Global DNA Methylation Levels Research Articles

Differential Lysyl oxidase like 1 expression in pseudoexfoliation glaucoma is orchestrated via DNA methylation

Pseudoexfoliation syndrome (PXF) is the most common cause of secondary open angle glaucoma worldwide. Single nucleotide polymorphisms (SNPs) in the gene Lysyl oxidase like 1 (LOXL1) are strongly associated with the development of pseudoexfoliation glaucoma (PXFG). However, these SNPs are also present in 50–80% of the general population, suggestive of other factors being involved in the pathogenesis of PXFG. In this study, we aimed to investigate the influence of epigenetic regulation, specifically DNA methylation, on LOXL1 expression in PXFG using human tenons fibroblasts (HTFs), aqueous humour and serum samples from donors with and without PXFG. LOXL1 expression in HTFs was measured by qPCR and Western Blotting and LOXL1 concentration in aqueous humour was determined by ELISA. Global DNA methylation levels were quantified using an ELISA for 5-methylcytosine. MeDIP assays assessed the methylation status of the LOXL1 promoter region. Expression of methylation-associated enzymes (DNMT1, DNMT3a and MeCP2) were determined by qPCR and inhibited by 0.3 μM 5-azacytidine (5-aza). Results showed that LOXL1 expression was significantly decreased in PXFG HTFs compared with Control HTFs at gene (Fold change 0.37 ± 0.05, P < 0.01) level and showed a decrease, when measured at the protein level (Fold change 0.65 ± 0.42, P = 0.22), however this was not found to be significant. LOXL1 concentration was increased in the aqueous of PXFG patients compared with Controls (2.76 ± 0.78 vs. 1.79 ± 0.33 ng/ml, P < 0.01). Increased global methylation (56.07% ± 4.87% vs. 32.39% ± 4.29%, P < 0.01) was observed in PXFG HTFs compared with Control HTFs, as was expression of methylation-associated enzymes (DNMT1 1.58 ± 0.30, P < 0.05, DNMT3a 1.89 ± 0.24, P < 0.05, MeCP2 1.63 ± 0.30, P < 0.01). Methylation-associated enzymes were also increased when measured at protein level (DNMT1 5.70 ± 2.64, P = 0.04, DNMT3a 1.79 ± 1.55, P = 0.42, MeCP2 1.64 ± 1.33, P = 0.45). LOXL1 promoter methylation was increased in patients with PXFG compared to Control patients in both blood (3.98 ± 2.24, 2.10 ± 1.29, P < 0.05) and HTF cells (37.31 ± 22.0, 8.66 ± 10.40, P < 0.01). Treatment of PXFG HTFs with in 5-azacytidine increased LOXL1 expression when compared with untreated PXFG HTFs (Fold change 2.26 ± 0.67, P < 0.05). These data demonstrate that LOXL1 expression is altered in PXFG via DNA methylation and that reversal of these epigenetic changes may represent future potential therapeutic targets in the management of PXFG.

An Engineered RNA Platform to Neutralize DNMT1 Function and Control DNA Methylation for Myelodysplastic Syndrome

DNA methylation is a major signature involved in the regulation of gene expression. Numerous studies have established a link between aberrant DNA methylation and cancer (Herman and Baylin 2003, Baylin and Jones 2011, Feinberg 2018). Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal hematopoietic malignancies, characterized by ineffective hematopoiesis, cytopenia and risk of progression to acute myeloid leukemia (AML) in approximately 30% of the cases (Khan, Vale et al. 2013, Arber, Orazi et al. 2016). Abnormal DNA methylation is considered the molecular lesion leading to tumor suppressor gene silencing and clonal variation in MDS and evolution to AML (Figueroa, Skrabanek et al. 2009, Jiang, Dunbar et al. 2009, Feinberg 2018). In the past decades two nucleoside-based compounds, 5-azacytidine and 5-aza 2'-deoxycytidine have been extensively tested to reduce global DNA methylation levels and received approval by the U.S. Food and Drug Administration (FDA) for the treatment of MDS. Both drugs have been used as the frontline therapy for the management of higher-risk-to-transform MDS, that are ineligible for more aggressive treatment such as: allogenic transplants and standard chemotherapy. Unfortunately, cytotoxic and global non-specific demethylation effects, have limited their clinical application, revealing the need for smarter and safer epigenetic drugs. Nucleic-acid aptamers are a new class of specific targeting molecules and represent high affinity ligands and potential antagonists of disease-associated proteins. Herein, we present an innovative RNA aptamer-based approach to target DNMT1 function by translating the inherent neutralizing properties of RNA (Di Ruscio, Ebralidze et al. Nature, 2013, 503(7476):371-6) into an aptamer platform. Through a combinatorial chemistry strategy named SELEX (Systematic Evolution of Ligands by EXponential enrichment) (Mercier, Dontenwill et al. 2017), we shortlisted the most specific DNMT1-neutralizing RNA aptamers. These molecules display high specificity and serum stability and reveal strong structural affinity in targeting DNMT1, by molecular modelling and dynamic simulations. Foremost, we found that the selected aptamers specifically inhibit DNMT1 enzymatic activity both in vitro and in cells, thus impairing cell viability and leading to global demethylation. Collectively, this study provides a proof-of-concept for the generation of the first RNA-based epigenetic therapy as well as an attractive and scalable tool of precision medicine. Such a tool will overcome the toxicity of in-use demethylating protocols and greatly improve care and treatment of patients with MDS and other disease triggered by aberrant DNA methylation. Figure Disclosures No relevant conflicts of interest to declare.

