Degree Of DNA Methylation Research Articles

DNA Methylation Variation Trends during the Embryonic Development of Chicken.

The embryogenesis period is critical for epigenetic reprogramming and is thus of great significance in the research field of poultry epigenetics for elucidation of the trends in DNA methylation variations during the embryonic development of birds, particularly due to differences in embryogenesis between birds and mammals. Here, we first examined the variations in genomic DNA methylation during chicken embryogenesis through high-performance liquid chromatography using broilers as the model organism. We then identified the degree of DNA methylation of the promoters and gene bodies involved in two specific genes (IGF2 and TNF-α) using the bisulfite sequencing polymerase chain reaction method. In addition, we measured the expression levels of IGF2, TNF-α and DNA methyltransferase (DNMT) 1, 3a and 3b. Our results showed that the genomic DNA methylation levels in the liver, heart and muscle increased during embryonic development and that the methylation level of the liver was significantly higher in mid-anaphase. In both the muscle and liver, the promoter methylation levels of TNF-α first increased and then decreased, whereas the gene body methylation levels remained lower at embryonic ages E8, 11 and 14 before increasing notably at E17. The promoter methylation level of IGF2 decreased persistently, whereas the methylation levels in the gene body showed a continuous increase. No differences in the expression of TNF-α were found among E8, 11 and 14, whereas a significant increase was observed at E17. IGF2 showed increasing expression level during the examined embryonic stages. In addition, the mRNA and protein levels of DNMTs increased with increasing embryonic ages. These results suggest that chicken shows increasing genomic DNA methylation patterns during the embryonic period. Furthermore, the genomic DNA methylation levels in tissues are closely related to the genes expression levels, and gene expression may be simultaneously regulated by promoter hypomethylation and gene body hypermethylation.

Rb silencing mediated by the down-regulation of MeCP2 is involved in cell transformation induced by long-term exposure to hydroquinone.

Hydroquinone (HQ), a metabolite of benzene, is a well-known human carcinogen; however, its molecular mechanisms of action remain unclear. MeCP2 has been traditionally described as a transcriptional repressor, though growing evidence indicates that it also activates gene expression. Here, we investigated whether some epigenetic machinery genes are aberrantly expressed as target tumor suppressor genes in HQ-transformed TK6 lymphoblastoid cells. Our results showed that treatment with 5-Aza-2'-deoxycytidine or trichostatin A enhanced the expression of Rb, resulting in cell arrest in G1-phase, and subsequently, an increase in apoptosis and a decrease in cell growth. Moreover, we hypothesised that Rb was silenced by the down-regulation of MeCP2 in HQ-transformed cells, resulting in the dynamic expression of Rb and epigenetic machinery proteins in HQ-transformed cells at different time points. The expression of Rb and MeCP2 in patients with B-cell non-Hodgkin's lymphoma (B-NHL) showed that positive staining for MeCP2 or Rb was significantly lower in B-NHL tumor tissues, and these changes were significantly and negatively correlated with the grade of B-NHL. The restoration of MeCP2 in HQ-transformed cells enhanced the expression of Rb, promoted cell apoptosis, and inhibited cell growth. The changes in the expression patterns of MeCP2 and Rb were inversely correlated with the degree of DNA methylation. A ChiP assay revealed that MeCP2 proteins were recruited to the Rb promoter with lower 5'-methylcytosine levels. In conclusion, we demonstrated that the down-regulation of MeCP2 silences Rb, a process involved in cell transformation resulting from long-term exposure to HQ. © 2016 Wiley Periodicals, Inc.

