Analysis of Developmental Changes in Avian DNA Methylation Using a Novel Method for Quantifying Genome-wide DNA Methylation

Although the factors responsible for the recent increase in the prevalence of diabetes worldwide are not entirely known, the morbidity associated with this disease results in substantial health and economic burden on society. Epigenetic modifications, including DNA methylation have been identified as one mechanism by which the environment interacts with the genome and there is evidence that alterations in DNA methylation may contribute to the increased prevalence of both type 1 and type 2 diabetes. This review provides a summary of DNA methylation and its role in gene regulation, and includes descriptions of various techniques to measure site-specific and genome-wide DNA methylation changes. In addition, we review current literature highlighting the complex relationship between DNA methylation, gene expression, and the development of diabetes and related complications. In studies where both DNA methylation and gene expression changes were reported, DNA methylation status had a strong inverse correlation with gene expression, suggesting that this interaction may be a potential future therapeutic target. We highlight the emerging use of genome-wide DNA methylation profiles as a biomarker to predict patients at risk of developing diabetes or specific complications of diabetes. The development of a predictive model that incorporates both genetic sequencing and DNA methylation data may be an effective diagnostic approach for all types of diabetes and could lead to additional innovative therapies.

DNA methylation programs gene expression and is involved in numerous biological processes. Accumulating evidence supports transgenerational inheritance of DNA methylation changes in mammals via germ cells. Our aim was to determine the effect of exercise on sperm DNA methylation. Twenty-four men were recruited and assigned to an exercise intervention or control group. Clinical parameters were measured and sperm samples were donated by subjects before and after the 3-month time-period. Mature sperm global and genome-wide DNA methylation was assessed using an ELISA assay and the 450K BeadChip (Illumina). Global and genome-wide sperm DNA methylation was altered after 3 months of exercise training. DNA methylation changes occurred in genes related to numerous diseases such as schizophrenia and Parkinson's disease. Our study provides the first evidence showing exercise training reprograms the sperm methylome. Whether these DNA methylation changes are inherited to future generations warrants attention.

An expanding range of genetic syndromes are characterized by genome-wide disruptions in DNA methylation profiles referred to as episignatures. Episignatures are distinct, highly sensitive, and specific biomarkers that have recently been applied in clinical diagnosis of genetic syndromes. Episignatures are contained within the broader disorder-specific genome-wide DNA methylation changes, which can share significant overlap amongdifferent conditions. In this study, we performed functional genomic assessment and comparison of disorder-specific and overlapping genome-wide DNA methylation changes related to 65 genetic syndromes with previously described episignatures. We demonstrate evidence of disorder-specific and recurring genome-wide differentially methylated probes (DMPs) and regions (DMRs). The overall distribution of DMPs and DMRs across the majority of the neurodevelopmental genetic syndromes analyzed showed substantial enrichment in gene promoters and CpG islands, and under-representation of the more variable intergenic regions. Analysis showed significant enrichment of the DMPs and DMRs in gene pathways and processes related to neurodevelopment, including neurogenesis, synaptic signaling and synaptic transmission. This study expands beyond the diagnostic utility of DNA methylation episignatures by demonstrating correlation between the function of the mutated genes and the consequent genomic DNA methylation profiles as a key functional element in the molecular etiology of genetic neurodevelopmental disorders.

