Changes In Methylation Status Research Articles

Betaine supplementation alleviates corticosterone-induced hepatic cholesterol accumulation through epigenetic modulation of HMGCR and CYP7A1 genes in laying hens

Excessive corticosterone (CORT) exposure could cause hepatic cholesterol accumulation in chickens and maternal betaine supplementation could decrease hepatic cholesterol deposition through epigenetic modifications in offspring chickens. Nevertheless, it remains uncertain whether providing betaine to laying hens could protect CORT-induced hepatic cholesterol accumulation via epigenetic mechanisms. This study aimed to examine the effects of dietary betaine on plasma and hepatic cholesterol contents, expression of cholesterol metabolic genes, as well as DNA methylation on their promoters in the liver of laying hens exposed to CORT. A total of 72 laying hens at 130 d of age were randomly divided into 3 groups: control (CON), CORT, and CORT+betaine (CORT+BET) groups. The experiment lasted for 35 d. Chickens in CON and CORT groups were fed a basal diet, whereas the CORT+BET group chickens were fed the basal diet supplemented with 0.1% betaine for 35 d. On d 28 of the experiment, chickens in CORT and CORT+BET groups received daily subcutaneous injections of CORT (4.0 mg/kg body weight), whereas the CON group chickens were injected with an equal volume of solvent for 7 d. The results showed that CORT administration led to a significant increase (P < 0.05) in the contents of cholesterol in plasma and liver, associated with activation (P < 0.05) of sterol regulatory element binding transcription factor 2 (SREBP2), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), lecithin-cholesterol acyltransferase (LCAT) and low-density lipoprotein receptor (LDLR) genes expression, and inhibition of cholesterol-7-alpha hydroxylase (CYP7A1) and sterol 27-hydroxylase (CYP27A1) genes expression in the liver compared to the CON. In contrast, CORT-induced up-regulation of HMGCR mRNA and protein abundances and downregulation of CYP7A1 mRNA and protein abundances were completely normalized (P < 0.05) by betaine supplementation. Besides, CORT injection led to significant hypomethylation (P < 0.05) on HMGCR promoter and hypermethylation (P < 0.05) on CYP7A1 promoter. Moreover, dietary betaine rescued (P < 0.05) CORT-induced changes in methylation status of HMGCR and CYP7A1 genes promoters. These results indicate that dietary betaine addition protects laying hens from CORT-induced hepatic cholesterol accumulation via epigenetic modulation of HMGCR and CYP7A1 genes.

DNMTs-mediated SOCS3 methylation promotes the occurrence and development of AML.

As a tumor suppressor gene, SOCS3 inhibits the growth of tumor cells by regulating JAK/STAT signaling pathway through negative feedback. This study aimed to investigate the biological function and mechanism of SOCS3 methylation mediated by DNMTs in the development of AML. Bone marrow samples were collected from 70 AML patients and 20 healthy volunteers. The expression and methylation status of each gene were detected by RT-qPCR, western blot and MS-PCR, and the growth and apoptosis rate of leukemia cell lines were detected by CCK-8 and flow cytometry. The effects of changes in SOCS3 gene expression and methylation status of AML cell lines were observed by gene transfection and gene knockdown. The methylation rate of SOCS3 in AML initial treatment group was significantly higher than that in the remission group and the normal control group (60% vs. 0%, 0%). The expression of SOCS3 in the SOCS3 methylation group was significantly lower than that in the non-methylated group and control group, while the expression of DNMT1, DNMT3a, p-JAK2, p-STAT3 and p-STAT5 were significantly higher than those in the non-methylated group and control group. Demethylation treatment, SOCS3 transfection and DNMT3a knockdown could up-regulate the expression of SOCS3, which decreased the proliferation and increased the apoptosis of leukemia cell lines. SOCS3 methylation mediated by DNMTs promotes the occurrence and development of AML and can be used as a potential biomarker for the diagnosis and efficacy evaluation of AML.

Retraction: Zinc sulfate contributes to promote telomere length extension via increasing telomerase gene expression, telomerase activity and change in the TERT gene promoter CpG island methylation status of human adipose-derived mesenchymal stem cells.

