Epigenetic and sex differences in opioid use disorder in chronic pain: A real-world study linked with OPRM1 DNA methylation.

Opioid use disorder (OUD) is a multifaceted condition influenced by sex, genetic and environmental factors that could be linked with epigenetic changes. Understanding how these factors interact is crucial to understand and address the development and progression of this disorder. Our aim was to elucidate different potential epigenetic and genetic mechanisms between women and men that correlate with OUD under real-world pain unit conditions. Associations between analgesic response and the DNA methylation level of the opioid mu receptor (OPRM1) gene (CpG sites 1-5 selected in the promoter region) were evaluated in 345 long opioid-treated chronic non cancer pain: cases with OUD (n = 67) and controls (without OUD, n = 278). Cases showed younger ages, low employment status and quality of life, but higher morphine equivalent daily dose and psychotropic use, compared to the controls. The patients with OUD showed a significant decrease in OPRM1 DNA methylation, which correlated with clinical outcomes like pain relief, depression and different adverse events. Significant differences were found at the five CpG sites studied for men, and exclusively in women for CpG site 3, in relation to OUD diagnosis. These findings support the importance of epigenetics and sex as biological variables to be considered toward efficient OUD understanding and therapy development.

Role of ZNF334 in cervical cancer: implications for EMT reversal and tumor suppression.

Zinc-finger proteins are involved in many biological processes. However, the role of Zinc-finger protein 334 (ZNF334) in cervical cancer remains unidentified. This study showed that promoter methylation of ZNF334 was responsible for its reduced expression. ZNF334 suppressed malignant biological behaviors in cervical cancer. Notably, ZNF334 reversed the EMT process both in vitro and in vivo. RNA-seq coupled with bioinformatics analysis caught P3H3 which is upregulated by ZNF334. Dual-luciferase reporter and Chromatin immunoprecipitation assays illustrated that ZNF334 directly regulate P3H3. Knockdown of P3H3 attenuated the reversal of EMT induced by ZNF334. Additionally, ZNF334 overexpression sensitized cervical cancer cells to the cytotoxic effects of paclitaxel, cyclosporine and sunitinib. In conclusions, this study illustrated that DNA methylation-based silencing ZNF334 played a vital role in cervical cancer, by regulating P3H3 in turn affects EMT. ZNF334 has the potential to become a novel diagnostic biomarker and a potential treatment target for cervical cancer.

Transcriptome Dynamics and Cell Dialogs Between Oocytes and Granulosa Cells in Mouse Follicle Development.

The development and maturation of follicles is a sophisticated and multistage process. The dynamic gene expression of oocytes and their surrounding somatic cells and the dialogs between these cells are critical to this process. In this study, we accurately classified the oocyte and follicle development into nine stages and profiled the gene expression of mouse oocytes and their surrounding granulosa cells and cumulus cells. The clustering of the transcriptomes showed the trajectories of two distinct development courses of oocytes and their surrounding somatic cells. Gene expression changes precipitously increased at Type 4 stage and drastically dropped afterward within both oocytes and granulosa cells. Moreover, the number of differentially expressed genes between oocytes and granulosa cells dramatically increased at Type 4 stage, most of which persistently passed on to the later stages. Strikingly, cell communications within and between oocytes and granulosa cells became active from Type 4 stage onward. Cell dialogs connected oocytes and granulosa cells in both unidirectional and bidirectional manners. TGFB2/3, TGFBR2/3, INHBA/B, and ACVR1/1B/2B of TGF-ß signaling pathway functioned in the follicle development. NOTCH signaling pathway regulated the development of granulosa cells. Additionally, many maternally DNA methylation- or H3K27me3-imprinted genes remained active in granulosa cells but silent in oocytes during oogenesis. Collectively, Type 4 stage is the key turning point when significant transcription changes diverge the fate of oocytes and granulosa cells, and the cell dialogs become active to assure follicle development. These findings shed new insights on the transcriptome dynamics and cell dialogs facilitating the development and maturation of oocytes and follicles.

