Erythropoietin (EPO) is the primary cytokine regulator of erythropoiesis. Fundamental to this action is the ability of EPO to bind the EPO receptor (EPO-R), and activate the primary associated tyrosine kinase, JAK2. The critical importance of EPO, EPO-R and JAK2 to erythropoiesis is demonstrated by the fatal embryonic anemia that develops upon EPO, EPO-R or JAK2 deletion. Positive regulation of intracellular signal transduction pathways downstream of EPO-R and JAK2 are well documented, but less is known about negative regulation of EPO-R signaling. Two distinct experimental strategies were utilized to examine a subset of the negative regulatory mechanisms underlying EPO-R signaling and erythropoiesis. Mice deficient in the E3 ubiquitin ligase, Cbl, were generated previously and displayed elevated platelet numbers, expansion of splenic red pulp and splenomegaly, suggesting that Cbl-/- mice have defects in megakaryocyte/erythrocyte progenitors or more committed cells of each lineage. Our studies illustrated that genetic ablation of Cbl resulted in elevated total numbers of Burst Forming Unit-Erythroid and Colony Forming Unit-Erythroid, but decreased bone marrow-derived late erythroblasts. Cbl-deficient late erythroblasts displayed elevated apoptosis, as well as increased expression of Foxo3a and increased mRNA levels of the pro-apoptotic genes, Bim and FasL. These studies implicate Cbl as an important negative regulator of multiple facets of erythroid signaling. The discovery that EPO-R is ubiquitinated and degraded by the proteasome and lysosome led us to examine the role of EPO-R ubiquitination on signal transduction and proliferation. Lysine mutagenesis of EPO-R showed that K348, K388 and K428 were the primary ubiquitin acceptor sites when EPO-R mutants were expressed in HEK 293T cells. BaF3 cells expressing an EPO-R deficient in cytoplasmic lysines displayed diminished EPO-mediated EPO-R, JAK2, PKB and STAT5 phosphorylation and could not proliferate in response to EPO. The membrane proximal lysines of EPO-R, K256 and K276, were necessary for proliferation at physiologic EPO concentrations but were not required at saturating EPO concentrations. Single lysine EPO-R add-back mutants restored signaling and proliferation to BaF3 cells at physiologically elevated EPO concentrations, signifying that EPO-R lysines finely mediate EPO-dependent proliferation and signal transduction. These analyses demonstrate a positive regulatory role for lysines in signal transduction and proliferation.

Inheritance of one mutant von Hippel-Lindau (VHL) allele gives rise to the development of the autosomal dominant VHL disease, which affects approximately 1 in 36 000 individuals. The VHL tumour suppressor protein plays a critical role in the E3 ubiquitin ligase-mediated destruction of hypoxia-inducible factor (HIF) and the promotion of fibronectin extracellular matrix assembly. A failure in either process is associated with oncogenic progression. Work included in this thesis provides evidence that these tumour suppressor functions are mutually exclusive. Additionally, post-translational modification of VHL by NEDD8 is shown to act as a ‘molecular switch’, altering VHL protein associations and providing a mechanism of pathway segregation. As a result of HIF stabilization, the expression of a homophilic adhesion molecule E-cadherin is significantly down-regulated in primary renal clear-cell carcinoma (RCC) upon VHL loss. E-cadherin down-regulation is shown to increase the invasive potential and is of prognostic value in RCC. Finally, VHL and SOCS1 are shown to dimerize and negatively regulate the JAK2-STAT5 signalling cascade. Defects in this dimerization are shown to underlie Chuvash polycythemia and provide a molecular understanding of the phenotypic observations associated with VHL-related polycythemias.

MicroRNA is a single-stranded RNA molecule of about 22 nucleotides in length and is expressed endogenously. It functions as a gene regulator by pairing imperfectly with 3’ untranslated region (3’UTR) of target mRNAs, leading to translational inhibition. MicroRNA is implicated in many regulatory pathways and hence affects various cellular activities. In the development of cancer, genetic alterations occurred at miRNA locus and its expression level is dysregulated in various cancers versus normal tissue counterparts. It is thus important to find the targets of dysregulated microRNAs contributing to progression of cancer. To facilitate long term functional studies, a microRNA expression construct with unique futures was generated. Stable expression of miR-378 enhanced cell survival, reduced caspase-3 activity, and promoted tumor growth and angiogenesis. By algorithmic predictions and proteomic analysis, two tumor suppressors, SuFu and Fus-1, were found to be translationally regulated by miR-378. Target validation was confirmed by co-transfection experiments and luciferase activity assays, reassuring its oncogenic role by regulating two tumor suppressor genes simultaneously. Conversely, microRNA can also function as a tumor suppressor by modulating expression of Versican, an extracellular matrix protein known to facilitate tumorigenesis and angiogenesis. By a novel PCR method, more than one microRNA were found to bind to Versican 3’UTR. iii Among these microRNAs, targeting of Versican and Fibronectin by miR199a-3p was validated. Expression of a fragment of Versican 3’UTR was expected to antagonize the function of miR-199a-3p. Stable expression of Versican 3’UTR resulted change in cell morphology and increased cell-cell adhesion. Analysis of primary tissues from transgenic mice expressing versican 3’UTR showed an increase expression of Versican and Fibronectin, and organ adhesion was found between liver and its surrounding tissues. In addition, 3’UTR also modulated the level of miR-199a-3p and miR-136, alleviating translation of negative cell cycle regulators, PTEN and Rb1. This resulted in reduced cell proliferation and hence diminished tumor growth. These findings suggest a role of microRNA in tumor growth, providing a valuable target for therapeutic intervention.

