Luminometric Methylation Assay Research Articles

Trophectoderm biopsy of blastocysts following IVF and embryo culture increases epigenetic dysregulation in a mouse model.

Does trophectoderm biopsy (TEBx) of blastocysts for preimplantation genetic testing in the clinic affect normal placental and embryo development and offspring metabolic outcomes in a mouse model? TEBx impacts placental and embryonic health during early development, with some alterations resolving and others worsening later in development and triggering metabolic changes in adult offspring. Previous studies have not assessed the epigenetic and morphological impacts of TEBx either in human populations or in animal models. We employed a mouse model to identify the effects of TEBx during IVF. Three groups were assessed: naturally conceived (Naturals), IVF, and IVF + TEBx, at two developmental timepoints: embryonic day (E)12.5 (n = 40/Naturals, n = 36/IVF, and n = 36/IVF + TEBx) and E18.5 (n = 42/Naturals, n = 30/IVF, and n = 35/IVF + TEBx). Additionally, to mimic clinical practice, we assessed a fourth group: IVF + TEBx + Vitrification (Vit) at E12.5 (n = 29) that combines TEBx and vitrification. To assess the effect of TEBx in offspring health, we characterized a 12-week-old cohort (n = 24/Naturals, n = 25/IVF and n = 25/IVF + TEBx). Our mouse model used CF-1 females as egg donors and SJL/B6 males as sperm donors. IVF, TEBx, and vitrification were performed using standardized methods. Placenta morphology was evaluated by hematoxylin-eosin staining, in situ hybridization using Tpbpa as a junctional zone marker and immunohistochemistry using CD34 fetal endothelial cell markers. For molecular analysis of placentas and embryos, DNA methylation was analyzed using pyrosequencing, luminometric methylation assay, and chip array technology. Expression patterns were ascertained by RNA sequencing. Triglycerides, total cholesterol, high-, low-, and very low-density lipoprotein, insulin, and glucose were determined in the 12-week-old cohort using commercially available kits. We observed that at E12.5, IVF + TEBx had a worse outcome in terms of changes in DNA methylation and differential gene expression in placentas and whole embryos compared with IVF alone and compared with Naturals. These changes were reflected in alterations in placental morphology and blood vessel density. At E18.5, early molecular changes in fetuses were maintained or exacerbated. With respect to placentas, the molecular and morphological changes, although different compared to Naturals, were equivalent to the IVF group, except for changes in blood vessel density, which persisted. Of note is that most differences were sex specific. We conclude that TEBx has more detrimental effects in mid-gestation placental and embryonic tissues, with alterations in embryonic tissues persisting or worsening in later developmental stages compared to IVF alone, and the addition of vitrification after TEBx results in more pronounced and potentially detrimental epigenetic effects: these changes are significantly different compared to Naturals. Finally, we observed that 12-week IVF + TEBx offspring, regardless of sex, showed higher glucose, insulin, triglycerides, lower total cholesterol, and lower high-density lipoprotein compared to IVF and Naturals, with only males having higher body weight compared to IVF and Naturals. Our findings in a mouse model additionally support the need for more studies to assess the impact of new procedures in ART to ensure healthy pregnancies and offspring outcomes. Data reported in this work have been deposited in the NCBI Gene Expression Omnibus under accession number GSE225318. This study was performed using a mouse model that mimics many clinical IVF procedures and outcomes observed in humans, where studies on early embryos are not possible. This study highlights the importance of assaying new procedures used in ART to assess their impact on placenta and embryo development, and offspring metabolic outcomes. This work was funded by a National Centers for Translational Research in Reproduction and Infertility grant P50 HD068157-06A1 (M.S.B., C.C., M.M.), Ruth L. Kirschstein National Service Award Individual Postdoctoral Fellowship F32 HD107914 (E.A.R.-C.) and F32 HD089623 (L.A.V.), and National Institutes of Health Training program in Cell and Molecular Biology T32 GM007229 (C.N.H.). No conflict of interest.

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.

Urinary parabens and breast cancer risk: Modification by LINE-1 and LUMA global DNA methylation, and associations with breast cancer defined by tumor promoter methylation status.

