Age group DNA methylation differences in lemon sharks (Negaprion brevirostris): Implications for future age estimation tools.

IntroductionDNA methylation is regarded as a well-defined epigenetic mechanism and involved in biological processes including ageing and cancer. The purpose of this study is to find whether DNA methylation is...

Somatic cell NT (SCNT) efficiency remains poor, preventing the technology from being regularly used in the agricultural industry. It is believed that faulty epigenetic reprogramming of SCNT embryos leads to the low overall success. A clear apoptotic signature is associated with inappropriate gene expression and epigenomic aberrancies in many experimental cell culture systems, and we hypothesised that an apoptosis biomarker could be used to effectively separate properly reprogrammed porcine SCNT embryos from those that are destined to fail due to incomplete reprogramming. Therefore, our objective was to evaluate global gene expression and DNA methylation patterns in high- and low-apoptosis individual embryos in an effort to characterise the extent of genomic reprogramming that had taken place. Porcine SCNT blastocysts on Day 6 of development were stained with a nontoxic, noninvasive caspase activity reporter, and the top and bottom 20% of detected caspase activity were classified as high and low apoptosis, respectively (3 replicate cloning sessions; n=13 embryos per group). Genomic DNA and total RNA were isolated from each individual blastocyst. The RNA sequencing libraries were prepared using the Ovation SoLo RNA-Seq system (NuGen, San Carlos, CA, USA). Reduced representation bisulfite sequencing libraries were prepared for DNA methylation analysis using a modification of the single-cell reduced representation bisulfite sequencing global DNA methylation analysis approach detailed by Guo et al. (2015 Nat. Protoc. 10, 645-59). The RNA sequencing analysis using EdgeR (https://bioconductor.org/packages/release/bioc/html/edgeR.html) revealed 175 total differentially expressed genes (fold change ≥1.5; false discovery rate ≤0.05) between the high- and low-apoptosis SCNT embryos. This list of differentially expressed genes was used to perform enrichment analysis to identify overrepresented Gene Ontology (GO) terms or Kyoto Encyclopedia of Genes and Genomes pathways (DAVID Ease version 6.8 (https://david.ncifcrf.gov/) against the Sus scrofa background genome). However, no significantly enriched GO terms or pathways were identified (false discovery rate P>0.05). Analysis of global DNA methylation patterns between high- and low-apoptosis SCNT embryos using MethylKit (Akalin et al. 2012Genome Biol. 13, R87) revealed 335 differentially methylated 100-bp regions with at least 25% difference in methylation (adjusted P ≤ 0.01). Gene transcription start sites associated with these regions were used for enrichment analysis; again, no significant enrichment of GO terms or Kyoto Encyclopedia of Genes and Genomes pathways was identified. Principal component analysis of CpG methylation showed the low-apoptosis embryos clustering more tightly than the high-apoptosis embryos, which were highly scattered. Ongoing comparisons of high- and low-apoptosis cloned embryos with naturally fertilized embryos produced invivo may provide more information about which embryos were properly reprogrammed. Although we are still pursuing a link between reprogramming and gene expression in high- and low-apoptosis embryos, we conclude that these data support a model of stochastic epigenetic reprogramming following SCNT and reinforce the necessity of identifying embryos most likely to be successful due to proper epigenetic reprogramming in order to increase SCNT efficiency.

[This corrects the article DOI: 10.1186/s13148-016-0273-7.].

Decision letter: Epigenetic conservation at gene regulatory elements revealed by non-methylated DNA profiling in seven vertebrates

Recent studies have shown a genetic influence on gene expression variation, chromatin, and DNA methylation. However, the effects of genetic background and tissue types on DNA methylation at the genome-wide level have not been characterized extensively. To study the effect of genetic background and tissue types on global DNA methylation, we performed DNA methylation analysis using the Affymetrix 500K SNP array on tumor, adjacent normal tissue, and blood DNA from 30 patients with esophageal squamous cell carcinoma (ESCC). The use of multiple tissues from 30 individuals allowed us to evaluate variation of DNA methylation states across tissues and individuals. Our results demonstrate that blood and esophageal tissues shared similar DNA methylation patterns within the same individual, suggesting an influence of genetic background on DNA methylation. Furthermore, we showed that tissue types are important contributors of DNA methylation states.

Effects of DNA Methylation on Progression to Interstitial Fibrosis and Tubular Atrophy in Renal Allograft Biopsies: A Multi-Omics Approach.

