Breast cancer is the most frequently diagnosed form of cancer in women and the second leading cause of cancer-related deaths. Breast cancer is a heterogeneous disease consisting of many types of tissue neoplasia, and there appears to be no model of how a particular lesion develops into an aggressive, malignant, invasive carcinoma. Genetic mutation and aberrant epigenetic regulation are among the most common events that lead to neoplasia. In breast cancer, p53 mutation is the most common genetic defect related to a single gene. Therefore, this dissertation focuses on the mechanisms and consequences of p53 mutation during breast tumorigenesis. Genome-wide analysis of gene expression and epigenetic modifications in a panel of breast cancer cell lines suggested that p53 mutation and aberrant epigenetic silencing were cooperating mechanisms in the silencing of wild-type p53 target genes during cancer progression. Therefore, models of p53 inactivation were created in non-malignant human mammary epithelial cells to determine the role of p53 mutation on the epigenetic status of its target genes and the acquisition of malignant phenotypes. Comparisons of each model demonstrated that differing modes of p53 inactivation produced different functional consequences. Loss of wild-type p53 function alone ablated the normal cellular response to external stress stimuli, but had no affect on the expression of genes or epigenetic status in untreated cells. Introduction of missense mutant p53 protein caused very few changes when the protein was expressed at low levels. However, accumulation of mutant p53 caused a variety of gene expression changes and interfered with endogenous wild-type p53. The accumulation of mutant p53 also caused an increase in migration and invasion of the cells that expressed it. Interestingly, epigenetic aberrations were not detected in response to any of the p53 manipulations. These data suggest that accumulation of missense mutation is particularly dangerous to normal cells. They also suggest that p53 mutation and epigenetic aberration are two distinct mechanisms, which overlap and cooperate during tumorigenesis. These data suggest that treatment strategies for human breast cancer should include modalities to target both defects for increased efficacy.

Although structurally unrelated, the human killer cell immunoglobulin-like (KIR) and the rodent lectin-like Ly49 receptors serve similar functional roles in natural killer (NK) cells. Moreover, both gene families display variegated and mostly mono-allelic expression patterns established at the transcriptional level. DNA methylation, but not histone modifications, has recently been shown to play an important role in maintenance of the expression patterns of KIR genes but the potential role of DNA methylation in the expression of Ly49 genes was unknown. My thesis focuses on the role of epigenetic modifications, especially DNA methylation, in the maintenance of mouse Ly49 gene expression. I show that hypomethylation of the region encompassing the main promoter of Ly49a and Ly49c in primary C57BL/6 (B6) mouse NK cells correlates with expression of these genes. Using B6 x BALB/c Fl hybrid mice, I demonstrate that the expressed allele of Ly49a is hypomethylated while the non-expressed allele is heavily methylated, indicating a role for epigenetics in maintaining mono-allelic Ly49 gene expression. Furthermore, the Ly49a promoter region is heavily methylated in fetal NK cells but variably methylated in non-lymphoid tissues. In apparent contrast to the KIR genes, I show that histone acetylation state of the promoter region strictly correlate with Ly49A and Ly49G expression status. Also, the instability of Ly49G expression on some lymphoid cell lines is at least in part due to changes in the level of histone acetylation of the promoter region. As for the activating Ly49 receptors, it seems that although DNA methylation levels of the promoter regions do correlate with the state of expression of these receptors, the pattern of DNA methylation is different from that of the inhibitory Ly49a and c genes. In conclusion, my results support a role for epigenetic mechanisms in the maintenance of Ly49 expression. Moreover, these epigenetic mechanisms appear to vary among the Ly49 genes and also differ from those governing KIR expression.

