Global DNA Methylation Levels Research Articles

Effect of hubble-bubble smoking on global DNA methylation and transcription levels of protamine and histone genes in human spermatozoa

This study was conducted to assess the impact of hubble-bubble smoking on global DNA methylation, DNA fragmentation; protamine deficiency of spermatozoa, and to determine whether the transcription levels of the protamine and histone genes are different in hubble-bubble smokers compared to nonsmokers. Five hundred semen samples were collected from males with an average age of 32.2 ± 6.1 years (300 hubble-bubble smokers “60%” and 200 nonsmokers “40%”). The nucleic acid was isolated from purified sperm, then ELISA and qPCR were used to evaluate the global DNA methylation and transcription level of protamine and histone, respectively. A significant elevation in global DNA methylation, protamine deficiency, and DNA fragmentation was found in hubble-bubble smokers compared to nonsmokers (P < 0.0001). A significant decline was shown in transcription levels of protamine and histone genes in hubble-bubble compared to nonsmokers (P < 0.0001). Additionally, a down-regulation in the transcription levels of protamine and histone was revealed in hubble-bubble compared to nonsmokers with fold change (0.0001 and 0.007, respectively). In conclusion, this study provided proof that hubble-bubble smoking has a negative impact on global DNA methylation, DNA fragmentation, protamine deficiency, and the transcription of protamine and histone genes in spermatozoa, and these findings influence negatively males’ fecundity.

Abstract C080: Neighborhood disadvantage and biological aging biomarkers among breast cancer survivors

Abstract Living in a disadvantaged neighborhood is associated with adverse outcomes among breast cancer patients, but the underlying pathway is still unclear. Limited evidence has suggested that accelerated biological aging may play an important role. In this study, we attempted to take the first step by evaluating the relationship between neighborhood disadvantage and biological aging biomarkers among breast cancer patients. Using a sub-sample of 906 women with newly diagnosed breast cancer at M.D. Anderson, we examined whether levels of selected biological aging biomarkers (allostatic load (AL), telomere length, and global DNA methylation) were affected by neighborhood disadvantage. The Area Deprivation Index (ADI) was used to measure the levels of neighborhood disadvantage. Telomere length and global DNA methylation in leukocytes were measured by qPCR and ELISA, respectively. We used 17 factors that represent the activity of five physiological systems to construct the AL score. Based on the median ADI at the national level, the study population was divided into low and high ADI groups. Overall, breast cancer patients from the high ADI group were more likely to be younger and non-Hispanic Black than those from the low ADI group (P&amp;lt;0.001, respectively). They were also more likely to have higher stage and poorly differentiated breast tumors (P=0.029 and 0.019, respectively). For the relationship with markers, compared to the low ADI group, high ADI group had higher median levels of AL (P=0.046) and lower median levels of global DNA methylation (P&amp;lt;0.001). Compared to their counterparts, those from the high ADI group were 20% more likely to have increased AL and 51% less likely to have increased levels of global DNA methylation. Additionally, we found that the significant associations of AL and global DNA methylation with ADI group were observed in non-Hispanic Whites and Blacks, but not in Hispanics. Lastly, using the mediation analysis, we found that decreased global DNA methylation mediated 6.52 and 7.98% of the association between higher ADI group with stage III and poorly differentiated tumor (P = 0.037 and 0.023), respectively. In summary, we observed that neighborhood disadvantage was associated with more aggressive breast tumor characteristics, and influenced the levels of AL and global DNA methylation among breast cancer patients. Our findings suggest that neighborhood disadvantage is biologically embedded in molecular level and associated with accelerated biological aging among breast cancer patients. Citation Format: Jie Shen, Hua Zhao, Bernard Fuemmeler, Vanessa Sheppard, Harry Bear. Neighborhood disadvantage and biological aging biomarkers among breast cancer survivors [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C080.

