In mammals, epigenetic modifications and trans-acting effectors coordinate gene expression during development and impose transcriptional memories that define specific cell lineages and cell-types. Methylation at CpG dinucleotides is an epigenetic mechanism through which transcriptional silencing is established and heritably maintained through development. Functionally, DNA methylation regulates key biological processes such as X-chromosome inactivation, transposon repression and genomic imprinting. However, the extent to which DNA methylation is the primary regulator of single-copy gene expression and the precise mechanism of methylation-dependent silencing remain undetermined. Here, I identify a novel set of germline-specific candidate genes putatively regulated by DNA methylation. Analysis of one candidate gene, Tex19, demonstrates that promoter CpG methylation is the primary and exclusive mechanism for regulating developmental silencing in somatic lineages. Genetic or pharmacological removal of CpG methylation triggers robust de-repression of Tex19 and loss of transcriptional memory. Moreover, Tex19 critically relies on de novo methylation, mediated by Dnmt3b, to impose silencing in differentiating ES cells and somatic cells in vivo from embryonic day (E)7.5. Reporter gene and ChIP analysis demonstrate that Tex19 is strongly activated by general transcription factors and is not marked by repressive histone modifications in somatic lineages, consistent with differential DNA methylation per se being the primary mechanism of regulating expression. Full transcriptional silencing of Tex19 is critically dependent on the methyl-binding protein (MBP) Kaiso, which is only recruited to methylated Tex19 promoter. The reliance on DNA methylation and Kaiso for silencing in somatic cells establishes an epigenetic memory responsible for maintaining expression in germline and pluripotent cell types through successive developmental cycles. This thesis represents the first causal report of lineagespecific promoter DNA methylation directing silencing of an in vivo gene through recruitment of an MBP.

Chromatin structure and epigenetic mechanisms play an important role in initiating and maintaining the intricate patterns of gene expression required for embryonic development. One such mechanism, DNA methylation (5mC), involves the chemical modification of cytosine bases in DNA and is implicated in maintaining patterns of transcription. However, many fundamental aspects of DNA methylation are not fully understood, including the mechanisms by which it influences transcriptional states. Recent data suggest functional links between DNA methylation and a second epigenetic mechanism that has important roles in transcriptional repression, the polycomb group (PcG) repressor system. Here, I suggest that an intact DNA methylation system is required for the repression of many PcG target genes by influencing the genomic targeting of the polycomb repressor 2 complex (PRC2) and its signature histone modification, H3K27me3 (K27me3). I demonstrate differential genomic localisation of K27me3 at gene promoter regions in hypomethylated mouse embryonic fibroblast (MEF) cells deficient for the major maintenance DNA methyltransferase, Dnmt1. Globally, Dnmt1-/- MEFs have a higher level of the K27me3 mark than controls, as assessed by western blot and immunofluorescence. I observe increased K27me3 at a relatively small number of gene promoters in Dnmt1-/- MEFs that often are associated with high levels of DNA methylation in wildtype MEFs, consistent with the notion that DNA methylation is capable of antagonising PRC2 binding at certain loci. Conversely, I show that a large number of developmentally important genes that are normally repressed and highly bound by K27me3, including classic polycomb targets, the Hox genes, display dramatically reduced association with K27me3 in Dnmt1-/- MEFs. Many of these genes, but not all, show reciprocal increases in promoter H3K4me3 modification and are transcriptionally de-repressed in Dnmt1-/- MEFs. I suggest that these genes are mostly associated with CpG-rich promoters with low levels of DNA methylation in wildtype cells, implying that their silencing is not dependent on the canonical role of DNA methylation. Consistent with the findings of recently published work, I suggest a working model where PRC2 binding in wildtype cells is restricted by CpG methylation. According to this model, the differential genomic location of K27me3 in hypomethylated Dnmt1-/- MEFs is explained by a redistribution of PRC2 to normally DNA methylated, unbound loci, resulting in a titration effect and coincident loss of K27me3 from normal targets. It was also apparent that certain PRC2-target genes, including the developmentally important Hox gene clusters, are strongly affected in Dnmt1-/- MEFs, displaying striking loss of K27me3. As intergenic transcription has been implicated in relief from polycomb silencing and abundant intergenic transcription has been reported within Hox clusters, I measured RNA expression at Hox clusters and a small number of other PcG target genes in Dnmt1-/- MEFs using highdensity tiling arrays. In Dnmt1-deficient MEFs, widespread increases in intergenic transcription were observed within Hox clusters. In addition, mapping of the elongatingpolymerase- associated H3K36me3 histone modification showed widespread increases in this mark at intergenic and promoter regions in Dnmt1-/- MEFs. Increased local intergenic RNA and H3K36me3 were found to correlate with K27me3 loss for this cohort of genes. I suggest a working model where increased intergenic transcription and H3K36me3 in Dnmt1-/- MEFs leads to accelerated loss of K27me3 at certain loci, including Hox clusters. Taken together with recently published data, this work suggests that a major role of DNA methylation is in shaping the PRC2/K27me3 landscape. The potential implications of this putative role for DNA methylation are widespread, including our knowledge of how DNA methylation influences transcriptional regulation, and the consequence of rearranged DNA methylation patterns that are observed in many diseases including cancers.