Retraction: Zinc sulfate contributes to promote telomere length extension via increasing telomerase gene expression, telomerase activity and change in the TERT gene promoter CpG island methylation status of human adipose-derived mesenchymal stem cells.

LINE-1 Methylation Status in Canine Splenic Hemangiosarcoma Tissue and Cell-Free DNA.

Splenic hemangiosarcoma is one of the most common malignant tumors in dogs, and early diagnosis is of great importance for achieving a good prognosis. DNA methylation plays an important role in cancer development. Long interspersed nuclear element 1 (LINE-1) is the most abundant repetitive element in the genome. LINE-1 hypomethylation has been shown to be related to carcinogenesis in humans, and it has been used as a novel cancer biomarker. This study aimed to evaluate the methylation status of LINE-1 in tumor tissue and circulating cell-free DNA and assess its clinical significance in canine splenic hemangiosarcoma. Genomic DNA was isolated from splenic masses of 13 dogs with hemangiosarcoma, 11 with other malignant tumors, and 15 with benign lesions. LINE-1 methylation was quantified using methylation-sensitive and -insensitive restriction enzyme digestion followed by real-time polymerase chain reaction. Additionally, blood samples were collected from eight patients to isolate cell-free DNA to determine LINE-1 methylation status changes during the treatment course. LINE-1 methylation in tumor samples was significantly lower in patients with hemangiosarcoma than in those with other malignant tumors and benign lesions. Non-significant but similar results were observed for the cell-free DNA samples. Our results demonstrate that LINE-1 methylation status is a potential biomarker for splenic hemangiosarcoma.

Monitoring the macrophage response towards biomaterial implants using label-free imaging.

Understanding the immune system's foreign body response (FBR) is essential when developing and validating a biomaterial. Macrophage activation and proliferation are critical events in FBR that can determine the material's biocompatibility and fate in vivo. In this study, two different macro-encapsulation pouches intended for pancreatic islet transplantation were implanted into streptozotocin-induced diabetes rat models for 15 days. Post-explantation, the fibrotic capsules were analyzed by standard immunohistochemistry as well as non-invasive Raman microspectroscopy to determine the degree of FBR induced by both materials. The potential of Raman microspectroscopy to discern different processes of FBR was investigated and it was shown that Raman microspectroscopy is capable of targeting ECM components of the fibrotic capsule as well as pro and anti-inflammatory macrophage activation states, in a molecular-sensitive and marker-independent manner. In combination with multivariate analysis, spectral shifts reflecting conformational differences in Col I were identified and allowed to discriminate fibrotic and native interstitial connective tissue fibers. Moreover, spectral signatures retrieved from nuclei demonstrated changes in methylation states of nucleic acids in M1 and M2 phenotypes, relevant as indicator for fibrosis progression. This study could successfully implement Raman microspectroscopy as complementary tool to study in vivo immune-compatibility providing insightful information of FBR of biomaterials and medical devices, post-implantation.

Single-cell methylation sequencing data reveal succinct metastatic migration histories and tumor progression models.

Recent studies exploring the impact of methylation in tumor evolution suggest that although the methylation status of many of the CpG sites are preserved across distinct lineages, others are altered as the cancer progresses. Because changes in methylation status of a CpG site may be retained in mitosis, they could be used to infer the progression history of a tumor via single-cell lineage tree reconstruction. In this work, we introduce the first principled distance-based computational method, Sgootr, for inferring a tumor's single-cell methylation lineage tree and for jointly identifying lineage-informative CpG sites that harbor changes in methylation status that are retained along the lineage. We apply Sgootr on single-cell bisulfite-treated whole-genome sequencing data of multiregionally sampled tumor cells from nine metastatic colorectal cancer patients, as well as multiregionally sampled single-cell reduced-representation bisulfite sequencing data from a glioblastoma patient. We show that the tumor lineages constructed reveal a simple model underlying tumor progression and metastatic seeding. A comparison of Sgootr against alternative approaches shows that Sgootr can construct lineage trees with fewer migration events and with more in concordance with the sequential-progression model of tumor evolution, with a running time a fraction of that used in prior studies. Lineage-informative CpG sites identified by Sgootr are in inter-CpG island (CGI) regions, as opposed to intra-CGIs, which have been the main regions of interest in genomic methylation-related analyses.

