Number Of Differentially Methylated Regions Research Articles

Epigenetic modification of the hypothalamic–pituitary–adrenal (HPA) axis during development in the house sparrow (Passer domesticus)

Epigenetic modifications such as DNA methylation are important mechanisms for mediating developmental plasticity, where ontogenetic processes and their phenotypic outcomes are shaped by early environments. In particular, changes in DNA methylation of genes within the hypothalamic–pituitary–adrenal (HPA) axis can impact offspring growth and development. This relationship has been well documented in mammals but is less understood in other taxa. Here, we use target-enriched enzymatic methyl sequencing (TEEM-seq) to assess how DNA methylation in a suite of 25 genes changes over development, how these modifications relate to the early environment, and how they predict differential growth trajectories in the house sparrow (Passer domesticus). We found that DNA methylation changes dynamically over the postnatal developmental period: genes with initially low DNA methylation tended to decline in methylation over development, whereas genes with initially high DNA methylation tended to increase in methylation. However, sex-specific differentially methylated regions (DMRs) were maintained across the developmental period. We also found significant differences in post-hatching DNA methylation in relation to hatch date, with higher levels of DNA methylation in nestlings hatched earlier in the season. Although these differences were largely absent by the end of development, a number of DMRs in HPA-related genes (CRH, MC2R, NR3C1, NR3C2, POMC)—and to a lesser degree HPG-related genes (GNRHR2)—predicted nestling growth trajectories over development. These findings provide insight into the mechanisms by which the early environment shapes DNA methylation in the HPA axis, and how these changes subsequently influence growth and potentially mediate developmental plasticity.

Identification of DNA Methylation and Transcriptomic Profiles Associated With Fruit Mealiness in Prunus persica (L.) Batsch.

Peach (Prunus persica) fruits have a fast ripening process and a shelf-life of days, presenting a challenge for long-distance consuming markets. To prolong shelf-life, peach fruits are stored at low temperatures (0 to 7 °C) for at least two weeks, which can lead to the development of mealiness, a physiological disorder that reduces fruit quality and decreases consumer acceptance. Several studies have been made to understand this disorder, however, the molecular mechanisms underlying mealiness are not fully understood. Epigenetic factors, such as DNA methylation, modulate gene expression according to the genetic background and environmental conditions. In this sense, the aim of this work was to identify differentially methylated regions (DMRs) that could affect gene expression in contrasting individuals for mealiness. Peach flesh was studied at harvest time (E1 stage) and after cold storage (E3 stage) for 30 days. The distribution of DNA methylations within the eight chromosomes of P. persica showed higher methylation levels in pericentromeric regions and most differences between mealy and normal fruits were at Chr1, Chr4, and Chr8. Notably, differences in Chr4 co-localized with previous QTLs associated with mealiness. Additionally, the number of DMRs was higher in CHH cytosines of normal and mealy fruits at E3; however, most DMRs were attributed to mealy fruits from E1, increasing at E3. From RNA-Seq data, we observed that differentially expressed genes (DEGs) between normal and mealy fruits were associated with ethylene signaling, cell wall modification, lipid metabolism, oxidative stress and iron homeostasis. When integrating the annotation of DMRs and DEGs, we identified a CYP450 82A and an UDP-ARABINOSE 4 EPIMERASE 1 gene that were downregulated and hypermethylated in mealy fruits, coinciding with the co-localization of a transposable element (TE). Altogether, this study indicates that genetic differences between tolerant and susceptible individuals is predominantly affecting epigenetic regulation over gene expression, which could contribute to a metabolic alteration from earlier stages of development, resulting in mealiness at later stages. Finally, this epigenetic mark should be further studied for the development of new molecular tools in support of breeding programs.

