at various stages (immature oocyte, IM; mature oocyte, MII; zygote, ZY; 4-cells, 4C; 16-cells, 16C and blastocysts, BL). Total DNA (including mtDNA) was used for Whole Genome Enzymatic Methyl Sequencing and for quantification of mtDNA copy number. Extracted RNA was used for quantification of mitochondrial transcripts using Droplet Digital PCR. We selected ND6, CYTB, tRNA-Phe and tRNA-Gln based on their location in the mitochondrial genome, functionality and/or previous literature associating these regions with cytosine methylation. The number of mtDNA copies per oocyte/embryo was found to be similar, while methylation levels in mtDNA varied among stages. Higher total methylation levels were found mainly at 4C and 16C. In specific gene regions, higher methylation levels were also observed at 4C and 16C (ND6, CYTB and tRNA-Phe), as well as an inverse correlation with the quantity of transcripts for these regions. This is a first description of epigenetic changes occurring in mtDNA during early embryonic development. Our results indicate that methylation might regulate the mtDNA transcription at a local level, particularly around the time of embryonic genome activation.

Genome-wide analysis of DNA methylation reveals significant impact of two typical high temperature stress on biological functions in Apostichopus japonicus

Sea cucumber Apostichopus japonicus, an important marine benthic animal, is particularly sensitive to environmental temperature stress, which affected the growth, metabolism, and reproduction even survival. In this study, we used whole genome bisulfite sequencing (WGBS) to perform a comprehensive comparative analysis of the DNA methylation profiles of A. japonicus at three temperatures: normal (20 °C), aestivation (26 °C), and lethal (32 °C). The results showed that the total methylation level was highest in group T26 and lowest in group T32. Most of the DNA methylation occurred on the C bases of the CG type, and the degree of DNA methylation on the CG-type C bases was 13.32–14.99%, reflecting a moderate level of DNA methylation. Comparison of differentially methylated regions (DMRs) in the CG sequence context, we identified 17,174 DMRs in group T26_vs_T20 and 16,216 DMRs in group T32_vs_T26. There were more hypermethylated regions than hypomethylated regions, and this difference was more obvious in group T32_vs_T26. DMRs analysis of the promoter regions and the gene body regions identified more DMR-related genes in the gene body region than in the promoter region in both comparison groups (T26_vs_T20 and T32_vs_T26). GO and KEGG analysis showed that pathways such as protein processing in the endoplasmic reticulum, ubiquitin mediated proteolysis, and endocytosis, etc., were highly enriched in the DMR-related genes in the comparison groups. Some of these pathways that were significantly enriched in the hypermethylated genes in group T26_vs_T20 but were enriched in the hypomethylated genes in group T32_vs_T26. Enrichment analysis of genes both differentially methylated and differentially expressed showed that downregulated genes in group T32_vs_T26 that were also hypomethylated in the gene body region were significantly enriched in the metabolic and the antioxidant-related glutathione metabolism pathway. Our results suggested that DNA methylation develop a lot of variation in the regulation of the response of A. japonicus to two levels of heat stress: at aestivation temperature and at lethal temperature, and there are many differences between the two. The results of this study provide important insights into the epigenetic mechanisms underlying the response of A. japonicus to the pressure of environmental temperature.

Physiological and DNA methylation analysis provides epigenetic insights into kenaf cadmium tolerance heterosis