p-Sulfonatocalix[X]arene (X = 4 and 6) was explored as a host for trimethyllyated lysine. We found by 1H NMR and ITC titrations that p-sulfonatocalix[4]arene (PSC) bound the trimethyllysine amino acid with high affinity and good selectivity over dimethyllysine and similar dimethylated arginines. When trimethyllysine was in the context of a peptide of the histone 3 tail, affinities increased and PSC was up to 20 -fold selective over identical unmethylated peptides. Multiple scaffolds were synthetically explored as derivatives of PSC. I created five different scaffolds and synthesized a small library of compounds derived from these scaffolds as hosts for a variety of histone 3 peptides containing biologically important post-translationally modified amino acids. This library was tested using a high-throughput indicator displacement assay and I found three hosts that displayed tuned affinities and selectivities for post-translationally modified amino acids we had not previously targeted. I studied the ability of these synthetically elaborated calix[4]arenes to identify histone PTMs and monitor an enzymatic reaction. I found covalently linked fluorescent calixarenes were able to accomplish this goal. Furthermore, we studied the ability of these calix[4]arenes to disrupt protein-protein interactions that occur between the trimethyllyated lysine on histone tails and proteins that read these sites. I found that these calixarenes could disrupt these interactions between a variety of proteins and trimethyllyated lysine sites. These calix[4]arenes show promise as chemical tools that could be used to further probe epigenetic pathways in vitro and further work is needed to explore their utility in cellular assays and in vivo. / Graduate / 0490 / 0487 / kddaze@gmail.com

The dynamic changes in chromatin play a significant role in lineage commitment and differentiation. These epigenetic modifications control gene expression through recruitment of transcription factors. While the active mark H3K4me3 is present around the transcription start site on the gene, the function of the H3K36me3 mark is unknown. A number of H3K36-specific histone methyltransferases (HMTs) have been identified, however the focus of this study is the HMT Hypb. To elucidate the role of H3K36me3 in mediating expression of developmentally-regulated loci, native chromatin immunoprecipitation (N-ChIP) was performed at a subset of genes. Upon differentiation, we observe that H3K36me3 becomes enriched at the 3’ end of several muscle-specific genes. To further investigate the role of H3K36me3 in myogenesis, a lentiviral-mediated knockdown of the H3K36 HMT Hypb was performed in muscle myoblasts using shRNA. Upon Hypb knockdown, we were surprised to observe enhanced myogenesis. N-ChIP was also performed on differentiated Hypb knockdown cell lines in order to look at H3K36me3 enrichment on genes involved in muscle differentiation. N-ChIP data show a drop in H3K36me3 enrichment levels on myogenin and Ckm genes. The possible occupancy of Hypb on the coding regions of muscle-specific genes was experimentally observed by cross-linked chromatin immunoprecipitation (X-ChIP) on differentiated C2C12 cells and subsequently confirmed by X-ChIP on knockdown lines where the occupancy was lost. A model is proposed that links the observed phenotype with H3K36me3.

High throughput transcriptomic analyses have realized us with the fact that eukaryotic genome encodes thousands of noncoding RNAs (ncRNAs) with unknown function. In my thesis, I sought to address epigenetic regulation of transcription by ncRNA using the Kcnq1 imprinted cluster as a model system. Genomic imprinting is an epigenetic phenomenon whereby one of the parental alleles is silenced by epigenetic mechanism in a parent of origin-specific manner. A long ncRNA Kcnq1ot1 regulates imprinting of nearly 8 protein coding genes in the Kcnq1 imprinted cluster. Expression of Kcnq1ot1 is restricted to the paternal chromosome while that of protein-coding genes to the maternal chromosome. Kcnq1ot1 is a 91kb long, moderately stable, nuclear localized and RNAPII encoded transcript. We demonstrated that Kcnq1ot1 RNA itself mediates lineage specific silencing on the paternal chromosome by interacting with chromatin and recruiting the repressive chromatin modifiers to the imprinted gene promoters. Previously we identified an 890bp silencing domain (SD) at the 5´end of the Kcnq1ot1 RNA which is responsible for gene silencing. Targeted deletion of the 890SD in mouse resulted in specific loss of silencing of ubiquitously imprinted genes. We have further shown that Kcnq1ot1 interacts with Dnmt1 and recruit Dnmt1 at the somatic DMRs flanking some of the ubiquitously imprinted genes. We next addressed the stability of the Kcnq1ot1 mediated epigenetic silencing using transgenic mouse where we have conditionally deleted the Kcnq1ot1 RNA at different developmental stages and we found that Kcnq1ot1 RNA is required to maintain the silencing of the ubiquitously imprinted genes. In addition, DNA methylation, which controls imprinting of the ubiquitous genes require Kcnq1ot1 for its maintenance. To characterize the ncRNAs that mediate gene regulation through chromatin interaction we have isolated chromatin associated RNAs (CARs) from sucrose gradient fractioned chromatin. High-throughput sequencing of the CARs resulted in the identification of the 141 intronic and 74 intergenic regions harboring CARs. We characterized one of the intergenic CARs which regulate the transcription of the two neighboring genes by modulating the chromatin marks. In summary current thesis has uncovered unprecedented role of ncRNAs in gene expression via chromatin level regulation.