Evaluation of oxidative stress activity and the levels of homocysteine, vitamin B12, and DNA methylation among women with breast cancer

Objective: This study aims to identify the relationship between oxidative stress, vitamin B12, Homocysteine, and DNA methylation to evaluate their role in the progression of B.C. disease. Methodology: The 5mC global DNA methylation levels in 60 women diagnosed with breast cancer (age range 33–80 yrs.) and 30 age-matched healthy controls were assessed using Methyl Flash™ Methylated DNA Quantification Kit. Patients with B.C. were divided into two groups: group 1 consisted of stage II breast cancer women (Low level), and Group 2 consisted of patients in stage III and IV (High level). Malondialdehyde (MDA), Glutathione peroxidase (GPX), Homocysteine (HCY) and Vitamin B12 levels in the studied subjects were detected by measuring using ELISA. Results: The results showed a significant increase of HCY and MDA in breast cancer patients compared to healthy control, with apparent increases in patients with breast cancer in an advanced stage. They were accompanied by significantly reduced levels of 5mC with a positive correlation between 5mC and different stages of B.C. Also, the results showed that patients in advanced settings and those with a poor prognosis were exposed to low levels of Vit. B12 and GPX (except the patient in stage IV showed increased GPX level). Conclusion: In conclusion, our findings suggest an involvement of Glutathione peroxidase and Homocysteine in the progression of breast cancer, with the potential of utilizing the differences in the levels of global DNA methylation at the different stages as a risk factor for developing the breast cancer.

Involvement of 5mC DNA demethylation via 5-aza-2'-deoxycytidine in regulating gene expression during early somatic embryo development in white spruce (Picea glauca).

DNA methylation plays a crucial role in the development of somatic embryos (SEs) through the regulation of gene expression. To examine the impact of DNA methylation on gene expression during early SE development in Picea glauca, the demethylation reagent 5-aza-dC (5-aza-2'-deoxycytidine) was employed to modify DNA methylation regions and levels during the pre-maturation stage of somatic embryogenesis. The application of 2.0 µM 5-aza-dC did not induce toxicity to SEs in early development. Following treatment, the global DNA methylation level decreased significantly on the 7th day of pre-maturation and the 1st week of maturation. Methylated DNA immunoprecipitation (MeDIP) sequencing revealed that differentially methylated regions, as analyzed through Gene Ontology (GO), were related to plant development and reproduction and that they were hypomethylated on the 3rd day but hypermethylated on the 7th day in 5-aza-dC-treated embryogenic tissues. These findings indicate that 5-aza-dC treatment positively impacts early SE development, which was inhibited following 7 d of treatment. The expression of MSH7, JMJ14, and CalS10 was associated with DNA methylation, epigenetic regulation, and somatic embryogenesis. Further analysis of methylated regions revealed that the expression profiles of MSH7, JMJ14, and CalS10 were correlated with altered DNA methylation, suggesting DNA methylation at 5 mC may play a role in controlling the expression of these genes and regulating the early development of SEs in P. glauca. This study offers new insights into the regulation of somatic embryogenesis in conifers.

Impact of Methyl-Donor Micronutrient Supplementation on DNA Methylation Patterns: A Systematic Review and Meta-Analysis of in vitro, Animal, and Human Studies

Background: DNA methylation patterns are directly associated with diverse metabolic disorders. The status of methyl-donor micronutrients has been associated with DNA methylation levels, and altered ingestion of folate, choline, betaine, B vitamins and methionine may impact genes both globally and at the level of promoter regions. Despite this, the role of methyl-donor micronutrient supplementation on DNA methylation profiles is currently unclear. Objectives: The aims of this systematic review and meta-analysis were to identify and synthesize the evidence about methyl-donor nutrient supplementation on DNA methylation. Methods: A systematic literature search was performed in Medline, Embase, Scopus, and Web of Science databases with a combination of terms related to DNA methylation assessment, supplementation, and methyl-donor nutrients. Studies (in vitro, animal models, or human clinical trials) were included if DNA methylation levels after any kind of methyl-donor micronutrient supplementation or treatment was investigated. Studies were assessed for bias using Revised Cochrane risk-of-bias tool for randomized trials, risk-of-bias in Non-randomized Studies of Interventions or Systematic Review Centre for Laboratory Animal Experimentation tools. Data were extracted from studies measuring DNA methylation levels in any sample or tissue, following any kind of methyl-donor micronutrient supplementation or treatment. Separate random-effects meta-analyses were performed for animal model studies and human clinical trials that examined the effects of folic acid supplementation on DNA methylation. Results: Fifty-seven studies were included in this systematic review: 18 human clinical trials, 35 in animal model, and 4 in vitro studies. Concerning overall risk of bias, most of the studies were classified as “high risk” or “some concerns.” Meta-analysis with meta-regression from studies in animal models showed that folic acid dose significantly affected DNA methylation and that high and very high doses showed increases in DNA methylation when compared to low doses. However, meta-analysis of human clinical trials showed that folic acid supplementation did not promote significant changes in DNA methylation when compared to placebo. Conclusion: Folic acid supplementation may change global DNA methylation levels in animals supplemented with high, as compared to low, doses. Heterogeneity in studies and supplementation protocols make it difficult to establish clinical recommendations. However, these effects, even if small, might be of clinical importance in the management of patients with diseases related to DNA hypomethylation.