Virtually all multicellular organisms are capable of developing differently in response to environmental variation. At the molecular level, such developmental plasticity requires interpretation and perpetuation of environmental signals without changing the underlying genotype. Such non-genetic, heritable information is known as epigenetic information. This dissertation examines epigenetic information among social insects, and how differences in such information relate to phenotypic caste differences. The studies included herein primarily focus on one form of epigenetic information: DNA methylation. In particular, these studies explore DNA methylation as it relates to and impacts (i) alternative phenotype and particular gene expression differences in two social insect species, (ii) histone modifications, another important form of epigenetic information, in insect genomes, and (iii) molecular evolutionary rate of underlying actively transcribed gene sequences. We find that DNA methylation exhibits marked epigenetic and evolutionary associations, and is associated with alternative phenotype in multiple insect species. Thus, DNA methylation is emerging as one important epigenetic mediator of phenotypic plasticity in social insects.

Background: Gene expression is regulated via highly coordinated epigenetic changes, the most studied of which is DNA methylation (DNAm). Many studies have shown that DNAm is linearly associated with age, and some have even used DNAm data to build predictive models of human age, which are immensely important considering that DNAm can predict health outcomes, such as all-cause mortality, better than chronological age. Nevertheless, few studies have investigated non-linear relationships between DNAm and age, which could potentially improve these predictive models. While such investigations are relevant to predicting health outcomes, non-linear relationships between DNAm and age can also add to our understanding of biological responses to late-life events, such as diseases that afflict the elderly. Objectives: We aim to (1) examine non-linear relationships between DNAm and age at specific loci on the genome and (2) build upon regularization methods by comparing prediction errors between models with both non-transformed and square-root transformed predictors to models that include only non-transformed predictors. We used both the sparse partial least squares (SPLS) regression model and the lasso regression model to make our comparisons. Results: We found two age-differentially methylated sites implicated in the regulation of a gene known as KLF14, which could be involved in an immunosenescent phenotype. Inclusion of the square-root transformed variables had little effect on the prediction error of the SPLS model. On the other hand, the prediction error increased substantially in the lasso regression model, particularly when few predictors (70) were included. Conclusion: The growing amount and complexity of biological data coupled with advances in computational technology are indispensable to our understanding of biological pathways and perplexing biological phenomena. Moreover, high-dimensional biological data have enormous implications for clinical practice. Our findings implicate a possible biological pathway involved in immunosenescence. While we were unable to improve the predictive models of human age, future research should investigate other possible non-linear relationships between DNAm and human age, considering that such statistical methods can improve predictions of health outcomes.

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

Introduction: It has been reported that the principal source of exposure for humans to inorganic arsenic (As) comes from drinking water. It is known that selenium (Se) competes with the reductive metabolism and methylation of As and Se compete for the availability of glutathione. The overarching goal of this dissertation research is to assess relationships between arsenic intake from water and other fluids with urinary arsenic output and then to assess how urinary arsenic output is modified by selenium exposure. Methods: Households in the Binational Arsenic Exposure Survey (BAsES) were selected for their varying groundwater arsenic concentrations. A first morning urine void and water samples from all household drinking sources were collected for As quantification. Relationships were examined between various urinary arsenic biomarkers and estimated arsenic exposures. The association between urinary arsenic biomarkers and dietary intake and urinary output of selenium was also evaluated. Results: Arizonans reported consuming 18.5 mL/kg-day of water and 34.3 mL/kg-day from all fluids. In contrast, participants from Mexico reported 3.5 mL/kg-day of water and 12.3 mL/kg-day from all fluids. Median urinary inorganic As concentration among Arizona participants (ranging from 1.2 to 2.0 µg/L) was lower than among participants from Mexico (range 2.5 to 6.2 µg/L). Estimated arsenic intake from drinking water was associated with urinary total arsenic concentration (p<0.001), urinary inorganic arsenic concentration (p<0.001), and urinary sum of species (p<0.001). Urinary arsenic concentrations increased between 7% and 12% for each one percent increase in arsenic consumed from drinking water. No statistically significant relationships were seen between urinary methylated arsenic biomarkers with either dietary intake of selenium or the urinary selenium concentration. Conclusion: Water was the primary contributor to total fluid intake among Arizonans while Mexico participants primarily consumed carbonated beverages. Arsenic intake from water was significantly associated with urinary arsenic output; however, the concentration of arsenic consumed explained a small fraction of urinary arsenic levels. While selenium can biologically interact with arsenic in the liver, no relationship between urinary arsenic biomarkers were identified with either dietary intake of selenium or urinary output of selenium.