The role of epigenetic modifications in cardiovascular disease: A systematic review

BackgroundEpigenetic modifications of the genome, such as DNA methylation and histone modifications, have been reported to play a role in processes underlying cardiovascular disease (CVD), including atherosclerosis, inflammation, hypertension and diabetes. MethodsEleven databases were searched for studies investigating the association between epigenetic marks (either global, site-specific or genome-wide methylation of DNA and histone modifications) and CVD. ResultsOf the 3459 searched references, 31 studies met our inclusion criteria (26 cross-sectional studies and 5 prospective studies). Overall, 12,648 individuals were included, with total of 4037 CVD events. The global DNA methylation assessed at long-interspersed nuclear element (LINE-1) was inversely associated with CVD, independent of established cardiovascular risk factors. Conversely, a higher degree of global DNA methylation measured at Alu repeats or by the LUMA method was associated with the presence of CVD. The studies reported epigenetic regulation of 34 metabolic genes (involved in fetal growth, glucose and lipid metabolism, inflammation, atherosclerosis and oxidative stress) in blood cells to be related with CVD. Among them, 5 loci were validated and methylation at F2RL3 was reported in two large prospective studies to predict cardiovascular disease beyond the traditional risk factors. ConclusionsCurrent evidence supports an association between genomic DNA methylation and CVD. However, this review highlights important gaps in the existing evidences including lack of large-scale epigenetic investigations, needed to reliably identify genomic loci where DNA methylation is related to risk of CVD.

EGTA, a calcium chelator, affects cell cycle and increases DNA methylation in root tips of Triticum aestivum L.

In this study, when germinated <em>Triticum aestivum</em> L. seeds were treated with 0, 2, 4 and 6 mM ethyl glycol tetraacetic acid (EGTA), root growth was suppressed and the mitotic index decreased. These inhibitory effects were positively correlated with EGTA concentration. RT-PCR analysis revealed that the expression of several gene markers related to the G1/S transition of the cell cycle were significantly downregulated. Confocal microscopy of Fluo-3/AM-stained roots showed chelation of nearly all of the Ca<sup>2+</sup> within the root meristematic regions. Both random amplified polymorphic DNA (RAPD) and coupled restriction enzyme digestion-random amplification (CRED-RA) techniques showed significant increases in the levels of genomic DNA polymorphisms and degree of DNA methylation. The study provides information concerning the impact of Ca<sup>2+</sup> chelator, EGTA, on the growth, expression of cell cycle transition marker genes, and changes in DNA structure and methylation in the wheat roots.

Prediction efficiency of PITX2 DNA methylation for prostate cancer survival.

This study determined the level of PITX2 methylation in prostate cancer and benign tissues and its relationship with the postoperative survival rate. Forty-four patients with prostate cancer who underwent radical prostatectomy and 43 patients with benign prostatic hyperplasia were selected. DNA was extracted from the tissues and PITX2 methylation status was quantitatively analyzed by using the EpiTect MethyLight method. The median follow-up time of the patients was 63 months and was used to analyze the relationship between PITX2 methylation status with tumor stage and survival rates. Median PITX2 gene expression in benign tissues was 1.46, which was higher than that of tumor tissues with a median of 0.01 (P < 0.001). The median methylation in the controls was less than 0.001%, while the median methylation in the test group was 23.3% (P = 0.000). The number of patients with low methylation level in T2 stage was 15, which was more than that in T3 and T4 stages (8 patients); while the number of patients with high methylation levels in T2 stage was 6, which was less than that in T3 and T4 stages (15 patients) (P = 0.035). The PITX2 gene expression level in prostate cancer tissues was lower than that in benign tissues. A higher degree of PITX2 DNA methylation was associated with higher tumor stage and lower survival rates. PITX2 DNA methylation presents a good predictive value for prostate cancer survival.

Low expression of N-myc downstream-regulated gene 2 (NDRG2) correlates with poor prognosis in hepatoblastoma

Despite tremendous progress in therapy, about 30% of patients with hepatoblastoma still succumb to the disease. Thus, the development of improved therapies as well as the identification of prognostic factors are urgently needed. In the present study, expression and promoter methylation of the N-myc downstream-regulated gene (NDRG2), a tumor suppressor gene contributing to the regulation of the Wnt signalling pathway, was analysed in 38 hepatoblastoma samples by real-time reverse transcription-PCR and pyrosequencing, respectively. The NDRG2 gene was highly expressed in normal pediatric liver tissue, but was significantly downregulated in heptoblastoma primary tumors. Detailed methylation analysis of CpG sites in the NDRG2 promoter region revealed a general high degree of DNA methylation in hepatoblastoma, which correlated with the suppression of NDRG2. By analyzing clinicopathological features we could demonstrate a strong association between low NDRG2 expression and tumor metastasis. Importantly, the overall survival analysis by Kaplan-Meier revealed that high NDRG2 expression was correlated with a higher survival rate in hepatoblastoma patients. Our data show that downregulation of NDRG2 may play an important role in advanced hepatoblastomas.