[Clinicopathological features and prognosis of sporadic mismatch repair deficient colorectal cancer].

Objective: To investigate the clinicopathological characteristics and prognostic factors of sporadic mismatch repair deficient (dMMR) colorectal cancer. Methods: A total of 120 cases of sporadic dMMR colorectal cancer from July 2015 to April 2021 were retrospectively collected in Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College. Patients with Lynch syndrome; synchronous multiple colorectal cancers; preoperative anti-tumor treatments such as chemotherapy and radiotherapy; and those with incomplete follow-up information were excluded based on family history and next-generation sequencing (NGS) test results. Immunohistochemical stains were used to detect the expression of mismatch repair proteins, methylation-specific PCR for methylation testing, and fluorescent PCR for BRAF V600E gene mutation detection. The clinical and pathological data, and gene mutation status were analyzed. Follow-up was done to assess survival and prognosis including progression-free survival and overall survival rate. Results: Sporadic dMMR colorectal cancer occurred more frequently in the right side of the colon, in females, and in the elderly. Morphologically, it was mostly moderately-differentiated, and most patients had low-grade tumor budding. In terms of immunohistochemical expression, MLH1 and PMS2 loss were dominant, and there were age and location-specificities in protein expression. MLH1 methylation was commonly detected in elderly female patients and rare in young male patients; while MLH1 and PMS2 deficiency, and BRAF V600E mutation occurred more often on the right side (P<0.05). The 3-year and 5-year progression-free survival rates were 90.7% and 88.7% respectively, and the 3-year and 5-year overall survival rates were 92.8% and 90.7% respectively. Tumor budding status was an independent risk factor affecting patient recurrence (hazard ratio=3.375, 95% confidence interval: 1.060-10.741, P=0.039), patients with low-grade tumor budding had better prognosis, and those with medium or high-grade tumor budding had poor prognosis. Conclusion: For dMMR colorectal cancer patients, tumor budding status is an independent risk factor for recurrence.

Epigenetic scores derived in saliva are associated with gestational age at birth.

BACKGROUND: Epigenetic scores (EpiScores), reflecting DNA methylation (DNAm)-based surrogates for complex traits, have been developed for multiple circulating proteins. EpiScores for pro-inflammatory proteins, such as C-reactive protein (DNAm CRP), are associated with brain health and cognition in adults and with inflammatory comorbidities of preterm birth in neonates. Social disadvantage can become embedded in child development through inflammation, and deprivation is overrepresented in preterm infants. We tested the hypotheses that preterm birth and socioeconomic status (SES) are associated with alterations in a set of EpiScores enriched for inflammation-associated proteins. RESULTS: In total, 104 protein EpiScores were derived from saliva samples of 332 neonates born at gestational age (GA) 22.14 to 42.14 weeks. Saliva sampling was between 36.57 and 47.14 weeks. Forty-three (41%) EpiScores were associated with low GA at birth (standardised estimates |0.14 to 0.88|, Bonferroni-adjusted p-value < 8.3 × 10-3). These included EpiScores for chemokines, growth factors, proteins involved in neurogenesis and vascular development, cell membrane proteins and receptors, and other immune proteins. Three EpiScores were associated with SES, or the interaction between birth GA and SES: afamin, intercellular adhesion molecule 5, and hepatocyte growth factor-like protein (standardised estimates |0.06 to 0.13|, Bonferroni-adjusted p-value < 8.3 × 10-3). In a preterm subgroup (n = 217, median [range] GA 29.29 weeks [22.14 to 33.0 weeks]), SES-EpiScore associations did not remain statistically significant after adjustment for sepsis, bronchopulmonary dysplasia, necrotising enterocolitis, and histological chorioamnionitis. CONCLUSIONS: Low birth GA is substantially associated with a set of EpiScores. The set was enriched for inflammatory proteins, providing new insights into immune dysregulation in preterm infants. SES had fewer associations with EpiScores; these tended to have small effect sizes and were not statistically significant after adjusting for inflammatory comorbidities. This suggests that inflammation is unlikely to be the primary axis through which SES becomes embedded in the development of preterm infants in the neonatal period.