Decreased expression and DNA hypermethylation of syncytin-1 in human villus tissues with unexplained recurrent spontaneous abortion

Recurrent spontaneous abortion (RSA) affects approximately 5 % of women of reproductive age worldwide. The etiology and pathogenesis of approximately 50 % of RSA cases currently remain unclear, which known as unexplained RSA (URSA). Syncytin-1, an envelope protein encoded by HERV-W gene, is essential for human embryonic development. The purpose of this study was to explore the correlation between syncytin-1 expression and URSA occurrence. The villi tissues of URSA patients and patients with voluntary termination of pregnancy for non-medical reasons in early pregnancy (Control group) were collected. Compared with the Control group, syncytin-1 was abnormally low expressed in URSA villus tissues, and the HERV-W gene promoter was hypermethylated. Compared with the control group, the global DNA methylation level and the expression level of DNA methylases in the villus tissues of the URSA group had no significant difference. In addition, compared with the Control group, URSA villus tissue showed obviously abnormal apoptosis. Overexpression of syncytin-1 promoted the proliferation of HTR-8 cells and inhibited their apoptosis; while knockdown of syncytin-1 inhibited cell proliferation and promoted cell apoptosis. URSA villus tissue exhibited hypermethylation of the HERV-W gene and down-regulation of syncytin-1 expression. Syncytin-1 has the potential to be a predictive and diagnostic biomarker for URSA.

The Matricellular Protein SPARC Decreases in the Lacrimal Gland At Adulthood and During Inflammation.

Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein abundantly expressed in basement membranes and capsules surrounding a variety of organs and tissues. It mediates extracellular matrix organization and has been implicated in cell contraction. Here, we evaluated the expression of SPARC in the murine lacrimal gland at adulthood and during inflammation. Lacrimal glands of young mice (4-6 weeks old) and adult mice (32-40 weeks old) were used for extraction of DNA, RNA, and protein. The presence of SPARC was assessed by quantitative PCR, ELISA, and immunofluorescence microscopy. 5-Methylcytosine and DNA methylation were evaluated using ELISA and bisulfite genomic sequencing, respectively. The effects of cytokines and inflammation in Sparc expression were evaluated in vitro and in the non-obese diabetic (NOD) mouse model of Sjögren's syndrome. The mRNA and protein levels of SPARC were downregulated in lacrimal glands of mature adult mice presenting age-related histological alterations such as increased deposition of lipofuscin and lipids. Epigenetic analyses indicated that glands in adult mice contain higher levels of global DNA methylation and show increased hypermethylation of specific CpG sites within the Sparc gene promoter. Analysis of smooth muscle actin (SMA)-green fluorescent protein (GFP) transgenic mice revealed that SPARC localizes primarily to myoepithelial cells within the gland. Treatment of myoepithelial cells with IL-1β or TNF-α and the development of inflammation in the NOD mice led to decreased transcription of Sparc. SPARC is a novel matricellular glycoprotein expressed by myoepithelial cells in the lacrimal gland. Loss of SPARC during adulthood and chronic inflammation might have detrimental consequences on myoepithelial cell contraction and the secretion of tear fluid.