Myelodysplastic Syndrome Patients Show Mutation-Specific DNA Methylation Patterns

Introduction: Myelodysplastic syndromes (MDS) are characterized by recurrent mutations, which contribute to the classification of patients into prognostic subgroups. Besides genetic lesions, MDS cells show aberrant DNA methylation, which is used as a therapeutic target for pharmacological DNA demethylation. Genetic and epigenetic aberrations are tightly linked: The most frequently mutated genes in MDS are key players in epigenetic pathways, including DNMT3A and TET2, which directly regulate DNA methylation. Additionally, other epigenetic master regulators (EZH2 and ASXL1) are recurrently mutated. Recent data in myeloid malignancies also suggested that cases with mutations in splicing factors (e.g. SRSF2) are characterized by a specific DNA methylation signature.Aim: To describe the role of recurrent mutations in MDS as prognostic markers and elucidate the mutation-specific DNA methylation pattern.Patients and Methods: By next-generation sequencing 786 bone marrow samples were analyzed for mutations in 14 genes. Patients (488 males, 298 females) had a median age of 73 (23- 91) years. The cohort was balanced according to WHO classification subtypes, karyotype risk groups and IPSS-R risk groups. For DNA methylation analysis, 196 samples were analyzed on the 450 K platform (Illumina). Differences between groups were calculated for individual probes by t-test with IMA script; differences with an adjusted p-value <0.01 were considered significant.Results: We recently suggested four prognostic subgroups in MDS (Haferlach et al.; Leukemia; 2014), defined by combining the mutation status of 14 genes (including the aforementioned ASXL1, EZH2 and TET2) with clinical parameters. First, we re-analyzed the data set with an extended follow-up time (median 45 vs. 32 months) and confirmed separation of the subgroups regarding overall survival (OS, p<0.001) in 786 patients.Of our initial cohort, we now selected 196 cases for a comparison of DNA methylation patterns in patients with and without different mutations. In 70/196 cases (36%) with at least one TET2 mutation we identified 2% aberrant DNA methylation. In line with the function of TET2 in DNA demethylation, 90% of aberrant DNA methylation was hypermethylation. Importantly, hypermethylation almost exclusively located outside of promoters (only 3% of fraction expected by array design), and was overrepresented by twofold in regions with enhancer function. Only 6% of hypermethylated CpGs were located in CpG Islands (expected background: 31%). In contrast, in 31/196 (16%) DNMT3A mutated cases, only 0.02% abnormal DNA methylation was observed and 94% was hypomethylation, as expected by the role of DNMT3A to regulated DNA de novo methylation.In 42/196 (21%) of ASXL1 mutated cases, a methylation difference of 2% was identified with 67% hypermethylation, whereas in 13/196 (7%) EZH2 mutated cases only minute recurrent DNA methylation differences were observed (0.01%). Next, we analyzed the effect of mutated splicing factors on DNA methylation. In both 67/196 (34%) SF3B1 and 39/196 (20%) SRSF2 mutated cases, we identified strong DNA methylation differences (>3%), however with opposite direction (SF3B1: 99% hypomethylation, SRSF2: 91% hypermethylation). Finally, in 58/196 (30%) patients with an aberrant karyotype we could identify that 1% of probes showed DNA methylation changes, which was almost exclusively hypermethylation.Conclusion: 1) We confirmed the prognostic capacity of our previously suggested scoring model including the mutation status of 14 genes in MDS. 2) The mutation differences resulted in a mutation specific epigenetic signature regarding the degree, direction and localization of aberrant DNA methylation. 3) For the most frequently mutated gene in MDS, TET2, DNAhypermethylation is found outside of the classically analyzed promoter regions and CpG Islands, and enriched in enhancer regions. 4) MDS is treated with drugs that alter DNA methylation. The understanding of mutation specific DNA methylation patterns would allow to choose the right genomic loci to monitor the DNA methylation reduction under treatment. This would be a key step toward a deeper understanding of drug function or response. DisclosuresBaer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Role of Glutathione-S-Transferase Polymorphism on Arsenic Induced DNA Hypermethylation

