Higher DNA Methylation Research Articles

DNA Methylation Levels of the TBX5 Gene Promoter Are Associated with Congenital Septal Defects in Mexican Paediatric Patients.

Simple SummaryOne of the most important health problems in the paediatric population, due to its prevalence worldwide, is the occurrence of congenital heart defects. The integration of multidisciplinary approaches to clinical diagnosis will allow us to detect this type of problem in patients in a timely way. The TBX5 gene has an important participation in cardiogenesis. Therefore, we performed a case-control study, that involved the DNA methylation assessment of the TBX5 gene promoter region in patients that were non-syndromic with congenital septal defects, to identify an epigenetic marker. Moreover, we evaluated the exposure to environmental factors during pregnancy in mothers of these patients. Additionally, we used bioinformatic tools to identify transcription factors binding to the TBX5 gene region of interest and to learn their possible functional effect. These results could help to clarify the mechanisms that regulate these pathologies and establish risk markers, which can be used in future in clinical practice.The TBX5 gene regulates morphological changes during heart development, and it has been associated with epigenetic abnormalities observed in congenital heart defects (CHD). The aim of this research was to evaluate the association between DNA methylation levels of the TBX5 gene promoter and congenital septal defects. DNA methylation levels of six CpG sites in the TBX5 gene promoter were evaluated using pyrosequencing analysis in 35 patients with congenital septal defects and 48 controls. Average methylation levels were higher in individuals with congenital septal defects than in the controls (p < 0.004). In five CpG sites, we also found higher methylation levels in patients than in the controls (p < 0.05). High methylation levels were associated with congenital septal defects (OR = 3.91; 95% CI = 1.02–14.8; p = 0.045). The analysis of Receiver Operating Characteristic (ROC) showed that the methylation levels of the TBX5 gene could be used as a risk marker for congenital septal defects (AUC = 0.68, 95% CI = 0.56–0.80; p = 0.004). Finally, an analysis of environmental factors indicated that maternal infections increased the risk (OR = 2.90; 95% CI = 1.01–8.33; p = 0.048) of congenital septal defects. Our data suggest that a high DNA methylation of the TBX5 gene could be associated with congenital septal defects.

The Effect of Methylation Modification of MDD on the Expression of Key Genes in the Process of Saponin Synthesis in Eleutherococcus senticosus

Mevalonate pyrophosphate decarboxylase is a kind of key enzyme in the terpenoid synthesis pathway in Eleutherococcus senticosus. The results of bisulfite sequencing showed that there were three kinds of samples with a low (0.68%), medium (0.72%) and high (0.79%) DNA methylation ratio in the promoter of MDD in E. senticosus, respectively. The transcriptome sequencing results showed that the expression of MDD in E. senticosus was significantly up-regulated in the types with low DNA methylation ratios of MDD (P < 0.05). There was a significant negative correlation between the saponin content in E. senticosus and the DNA methylation ratio of MDD promoter (P < 0.05). The screening results of differentially expressed transcription factors among the three groups with different DNA methylation ratio showed that a total of 4 transcription factors could bind to 6 CpG sites. Protein-protein interaction analysis showed that E. senticosus MDD could interacted with other key enzymes in the process of terpenoid synthesis. In addition, it was found that the DNA methylation of MDD promoter was mainly regulated by DNA methyltransferase. These results demonstrated that under the action of DNA methyltransferase, the changes of DNA methylation of MDD promoter could regulate its own expression level by affecting the combination ability of transcription factors, and then affected the expression of other genes in terpenoid synthesis pathway. The synthesis and accumulation of saponins in E. senticosus was also changed.

Resetting of the 24-nt siRNA landscape in rice zygotes.