GLOBAL DNA METHYLATION LEVELS IN MIGRAINE PATIENTS FROM GEORGIA

Introduction: Migraine is a common, complex neurological disease characterized by the presence of a strong genetic component. Genome-wide association studies (GWAS) have identified gene variants involved in migraine development. In addition, it is known that epigenetic mechanisms, such as DNA methylation, play an important role in inflammation disorders. The aim of our study was to investigate global DNA methylation levels in migraine patients with C677T polymorphism of the MTHFR gene. Materials and methods: MTHFR C677T genotyping analysis (36 patients and 32 controls) was performed using TaqMan technology (Thermo Scientific, USA). Global DNA methylation was determined by the quantitative analysis of 5-methylcytosine (Zymo Research, Germany). Results: In individuals with TT genotypes, the percentage of methylated cytosine (5mC) was significantly lower in the migraine group compared to the control group (P=0.001). Conclusions: Our results suggest that epigenetic approaches can be used to study the migraine pathogenesis. In addition, reduced methylation levels can be ameliorated by the dietary supplementation with vitamins B6, B9, B12, which may be a strategy for migraine prevention and management in patients with MTHFR C677T polymorphism.

Young plasma transfer recovers decreased sperm counts and restores epigenetics in aged testis

Background and aimAging is one of the causes of male infertility, and abnormal global DNA methylation and imprinting defects have been characterized in testis during biological aging. One of the important emerging approaches aims to take advantage of the healing properties of young blood plasma to limit the progression of aging in various organs in the body. We aimed to show whether blood plasma transfer has an effect on DNA methylation and spermatogenetic cell development. In addition, we aimed to show whether the young plasma transfer to old mice has an effect on the rejuvenation of the old and whether the impaired DNA methylation and PCNA expression in old age can be restored. MethodsGroups were (i) young control, (ii) young plasma transfer to aged, (iii) aged control, (iv) aged plasma transfer to young. We utilized IHC and WB in protein level of Dnmts. For the global DNA methylation level, we used 5-methylcytosine staining. We also analyzed PCNA protein expressions in all groups by IHC. ResultsWe found that transfusion of young plasma into the old animal restored DNA methylation and PCNA expression as it did in the young animal. Most importantly, we observed an increase in spermatogonia and spermatid counts in older animals after young blood plasma transfer. ConclusionsOur findings show that young plasma transfer can restore epigenetic disorders that occur with aging and solve infertility problems by increasing the sperm count that decreases. It needs to be supported by different studies, especially human studies.

Association of nutritional glycaemic indices with global DNA methylation patterns: results from the Moli-sani cohort

BackgroundHigh dietary glycaemic index (GI) and load (GL) have been associated with increased risk of various cardiometabolic conditions. Among the molecular potential mechanisms underlying this relationship, DNA methylation has been studied, but a direct link between high GI and/or GL of diet and global DNA methylation levels has not been proved yet. We analyzed the associations between GI and GL and global DNA methylation patterns within an Italian population.ResultsGenomic DNA methylation (5mC) and hydroxymethylation (5hmC) levels were measured in 1080 buffy coat samples from participants of the Moli-sani study (mean(SD) = 54.9(11.5) years; 52% women) via ELISA. A 188-item Food Frequency Questionnaire was used to assess food intake and dietary GI and GL for each participant were calculated. Multiple linear regressions were used to investigate the associations between dietary GI and GL and global 5mC and 5hmC levels, as well as the proportion of effect explained by metabolic and inflammatory markers. We found negative associations of GI with both 5mC (β (SE) = − 0.073 (0.027), p = 0.007) and 5hmC (− 0.084 (0.030), p = 0.006), and of GL with 5mC (− 0.14 (0.060), p = 0.014). Circulating biomarkers did not explain the above-mentioned associations. Gender interaction analyses revealed a significant association of the gender-x-GL interaction with 5mC levels, with men showing an inverse association three times as negative as in women (interaction β (SE) = − 0.16 (0.06), p = 0.005).ConclusionsOur findings suggest that global DNA methylation and hydroxymethylation patterns represent a biomarker of carbohydrate intake. Based on the differential association of GL with 5mC between men and women, further gender-based separate approaches are warranted.