Parabens are a group of alkyl esters of p-hydroxybenzoic acid added to consumer products to prevent the growth of harmful bacteria and molds. Parabens are hypothesized to increase the risk of breast cancer (BC); however, no study has examined the interactions between parabens, global DNA methylation (DNAm), and BCrisk. We examined the modifying effects of DNAm on the associations between parabens and BC, and whether parabens were associated with BCdefined by tumor promoter methylation status. Participants included 708 cases and 598 controls from the Long Island Breast CancerStudy Project. Methylparaben (MPB), propylparaben, and butylparabenlevels were measured in spot urine samples. Global DNAm was measured by analysis of long interspersed elementes-1(LINE-1) and the luminometric methylation assay (LUMA). The promoter methylation status of 13 genes was measured in tumor samples from 509 cases. We used logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the associations between parabens and BC stratified by LINE-1/LUMA, and between parabens and gene-specific promoter methylation-defined BC. Outcome heterogeneity was evaluated using ratios of ORs (RORs). We assessed the joint effects of the multiple parabens using quantile g-computation. The highest versus lowest tertile of MPB and a one-quantile increase in all parabens were associated with ORs of 1.46 (95%CI = 0.96-2.23) and 1.32 (95%CI = 1.02-1.71), respectively, among women with hypomethylated LINE-1. A one-ln unit increase in MPB was associated with a 25% increase in the odds of hypomethylated (vs. hypermethylated) CCND2 promoter-defined BC (ROR = 1.25, 95%CI = 1.06-1.48), and a one-quantile increase in all parabens was associated with a 55% increase in the odds of hypomethylated (vs. hypermethylated) CCND2 promoter-defined BC (ROR = 1.55, 95%CI = 1.04-2.32). Exposure to parabens may increase the risk of BC among women with hypomethylated global DNAm and may increase the risk of tumors with gene-specific hypomethylated promoter regions.

Sources of variation of DNA methylation in rainbow trout: combined effects of temperature and genetic background

ABSTRACT Phenotypic plasticity is a key component of the ability of organisms to respond to changing environmental conditions. In this study, we aimed to study the establishment of DNA methylation marks in response to an environmental stress in rainbow trout and to assess whether these marks depend on the genetic background. The environmental stress chosen here was temperature, a known induction factor of epigenetic marks in fish. To disentangle the role of epigenetic mechanisms such as DNA methylation in generating phenotypic variations, nine rainbow trout isogenic lines with no genetic variability within a line were used. For each line, half of the eggs were incubated at standard temperature (11°C) and the other half at high temperature (16°C), from eyed-stage to hatching. In order to gain a first insight into the establishment of DNA methylation marks in response to an early temperature regime (control 11°C vs. heated 16°C), we have studied the expression of 8 dnmt3 (DNA methyltransferase) genes, potentially involved in de novo methylation, and analysed global DNA methylation in the different rainbow trout isogenic lines using LUMA (LUminometric Methylation Assay). Finally, finer investigation of genome-wide methylation patterns was performed using EpiRADseq, a reduced-representation library approach based on the ddRADseq (Double Digest Restriction Associated DNA) protocol, for six rainbow trout isogenic lines. We have demonstrated that thermal history during embryonic development alters patterns of DNA methylation, but to a greater or lesser extent depending on the genetic background.

Effects of vanadium (sodium metavanadate) and aflatoxin-B1 on cytochrome p450 activities, DNA damage and DNA methylation in human liver cell lines

Vanadium is considered as “possibly carcinogenic to humans” (V2O5, IARC Group 2B), yet uncertainties persist related to the toxicity mechanisms of the multiple forms of vanadium. Exposure to vanadium often co-occurs with other metals or with organic compounds that can be transformed by cytochrome p450 (CYP) enzymes into DNA-reactive carcinogens. Therefore, effects of a soluble form of vanadium (sodium metavanadate, NaVO3) and aflatoxin-B1 (AFB1) were tested separately and together, for induction of CYP activities, DNA damage (γH2AX and DNA alkaline unwinding assays), and DNA methylation changes (global genome and DNA repeats) in HepaRG or HepG2 liver cell lines. NaVO3 (≥ 2.3 μM) reduced CYP1A1 and CYP3A4 activities and induced DNA damage, butcaused important cell proliferation only in HepaRG cells. As a binary mixture, NaVO3 did not modify the effects of AFB1. There was no reproducible effect of NaVO3 (<21 μM) on DNA methylation in AluYb8, satellite-α, satellite-2, and by the luminometric methylation assay, but DNA methylation flow-cytometry signals in HepG2 cells (25–50 μM) increased at the G1 and G2 cell cycle phases. In conclusion, cell lines responded differently to NaVO3 supporting the importance of investigating more than one cell line, and a carcinogenic role of NaVO3 might reside at low concentrations by stimulating the proliferation of tumorigenic cells.