The neuroendocrine modulation of global DNA methylation in neuropsychiatric disorders.

There is an increasing body of knowledge on the influence of differential DNA methylation of specific genomic regions in psychiatric disorders. However, fewer studies have addressed global DNA methylation (GMe) levels. GMe is an estimative of biological functioning that is regulated by pervasive mechanisms able to capture the big picture of metabolic and environmental influences upon gene expression. In the present perspective article, we highlighted evidence for the relationships between cortisol and sex hormones and GMe in psychiatric disorders. We argue that the far-reaching effects of cortisol and sexual hormones on GMe may lie on the pathways linking stress and mental health. Further research on these endocrine-epigenetic links may help to explain the role of environmental stress as well as sex differences in the prevalence of psychiatric disorders.

Evaluation of the influence of global DNA methylation level in patients with acute coronary syndrome

DNA methylation is one of the mechanisms of epigenetic regulation and is observed in mammals to maintain a normal expression pattern of the genes. Aberrant profiles of DNA methylation have already been associated with cardiovascular diseases. We evaluated 190 patients with Acute Coronary Syndrome (ACS) and 75 patients without ACS (non-ACS). Patient severity was assessed by the TIMI risk score, and both levels of global DNA methylation (ACS = 190; non-ACS = 75), stratified in expected group (male ≥ 65 years; female ≥ 55 years) and early group (male < 65 years; female < 55 years). As results, the ACS and non-ACS groups showed different levels of global DNA methylation, and patients with ACS were more methylated (p = 0.0121). Patients with ACS, showed a difference (p < 0.0001) in methylation profiles between groups. The low TIMI group had a higher level of DNA methylation, while the intermediate / high group showed a decreased methylation pattern. A negative correlation was observed between the level of global methylation and the increase in age (p = 0.0387; r = −0.15), which became hypomethylated over the years. The hypermethylated global DNA profile by its association with the development of ACS can be a potential biomarker.

Dose-dependent genomic DNA hypermethylation and mitochondrial DNA damage in Japanese tree frogs sampled in the Fukushima Daiichi area

The long-term consequences of the nuclear disaster at the Fukushima Daiichi Nuclear Power Plant (FDNPP) that occurred on March 2011, have been scarcely studied on wildlife. We sampled Japanese tree frogs (Dryophytes japonicus), in a 50 –km area around the FDNPP to test for an increase of DNA damages and variation of DNA methylation level. The ambient dose rate ranged between 0.4 and 2.8 μGy h−1 and the total estimated dose rate absorbed by frogs ranged between 0.3 and 7.7 μGy h−1. Frogs from contaminated sites exhibited a dose-dependent increase of global genomic DNA methylation level (5-mdC and 5-hmdC) and of mitochondrial DNA damages. Such DNA damages may indicate a genomic instability, which may induce physiological adaptations governed by DNA methylation changes.This study stresses the need for biological data combining targeted molecular methods and classic ecotoxicology, in order to better understand the impacts on wildlife of long term exposure to low ionizing radiation levels.

Effect of neonatal exposure to endocrine-active compounds on epigenetic regulation of gene expression in corpus luteum of gilts