The CTCF protein is involved in several important aspects of gene regulation including transcriptional activation, transcriptional repression and insulator ability. It is also involved in the regulation of epigenetic processes including X-chromosome inactivation and the maintenance of genomic imprinting. CTCF has been shown to bind to approximately 15 000 sites in the mammalian genome and has been implicated in nuclear organization. The CTCF protein mediates long-range chromatin interactions and is believed to form DNA loops. It also acts to block the communication of an enhancer with a promoter by acting as an insulator. Despite its importance in gene regulation, the molecular mechanisms that govern CTCF’s ability to perform its myriad functions remain enigmatic. In this thesis, I add insight into our understanding of the mechanisms behind CTCF’s function. I show that CTCF is post-translationally modified by the Small Ubiquitin-like Modifier proteins and that this post-translational modification contributes to its repressive ability at the c-myc P2 promoter. I also show that CTCF is localized to the sub-nuclear compartment called the Polycomb bodies. The Polycomb protein Pc2 acts as an E3 ligase to enhance the SUMOylation of CTCF by SUMOs 2 and 3. These findings help to explain CTCF’s ability to act as a transcriptional repressor. I also report biochemical evidence to support the role for CTCF in forming an unusual DNA structure, possibly a loop. I hypothesize that a single CTCF binding site is able to form DNA loops. These findings suggest mechanisms by which CTCF is able to organize the mammalian genome and to function as an insulator protein. In addition to these findings I have also purified CTCF interacting proteins through the use of the tandem affinity purification technique. The interacting proteins contain many chromatin and DNA binding proteins further suggesting a role for CTCF in chromatin organization. The results in this thesis enhance our knowledge of the molecular mechanisms of CTCF function and provide a basis for the improved understanding of CTCF mediated gene expression.

The Varkud satellite (VS) plasmid is found in the mitochondria of some strains of Neurospora, and exhibits properties that may allow it to be developed as a genetic transformation vector to study mitochondrial molecular biology. An ideal transformation vector would not confer a phenotype. The overall goal of my thesis was to examine interactions of VS with its Neurospora host to identify possible phenotypes, using biochemical and proteomic approaches. Biochemical experiments provided evidence consistent with the plasmid transcript, VS RNA, being present as a ribonucleoprotein particle that can be separated from ribosomal subunits by sucrose density gradient centrifugation; however, no VS-specific proteins were identified under the purification conditions examined. During the analyses of proteomic data I obtained new insights into the consequences of the statistical methods commonly used to normalize quantitative 2D gel data. However, irrespective of the method used, the fraction of the proteome amenable to 2D gel-based proteomics revealed, at most, subtle effects of VS on the abundance of a few proteins. I also observed no differences in growth rate between strains differing by the presence or absence of VS when grown in the presence of inhibitors and stressors affecting a wide range of mitochondrial and other cellular functions. Overall, despite VS RNA being as abundant as the large and small mitochondrial ribosomal RNAs, my genetic, biochemical and proteomic investigations of the effect of VS on its host strain provides evidence that VS is a phenotypically neutral element.

Bioremediation of chlorinated solvents to a non-toxic end product can be achieved with Dehalococcoides sp., through reductive dehalogenation of the chlorinated organics. Dehalococcoides sp. are typically maintained in enrichment cultures containing multiple microorganisms, which often exhibit better growth and dechlorination rates than Dehalococcoides isolates. This thesis examines the nature of the relationships between the Dehalococcoides and the non-dechlorinating organisms in enrichment cultures. Comparative metagenomics revealed differences and similarities in taxonomy and functional gene complements between three Dehalococcoides-containing enrichment cultures. This allowed identification of pivotal supporting organisms involved in maintaining dechlorination activity through provision of nutrients and other factors to the Dehalococcoides. A Dehalococcoides pan-genus microarray was designed using available sequenced genomes as well as a draft genome generated from an in-house metagenome sequence. The array leverages homolog clustering during probe design to improve detection of the Dehalococcoides genus, including strains not utilized in the array design. A phylogenetic examination of the reductive dehalogenase gene family showed that organism and gene phylogenies are not linked, indicating vertical inheritance of reductive dehalogenases is not a primary mechanism of acquisition. Design of a universal PCR primer suite targeting a curated database of reductive dehalogenase homologous genes was used to characterize the reductive dehalogenase complement of four environmental sites and two enrichment cultures. Using an enrichment culture containing three phylogenetically distinct dechlorinating organisms, the interactions of niche-specific organisms were examined through single-cell genome sequencing. From the partial genome sequences, novel reductive dehalogenase genes were identified, as well as evidence of lateral gene transfer between the three dechlorinating organisms. This research primarily utilizes genomic and metagenomic datasets, which serve as metabolic blueprints for prediction of organisms’ functions. The results presented in this thesis advocate in favour of phylogenetic diversity within enrichment cultures to maintain functional redundancy, leading to more robust cultures for bioremediation efforts.