Non-additive effect of the DNA methylation inhibitor, 5-Aza-dC, and glass as a culture surface on osteogenic differentiation

The clinical need for bone regenerative solutions is expanding with increasing life expectancy and escalating incidence of accidents. Several strategies are being investigated to enhance the osteogenic differentiation of stem cells. We previously reported two different approaches for this purpose, in monolayer and three-dimensional cell culture. The first approach was based on pretreating cells with 5-Aza-dC, a DNA methylation inhibitor, before the applying the differentiation media. The second approach was based on culturing cells on a glass surface during differentiation. In this study, we investigated the potential effect of combining both methods. Our results suggested that both approaches were associated with decreasing global DNA methylation levels. Cells cultured as a monolayer on glass surface showed enhancement in alkaline phosphatase activity at day 10, while 5-Aza-dC pretreatment enhanced the activity at day 5, irrespective of the culture surface. In three-dimensional pellet culture, 5-Aza-dC pretreatment enhanced osteogenesis through Runx-2 and TGF-β1 upregulation while the glass surface induced Osterix.Furthermore, pellets cultured on glass showed upregulation of a group of miRNAs, including pro-osteogenesis miR- 20a and miR -148b and anti-osteogenesis miR -125b, miR -31, miR -138, and miR -133a. Interestingly, 5-Aza-dC was not associated with a change of miRNAs in cells cultured on tissue culture plastic but reverted the upregulated miRNAs on the glass to the basal level. This study confirms the two approaches for enhancing osteogenic differentiation and contradicts their combination.

Diabetic Hearts Exhibit Global DNA Hypermethylation That Alter the Mitochondrial Functional Genes to Enhance the Sensitivity of the Heart to Ischemia Reperfusion Injury.

A recent study has shown that DNA hypermethylation can promote ischemia reperfusion (I/R) injury by regulating the mitochondrial function. Diabetes mellitus (DM) is reported to induce DNA hypermethylation, but whether this prior DNA methylation in DM I/R heart inflicts a beneficial or detrimental effect is not known and is addressed in this study. DM was induced in 6-week-old male Wistar rats with streptozotocin (65 mg/kg b.wt). After 24 weeks on a normal diet, I/R was induced in rat heart using a Langendorff perfusion system and analyzed the myocardium for different parameters to measure hemodynamics, infarct size, DNA methylation and mitochondrial function. Diabetic heart exhibited DNA hypermethylation of 39% compared to the control, along with DNMT expression elevated by 41%. I/R induction in diabetic heart promoted further DNA hypermethylation (24%) with aggravated infarct size (21%) and reduced the cardiac rate pressure product (43%) from I/R heart. Importantly, diabetic I/R hearts also experienced a decline in the mitochondrial copy number (60%); downregulation in the expression of mitochondrial bioenergetics (ND1, ND2, ND3, ND4, ND5, ND6) and mitofusion (MFN1, MFN2) genes and the upregulation of mitophagy (PINK, PARKIN, OPTN) and mitofission (MFF, DNM1, FIS1) genes that reduce the dp/dt contribute to the contractile dysfunction in DM I/R hearts. Besides, a negative correlation was obtained between mitochondrial PGC1α, POLGA, TFAM genes and DNA hypermethylation in DM I/R hearts. Based on the above data, the elevated global DNA methylation level in diabetic I/R rat hearts deteriorated the mitochondrial function by downregulating the expression of POLGA, TFAM and PGC1α genes and negatively contributed to I/R-associated increased infarct size and altered hemodynamics.

Status of TWEAK DNA methylation and mRNA expression in systemic lupus erythematosus.

Draw upon research into the serum concentration, mRNA expression, and DNA methylation of TNF-like weak inducer of apoptosis (TWEAK) in the peripheral blood of systemic lupus erythematosus patients and healthy controls in an attempt to investigate the epigenetics associated with TWEAK in the pathogenesis of systemic lupus erythematosus (SLE). A total of 178 SLE patients (SLE group) and 131 sex-age matched healthy controls (HC group) were recruited. Enzyme-linked immunosorbent assays (ELISA) was used to detect serum protein concentration of TWEAK. TWEAK mRNA expression was analyzed by Real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Methylation levels of the promotor of TWEAK were measured using quantitative DNA methylation analysis on the MassARRAY spectrometry. Serum TWEAK concentrations were not statistically significant in SLE patients and HCs. Nevertheless, serum TWEAK concentrations were significantly lower in patients with renal involvement when compared to those without it. Serum TWEAK concentrations were reduced in clinically active patients (SLEDAI ≥ 10) compared with clinically stable patients (SLEDAI < 10). It was also significantly associated with SLEDAI. Compared with the HC group, the TWEAK mRNA expression in the SLE group was significantly lower. The global DNA methylation levels of TWEAK in the SLE group were observed to be significantly higher than the HC group. SLE patients with renal involvement, and the clinically active patients had higher TWEAK global methylation as well as exhibited variation in certain CpG island methylation. Furthermore, TWEAK methylation negatively correlated with TWEAK mRNA expression. This study suggests that TWEAK DNA methylation is a valuable as a focus for epigenetic studies because of it potentially influencing TWEAK gene expression in SLE patients. Aberrant DNA methylation of TWEAK may be involved in the initiation and development of SLE.