Recently, we have demonstrated that neonatal exposure to environmental endocrine-active compounds (EACs) with androgenic/antiandrogenic and estrogenic/antiestrogenic activities led to morphological and functional changes in the porcine corpus luteum (CL). To gain insight into the regulatory mechanisms of the long-term effects of EACs, we analyzed the impact of neonatal exposure of such compounds on global DNA methylation and the expression of miRNA biogenesis components in the porcine CL. Piglets were injected subcutaneously with testosterone propionate (TP, an androgen), flutamide (FLU, an antiandrogen), 4-tert-octylphenol (OP, an estrogenic compound), ICI 182,780 (ICI, an antiestrogen), methoxychlor (MXC, a compound with mixed activities) or corn oil (control) between postnatal days 1 and 10 (n = 5/group). The CLs from sexually mature gilts were examined for global DNA methylation and for the abundance of proteins related to DNA methylation (DNMT1, DNMT3A and DNMT3B) and miRNA biogenesis (DROSHA, XPO5, DICER1, AGO2) using an enzyme-linked immunosorbent assay, Western blotting and immunohistochemical staining. ICI and MXC increased the global DNA methylation levels and DNMT1 protein abundance in the luteal tissue. OP treatment led to a lower DROSHA protein abundance, while ICI treatment resulted in a greater DROSHA protein abundance. Both FLU and ICI increased DICER1 protein abundance in the luteal tissue. In addition, XPO5 showed immunolocalization exclusively in small luteal cells in the OP-treated pigs, in contrast to localization in both small and large luteal cells in the controls. In conclusion, the changes in DNA methylation, as well as the altered miRNA biogenesis components, seem to be a part of the regulatory network that mediates the long-term effects of EACs on CL function in pigs.

What you remember saves you.

What you remember saves you.

Identification of Differentially Methylated miRNA Genes During Compatible and Incompatible Interactions Between Soybean and Soybean Cyst Nematode.

DNA methylation is a widespread epigenetic mark that affects gene expression and transposon mobility during plant development and stress responses. However, the role of DNA methylation in regulating the expression of microRNA (miRNA) genes remains largely unexplored. Here, we analyzed DNA methylation changes of miRNA genes using a pair of soybean (Glycine max) near-isogenic lines (NILs) differing in their response to soybean cyst nematode (SCN; Heterodera glycines). Differences in global DNA methylation levels over miRNA genes in response to SCN infection were observed between the isogenic lines. miRNA genes with significant changes in DNA methylation levels in the promoter and primary transcript-coding regions were detected in both lines. In the susceptible isogenic line (NIL-S), 82 differentially methylated miRNAs were identified in response to SCN infection whereas, in the resistant isogenic line (NIL-R), only 16 differentially methylated miRNAs were identified. Interestingly, gma-miR5032, gma-miR5043, gma-miR1520b, and gma-2107-ch16 showed opposite methylation patterns in the isogenic lines. In addition, the miRNA paralogs gma-miR5770a and gma-miR5770b showed hypermethylation and hypomethylation in NIL-S and NIL-R, respectively. Gene expression quantification of gma-miR5032, gma-miR5043, gma-miR1520b, and gma-miR5770a/b and their confirmed targets indicated a role of DNA methylation in regulating miRNA expression and, thus, their targets upon SCN infection. Furthermore, overexpression of these four miRNAs in NIL-S using transgenic hairy root system enhanced plant resistance to SCN to various degrees with a key role observed for miR5032. Together, our results provide new insights into the role of epigenetic mechanisms in controlling miRNA regulatory function during SCN-soybean interactions.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Investigation of genomic DNA methylation by ultraviolet resonant Raman spectroscopy.

Cytosine plays a preeminent role in DNA methylation, an epigenetic mechanism that regulates gene expression, the misregulation of which can lead to severe diseases. Several methods are nowadays employed for assessing the global DNA methylation levels, but none of them combines simplicity, high sensitivity, and low operating costs to be translated into clinical applications. Ultraviolet (UV) resonant Raman measurements at excitation wavelengths of 272 nm, 260 nm, 250 nm, and 228 nm have been carried out on isolated deoxynucleoside triphosphates (dNTPs), on a dNTP mixture as well as on genomic DNA (gDNA) samples, commercial from salmon sperm and non-commercial from B16 murine melanoma cell line. The 228 nm excitation wavelength was identified as the most suitable energy for enhancing cytosine signals over the other DNA bases. The UV Raman measurements performed at this excitation wavelength on hyper-methylated and hypo-methylated DNA from Jurkat leukemic T-cell line have revealed significant spectral differences with respect to gDNA isolated from salmon sperm and mouse melanoma B16 cells. This demonstrates how the proper choice of the excitation wavelength, combined with optimized extraction protocols, makes UV Raman spectroscopy a suitable technique for highlighting the chemical modifications undergone by cytosine nucleotides in gDNA upon hyper- and hypo-methylation events.

Stearoyl-CoA Desaturase 1 Activity Determines the Maintenance of DNMT1-Mediated DNA Methylation Patterns in Pancreatic β-Cells.