Application of the LUminometric Methylatoion Assay for plant ecological researches: the study of global DNA methylation in leaves of Elodea canadensis under laboratory conditions and in leaves of fen orchid from wild populations

The epigenetic changes in the genome of plants are one of the important regulatory mechanisms in response to the environmental factors. The LUminometric Methylation Assay (LUMA) requires a relatively small DNA amount, a short processing time and is easily adapted for species with a non-resolved genome. The LUMA has not been previously used for ecological research of plants. In this research, LUMA was used for the first time to investigate the changes of global DNA methylation under different environmental factors in the leaves of different plants. The influence of salinity on global DNA methylation was studied on aquatic macrophyte Elodea canadensis Michx, which grew in aquatic tanks under different NaCl concentrations. After the third week of growth, the HpaII/MspI ratio was measured by LUMA and global DNA methylation percentages were calculated. The results showed salt stress-induced changes in the global DNA methylation level in E.canadensis leaves, compared to control. The response was salt dose-dependent. The changes of global DNA methylation in wildlife plant populations were analogically assessed on fen orchid Liparis loeselii (L.) Rich. It was shown that global DNA methylation level was higher in leaves of these plants in Engure Lake, where there are temporary changes in water regime, compared to leaves of plants from other places. It was assumed that global GC-DNA methylation plays an essential role in the survival of this plant. Therefore, we show the possibilities of using the LUMA method for epigenetic study of different plants ecological researches.

DNA methylation stability in fish spermatozoa upon external constraint: Impact of fish hormonal stimulation and sperm cryopreservation.

Highly differentiated mature spermatozoa carry not only genetic but also epigenetic information that is to be transmitted to the embryo. DNA methylation is one epigenetic actor associated with sperm nucleus compaction, gene silencing, and prepatterning of embryonic gene expression. Therefore, the stability of this mark toward reproductive biotechnologies is a major issue in animal production. The present work explored the impact of hormonal induction of spermiation and sperm cryopreservation in two cyprinids, the goldfish (Carassius auratus) and the zebrafish (Danio rerio), using LUminometric Methylation Assay (LUMA). We showed that while goldfish hormonal treatment did increase sperm production, it did not alter global DNA methylation of spermatozoa. Different sperm samples repeatedly collected from the same males for 2 months also showed the same global DNA methylation level. Similarly, global DNA methylation was not affected after cryopreservation of goldfish spermatozoa with methanol, whereas less efficient cryoprotectants (dimethylsulfoxide and 1,2-propanediol) decreased DNA methylation. In contrast, cryopreservation of zebrafish spermatozoa with methanol induced a slight, but significant, increase in global DNA methylation. In the less compact nuclei, that is, goldfish fin somatic cells, cryopreservation did not change global DNA methylation regardless of the choice of cryoprotectant. To conclude, global DNA methylation is a robust parameter with respect to biotechnologies such as hormonal induction of spermiation and sperm cryopreservation, but it can be altered when the best sperm manipulation conditions are not met.

Prediagnosis aspirin use, DNA methylation, and mortality after breast cancer: A population‐based study

