Transgenic organisms are a valuable tool for studying epigenetics, as they provide significant insight into the evolutionary conservation of epigenetic control sequences, the interacting proteins, and the underlying molecular mechanisms. Paramutation is an epigenetic phenomenon in which the epigenetic status and expression level of one allele is heritably altered after pairing with another. At the b1 locus in maize, a control region consisting of seven 853 bp tandem repeats is required for paramutation. To study the conservation of the epigenetic mechanisms underlying maize b1 paramutation, I created transgenic Drosophila carrying the maize b1 control region flanked by FRT sites and adjacent to the Drosophila white reporter gene. The maize b1 tandem repeats caused epigenetic silencing in Drosophila, as white expression consistently increased following repeat removal. A single copy of the tandem repeat sequence was sufficient to cause silencing, and silencing strength increased as the number of repeats increased. Trans interactions, such as pairing-sensitive silencing, were also observed and appear to require a threshold number of b1 tandem repeats, similar to paramutation in maize. Analysis of transcription from the repeats showed that the b1 tandem repeats are transcribed from both strands in Drosophila, as they are in maize. Bidirectional transcription was found to extend to the regions flanking the repeats, and persisted in “repeats-out” transgenes following repeat removal. However, aberrant transcription was lost when a zero-repeat transgene was moved to a new genomic position, suggesting that it may be due to an epigenetic mark that is retained from the previous silenced state. A search for modifiers of b1 repeat-mediated silencing demonstrated that Polycomb group proteins are involved. Together, these results indicate considerable conservation of an epigenetic silencing process between the plant and animal kingdoms. Genomic imprinting is a related epigenetic process in which parent-specific epigenetic states are inherited and maintained in progeny. The conservation of epigenetic mechanisms was further explored via an in-depth review of the molecular mechanisms underlying genomic imprinting in plants, mammals and insects, and identification of potentially imprinted genes in Drosophila by microarray analysis.

Separate domestication events and strong selective pressures have created diverse phenotypes among existing cattle populations; however, the genetic determinants underlying most phenotypes are currently unknown. Bos taurus taurus (Bos taurus) and Bos taurus indicus (Bos indicus) cattle are subspecies of domesticated cattle that are characterized by unique morphological and metabolic traits. Because of their divergence, they are ideal model systems to understand the genetic basis of phenotypic variation. Here, we developed DNA and structural variant maps of cattle genomes representing the Bos taurus and Bos indicus breeds. Using this data, we identified genes under selection and biological processes enriched with functional coding variants between the two subspecies. Furthermore, we examined genetic variation at functional non-coding regions, which were identified through epigenetic profiling of indicative histone- and DNA-methylation modifications. Copy number variants, which were frequently not imputed by flanking or tagged SNPs, represented the largest source of genetic divergence between the subspecies, with almost half of the variants present at coding regions. We identified a number of divergent genes and biological processes between Bos taurus and Bos indicus cattle; however, the extent of functional coding variation was relatively small compared to that of functional non-coding variation. Collectively, our findings suggest that copy number and functional non-coding variants may play an important role in regulating phenotypic variation among cattle breeds and subspecies.

Exposure to the anticonvulsant valproic acid (VPA) is associated with a 7.5% rate of major malformations and a 1-2% incidence of neural tube defects (NTDs). Although the teratogenic outcomes resulting from VPA use during pregnancy were first identified in the 1980s, the mechanisms by which VPA induces birth defects are not fully elucidated. Based on evidence in the literature, the studies in this thesis examined the role of in utero VPA exposure on oxidative stress and epigenetic alterations in the developing embryo to provide further mechanistic insight into VPA’s teratogenic pathway. The first study investigated the role of oxidative stress in VPA-induced teratogenesis. Using CD-1 mice, catalase was shown to protect against VPA-induced effects on developmental and morphological parameters in both whole embryo culture and in vivo models. Studies in whole embryo culture demonstrated that markers of oxidative damage were not altered by VPA; however, VPA increased apoptosis in the neuroepithelium, which was attenuated by the addition of catalase. The second objective addressed epigenetic modifications induced by VPA in an in vivo mouse model. Maternal administration of VPA resulted in increased acetylation of histones H3 and H4, increased methylation of histone H3K4, and decreased methylation of histone H3K9. Furthermore, these changes were localized to VPA target tissues including the neuroepithelium, heart, and somites. Global DNA methylation in the embryo was not altered by VPA. The final objective was to determine VPA’s effect on a marker of DNA damage, markers of cell cycle proteins, and a marker of apotosis in vivo. Maternal administration of VPA resulted in a rapid increase of γH2A.X, a marker of DNA double strand breaks (DSBs). Increased expression of p27KIP1, a cyclin-dependent kinase inhibitor, and activated caspase-3, a marker of apoptosis, were observed and these changes were localized to the neuroepithelium of developing embryos. In conclusion, this thesis supports the hypothesis that VPA-induced increases in ROS production and HDAC inhibition may lead to altered gene expression patterns and consequently teratogenic effects, namely NTDs. / Thesis (Ph.D, Pharmacology & Toxicology) -- Queen's University, 2011-05-24 13:12:33.778

