The stochastic nature of intracellular chemistry guarantees that even genetically identical cells sharing an environment will differ in composition. The question of whether this chemical diversity translates into significant phenotypic individuality is tied to the relative timescales of the processes involved. In order for cells in a population to have distinct functional identities, they must maintain their states for an appreciable period of time. Quantification of these timescales requires accurate time-lapse measurements covering tens or even hundreds of generations, a technical hurdle that has left these questions largely underexplored. In this thesis I present three pieces of work that aim to provide a foundation for the study of fluctuation timescales in bacteria. In the first part, I describe modifications to a recently developed microfluidic platform for continuous culture of cells under constant conditions. These revised devices enable the high-throughput, long-term measurement of gene expression dynamics while eliminating several confounding experimental factors that interfere with timescale measurements. In the second part, I employ one of these devices to survey fluctuation timescales in ~50 reporters for Eshcerichia coli gene expression. Under rich conditions, all reporters exhibited nearly identical, rapid fluctuation dynamics that were captured by a simple model of gene expression. In contrast, under poor nutritional conditions gene expression states became correlated over several cell divisions. However, accounting for instantaneous growth rate fluctuations eliminated these slow timescales, revealing an exceedingly simple behavior. In the third part, I describe our work to dissect the stochastic transition between the solitary motile state and sessile multicellular state in exponentially growing Bacillus subtilis</italic.. By enforcing static environmental conditions, we uncover the cell's internal strategies for state switching. The transition to the multicellular state occurs without regard to the cell's state history, whereas commitment to the multicellular state is tightly timed. By manipulating the genetic circuit responsible for the switch, we also expose surprising functional modularity in the commitment. I believe that the striking range of gene expression timescales we observe--from the fast fluctuations in E. coli gene expression to the feedback-amplified noise in B. subtilis--will serve as a useful starting point for future studies.

<p>Neural tube defects (NTDs) are a common class of human birth defects with a complex, multifactorial etiology. Although many contributing factors have been identified, an estimated 60% of human population risk remains unexplained. A portion of that risk is likely attributable to gene-gene and gene-environment interactions which have yet to be fully elucidated. In one project, we used whole-exome sequencing to identify candidate genetic factors in a multiplex anencephaly family, revealing an aggregation of rare and common variants in planar cell polarity genes among the affecteds. In the second project, we profiled the methylomes of a pair of monozygotic twins discordant for anencephaly and identified several differentially methylated sites which could contribute to NTD risk, particularly the <italic>mir-886</italic> locus. Finally, we performed whole-exome and whole-methylome sequencing of mouse strains with differential susceptibility to fumonisin-induced NTDs, in combination with a human SNP association study. We identified epigenetic changes and variant associations which implicate Wnt and Hippo signaling genes as modifiers of the metabolic impacts of fumonisin exposure. These findings underscore the complexity of NTD pathogenesis and highlight the need to elucidate gene-gene and gene-environment interactions contributing to NTD etiology.</p> / Dissertation

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Multiple computational algorithms were developed for analyzing ChIP-seq datasets of histone modifications. For basic ChIP-seq data processing, the problems of ambiguous short sequence read mapping and broad peak calling of diffuse ChIP-seq signals were solved by novel statistical methods. Their performance was systematically evaluated compared with existing approaches. The potential utility of finding meaningful biological information was demonstrated by the applications on real datasets. For biological question driven data mining, several important topics were selected for algorithm developments, including hypothesis-driven insulator prediction, unbiased chromatin boundary element discovery and combinatorial histone modification signature inference. The integrative computational pipeline for insulator prediction not only produced a list of putative insulators but also recovered specific associated chromatin and functional features. Selected predictions have been experimentally validated. The unbiased chromatin boundary element prediction algorithm was feature-free and had the capability to discover novel types of boundary elements. The predictions found a set of chromatin features and provided the first report of tRNA-derived boundary elements in the human genome. The combinatorial chromatin signature algorithm employed chromatin profile alignments for unsupervised inferences of histone modification patterns. The signatures were associated with various regulatory elements and functional activities. Both the computational advantages and the biological discoveries were discussed.