Status of estrogen receptor expression and epigenetic methylation in Leydig cells after exposure to metalloestrogen - selenium

The trace element selenium (Se) is essential for the maintenance of spermatogenesis and fertility. A growing volume of evidence shows that Se is necessary for testosterone synthesis, and Se can stimulate Leydig cell proliferation. However, Se can also act as a metalloestrogen, which can mimic estrogen and activate the estrogen receptors. This study aimed to investigate Se effect on estrogen signaling and the epigenetic status of Leydig cells. Mouse Leydig cells (MA-10) were cultured in a medium supplemented with different Se concentrations (4, 8 µM) for 24 h. Next, cells were assessed for morphological and molecular (qRT PCR, western blot, immunofluorescence) analyses. Immunofluorescence revealed strong immunosignal for 5-methylcytosine in both control and treated cells, with a stronger signal in the 8 μM treated group. qRT-PCR confirmed an increased expression of methyltransferase 3 beta (Dnmt3b) in 8 μM cells. Analysis of the expression of γH2AX (a marker for double-stranded DNA breaks) revealed an increase in the DNA breaks in cells exposed to 8 μM Se. Selenium exposure did not affect the expression of canonical estrogen receptors (ERα and ERβ), however, an increase in membrane estrogen receptor G-protein coupled (GPER) protein expression was observed.To sum up, in a high concentration (8 μM) Se affects GPER expression (non-genomic estrogen signaling) in Leydig cells possibly via acting on receptor protein and/or its binding. This causes DNA breaks and induces changes in Leydig cell methylation status, especially in de novo methylation which is mediated by Dnmt3b.

Abstract 127: Single-cell methylation sequencing data reveals succinct metastatic migration histories and tumor progression models

Abstract Recent studies exploring the impact of methylation in tumor evolution suggest that while the methylation status of many of the CpG sites are preserved across distinct lineages, others are altered as the cancer progresses. Since changes in methylation status of a CpG site may be retained in mitosis, they could be used to infer the progression history of a tumor via single-cell lineage tree reconstruction. In this work, we introduce the first principled distance-based computational method, Sgootr, for inferring a tumor's single-cell methylation lineage tree and jointly identifying lineage-informative CpG sites which harbor changes in methylation status that are retained along the lineage. We apply Sgootr on the single-cell bisulfite-treated whole genome sequencing data of multiregionally-sampled tumor cells from 9 metastatic colorectal cancer patients made available by Bian et al., as well as multiregionally-sampled single-cell reduced-representation bisulfite sequencing data from a glioblastoma patient made available by Chaligne et al. We demonstrate that the tumor lineages constructed reveal a simple model underlying colorectal tumor progression and metastatic seeding. A comparison of Sgootr against alternative approaches shows that Sgootr can construct lineage trees with fewer migration events and more in concordance with the sequential-progression model of tumor evolution, in time a fraction of that used in prior studies. Interestingly, lineage-informative CpG sites identified by Sgootr are in inter-CpG island (CGI) regions, as opposed to CGI's, which have been the main regions of interest in genomic methylation-related analyses. Sgootr is implemented as a Snakemake workflow, available at https://github.com/liuy0421/Sgootr. Citation Format: Xuan C. Li, Yuelin Liu, Farid Rashidi Mehrabadi, Alejandro A. Schäffer, Drew Pratt, David R. Crawford, Salem Malikić, Erin K. Molloy, Vishaka Gopalan, Stephen M. Mount, Eytan Ruppin, Kenneth Aldape, S. Cenk Sahinalp. Single-cell methylation sequencing data reveals succinct metastatic migration histories and tumor progression models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 127.