Soil heavy metal pollution is one of the most challenging problems. Kenaf is an important natural fiber crop with strong heterosis and a higher tolerance to heavy metals. However, little is known about the molecular mechanisms of kenaf heavy metal tolerance, especially the mechanism of genomic DNA methylation regulating heterosis. In this study, kenaf cultivars CP085, CP089, and their hybrid F1 seedlings were subjected to 300 µM cadmium stress and found obvious heterosis of cadmium resistance in morphology and antioxidant enzyme activity of F1 hybrid seedlings. Through methylation-sensitive amplification polymorphism (MSAP) analysis, we highlighted that the total DNA methylation level under cadmium decreased by 16.9 % in F1 and increased by 14.0 % and 3.0 % in parents CP085 and CP089, respectively. The hypomethylation rate was highest (21.84 %), but hypermethylation was lowest (17.24 %) in F1 compared to parent cultivars. In particular, principal coordinates analysis (PCoA) indicates a significant epigenetic differentiation between F1 and its parents under cadmium. Furthermore, 21 differentially methylated DNA fragments (DMFs) were analyzed. Especially, the expression of NPF2.7, NADP-ME, NAC71, TPP-D, LRR-RLKs, and DHX51 genes were changed due to cadmium stress and related to cytosine methylation regulation. Finally, the knocked-down of the differentially methylated gene NPF2.7 by virus-induced gene silencing (VIGS) resulted in increased sensitivity of kenaf seedlings under cadmium stress. It is speculated that low DNA methylation levels can regulate gene expression that led to the heterosis of cadmium tolerance in kenaf.

Comparing the Biological Properties of Double-Stranded DNA Extracted from Human and Porcine Placenta and Salmon Sperm.

Double-stranded fragmented extracellular DNA is a participant, inducer, and indicator of various processes occurring in the organism. When investigating the properties of extracellular DNA, the question regarding the specificity of exposure to DNA from different sources has always been raised. The aim of this study was to perform comparative assessment of biological properties of double-stranded DNA obtained from the human placenta, porcine placenta and salmon sperm. The intensity of leukocyte-stimulating effect of different dsDNA was assessed in mice after cyclophosphamide-induced cytoreduction. The stimulatory effect of different dsDNA on maturation and functions of human dendritic cells and the intensity of cytokine production by human whole blood cells was analyzed ex vivo. The oxidation level of the dsDNA was also compared. Human placental DNA exhibited the strongest leukocyte-stimulating effect. DNA extracted from human and porcine placenta exhibited similar stimulatory action on maturation of dendritic cells, allostimulatory capacity, and ability of dendritic cells to induce generation of cytotoxic CD8+CD107a+ T cells in the mixed leukocyte reaction. DNA extracted from salmon sperm stimulated the maturation of dendritic cells, while having no effect on their allostimulatory capacity. DNA extracted from human and porcine placenta was shown to exhibit a stimulatory effect on cytokine secretion by human whole blood cells. The observed differences between the DNA preparations can be caused by the total methylation level and are not related to differences in oxidation level of DNA molecules. Human placental DNA exhibited the maximum combination of all biological effects.

N6-methyladenosine-modified lncRNA and mRNA modification profiles in cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion injury (CIRI) is common in ischemic stroke and seriously affects the prognosis of patients. At present, N6-methyladenosine (m6A) modification of lncRNAs and mRNAs has been reported in other diseases, such as cancer, but its role in CIRI has not been clarified. In this study, we aimed to investigate the m6A lncRNA and m6A mRNA modification profiles in CIRI. First, we detected the total level of m6A and the changes in related m6A methyltransferases and demethylases in the brain tissue of rats with CIRI and then identified differentially modified lncRNAs and mRNAs in CIRI by lncRNA and mRNA epigenetic transcriptomic microarray. In addition, bioinformatics analysis was used to predict the underlying functions and related pathways of related lncRNAs and mRNAs. We found that the total m6A methylation level was significantly increased, and the expression of fat mass and obesity-associated protein (FTO) was downregulated after CIRI. In addition, a large number of m6A-modified lncRNAs and mRNAs appeared after CIRI, and these genes were mainly enriched for the Toll-like receptor signaling pathway, peroxisome proliferator-activated receptor (PPAR) signaling pathway, and mitogen-activated protein kinase (MAPK) signaling pathway. Our findings provide the basis and insights for further studies on m6A modification in CIRI.