The zygote, a totipotent stem cell, is crucial to the life cycle of sexually reproducing organisms. It is produced by the fusion of two differentiated cells—the egg and sperm, which in plants have radically different siRNA transcriptomes from each other and from multicellular embryos. Owing to technical challenges, the epigenetic changes that accompany the transition from differentiated gametes to totipotent zygote are poorly understood. Because siRNAs serve as both regulators and outputs of the epigenome, we characterized small RNA transcriptomes of zygotes from rice. Zygote small RNAs exhibit extensive maternal carryover and an apparent lack of paternal contribution, indicated by absence of sperm signature siRNAs. Zygote formation is accompanied by widespread redistribution of 24-nt siRNAs relative to gametes, such that ∼70% of the zygote siRNA loci do not overlap any egg cell siRNA loci. Newly detected siRNA loci in zygote are gene-proximal and not associated with centromeric heterochromatin, similar to canonical siRNAs, in sharp contrast to gametic siRNA loci that are gene-distal and heterochromatic. In addition, zygote but not egg siRNA loci are associated with high DNA methylation in the mature embryo. Thus, the zygote begins transitioning before the first embryonic division to an siRNA profile that is associated with future RdDM in embryogenesis. These findings indicate that, in addition to changes in gene expression, the transition to totipotency in the plant zygote is accompanied by resetting of the epigenetic reprogramming that occurred during gamete formation.

Offspring from trained male mice inherit improved muscle mitochondrial function through PPAR co-repressor modulation

AimInvestigate whether inheritance of improved skeletal muscle mitochondrial function and its association with glycemic control are multigenerational benefits of exercise. Main methodsMale Swiss mice were subjected to 8 weeks of endurance training and mated with untrained females. Key findingsTrained fathers displayed typical endurance training-induced adaptations. Remarkably, offspring from trained fathers also exhibited higher endurance performance, mitochondrial oxygen consumption, glucose tolerance and insulin sensitivity. However, PGC-1α expression was not increased in the offspring. In the offspring, the expression of the co-repressor NCoR1 was reduced, increasing activation of PGC-1α target genes. These effects correlated with higher DNA methylation at the NCoR1 promoter in both, the sperm of trained fathers and in the skeletal muscle of their offspring. SignificanceHigher skeletal muscle mitochondrial function is inherited by epigenetic de-activation of a key PGC-1α co-repressor.

DNA methylation of ARHGAP30 is negatively associated with ARHGAP30 expression in lung adenocarcinoma, which reduces tumor immunity and is detrimental to patient survival.

Rho-GTPase activating protein 30 (ARHGAP30) can enhance the intrinsic hydrolysis of GTP and regulates Rho-GTPase negatively. The relationship between ARHGAP30 expression and lung adenocarcinoma is unclear. Therefore, the present study aimed to assess the differences in expression of ARHGAP30 between lung adenocarcinoma tissues and normal tissues and the relationship between DNA methylation and ARHGAP30 expression in lung adenocarcinoma. To determine the role of ARHGAP30 expression in the prognosis and survival of patients with lung adenocarcinoma, gene set enrichment analysis of ARHGAP30 was performed, comprising analyses of Kyoto Encyclopedia of Genes and Genomes pathways, Panther pathways, Reactome pathways, Wikipathways, Gene Ontology, Kinase Target Network, Transcription Factor Network, and a protein-protein interaction network. The association of ARHGAP30 expression with tumor-infiltrating lymphocytes, immunostimulators, major histocompatibility complex molecules, chemokines, and chemokine receptors in lung adenocarcinoma tissues was also analyzed. DNA methylation of ARHGAP30 correlated negatively with ARHGAP30 expression. Patients with lung adenocarcinoma with high DNA methylation of ARHGAP30 had poor prognosis. The prognosis of patients with lung adenocarcinoma with low ARHGAP30 expression was also poor. ARHGAP30 expression in lung adenocarcinoma correlated positively, whereas methylation of ARHGAP30 correlated negatively, with levels of tumor infiltrating lymphocytes. Gene set enrichment analysis revealed that many pathways associated with ARHGAP30 should be studied to improve the diagnosis, treatment, and prognosis of lung adenocarcinoma. We speculated that DNA methylation of ARHGAP30 suppresses ARHGAP30 expression, which reduces tumor immunity, leading to poor prognosis for patients with lung adenocarcinoma.

Leukocyte methylomic imprints of exposure to the genocide against the Tutsi in Rwanda: a pilot epigenome-wide analysis.