Heterogeneity and Evolution Of DNA Methylation In Chronic Lymphocytic Leukemia

Study questionIs there a difference in DNA methylation status of imprinted genes in placentas derived from IVF conceptions where embryo culture was performed in human tubal fluid (HTF) versus G5 culture medium?Summary answerWe found no statistically significant differences in the mean DNA methylation status of differentially methylated regions (DMRs) associated with parentally imprinted genes in placentas derived from IVF conceptions cultured in HTF versus G5 culture medium.What is known alreadyAnimal studies indicate that the embryo culture environment affects the DNA methylation status of the embryo. In humans, birthweight is known to be affected by the type of embryo culture medium used. The effect of embryo culture media on pregnancy, birth and child development may thus be mediated by differential methylation of parentally imprinted genes in the placenta.Study design, size, durationTo identify differential DNA methylation of imprinted genes in human placenta derived from IVF conceptions exposed to HTF or G5 embryo culture medium, placenta samples (n = 43 for HTF, n = 54 for G5) were collected between 2010 and 2012 s as part of a multi-center randomized controlled trial in the Netherlands comparing these embryo culture media. Placenta samples from 69 naturally conceived (NC) live births were collected during 2008–2013 in the Netherlands as reference material.Participants/materials, setting, methodsTo identify differential DNA methylation of imprinted genes, we opted for an amplicon-based sequencing strategy on an Illumina MiSeq sequencing platform. DNA was isolated and 34 DMRs associated with well-defined parentally imprinted genes were amplified in a two-step PCR before sequencing using MiSeq technology. Sequencing data were analyzed in a multivariate fashion to eliminate possible confounding effects.Main results and the role of chanceWe found no statistically significant differences in the mean DNA methylation status of any of the imprinted DMRs in placentas derived from IVF conceptions cultured in HTF or G5 culture medium. We also did not observe any differences in the mean methylation status per amplicon nor in the variance in methylation per amplicon between the two culture mediumgroups. A separate surrogate variable analysis also demonstrated that the IVF culture medium was not associated with the DNA methylation status of these DMRs. The mean methylation level and variance per CpG was equal between HTF and G5 placenta. Additional comparison of DNA methylation status of NC placenta samples revealed no statistically significant differences in mean amplicon and CpG methylation between G5, HTF and NC placenta; however, the number of placenta samples exhibiting outlier methylation levels was higher in IVF placenta compared to NC (P < 0.00001). Also, we were able to identify 37 CpG sites that uniquely displayed outlier methylation in G5 placentas and 32 CpG sites that uniquely displayed outlier methylation in HTF. In 8/37 (G5) and 4/32 (HTF) unique outliers CpGs, a medium-specific unique outlier could be directly correlated to outlier methylation of the entire amplicon.Limitations, reasons for cautionDue to practical reasons, not all placentas were collected during the trial, and we collected the placentas from natural conceptions from a different cohort, potentially creating bias. We limited ourselves to the DNA methylation status of 34 imprinted DMRs, and we studied only the placenta and no other embryo-derived tissues.Wider implications of the findingsIt has often been postulated, but has yet to be rigorously tested, that imprinting mediates the effects of embryo culture conditions on pregnancy, birth and child development in humans. Since we did not detect any statistically significant effects of embryo culture conditions on methylation status of imprinted genes in the placenta, this suggests that other unexplored mechanisms may underlie these effects. The biological and clinical relevance of detected outliers with respect to methylation levels of CpGs and DMR require additional analysis in a larger sample size as well. Given the importance and the growing number of children born through IVF, research into these molecular mechanisms is urgently needed.Study funding/competing interest(s)This study was funded by the March of Dimes grant number #6-FY13-153. The authors have no conflicts of interest.Trial registration numberPlacental biopsies were obtained under Netherlands Trial Registry number 1979 and 1298.

Metabolic syndrome (MetS) has been postulated to increase the risk for type 2 diabetes, cardiovascular disease and cancer. Adipose tissue (AT) plays an important role in metabolic homeostasis, and AT dysfunction has an active role in metabolic diseases. MetS is closely related to lifestyle and environmental factors. Epigenetics has emerged as an interesting landscape to evaluate the possible interconnection between AT and metabolic disease, since it can be modulated by environmental factors and metabolic status. The aim of this study was to determine whether MetS has an impact on the global DNA methylation pattern and the DNA methylation of several genes related to adipogenesis (PPARG, PPARA), lipid metabolism (RXRA, SREBF2, SREBF1, SCD, LPL, LXRb), and inflammation (LRP1 C3, LEP and TNF) in visceral adipose tissue. LPL and TNF DNA methylation values were significantly different in the control-case comparisons, with higher and lower methylation respectively in the MetS group. Negative correlations were found between global DNA methylation (measured by LINE-1 methylation levels) and the metabolic deterioration and glucose levels. There were associations among variables of MetS, BMI, and HOMA-IR with DNA methylation at several CpG positions for the studied genes. In particular, there was a strong positive association between serum triglyceride levels (TG) with PPARA and LPL methylation levels. TNF methylation was negatively associated with the metabolic worsening and could be an important factor in preventing MetS occurrence according to logistic regression analysis. Therefore, global DNA methylation and methylation at specific genes related to adipogenesis, lipid metabolism and inflammation are related to the etiology of MetS and might explain in part some of the features associated to metabolic disorders.