Mecp2 promotes the anti-inflammatory effect of alpinetin via epigenetic modification crosstalk.

In recent years, inflammatory disorders have emerged as a significant concern for human health. Through ongoing research on anti-inflammatory agents, alpinetin has shown promising anti-inflammatory properties, including involvement in epigenetic modification pathways. As a crucial regulator of epigenetic modifications, Mecp2 may play a role in modulating the epigenetic effects of alpinetin, potentially impacting its anti-inflammatory properties. To test this hypothesis, two key components, p65 (a member of NF-KB family) and p300 (a type of co-activator), were screened by the expression profiling microarray, which exhibited a strong correlation with the intensity of LPS stimulation in mouse macrophages. Meanwhile, alpinetin demonstrates the anti-inflammatory properties through its ability to disrupt the synthesis of p65 and its interaction with promoters of inflammatory genes, yet it did not exhibit similar effects on p300. Additionally, Mecp2 can inhibit the binding of p300 by attaching to the methylated inflammatory gene promoter induced by alpinetin, leading to obstacles in promoter acetylation and subsequently impacting the binding of p65, ultimately enhancing the anti-inflammatory capabilities of alpinetin. Similarly, in a sepsis mouse model, it was observed that homozygotes overexpressing Mecp2 showed a greater reduction in organ damage and improved survival rates compared to heterozygotes when administered by alpinetin. However, blocking the expression of DNA methyltransferase 3A (DNMT3A) resulted in the loss of Mecp2's anti-inflammatory assistance. In conclusion, Mecp2 may augment the anti-inflammatory effects of alpinetin through epigenetic 'crosstalk', highlighting the potential efficacy of a combined therapeutic strategy involving Mecp2 and alpinetin for anti-inflammatory intervention.

Epigenetic dysregulation in cancers by isocitrate dehydrogenase 2 (IDH2).

Recent advances in genome-wide studies have revealed numerous epigenetic regulations brought about by genes involved in cellular metabolism. Isocitrate dehydrogenase (IDH), an essential enzyme, that converts isocitrate into -ketoglutarate (KG) predominantly in the tricarboxylic acid (TCA) cycle, has gained particular importance due to its cardinal role in the metabolic pathway in cells. IDH1, IDH2, and IDH3 are the three isomeric IDH enzymes that have been shown to regulate cellular metabolism. Of particular importance, IDH2 genes are associated with several cancers, including gliomas, oligodendroglioma, and astrocytomas. These mutations lead to the production of oncometabolite D-2-hydroxyglutarate (D-2-HG), which accumulates in cells promoting tumor growth. The enhanced levels of D-2-HG competitively inhibit α-KG dependent enzymes, inhibiting cell TCA cycle, upregulating the cell growth and survival relevant HIF-1α pathway, promoting DNA hypermethylation related epigenetic activity, all of which synergistically contribute to carcinogenesis. The present review discusses epigenetic mechanisms inIDH2 regulation in cells and further its clinical implications.

Research progress of DNA methylation in colorectal cancer (Review).

DNA methylation is one of the earliest and most significant epigenetic mechanisms discovered. DNA methylation refers, in general, to the addition of a methyl group to a specific base in the DNA sequence under the catalysis of DNA methyltransferase, with S­adenosine methionine as the methyl donor, via covalent bonding and chemical modifications. DNA methylation is an important factor in inducing cancer. There are different types of DNA methylation, and methylation at different sites plays different roles. It is well known that the progression of colorectal cancer (CRC) is affected by the methylation of key genes. The present review did not only discuss the potential relationship between DNA methylation and CRC but also discussed how DNA methylation affects the development of CRC by affecting key genes. Furthermore, the clinical significance of DNA methylation in CRC was highlighted, including that of the therapeutic targets and biomarkers of methylation; and the importance of DNA methylation inhibitors was discussed as a novel strategy for treatment of CRC. The present review did not only focus upon the latest research findings, but earlier reviews were also cited as references to older literature.