Metabolic stress, such as lipotoxicity, affects the DNA methylation profile in pancreatic β-cells and thus contributes to β-cell failure and the progression of type 2 diabetes (T2D). Stearoyl-CoA desaturase 1 (SCD1) is a rate-limiting enzyme that is involved in monounsaturated fatty acid synthesis, which protects pancreatic β-cells against lipotoxicity. The present study found that SCD1 is also required for the establishment and maintenance of DNA methylation patterns in β-cells. We showed that SCD1 inhibition/deficiency caused DNA hypomethylation and changed the methyl group distribution within chromosomes in β-cells. Lower levels of DNA methylation in SCD1-deficient β-cells were followed by lower levels of DNA methyltransferase 1 (DNMT1). We also found that the downregulation of SCD1 in pancreatic β-cells led to the activation of adenosine monophosphate-activated protein kinase (AMPK) and an increase in the activity of the NAD-dependent deacetylase sirtuin-1 (SIRT1). Furthermore, the physical association between DNMT1 and SIRT1 stimulated the deacetylation of DNMT1 under conditions of SCD1 inhibition/downregulation, suggesting a mechanism by which SCD1 exerts control over DNMT1. We also found that SCD1-deficient β-cells that were treated with compound c, an inhibitor of AMPK, were characterized by higher levels of both global DNA methylation and DNMT1 protein expression compared with untreated cells. Therefore, we found that activation of the AMPK/SIRT1 signaling pathway mediates the effect of SCD1 inhibition/deficiency on DNA methylation status in pancreatic β-cells. Altogether, these findings suggest that SCD1 is a gatekeeper that protects β-cells against the lipid-derived loss of DNA methylation and provide mechanistic insights into the mechanism by which SCD1 regulates DNA methylation patterns in β-cells and T2D-relevant tissues.

Neuroprotective Effect of Piracetam against Cocaine-Induced Neuro Epigenetic Modification of DNA Methylation in Astrocytes.

Cocaine abuse is known to alter mitochondrial biogenesis and induce epigenetic modification linked with neuronal dysfunction. Cocaine-induced epigenetic modification of DNA methylation and the mitochondrial genome may affect mitochondrial DNA (mtDNA) and nuclear DNA (nDNA), as epigenetic DNA methylation is key to maintaining genomic integrity in the central nervous system (CNS). However, the impact of cocaine-mediated epigenetic changes in astrocytes has not yet been elucidated. In this study, we explored the neuroprotective effect of piracetam against cocaine-induced epigenetic changes in DNA methylation in astrocytes. To study our hypothesis, we exposed human astrocytes to cocaine alone or in combination with the nootropic drug piracetam. We examined the expression of the DNA methyltransferases (DNMTs) DNMT-1, DNMT-3A, and DNMT-3B; global DNA methylation levels of 5-methycytosine (5-mC); and induction of ten–eleven translocation (TET) enzymes in astrocytes. In addition, we analyzed mtDNA methylation by targeted next-generation bisulfite sequencing. Our data provide evidence that cocaine impairs DNMT activity and thereby has impacts on mtDNA, which might contribute to the neurodegeneration observed in cocaine users. These effects might be at least partially prevented by piracetam, allowing neuronal function to be maintained.

Exposure to different early-life stress experiences results in differentially altered DNA methylation in the brain and immune system

The existence of a proportional relationship between the number of early-life stress (ELS) events experienced and the impoverishment of child mental health has been hypothesized. However, different types of ELS experiences may be associated with different neuro-psycho-biological impacts, due to differences in the intrinsic nature of the stress. DNA methylation is one of the molecular mechanisms that have been implicated in the “translation” of ELS exposure into neurobiological and behavioral abnormalities during adulthood. Here, we investigated whether different ELS experiences resulted in differential impacts on global DNA methylation levels in the brain and blood samples from mice and humans. ELS exposure in mice resulted in observable changes in adulthood, with exposure to social isolation inducing more dramatic alterations in global DNA methylation levels in several brain structures compared with exposure to a social threatening environment. Moreover, these two types of stress resulted in differential impacts on the epigenetic programming of different brain regions and cellular populations, namely microglia. In a pilot clinical study, blood global DNA methylation levels and exposure to childhood neglect or abuse were investigated in patients presenting with major depressive disorder or substance use disorder. A significant effect of the mental health diagnosis on global methylation levels was observed, but no effect of either childhood abuse or neglect was detected. These findings demonstrate that different types of ELS have differential impacts on epigenetic programming, through DNA methylation in specific brain regions, and that these differential impacts are associated with the different behavioral outcomes observed after ELS experiences.

TET3 overexpression facilitates DNA reprogramming and early development of bovine SCNT embryos.