Recent evidence has indicated an important role for DNA methylation in the control of gene expression, silencing of transposons and maintenance of genome stability. The current study utilised the Amplified Methylation Polymorphism (AMP) technique to investigate the extent of DNA methylation polymorphisms and their biological importance in several plant species. DNA methylation patterns were examined in response to different environments and propagation through tissue culture, and in different tissue types of one plant species. The technique was also used to investigate the extent of DNA loss in transgenic sugarcane as a result of genetic engineering procedures. Widespread DNA methylation changes were detected in response to the environment in both species investigated, namely sugarcane (a complex monocot polyploid) and Arabidopsis thaliana (the simple dicot diploid). Sugarcane cultivar Q155 grown at three diverse locations in Queensland, Australia, showed location-specific DNA methylation patterns, and when clones were moved to common glasshouses they showed new glasshouse-specific DNA methylation patterns whilst generally retaining the older location-specific DNA methylation signatures. In addition, prolonged cold treatment (vernalisation) in Arabidopsis induced DNA methylation changes. These findings indicate that DNA methylation may be a general control mechanism for mediating phenotypic responses to environmental change. Plant genome instability with passage through tissue culture was shown to vary greatly between plant species, with wheat cultivar Hartog being particularly stable in comparison to sugarcane cultivars. It was also demonstrated that the process of producing double haploids of Hartog resulted in few, if any DNA methylation or sequence changes, thereby validating this approach for producing mapping populations for mapping important traits in wheat. In sugarcane, on the other hand, genome instability in conventionally tissue culture-propagated plants was demonstrated to be a major problem. Furthermore, gene transfer and/or selection processes increased the extent of genome instability in the form of DNA loss compared to that resulting from the conventional tissue culture-propagation process alone. While genome instability in conventionally tissue culture-propagated and genetically engineered sugarcane is currently a major problem, AMP analysis of non-transgenic sugarcane plants propagated by a new direct regeneration method indicated that the prospects of overcoming this problem of genome instability in sugarcane are encouraging. AMP profiles were shown to be distinguishable between leaf and pollen DNA in lily (Lilium longiforum), however, the extent of tissue-specific DNA methylation changes was shown to be less than that reported for mammals. Throughout the present study, the AMP technique has been proven to be a robust and efficient tool for analysing plant genome instability and assessing the DNA methylation status of specific loci throughout the genome. This study has also demonstrated the potential importance of DNA methylation in the plant response mechanism to changing environments, including responses to stresses imposed by the tissue culture process. However, more studies are necessary to elucidate the environmentally-induced DNA methylation changes. In particular, the priority would be the cloning, sequencing and mapping of the environmentally-induced DNA methylation changes. In view of the fact that the Arabidopsis genome is completely sequenced, mapping of the vernalisation-induced DNA methylation markers is currently achievable in this species and could greatly enhance our understanding of vernalisation and the control of flowering time in plants. Future applications of the AMP protocol would include efficient screening for genome integrity in tissue culture-derived and genetically engineered plants, assessing the effect of tissue culture conditions and gene transfer and selection procedures on genome instability, and further identification and characterisation of genomic regions displaying environment-specific DNA methylation.

Energy-dense diets that are high in fat are associated with a risk of metabolic diseases. The underlying molecular mechanisms could involve epigenetics, as recent data show altered DNA methylation of putative type 2 diabetes candidate genes in response to high-fat diets. We examined the effect of a short-term high-fat overfeeding (HFO) diet on genome-wide DNA methylation patterns in human skeletal muscle. Skeletal muscle biopsies were obtained from 21 healthy young men after ingestion of a short-term HFO diet and a control diet, in a randomised crossover setting. DNA methylation was measured in 27,578 CpG sites/14,475 genes using Illumina's Infinium Bead Array. Candidate gene expression was determined by quantitative real-time PCR. HFO introduced widespread DNA methylation changes affecting 6,508 genes (45%), with a maximum methylation change of 13.0 percentage points. The HFO-induced methylation changes were only partly and non-significantly reversed after 6-8weeks. Alterations in DNA methylation levels primarily affected genes involved in inflammation, the reproductive system and cancer. Few gene expression changes were observed and these had poor correlation to DNA methylation. The genome-wide DNA methylation changes induced by the short-term HFO diet could have implications for our understanding of transient epigenetic regulation in humans and its contribution to the development of metabolic diseases. The slow reversibility suggests a methylation build-up with HFO, which over time may influence gene expression levels.