The tumor microenvironment consists of multiple cells types, including endothelial cells that line the tumor vasculature. Tumor vasculature is often abnormal and results in development of tissue ischemia, another contributing factor to the tumor microenvironment. Previous studies have demonstrated that ischemia influences epigenetic programming, but the mechanisms remained unclear and required further investigation. First, we profiled DNA methyltransferase (DNMT) expression and activity in human colorectal cancer cells (HCT116) under hypoxia or hypoglycaemia (mimicking ischemia). We found that DNMT1 and DNMT3b were significantly downregulated by hypoxia and hypoglycaemia, and DNMT3a was downregulated by hypoglycaemia. However, DNMT1 downregulation was p53-dependent. To examine if the changes in DNMT expression and activity translated to changes in DNA methylation patterns, we used bisulfite sequencing and examined the promoter region of p16. Hypoglycaemia significantly demethylated this region in both p53 wild-type and p53-null cells. Next, we used a genome-wide approach to discover what additional genes are hypomethylated by ischemia. Methylated DNA was immunoprecipitated and analysed with an Affymetrix promoter array, in parallel with an expression array. Ingenuity pathway analysis software revealed that a significant proportion of genes which were hypomethylated and upregulated were involved in cellular movement, including PLAUR and CYR61. We believe that hypoxia and hypoglycaemia may be driving changes in DNA methylation through dysregulation of DNMTs, resulting in cells acquiring a more mobile phenotype in ischemic regions. DNMT and histone deacetylase inhibitors are commonly used in research and some cancer therapies. Modifying epigenetic patterning with these inhibitors has been widely studied in cancer cells, but only briefly explored in the tumor’s vascular endothelium. We profiled the effect of these inhibitors on endothelial cell (EC) behaviour, and tested if combining them with a targeted anti-angiogenic therapy would augment the inhibition of angiogenesis. When the DNMT inhibitor 5-aza-2’-deoxycytidine was combined with sunitinib, inhibition of EC proliferation was enhanced compared to treatment with sunitinib alone. EC migration was also inhibited by the combination of these two inhibitors, but not in an additive manner. These studies have improved our understanding of how altering epigenetic patterning with ischemia and therapeutic inhibitors can influence colorectal cancer and endothelial cell behaviour. / Canadian Cancer Society Research Institute. The Cancer Research Society.

Cytotoxic chemotherapy is extremely important in adjuvant treatment of breast cancer. Yet, tumours frequently acquire chemoresistance that correlates with increased aggressiveness and poor prognosis. Three theories exist describing how the resistance develops: genetic, epigenetic and karyotypic theory. The epigenetic theory is the least explored. Here we analyzed the role of the epigenetic phenomena in the acquisition of drug resistance. To do so, we employed genome wide screens of microRNA and gene expression, DNA methylation and complete genome hybridization. We identified three novel microRNA interactions involved in the chemoresistant phenotype. These three microRNAs displayed depressed expression in the resistant cell lines and we were able to re-establish some level of drug sensitivity through ectopic expression of these under expressed microRNAs. In addition, we described the role of DNA methylation in impacting expression of a wide range of genes, thus, contributing to the phenotype of chemoresistance. Furthermore, we revealed a distorted global DNA methylation pattern that coincides with massive instability of the resistant genome. Finally, our results present a striking similarity between gene expression, epigenetic profiles and chromosomal aberrations in two different drug resistant cell lines. Taken together, this project suggests that the acquisition of chemoresistant phenotype is epigenetic in nature and may arise with a predictable pattern. Elucidating the specifics of this pattern may in the future prove useful in developing treatment and prognostic chemoresistance biomarkers. / xiii, 116 leaves : ill. (some col.) ; 29 cm