Somatic cell nuclear transfer (SCNT) has been successfully used for cloning in a variety of mammalian species. However, SCNT reprogramming efficiency is relatively low, in part, due to incomplete DNA methylation reprogramming of donor cell nuclei. We previously showed that ten-eleven translocation 3 (TET3) is responsible for active DNA demethylation during preimplantation embryonic development in bovines. In this study, we constructed TET3-overexpressing cell lines in vitro and observed that the use of these fibroblasts as donor cells increased the blastocyst rate by approximately 18 percentage points compared to SCNT. The overexpression of TET3 in bovine SCNT embryos caused a decrease in the global DNA methylation level of the pluripotency genes Nanog and Oct-4, ultimately resulting in an increase in the transcriptional activity of these pluripotency genes. Moreover, the quality of bovine TET3-NT embryos at the blastocyst stage was significantly improved, and bovine TET3-NT blastocysts possessed more total number of cells and fewer apoptotic cells than the SCNT blastocysts, similar to in vitro fertilization (IVF) embryos. Nevertheless, DNA methylation of the imprinting control region (ICR) for the imprinted genes H19-IGF2 in SCNT embryos remained unaffected by TET3 overexpression, maintaining parent-specific activity for further development. Thus, the results of our study provide a promising approach to rectify incomplete epigenetic reprogramming and achieve higher cloning efficiency.

A king and vassals' tale: Molecular signatures of clonal integration in Posidonia oceanica under chronic light shortage

Abstract Under unfavourable conditions, clonal plants benefit from physiological integration among ramets, sharing resources and information. Clonal integration can buffer against environmental changes and lets the plant clone work as a ‘macro’ organism. Molecular signals that regulate this phenomenon are completely unknown in marine plants. Here we present a first comprehensive study providing insights into the metabolic role of different types of ramets (i.e. apical vs. vertical) in the foundation species Posidonia oceanica. Plants were exposed to 80% diminishing irradiance level (LL) in a controlled mesocosm system. Subsequent multiscale variations in whole transcriptome expression, global DNA methylation level, photo‐physiology, morphology and fitness‐related traits, were explored at different exposure times. We tested the hypothesis that vertical shoots (the ‘vassals’) can provide vital resources to apical shoots (the ‘kings’) under energy shortage, thus safeguarding the whole clone survival. Whole transcriptome analysis of leaves and shoot‐apical meristems (SAMs) emphasized signatures of molecular integration among ramets, which strongly correlated with higher organization‐level responses. In both shoots types, the exposure to LL resulted in a growth slowdown throughout the experiment, which started from immediate signals in SAMs. In apical shoots, this was linked to an acclimative response, where they were suffering a mild stress condition, while in vertical ones it fell in a more severe stress response. Yet, they suffered from sugar starvation and showed a clear cellular stress response in terms of protein refolding and DNA repair mechanisms. Several epigenetic mechanisms modulated the observed gene‐expression patterns and the cross‐talk between DNA methylation and the cellular energetic status appeared to regulate shoot metabolism under LL. Synthesis. Our results demonstrate a high level of specialization of integrated ramets within seagrass clones and a ‘division of labour’ under adverse conditions. Vertical shoots appear to do ‘most of the job’ especially in terms of resource providing, whereas activated functions in apical shoots were restricted to few important processes, according to an ‘energy‐saving’ strategy. The response of vertical shoots could be seen as a ‘sacrificing response’ allowing the survival of ‘the king’ that is key for ensuring propagation and population maintenance, and for the colonization of new environments.

Assessment of DNA Methylation and Oxidative Changes in the Heart and Brain of Rats Receiving a High-Fat Diet Supplemented with Various Forms of Chromium.

Simple SummaryObesity is one of the most prevalent diseases of civilization in the 21st century. This may be due to an increase in the consumption of high-fat diets. For the treatment of obesity, various supplements with chromium (III) are used. Chromium has the ability to regulate carbohydrate and lipid metabolism, which may result in weight loss. Our studies compared the effects of a high-fat diet supplemented with three different forms of chromium-chromium (III) picolinate, chromium (III) -methionine, and nano-sized chromium on oxidative damage of the DNA, proteins, and lipids in the heart and brain of rats. The conducted study showed that the use of a high-fat diet results in oxidation of the DNA, proteins, and lipids in the brain and heart, and the addition of chromium additionally intensifies these processes, especially when used in the form of nanoparticles. Therefore, the results of these studies suggest that one should be careful when using chromium supplementation to counteract obesity, because it may be associated with the risk of deterioration of the functioning of the heart and brain.The aim of the study was to determine how feeding rats a high-fat diet supplemented with various forms of chromium affects DNA methylation and oxidation reactions as well as the histology of heart and brain tissue. The rats received standard diet or high-fat diet and chromium at 0.3 mg/kg body weight (BW) in form of chromium (III) picolinate, chromium (III)-methionine, or nano-sized chromium. The content of malondialdehyde (MDA), protein carbonyl (PC), and 8-hydroxydeoxyguanosine (8-OHDG), the level of global DNA methylation and the activity of selected DNA repair enzymes were determined in the blood. In the brain and heart, the content of MDA, PC, 8-OHDG, and levels of global DNA methylation were determined. The brain was subjected to histological examination. The use of a high-fat diet was found to intensify epigenetic changes and oxidation reactions in the heart and brain. It was concluded that epigenetic changes and oxidation of lipids, proteins, and DNA in the heart and brain of rats resulting from the use of a high-fat diet cannot be limited by supplementing the diet with chromium. It was established that the use of chromium to supplement a high-fat diet intensifies the negative epigenetic and oxidative changes in the heart and brain, especially in the case of chromium nanoparticles.