Doctor of Philosophy (PhD) / The field of epigenetics emerged primarily from studies in Drosophila, and is now being studied intensively by mammalian biologists. In order to increase our knowledge of epigenetic gene control in the mouse, I have studied modifiers of epigenetic gene silencing. My main method of investigation involved the characterisation of mutants from a sensitised ENU mutagenesis screen performed previously in our laboratory. The screen was carried out in an FVB/NJ strain carrying a variegating GFP transgene expressed in erythrocytes. To date we have recovered 12 dominant (D) and seven recessive (R) mutant mouse lines from this screen that display altered transgene expression. We have named these Mommes (Modifiers of murine metastable epialleles). I investigated the phenotype and attempted to identify the underlying causative mutation of two of these Momme mutants. MommeD6 is a semi-dominant, homozygous lethal mutation that acts as a suppressor of variegation with respect to the GFP transgene. This mutation has a large effect on the level of expression of the transgene in expressing cells, but little effect on the percentage of cells expressing the transgene. MommeD6 is linked to a 2.5 Mbp interval on chromosome 14. MommeD9 is a semi-dominant, homozygous lethal mutation that acts as an enhancer of variegation with respect to the GFP transgene. Mutants have a tendency to become obese as they age, show abnormal haematology profiles, and females develop infertility. MommeD9 is linked to a 17.4 Mbp region on chromosome 7. I produced and studied a strain carrying the same GFP transgene but in a new strain background, C57BL/6J. This strain provided an opportunity to look for strain-specific modifiers of expression of the GFP transgene. Several regions were mapped to chromosomal locations. Further work will be needed to identify the genes involved. This mouse will be useful in future mutagenesis screens of this type.

The primary objective of this DPhil research project was to develop selective and cell-active inhibitors of the histone lysine demethylase KDM2A, which could potentially lead to the discovery of a novel chemical probe. Chapter one of this thesis introduces the role of histone lysine demethylases (KDMs) in the epigenetic regulation of gene expression and discusses the value of chemical probes as tools to study these enzymes. Chapter two describes the synthesis of a library of indoline-based KDM2A inhibitors using a modular synthetic approach to explore key structure-activity relationships and a chiral counterion-mediated strategy to synthesize lead candidates enantioselectively. Chapter three discusses investigations into the cellular activity of lead compounds and explores strategies to address limitations associated with cytotoxicity and promiscuity. Chapter four describes the application of a variety of experimental techniques to identify the mode of target inhibition. Finally, chapter five focuses on the development of an enantioselective C-acylation reaction to access spirocyclic fragments asymmetrically.