P02.09.B Valproic acid changes total DNA methylation level and influences temozolomide effect in glioblastoma cell lines

Abstract Background Valproic acid (VPA) is a first-line antiepileptic drug for glioblastoma (GBM) patients. There is also some evidence it improves the clinical outcome in those patients. However, the exact mechanism of VPA action is vague. Temozolomide (TMZ) is a first-line chemotherapeutic in GBM. Epigenetics offers a connection between genetic and environmental factors that influence the development of the disease. The best-characterized epigenetic mark is 5-methylcytosine (m5C) in DNA. The aim of that project is to show the effects of VPA administration on the total DNA methylation level. Material and Methods Using the nucleotide post-labeling method, we analyzed the total amount of m5C in DNA of GBM (T98G, U118, U138), cancer (HeLa) and normal (HaCaT) cell lines treated with VPA, and a combination of VPA and with TMZ. Results We adjusted the VPA doses to the ones achieved in the central nervous system during treatment. We observed dose-dependent changes in the total DNA methylation in neoplastic cell lines and the lack of such effect in a normal cell line. VPA alone produced a clear dose-dependent increase in total DNA methylation in GBM cell lines and scarce in the non-neoplastic cell line. In GBM cell lines, TMZ decreased the level of m5C. However, the exposition of GBM cells to the combination of VPA and TMZ caused an adverse synergistic effect resulting in DNA demethylation. The highest loss of m5C was observed at the highest concentration of TMZ (100 μM) and VPA (350 μM), after 3 h of incubation in the T98G cell line. Conclusion Total DNA methylation changes in glioma cell lines under VPA treatment suggest the new mechanism of that drug action and promote clinical implications for adjusting VPA and TMZ therapy in GBM patients. Our results show the potential and possible obstacles of the combined therapy of TMZ with VPA. Our experiments show that combined therapy with both drugs leads to total DNA hypomethylation. Therefore the conclusion would be to stop VPA administration during TMZ chemotherapy temporarily.

Differential Epigenetic Status and Responses to Stressors between Retinal Cybrids Cells with African versus European Mitochondrial DNA: Insights into Disease Susceptibilities.

Mitochondrial (mt) DNA can be classified into haplogroups, which represent populations with different geographic origins. Individuals of maternal African backgrounds (L haplogroup) are more prone to develop specific diseases compared those with maternal European-H haplogroups. Using a cybrid model, effects of amyloid-β (Amyβ), sub-lethal ultraviolet (UV) radiation, and 5-Aza-2′-deoxycytidine (5-aza-dC), a methylation inhibitor, were investigated. Amyβ treatment decreased cell metabolism and increased levels of reactive oxygen species in European-H and African-L cybrids, but lower mitochondrial membrane potential (ΔΨM) was found only in African-L cybrids. Sub-lethal UV radiation induced higher expression levels of CFH, EFEMP1, BBC3, and BCL2L13 in European-H cybrids compared to African-L cybrids. With respect to epigenetic status, the African-L cybrids had (a) 4.7-fold higher total global methylation levels (p = 0.005); (b) lower expression patterns for DNMT3B; and (c) elevated levels for HIST1H3F. The European-H and African-L cybrids showed different transcription levels for CFH, EFEMP1, CXCL1, CXCL8, USP25, and VEGF after treatment with 5-aza-dC. In conclusion, compared to European-H haplogroup cybrids, the African-L cybrids have different (i) responses to exogenous stressors (Amyβ and UV radiation), (ii) epigenetic status, and (iii) modulation profiles of methylation-mediated downstream complement, inflammation, and angiogenesis genes, commonly associated with various human diseases.