Monozygotic (MZ) twins are currently used in epigenetic studies as one homogenous group. However, there are two MZ twin types: dichorionic (DC) and monochorionic (MC). We hypothesize that DCMZ twins are more similar epigenetically, compared to MCMZ twins, due to earlier zygote splitting, a higher degree of birth outcome similarity, or both. We recruited 220 newborn twins and obtained genome-wide DNA methylation profiles for 48 twins. Intraclass correlation coefficients (ICC) and linear mixed models were used to investigate the relationships between DNA methylation levels and chorionicity. DCMZ twins tended to have longer gestational age, larger birth weight, and smaller birth length discordance. DCMZ twins had more similar DNA methylation profiles than MCMZ twins (ICC=0.21 vs. 0.13), after adjusting for birth outcomes. Additionally, we identified 5,170 CpG sites with different DNA methylation levels between DCMZ and MCMZ twins. This study highlights the importance of incorporating chorionicity information in epigenetic twin studies.

Ascorbic acid (AA), in popular speech vitamin C, is a commonly known nutrient. It is involved in several biological processes and deficiency can lead to scurvy. Recent publications have shown the impact of AA on epigenetic regulation in mice. Addition of AA, via enzymatic activity, enhances the generation of 5-hydroxymethylcytosine (5hmC), which is an intermediate in active demethylation of DNA. The role of AA on epigenetic changes in humans has to our knowledge never been studied. In this study, naïve CD4+ T cells from blood donors were used as a model system to investigate AAs possible role in methylation changes in the immune system. By using dot-blot assay, hydroxymethylated DNA immunoprecipitation (hmeDIP) and qPCR, changes in methylation executed by AA could be detected. A confirmation of AAs impact on epigenetic changes in mice was observed. AA enhanced the levels of 5hmC compared to untreated cells. The Jurkat cell line, a human T lymphocyte cell line, showed an opposite result. Treatment with AA decreased the levels of 5hmC compared to untreated cells. When comparing this result with the results obtained in human naïve T cells, the same observation was made. The difference between mouse and human in the ability of producing and metabolize AA could be a reason for this opposite result. Since AA had the ability to modify epigenetic changes in primary human CD4+ T cells, the results suggest that AA may have a function in the human immune system.

Infection of the human urinary tract is one of the commonest bacterial infections, with uropathogenic E.coli (UPEC) being responsible for 90% of the diagnosed cases, with significant morbidity and mortality. The urinary bladder is a remarkable autonomic musculomembranous organ under conscious control. Its two main functions are, storage and voiding of urine. Any disturbance to normal urination leads to various clinical conditions, such as urinary incontinence, bladder retention, overactive bladder syndrome, prostatitis in men and urinary tract infections (UTI). Determining the predisposition of an individual to UTI by discovering a biomarker would allow for a more rational selection of patients who might best benefit from either antibiotic prophylaxis or preemptive surgical intervention. The purpose of this study was to examine the epigenetic effects of UPEC infection directly, or indirectly in the bladder. The study also identified potential gene candidates, such as TLR4 and CTCF, for development of DNA methylation biomarker targets.