BackgroundProton irradiation poses a potential hazard to astronauts during and following a mission, with post-mitotic cells at most risk because they cannot dilute resultant epigenetic changes via cell division. Persistent epigenetic changes that result from environmental exposures include gains or losses of DNA methylation of cytosine, which can impact gene expression. In the present study, we compared the long-term epigenetic effects of whole body proton irradiation in the mouse hippocampus and left ventricle. We used an unbiased genome-wide DNA methylation study, involving ChIP-seq with antibodies to 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) to identify DNA regions in which methylation levels have changed 22 weeks after a single exposure to proton irradiation. We used DIP-Seq to profile changes in genome-wide DNA methylation and hydroxymethylation following proton irradiation. In addition, we used published RNAseq data to assess whether differentially methylated regions were linked to changes in gene expression.ResultsThe DNA methylation data showed tissue-dependent effects of proton irradiation and revealed significant major pathway changes in response to irradiation that are related to known pathophysiologic processes. Many regions affected in the ventricle mapped to genes involved in cardiovascular function pathways, whereas many regions affected in the hippocampus mapped to genes involved in neuronal functions. In the ventricle, increases in 5hmC were associated with decreases in 5mC. We also observed spatial overlap for regions where both epigenetic marks decreased in the ventricle. In hippocampus, increases in 5hmC were most significantly correlated (spatially) with regions that had increased 5mC, suggesting that deposition of hippocampal 5mC and 5hmC may be mechanistically coupled.ConclusionsThe results demonstrate long-term changes in DNA methylation patterns following a single proton irradiation, that these changes are tissue specific, and that they map to pathways consistent with tissue specific responses to proton irradiation. Further, the results suggest novel relationships between changes in 5mC and 5hmC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2581-x) contains supplementary material, which is available to authorized users.

Renal involvement in lupus is characterized by unique DNA methylation changes in naïve CD4+ T cells

Williams-Beuren syndrome (WBS) and 7q11.23 duplication syndrome (Dup7) are rare neurodevelopmental disorders caused by deletion and duplication of a 1.5 Mb region that includes at least five genes with a known role in epigenetic regulation. We have shown that CNV of this chromosome segment causes dose-dependent, genome-wide changes in DNA methylation, but the specific genes driving these changes are unknown. We measured genome-wide whole blood DNA methylation in six participants with atypical CNV of 7q11.23 (three with deletions and three with duplications) using the Illumina HumanMethylation450k array and compared their profiles with those from groups of individuals with classic WBS or classic Dup7 and with typically developing (TD) controls. Across the top 1000 most variable positions we found that only the atypical rearrangements that changed the copy number of GTF2IRD1 and/or GTF2I (coding for the TFII-IRD1 and TFII-I proteins) clustered with their respective syndromic cohorts. This finding was supported by results from hierarchical clustering across a selection of differentially methylated CpGs, in addition to pyrosequencing validation. These findings suggest that CNV of the GTF2I genes at the telomeric end of the 7q11.23 interval is a key contributor to the large changes in DNA methylation that are seen in blood DNA from our WBS and Dup7 cohorts, compared to TD controls. Our findings suggest that members of the TFII-I protein family are involved in epigenetic processes that alter DNA methylation on a genome-wide level.