Inhibition of FBP1 expression by KMT5A through TWIST1 methylation is one of the mechanisms leading to chemoresistance in breast cancer.

Lysine methyltransferase 5A (KMT5A) is the sole mammalian enzyme known to catalyse the mono­methylation of histone H4 lysine 20 and non­histone proteins such as p53, which are involved in the occurrence and progression of numerous cancers. The present study aimed to determine the function of KMT5A in inducing docetaxel (DTX) resistance in patients with breast carcinoma by evaluating glucose metabolism and the underlying mechanism involved. The upregulation or downregulation of KMT5A­related proteins was examined after KMT5A knockdown in breast cancer (BRCA) cells by Tandem Mass Tag proteomics. Through differential protein expression and pathway enrichment analysis, the upregulated key gluconeogenic enzyme fructose­1,6­bisphosphatase 1 (FBP1) was discovered. Loss of FBP1 expression is closely related to the development and prognosis of cancers. A dual­luciferase reporter gene assay confirmed that KMT5A inhibited the expression of FBP1 and that overexpression of FBP1 could enhance the chemotherapeutic sensitivity to DTX through the suppression of KMT5A expression. The KMT5A inhibitor UNC0379 was used to verify that DTX resistance induced by KMT5A through the inhibition of FBP1 depended on the methylase activity of KMT5A. According to previous literature and interaction network structure, it was revealed that KMT5A acts on the transcription factor twist family BHLH transcription factor 1 (TWIST1). Then, it was verified that TWSIT1 promoted the expression of FBP1 by using a dual­luciferase reporter gene experiment. KMT5A induces chemotherapy resistance in BRCA cells by promoting cell proliferation and glycolysis. After the knockdown of the KMT5A gene, the FBP1 related to glucose metabolism in BRCA was upregulated. KMT5A knockdown expression and FBP1 overexpression synergistically inhibit cell proliferation and block cells in the G2/M phase. KMT5A inhibits the expression of FBP1 by methylating TWIST1 and weakening its promotion of FBP1 transcription. In conclusion, KMT5A was shown to affect chemotherapy resistance by regulating the cell cycle and positively regulate glycolysis­mediated chemotherapy resistance by inhibiting the transcription of FBP1 in collaboration with TWIST1. KMT5A may be a potential therapeutic target for chemotherapy resistance in BRCA.

Dulaglutide reduces oxidative DNA damage and hypermethylation in the somatic cells of mice fed a high-energy diet by restoring redox balance, inflammatory responses, and DNA repair gene expressions.

Obesity is an established risk factor for numerous malignancies, although it remains uncertain whether the disease itself or weight-loss drugs are responsible for a greater predisposition to cancer. The objective of the current study was to determine the impact of dulaglutide on genetic and epigenetic DNA damage caused by obesity, which is a crucial factor in the development of cancer. Mice were administered a low-fat or high-fat diet for 12 weeks, followed by a 5-week treatment with dulaglutide. Following that, modifications of the DNA bases were examined using the comet assay. To clarify the underlying molecular mechanisms, oxidized and methylated DNA bases, changes in the redox status, levels of inflammatory cytokines, and the expression levels of some DNA repair genes were evaluated. Animals fed a high-fat diet exhibited increased body weights, elevated DNA damage, oxidation of DNA bases, and DNA hypermethylation. In addition, obese mice showed altered inflammatory responses, redox imbalances, and repair gene expressions. The findings demonstrated that dulaglutide does not exhibit genotoxicity in the investigated conditions. Following dulaglutide administration, animals fed a high-fat diet demonstrated low DNA damage, less oxidation and methylation of DNA bases, restored redox balance, and improved inflammatory responses. In addition, dulaglutide treatment restored the upregulated DNMT1, Ogg1, and p53 gene expression. Overall, dulaglutide effectively maintains DNA integrity in obese animals. It reduces oxidative DNA damage and hypermethylation by restoring redox balance, modulating inflammatory responses, and recovering altered gene expressions. These findings demonstrate dulaglutide's expediency in treating obesity and its associated complications.