Methyl-Sensitive Amplification Polymorphism (MSAP) Analysis Provides Insights into the DNA Methylation Underlying Heterosis in Kenaf (Hibiscus Cannabinus L.) Drought Tolerance

ABSTRACT Kenaf (Hibiscus cannabinus L.) is an industrial fiber crop with significant heterosis and tolerance to abiotic stresses. However, the mechanism underlying heterosis in kenaf under drought conditions is completely unknown. In this study, F1 hybrids were remarkably tolerant to drought stress. Through methylation-sensitive amplification polymorphism (MSAP) analysis, we highlighted that total DNA methylation level under drought decreased by 11.2% in F1 and increased by 29.7% and 13.1% in parents CP079 and CP071, respectively. Unlike parents, the hypomethylation rate was 38% higher than hypermethylation in F1, the overall change in methylation pattern was higher in F1 (1.6%) than parents (0.4%). Furthermore, 35 key DNA fragments showing different methylation patterns were analyzed and found that the cytosine methylation status of DnaJ, ERF5, ZIP2, and PATL3 revealed changes in DNA methylation that may affect their respective expression level. Subsequently, virus-induced gene silencing (VIGS) mediated knockdown of the DnaJ, significantly increased the sensitivity of kenaf seedlings to drought. Overall, this study provides an epigenetic mechanism that could be used to improve the breeding strategy in kenaf.

Sheep IGFBP2 and IGFBP4 promoter methylation regulates gene expression and hair follicle development

BackgroundHair follicle development is important for the fineness of wool. Our previous methylated DNA immunoprecipitation sequencing showed that IGFBP2 and IGFBP4 were differentially methylated at different development stages of the hair follicle of Super Merino sheep. This study selected these two genes as candidates to uncover the influence of their promoter methylation on hair follicle development. ResultsThe results showed that the expression levels of IGFBP2 and IGFBP4 in the skin of very fine wool Super Merino sheep were higher than that in the very thick wool group, and the difference for IGFBP4 was significant. The total methylation rate of IGFBP2 promoter was extremely low, and no significant difference was found in the skin tissue of the Super Merino sheep at different fineness groups. However, the total methylation rate of IGFBP4 promoter in the very thick group was significantly higher than that in the very fine group, and the total DNA methylation levels of the IGFBP4 were significantly negatively correlated with mRNA expression. Furthermore, the transcription factor prediction results showed 104 and 91 candidate binding sites for transcription factors in the promoter regions of IGFBP2 and IGFBP4, respectively. Among these, JUN, EP300, SP1, PAX6, ETS1, and MYOD1 may be related to hair follicle development. ConclusionsThe total methylation level of IGFBP4 promoter was negatively correlated with its mRNA expression in Super Merino sheep skin tissues with different degrees of fineness, which could be used as an epigenetic marker for wool breeding in Super Merino sheep.How to cite: Tian Y, Du J, Yang X, et al. Sheep IGFBP2 and IGFBP4 promoter methylation regulates gene expression and hair follicle development. Electron J Biotechnol 2022;59. https://doi.org/10.1016/j.ejbt.2022.07.001.

Sequence, Expression, and Anti-GCRV Function of the Ferritin from the Grass Carp, Ctenopharyngodon idellus.