Memory formation is a crucial task of the brain. It allows animals to dynamically respond to a changing environment by combining information about current and previous experiences. Thus, it promotes complex behaviours such as living in social groups and elaborate foraging tactics. The ability to form memories is present across the animal kingdom in a variety of forms. The honeybee - an eusocial insect - is capable of both ’simple’ associative and complex rule learning. Despite decades of research into the mechanisms of memory formation, however, much remains unknown. One little- understood aspect is the regulation of molecular mechanisms during memory formation. Understanding regulation of transcription is particularly important in this context, as transcription is a requirement for any stable memory. Epigenetic mechanisms regulate transcription by directly interacting with chromatin. Importantly, epigenetic mechanisms are conserved across species as distinct as humans and honeybees. This thesis aims to investigate one particular epigenetic mechanism - DNA methylation - and its role in honey bee memory formation by using behavioural, physiological and molecular assays.First, I studied the role of DNA methyltransferases (Dnmts), which catalyse DNA methylation, after olfactory reward learning. Bees were trained to associate an odour with a sugar reward. Dnmts were then blocked after conditioning using a pharmacological inhibitor. 24 hours later, bees were tested for memory retention and generalisation. Dnmt inhibition increased the generalisation to an odour that was not present during the training, thus impairing stimulus-specific memory. This effect was learning-dependent, as bees’ response to odours or sugar water alone did not change after treatment. Furthermore, this effect was robust against alterations in the training paradigm, but the directionality depended on the number of training trials.Next, I used Ca2+-imaging of the bees’ primary olfactory centre (i.e. antennal lobe, AL) to investigate whether Dnmts affect changes in AL processing established during memory formation. The AL is involved in odour discrimination and olfactory learning; both processes are also crucial for stimulus-specific memory formation. If Dnmts were inhibited after olfactory reward learning, the AL response to a new odour changed 48 hours later. This effect potentially serves as a functional explanation for the behavioural phenotype observed after Dnmt inhibition. Furthermore, this study provides the first in vivo evidence for Dnmt-mediated regulation of neuronal networks during memory formation.As DNA methylation regulates transcription, I next investigated the effect of Dnmt inhibition on gene expression. Using qPCR I studied 30 memory-associated genes. In response to Dnmt inhibition, 9 genes were upregulated after conditioning. For some of these genes I then investigated their methylation patterns using a bisulfite conversion based Mass spectrometry approach. Memory formation changed the methylation pattern compared to both unpaired (i.e. odour and sugar stimulation) and naive bees in these genes. Furthermore, I analysed the expression of Dnmts and Tet, which catalyses demethylation, after olfactory reward conditioning. Dnmt1b and Tet were upregulated after 1 hour, whereas Dnmt3 was only upregulated 5 hours after conditioning. Thus, Dnmts and Tet are likely active - in a specific temporal order - during a phase crucial for memory consolidation.Finally, I investigated genome-wide changes in DNA methylation and hydroxymethylation (i.e. intermediate of demethylation pathway) 24 hours after olfactory reward conditioning using MeDIP and hMeDIP sequencing. In order to connect and interpret the findings of my previous studies it is crucial to know which genomic regions Dnmts and Tet target during memory formation. Most memory-associated changes were located in the gene body; specifically, they were enriched in regionsflanking the transcription start and termination sites. This suggests a possible impact of DNA methylation and hydroxymethylation levels on transcription initiation and termination. Furthermore, transcription factors were enriched among the genes associated with changes in DNA methylation and hydroxymethylation. Thus, regulatory events are potentially multiplied by indirectly affecting genes targeted by these transcription factors.Overall, the results of this thesis reveal important novel aspects of DNA methylation and demethylation mediated regulation of memory formation in honey bees: (1) Dnmts are crucial for stimulusspecific memory formation and regulate cognitive, rather than perceptual, generalisation. (2) Dnmts regulate changes in odour processing in the primary olfactory centre during memory formation; likely promoting better discrimination between trained and novel odours. (3) Dnmts affect gene expression of a subgroup of memory-associated genes; but only in a few of these genes did methylation levels change as well, suggesting that Dnmts have a strong indirect effect on gene expression of memory-associated genes. This assumption is supported by the enrichment of transcription factors among genes associated with methylation or hydroxymethylation changes. (4) Dnmts and Tet are upregulated and thus likely active during a period crucial for memory consolidation. In conclusion, both DNA methylation and demethylation seem to play an important role in honey bee stimulusspecific memory formation by affecting the transcriptional landscape in neurons and consequently changing early odour processing and generalisation patterns.