Myc deregulation critically contributes to many cancer etiologies. Recent work suggests that Myc and its direct interactors can confer a distinct epigenetic state. Our goal is to better understand the Myc-conferred epigenetic status of cells. We have previously identified the long non-coding RNA (lncRNA), H19, as a target of Myc regulation and shown it to be important for transformation in lung and breast cells. These results prompted further analysis to identify similarly important Myc-regulated lncRNAs. Myc-regulated lncRNAs associated with the cell cycle and transformation have been identified by microarray analysis. A small number of candidate lncRNAs that were differentially expressed in both the cell cycle and transformation have been validated. Given the increasing importance of lncRNAs and epigenetics to cancer biology, the discovery of Myc-induced, growth associated lncRNAs could provide insight into the mechanisms behind Myc-related epigenetic signatures in both normal and disease states.

Cancer is the second leading cause of death in the United States (more than half a million people each year), and even with billions of dollars in medical effort patients are rarely cured. This dissertation research is devoted to meeting this medical need by providing new cancer therapeutics that are more potent and safer than current chemotherapies. This is achieved by using two state of the art anticancer “warheads”: 1) gold nanoparticle (AuNP) technology and 2) a new class of epigenetic anticancer small molecules, histone deacetylase inhibitors (HDACi). These warheads are then selectively delivered to cancer cells via “homing devices” targeted to receptors that are overexpressed in the cancers. This work primarily focuses on the androgen receptor (AR) to target prostate cancer. The 1st chapter sets the stage, providing scientific rationale and background for the central hypothesis: small molecules that engage the AR can, upon conjugation to a therapeutic agent, enable selective delivery of that agent to prostate cancer cells. Chapter 2 delves into the structural molecular biology of the androgen receptor. There is a survey of the crystallographic data for all nuclear receptors, providing structural information which is used to build AR homology models for antagonist and inverse agonist modes of ligand binding. These models are used to design AR targeting ligands (Chapters 3, 5, 6 and 7). The application of the targeting technology is illustrated by attaching them to AuNPs for selective delivery to prostate cancer cells (Chapter 3). Next, in order to appreciate the importance of using targeting agents in HDACi cancer therapeutics, we reviewed this recently emerged field in Chapter 4. In this chapter we argue that the failure of HDACi in solid tumors, despite more than 500 clinical trials in the last decade, is primarily due to an inability of these small molecules to accumulate at effective concentrations in the cancer. We provide an analysis of the paradigms being pursued to overcome this barrier, including HDAC isoform selectivity, localized administration, and targeting cap groups to achieve selective tissue and cell type distribution. In Chapter 5, this last approach (targeting cap groups, or a “homing device”) is illustrated with HDACi targeted to prostate cancer via antiandrogens that bind the AR. The second generation of improved “homing devices” is disclosed in Chapter 6 (for both AuNPs and HDACi), in addition to preliminary ADMET data and safety studies in mice. Excitingly, our three dimensional understanding of binding to the AR allowed design and structure-activity-relationship studies that lead to the first reported examples of AR inverse agonists (Chapter 7) Several points of significance: • AuNP targeted to AR ∙ have the strongest binding affinity ever reported (IC50 ~14 picomolar) ∙ are actively recruited to prostate cancer cells ∙ overcome treatment resistance in advanced prostate cancer cells ∙ exhibit nanomolar anticancer potency ∙ resolved the identity of the “membrane AR” as the GPRC6A • HDACi targeted to AR ∙ have HDACi activity and AR binding affinity superior to their clinical precursors ∙ exhibit potent AR antagonist activity ∙ induce AR translocation to the nucleus in a HDACi dependent fashion ∙ selectively and potently kill prostate cancer cells that express AR ∙ are safer than Tylenol®, as tested in small animals • Pure AR binding ligand studies ∙ resulted in the discovery of the first examples of AR inverse agonists, which are vastly more potent that clinically available antiandrogens for prostate cancer ∙ work via a never-before-seen mechanism of action, by localizing to the nucleus and recruiting corepressors to actively shut off AR genes

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No description available.