Nutrition has powerful impacts on our health and longevity. One of the mechanisms by which nutrition might influence our health is by inducing epigenetic modifications, modulating the molecular mechanisms that regulate aging. Observational studies have provided evidence of a relationship between nutrition and differences in DNA methylation. However, these studies are limited in that they might not provide an accurate control of the interactions between different nutrients, or between nutrition and other lifestyle behaviors. Here we systematically reviewed clinical studies examining the impact of nutrition strategies on DNA methylation. We examined clinical studies in community-dwelling adults testing the effects of nutrition interventions on i) global DNA methylation and its proxies, and ii) epigenetic clocks. We included 21 intervention studies that focused on the effects of healthy nutrition patterns, specific foods or nutrients, as well as the effect of multivitamin or multimineral supplements. In four studies on the methylation effects of healthy dietary patterns, as defined by being rich in vegetables, fruits, whole-grains, and nuts and reduced in the intake of added sugars, saturated fat, and alcohol, two of them suggested that a healthy diet, is associated with lower epigenetic age acceleration, one of them reported increases in global DNA methylation, while another one found no diet effects. Studies examining epigenetic effects of specific foods, nutrients, or mixtures of nutrients were scarce. For both folic acid and polyunsaturated fatty acids, the available independent studies produced conflicting findings. Although more evidence is still needed to draw firm conclusions, results begin to suggest that healthy dietary patterns have positive effects on DNA methylation. Additional evidence from large randomized-controlled clinical trials is needed to support the effects of healthy nutrition on the DNA methylome.

In this investigation, global DNA methylation patterns and the specific methylation status of 5 genes were studied in DNA from peripheral blood (PB) and impact on progression free survival (PFS) and overall-survival (OS) in patients with de novo or relapsed acute myeloid leukemia (AML) treated with decitabine-based regimens waas assessed. DNA was isolated from PB samples at the time of -1, 1, and 7 days of chemotherapy. Global methylation was determined by ELISA, and the CpG island DNA methylation profile of 5 genes using a DNA methylation PCR system. Our data demonstrated that patients with a high level of 5-mC had a poor prognosis after demethylation therapy and those who have low levels of 5-mC in PB achieved higher CR and better SO, but there was no significant correlation found between the 5-mC levels and other clinical features before treatment except the disease status. Higher methylation status of Sox2 and Oct4 genes was associated with differential response to demethylation therapy. A relatively low methylation percentage in one or both of these two genes was also associated with longer OS after decitabine based chemotherapy. We also suggest that global DNA and Oct-4/Sox2 methylation might impact on the pathogenesis of leukemia and play an important role in the initiation and progression. Moreover, dynamic analysis of 5-mC and Oct-4/Sox2 in peripheral blood nucleated cells of leukemia patients may provide clues to important molecular diagnostic and prognostic targets.