Aim & methods: We conducted a pilot epigenome-wide association study of women from Tutsi ethnicity exposed to the genocide while pregnant and their resulting offspring, and a comparison group of women who were pregnant at the time of the genocide but living outside of Rwanda.Results: Fifty-nine leukocyte-derived DNA samples survived quality control: 33 mothers (20 exposed, 13 unexposed)and 26 offspring (16 exposed, 10 unexposed). Twenty-four significant differentially methylated regions (DMRs) were identified in mothers and 16 in children. Conclusions:In utero genocide exposure was associated with CpGs in three of the 24 DMRs: BCOR, PRDM8and VWDE, with higher DNAmethylation in exposed versusunexposed offspring. Of note, BCOR and VWDE show significant correlation between brain and blood DNA methylation within individuals, suggesting these peripherally derived signals of genocide exposure may have relevance to the brain.

Longitudinal DNA methylation dynamics as a practical indicator in clinical epigenetics

BackgroundOne of the fundamental assumptions of DNA methylation in clinical epigenetics is that DNA methylation status can change over time with or without interplay with environmental and clinical conditions. However, little is known about how DNA methylation status changes over time under ordinary environmental and clinical conditions. In this study, we revisited the high frequency longitudinal DNA methylation data of two Japanese males (24 time-points within three months) and characterized the longitudinal dynamics.ResultsThe results showed that the majority of CpGs on Illumina HumanMethylation450 BeadChip probe set were longitudinally stable over the time period of three months. Focusing on dynamic and stable CpGs extracted from datasets, dynamic CpGs were more likely to be reported as epigenome-wide association study (EWAS) markers of various traits, especially those of immune- and inflammatory-related traits; meanwhile, the stable CpGs were enriched in metabolism-related genes and were less likely to be EWAS markers, indicating that the stable CpGs are stable both in the short-term within individuals and under various environmental and clinical conditions.ConclusionsThis study indicates that CpGs with different stabilities are involved in different functions and traits, and thus, they are potential indicators that can be applied for clinical epigenetic studies to outline underlying mechanisms.

Iron homeostasis pathway DNA methylation trajectories reveal a role for STEAP3 metalloreductase in patient outcomes after aneurysmal subarachnoid hemorrhage

BackgroundFollowing aneurysmal subarachnoid hemorrhage (aSAH), the brain is susceptible to ferroptosis, a type of iron-dependent cell death. Therapeutic intervention targeting the iron homeostasis pathway shows promise for mitigating ferroptosis and improving recovery in animal models, but little work has been conducted in humans. DNA methylation (DNAm) plays a key role in gene expression and brain function, plasticity, and injury recovery, making it a potentially useful biomarker of outcomes or therapeutic target for intervention. Therefore, in this longitudinal, observational study, we examined the relationships between trajectories of DNAm in candidate genes related to iron homeostasis and acute (cerebral vasospasm and delayed cerebral ischemia) and long-term (Glasgow Outcome Scale [GOS, unfavorable = 1–3] and death) patient outcomes after aSAH.ResultsLongitudinal, genome-wide DNAm data were generated from DNA extracted from post-aSAH cerebrospinal fluid (n = 260 participants). DNAm trajectories of 637 CpG sites in 36 candidate genes related to iron homeostasis were characterized over 13 days post-aSAH using group-based trajectory analysis, an unsupervised clustering method. Significant associations were identified between inferred DNAm trajectory groups at several CpG sites and acute and long-term outcomes. Among our results, cg25713625 in the STEAP3 metalloreductase gene (STEAP3) stood out. Specifically, in comparing the highest cg25713625 DNAm trajectory group with the lowest, we observed significant associations (i.e., based on p-values less than an empirical significance threshold) with unfavorable GOS at 3 and 12 months (OR = 11.7, p = 0.0006 and OR = 15.6, p = 0.0018, respectively) and death at 3 and 12 months (OR = 19.1, p = 0.0093 and OR = 12.8, p = 0.0041, respectively). These results were replicated in an independent sample (n = 100 participants) observing significant associations with GOS at 3 and 12 months (OR = 8.2, p = 0.001 and OR = 6.3, p = 0.0.0047, respectively) and death at 3 months (OR = 2.3, p = 0.008) and a suggestive association (i.e., p-value < 0.05 not meeting an empirical significance threshold) with death at 12 months (OR = 2.0, p = 0.0272). In both samples, an additive effect of the DNAm trajectory group was observed as the percentage of participants with unfavorable long-term outcomes increased substantially with higher DNAm trajectory groups.ConclusionOur results support a role for DNAm of cg25713625/STEAP3 in recovery following aSAH. Additional research is needed to further explore the role of DNAm of cg25713625/STEAP3 as a biomarker of unfavorable outcomes, or therapeutic target to improve outcomes, to translate these findings clinically.