Tissue of origin detection for cancer tumor using low-depth cfDNA samples through combination of tumor-specific methylation atlas and genome-wide methylation density in graph convolutional neural networks.

BACKGROUND: Cell free DNA (cfDNA)-based assays hold great potential in detecting early cancer signals yet determining the tissue-of-origin (TOO) for cancer signals remains a challenging task. Here, we investigated the contribution of a methylation atlas to TOO detection in low depth cfDNA samples. METHODS: We constructed a tumor-specific methylation atlas (TSMA) using whole-genome bisulfite sequencing (WGBS) data from five types of tumor tissues (breast, colorectal, gastric, liver and lung cancer) and paired white blood cells (WBC). TSMA was used with a non-negative least square matrix factorization (NNLS) deconvolution algorithm to identify the abundance of tumor tissue types in a WGBS sample. We showed that TSMA worked well with tumor tissue but struggled with cfDNA samples due to the overwhelming amount of WBC-derived DNA. To construct a model for TOO, we adopted the multi-modal strategy and used as inputs the combination of deconvolution scores from TSMA with other features of cfDNA. RESULTS: Our final model comprised of a graph convolutional neural network using deconvolution scores and genome-wide methylation density features, which achieved an accuracy of 69% in a held-out validation dataset of 239 low-depth cfDNA samples. CONCLUSIONS: In conclusion, we have demonstrated that our TSMA in combination with other cfDNA features can improve TOO detection in low-depth cfDNA samples.

Respiratory infection- and asthma-prone, low vaccine responder children demonstrate distinct mononuclear cell DNA methylation pathways.

BACKGROUND: Infants with frequent viral and bacterial respiratory infections exhibit compromised immunity to routine immunizations. They are also more likely to develop chronic respiratory diseases in later childhood. This study investigated the feasibility of epigenetic profiling to reveal endotype-specific molecular pathways with potential for early identification and immuno-modulation. Peripheral blood mononuclear cells from respiratory infection allergy/asthma-prone (IAP) infants and non-infection allergy/asthma prone (NIAP) were retrospectively selected for genome-wide DNA methylation and single nucleotide polymorphism analysis. The IAP infants were enriched for the low vaccine responsiveness (LVR) phenotype (Fisher's exact p-value = 0.02). RESULTS: An endotype signature of 813 differentially methylated regions (DMRs) comprising 238 lead CpG associations (FDR < 0.05) emerged, implicating pathways related to asthma, mucin production, antigen presentation and inflammasome activation. Allelic variation explained only a minor portion of this signature. Stimulation of mononuclear cells with monophosphoryl lipid A (MPL), a TLR agonist, partially reversed this signature at a subset of CpGs, suggesting the potential for epigenetic remodeling. CONCLUSIONS: This proof-of-concept study establishes a foundation for precision endotyping of IAP children and highlights the potential for immune modulation strategies using adjuvants for future investigation.

SETDB1 regulates short interspersed nuclear elements and chromatin loop organization in mouse neural precursor cells.

BACKGROUND: Transposable elements play a critical role in maintaining genome architecture during neurodevelopment. Short Interspersed Nuclear Elements (SINEs), a major subtype of transposable elements, are known to harbor binding sites for the CCCTC-binding factor (CTCF) and pivotal in orchestrating chromatin organization. However, the regulatory mechanisms controlling the activity of SINEs in the developing brain remains elusive. RESULTS: In our study, we conduct a comprehensive genome-wide epigenetic analysis in mouse neural precursor cells using ATAC-seq, ChIP-seq, whole genome bisulfite sequencing, in situ Hi-C, and RNA-seq. Our findings reveal that the SET domain bifurcated histone lysine methyltransferase 1 (SETDB1)-mediated H3K9me3, in conjunction with DNA methylation, restricts chromatin accessibility on a selective subset of SINEs in neural precursor cells. Mechanistically, loss of Setdb1 increases CTCF access to these SINE elements and contributes to chromatin loop reorganization. Moreover, de novo loop formation contributes to differential gene expression, including the dysregulation of genes enriched in mitotic pathways. This leads to the disruptions of cell proliferation in the embryonic brain after genetic ablation of Setdb1 both in vitro and in vivo. CONCLUSIONS: In summary, our study sheds light on the epigenetic regulation of SINEs in mouse neural precursor cells, suggesting their role in maintaining chromatin organization and cell proliferation during neurodevelopment.