Nonhomologous end-joining (NHEJ) is a major DNA double-strand break (DSB) repair pathway. NHEJ is initiated through DSB recognition by the DNA end-binding heterodimer, Ku, while end-joining is accomplished by the XRCC4-DNA ligase IV (X4L4) complex. This thesis reports that APLF (Aprataxin and Polynucleotide kinase-Like Factor), an endo/exonuclease with a forkhead-associated (FHA) domain and two unique zinc fingers (ZF), interacts with both Ku and X4L4. The APLF-X4L4 interaction is FHA- and phospho-dependent, and is mediated by CK2 phosphorylation of XRCC4 in vitro. APLF binds Ku independently of the FHA and ZF domains, and complexes with Ku at DNA ends. APLF undergoes ionizing radiation induced ATM-dependent hyperphosphorylation and ATM phosphorylates APLF in vitro. Downregulation of APLF is associated with defective NHEJ and impaired DSB repair kinetics. These results suggest that APLF is an ATM target that is involved in NHEJ and facilitates DSB repair, likely via interactions with Ku and X4L4.

Wnt signalling is involved throughout development and is inappropriately activated in a variety of human cancers. In the canonical pathway, secreted Wnt proteins induce the stabilization of b-catenin. Drosophila Naked (Nkd), or Nkd1 and Nkd2 in vertebrates, is believed to antagonize canonical Wnt signalling through an interaction with Dishevelled (Dvl). Analysis of a high-throughput protein-protein interaction screen conducted in our lab led to the identification of novel Nkd1 interacting proteins, including Nkd1/2 and Axin1. Mapping of Nkd1 regions required for these novel interactions and functional studies, including transcriptional and siRNA mediated knockdown assays, were performed to examine the role of Nkd1 in canonical Wnt signalling. Previous work suggests that Nkd1 functions only through Dvl, but this work suggests that Nkd1 acts via a more complex mechanism. In addition to serving as an antagonist to regulate the canonical Wnt pathway, we propose that Nkd1 may also act positively to promote signalling.

Hyperosmotic stress represents a major threat to cellular integrity and may lead to cell death via apoptosis. Accordingly, each cell reacts to hyperosmolarity with a set of functional and structural compensatory responses. Recently it has been shown that the mitochondria remodel during hyperosmotic stress. Although changes in mitochondrial dynamics could be crucial for both adaptation and apoptosis, hyperosmolarity-induced mitochondrial remodeling has not been characterized. We found that hyperosmotic stress translocates dynamin like protein 1 (DLP-1) to the mitochondria and induces DLP-1 mediated, F-actin-modulated, Rac-dependent fragmentation of these organelles in LLC-PK1 cells. Downregulation of DLP-1 mitigates the activation of the osmotic response element and increases the susceptibility of tubular cells to hyperosmotically-induced apoptosis, suggesting that DLP-1 (or mitochondrial fragmentation) may have a protective role during osmotic stress. The hyperosmolarity-triggered remodeling of the mitochondrion represents a hitherto unrecognized response to osmotic shock, which may have significant impact on adaptation and apoptosis.

A DNA palindrome is a sequence of DNA followed by an exact inverted copy of itself. Palindromes are associated with gross chromosomal instability in mammalian cells. This may be related to their ability to extrude a double-stranded cruciform structure. In mammalian cells, palindromes have been shown to undergo centre-directed rearrangements resulting in a central region of asymmetry. This process occurs via a mechanism termed â centre break palindrome revisionâ . In this thesis, I have investigated palindrome revision in mammalian cells using two existing assays. In the first, performed by transfection of an extrachromosomal palindromic dimer, I have shown that joining of palindrome-mediated double-strand breaks does not depend solely on NHEJ and instead relies heavily on an alternate end-joining pathway. Using the second assay, the Line78 mouse model which contains a 15.4kb transgenic palindrome, I have shown that small modifications near the centre of the palindrome prevent these centre-directed rearrangements possibly by inhibiting cruciform formation.