People chronically exposed to environmental arsenic through their drinking water experience various arsenic induced clinical manifestations and there is modulation of methylation level in their genome. However the response varies widely among persons. The present manuscript deals with the evaluation of role of individual's GST status in altering the degree of DNA methylation after chronic arsenic exposure in human. To study whether Glutathione-S-Transferase (GST) gene polymorphism plays any role in this variation, a total of 92 study subjects were recruited from the villages of southern region of West Bengal, India. Concentration of arsenic in their urine and water, extent of clinical manifestation, GST status and p53 and p16 gene promoter methylation status were determined. Results showed that genetic polymorphism of GSTM1 and T1 were significantly associated (p<0.05) with degree of methylation in p53 and p16 gene promoter region and urinary arsenic in higher exposure group. Persons having null genotype have significantly decreased urinary arsenic and increased DNA methylation level relative to persons with GSTM1 or GSTT1 nonnull genotype of same arsenic exposure group. In the present work we are therefore trying to show that individual's GST polymorphic status may alter the degree of DNA methylation after arsenic exposure.

Epigenetic control of phospholipase A2 receptor expression in mammary cancer cells.

BackgroundIt has recently been proposed that the M-type phospholipase A2 receptor (PLA2R1) acts as a tumour suppressor in certain malignancies including mammary cancer. Considering that DNA methylation is an important regulator of gene transcription during carcinogenesis, in the current study we analyzed the PLA2R1 expression, PLA2R1 promoter methylation, and selected micro RNA (miRNA) levels in normal human mammary epithelial cells (HMEC) and cancer cell lines.MethodsLevels of PLA2R1 and DNA methyltransferases (DNMT) specific mRNA were determined using real-time RT-PCR. Methylation specific-high resolution melting (MS-HRM) analysis was utilized to quantify the methylation degree of selected CpG sites localized in the promoter region of the PLA2R1 gene. Expression of miRNA was tested using miScript Primer Assay system.ResultsNearly complete methylation of the analyzed PLA2R1 promoter region along with PLA2R1 gene silencing was identified in MDA-MB-453 mammary cancer cells. In MCF-7 and BT-474 mammary cancer cell lines, a higher DNA methylation degree and reduced PLA2R1 expression were found in comparison with those in normal HMEC. Synergistic effects of demethylating agent (5-aza-2′-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) on PLA2R1 transcription in MDA-MB-453 cells confirmed the importance of DNA methylation and histone modification in the regulation of the PLA2R1 gene expression in mammary cells. Furthermore, significant positive correlation between the expression of DNMT1 and PLA2R1 gene methylation and negative correlation between the cellular levels of hsa-mir-141, −181b, and -181d-1 and the expression of PLA2R1 were identified in the analyzed cells. Analysis of combined z-score of miR-23b, −154 and -302d demonstrated a strong and significant positive correlation with PLA2R1 expression.ConclusionsOur data indicate that (i) PLA2R1 expression in breast cancer cells is controlled by DNA methylation and histone modifications, (ii) hypermethylation of the PLA2R1 promoter region is associated with up-regulation of DNMT1, and (iii) hsa-miR-23b, −154, and −302d, as well as hsa-miR-141, −181b, and −181d-1 are potential candidates for post-transcriptional regulation of PLA2R1 expression in mammary cancer cells.

DNA Methylation and Methylation Polymorphism in Genetically Stable In vitro Regenerates of Jatropha curcas L. Using Methylation-Sensitive AFLP Markers.