A significant challenge of the 21st century is the growing health threat stemming from drug-resistant infectious diseases. There is an undeniable need to discover new, safe and effective drugs with novel mechanisms of action to combat this threat. A study of drugs currently on the market showed that natural products account for approximately 75% of new anti-infective drugs, either as new agents or analogs based upon their structure. Unfortunately, major pharmaceutical companies have cut back tremendously in natural products research in part due to the frustrating obstacle of frequent rediscovery of compounds. Fungi in particular are difficult to work with in that they do not always produce the same variety and quantities of secondary metabolites under laboratory conditions. One of the groundbreaking discoveries evolving from genomics research is the observation that many fungi possess more gene clusters encoding for the production of secondary metabolites than the reported number of natural products isolated from those organisms. Simple epigenetic modifications such as DNA methlytransferase or histone deacetylase inhibition can activate silenced genes leading to the genesis of novel chemistry from the fungus. The work presented herein is a study of the isolation and characterization of anti-infective compounds from Floridian mangrove endophytes. In addition, epigenetic modifications were explored in order to increase the production of secondary metabolites as well as for the purpose of generating new analogs not found in the controls. Finally, structure activity relationship studies were performed in order to maximize the anti-malarial and antibiotic activity of cytosporone E.

The histone acetyltransferase protein, Tip60, has many important functions in epigenetic regulation of gene expression and DNA repair. Our objective is to design, synthesize, and evaluate potent inhibitors for Tip60. Full-length Tip60 (fl-Tip60) and catalytic domain of Tip60 (cat-Tip60) were expressed in E. coli and purified with nickel affinity chromatography. Quantitative analysis of enzyme activities demonstrated that both enzyme forms had very high activity with the substrate H4. To create new Tip60 inhibitors, various histone H3 peptides conjugated with CoA were synthesized using the Fmoc solid-phase peptide synthesis and solution phase synthesis protocols. The results from the inhibition radioactive assay showed that the synthetic H3-CoA conjugates inhibited effectively the enzymatic activity of both fl-Tip60 and cat-Tip60; and the addition of methyl groups to the Lys-4 or Lys-9 residue of H3 aided in a 7-9 fold enhancement in potency in comparison to nascent H3-CoA inhibitor.

Chromatin remodeling enzymes are instrumental for neural development as evidenced by their identification as disease genes underlying human disorders characterized by intellectual-disability. In this regard, the murine Snf2h and Snf2l genes show differential expression patterns during embryonic development, with a unique pattern in the brain where Snf2h is predominant in neural progenitors, while Snf2l expression peaks at the onset of differentiation. These observations led me to investigate the role of Snf2h and Snf2l in brain development by using conditionally targeted Snf2h and Snf2l mice. I selectively ablated Snf2h expression in cortical progenitors, cerebellar progenitors, or postmitotic Purkinje neurons of the cerebellum, while Snf2l was deleted in the germline. I found that Snf2h plays diverse roles in neural progenitor expansion and postmitotic gene expression control, while Snf2l is involved in the precise timing of neural differentiation onset. Gene expression studies revealed that Snf2h and Snf2l co-modulate the FoxG1 and En1 transcription factors during cortical and cerebellar neurogenesis, respectively, to precisely control the transition from a progenitor to a differentiated neuron. Moreover, Snf2h is essential for the postmitotic neural activation of the clustered protocadherin genes, and does so by functionally interacting with the matrix-attachment region protein Satb2. My neurobehavioral studies also provided insight into how Snf2h loss in cerebellar progenitors results in cerebellar ataxia, while Snf2h loss in cortical progenitors, or in postmitotic Purkinje neurons of the cerebellum, resulted in learning and memory deficits, and hyperactive-like behavior. Molecularly, Snf2h plays an important role in linker histone H1e dynamics and higher order chromatin packaging, as evidenced by loss of chromatin ultrastructure upon Snf2h deletion in progenitor and postmitotic neurons. I further demonstrated that Snf2h loss in a neuronal cell culture model results in reduced H1e deposition, and that overexpression of human SNF2H or SNF2L upon Snf2h knockdown rescues this biochemical dysfunction. My experiments suggest that Snf2h and Snf2l are regulatory nucleosome remodeling engines that co-modulate the gene expression programs necessary for proper brain development, maturation and function.

The covalent modifications to the histone 2A, 2B, 3, and 4 N-terminal tails that affect gene expression have been deemed the “Histone Code.” Mis-regulation of these signalling pathways is of great interest as are important in human disease. A variety of peptides containing post-translationally modified histone 3 and 4 sequences were read using a supramolecular sensor array approach, where two or three sensors gave a unique response for each analyte when compared to others. These sequences were chosen to determine what type of modifications could be read (phosphorylation, acetylation, methylation) and if this type of array would be suitable for reading analytes on which antibodies—the leading technology—typically perform poorly. It was found that three sensors, which operate in neutral aqueous solution, were able to discriminate 16 different histone analytes. Additionally, it was shown that this array could report simultaneously on both concentration and the identities of histone analytes. / Graduate