Ferritin possesses an immune function to defend against pathogen infection. To elucidate the immunity-protecting roles of ferritin from Ctenopharyngodon idellus (Ciferritin) against virus infection, the cDNA and promoter sequences of Ciferritin were determined, and the correlations between Ciferrtin expressions and promoter methylation levels were analyzed. In addition, the functional role of Ciferrtin on GCRV (grass carp reovirus) infection was assessed. The full-length cDNA of Ciferritin is 1053 bp, consists of a 531 bp open-reading frame, and encodes 176 amino acids. Ciferritin showed the highest sequence identity with the ferritin middle subunit of Mylopharyngodon piceus (93.56%), followed by the subunits of Megalobrama amblycephala and Sinocyclocheilus rhinocerous. Ciferritin contains a conserved ferritin domain (interval: 10–94 aa), and the caspase recruitment domain (CARD) and Rubrerythrin domain were also predicted. In the spleen and kidney, significantly higher Ciferritin expressions were observed at 6, 12, 24, or 168 h post GCRV infection than those in the PBS injection group (p < 0.05). The Ciferrtin expression level in the progeny of maternal-immunized grass carp was significantly higher than that in the progeny of common grass carp (p < 0.05). Ciferritin promoter methylation level in the progeny from common grass carp was 1.27 ± 0.15, and in the progeny of the maternal-immunized group was 1.00 ± 0.14. In addition, methylation levels of “CpG9” and “CpG10” loci were significantly lower in the progeny of maternal-immunized fish than those in the common group. Except for the “CpG5”, methylation levels of all other detected “CpG” loci negatively correlated with Ciferritin expression levels. Furthermore, the total methylation level of “CpG1–10” negatively correlated with the Ciferritin expressions. The Ciferritin expression level was significantly up-regulated, and the VP7 protein levels were significantly reduced, at 24 h post GCRV infection in the Ciferritin over-expression cells (p < 0.05). The results from the present study provide sequence, epigenetic modification and expression, and anti-GCRV functional information of Ciferritin, which provide a basis for achieving resistance to GCRV in grass carp breeding.

Epigenome-Wide Association Study Identified VTI1A DNA Methylation Associated With Accelerometer-Assessed Physical Activity.

Health benefits of physical activity (PA) may be mediated by DNA methylation alterations. The purpose of the current study was to comprehensively identify CpG sites whose methylation levels were associated with accelerometer-assessed total PA in a general Japanese population. The study participants were from the baseline survey of Saga Japan Multi-institutional Collaborative Cohort. PA was objectively measured by a single-axis accelerometer for 7 d. We used a two-stage strategy. In the discovery stage, we performed a meta-analysis of two epigenome-wide association studies of total PA in 898 individuals (a combination of random sample ( n = 507) and case-control study sample ( n = 391)). Peripheral blood DNA methylation levels were measured using Infinium EPIC or HM450 arrays. In the replication stage, we subsequently examined whether CpG sites significantly associated ( P < 1 × 10 -5 ) with total PA were replicated in another sample ( n = 1711), in which methylation levels were measured by pyrosequencing. A multiple linear regression was performed to determine the cross-sectional association between total PA and methylation levels with adjustment for potential confounders, including body mass index. A fixed-effects model was used in the meta-analysis. Correlations between total PA-associated DNA methylation and several inflammatory markers, such as high-sensitivity C-reactive protein, were also conducted. In the meta-analysis, nine CpG sites were significantly associated with total PA ( P < 1 × 10 -5 ). Among the nine sites, one site cg07030336 (annotated to VTI1A/ZDHHC6 gene) was successfully replicated ( P = 0.009). The current study showed that greater accelerometer-assessed total PA was associated with higher DNA methylation levels at cg07030336 ( VTI1A/ZDHHC6 ) in the general population. In addition, we found a divergent relationship between the methylation levels at cg07030336 and several inflammatory biomarkers.

Dexmedetomidine prevents cardiomyocytes from hypoxia/reoxygenation injury via modulating tetmethylcytosine dioxygenase 1-mediated DNA demethylation of Sirtuin1