No description available.

No description available.

One of the most frequently reported deficits seen in individuals with Fetal Alcohol Spectrum Disorder (FASD) is impairments in learning and memory, which is likely attributed to the teratogenic effects of ethanol on the developing hippocampus. TGF-β (transforming growth factor-β), hedgehog and Wnt signaling pathways have been identified as high probability candidate pathways associated with brain deficits seen in FASD. Increasing evidence indicates that ethanol may induce changes in DNA methylation that could alter transcription regulating factors within signaling pathways critical in brain development. The purpose of this study was to test the hypotheses that prenatal ethanol exposure during i) the first trimester-equivalent period, or ii) throughout the entire gestational period induces changes in DNA methylation and alters the transcription/translation of TGF-β1, SHH (sonic hedgehog) and Wnt3a within the developing hippocampus. Pregnant Dunkin-Hartley-strain guinea pigs were assigned to one of three groups: ethanol (4 g/kg maternal body weight), isocaloric-sucrose/pair-feeding, or no treatment. Embryonic telencephalon tissue (which gives rise to the hippocampus) and fetal hippocampus were collected at gestational day (GD) 23 or GD 65, respectively. GD 23 ethanol-exposed and nutritional control embryos exhibited decreased crown-rump and head lengths. GD 65 ethanol-exposed fetuses exhibited decreased body and brain weights compared with the control groups. Ethanol exposure during the first trimester-equivalent period, but not during the entire gestational period, resulted in an increase in global DNA methylation. First trimester-equivalent ethanol exposure did not alter TGF-β1, SHH and Wnt3a gene expression within the GD 23 telencephalon. However, ethanol exposure throughout the entire pregnancy led to an increase in the expression of all three genes within the GD 65 hippocampus. No change in TGF-β1 protein was seen in the hippocampus of ethanol-treated fetuses. Post-translationally modified (ptm) SHH, but not unmodified SHH protein, was decreased in the hippocampus of ethanol-exposed fetuses. A decrease in unmodified, but not ptm Wnt3a protein, was observed in both ethanol-exposed and nutritional control hippocampus. These results suggest that prenatal ethanol exposure may affect hippocampal development through alterations in i) DNA methylation as shown at early gestation and ii) the expression of transcription regulating factors, especially SHH, as shown at term. / Thesis (Master, Pharmacology & Toxicology) -- Queen's University, 2012-12-03 12:36:33.035

Background: Obesity is a metabolic disease caused by environmental and genetic factors. However, the epigenetic mechanisms of obesity are incompletely understood. The aim of our study was to investigate the role of skeletal muscle DNA methylation in combination with transcriptomic changes in obesity. Results: Muscle biopsies were obtained basally from lean (n = 12; BMI = 23.4 +/- 0.7 kg/m(2)) and obese (n = 10; BMI = 32.9 +/- 0.7 kg/m(2)) participants in combination with euglycemic-hyperinsulinemic clamps to assess insulin sensitivity. We performed reduced representation bisulfite sequencing (RRBS) next-generation methylation and microarray analyses on DNA and RNA isolated from vastus lateralis muscle biopsies. There were 13,130 differentially methylated cytosines (DMC; uncorrected P < 0.05) that were altered in the promoter and untranslated (5' and 3'UTR) regions in the obese versus lean analysis. Microarray analysis revealed 99 probes that were significantly (corrected P < 0.05) altered. Of these, 12 genes (encompassing 22 methylation sites) demonstrated a negative relationship between gene expression and DNA methylation. Specifically, sorbin and SH3 domain containing 3 (SORBS3) which codes for the adapter protein vinexin was significantly decreased in gene expression (fold change -1.9) and had nine DMCs that were significantly increased in methylation in obesity (methylation differences ranged from 5.0 to 24.4 %). Moreover, differentially methylated region (DMR) analysis identified a region in the 5' UTR (Chr. 8: 22,423,530-22,423,569) of SORBS3 that was increased in methylation by 11.2 % in the obese group. The negative relationship observed between DNA methylation and gene expression for SORBS3 was validated by a site-specific sequencing approach, pyrosequencing, and qRT-PCR. Additionally, we performed transcription factor binding analysis and identified a number of transcription factors whose binding to the differentially methylated sites or region may contribute to obesity. Conclusions: These results demonstrate that obesity alters the epigenome through DNA methylation and highlights novel transcriptomic changes in SORBS3 in skeletal muscle.