Current evidence supporting associations of DNA methylation measurements with survivorship burdens in cancer survivors: A scoping review.

INTRODUCTION: Identifying reliable biomarkers that reflect cancer survivorship symptoms remains a challenge for researchers. DNA methylation (DNAm) measurements reflecting epigenetic changes caused by anti-cancer therapy may provide needed insights. Given lack of consensus describing utilization of DNAm data to predict survivorship issues, a review evaluating the current landscape is warranted. OBJECTIVE: Provide an overview of current studies examining associations of DNAm with survivorship burdens in cancer survivors. METHODS: A literature review was conducted including studies if they focused on cohorts of cancer survivors, utilized peripheral blood cell DNAm data, and evaluated the associations of DNAm and survivorship issues. RESULTS: A total of 22 studies were identified, with majority focused on breast (n = 7) or childhood cancer (n = 9) survivors, and half studies included less than 100 patients (n = 11). Survivorship issues evaluated included those related to neurocognition (n = 5), psychiatric health (n = 3), general wellness (n = 9), chronic conditions (n = 5), and treatment specific toxicities (n = 4). Studies evaluated epigenetic age metrics (n = 10) and DNAm levels at individual CpG sites or regions (n = 12) for their associations with survivorship issues in cancer survivors along with relevant confounding factors. Significant associations of measured DNAm in the peripheral blood samples of cancer survivors and survivorship issues were identified. DISCUSSION/CONCLUSION: Studies utilizing epigenetic age metrics and differential methylation analysis demonstrated significant associations of DNAm measurements with survivorship burdens. Associations were observed encompassing diverse survivorship outcomes and timeframes relative to anti-cancer therapy initiation. These findings underscore the potential of these measurements as useful biomarkers in survivorship care and research.

Race and Ethnicity, Socioeconomic Factors, and Epigenetic Age Acceleration in Survivors of Childhood Cancer.

Importance: Current research in epigenetic age acceleration (EAA) is limited to non-Hispanic White individuals. It is imperative to improve inclusivity by considering racial and ethnic minorities in EAA research. Objective: To compare non-Hispanic Black with non-Hispanic White survivors of childhood cancer by examining the associations of EAA with cancer treatment exposures, potential racial and ethnic disparity in EAA, and mediating roles of social determinants of health (SDOH). Design, Setting, and Participants: In this cross-sectional study, participants were from the St Jude Lifetime Cohort, which was initiated in 2007 with ongoing follow-up. Eligible participants included non-Hispanic Black and non-Hispanic White survivors of childhood cancer treated at St Jude Children's Research Hospital between 1962 and 2012 who had DNA methylation data. Data analysis was conducted from February 2023 to May 2024. Exposure: Three treatment exposures for childhood cancer (chest radiotherapy, alkylating agents, and epipodophyllotoxin). Main Outcomes and Measures: DNA methylation was generated from peripheral blood mononuclear cell-derived DNA. EAA was calculated as residuals from regressing Levine or Horvath epigenetic age on chronological age. SDOH included educational attainment, annual personal income, and the socioeconomic area deprivation index (ADI). General linear models evaluated cross-sectional associations of EAA with race and ethnicity (non-Hispanic Black and non-Hispanic White) and/or SDOH, adjusting for sex, body mass index, smoking, and cancer treatments. Adjusted least square means (ALSM) of EAA were calculated for group comparisons. Mediation analysis treated SDOH as mediators with average causal mediation effect (ACME) calculated for the association of EAA with race and ethnicity. Results: Among a total of 1706 survivors including 230 non-Hispanic Black survivors (median [IQR] age at diagnosis, 9.5 [4.3-14.3] years; 103 male [44.8%] and 127 female [55.2%]) and 1476 non-Hispanic White survivors (median [IQR] age at diagnosis, 9.3 [3.9-14.6] years; 766 male [51.9%] and 710 female [48.1%]), EAA was significantly greater among non-Hispanic Black survivors (ALSM = 1.41; 95% CI, 0.66 to 2.16) than non-Hispanic White survivors (ALSM = 0.47; 95% CI, 0.12 to 0.81). Among non-Hispanic Black survivors, EAA was significantly increased among those exposed to chest radiotherapy (ALSM = 2.82; 95% CI, 1.37 to 4.26) vs those unexposed (ALSM = 0.46; 95% CI, -0.60 to 1.51), among those exposed to alkylating agents (ALSM = 2.33; 95% CI, 1.21 to 3.45) vs those unexposed (ALSM = 0.95; 95% CI, -0.38 to 2.27), and among those exposed to epipodophyllotoxins (ALSM = 2.83; 95% CI, 1.27 to 4.40) vs those unexposed (ALSM = 0.44; 95% CI, -0.52 to 1.40). The association of EAA with epipodophyllotoxins differed by race and ethnicity (ß for non-Hispanic Black survivors, 2.39 years; 95% CI, 0.74 to 4.04 years; ß for non-Hispanic White survivors, 0.68; 95% CI, 0.05 to 1.31 years) and the difference was significant (1.77 years; 95% CI, 0.01 to 3.53 years; P for interaction = .049). Racial and ethnic disparities in EAA were mediated by educational attainment (