DNA methylation dynamics underlie metamorphic gene regulation programs in Xenopus tadpole brain

Methylation of cytosine residues in DNA influences chromatin structure and gene transcription, and its regulation is crucial for brain development. There is mounting evidence that DNA methylation can be modulated by hormone signaling. We analyzed genome-wide changes in DNA methylation and their relationship to gene regulation in the brain of Xenopus tadpoles during metamorphosis, a thyroid hormone-dependent developmental process. We studied the region of the tadpole brain containing neurosecretory neurons that control pituitary hormone secretion, a region that is highly responsive to thyroid hormone action. Using Methylated DNA Capture sequencing (MethylCap-seq) we discovered a diverse landscape of DNA methylation across the tadpole neural cell genome, and pairwise stage comparisons identified several thousand differentially methylated regions (DMRs). During the pre-to pro-metamorphic period, the number of DMRs was lowest (1,163), with demethylation predominating. From pre-metamorphosis to metamorphic climax DMRs nearly doubled (2,204), with methylation predominating. The largest changes in DNA methylation were seen from metamorphic climax to the completion of metamorphosis (2960 DMRs), with 80% of the DMRs representing demethylation. Using RNA sequencing, we found negative correlations between differentially expressed genes and DMRs localized to gene bodies and regions upstream of transcription start sites. DNA demethylation at metamorphosis revealed by MethylCap-seq was corroborated by increased immunoreactivity for the DNA demethylation intermediates 5-hydroxymethylcytosine and 5-carboxymethylcytosine, and the methylcytosine dioxygenase ten eleven translocation 3 that catalyzes DNA demethylation. Our findings show that the genome of tadpole neural cells undergoes significant changes in DNA methylation during metamorphosis, and these changes likely influence chromatin architecture, and gene regulation programs occurring during this developmental period.

55 Peri-conceptional undernourishment perturbs offspring sperm methylome

Whereas an organism’s genotype is relatively static throughout life, the epigenome is highly dynamic and can adapt, or be altered, in response to the external environment such as diet. Intrauterine exposure to nutrient availability can alter the establishment of epigenetic marks, not only in the exposed individuals, but also in their offspring. Inheritance of such an environmentally acquired phenotype by the subsequent generation occurs through epigenetic modifications in the germline. Here, using a genome-wide approach, we evaluate how modification of the maternal diet pre- (14 days) and post- (28 days) conception can affect methylation status of the sperm of male offspring. Specifically, using a sheep model, we focused on the effect of maternal undernutrition on adult sperm methylation and its long-term consequences on sperm physiology and quality. Moreover, we investigated if supplementation of folic acid, to increase the availability of methyl donors, could prevent or ameliorate the adverse uterine environment caused by maternal undernutrition. Male lambs obtained from mothers subjected to different nutritional regimens (UND: undernutrition; FA: undernutrition and folic acid supplementation) appeared normal at birth, with a comparable body weight until Day 30 postpartum. Sperm DNA methylation, obtained by reduced representation bisulfite sequencing, differed in offspring that experienced in utero undernutrition (UND and FA) compared with the control group (CTR). In particular, the number of differentially methylated regions (DMR) was lower when UND and FA groups were compared, whereas a higher number of DMR was observed by comparison of CTR with both experimental groups. In addition, a high percentage of DMR were shared between UND and FA groups when compared with CTR, clearly indicating a influence of maternal nutrition on the offspring sperm DNA methylation rearrangement. Gene ontology (GO) analysis showed variation in functional categories related to sperm functionality such as chondroitin sulfate synthesis, potassium ion import, and others related to metabolism (biotin and glucagon). Furthermore, using computer-assisted semen analysis and flow cytometric measurement, we observed lower a sperm motility index and higher incidence of chromatin structure alterations in spermatozoa collected from UND and FA groups compared with CTR. Finally, to verify the effect of such reported abnormality on lamb fertility, we used the semen for in vitro embryo production. While we obtained good quality blastocysts from all 3 groups, a reduction in the percentage of embryo development, partially compensated in the FA group, was found using spermatozoa from UND rams. Taken together, our results confirm that a nutritional stress during early mammalian development can lead to epigenetic modification in the offspring. This damage can be partially ameliorated with folic acid supplementation; however, some alteration still persists in the germline and could be passed to the next generation, with as yet unknown consequences.

Effect of prenatal DHA supplementation on the infant epigenome: results from a randomized controlled trial.

