A Sustained Dietary Change Increases Epigenetic Variation in Isogenic Mice

Epigenetic changes can be induced by adverse environmental exposures, such as nutritional imbalance, but little is known about the nature or extent of these changes. Here we have explored the epigenomic effects of a sustained nutritional change, excess dietary methyl donors, by assessing genomic CpG methylation patterns in isogenic mice exposed for one or six generations. We find stochastic variation in methylation levels at many loci; exposure to methyl donors increases the magnitude of this variation and the number of variable loci. Several gene ontology categories are significantly overrepresented in genes proximal to these methylation-variable loci, suggesting that certain pathways are susceptible to environmental influence on their epigenetic states. Long-term exposure to the diet (six generations) results in a larger number of loci exhibiting epigenetic variability, suggesting that some of the induced changes are heritable. This finding presents the possibility that epigenetic variation within populations can be induced by environmental change, providing a vehicle for disease predisposition and possibly a substrate for natural selection.This work was supported by the Australian Research Council (DP0771859) and the National Health and Medical Research Council (#459412, #635510)

High-Resolution Analysis of Cytosine Methylation in Ancient DNA

Epigenetic changes to gene expression can result in heritable phenotypic characteristics that are not encoded in the DNA itself, but rather by biochemical modifications to the DNA or associated chromatin proteins. Interposed between genes and environment, these epigenetic modifications can be influenced by environmental factors to affect phenotype for multiple generations. This raises the possibility that epigenetic states provide a substrate for natural selection, with the potential to participate in the rapid adaptation of species to changes in environment. Any direct test of this hypothesis would require the ability to measure epigenetic states over evolutionary timescales. Here we describe the first single-base resolution of cytosine methylation patterns in an ancient mammalian genome, by bisulphite allelic sequencing of loci from late Pleistocene Bison priscus remains. Retrotransposons and the differentially methylated regions of imprinted loci displayed methylation patterns identical to those derived from fresh bovine tissue, indicating that methylation patterns are preserved in the ancient DNA. Our findings establish the biochemical stability of methylated cytosines over extensive time frames, and provide the first direct evidence that cytosine methylation patterns are retained in DNA from ancient specimens. The ability to resolve cytosine methylation in ancient DNA provides a powerful means to study the role of epigenetics in evolution

CpG Methylation of a Silent Controlling Element in the Murine Avy Allele Is Incomplete and Unresponsive to Methyl Donor Supplementation

Background: The viable yellow allele of agouti (A vy) is remarkable for its unstable and partially heritable epigenetic state, which produces wide variation in phenotypes of isogenic mice. In the A vy allele an inserted intracisternal A particle (IAP) acts as a controlling element which deregulates expression of agouti by transcription from the LTR of the IAP; the phenotypic state has been linked to CpG methylation of the LTR. Phenotypic variation between A vy mice indicates that the epigenetic state of the IAP is unstable in the germline. Principal Findings: We have made a detailed examination of somatic methylation of the IAP using bisulphite allelic sequencing, and find that the promoter is incompletely methylated even when it is transcriptionally silent. In utero exposure to supplementary methyl donors, which alters the spectrum of A vy phenotypes, does not increase the density of CpG methylation in the silent LTR. Conclusions: Our findings suggest that, contrary to previous supposition, methyl donor supplementation acts through an indirect mechanism to silence A vy. The incomplete cytosine methylation we observe at the somatically silent A vy allele ma

Cytosine Methylation Dysregulation in Neonates Following Intrauterine Growth Restriction

Perturbations of the intrauterine environment can affect fetal development during critical periods of plasticity, and can increase susceptibility to a number of age-related diseases (e.g., type 2 diabetes mellitus; T2DM), manifesting as late as decades later. We hypothesized that this biological memory is mediated by permanent alterations of the epigenome in stem cell populations, and focused our studies specifically on DNA methylation in CD34+ hematopoietic stem and progenitor cells from cord blood from neonates with intrauterine growth restriction (IUGR) and control subjects.Our epigenomic assays utilized a two-stage design involving genome-wide discovery followed by quantitative, single-locus validation. We found that changes in cytosine methylation occur in response to IUGR of moderate degree and involving a restricted number of loci. We also identify specific loci that are targeted for dysregulation of DNA methylation, in particular the hepatocyte nuclear factor 4alpha (HNF4A) gene, a well-known diabetes candidate gene not previously associated with growth restriction in utero, and other loci encoding HNF4A-interacting proteins.Our results give insights into the potential contribution of epigenomic dysregulation in mediating the long-term consequences of IUGR, and demonstrate the value of this approach to studies of the fetal origin of adult disease