Abstract 1276: Analyses of cell-free DNA alterations in physiological conditions and colon diseases for screening, diagnostics and therapy follow-up

Abstract Cell-free DNA (cfDNA) level increases in several physiological conditions, such as pregnancy, aging or high physical activity. Furthermore, alterations of cfDNA concentration and genetic or epigenetic (e.g. DNA methylation) patterns can also be observed in pathological processes, as inflammatory diseases or different cancer types including colorectal cancer (CRC). However, studies about quality changes of cfDNA aimed to analyze its fragment length profile and DNA methylation pattern are lacking. We aimed to define the quantity and quality changes of cfDNA, including concentration, fragment length distribution and global DNA methylation pattern during high physical exercise, and in colorectal adenoma and cancer patients. Moreover, we aimed to investigate the treatment response in plasma of metastatic CRC (mCRC) patients analyzing the above-mentioned parameters and the DNA methylation level of SFRP2 gene during therapy. Plasma fraction was separated from blood samples of 6 healthy athletes before, during and after physical training, and from healthy (n=16), adenoma (n=13), IBD (n=19), and CRC (n=16) patients. Moreover, plasma longitudinal assessment was performed in the case of 20 mCRC patients treated with chemotherapy. CfDNA level was quantified with Qubit 1.0 (Thermo Fisher Scientific), fragment length distribution was examined by 2100 Bioanalyzer instrument (Agilent), and global DNA methylation level was measured with bisulfite pyrosequencing of long interspersed nuclear element-1 (LINE-1) (Qiagen) in all samples. Besides, DNA methylation status of SFRP2 gene was determined with droplet digital PCR (Bio-Rad) in mCRC plasma samples. Increased cfDNA amount was observed during physical exercise (198.5±90ng), in comparison with control phase (86.3±40.6ng), and then in restitution period, lower cfDNA level (155.6±119.3ng) was detected. Elevated cfDNA amounts have been found in plasma of adenoma (72.1±37.5ng), IBD (78.0±50.9ng) and CRC (84.5±70.1ng) patients compared to controls (36.2±11.3ng). CfDNA fragment length distribution showed different patterns in each sample group, longer fragments (200-600bp) enriched in AD and CRC samples in addition to short (&amp;lt;200bp) fragments. Slightly lower level of global DNA methylation was noticed in AD (79,5%) and CRC (79,2%) compared to normal (83,8%) samples. Interestingly, the amount and global methylation level alteration of cfDNA revealed negative correlation in CRC patients during therapy and progression. Furthermore, the amount of methylated SFRP2 copies (5-500copies) was increased in the case of progressive disease, while were undetectable in patients achieved complete remission. Characteristic cfDNA level, fragment length, and global DNA methylation level were identified in the different sample groups. The above-mentioned parameters can be applied for the follow-up of mCRC patients during chemotherapy and could be integrated into current follow-up methods. Citation Format: Barbara K. Bartak, Krisztina Andrea Szigeti, Alexandra Kalmar, Orsolya Galamb, Zsófia Brigitta Nagy, Sara Zsigrai, Zsolt Tulassay, Peter Igaz, Magdolna Dank, Bela Molnar. Analyses of cell-free DNA alterations in physiological conditions and colon diseases for screening, diagnostics and therapy follow-up [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1276.