The present investigation aimed to evaluate the degree and pattern of DNA methylation using methylation-sensitive AFLP (MS-AFLP) markers in genetically stable in vitro regenerates of Jatropha curcas L.. The genetically stable in vitro regenerates were raised through direct organogenesis via enhanced axillary shoot bud proliferation (Protocol-1) and in vitro-derived leaf regeneration (Protocol-2). Ten selective combinations of MS-AFLP primers produced 462 and 477 MS-AFLP bands in Protocol-1 (P-1) and Protocol-2 (P-2) regenerates, respectively. In P-1 regenerates, 15.8-31.17% DNA was found methylated with an average of 25.24%. In P-2 regenerates, 15.93-32.7% DNA was found methylated with an average of 24.11%. Using MS-AFLP in P-1 and P-2 regenerates, 11.52-25.53% and 13.33-25.47% polymorphism in methylated DNA was reported, respectively. Compared to the mother plant, P-1 regenerates showed hyper-methylation while P-2 showed hypo-methylation. The results clearly indicated alternation in degree and pattern of DNA methylation; hence, epigenetic instability in the genetically stable in vitro regenerates of J. curcas, developed so far using two different regeneration systems and explants of two different origins. The homologous nucleotide fragments in genomes of P-1 and P-2 regenerates showing methylation re-patterning might be involved in immediate adaptive responses and developmental processes through differential regulation of transcriptome under in vitro conditions.

Genome-wide DNA methylation profiling by modified reduced representation bisulfite sequencing in Brassica rapa suggests that epigenetic modifications play a key role in polyploid genome evolution.

Brassica rapa includes some of the most important vegetables worldwide as well as oilseed crops. The complete annotated genome sequence confirmed its paleohexaploid origins and provides opportunities for exploring the detailed process of polyploid genome evolution. We generated a genome-wide DNA methylation profile for B. rapa using a modified reduced representation bisulfite sequencing (RRBS) method. This sampling represented 2.24% of all CG loci (2.5 × 105), 2.16% CHG (2.7 × 105), and 1.68% CHH loci (1.05 × 105) (where H = A, T, or C). Our sampling of DNA methylation in B. rapa indicated that 52.4% of CG sites were present as 5mCG, with 31.8% of CHG and 8.3% of CHH. It was found that genic regions of single copy genes had significantly higher methylation compared to those of two or three copy genes. Differences in degree of genic DNA methylation were observed in a hierarchical relationship corresponding to the relative age of the three ancestral subgenomes, primarily accounted by single-copy genes. RNA-seq analysis revealed that overall the level of transcription was negatively correlated with mean gene methylation content and depended on copy number or was associated with the different subgenomes. These results provide new insights into the role epigenetic variation plays in polyploid genome evolution, and suggest an alternative mechanism for duplicate gene loss.

The Impact of Different Preservation Conditions and Freezing-Thawing Cycles on Quality of RNA, DNA, and Proteins in Cancer Tissue.

High-quality cancer tissues are essential for future research, especially molecular research. For the sake of better quality of tissues, some storage methods are chosen according to lab conditions. But the impact of different storing conditions on the quality of RNA, DNA (especially the degree of DNA methylation), and protein of tissues that have undergone a thawing process, is not clear. We analyzed the influence of different storage conditions including in RNALater solution, normal saline, Opti-mum Cutting Temperature compound (OCT), and snap frozen with no protective reagent (as control) in paired tissue samples on the quality of RNA (RNA Integrity Number value and mRNA expression), DNA quality (DNA amplification and DNA methylation degree of gene RASSF1a), and protein quality. Further, we analyzed the RNA quality of tissues that underwent three freeze-thaw cycles. The RNALater-treated group retained good RNA quality as expected on three repeated freeze-thaw cycles (RIN>8), but the snap-frozen tissues showed relatively poor results after one freeze-thaw cycle (RIN<7) and three times repeated freeze-thaw cycles (RIN<6). RNA from saline- and OCT-treated groups also yielded good results when we repeated freezing and thawing one time (RIN>7) and two times (RIN>6). The impact of different storing conditions on DNA amplification is small. However, DNA methylation and protein quality are different with different storing conditions. OCT seems to be more secure and stable compared with other two experimental groups, and show a similar trend with control group. In consideration of budget and efficiency, we suggest OCT as the best storing method that not only preserves RNA quality during the freezing-thawing process well, but also ensures more secure and stable DNA and protein.