BackgroundEvidence is accumulating that nutritional exposures in utero can influence health outcomes in later life. Animal studies and human epidemiological studies have implicated epigenetic modifications as playing a key role in this process, but there are limited data from large well-controlled human intervention trials.This study utilized a large double-blind randomized placebo-controlled trial to test whether a defined nutritional exposure in utero, in this case docosahexaenoic acid (DHA), could alter the infant epigenome. Pregnant mothers consumed DHA-rich fish oil (800 mg DHA/day) or placebo supplements from 20 weeks’ gestation to delivery. Blood spots were collected from the children at birth (n = 991) and blood leukocytes at 5 years (n = 667). Global DNA methylation was measured in all samples, and Illumina HumanMethylation450K BeadChip arrays were used for genome-wide methylation profiling in a subset of 369 children at birth and 65 children at 5 years.ResultsThere were no differences in global DNA methylation levels between the DHA and control group either at birth or at 5 years, but we identified 21 differentially methylated regions (DMRs) at birth, showing small DNA methylation differences (<5%) between the treatment groups, some of which seemed to persist until 5 years. The number of DMRs at birth was greater in males (127 DMRs) and in females (72 DMRs) separately, indicating a gender-specific effect.ConclusionMaternal DHA supplementation during the second half of pregnancy had small effects on DNA methylation of infants. While the potential functional significance of these changes remains to be determined, these findings further support the role of epigenetic modifications in developmental programming in humans and point the way for future studies.Trial registrationAustralian New Zealand Clinical Trials Registry (ANZCTR), ACTRN12605000569606 and ACTRN12611001127998 Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0281-7) contains supplementary material, which is available to authorized users.

Evaluation of 5-methylcytosine and 5-hydroxymethylcytosine as potential biomarkers for characterisation of chemical allergens

Epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune responses. Chemical allergens form two categories: skin sensitizing chemicals associated with allergic contact dermatitis, and chemicals that cause sensitization of the respiratory tract and occupational asthma. In mice these are characterized by different T helper (Th) cell responses. Changes in DNA methylation in particular have been implicated in the in vivo responses to chemical allergy. As such it was hypothesised that differentially methylated regions (DMR) may provide candidates biomarkers of chemical allergy To examine this, mice were exposed to 2,4-dinitrochlorobenzene (DNCB; a contact allergen) or trimellitic anhydride (TMA; a respiratory allergen). DNA from draining lymph nodes was processed for methylated (5mC) and hydroxymethylated (5hmC) DNA immunoprecipitation (MeDIP/hMeDIP) then selected DMR analysed by qPCR. We describe a number of DMRs which, by combined analysis of 5mC and 5hmC, differentiate between responses induced by DNCB and those by TMA. Furthermore, these changes in methylation are specific to the draining lymph node. The Gmpr DMR is suggested as a possible biomarker for contact allergen-induced immune responses; it is characterised by divergent levels of 5mC and 5hmC DNCB-treated mice only. In contrast, the Nwc DMR was characterised by divergent 5mC and 5hmC specifically in response to TMA, highlighting its possible utility as a biomarker for responses induced by chemical respiratory allergens. These data not only represent novel analysis of 5hmC in response to chemical allergy in vivo, but with further investigation, may also provide a possible basis for differentiation between classes of chemical allergens.

Genome-wide profiling of DNA methylation in human cancer cells

Global changes in DNA methylation correlate with altered gene expression and genomic instability in cancer. We have developed a methylation-specific digital sequencing (MSDS) method that can assess DNA methylation on a genomic scale. MSDS is a simple, low-cost method that combines the use of methylation-sensitive restriction enzymes with second generation sequencing technology. DNA methylation in two colon cancer cell lines, HT29 and HCT116, was measured using MSDS. When methylation levels were compared between the two cell lines, many differentially methylated regions (DMRs) were identified in CpG island shore regions (located within 2kb of a CpG island), gene body regions and intergenic regions. The number of DMRs in the vicinity of gene transcription start sites correlated with the level of expression of TACC1, CLDN1, and PLEKHC1 (FERMT2) genes, which have been linked to carcinogenesis. The MSDS method has the potential to provide novel insight into the functional complexity of the human genome.