Abstract 6023: Plasma methylome landscape across the spectrum of prostate cancer progression, and differentially methylated region (DMR) based biomarkers of hormonal therapy failure in metastatic hormone-sensitive prostate cancer

Abstract Background: DNA silencing of gene expression can affect tumorigenesis, treatment outcomes and prognosis. Epigenetic silencing of tumor suppressor genes in prostate cancer detected in plasma at different stages of progression and its effect on clinical outcomes has not been determined. In a cohort study across different stages of prostate cancer (PC) progression, we performed plasma cell-free DNA (cfDNA)-based epigenomic profiling for determining the landscape of methylation and Differentially Methylated Region (DMR) classifiers and the potential impact on clinical outcomes in metastatic hormone-sensitive prostate cancer (mHSPC) state. Methods: Plasma was collected prospectively in a longitudinal cohort study of 108 PC patients (pts) of which 24 had non-metastatic Local PC; 28 had mHSPC; 56 had mCRPC. cfDNA was used for enzymatic methylation sequencing (EM-seq) of a targeted panel of genes (N=441) implicated in prostate cancer biology. Methylation status at CpG islands of these genes were examined using Twist targeted cfDNA methylation assay. The Bismark program was used for methylation calls. Temporal trends of differentially methylated regions (DMRs) were identified in different states of progression by comparing the methylation status of target genes in localized PC, mHSPC and mCRPC states using a False Discovery Rate (FDR-q-value) of 0.2 or less. DMRs uniquely associated with mHSPC state were identified and determination of genes with DMRs in pts experiencing early failure of androgen deprivation therapy (ADT)-based therapies was performed. Early failure was defined as progression on ADT within 12 months. Results: We identified 304/441 genes that were differentially methylated between localized PC and advanced PC (mHSPC and mCRPC) (FDR&amp;lt;0.2), of which 237 genes differed between mHSPC and mCRPC (p&amp;lt;0.05) states. mHSPC pts were serially profiles before and after 3 months of ADT-based therapies. All 28 pts experienced a PSA response at 3-months and 112/237 genes showed significant change in methylation status in these serial samples taken before/after 3 months of ADT-based therapies. The median follow up of the cohort was 18 months (range 10-24). 6/28 mHSPC pts experienced early ADT failure (&amp;lt;12 months; median 10 months). 8/112 genes were significantly associated with early failures and included DMRs in RB1, FOXA1, SOX11. GSC, ALDH1L1, SCGB3A1, FLT4, and PTPRN2. Conclusions. A targeted cfDNA plasma liquid-biopsy based epigenomic profiling of PC cancer related genes was able to capture significant differences in DMRs across different states of cancer progression. Candidate differentially expressed CpG methylome gene biomarkers detected in plasma also identified biomarkers associated with early ADT-based treatment failure in mHSPC. Citation Format: Manish Kohli, Jodie Wong, Yijun Tian, Amir Bitaraf, Claire Hanson, Matt Larsen, Jennifer Lloyd, Julia Batten, Neeraj Agarwal, Umang Swami, Benjamin Maughan, Sumati Gupta, Jonathan Tward. Plasma methylome landscape across the spectrum of prostate cancer progression, and differentially methylated region (DMR) based biomarkers of hormonal therapy failure in metastatic hormone-sensitive prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6023.

Quantitative analysis of MGMT promoter methylation status changes by pyrosequencing in recurrent glioblastoma.

MGMT promoter methylation status can change in response to several factors, treatment with alkylating therapy being the mechanism more commonly cited in the literature. Some authors have attempted to quantify these alterations, with inconsistent results. This study aims to determine changes in MGMT promoter methylation status by pyrosequencing, which quantitatively yields results, in a cohort of patients reoperated for recurrent glioblastoma and having previously completed the Stupp protocol. Methylation status of the MGMT promoter gene of a total of 24 pairs of glioblastoma preselected tumor samples was retrospectively analyzed using pyrosequencing and depicted as percentages or categories (hypermethylated, intermediate methylation, unmethylated). Matched samples were compared using Wilcoxon signed-rank test, and log-rank test was used to establish a correlation with survival data. The median value of MGMT promoter methylation status declined after adjuvant treatment from 20.35% to 14.25% (p= 0.346). A significant correlation between methylation in primary samples and overall survival (p= 0.05) and progression-free survival (p= 0.024) was found. Intermediate methylation status at recurrence was linked to greater survival after progression, without reaching statistical significance (post-progression survival [PPS]) (p= 0.217). Although treatment with alkylating chemotherapy was a common feature in all patients of our cohort, switching in both directions was observed when MGMT promoter methylation status was analyzed as a continuous variable. These data suggest that the dynamics of epigenetics may be very complex and not entirely explained by clonal selection influenced by temozolomide.