ABSTRACT Myocardial hypoxia/reoxygenation (H/R) injury is a common pathological change in patients with acute myocardial infarction undergoing reperfusion therapy. Dexmedetomidine (DEX) has been found to substantially improve ischemia-mediated cell damage. Here, we focus on probing the role and mechanism of DEX in ameliorating myocardial H/R injury. Oxygen–glucose deprivation and reoxygenation (OGD/R) were applied to construct the H/R injury model in human myocardial cell lines. After different concentrations of DEX’s treatment, cell counting kit-8 (CCK-8) assay and BrdU assay were employed to test cell viability. The profiles of apoptosis-related proteins Bcl2, Bax, Bad and Caspase3, 8, 9 were determined by Western blot (WB). The expression of inflammatory factors interleukin 1β (IL-1β) and tumor necrosis factor-α (TNF-α) was checked by reverse transcription-polymerase chain reaction (RT-PCR). By conducting WB, we examined the expression of NF-κB, Sirt1, Tet methylcytosine dioxygenase 1 (TET1) and DNA methylation-related proteins (DNA methyltransferase 1, DNMT1; DNA methyltransferase 3 alpha, DNMT3A; and DNA methyltransferase 3 beta, DNMT3B). Our data showed that OGD/R stimulation distinctly hampered the viability and elevated apoptosis and inflammatory factor expression in cardiomyocytes. DEX treatment notably impeded myocardial apoptosis and inflammation and enhanced cardiomyocyte viability. OGD/R enhanced total DNA methylation levels in cardiomyocytes, while DEX curbed DNA methylation. In terms of mechanism, inhibiting TET1 or Sirtuin1 (Sirt1) curbed the DEX-mediated myocardial protection. TET1 strengthened demethylation of the Sirt1 promoter and up-regulated Sirt1. DEX up-regulates Sirt1 by accelerating TET1 and mediating demethylation of the Sirt1 promoter and improves H/R-mediated myocardial injury.

Maslinic acid protects against pressure-overload-induced cardiac hypertrophy by blocking METTL3-mediated m6A methylation.

Coordinated response of the heart to physiological stressors (including stress overload, ischemia, hypothyroidism, and metabolic signals) is a hallmark of heart disease. However, effective treatment and its molecular targets are unknown. Although Maslinic Acid (MA) has been shown to inhibit inflammatory responses with strong anti-tumor, anti-bacterial, and antioxidant effects, information on its role and underlying mechanism in cardiac hypertrophy are scanty. The present study revealed that 10-103 μg/ml MA treatment significantly inhibited Ang-II induced hypertrophy in NMCMs and the dosage did not influence the cell viability of H9C2 and NCMCs. Moreover, the anti-hypertrophy effect of MA (30 mg/kg·day) was verified in the TAC-induced hypertrophy mouse model in vivo. Further analysis showed that MA administration decreased the total RNA m6A methylation and METTL3 levels in Ang-II treated NMCMs and TAC stressed hearts. Rescue experiments under adenovirus-mediated myocardial METTL3 overexpression confirmed that METTL3-mediated m6A methylation is essential in M-driven inhibition of myocardial hypertrophy. Collectively, MA exerts a significant anti-hypertrophy effect by regulating the modification of METTL3-mediated m6A methylation in vitro and in vivo. These findings may provide a platform for establishing a new target and strategy for cardiac hypertrophy treatment.

Untargeted Metabolomics Showed Accumulation of One-Carbon Metabolites to Facilitate DNA Methylation during Extracellular Matrix Detachment of Cancer Cells.

Tumor cells detached from the extracellular matrix (ECM) undergo anoikis resistance and metabolic reprogramming to facilitate cancer cell survival and promote metastasis. During ECM detachment, cancer cells utilize genomic methylation to regulate transcriptional events. One-carbon (1C) metabolism is a well-known contributor of SAM, a global substrate for methylation reactions, especially DNA methylation. DNA methylation-mediated repression of NK cell ligands MICA and MICB during ECM detachment has been overlooked. In the current work, we quantitated the impact of ECM detachment on one-carbon metabolites, expression of 1C regulatory pathway genes, and total methylation levels. Our results showed that ECM detachment promotes the accumulation of one-carbon metabolites and induces regulatory pathway genes and total DNA methylation. Furthermore, we measured the expression of well-known targets of DNA methylation in NK cell ligands in cancer cells, namely, MICA/B, during ECM detachment and observed low expression compared to ECM-attached cancer cells. Finally, we treated the ECM-detached cancer cells with vitamin C (a global methylation inhibitor) and observed a reduction in the promoter methylation of NK cell ligands, resulting in MICA/B re-expression. Treatment with vitamin C was also found to reduce global DNA methylation levels in ECM-detached cancer cells.