Imprinted lncRNA Dio3os preprograms intergenerational brown fat development and obesity resistance

Maternal obesity (MO) predisposes offspring to obesity and metabolic disorders but little is known about the contribution of offspring brown adipose tissue (BAT). We find that MO impairs fetal BAT development, which persistently suppresses BAT thermogenesis and primes female offspring to metabolic dysfunction. In fetal BAT, MO enhances expression of Dio3, which encodes deiodinase 3 (D3) to catabolize triiodothyronine (T3), while a maternally imprinted long noncoding RNA, Dio3 antisense RNA (Dio3os), is inhibited, leading to intracellular T3 deficiency and suppression of BAT development. Gain and loss of function shows Dio3os reduces D3 content and enhances BAT thermogenesis, rendering female offspring resistant to high fat diet-induced obesity. Attributing to Dio3os inactivation, its promoter has higher DNA methylation in obese dam oocytes which persists in fetal and adult BAT, uncovering an oocyte origin of intergenerational obesity. Overall, our data uncover key features of Dio3os activation in BAT to prevent intergenerational obesity and metabolic dysfunctions.

Dietary with proper ratio of alpha‐linolenic acid to linoleic acid enhanced the unsaturated fatty acids deposition of Chinese perch ( Siniperca Chuatsi )

The study explored the effect of increasing α-linolenic acid (LNA; 18:3n-3)-to-linoleic acid (LA; 18:2n6) ratios on the growth, fatty acid (FA) composition/deposition, hepatic expression of various genes responsible for fatty acid metabolism, fatty acid binding proteins (fabp1) from the level of DNA methylation and liver/intestinal histology in Chinese perch, Siniperca chuatsi. A total of 5 isonitrogenous and isolipidic diets were formulated at LNA/LA ratios of 0.15, 0.38, 0.94, 1.43 and 2.94, hereafter referred to as D1, D2, D3, D4 and D5, respectively, and fed to the fish that could accept artificial diets successfully for 90 days. Results showed that the D3 diet led to significantly higher growth than all other treatments. Additionally, the D3 diet led to significantly longer intestinal villi, greater number of hepatic lipid droplets and higher hepatic triglyceride (TG) content. With increasing LNA/LA ratios, the LNA content increased and LA decreased in both the liver and muscle. Dietary LNA/LA ratios markedly affected the relative expression level of some genes of FA metabolism in hepatic, including fatty acid synthase (fas), fatty acid binding proteins (fabp1), fatty acid desaturase 2 (fads2) and fatty acid elongase 5 (elovl5). The D3 diet enhanced hepatic FA metabolism in hepatic markedly and thus promoted the arachidonic acids (ARA) deposition. In addition, it can promote the deposition of eicosapentaenoic acids (EPA) in muscle. It was shown that dietary with the ratio of LNA to LA being 0.94 can enhance the FA metabolism in hepatic markedly and thus promoted the polyunsaturated fatty acid (PUFA) deposition. And we found that there was high DNA methylation in six CpG sites of fabp11 for D1 group and D3 group, but D3 group had no marked difference with control group (D1). Finally, through the broken-line regression analysis, it was found that the optimal dietary LNA/LA ratio of Chinese perch was 0.81.

Prediction and prognostic significance of ALOX12B and PACSIN1 expression in gastric cancer by genome-wide RNA expression and methylation analysis.