Complexity of murine cardiomyocyte miRNA biogenesis, sequence variant expression and function

microRNAs (miRNAs) are critical to heart development and disease. Emerging research indicates that regulated precursor processing can give rise to an unexpected diversity of miRNA variants. We subjected small RNA from murine HL-1 cardiomyocyte cells to next generation sequencing to investigate the relevance of such diversity to cardiac biology. ∼40 million tags were mapped to known miRNA hairpin sequences as deposited in miRBase version 16, calling 403 generic miRNAs as appreciably expressed. Hairpin arm bias broadly agreed with miRBase annotation, although 44 miR* were unexpectedly abundant (>20% of tags); conversely, 33 -5p/-3p annotated hairpins were asymmetrically expressed. Overall, variability was infrequent at the 5' start but common at the 3' end of miRNAs (5.2% and 52.3% of tags, respectively). Nevertheless, 105 miRNAs showed marked 5' isomiR expression (>20% of tags). Among these was miR-133a, a miRNA with important cardiac functions, and we demonstrated differential mRNA targeting by two of its prevalent 5' isomiRs. Analyses of miRNA termini and base-pairing patterns around Drosha and Dicer cleavage regions confirmed the known bias towards uridine at the 5' most position of miRNAs, as well as supporting the thermodynamic asymmetry rule for miRNA strand selection and a role for local structural distortions in fine tuning miRNA processing. We further recorded appreciable expression of 5 novel miR*, 38 extreme variants and 8 antisense miRNAs. Analysis of genome-mapped tags revealed 147 novel candidate miRNAs. In summary, we revealed pronounced sequence diversity among cardiomyocyte miRNAs, knowledge of which will underpin future research into the mechanisms involved in miRNA biogenesis and, importantly, cardiac function, disease and therapy.This work was supported by by the Victor Chang Cardiac Research Institute and grants 573726, 573731 and 514904 from the National Health & Medical Research Council awarded to TP

Role for DNA Methylation in the Regulation of miR-200c and miR-141 Expression in Normal and Cancer Cells

The microRNA-200 family participates in the maintenance of an epithelial phenotype and loss of its expression can result in epithelial to mesenchymal transition (EMT). Furthermore, the loss of expression of miR-200 family members is linked to an aggressive cancer phenotype. Regulation of the miR-200 family expression in normal and cancer cells is not fully understood.Epigenetic mechanisms participate in the control of miR-200c and miR-141 expression in both normal and cancer cells. A CpG island near the predicted mir-200c/mir-141 transcription start site shows a striking correlation between miR-200c and miR-141 expression and DNA methylation in both normal and cancer cells, as determined by MassARRAY technology. The CpG island is unmethylated in human miR-200/miR-141 expressing epithelial cells and in miR-200c/miR-141 positive tumor cells. The CpG island is heavily methylated in human miR-200c/miR-141 negative fibroblasts and miR-200c/miR-141 negative tumor cells. Mouse cells show a similar inverse correlation between DNA methylation and miR-200c expression. Enrichment of permissive histone modifications, H3 acetylation and H3K4 trimethylation, is seen in normal miR-200c/miR-141-positive epithelial cells, as determined by chromatin immunoprecipitation coupled to real-time PCR. In contrast, repressive H3K9 dimethylation marks are present in normal miR-200c/miR-141-negative fibroblasts and miR-200c/miR-141 negative cancer cells and the permissive histone modifications are absent. The epigenetic modifier drug, 5-aza-2′-deoxycytidine, reactivates miR-200c/miR-141 expression showing that epigenetic mechanisms play a functional role in their transcriptional control.We report that DNA methylation plays a role in the normal cell type-specific expression of miR-200c and miR-141 and this role appears evolutionarily conserved, since similar results were obtained in mouse. Aberrant DNA methylation of the miR-200c/141 CpG island is closely linked to their inappropriate silencing in cancer cells. Since the miR-200c cluster plays a significant role in EMT, our results suggest an important role for DNA methylation in the control of phenotypic conversions in normal cells

Impact of periconceptional and preimplantation undernutrition on factors regulating myogenesis and protein synthesis in muscle of singleton and twin fetal sheep.