BackgroundDistinct subpopulations of neoplastic cells within tumors, including hepatocellular carcinoma (HCC), display pronounced ability to initiate new tumors and induce metastasis. Recent evidence suggests that signals from transforming growth factor beta (TGF-β) may increase the survival of these so called tumor initiating cells leading to poor HCC prognosis. However, how TGF-β establishes and modifies the key features of these cell subpopulations is not fully understood.ResultsIn the present report we describe the differential DNA methylome of CD133-negative and CD133-expressing liver cancer cells. Next, we show that TGF-β is able to increase the proportion of CD133+ cells in liver cancer cell lines in a way that is stable and persistent across cell division. This process is associated with stable genome-wide changes in DNA methylation that persist through cell division. Differential methylation in response to TGF-β is under-represented at promoter CpG islands and enriched at gene bodies, including a locus in the body of the de novo DNA methyl-transferase DNMT3B gene. Moreover, phenotypic changes induced by TGF-β, including the induction of CD133, are impaired by siRNA silencing of de novo DNA methyl-transferases.ConclusionsOur study reveals a self-perpetuating crosstalk between TGF-β signaling and the DNA methylation machinery, which can be relevant in the establishment of cellular phenotypes. This is the first indication of the ability of TGF-β to induce genome-wide changes in DNA methylation, resulting in a stable change in the proportion of liver cancer cell subpopulations.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-435) contains supplementary material, which is available to authorized users.

A mammalian cell utilizes DNA methylation to modulate gene expression in response to environmental changes during development and differentiation. Aberrant DNA methylation changes as a correlate to diseased states like cancer, neurodegenerative conditions and cardiovascular diseases have been documented. Here we show genome-wide DNA methylation changes in macrophages infected with the pathogen M. tuberculosis. Majority of the affected genomic loci were hypermethylated in M. tuberculosis infected THP1 macrophages. Hotspots of differential DNA methylation were enriched in genes involved in immune response and chromatin reorganization. Importantly, DNA methylation changes were observed predominantly for cytosines present in non-CpG dinucleotide context. This observation was consistent with our previous finding that the mycobacterial DNA methyltransferase, Rv2966c, targets non-CpG dinucleotides in the host DNA during M. tuberculosis infection and reiterates the hypothesis that pathogenic bacteria use non-canonical epigenetic strategies during infection.

Parent specific-DNA methylation is the genomic imprint that induces mono-allelic gene expression dependent on parental origin. Resetting of DNA methylation in the germ line is mediated by a genome-wide re-methylation following demethylation known as epigenetic reprogramming. Most of our understanding of epigenetic reprogramming in germ cells is based on studies in mice, but little is known about this in marsupials. We examined genome-wide changes in DNA methylation levels by measuring 5-methylcytosine expression, and mRNA expression and protein localization of the key enzyme DNA methyltransferase 3 L (DNMT3L) during germ cell development of the marsupial tammar wallaby, Macropus eugenii. Our data clearly showed that the relative timing of genome-wide changes in DNA methylation was conserved between the tammar and mouse, but in the tammar it all occurred post-natally. In the female tammar, genome-wide demethylation occurred in two phases, I and II, suggesting that there is an unidentified demethylation mechanism in this species. Although the localization pattern of DNMT3L in male germ cells differed, the expression patterns of DNMT3L were broadly conserved between tammar, mouse and human. Thus, the basic mechanisms of DNA methylation-reprogramming must have been established before the marsupial-eutherian mammal divergence over 160 Mya.

Fructose-induced prediabetes causes persistent DNA methylation changes in white adipose tissue despite metabolic normalization.

BackgroundDietary restriction (DR), a reduction in food intake without malnutrition, increases most aspects of health during aging and extends lifespan in diverse species, including rodents. However, the mechanisms by which DR interacts with the aging process to improve health in old age are poorly understood. DNA methylation could play an important role in mediating the effects of DR because it is sensitive to the effects of nutrition and can affect gene expression memory over time.ResultsHere, we profile genome-wide changes in DNA methylation, gene expression and lipidomics in response to DR and aging in female mouse liver. DR is generally strongly protective against age-related changes in DNA methylation. During aging with DR, DNA methylation becomes targeted to gene bodies and is associated with reduced gene expression, particularly of genes involved in lipid metabolism. The lipid profile of the livers of DR mice is correspondingly shifted towards lowered triglyceride content and shorter chain length of triglyceride-associated fatty acids, and these effects become more pronounced with age.ConclusionsOur results indicate that DR remodels genome-wide patterns of DNA methylation so that age-related changes are profoundly delayed, while changes at loci involved in lipid metabolism affect gene expression and the resulting lipid profile.

Acute changes in DNA methylation in relation to 24 h personal air pollution exposure measurements: A panel study in four European countries