ZMP recruits and excludes Pol IV-mediated DNA methylation in a site-specific manner.

In plants, RNA-directed DNA methylation (RdDM) uses small interfering RNAs (siRNAs) to target transposable elements (TEs) but usually avoids genes. RNA polymerase IV (Pol IV) shapes the landscape of DNA methylation through its pivotal role in siRNA biogenesis. However, how Pol IV is recruited to specific loci, particularly how it avoids genes, is poorly understood. Here, we identified a Pol IV-interacting protein, ZMP (zinc finger, mouse double-minute/switching complex B, Plus-3 protein), which exerts a dual role in regulating siRNA biogenesis and DNA methylation at specific genomic regions. ZMP is required for siRNA biogenesis at some pericentromeric regions and prevents Pol IV from targeting a subset of TEs and genes at euchromatic loci. As a chromatin-associated protein, ZMP prefers regions with depleted histone H3 lysine 4 (H3K4) methylation abutted by regions with H3K4 methylation, probably monitoring changes in local H3K4 methylation status to regulate Pol IV's chromatin occupancy. Our findings uncover a mechanism governing the specificity of RdDM.

Biomarkers of Clinical Resistance to Decitabine and Venetoclax Therapy Inferred By Transcriptome and Methylation Analyses

The combination of the selective BCL-2 inhibitor venetoclax (VEN) with the hypomethylating agent (HMA) decitabine (DEC) is highly effective in treating older ineligible for intensive chemotherapy AML patients. However, approximately 15-34% of newly diagnosed (ND) and 60% of relapsed/refractory (R/R) patients fail to achieve complete remission (CR) (DiNardo et al. New Engl J Med. 2020, DiNardo et al. Lancet Haematol. 2020). Factors that reduce efficacy remain to be elucidated. To dissect underlying mechanisms of HMA-VEN resistance, we performed RNA-seq utilizing primary samples from patients treated on a prospective clinical trial of 10-day DEC with VEN (NCT03404193). Previously, we reported that alterations of transcriptome signatures of energy-metabolism and cell migration/adhesion pathways were upregulated in primary resistant ND group, and immune-system-associated genes were downregulated in R/R group (Yamatani et al. ASH 2021). This study investigated transcriptomic and DNA methylation signatures associated with DEC10-VEN response or resistance in ND and R/R AML. Because DEC was reported to strongly induce interferon-stimulated genes (ISGs) through gene methylation modifications, we focused on 116 ISGs. An integrated analysis of DNA methylation status around the transcription start site (TSS) was performed using the Infinium methylated EPIC array (Illumina). Forty-four pre-treatment samples from 32 patients with AML from DEC-VEN clinical trial, ND patient group (CR/CRi 14; non-responder, NR 11) and R/R patient group (CR/CRi 6; NR 13), were analyzed. First, we performed the comparative analysis of NR vs CRs. In the ND group, NR cases showed significantly higher gene expression and lower TSS methylation of an urea cycle enzyme, ASS1 (Argininosuccinate Synthase 1) with RNA-seq Log2 fold-change (FC) 3.6 and methylation array Δβ (differential methylation β-value) -0.11, p < 0.05, and of an actin-based molecular motor, MYO1C (Myosin 1C), Log2FC 2.5, Δβ = -0.18, p< 0.05, than in CR cases. In the R/R group, gene expression levels of seven ISGs, including CCL3 (C-C motif chemokine 3), were significantly lower in NR than in CRs, with no difference in methylation status. Next, changes in gene expression levels and TSS methylation status after DEC-VEN treatment were investigated in non-responders in ND group (pre 19, post 12) and R/R group (pre 16, post 4). After DEC-VEN treatment, PPARG (Peroxisome Proliferator-Activated Receptor γ), a transcription factor that activates fatty acid metabolism and negatively regulates subsets of IFN-γ target genes, was significantly upregulated along with its TSS demethylation both in ND (Log2FC 4.9, Δβ = -0.14, p < 0.05), and R/R groups (Log2FC 3.7, Δβ = -0.06, p < 0.05). CDC42EP4, an effector protein of CDC42 involved in the organization of the actin cytoskeleton, was upregulated after the treatment in the R/R group (Log2FC 2.7, Δβ = -0.14, p < 0.05). Based on these findings, we validated transcriptional and methylation changes of ASS1, PPARG, MYO1C and CDC42EP4 by RT-qPCR and bisulfite pyrosequencing using HL60 and MV4-11 AML cell lines in which DEC (IC50, HL60, 249 nM; MV4-11, 2090 nM) and VEN (IC50, HL60, 18 nM; MV4-11, 64 nM) produced additive or synergistic combination effect. Cells were treated by DEC and/or VEN under BM-derived mesenchymal stem cells (MSCs) co-culture conditions to mimic the BM microenvironment. DEC upregulated gene expression levels with TSS demethylation of PPARG, MYO1C and CDC42EP4 both in HL60 and MV4-11, and of ASS1 only in HL-60. VEN treatment alone or in combination with DEC did not induce significant changes in the expression of these tested genes. Of note, PPARG expression was further upregulated under MSCs co-culture condition without TSS methylation status change in AML cells. DEC treatment induced no significant changes of PPARG in MSC. These findings suggest that not only TSS demethylation but also interaction with BM stromal cells may be involved in PPARG upregulation associated with energy production in AML cells. Taken together, DEC-VEN treatment stimulates two key factors, energy metabolism and interaction with the BM microenvironment, which may reduce therapeutic efficacy by increasing gene expression of PPARG,MYO1C,ASS1, and CDC42EP4 through TSS demethylation and/or interaction with BM stromal cells. Experimentally, the consequences of overexpression / knockdown of these genes will be further validated.