DNA methylation as a potential diagnosis indicator for rapid discrimination of rare cancer cells and normal cells.

The global DNA methylation degree may be a ubiquitous and early biomarker to distinguish cancer cells from benign cells. However, its usefulness in clinical diagnosis was scarcely demonstrated, because the cancer cells isolated from patients were usually very rare. Even if 10 mL of peripheral blood was sampled from a patient, only tens of cancer cells could be isolated. So a method to quantify DNA methylation from small number of cells was needed to apply DNA methylation in clinical environment. In this study, we found that normal breast cell line MCF10A and breast cancer cell line MCF7 cells present significantly different percentage of genomic 5-methylcytosine (p < 0.02, n = 8), it could be a potential indicator for rapid discrimination of rare cancer cells from normal cells. However, conventional mass spectrometry needs usually ~106 cells to quantify DNA methylation degree, which was too large to be applied in clinical diagnosis. Here we developed a fast mass spectrometry-based method capable of analyzing the DNA methylation degree from only ~100 human cells. Our method could reveal the different DNA methylation degree between MCF10A and MCF7 cells in less than two hours, having the potential to provide reliable information for clinical application.

The fat cell epigenetic signature in post-obese women is characterized by global hypomethylation and differential DNA methylation of adipogenesis genes.

Obese subjects have increased number of enlarged fat cells that are reduced in size but not in number in post-obesity. We performed DNA methylation profiling in fat cells with the aim of identifying differentially methylated DNA sites (DMS) linked to adipose hyperplasia (many small fat cells) in post-obesity. Genome-wide DNA methylation was analyzed in abdominal subcutaneous fat cells from 16 women examined 2 years after gastric bypass surgery at a post-obese state (body mass index (BMI) 26±2 kg m(-2), mean±s.d.) and from 14 never-obese women (BMI 25±2 kg m(-2)). Gene expression was analyzed in subcutaneous adipose tissue from nine women in each group. In a secondary analysis, we examined DNA methylation and expression of adipogenesis genes in 15 and 11 obese women, respectively. The average degree of DNA methylation of all analyzed CpG sites was lower in fat cells from post-obese as compared with never-obese women (P=0.014). A total of 8504 CpG sites were differentially methylated in fat cells from post-obese versus never-obese women (false discovery rate 1%). DMS were under-represented in CpG islands and surrounding shores. The 8504 DMS mapped to 3717 unique genes; these genes were over-represented in cell differentiation pathways. Notably, 27% of the genes linked to adipogenesis (that is, 35 of 130) displayed DMS (adjusted P=10(-8)) in post-obese versus never-obese women. Next, we explored DNA methylation and expression of genes linked to adipogenesis in more detail in adipose tissue samples. DMS annotated to adipogenesis genes were not accompanied by differential gene expression in post-obese compared with never-obese women. In contrast, adipogenesis genes displayed differential DNA methylation accompanied by altered expression in obese women. Global CpG hypomethylation and over-representation of DMS in adipogenesis genes in fat cells may contribute to adipose hyperplasia in post-obese women.

Overexpression of mutant Ptch in rhabdomyosarcomas is associated with promoter hypomethylation and increased Gli1 and H3K4me3 occupancy.

Mice with heterozygous loss of the tumor suppressor Patched1 (Ptch) develop rhabdomyosarcoma (RMS)-like tumors. However, Ptch transcripts are consistently overexpressed in these tumors. We have recently shown that the upregulated transcripts are derived from the mutated Ptch allele thus leading to the hypothesis that the wild-type allele is repressed during RMS development. Here we describe epigenetic changes taking place at the Ptch locus during RMS development. We showed a lower degree of DNA-methylation in methylation-sensitive CpG regions of the Ptch promoter in RMS compared to normal muscle from heterozygous Ptch animals. In agreement with these results, treatment of heterozygous Ptch mice with the DNA demethylating agent 5-aza-2-deoxycytidine (5-aza-dC) between embryonic days E9.5-E11.5 significantly accelerated RMS formation. Since Ptch promoter methylation occurs after/around E13.5, the window for RMS initiation during embryogenesis, these results provide additional evidence that Ptch promoter hypomethylation may contribute to RMS formation. We have also demonstrated increased trimethylation of histone H3 lysine 4 (H3K4me3) and preferential binding of Gli1, a known Ptch activator, to the mutant locus in RMS. Together, these findings support an alternative model for RMS formation in heterozygous Ptch mice including loss of methylation and concomitant occupancy by activating histone marks of mutant Ptch.