The authors hypothesized that epigenetic changes may help to clarify the underlying biologic mechanism linking aspirin use to breast cancer prognosis. To the authors' knowledge, this is the first epidemiologic study to examine whether global methylation and/or tumor promoter methylation of breast cancer-related genes interact with aspirin use to impact mortality after breast cancer. Prediagnosis aspirin use was assessed through in-person interviews within a population-based cohort of 1508 women diagnosed with a first primary breast cancer in 1996 and 1997. Global methylation in peripheral blood was assessed by long interspersed elements-1 (LINE-1) and the luminometric methylation assay. Promoter methylation of 13 breast cancer-related genes was measured in tumor by methylation-specific polymerase chain reaction and the MethyLight assay. Vital status was determined by the National Death Index through December 31, 2014 (N=202/476 breast cancer-specific/all-cause deaths identified among 1266 women with any methylation assessment and complete aspirin data). Cox proportional hazards regression was used to estimate hazard ratios (HRs) and 95% CIs, and the likelihood ratio test was used to evaluate multiplicative interactions. All-cause mortality was elevated among aspirin users who had methylated promotor of BRCA1 (HR, 1.67; 95% CI, 1.26-2.22), but not among those with unmethylated promoter of BRCA1 (HR, 0.99; 95% CI, 0.67-1.45; P for interaction ≤.05). Decreased breast cancer-specific mortality was observed among aspirin users who had unmethylated promotor of BRCA1 and PR and global hypermethylation of LINE-1 (HR, 0.60, 0.78, and 0.63, respectively; P for interaction ≤.05), although the 95% CIs included the null. The current study suggests that the LINE-1 global methylation and promoter methylation of BRCA1 and PR in tumor may interact with aspirin use to influence mortality after breast cancer.

Early life social and ecological determinants of global DNA methylation in wild spotted hyenas.

Environmental factors early in life can have lasting influence on the development and phenotypes of animals, but the underlying molecular modifications remain poorly understood. We examined cross-sectional associations among early life socioecological factors and global DNA methylation in 293 wild spotted hyenas (Crocuta crocuta) in the Masai Mara National Reserve, Kenya, grouped according to three age classes (cub, subadult and adult). Explanatory variables of interest included annual maternal rank based on outcomes of dyadic agonistic interactions, litter size, wild ungulate prey density and anthropogenic disturbance in the year each hyena was born based on counts of illegal livestock in the Reserve. The dependent variable of interest was global DNA methylation, assessed via the LUminometric Methylation Assay, which provides a percentage methylation value calculated at CCGG sites across the genome. Among cubs, we observed approximately 2.75% higher CCGG methylation in offspring born to high- than low-ranking mothers. Among cubs and subadults, higher anthropogenic disturbance corresponded with greater %CCGG methylation. In both cubs and adults, we found an inverse association between prey density measured before a hyena was 3months old and %CCGG methylation. Our results suggest that maternal rank, anthropogenic disturbance and prey availability early in life are associated with later life global DNA methylation. Future studies are required to understand the extent to which these DNA methylation patterns relate to adult phenotypes and fitness outcomes.

Multiwalled Carbon Nanotubes of Varying Size Lead to DNA Methylation Changes That Correspond to Lung Inflammation and Injury in a Mouse Model.

Diversity in physicochemical properties of engineered multiwalled carbon nanotubes (MWCNTs) increases the complexity involved in interpreting toxicity studies of these materials. Studies indicate that epigenetic changes could be at least partially involved in MWCNTs-induced pro-inflammatory and fibrotic lung pathology. Therefore, we examined distinct methylation changes in response to MWCNTs of varied sizes to identify potential epigenetic biomarkers of MWCNTs exposure and disease progression. C57BL/6 mice were exposed via oropharyngeal instillation to a single dose (50 μg) to one of three differently sized MWCNTs: "narrow short" (NS), "wide short" (WS), and "narrow long" (NL). Vehicle-treated control mice received dispersion media (DM) only. Whole lung lavage fluid (LLF) and lung tissue were collected 24 h and 7 days postexposure to evaluate pro-inflammatory cytokines, epigenetic, or histological responses at acute and subchronic intervals, respectively. Luminometric methylation assay and pyrosequencing were used to measure global DNA methylation as well as promoter methylation of inflammation and fibrosis-related genes, respectively. Pro-inflammatory cytokines, including IL-1ß, IL-6, and TNF-α, were measured using enzyme-linked immunosorbant assay, while airway thickening and interstitial collagen accumulation were measured in 7-day lung tissue using laser scanning cytometry. Distinct patterns of methylation (i.e., IL-1ß, IL-6, and TNF-α) among the different sized MWCNTs at 24 h postexposure corresponded to some pro-inflammatory cytokine measurements from whole LLF. Fibrosis-related gene, Thy-1, was significantly hypermethylated after exposures to WS and NL MWCNTs, while only NL MWCNTs induced significantly lower global DNA methylation. After 7 days, a hierarchy in airway thickness and interstitial collagen deposition was observed: NS < WS < NL. However, only airway thickness was significantly greater in the WS and NL MWCNTs-exposed groups than the DM-exposed group. These data suggest that methylation changes could be involved in the initial immune response of inflammation and tissue remodeling that precedes lung disease in response to different MWCNTs sizes.