Stomach adenocarcinoma (STAD) is one of the common gastrointestinal cancers, characterized by late discovery and metastasis. However, research of gene methylation and expression in gastric cancer (GC) metastasis has been quite limited. This study aimed to investigate the altered gene expression patterns between metastasis and non-metastasis samples using high-throughput RNA and methylation profiles from a large number of patients. Another aim was to identify a specific potential metastasis biomarker, with the ability to predict the metastasis possibility and prognosis of patients with STAD. In this study, we integrated The Cancer Genome Atlas (TCGA) program STAD datasets, analyzed the RNA expression and DNA methylation data between non-metastasis (M0) and distant metastasis (M1) samples, and evaluated the candidate biomarker in survival and prognosis of GC. Among all patients enrolled, 329 with M0 and M1 information were positive for RNA analysis, and 353 with M0 and M1 information were positive for methylation analysis. We found 29 upregulated and 200 downregulated genes in RNA level, and 5,046 hypermethylated and 8,563 hypomethylated probes in methylation level. Among these genes, we found high RNA expression level and low DNA methylation level of ALOX12B and PACSIN1 in GC metastasis samples. Patients with high expression of these 2 genes had poor overall survival (OS), progression-free survival (PFS), and post-progression survival (PPS). The expression levels of ALOX12B and PACSIN1 were higher in the metastasis than non-metastasis group, and participants with high expression of these 2 genes were found to have poor survival. The genes ALOX12B and PACSIN1 are potential biomarkers of metastasis and poor prognosis, especially in early stage GC, and provide additional information for subsequent comprehensive treatment of GC.

High cytokinin concentration and nutrient starvation trigger DNA methylation changes in somaclonal variants of Agave angustifolia Haw

Somaclonal variation (SV) is observed during micropropagation, especially in organogenic processes when differentiated somatic cells are induced into a pluripotent state. However, the clones obtained show differences in phenotype from each other and from the mother plants, which could be an agricultural problem or an ornamental advantage. Few of the mechanisms associated with how a somatic cell generates phenotypic variation have been elucidated. Recently, our group demonstrated that an albino phenotype produced in Agave in in vitro culture was linked to changes in DNA methylation through the micropropagation process, suggesting a new way to investigate the mechanism. Therefore, here we evaluate the effect of stressful conditions in the culture media on three different phenotypes (green, variegated and albino) of Agaveangustifolia Haw. to determine the changes in DNA methylation linked to SV in the new formed shoots. The effects of nutrient availability and exogen cytokinin concentration in the culture media were evaluated for five months in each phenotype. We observed that a reduction of 25 % in the nutrients in the culture media affected the coloration of green plantlets. On the other hand, a high 6-benzyladenine (BA) concentration exposure (88.8 μM) in the variegated phenotype enhanced organogenic capability, increased phenotypic variation, and changed DNA methylation levels. We have demonstrated that long periods of time under nutrient-limited conditions or high cytokinin concentration provoke changes in DNA methylation levels that increase SV frequency in the newly formed shoots. Epigenetic changes may thus be an overlooked factor in SV induced by culture media stress.

Determination of Genetic and Epigenetic Modifications-Related Prognostic Biomarkers of Breast Cancer: Genome High-Throughput Data Analysis.

The high heterogeneity of breast cancer (BRCA) makes it more challenging to interpret the genetic variation mechanisms involved in BRCA pathogenesis and prognosis. Areas with high DNA methylation (such as CpG islands) were accompanied by copy number variation (CNV), and these genomic variations affected the level of DNA methylation. In this study, we characterized intertumor heterogeneity and analyzed the effects of CNV on DNA methylation and gene expression. In addition, we performed a Genetic Set Enrichment Analysis (GSEA) to identify key pathways for changes between patients with low and high expression of genes. Our analysis found two key genes, namely, HPDL and SOX17. The protein expressed by HPDL is 4-hydroxyphenylpyruvate dioxygenase-like protein, which has dioxygenase activity. SOX17 is a transcription factor that can inhibit Wnt signaling, promote the degradation of activated CTNNB1, and participate in cell proliferation. Our analysis found that the CNV of HPDL and SOX17 is not only related to the patient's prognosis, but also related to gene methylation and expression levels affecting the patient's survival time. Among them, the high-methylation, low-expression HPDL and SOX17 showed poor prognosis. And the addition of two copies of SOX17 is associated with a lower survival rate, while a decrease in the copy number of HPDL also suggests a poor prognosis. This study provided an effective bioinformatics basis for further exploration of molecular mechanisms related to BRCA and assessment of patient prognosis, but the development of biomarkers for diagnosis and treatment still requires further clinical data validation.