Methylome Analysis of Epstein-Barr Virus with Nanopore Sequencing Reveals Viral State Dynamics

Epstein Barr Virus (EBV) is a gammaherpesvirus responsible for a broad spectrum of human diseases, including hematologic malignancies and autoimmune disorders. EBV maintains lifelong infection by entering an endosomal state of latency inside its host cell. In response to external stimuli EBV undergoes lytic activation, resulting in vast gene expression changes, DNA replication, and eventually release of infectious viral particles. DNA methylation of the EBV genome is essential to regulating its life cycle. Both lytic and latent life stages are key determinants of EBV-driven disease, but current clinical assays for EBV are limited to detecting the presence of EBV and do not provide important information on EBV lytic state. Thus, making clinical decisions based on EBV detection alone has remained challenging. To address these limitations, we have developed an EBV analysis pipeline utilizing nanopore sequencing from amplification-free, native DNA that directly interrogates CpG dinucleotide DNA methylation on the EBV genome. Our workflow generates highly accurate EBV methylation calls for the majority of CpGs across the EBV genome. We also deployed a quantitative mass-spectrometry-based methylation assay (EpiTYPER) to validate our data and provide broader analysis of a representative subset of methylation sites (n=123 methylation sites from n=25 EBV loci). To initially investigate the relationship between locus-specific EBV DNA methylation and EBV gene expression, we compared DNA methylation and RNA sequencing profiles in EBV-positive cell lines (n=7). We correlated global EBV DNA methylation levels and the overall patterns of latent versus lytic EBV gene expression. We found that average methylation levels are related to the ratio of latent versus lytic gene expression, establishing nanopore-derived DNA methylation as a method to quantify latent and lytic states in a given sample. We next shifted our approach to bin nanopore reads based on the average methylation of all CpGs per read, hypothesizing that latent/lytic viral states derive from distinct subpopulations of EBV endosomes that contain methylated versus unmethylated DNA. We uncovered widespread co-occurrence of high and low methylated viral DNA in cell lines, indicating that EBV methylation states exist in defined subfractions with either present or absent DNA methylation. In cell models of latent EBV infection (Akata, Kem, MEC1, Mutu, Rael) this approach showed that methylation states dynamically responded to lytic induction and selective pressure via antiviral treatment with ganciclovir. Of note, we observed a shift in methylation without the appearance of partially methylated DNA, indicating that EBV lytic activation/replication is associated with the appearance of unmethylated EBV DNA, and not gradual demethylation of endosomes in the latent viral state. Finally, we aimed to obtain measurements of overall EBV status in patients, using nanopore sequencing of cell-free DNA (cfDNA) obtained from patient plasma. We analyzed patients diagnosed with different EBV-positive diseases from The Ohio State University Medical Center, including n=3 patients with lymphomas (Burkitt, plasmablastic and diffuse large B cell lymphoma) and n=2 patients with post-transplant lymphoproliferative disorder (PTLD). The patient samples with lymphoma displayed a near uniformly high methylation profile, with little evidence of unmethylated DNA/lytic replication. One of the PTLD patients analyzed showed a distinct bi-modal distribution of per read EBV methylation values, indicating the presence of lytic replication in a significant proportion of cells. In conclusion, nanopore sequencing analysis of methylated versus unmethylated EBV DNA subfractions is a quantitative method for determining viral states. In patient samples, nanopore sequencing revealed methylation heterogeneity in cfDNA and may be a useful advancement compared to standard clinical EBV detection. Quantifying the relative ratio of latent versus lytic virus in a liquid biopsy sample could be used to guide therapy selection for patients displaying EBV-positivity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Comparison of global DNA methylation analysis by whole genome bisulfite sequencing and the Infinium Mouse Methylation BeadChip using fresh and fresh-frozen mouse epidermis