Combined exercise training decreases blood pressure in OLDER women with NOS3 polymorphism providing changes in differentially methylated regions (DMRs).

The mechanisms by which the ageing process is associated to an unhealthy lifestyle and how they play an essential role in the aetiology of systemic arterial hypertension have not yet been completely elucidated. Our objective is to investigate the influence of NOS3 polymorphisms [-786T > C and (Glu298Asp)] on systolic blood pressure (SBP) and diastolic blood pressure (DBP) response, differentially methylated regions (DMRs), and physical fitness of adult and older women after a 14-week combined training intervention. The combined training was carried out for 14 weeks, performed 3 times a week, totalling 180 minutes weekly. The genotyping experiment used Illumina Infinium Global Screening Array version 2.0 (GSA V2.0) and Illumina's EPIC Infinium Methylation BeadChip. The participants were separated into SNP rs2070744 in TT (59.7 ± 6.2 years) and TC + CC (60.0 ± 5.2 years), and SNP rs17999 in GluGlu (58.8 ± 5.7 years) and GluAsp + AspAsp (61.6 ± 4.9 years). We observed an effect of time for variables BP, physical capacities, and cholesterol. DMRs related to SBP and DBP were identified for the rs2070744 and rs17999 groups pre- and decreased numbers of DMRs post-training. When we analysed the effect of exercise training in pre- and post-comparisons, the GluGlu SNP (rs17999) showed 10 DMRs, and after enrichment, we identified several biological biases. The combined training improved the SBP and DBP values of the participants regardless of the SNPs. In addition, exercise training affected DNA methylation differently between the groups of NOS3 polymorphisms.

Direction-aware functional class scoring enrichment analysis of infinium DNA methylation data.

Infinium Methylation BeadChip arrays remain one of the most popular platforms for epigenome-wide association studies, but tools for downstream pathway analysis have their limitations. Functional class scoring (FCS) is a group of pathway enrichment techniques that involve the ranking of genes and evaluation of their collective regulation in biological systems, but the implementations described for Infinium methylation array data do not retain direction information, which is important for mechanistic understanding of genomic regulation. Here, we evaluate several candidate FCS methods that retain directional information. According to simulation results, the best-performing method involves the mean aggregation of probe limma t-statistics by gene followed by a rank-ANOVA enrichment test using the mitch package. This method, which we call 'LAM,' outperformed an existing over-representation analysis method in simulations, and showed higher sensitivity and robustness in an analysis of real lung tumour-normal paired datasets. Using matched RNA-seq data, we examine the relationship of methylation differences at promoters and gene bodies with RNA expression at the level of pathways in lung cancer. To demonstrate the utility of our approach, we apply it to three other contexts where public data were available. First, we examine the differential pathway methylation associated with chronological age. Second, we investigate pathway methylation differences in infants conceived with in vitro fertilization. Lastly, we analyse differential pathway methylation in 19 disease states, identifying hundreds of novel associations. These results show LAM is a powerful method for the detection of differential pathway methylation complementing existing methods. A reproducible vignette is provided to illustrate how to implement this method.