Integrated Analysis of the Expression Characteristics, Prognostic Value, and Immune Characteristics of PPARG in Breast Cancer.

Background: Breast cancer (BRCA) is the most frequent malignancy. Identification of potential biomarkers could help to better understand and combat the disease at early stages.Methods: We selected the overlapping genes of differential expressed genes and genes in BRCA-highly correlated modules by Weighted Gene Co-Expression Network Analysis (WGCNA) in TCGA and GEO data and performed KEGG and GO enrichment. PPARG was achieved from Protein-Protein Interaction (PPI) network analysis and prognostic analysis. TIMER, UALCAN, GEO, TCGA, and western blot analysis were used to validate the expression of PPARG in BRCA. PPARG was further analyzed by DNA methylation, immune parameters, and tumor mutation burden.Results: Among 381 overlapping genes, the lipid metabolic process was identified as highly enriched pathways in BRCA by TCGA and GEO data. When the prognostic analysis of 10 core genes by PPI network was performed, results revealed that high expression of PPARG was significantly correlated to a better prognosis. PPARG was lesser expression in BRCA according to TIMER, UALCAN, GEO, TCGA, and western blot in both mRNA level and protein level. PPARG had several high DNA methylation level sites and the methylation level is negatively correlated to expression. PPARG is also correlated to TNM stages, tumor microenvironment, and tumor burden.Conclusions: Findings of our study identified the PPARG as a potential biomarker by confirming its low expression in BRCA and its correlation to prognosis. Moreover, its correlation to DNA methylation and tumor microenvironment may guide new therapeutic strategies for BRCA patients.

Interplay between RNA Methylation Eraser FTO and Writer METTL3 in Renal Clear Cell Carcinoma Patient Survival.

m6A-methyltransferase METTL3 and demethylase FTO regulate gene expression by dynamically modifying RNA methylation. However, their clinical relevance in renal Clear Cell Carcinoma (CCRCC) has not been well elucidated. This study aims to investigate prognostic values of FTO and METTL3 mRNA and DNA methylation, their differential expression and associations with chemokines and inflammation-related genes in CCRCC. Kaplan-Meier survival curves and multivariate cox regression were performed for survival analyses, and random-effects meta-analysis was conducted for differential expression of FTO and METTL3 in CCRCC. A significantly negative correlation was observed between mRNA and DNA methylation for both FTO and METTL3. Survival analysis showed a superior survival in patients with either high FTO mRNA or low DNA methylation, or low METTL3 mRNA or high DNA methylation. The adjusted hazard ratios were 0.67 (95% CI: 0.49-0.91, p = 0.01) for high vs. low FTO mRNA, 2.17 (1.38-3.42, p = 0.0008) for high vs. low FTO DNA methylation, 1.97 (1.45-2.68, p < 0.0001) for high vs. low METTL3 mRNA, and 0.49 (0.31-0.79, p = 0.003) for high vs. low METTL3 DNA methylation, respectively. There was a significant interaction between FTO and METTL3 mRNA levels (p = 0.024). Upregulation of FTO and METTL3 with 1.64 folds (95% CI: 1.43-1.89) and 1.17 folds (1.02-1.35), respectively, was observed in CCRCC vs. normal kidney tissues. FTO and METTL3 mRNA showed opposite expressions of CD8+ T cell migration-related chemokines. Dysregulated FTO and METTL3 may be involved in the disease development and progression, affecting immune response in CCRCC. FTO and METTL3 expression and DNA methylation are potential prognostic markers of CCRCC.

Dietary fat intake during early pregnancy is associated with cord blood DNA methylation at IGF2 and H19 genes in newborns.