DNA methylation dynamics at imprinted genes during bovine pre-implantation embryo development

BackgroundIn mammals, maternal differentially methylated regions (DMRs) acquire DNA methylation during the postnatal growth stage of oogenesis, with paternal DMRs acquiring DNA methylation in the perinatal prospermatagonia. Following fusion of the male and female gametes, it is widely accepted that murine DNA methylation marks at the DMRs of imprinted genes are stable through embryogenesis and early development, until they are reprogrammed in primordial germ cells. However, the DNA methylation dynamics at DMRs of bovine imprinted genes during early stages of development remains largely unknown. The objective of this investigation was to analyse the methylation dynamics at imprinted gene DMRs during bovine embryo development, from blastocyst stage until implantation.ResultsTo this end, pyrosequencing technology was used to quantify DNA methylation at DMR-associated CpG dinucleotides of six imprinted bovine genes (SNRPN, MEST, IGF2R, PLAGL1, PEG10 and H19) using bisulfite-modified genomic DNA isolated from individual blastocysts (Day 7); ovoid embryos (Day 14); filamentous embryos (Day 17) and implanting conceptuses (Day 25). For all genes, the degree of DNA methylation was most variable in Day 7 blastocysts compared to later developmental stages (P < 0.05). Furthermore, mining of RNA-seq transcriptomic data and western blot analysis revealed a specific window of expression of DNA methylation machinery genes (including DNMT3A, DNMT3B, TRIM28/KAP1 and DNMT1) and proteins (DNMT3A, DNMT3A2 and DNMT3B) by bovine embryos coincident with imprint stabilization.ConclusionThe findings of this study suggest that the DNA methylation status of bovine DMRs might be variable during the early stages of embryonic development, possibly requiring an active period of imprint stabilization.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-015-0060-2) contains supplementary material, which is available to authorized users.

Plasma Homocysteine and Asymmetrical Dimethyl-l-Arginine (ADMA) and Whole Blood DNA Methylation in Early and Neovascular Age-Related Macular Degeneration: A Pilot Study

Purpose/Aim: To compare the plasma levels of homocysteine and asymmetrical dimethyl-l-arginine (ADMA) and the degree of whole blood DNA methylation in patients with early and neovascular age-related macular degeneration (AMD) and in controls without maculopathy of any sort.Materials and methods: This observational case–control pilot study included 39 early AMD patients, 27 neovascular AMD patients and 132 sex- and age-matched controls without maculopathy. Plasma homocysteine and ADMA concentrations and the degree of whole blood DNA methylation were measured. Quantitative variables were compared by Student’s t-test or Mann–Whitney test. Logistic regression models were used to investigate the significance of the association between early or wet AMD and some variables.Results: There were no significant differences in mean plasma homocysteine and ADMA concentrations and in the degree of whole blood DNA methylation between patients with early or neovascular AMD and their controls. Similarly, logistic regression analysis disclosed that plasma homocysteine and ADMA levels were not associated with an increased risk for early or neovascular AMD.Conclusions: We failed to demonstrate an association between early or neovascular AMD and increased plasma homocysteine and/or ADMA. Results also suggest that the degree of whole blood DNA methylation is not a marker of AMD.

Examination of DNA methylation status of the ELOVL2 marker may be useful for human age prediction in forensic science.