Abstract 1265: The effects of ancestry and histology on the lung tumor methylome

Abstract Methylation-based biomarkers can provide molecular insight on the contribution of ancestry and histology to lung tumor biology. Loss of global methylation is a benchmark of aging and a signal of declining biological maintenance. In addition to aging, global loss of methylation occurs in response to some cancer-promoting exposures, including tobacco use. Reduced levels of global DNA methylation are associated with increased risk of several cancers, including lung. In lung tumors, global DNA hypomethylation is often associated with a more aggressive diseases and shorter survival time. Several studies have identified population-level differences in global methylation present at birth. Additionally, gender and ancestry influence DNA methylation age (DNAm) and may predispose some individuals to accelerated aging. We hypothesized that differences in global hypomethylation and accelerated aging may contribute to differences in lung tumor biology by genetic ancestry. Therefore, we investigated methylome differences between European Americans (EA) and African Americans (AA) from the NCI-Maryland Case Control Study, which is overrepresented with AA patients (~40%) from the Baltimore area. We assessed lung tumor and paired non-involved adjacent tissue (NAT) methylation (n = 77 EA, n = 39 AA) using the Illumina EPIC methylation array. We used the Repetitive Element Methylation Prediction (REMP) package in R to identify roughly 20,000 predicted LINE-1 CpG's. We observed significantly decreased LINE-1 methylation in tumor tissue compared with NAT in both AA and EA. Additionally, among tumor samples we observed significantly decreased LINE-1 methylation in AA LUSC samples compared to EA LUSC, with only modest differences between AA LUAD and EA LUAD. In addition to decreased levels, LINE-1 methylation in AA LUSC tumors also had the greatest heterogeneity within CpG's of the same sample and between other AA LUSC samples. We observed accelerated aging (increased DNAm relative to chronological age) in 57/67 EA NAT samples and 28/38 AA NAT samples with no significant differences in age acceleration between ancestry or histology. Our results show global hypomethylation of LINE-1 is a distinctive feature of LUSC in AA compared to EA, which is consistent with the enhanced genomic instability observed in AA LUSC relative to EA LUSC. We also observed accelerated aging in the vast majority of the NAT samples, which may implicate accelerated age with lung cancer risk. Future analysis will work towards completing a comprehensive analysis of the epigenetic landscape of lung cancer in AA and EA patients. We plan to study the relationship between global hypomethylation and genetic variants, smoking behaviors, and patient survival. Citation Format: Emily L. Rossi, Isaac Zhao, Rony F. Arauz, Adriana Zingone, Bríd M. Ryan. The effects of ancestry and histology on the lung tumor methylome [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1265.

Abstract 164: Alteration of DNA methylation, DNA repair and epithelial-mesenchymal transition in colorectal cancer cell lines by S-adenosylmethionine treatment

Abstract Global DNA hypomethylation can be observed along with the aging of normal cells, and it is also related to tumor initiation and progression. S-adenosylmethionine (SAM) is a universal methyl donor molecule, used as a dietary supplement. SAM is involved in DNA methylation processes, thereby it may have a favorable effect on gene expression of cancer-associated genes through epigenetic modifications, but may also influence DNA folding during repair processes. Our aim was to analyze the effect of SAM treatment on global and promoter-specific DNA methylation level, gene expression, DNA integrity, cell cycle and the proliferation of two different colorectal cancer cell lines (HT-29, SW480). HT-29 and SW480 cells were treated with SAM in different concentrations (0, 0.5, 1 mmol/l) for 48 hours. Global DNA methylation status was analyzed by bisulfite pyrosequencing of long interspersed nuclear element-1 (LINE-1) retrotransposons. Promoter-specific DNA methylation alterations were determined by Reduced Representation Bisulfite Sequencing (RRBS) method. Gene expression changes were detected using Human Transcriptome Array 2.0 (HTA 2.0). DNA integrity analysis was performed with γH2AX ELISA, immunostaining and Comet Assay. Flow cytometry measurement and Sulforhodamine B (SRB) assay were assessed for cell cycle and proliferation determination. Global and promoter-specific DNA methylation alterations, as well as decreased expression (p&amp;lt; 0.05) of genes, that are involved in epithelial-mesenchymal transition were observed after SAM treatment. Increased phosphorylation of H2AX (74.9, 166.5, 200.6 pM) and decreased micronucleus number (1.47, 0.76, 0.45% of cells) were referred to the activation of reparative processes, that was supported by the changes of comet tail lengths. Proportion of cells was decreased in the G0/G1 (48.4, 28.5, 20.4%) phase; however, it was increased in both S (45.7, 61.7, 67.0%) and G2/M (6.0, 10.7, 12.5%) phases. Significant (p&amp;lt; 0.05) decrease of cell proliferation (99.5, 78.0, 70.6%) was also detected with SRB assay. SAM is able to alter the DNA methylation pattern of tumor cells and can induce DNA repair. Activation of these processes can lead to cell cycle arrest, decreased proliferation, and inhibition of epithelial-mesenchymal transition. Tumor cells could be targeted by SAM through different pathways; therefore, it may enhance the effect of chemotherapeutic agents. Citation Format: Sára Zsigrai, Alexandra Kalmár, Krisztina Andrea Szigeti, Zsófia Brigitta Nagy, Barbara K. Barták, Gábor Valcz, Orsolya Galamb, Titanilla Dankó, Zsolt Tulassay, Péter Igaz, Béla Molnár. Alteration of DNA methylation, DNA repair and epithelial-mesenchymal transition in colorectal cancer cell lines by S-adenosylmethionine treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 164.