Maternal fat intake during pregnancy affects fetal growth, but mechanisms underlying this relationship are unclear. We performed an exploratory study of the associations of fat consumption during pregnancy with cord blood DNA methylation of the insulin‐like growth factor 2 (IGF2) and H19 genes. We used data from 96 uncomplicated full‐term pregnancies of mothers of whom majority had normal body mass index (BMI) (66%) in Project Viva, a prospective pre‐birth cohort. We assessed maternal diet with validated food frequency questionnaires during the first and second trimesters and measured DNA methylation in segments of the IGF2‐ and H19‐differentially methylated regions (DMRs) by pyrosequencing DNA extracted from umbilical cord blood samples. Mean (SD) age was 32.8 (4.1) years and prepregnancy BMI was 24.0 (4.4) kg/m2. Mean DNA methylation was 56.3% (3.9%) for IGF2‐DMR and 44.6% (1.9%) for H19‐DMR. Greater first trimester intake of omega‐6 polyunsaturated fat (effect per 1% of calories at the expense of carbohydrates) was associated with lower DNA methylation of IGF2‐DMR (−1.2%; 95% confidence interval [CI]: −2.2%, −0.2%) and higher DNA methylation at H19‐DMR (0.8%; 95% CI: 0.3%, 1.3%). On the other hand, greater first trimester intake of omega‐3 polyunsaturated fat was associated with lower DNA methylation of the H19‐DMR (−4.3%; 95% CI: −7.9%, −0.8%). We did not find significant associations of IGF2 and H19 methylation with IGF2 cord blood levels. Our findings suggest that early prenatal fat intake (omega‐3, omega‐6, and saturated fatty acids) may influence DNA methylation at the IGF2 and H19 locus, which could impact fetal development and long‐term health.

High throughput and low bias DNA methylation and hydroxymethylation analysis by direct injection mass spectrometry

We present a direct injection mass spectrometry (DI-MS) platform that accurately, precisely, and quickly quantitates global levels of DNA cytidine methylation (5 mC) and hydroxymethylation (5hmC). Our platform combines an Advion TriVersa NanoMate coupled online to a Thermo Scientific Orbitrap Fusion Lumos. Following digestion to nucleosides, the DNA samples are analyzed at the rate of <1 min per injection with comparable detection limits of 0.63 ng/μL and 0.31 ng/μL, respectively. In contrast, the detection limits for 5 mC and 5hmC in state-of-art nano liquid chromatography (LC) coupled to online mass spectrometry (nLC-MS) are notably different (0.04 ng/μL and 2.5 ng/μL, respectively). The high sensitivity of DI-MS is achieved by optimizing sample buffer composition, the source fragmentation energy, and the radio frequency of the instrument ion funnel. DI-MS accurately reports the relative abundance of 5 mC and 5hmC over a range of 1%–7% (R2 > 0.98) and 0.13%–1.75% (R2 > 0.99), respectively. Accurate measurement of C, 5 mC and 5hmC is achieved by optimizing in-source fragmentation to obtain a population of up to 93% of just the nucleoside base. This protocol minimizes base dimer formation and partial base-deoxyribose dissociation in gas phase and greatly improves modified base quantitation. We also demonstrate that DI-MS overcomes biases in differential chromatographic retention and issues of sample degradation in the autosampler due to its high throughput. Finally, we present an application of our workflow to quantify DNA modifications on a batch of 81 samples in about 1.5 h.

Physical exercise increases global and gene-specific (interleukin-17 and interferon-γ) DNA methylation in lymphocytes from aged women.