Age estimation in forensic investigations may complement the prediction of externally visible characteristics and the inference of biogeographical ancestry, thus allowing a better description of an unknown individual. Multiple CpG sites that show linear correlation between age and degree of DNA methylation have been identified in the human genome, providing a selection of candidates for age prediction. In this study, we optimized an assay based on bisulfite conversion and pyrosequencing of 7 CpG sites located in the ELOVL2 gene. Examination of 303 blood samples collected from individuals aged 2-75 years allowed selection of the most informative site, explaining 83% of variation in age. The final linear regression model included two CpG sites in ELOVL2 and enabled age prediction with R(2)=0.859, prediction error=6.85 and mean absolute deviation MAD=5.03. Examination of a testing set of 124 blood samples (MAD=5.75) showed that 68.5% of samples were correctly predicted, assuming that chronological and predicted ages matched ± 7 years. It was found that the ELOVL2 methylation status in bloodstains had not changed significantly after 4 weeks of storage in room temperature conditions. Analysis of 45 bloodstains deposited on tissue paper after 5, 10 and 15 years of storage in room conditions indicated that although a gradual decrease of positive PCR results was observed, the general age prediction success rate remained similar and equaled 60-78%. The obtained results show that the ELOVL2 locus provides a very good source of information about human chronological age based on analysis of blood, including bloodstains, and it may constitute a powerful and reliable predictor in future forensic age estimation models.

Evaluation of informativeness of DNA methylation degree of DKK4 gene as a diagnostic criterion in breast adenocarcinoma.

Abnormalities of DNA methylation play a significant role in initiation and progression of tumors. The aim of the study was the evaluation of the quantitative determination of hypermethylation of DKK4 gene first exon as a biomarker of the breast epithelial cells malignant transformation in patients with adenocarcinoma (2-3 stage). The analysis of DNA methylation was performed by quantitative pyrosequencing using the set PSQ96MA (Qiagen, USA). It was established that DKK4 methylated DNA content in breast adenocarcinoma tissue samples significantly exceeds the same indexes in the unchanged breast tissue taken from the same patients. The average methylated DNA level in conditions of breast cancer equals to 36,8±12,63 percents whereas the same normal breast indexes equal to 19,37±7,1 percents, р≤0,01. The increased content of methylated DNA in DKK4 gene first exon was revealed in 20 samples of breast adenocarcinoma tissues out of 23 (86.96%, P ≤ 0,01); in the rest 3 samples the investigated content of methylated DNA was comparable with the same index in the normal breast tissues.

Modification of epigenetic patterns in low birth weight children: importance of hypomethylation of the ACE gene promoter.

There is a growing body of evidence that epigenetic alterations are involved in the pathological mechanisms of many chronic disorders linked to fetal programming. Angiotensin-converting enzyme (ACE) appears as one candidate gene that brings new insights into the epigenetic control and later development of diseases. In this view, we have postulated that epigenetic modifications in the ACE gene might show different interactions between birth weight (BW), blood pressure levels, plasma ACE activity and ACE I/D polymorphism. To explore this hypothesis, we performed a cross-sectional study to evaluate the DNA methylation of 3 CpG sites using pyrosequencing within the ACE gene promoter of peripheral blood leukocytes from 45 LBW children compared with 70 NBW children. Our results have revealed that LBW children have lower methylation levels (P<0.001) in parallel with a higher ACE activity (P = 0.001). Adjusting for prematurity, gender, age, body mass index, and family history of cardiovascular disease did not alter these findings. We have also performed analyses of individual CpG sites. The frequency of DNA methylation was significantly different at two CpG sites (site 1: nucleotide position +555; and site 3: nucleotide position +563). In addition, we have found a significant inverse correlation between degree of DNA methylation and both ACE activity (P<0.001) and systolic blood pressure levels (P<0.001). We also observed that the methylation level was significantly lower in LBW children who are carriers of the DD genotype compared to NBW children with DD genotype (P<0.024). In conclusion, we are able to demonstrate that the hypomethylation in the 3 CpG sites of ACE gene promoter is associated with LBW in 6 to 12 year-old children. The magnitude of these epigenetic changes appears to be clinically important, which is supported by the observation that discrete changes in DNA methylation can affect systolic blood pressure and ACE protein activity levels.