Global Methylation Analysis Using MSAP Reveals Differences in Chilling-Associated DNA Methylation Changes during Dormancy Release in Contrasting Sweet Cherry Varieties

Dormancy is an adaptive strategy developed by temperate perennial crops to protect overwinter tissues from unfavorable environmental conditions. Sweet cherry (Prunus avium L.), a member of the Rosaceae family, requires chilling to overcome dormancy. The time of harvest is directly correlated with chilling requirements in sweet cherries. Consequently, early and late season varieties have low and high chilling requirements, respectively. There is evidence that the expression of dormancy-related genes is regulated by DNA methylation. In this work, methylation-sensitive amplified polymorphism (MSAP) was applied to study genome-wide DNA methylation changes associated with dormancy in two low-chill varieties, ‘Royal Dawn’ and ‘Glen Red’, and one high-chill variety, ‘Kordia’. Our primary results suggest that the occurrence of progressive DNA demethylation is associated with chilling accumulation during dormancy in the three varieties, independent of their chilling requirements. Genes were identified with different methylation status changes, detected by MSAP, related to cell wall remodeling and energy metabolism. Several MSAP profiles among the varieties were observed, suggesting that fine epigenetic control is required to coordinate hormonal and environmental signals that induce dormancy and its release.

Association of Accelerometer-Measured Physical Activity and Sedentary Time with Epigenetic Markers of Aging.

Physical activity may influence chronic disease risk, in part, through epigenetic mechanisms. Previous studies have demonstrated that an acute bout of physical activity can influence DNA methylation status. Few studies have explored the relationship between habitual, accelerometer-measured physical activity or sedentary time with epigenetic markers of aging. We used linear regression to examine cross-sectional associations of accelerometer-measured physical activity and sedentary time with extrinsic and intrinsic epigenetic age acceleration (EEAA and IEAA) models and GrimAge measured from blood samples from Framingham Heart Study participants with accelerometry and DNA methylation data ( n = 2435; mean age, 54.9 ± 14.3; 46.0% men). Residuals of Hannum-, Horvath-, and GrimAge-predicted epigenetic age were calculated by regressing epigenetic age on chronological age. We took into account blood cell composition for EEAA, IEAA, and AdjGrimAge. Moderate to vigorous physical activity was log-transformed to normalize its distribution. Adjustment models accounted for family structure, age, sex, smoking status, cohort-laboratory indicator, and accelerometer wear time. We additionally explored adjustment for body mass index (BMI). Walking 1500 more steps per day or spending 3 fewer hours sedentary was associated with >10 months lower GrimAge biological age (or ~1 month lower AdjGrimAge, after adjusting for blood cells, P < 0.05). Every 5 min·d -1 more moderate to vigorous physical activity was associated with 19-79 d of lower GrimAge (4-23 d lower using EEAA or AdjGrimAge, P < 0.01). Adjusting for BMI attenuated these results, but all statistically significant associations with AdjGrimAge remained. Greater habitual physical activity and lower sedentary time were associated with lower epigenetic age, which was partially explained by BMI. Further research should explore whether changes in physical activity influence methylation status and whether those modifications influence chronic disease risk.