An expedited screening platform for the discovery of anti-ageing compounds in vitro and in vivo.

BACKGROUND: Restraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan. METHODS: Using Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture. RESULTS: We developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo. CONCLUSIONS: Our method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors.

Integrated analysis of DNA methylome and transcriptome revealing epigenetic regulation of CRIR1-promoted cold tolerance.

BACKGROUND: DNA methylation contributes to the epigenetic regulation of nuclear gene expression, and is associated with plant growth, development, and stress responses. Compelling evidence has emerged that long non-coding RNA (lncRNA) regulates DNA methylation. Previous genetic and physiological evidence indicates that lncRNA-CRIR1 plays a positive role in the responses of cassava plants to cold stress. However, it is unclear whether global DNA methylation changes with CRIR1-promoted cold tolerance. RESULTS: In this study, a comprehensive comparative analysis of DNA methylation and transcriptome profiles was performed to reveal the gene expression and epigenetic dynamics after CRIR1 overexpression. Compared with the wild-type plants, CRIR1-overexpressing plants present gained DNA methylation in over 37,000 genomic regions and lost DNA methylation in about 16,000 genomic regions, indicating a global decrease in DNA methylation after CRIR1 overexpression. Declining DNA methylation is not correlated with decreased/increased expression of the DNA methylase/demethylase genes, but is associated with increased transcripts of a few transcription factors, chlorophyll metabolism and photosynthesis-related genes, which could contribute to the CRIR1-promoted cold tolerance. CONCLUSIONS: In summary, a first set of transcriptome and epigenome data was integrated in this study to reveal the gene expression and epigenetic dynamics after CRIR1 overexpression, with the identification of several TFs, chlorophyll metabolism and photosynthesis-related genes that may be involved in CRIR1-promoted cold tolerance. Therefore, our study has provided valuable data for the systematic study of molecular insights for plant cold stress response.

DNA methylation of exercise-responsive genes differs between trained and untrained men.

BACKGROUND: Physical activity is well known for its multiple health benefits and although the knowledge of the underlying molecular mechanisms is increasing, our understanding of the role of epigenetics in long-term training adaptation remains incomplete. In this intervention study, we included individuals with a history of > 15 years of regular endurance or resistance training compared to age-matched untrained controls performing endurance or resistance exercise. We examined skeletal muscle DNA methylation of genes involved in key adaptation processes, including myogenesis, gene regulation, angiogenesis and metabolism. RESULTS: A greater number of differentially methylated regions and differentially expressed genes were identified when comparing the endurance group with the control group than in the comparison between the strength group and the control group at baseline. Although the cellular composition of skeletal muscle samples was generally consistent across groups, variations were observed in the distribution of muscle fiber types. Slow-twitch fiber type genes MYH7 and MYL3 exhibited lower promoter methylation and elevated expression in endurance-trained athletes, while the same group showed higher methylation in transcription factors such as FOXO3, CREB5, and PGC-1α. The baseline DNA methylation state of those genes was associated with the transcriptional response to an acute bout of exercise. Acute exercise altered very few of the investigated CpG sites. CONCLUSIONS: Endurance- compared to resistance-trained athletes and untrained individuals demonstrated a different DNA methylation signature of selected skeletal muscle genes, which may influence transcriptional dynamics following a bout of acute exercise. Skeletal muscle fiber type distribution is associated with methylation of fiber type specific genes. Our results suggest that the baseline DNA methylation landscape in skeletal muscle influences the transcription of regulatory genes in response to an acute exercise bout.