What is the central question of this study? Is lymphocyte DNA methylation differentially modulated by resistance training and aerobic exercise in older women? What is the main finding and its importance? The practice of resistance training led to an increased global DNA methylation in lymphocytes. The exercise-induced increase of inflammatory genes methylation may be associated with immune function impairment during ageing. Ageing-induced increase in inflammatory gene expression through a reduction in DNA methylation might contribute to chronic diseases. Regular physical exercise practices, in turn, are associated with a decrease in the incidence of inflammatory diseases. We herein evaluated the effects of three exercise modalities on lymphocyte global and gene-specific (interferon γ (IFN-γ) and interleukin 17A (IL-17A) DNA methylation in aged women (68±7.5years). This cross-sectional study included 86 women, divided into four groups according to the physical exercise practice: 20 were practicing resistance training (RT); 24 were practicing water aerobics exercise (W); 22 were practicing water aerobics and resistance exercise (RWT), and 20 did not practice any physical exercise (CON). We evaluated volunteer functional capability using the Timed Up and Go (TUG) test, global lymphocyte DNA methylation by enzyme-linked immunosorbent assay, IFN-γ and IL-17A methylation by qPCR and CD4+ IFN-γ+ and CD4+ IL-17+ cell percentage by flow cytometry. The three physically exercised groups performed functional capability tests in a shorter period and showed a higher global lymphocyte DNA methylation and methylated CpGs of IL-17A and IFN-γ promoter regions than the control group. The practice of resistance training (RT and RWT groups) lead to high global DNA methylation. The combination of resistance training and aerobic exercise led to the increase of lymphocyte IL-17A and IFN-γ gene methylation induced by each separately. However, the percentage of IFN-γ+ and IL-17+ cells was lower only in the RT group. The exercise-induced increase of inflammatory-gene methylation may be associated with gene expression changes and immune function impairment during ageing.

Activation of Cryptic Secondary Metabolite Biosynthesis in Bamboo Suspension Cells by a Histone Deacetylase Inhibitor.

Plants have evolved a diverse array of secondary metabolite biosynthetic pathways. Undifferentiated plant cells, however, tend to biosynthesize secondary metabolites to a lesser extent and sometimes not at all. This phenomenon in cultured cells is associated with the transcriptional suppression of biosynthetic genes due to epigenetic alterations, such as low histone acetylation levels and/or high DNA methylation levels. Here, using cultured cells of bamboo (Bambusa multiplex; Bm) as a model system, we investigated the effect of histone deacetylase (HDAC) inhibitors on the activation of cryptic secondary metabolite biosynthesis. The Bm suspension cells cultured in the presence of an HDAC inhibitor, suberoyl bis-hydroxamic acid (SBHA), exhibited strong biosynthesis of some compounds that are inherently present at very low levels in Bm cells. Two major compounds induced by SBHA were isolated and were identified as 3-O-p-coumaroylquinic acid (1) and 3-O-feruloylquinic acid (2). Their productivities depended on the type of basal culture medium, initial cell density, and culture period, as well as the SBHA concentration. The biosynthesis of these two compounds was also induced by another HDAC inhibitor, trichostatin A. These results demonstrate the usefulness of HDAC inhibitors to activate cryptic secondary metabolite biosynthesis in cultured plant cells.

Acute Brain-Derived Neurotrophic Factor DNA Methylation Trajectories in Cerebrospinal Fluid and Associations With Outcomes Following Severe Traumatic Brain Injury in Adults

Background. Epigenetic biomarkers have the potential to explain outcome heterogeneity following traumatic brain injury (TBI) but are largely unexplored. Objective. This exploratory pilot study characterized brain-derived neurotrophic factor (BDNF) DNA methylation trajectories following severe TBI. Methods. Brain-derived neurotrophic factor DNA methylation trajectories in cerebrospinal fluid (CSF) over the first 5 days following severe TBI in 112 adults were examined in association with 3- and 12-month outcomes. Results. Group-based trajectory analysis revealed low and high DNA methylation groups at two BDNF cytosine-phosphate-guanine (CpG) targets that showed suggestive associations (P < .05) with outcomes. Membership in the high DNA methylation groups was associated with better outcomes after controlling for age, sex, and injury severity. Associations of age × trajectory group interactions with outcomes at a third CpG site revealed a pattern of the same or better outcomes with higher ages in the high DNA methylation group and worse outcomes with higher ages in the low DNA methylation group. Conclusions. Although no observed associations met the empirical significance threshold after correcting for multiple comparisons, suggestive associations of the main effect models were consistent in their direction of effect and were observed across two CpG sites and two outcome time points. Results suggest that higher acute CSF BDNF DNA methylation may promote recovery following severe TBI in adults, and this effect may be more robust with higher age. While the results require replication in larger and racially diverse independent samples, BDNF DNA methylation may serve as an early postinjury biomarker helping to explain outcome heterogeneity following TBI.