Abstract 161: xCT expression alters global DNA methylation levels in lung adenocarcinoma cells

Abstract Metabolic reprogramming and genomic instability are two key components in driving carcinogenesis. While it has been theorized that these two processes influence each other, the mechanisms by which metabolic reprogramming can induce genomic instability remain unknown. Our lab previously demonstrated xCT, a cystine/glutamate antiporter, is highly expressed in NSCLC and induces metabolic reprogramming. However, its influence on genomic instability is unknown. In this study, we aim to examine how xCT could potentially contribute to genomic instability by first investigating its influence on the epigenetic landscape, markedly DNA methylation. We hypothesize that changes in intracellular metabolite concentrations due to high xCT expression have a downstream effect on the levels of metabolites involved in DNA methylation, and such changes are significant enough to induce alterations in global DNA methylation. Global methylation levels were quantified through a colorimetric assay in A549 wildtype (WT) and xCT knockout (KO) cells. Next, xCT KO cells were treated with propargylglycine (PAG), a cystathionase inhibitor, and measured global methylation. By inhibiting cystathionase, we blocked cells' ability to synthesize cysteine through the utilization metabolites in the methionine cycle. Quantified methylation levels reported 51.2 ± 11.4% and 25.6 ± 11.0% in WT and xCT KO cells respectively- an average of a 50.0% decrease in methylation upon knocking out xCT. Furthermore, initial data showed DNA methylation more than tripling (234.8% increase) in xCT KO cells when treated with PAG, notably demonstrating 43.4% higher methylation levels than that of treated and untreated WT. Additionally, a western blot revealed that compared to WT, xCT KO cells express much higher levels of cystathionine beta synthase, another enzyme linking cysteine biosynthesis and the methionine cycle. These results support the notion that metabolic activity in the context of xCT can change DNA methylation, potentially through altered metabolic demand between the transsulfuration pathway and methionine cycle. Further studies investigating key metabolite concentrations in xCT KO versus WT cells, as well as analyzing deregulated genes resulting from the altered methylation landscape, will help elucidate the extent of xCT-induced metabolic reprogramming. Understanding the full scope xCT overexpression and the underlying mechanisms leading to epigenetic changes may provide crucial insight to the interplay of metabolic reprogramming and other cancer drivers like genomic instability. By understanding the true origin and influences of such hallmarks of carcinogenesis, more informed approaches can be applied to early preventative therapies. Citation Format: Dalton Hill, S.M. Jamshedur Rahman, Christien Kluwe, Pierre P. Massion. xCT expression alters global DNA methylation levels in lung adenocarcinoma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 161.

Sperm DNA Hypomethylation Proximal to Reproduction Pathway Genes in Maturing Elite Norwegian Red Bulls.

Genomic selection in modern farming demands sufficient semen production in young bulls. Factors affecting semen quality and production capacity in young bulls are not well understood; DNA methylation, a complicated phenomenon in sperm cells, is one such factors. In this study, fresh and frozen-thawed semen samples from the same Norwegian Red (NR) bulls at both 14 and 17 months of age were examined for sperm chromatin integrity parameters, ATP content, viability, and motility. Furthermore, reduced representation bisulfite libraries constructed according to two protocols, the Ovation® RRBS Methyl-Seq System (Ovation method) and a previously optimized gel-free method and were sequenced to study the sperm DNA methylome in frozen-thawed semen samples. Sperm quality analyses indicated that sperm concentration, total motility and progressivity in fresh semen from 17 months old NR bulls were significantly higher compared to individuals at 14 months of age. The percentage of DNA fragmented sperm cells significantly decreased in both fresh and frozen-thawed semen samples in bulls with increasing age. Libraries from the Ovation method exhibited a greater percentage of read loss and shorter read size following trimming. Downstream analyses for reads obtained from the gel-free method revealed similar global sperm DNA methylation but differentially methylated regions (DMRs) between 14- and 17 months old NR bulls. The majority of identified DMRs were hypomethylated in 14 months old bulls. Most of the identified DMRs (69%) exhibited a less than 10% methylation difference while only 1.5% of DMRs exceeded a 25% methylation difference. Pathway analysis showed that genes annotated with DMRs having low methylation differences (less than 10%) and DMRs having between 10 and 25% methylation differences, could be associated with important hormonal signaling and sperm function relevant pathways, respectively. The current research shows that RRBS in parallel with routine sperm quality analyses could be informative in reproductive capacity of young NR bulls. Although global sperm DNA methylation levels in 14 and 17 months old NR bulls were similar, regions with low and varying levels of DNA methylation differences can be identified and linked with important sperm function and hormonal pathways.