ABSTRACT Until recently, studying the murine methylome was restricted to sequencing-based methods. In this study we compared the global DNA methylation levels of hairless mouse epidermis using the recently released Infinium Mouse Methylation BeadChip from Illumina and whole genome bisulphite sequencing (WGBS). We also studied the effect of sample storage conditions by using fresh and fresh-frozen epidermis. The DNA methylation levels of 123,851 CpG sites covered by both the BeadChip and WGBS were compared. DNA methylation levels obtained with WGBS and the BeadChip were strongly correlated (Pearson correlation r = 0.984). We applied a threshold of 15 reads for the WGBS methylation analysis. Even at a threshold of 10 reads, we observed no substantial difference in DNA methylation levels compared with that obtained with the BeadChip. The DNA methylation levels from the fresh and the fresh-frozen samples were strongly correlated when analysed with both the BeadChip (r = 0.999) and WGBS (r = 0.994). We conclude that the two methods of analysis generally work equally well for studies of DNA methylation of mouse epidermis and find that fresh and fresh-frozen epidermis can generally be used equally well. The choice of method will depend on the specific study’s aims and the available resources in the laboratory.

Gestational exposure to triphenyl phosphate induces epigenetic modifications in C57Bl/6 fetal liver.

Triphenyl phosphate (TPHP) is a chemical flame retardant and plasticizer which is added to consumer and industrial products. The developmental origins of health and disease hypothesis postulate that in utero exposures can have later-in-life effects on the developing fetus and can alter fetal gene expression. This study aimed to determine whether epigenetic modifications occurred following in utero TPHP exposure in mice and whether these changes were dose and/or sex-dependent. Pregnant C57Bl/6 mice were treated with 0, 5, 25, or 50 mg/kg of TPHP on gestational days (GD) 8, 10, 12, and 14 via intraperitoneal injection and fetal livers were collected on GD 19. Changes in the levels of acetylation of H3 and H4, as well as methylation of H3K9 and global DNA methylation were assessed in the fetal livers by western blot. Results showed that there was a significant decrease in fetal DNA methylation following in utero exposure to 50 mg/kg TPHP compared to the control (0mg/kg) independent of the sex of the fetus. While there were no significant alterations compared to controls in any histone modifications at any dose or sex following in utero TPHP exposure, we did note a decrease (t test, p= .025) in the levels of acetylated H3 in males versus females following a maternal dose of 25 mg/kg. The monomethylated H3K9 levels were also increased in females versus males following exposure to TPHP at 5mg/kg (p= .018) and 25 mg/kg (p= .027) when analyzed via unpaired t tests, although not significantly different from controls. The results suggest that gestational TPHP exposure can induce epigenetic modifications in murine fetal tissue. Specifically, global DNA methylation levels were downregulated in response to TPHP. Additionally, males appear to be more sensitive to TPHP-induced histone modifications than females. These data support the need for further studies investigating the impacts of gestational TPHP exposure on the developing fetus.

Association between serum concentrations of perfluoroalkyl substances and global DNA methylation levels in peripheral blood leukocytes of Japanese women: A cross-sectional study

Global DNA methylation levels in peripheral blood leukocytes can be a biomarker for cancer risk; however, levels can be changed by various factors such as environmental pollutants. We investigated the association between serum concentrations of perfluoroalkyl substances (PFASs) and global DNA methylation levels of leukocytes in a cross-sectional study using the control group of a Japanese breast cancer case-control study [397 women with a mean age of 54.1 (SD 10.1) years]. Importantly, our analysis distinguished branched PFAS isomers as different from linear isomers. The serum concentrations of 20 PFASs were measured by in-port arylation gas-chromatography negative chemical ionization mass spectrometry. Global DNA methylation levels in peripheral blood leukocytes were measured using a luminometric methylation assay. Associations between log10-transformed serum PFAS concentrations and global DNA methylation levels were evaluated by regression coefficients in multivariable robust linear regression analyses. Serum concentrations of 13 PFASs were significantly associated with increased global DNA methylation levels in leukocytes. Global DNA methylation was significantly increased by 1.45 %–3.96 % per log10-unit increase of serum PFAS concentration. Our results indicate that exposure to PFASs may increase global DNA methylation levels in peripheral blood leukocytes of Japanese women.