P07.08.B Surgical management of recurrent lower-grade gliomas: analysis of oncological and functional outcomes and associated factors

Abstract Background Presently, there are no clear evidence-based recommendations for the surgical management of progressive or recurrent lower-grade gliomas (LGGs). In our work, we evaluated the oncological (PFS, OS) and functional (neurological, neuropsychological, quality of life evaluation) outcomes of a series of LGGs treated in a 5-year period. The aim was to identify clinical, imaging, and molecular factors, associated with better outcomes, to be implemented in the treatment decision making process. Material and Methods In our retrospective analysis, 738 recurrent LGGs were included (follow-up 7.9 years; IQR:6-9); 550 were surgically treated, 495 with open resection (with brain mapping techniques), and 55 with tumor biopsy (with frameless stereotactic technique). Results Overall, 521 patients (70.6%) recurred. Of those treated with open resection 63.5% relapsed. Tumor progression was histologically confirmed in 72.5% of cases, no valuable tumor (effect of treatment) in 27.5% of cases (particularly in cases previously treated with RT and CHT), histo-molecular conversion (astrocytoma instead of oligodendroglioma diagnosis) in 7.5% of cases, malignant transformation in 38.7% of cases. Among the clinical (patient functional status, previous PFS, previous EOR, previous CHT and duration of CHT, previous RT), imaging (type of recurrence, i.e. typical versus atypical, speed of growth, contrast enhancement), and molecular (IDH, codeletion, ATRX, MGMT, tumor grade, CDNK status) factors analyzed, longest PFS of recurrent tumors was associated with patient functional status, previous PFS, previous EOR, speed of growth (&amp;gt;6 mm/year), atypical type of recurrence, tumor grade, presence of codeletion, and CDNK status. Best functional outcome associated with surgical resection, EOR, and tumor grade; worst functional outcome with RT and malignant transformation. Longest OS associated with previous EOR, surgical resection, tumor grade, presence of co-deletion. Malignant transformation was associated to previous EOR. Among patients submitted solely to tumor biopsy, changes in MGMT methylation status and in IDH status were observed in 65.3% and 5% of cases, respectively. Modification in IDH status was associated with atypical (distant) type of recurrence. New histo-molecular profile helped in the treatment decision making process. Conclusion Surgical resection of recurrent LGGs is associated with longer PFS and OS. Tumor biopsy should be considered, when surgery considered unfeasible, to tailor adjuvant treatment.

Transcriptome-wide analyses of RNA m6A methylation in hexaploid wheat reveal its roles in mRNA translation regulation.

N6-methyladenosine (m6A) is the most abundant RNA modification in eukaryotic messenger RNAs. m6A was discovered in wheat about 40 years ago; however, its potential roles in wheat remain unknown. In this study, we profiled m6As in spikelets transcriptome at the flowering stage of hexaploid wheat and found that m6As are evenly distributed across the A, B, and D subgenomes but their extents and locations vary across homeologous genes. m6As are enriched in homeologous genes with close expression levels and the m6A methylated genes are more conserved. The extent of m6A methylation is negatively correlated with mRNA expression levels and its presence on mRNAs has profound impacts on mRNA translation in a location-dependent manner. Specifically, m6As within coding sequences and 3′UTRs repress the translation of mRNAs while the m6As within 5′UTRs and start codons could promote it. The m6A-containing mRNAs are significantly enriched in processes and pathways of “translation” and “RNA transport,” suggesting the potential role of m6As in regulating the translation of genes involved in translation regulation. Our data also show a stronger translation inhibition by small RNAs (miRNA and phasiRNA) than by m6A methylation, and no synergistical effect between the two was observed. We propose a secondary amplification machinery of translation regulation triggered by the changes in m6A methylation status. Taken together, our results suggest translation regulation as a key role played by m6As in hexaploid wheat.