In this study, we determined the effect of maternal undernutrition in the periconceptional (PCUN: ~80 days before to 6 days after conception) and preimplantation (PIUN: 0-6 days after conception) periods on the mRNA and protein abundance of key factors regulating myogenesis and protein synthesis, and on the relationship between the abundance of these factors and specific microRNA expression in the quadriceps muscle of singleton and twin fetal sheep at 135-138 days of gestation. PCUN and PIUN resulted in a decrease in the protein abundance of MYF5, a factor which determines the myogenic lineage, in singletons and twins. Interestingly, there was a concomitant increase in insulin-like growth factor-1 mRNA expression, a decrease in the protein abundance of the myogenic inhibitor, myostatin (MSTN), and an increase in the mRNA and protein abundance of the MSTN inhibitor, follistatin (FST), in the PCUN and PIUN groups in both singletons and twins. These promyogenic changes may compensate for the decrease in MYF5 protein abundance evoked by early embryonic undernutrition. PCUN and PIUN also increased the protein abundance of phosphorylated eukaryotic translation initiation factor binding protein 1 (EIF4EBP1; T70 and S65) in fetal muscle in singletons and twins. There was a significant inverse relationship between the expression of miR-30a-5p, miR-30d-5p, miR-27b-3p, miR106b-5p, and miR-376b and the protein abundance of mechanistic target of rapamycin (MTOR), FST, or MYF5 in singletons or twins. In particular, the expression of miR-30a-5p was increased and MYF5 protein abundance was decreased, in PCUN and PIUN twins supporting the conclusion that the impact of PCUN and PIUN is predominantly on the embryo

Telomerase activity is associated with an increase in DNA methylation at the proximal subtelomere and a reduction in telomeric transcription

Tumours and immortalized cells avoid telomere attrition by using either the ribonucleoprotein enzyme telomerase or a recombination-based alternative lengthening of telomeres (ALT) mechanism. Available evidence from mice suggests that the epigenetic state of the telomere may influence the mechanism of telomere maintenance, but this has not been directly tested in human cancer. Here we investigated cytosine methylation directly adjacent to the telomere as a marker of the telomere's epigenetic state in a panel of human cell lines. We find that while ALT cells show highly heterogeneous patterns of subtelomeric methylation, subtelomeric regions in telomerase-positive cells invariably show denser methylation than normal cells, being almost completely methylated. When compared to matched normal and ALT cells, telomerase-positive cells also exhibit reduced levels of the telomeric repeat-containing-RNA (TERRA), whose transcription originates in the subtelomere. Our results are consistent with the notion that TERRA may inhibit telomerase: the heavy cytosine methylation we observe in telomerase-positive cells may reflect selection for TERRA silencing in order to facilitate telomerase activity at the telomere. These data suggest that the epigenetic differences between telomerase-positive and ALT cells may underlie the mechanism of telomere maintenance in human tumorigenesis and highlight the broad reaching consequences of epigenetic dysregulation in cancer

Landscape of DNA methylation on the marsupial x

DNA methylation plays a key role in maintaining transcriptional silence on the inactive X chromosome of eutherian mammals. Beyond eutherians, there are limited genome wide data on DNA methylation from other vertebrates. Previous studies of X borne genes in various marsupial models revealed no differential DNA methylation of promoters between the sexes, leading to the conclusion that CpG methylation plays no role in marsupial X-inactivation. Using reduced representation bisulfite sequencing, we generated male and female CpG methylation profiles in four representative vertebrates (mouse, gray short-tailed opossum, platypus, and chicken). A variety of DNA methylation patterns were observed. Platypus and chicken displayed no large-scale differential DNA methylation between the sexes on the autosomes or the sex chromosomes. As expected, a metagene analysis revealed hypermethylation at transcription start sites (TSS) of genes subject to X-inactivation in female mice. This contrasted with the opossum, in which metagene analysis did not detect differential DNA methylation between the sexes at TSSs of genes subject to X-inactivation. However, regions flanking TSSs of these genes were hypomethylated. Our data are the first to demonstrate that, for genes subject to X-inactivation in both eutherian and marsupial mammals, there is a consistent difference between DNA methylation levels at TSSs and immediate flanking regions, which we propose has a silencing effect in both groups.This work was funded by Australian Research Council Discovery Projects DP0987091 and DP1094868