118 Contribution of sperm methylome to bull fertility and interactions with DNA polymorphism.

Spermatozoa have a remarkable epigenome in line with their degree of specialization, their unique nature and different requirements for successful fertilization. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bovine spermatozoa is still scarce. In the context of genomic selection, more information on the epigenetic features transferred to the embryo alongside the paternal genetic heritage is necessary, in order to improve semen quality control procedures and bull fertility. We characterized the bull sperm methylome relative to bovine fibroblasts and monocytes using reduced restriction bisulfite sequencing (RRBS). A wide majority of differentially methylated cytosines (DMCs) were less methylated in sperm than in somatic cells. Consistent with previous studies in other species, these hypomethylated DMCs were enriched for genes relevant to the germline differentiation program and sperm functions. The most remarkable observation was a dramatic enrichment for repeats and particularly for satellites, which may partly account for the lower global methylation we observed in bull sperm relative to sperm from other species (LUminometric Methylation Assay). We next investigated whether fertility biomarkers could be identified in the sperm methylome. Based on field fertility indicators, we compared fertile ejaculates, subfertile ejaculates and ejaculates from bulls with a disappointing career in contradiction with genomic predictions (Montbéliarde breed, n=10/group). The median age at production was consistent among groups and the phenotypic characterization revealed no significant differences except for the proportion of motile sperm, which was reduced in disappointing bulls. We identified 4823 DMCs targeting genes involved in signaling pathways, angiogenesis and the response to growth factors. Since DNA methylation is affected by the CpG content of the genome and its alteration by DNA polymorphism, an integrative approach was conducted to identify CpGs and SNPs in correlation, which revealed both direct and indirect interactions between the genome and the epigenome.

LINE-1 hypomethylation is associated with radiation-induced genomic instability in industrial radiographers.

Global DNA hypomethylation is proposed as a potential biomarker for cancer risk associated with genomic instability, which is an important factor in radiation-induced cancer. However, the associations among radiation exposure, changes in DNA methylation, and carcinogenesis are unclear. The aims of this study were (1) to examine whether low-level occupational radiation exposure induces genomic DNA hypomethylation; and (2) to determine the relationships between radiation exposure, genomic DNA hypomethylation and radiation-induced genomic instability (RIGI) in industrial radiographers. Genomic DNA methylation levels were measured in blood DNA from 40 radiographers and 28 controls using the LINE-1 pyrosequencing assay and the luminometric methylation assay. Further, the micronucleus-centromere assay was performed to measure aneuploidy of chromosomes 1 and 4 as a marker of delayed RIGI. Genomic DNA methylation levels were significantly lower in radiographers than those in controls. LINE-1 hypomethylation was not significantly correlated with recent 1-year, recent 3-year, or total cumulative radiation doses in radiographers; however, LINE-1 hypomethylation significantly correlated with the cumulative radiation dose without recent 3-year exposure data (D3dose, r = -0.39, P < 0.05). In addition, LINE-1 hypomethylation was a significant contributor to aneuploidy frequency by D3dose (F (2, 34) = 13.85, P < 0.001), in which a total of 45% of the variance in aneuploidy frequency was explained. Our results provide suggestive evidence regarding the delayed effects of low-dose occupational radiation exposure in radiographers and its association with LINE-1 hypomethylation; however, additional studies using more subjects are needed to fully understand the relationship between genomic DNA hypomethylation and RIGI. Environ. Mol. Mutagen. 60: 174-184, 2019. © 2018 Wiley Periodicals, Inc.