Early life vitamin D depletion and mechanical loading determine methylation changes in the RUNX2, RXRA, and osterix promoters in mice

BackgroundEarly life vitamin D exposure is linked to later skeletal health with maternal vitamin D status in pregnancy associated with neonatal bone mass. The MAVIDOS study has demonstrated that vitamin D supplementation leads to reduced RXRA DNA methylation. Mice exposed to early life vitamin D deficiency have reduced bone mass and bone accrual in response to mechanical loading. Using the tibiae of these mice, we have examined the effect of diet and mechanical loading on the DNA methylation of promoters of genetic loci important for bone growth and development and their association with bone strength.ResultsMechanical loading of mouse tibiae leads to a reduction of RXRA DNA methylation. Early life vitamin D deficiency is associated with altered methylation of osterix and Runx2 in these bones. Tibia strength was also demonstrated to be associated with a change in DNA methylation status in CpGs of the vitamin D receptor (VDR), ostrix, and RXRA genes.ConclusionsWe have shown for the first time that mechanical loading of bone and early life vitamin D deficiency leads to changes in the epigenome of this tissue in key genes in the vitamin D and osteoblast differentiation pathway.

Epigenetic disruption of placental genes by chronic maternal cafeteria diet in rats

Maternal diet has impact on reproduction, fetal development and offspring behavior, although molecular mechanisms remained unknown. Our aims were to assess (1) the effects of a cafeteria (CAF) diet (western diet habits) on female reproductive performance, fetal and placental parameters on gestational day 21 and litter size and pup weight at birth; and (2) placental messenger RNA (mRNA) expression and epigenetic regulation of Insulin-Like Growth Factor (Igf) and Vascular Endothelial Growth Factor (Vegf) and their receptors. Female Wistar rats were fed with control or CAF diet from weaning until parturition. At week 14 after diets started, females were mated and half of the animals were euthanized on gestational day 21 to evaluate reproductive parameters including the pregnancy rate, number of corpora lutea, implantation sites and resorption sites. Moreover, fetal weight and length, placental weight, and placental index were recorded. Placentas were collected for mRNA quantification and DNA methylation analysis. The remaining animals were allowed to give birth and the number and weight of the pups were evaluated.CAF diet did not affect reproductive performance or fetal weight and length. However, CAF-fed animals showed a decrease in placental weight and index and the pups exhibited a low birth weight. Additionally, we found an upregulation of Igf2 and a down regulation of Vegf placental mRNA expression in CAF dams, associated with methylation status changes of their promoters. We conclude that female chronic CAF diet consumption impairs feto-placental development and could be explained by an epigenetic disruption of Igf and Vegf systems.

Epstein-Barr virus miR-BART4-3p regulates cell proliferation, apoptosis, and migration by targeting AXL in gastric carcinoma.

To investigate the role of miR-BART4-3p in EBV-associated gastric cancer (EBVaGC) and its regulation of cell proliferation, apoptosis, and migration by targeting AXL in GC. Quantitative real-time PCR and western blot were used to detect the expression of AXL. The methylation status of AXL gene promoter region was determined by bisulfite sequencing PCR. Luciferase reporter assay was used to detect whether miR-BART4-3p targets AXL. The key molecules of EMT and PI3K/AKT pathway were used to examine by western blot. CCK8, Transwell, and flow cytometry were used to detect the phenotypic gastric cancer cells after interference with AXL and miR-BART4-3p. EBV infection inhibited the expression of AXL in GC cells and the inhibition was not caused by the change of promoter methylation status. MiR-BART4-3p directly targeted AXL. Moreover, both inhibition of miR-BART4-3p and AXL inhibited cell proliferation and migration and promoted cell apoptosis. In addition, E-cadherin, Vimentin, ZEB1, and p-AKT were found to be the downstream molecules of the miR-BART4-3p/AXL pathway. The change of promoter methylation status was not the reason for the downregulation of AXL expression in EBV-positive cells. MiR-BART4-3p may inhibit the proliferation and migration and promote apoptosis of GC cells by directly targeting AXL.

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.