L'appariement des chromatides sœurs est un prérequis fondamental pour la ségrégation fidèle du génome. Cet assemblage est précisément régulé par plusieurs facteurs modulant la solidarité entre le complexe formant la cohésine et les chromosomes. Un de ces facteurs, Pds5, engage la cohésine par le biais de SCC1 et participe à la fois au renforcement de la cohésion, et inversement à la libération de la cohésine de la chromatine. Dans cette thèse, la structure cristalline du complexe entre les protéines de levure Pds5 et SCC1 est présentée. Celle-ci permet la compréhension de la fonction moléculaire de Pds5. Pds5 forme un « heat-repeat » allongé qui se lie à SCC1 via une interface dont sa séquence reste conservée. Suite à la caractérisation biologique et biochimique de cette structure, cette thèse démontre que l'intégrité de l'interface entre Pds5 et SCC1 est indispensable pour le recrutement de Pds5 à la cohésine et que son abrogation conduit à la perte de la cohésion entre les chromatides sœurs ainsi que la perte de la viabilité cellulaire. Les résultats présentés dans cette thèse suggèrent donc que Pds5 est constitutivement lie au cœur de la sous-unité de la cohésine. / Sister chromatid cohesion is a fundamental prerequisite to faithful genome segregation. Cohesion is precisely regulated by accessory factors that modulate the stability with which the cohesin complex embraces chromosomes. One of these factors, Pds5, engages cohesin through Scc1 and participates both in the enhancement of cohesion, and conversely in mediating the release of cohesin from chromatin. In this thesis the crystal structure of a complex between budding yeast Pds5 and Scc1 is presented, thus elucidating the molecular basis of Pds5 function. Pds5 forms an elongated HEAT repeat that binds to Scc1 via a conserved surface patch. Through complementary cell biological and biochemical characterisation of this structure, the thesis demonstrates that the integrity of the Pds5–Scc1 interface is indispensable for the recruitment of Pds5 to cohesin, and that its abrogation results in loss of sister chromatid cohesion and cell viability. The results presented in this thesis therefore suggest that Pds5 is a constitutively bound, core subunit of cohesin.

One of the most important medical and public health issues of today is obesity, defined as abnormal and excess fat accumulation. Obesity is linked to many health problems including metabolic syndrome (MS), hypertension, type II diabetes mellitus (T2DM), and cardiovascular disease (CVD). Recently, the incidence of these conditions has surged to epidemic proportions, especially in Western societies. Research has also linked obesity to cancer and osteoarthritis. Preventing, diagnosing, and treating obesity is challenging. The diagnosis of obesity is often unclear when it is made with generalized criteria such as the Body Mass Index (BMI). Obesity interventions generally include the often difficult lifestyle change to healthy diets and adequate exercise, which depends heavily upon patient compliance and discipline. Today's society is pushing for the discovery of a shortcut or of a "magic pill" to cure obesity. Consequently, many studies aim to identify therapeutic targets. The majority of current obesity research is focused on discovering and revealing the underlying mechanisms and genetic risk factors. Certain stages of development, such as childhood, are especially susceptible times to be exposed to stressors that lead to obesity. A developing concept is the intergenerational transmission of risk of obesity through epigenetics. Epigenetics is the study of the heritable changes in gene regulation and expression not caused by mutations or changes in DNA sequence. A person's genes may increase or decrease his or her susceptibility to obesity. In addition to genetic inheritance, parents may pass non-genetic alterations to their children. Changes can be mediated through methylation of deoxyribonucleic acids (DNA) and modifications to histones. These epigenetic changes may alter gene expression patterns and "program" offspring towards developing chronic metabolic disease. Many models have begun to show the effects of environmental perturbations on individuals and on several generations of future descendants. This review will analyze the current literature on obesity and evaluate this rapidly evolving field. Current obesity preventions and treatments will be surveyed. In addition, the relative impact of different contributors to obesity risk will be examined. The crossover between obesity and epigenetics may provide a deeper understanding of disease risk and developmental origins. Future directions of study will be proposed such as large-scale prospective studies to further characterize intergenerational transmission of risk.

Alzheimer's disease (AD) is a neurodegenerative condition affecting millions of individuals worldwide and is a major source of mortality in elderly populations. While it is well established that there is a strong genetic basis for the disease, the epigenetic mechanism underlying the disease is largely unknown. The main purpose of this thesis is to understand the alteration of epigenetic modifications associated with the disease and its progression. In particular, we examine how alterations in the cytosine methylation and cytosine hydroxymethylation, two epigenetic modifications that are critically important for the development and function of the brain, are associated with advancing stages of Alzheimer's disease. Eight progressive AD brain samples were examined for changes in DNA methylation and hydroxymethylation by both dot blot analysis and a new oxidative bisulfite (OXBS) deep sequencing technology. The initial results of dot blot analysis reveal a statistically significant decrease in 5hmC associated with intermediate stage AD among the samples. This data suggests that the alterations in epigenetic modifications is likely associated with the pathophysiology of Alzheimer's disease, not only shedding new light on our understanding of the epigenetics of the disease, but also providing the basis for our future investigation on the exact cause and effect relationships of these epigenetic changes and their respective stages in Alzheimer's.