Paternal alcohol exposure has task- and sex-dependent behavioral effect in offspring.

Endophenotypes for alcohol use disorder are well known and may reflect paternal exposure effects passed down to offspring via epigenetic mechanisms. Previously, we showed that paternal alcohol exposure prior to conception attenuates the acquisition of operant alcohol self-administration. We now test whether paternal alcohol exposure alters their offsprings' behavioral responses to alcohol (endophenotypes) and global DNA methylation levels in reward-related brain regions. Adult male rats were exposed to alcohol vapors or air for 6 weeks and mated with alcohol-naïve females 8 weeks later. Adult male and female offspring of the alcohol- and control-sired litters were tested on three behaviors 30 m after gavage with water or alcohol (1.5 g/kg): open field, elevated plus maze, and accelerating rotarod. Global DNA methylation levels in sperm, nucleus accumbens, and prefrontal cortex were examined in male sires and in another group of offspring. Alcohol-sired males showed less anxiety-like behavior in the elevated plus maze that was not affected by alcohol administration. By contrast, alcohol had anxiolytic effects in the open field in male offspring only with no paternal alcohol effect. Neither paternal alcohol exposure nor alcohol administration altered locomotor activity in either sex. Sex-specific effects of paternal alcohol exposure were seen in the rotarod test. Alcohol-sired male offspring showed blunted sensitivity to the alcohol's motor-impairing effects, whereas alcohol-sired female offspring showed enhanced sensitivity. Global DNA methylation was altered in the sperm of alcohol-exposed males, but no changes were seen in their offspring. Paternal alcohol exposure prior to conception has sex- and task-dependent effects on unconditioned behaviors in their offspring.

Decitabine-induced DNA methylation-mediated transcriptomic reprogramming in human breast cancer cell lines; the impact of DCK overexpression.

Decitabine (DAC), a DNA methyltransferase (DNMT) inhibitor, is tested in combination with conventional anticancer drugs as a treatment option for various solid tumors. Although epigenome modulation provides a promising avenue in treating resistant cancer types, more studies are required to evaluate its safety and ability to normalize the aberrant transcriptional profiles. As deoxycytidine kinase (DCK)-mediated phosphorylation is a rate-limiting step in DAC metabolic activation, we hypothesized that its intracellular overexpression could potentiate DAC’s effect on cell methylome and thus increase its therapeutic efficacy. Therefore, two breast cancer cell lines, JIMT-1 and T-47D, differing in their molecular characteristics, were transfected with a DCK expression vector and exposed to low-dose DAC (approximately IC20). Although transfection resulted in a significant DCK expression increase, further enhanced by DAC exposure, no transfection-induced changes were found at the global DNA methylation level or in cell viability. In parallel, an integrative approach was applied to decipher DAC-induced, methylation-mediated, transcriptomic reprogramming. Besides large-scale hypomethylation, accompanied by up-regulation of gene expression across the entire genome, DAC also induced hypermethylation and down-regulation of numerous genes in both cell lines. Interestingly, TET1 and TET2 expression halved in JIMT-1 cells after DAC exposure, while DNMTs’ changes were not significant. The protein digestion and absorption pathway, containing numerous collagen and solute carrier genes, ranking second among membrane transport proteins, was the top enriched pathway in both cell lines when hypomethylated and up-regulated genes were considered. Moreover, the calcium signaling pathway, playing a significant role in drug resistance, was among the top enriched in JIMT-1 cells. Although low-dose DAC demonstrated its ability to normalize the expression of tumor suppressors, several oncogenes were also up-regulated, a finding, that supports previously raised concerns regarding its broad reprogramming potential. Importantly, our research provides evidence about the involvement of active demethylation in DAC-mediated transcriptional reprogramming.

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