Pregnancy-Induced Physiologic Adaptation of the Abdominal Aorta Is Associated with Changes in Gene Expression and Genomic Methylation

Background/Aims: Ten-eleven translocation 2 (Tet2), a DNA demethylase enzyme, has been identified as a master epigenetic regulator of vascular smooth muscle cell plasticity. We hypothesized that pregnancy will induce significant adaptive changes in aortic biomechanics that correlate with the Tet family gene expression. Methods: Abdominal aortas from pregnant and nonpregnant mice were dissected and cannulated. Intraluminal pressure was adjusted using a pressure-servo system while using a video dimension analyzer to measure the lumen diameter. Quantitative polymerase chain reaction and immunoblot was used to analyze the expression of Tet genes. Global genomic methylation was assessed with the luminometric methylation assay. Results: Compared to the nonpregnant (NP, 706 ± 8 µm) control group, the aortic luminal diameter was significantly increased in both E18.5 (836 ± 14 µm) and PP30 (889 ± 16 µm) mice. Distensibility was reduced in E18.5 (90 ± 4%) mice and returned to NP values (108 ± 2%) in PP30 (108 ± 3%) mice. Tet2 transcription decreased at the beginning of pregnancy and subsequently increased in late gestation, inversely corresponding to changes in global methylation. Conclusion: Physiologic changes in the aorta were accompanied by changes in gene expression and genomic methylation, suggesting an epigenetic component to maternal vascular remodeling during pregnancy.

Comparison of European eel sperm cryopreservation protocols with standardization as a target

The critical situation of the European eel (Anguilla anguilla) has urged the development of sperm cryopreservation protocols for reproduction in captivity and cryobanking. In the last years, two research groups have developed their own protocols in Spain and Hungary with positive results, but difficult to compare.Here, a series of experiments were conducted to test the quality of thawed sperm after using both protocols, determining which of them produce the best results and aiming for standardization. The quality of thawed sperm was assessed by studying the motility and kinetic values of thawed sperm from both cryopreservation protocols using a computer-assisted sperm analysis (CASA-Mot) system. In addition, a viability analysis was performed using flow cytometry to test if the cryoprotectants or the freezing-thawing process led to a reduction in spermatozoa survival. Furthermore, since during cryopreservation the sperm was treated with methylated cryoprotectants (DMSO or methanol) that may induce epigenetic changes in the sperm DNA (cytosine methylation) and could affect the offspring, we conducted a luminometric methylation assay (LUMA) to study the DNA methylation levels induced by both protocols.In this work, all the above-mentioned parameters were analyzed in fresh and frozen-thawed sperm samples. Our results showed that thawed sperm samples from both protocols presented lower sperm motility and velocity, and lower percentage of live cells than those shown in fresh sperm samples. Furthermore, sperm samples from the methanol based protocol showed significantly higher motility, velocity and percentage of live spermatozoa than the same sperm samples treated with the DMSO based protocol. In addition, the DMSO based protocol induced a hypomethylation of sperm DNA compared to fresh samples whereas the methanol based protocol did not alter sperm DNA methylation level. Our results indicate that the methanol based protocol is a more suitable protocol that preserves better the motility and genetic qualities of the European eel sperm.

A multi-scale analysis of bull sperm methylome revealed both species peculiarities and conserved tissue-specific features

BackgroundSpermatozoa have a remarkable epigenome in line with their degree of specialization, their unique nature and different requirements for successful fertilization. Accordingly, perturbations in the establishment of DNA methylation patterns during male germ cell differentiation have been associated with infertility in several species. While bull semen is widely used in artificial insemination, the literature describing DNA methylation in bull spermatozoa is still scarce. The purpose of this study was therefore to characterize the bull sperm methylome relative to both bovine somatic cells and the sperm of other mammals through a multiscale analysis.ResultsThe quantification of DNA methylation at CCGG sites using luminometric methylation assay (LUMA) highlighted the undermethylation of bull sperm compared to the sperm of rams, stallions, mice, goats and men. Total blood cells displayed a similarly high level of methylation in bulls and rams, suggesting that undermethylation of the bovine genome was specific to sperm. Annotation of CCGG sites in different species revealed no striking bias in the distribution of genome features targeted by LUMA that could explain undermethylation of bull sperm. To map DNA methylation at a genome-wide scale, bull sperm was compared with bovine liver, fibroblasts and monocytes using reduced representation bisulfite sequencing (RRBS) and immunoprecipitation of methylated DNA followed by microarray hybridization (MeDIP-chip). These two methods exhibited differences in terms of genome coverage, and consistently, two independent sets of sequences differentially methylated in sperm and somatic cells were identified for RRBS and MeDIP-chip. Remarkably, in the two sets most of the differentially methylated sequences were hypomethylated in sperm. In agreement with previous studies in other species, the sequences that were specifically hypomethylated in bull sperm targeted processes relevant to the germline differentiation program (piRNA metabolism, meiosis, spermatogenesis) and sperm functions (cell adhesion, fertilization), as well as satellites and rDNA repeats.ConclusionsThese results highlight the undermethylation of bull spermatozoa when compared with both bovine somatic cells and the sperm of other mammals, and raise questions regarding the dynamics of DNA methylation in bovine male germline. Whether sperm undermethylation has potential interactions with structural variation in the cattle genome may deserve further attention.