PurposeUveal melanoma (UM) is an aggressive malignancy, in which nearly 50% of the patients die from metastatic disease. Aberrant DNA methylation is recognized as an important epigenomic event in carcinogenesis. Formalin-fixed paraffin-embedded (FFPE) samples represent a valuable source of tumor tissue, and recent technology has enabled the use of these samples in genome-wide DNA methylation analyses. Our aim was to investigate differential DNA methylation in relation to histopathological classification and survival data. In addition we sought to identify aberrant DNA methylation of genes that could be associated with metastatic disease and poor survival. MethodsFFPE samples from UM patients (n = 23) who underwent enucleation of the eye in the period 1976–1989 were included. DNA methylation was assessed using the Illumina Infinium HumanMethylation450 array and coupled to histopathological data, Cancer Registry of Norway- (registered UM metastasis) and Norwegian Cause of Death Registry- (time and cause of death) data. Differential DNA methylation patterns contrasting histological classification, survival data and clustering properties were investigated. Survival groups were defined as “Early metastasis” (metastases and death within 2–5 years after enucleation, n = 8), “Late metastasis” (metastases and death within 9–21 years after enucleation, n = 7) and “No metastasis” (no detected metastases ≥18 years after enucleation, n = 8). A subset of samples were selected based on preliminary multi-dimensional scaling (MDS) plots, histopathological classification, chromosome 3 status, survival status and clustering properties; “Subset Early metastasis” (n = 4) vs “Subset No metastasis” (n = 4). Bioinformatics analyses were conducted in the R statistical software. Differentially methylated positions (DMPs) and differentially methylated regions (DMRs) in various comparisons were assessed. Gene expression of relevant subgroups was determined by microarray analysis and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). ResultsDNA methylation analyses identified 2 clusters that separated the samples according to chromosome 3 status. Cluster 1 consisted of samples (n = 5) with chromosome 3 disomy (D3), while Cluster 2 was comprised of samples (n = 15) with chromosome 3 monosomy (M3). 1212 DMRs and 9386 DMPs were identified in M3 vs D3. No clear clusters were formed based on our predefined survival groups (“Early”, “Late”, “No”) nor histopathological classification (Epithelioid, Mixed, Spindle). We identified significant changes in DNA methylation (beta FC ≥ 0.2, adjusted p < 0.05) between two sample subsets (n = 8). “Subset Early metastasis” (n = 4) vs “Subset No metastasis” (n = 4) identified 348 DMPs and 36 DMRs, and their differential gene expression by microarray showed that 14 DMPs and 2 DMRs corresponded to changes in gene expression (FC ≥ 1.5, p < 0.05). RNF13, ZNF217 and HYAL1 were hypermethylated and downregulated in “Subset Early metastasis” vs “Subset No metastasis” and could be potential tumor suppressors. TMEM200C, RGS10, ADAM12 and PAM were hypomethylated and upregulated in “Subset Early metastasis vs “Subset No metastasis” and could be potential oncogenes and thus markers of early metastasis and poor prognosis in UM. ConclusionsDNA methylation profiling showed differential clustering of samples according to chromosome 3 status: Cluster 1 (D3) and Cluster 2 (M3). Integrated differential DNA methylation and gene expression of two subsets of samples identified genes associated with early metastasis and poor prognosis. RNF13, ZNF217 and HYAL1 are hypermethylated and candidate tumor suppressors, while TMEM200C, RGS10, ADAM12 and PAM are hypomethylated and candidate oncogenes linked to early metastasis. UM FFPE samples represent a valuable source for methylome studies and enable long-time follow-up.

Following a variety of early environmental experiences and exposures, epigenetic modifications such as DNA methylation are proposed as candidate mechanisms that contribute to health and disease across the lifespan. Epigenetic clocks are a type of aging biomarker that can offer insight into age-related changes associated with early environmental exposures. This review provides a brief overview of epigenetic clocks that are readily available for use with perinatal and/or pediatric samples, as well as highlights some recent research that has studied the associations between early environmental chemical exposures and epigenetic aging rates. Broadly, the easily accessible epigenetic clocks can be categorized as chronological age estimators and gestational age estimators, but some clocks were developed for use with specific tissues and/or age groups. Previous environmental epidemiology studies have shown that early environmental exposures such as air pollutants and endocrine-disrupting chemicals are associated with altered epigenetic aging rates in perinatal and pediatric populations. However, more research is needed that examines how factors such as exposure level, timing of exposure, and sex may affect the direction and magnitude of associations. This review concludes with some recommendations and future directions for the use of epigenetic clocks in environmental epigenetics. Overall, epigenetic clocks are promising, non-causal biomarkers of early exposures that can be examined in relation to environmental chemicals, health and disease outcomes, and as biological mediators. Future research could help determine whether these clocks hold promise as informative biomarkers that reflect developmental epigenotoxicity following early exposure to environmental chemicals.

Accelerated aging in bipolar disorders: An exploratory study of six epigenetic clocks

The distribution of age classes is a key demographic parameter of populations and thus proper age estimation is crucial for fisheries management and for conservation biology. Age estimation in fishes has traditionally relied on the analysis of growth marks in hard structures such as otoliths. However, besides being lethal this method is time-consuming, can have low accuracy in some species and cannot be applied in others. Thus, there is a need for the development of new methods. DNA methylation is an epigenetic modification consisting in the addition of a methyl group in cytosine-guanine loci. Aging is associated with changes in DNA methylation. Among a background of global and weak genome hypomethylation, there are some loci in which age-associated DNA methylation changes are of a “clock-like” nature and thus predictable. Chronological age estimators built from DNA methylation are termed ‘epigenetic clocks’. Epigenetic clocks have been developed in the last ten years for many species, notably vertebrates, including already several fish species. Here, we review the piscine epigenetic clocks built so far and outline the major considerations to be taken into account for the development of new epigenetic clocks for additional species, which include the number of samples to be collected and tissues to be targeted. The steps on how to develop such a clock and the techniques available to do so are also discussed. Next, we focus on the features of epigenetic clocks as measuring devices, considering aspects such as accuracy, precision and reproducibility. Finally, we discuss the possibility of developing a multi-species piscine epigenetic clock and how processing automation can greatly reduce the cost per sample. One important knowledge gap is to determine how environmental changes, especially temperature and food availability, may affect the tick rate of piscine epigenetic clocks. Improved age prediction through the use of piscine epigenetic clocks can contribute to better fisheries management practices in a context of overexploited fish stocks worldwide, and in the estimation of age classes in endangered species.