DNA methylation in adults and during development of the self-fertilizing mangrove rivulus, Kryptolebias marmoratus.

In addition to genetic variation, epigenetic mechanisms such as DNA methylation might make important contributions to heritable phenotypic diversity in populations. However, it is often difficult to disentangle the contributions of genetic and epigenetic variation to phenotypic diversity. Here, we investigated global DNA methylation and mRNA expression of the methylation‐associated enzymes during embryonic development and in adult tissues of one natural isogenic lineage of mangrove rivulus fish, Kryptolebias marmoratus. Being the best‐known self‐fertilizing hermaphroditic vertebrate affords the opportunity to work with genetically identical individuals to examine, explicitly, the phenotypic effects of epigenetic variance. Using the LUminometric Methylation Assay (LUMA), we described variable global DNA methylation at CpG sites in adult tissues, which differed significantly between hermaphrodite ovotestes and male testes (79.6% and 87.2%, respectively). After fertilization, an immediate decrease in DNA methylation occurred to 15.8% in gastrula followed by re‐establishment to 70.0% by stage 26 (liver formation). Compared to zebrafish, at the same embryonic stages, this reprogramming event seems later, deeper, and longer. Furthermore, genes putatively encoding DNA methyltransferases (DNMTs), Ten‐Eleven Translocation (TET), and MeCP2 proteins showed specific regulation in adult gonad and brain, and also during early embryogenesis. Their conserved domains and expression profiles suggest that these proteins play important roles during reproduction and development. This study raises questions about mangrove rivulus’ peculiar reprogramming period in terms of epigenetic transmission and physiological adaptation of individuals to highly variable environments. In accordance with the general‐purpose genotype model, epigenetic mechanisms might allow for the expression of diverse phenotypes among genetically identical individuals. Such phenotypes might help to overcome environmental challenges, making the mangrove rivulus a valuable vertebrate model for ecological epigenetic studies. The mangrove rivulus, Kryptolebias marmoratus, is the best‐known self‐fertilizing hermaphroditic vertebrate that allows to work with genetically identical individuals to examine, explicitly, the phenotypic effects of epigenetic variance. The reprogramming event is later, more dramatic and longer than in other described vertebrates. High evolutionary conservation and expression patterns of DNMT, TET, and MeCP2 proteins in K. marmoratus suggest biological roles for each member in gametogenesis and development.

Correlation between global methylation level of peripheral blood leukocytes and serum C reactive protein level modified by MTHFR polymorphism: a cross-sectional study

BackgroundChronic inflammatory conditions are associated with higher tumor incidence through epigenetic and genetic alterations. Here, we focused on an association between an inflammation marker, C-reactive-protein (CRP), and global DNA methylation levels of peripheral blood leukocytes.MethodsThe subjects were 384 healthy Japanese women enrolled as the control group of a case-control study for breast cancer conducted from 2001 to 2005. Global DNA methylation was quantified by Luminometric Methylation Assay (LUMA).ResultsWith adjustment for lifestyle-related factors, including folate intake, the global DNA methylation level of peripheral blood leukocytes was significantly but weakly increased by 0.43% per quartile category for CRP (P for trend = 0.010). Estimated methylation levels stratified by CRP quartile were 70.0%, 70.8%, 71.4%, and 71.3%, respectively. In addition, interaction between polymorphism of MTHFR (rs1801133, known as C677T) and CRP was significant (P for interaction = 0.046); the global methylation level was significantly increased by 0.61% per quartile category for CRP in the CT/TT group (those with the minor allele T, P for trend = 0.001), whereas no association was observed in the CC group (wild type).ConclusionsOur study suggests that CRP concentration is weakly associated with global DNA methylation level. However, this association was observed more clearly in individuals with the minor allele of the MTHFR missense SNP rs1801133. By elucidating the complex mechanism of the regulation of DNA methylation by both acquired and genetic factors, our results may be important for cancer prevention.

No association between global DNA methylation in peripheral blood and lung cancer risk in nonsmoking women: results from a multicenter study in Eastern and Central Europe.

Alterations in global DNA methylation have been suggested to play an important role in cancer development. We evaluated the association of global DNA methylation in peripheral blood with the risk of lung cancer in nonsmoking women from six countries in Central and Eastern Europe. This multicenter case-control study included primary, incident lung cancer cases diagnosed from 1998 to 2001 and controls frequency-matched for geographic area, sex, and age. Global methylation was assessed in peripheral blood DNA from 83 nonsmoking female cases and 181 nonsmoking female controls using the luminometric methylation assay (LUMA). Unconditional logistic regression models were used to estimate associations between DNA methylation in the blood and the risk of lung cancer. LUMA methylation level was not associated with the risk of lung cancer in nonsmoking women. Associations were not significantly different according to different strata of age, BMI, alcohol drinking, or second-hand tobacco smoke exposure status. In our study of nonsmoking women, the LUMA methylation level in peripheral blood was not associated with the risk of lung cancer. Our findings do not support an association of global blood DNA methylation with the risk of lung cancer in nonsmoking women.

Two-color fluorescent cytosine extension assay for the determination of global DNA methylation.

Here, we present a DNA restriction enzyme-based, fluorescent cytosine extension assay (CEA) to improve normalization and technical variation among sample-to-sample measurements. The assay includes end-labeling of parallel methylation-sensitive and methylation-insensitive DNA restriction enzyme digests along with co-purification and subsequent co-measurement of incorporated fluorescence. This non-radioactive, two-color fluorescent CEA (TCF-CEA) was shown to be a relatively rapid and accurate, with 3-fold greater precision than the one-color CEA. In addition, TCF-CEA provided an index of global DNA methylation that was sensitive to differences >5%. TCF-CEA results were highly correlated with LUminometric Methylation Assay (LUMA) results using human liver cell lines (HepG2, HepaRG, HC-04) as well as a human liver primary cell culture. Hypomethylation was observed in cells treated with the de-methylating agent 5-aza-2'-deoxycytidine. These results demonstrate that TCF-CEA provides a simple method for measuring relative degrees of global DNA methylation that could potentially be scaled up to higher-throughput formats.

Modification of the association between recreational physical activity and survival after breast cancer by promoter methylation in breast cancer-related genes

BackgroundMechanisms underlying the inverse association between physical activity and survival after breast cancer are unresolved, but DNA methylation may play a role. We hypothesized that promoter methylation of breast cancer-related genes, as well as global methylation, may modify the association between prediagnostic recreational physical activity (RPA) and breast cancer mortality.MethodsUsing a population-based sample of 1254 women diagnosed with first primary breast cancer, we examined modification of the RPA-mortality association by gene-specific promoter methylation and global methylation. Average lifetime RPA was assessed from menarche to diagnosis through structured in-home interviews. Promoter methylation of 13 breast cancer-related genes was evaluated in archived tumor by methylation-specific polymerase chain reaction and MethyLight assay. Global methylation in white blood cell DNA was determined at long interspersed nucleotide element 1 and by the luminometric methylation assay. After approximately 15 years of follow-up, 486 patients had died, and 186 of the deaths were breast cancer-related. We used Cox proportional hazards regression to estimate HRs and 95% CIs as well as likelihood ratio tests to assess multiplicative interactions.ResultsAll-cause mortality was lower only among physically active women with methylated promoter of APC (HR 0.60, 95% CI 0.40–0.80), CCND2 (HR 0.56, 95% CI 0.32–0.99), HIN (HR 0.55, 95% CI 0.38–0.80), and TWIST1 (HR 0.28, 95% CI 0.14–0.56) in tumors, but not among those with unmethylated tumors (significant interaction p < 0.05). We found no interaction between RPA and global methylation.ConclusionsThe improved survival after breast cancer that is associated with RPA may be more pronounced in women with promoter tumor methylation in biologically plausible genes.