Glycogen Synthase Kinase-3 (GSK-3) is a highly conserved serine/threonine kinase comprised of two mammalian homologues, GSK-3α and β, encoded by independent genes. This thesis reports the characterization of GSK-3-null primary mouse embryonic fibroblasts (MEFs) generated by gene targeting to gain insight into the physiological functions of this protein kinase. Combined inactivation of both alleles of GSK-3α and GSK-β led to elevated sensitivity to TNFα-induced apoptosis, altered organization of focal adhesion complexes, defects in cell spreading on fibronectin, decreased cell growth associated with altered cell cycle progression through the G2/M phase and increased spontaneous apoptosis. Future work will focus on unraveling the molecular mechanisms responsible for these effects and identifying the common and distinct cellular roles for GSK-3α and β, and specific variants of these isoforms.

Hedgehog (Hh) signaling is essential for embryonic development and adult homeostasis. Aberrant pathway activity can result in various developmental disorders and cancers. The Hh receptor Patched1 (Ptc1) is a negative regulator of the pathway and acts as a tumor suppressor. Our lab and others have shown that Suppressor of fused (Su(fu)) and Kinesin family member 7 (Kif7) are major negative regulators of the pathway that function downstream of Ptc1. Medulloblastoma (MB) is the most common malignant pediatric brain tumor originating from the cerebellum. Several forms of MB have been identified, with abnormal activation of the Hh pathway associated with one major subtype. These tumors commonly show inactivating mutations in PTCH1, whereas mutations in SU(FU) are more rare. Mouse models with deletion of Ptc1 or expression of a constitutively active form of Smoothened (Smo) exhibit elevated Hh pathway activity, leading to MB formation. In this study, I examined the complex roles of Su(fu) in the formation of MB alongside the inactivation of Ptc1, Kif7 and p53 in the cerebellum. Unlike Ptc1+/- mice, Su(fu)+/- mice do not develop MB, even upon exposure to DNA damaging agents (X-ray irradiation or chemical carcinogenesis), which dramatically increases MB incidence in Ptc1+/- mutants. However, Su(fu)+/-;p53-/- mice develop MB and these MB tumor cells exhibit loss of heterozygosity of Su(fu), suggesting a protective role of p53 in tumor suppression in Su(fu) deficiency. Kif7+/- mice are not prone to tumorigenesis, even with deletion of p53. To bypass the embryonic lethality of Su(fu)- and Kif7-null mice, I generated neural stem cell-specific knockout mice, GFAP-Cre;Su(fu)f/f and GFAP-Cre;Kif7f/f. GFAP-Cre;Su(fu)f/f mice exhibit a severely disorganized cerebellum, with drastic up-regulation of p53 expression, and they survive past 1 year of age and do not develop MB. In contrast, GFAP-Cre;Kif7f/f mice appear grossly normal with only subtle cerebellar defects. These observations indicate that neither Su(fu) nor Kif7 inactivation is sufficient to drive tumorigenesis in the cerebellum. To investigate whether Su(fu) and Kif7 possess any overlapping tumor suppressor functions, I generated GFAP-Cre;Kif7f/f;Su(fu)f/f mice. Indeed, simultaneous loss of these negative regulators resulted in MB formation, which is correlated with increased Hh pathway activity as well as a lower level of p53 expression. Furthermore, I discovered a novel positive role for Su(fu) in MB development, as Su(fu) activity is required for robust formation of MB in Ptc1-deficient mice. Together, these data illustrate that Su(fu) plays a dual role in the genesis of MB, and suggest that the rare human MBs with SU(FU) mutations could be caused by simultaneous deletion of TP53 or KIF7.

Transient Receptor Potential Melastatin 2 (TRPM2) is a widely expressed, non-selective cationic channel with implicated roles in cell death, chemokine production and oxidative stress. This study characterizes a novel interactor of TRPM2. Using fusion proteins comprised of the TRPM2 C-terminus we established that tubulin interacted directly with the predicted C-terminal coiled-coil domain of the channel. In vitro studies revealed increased interaction between tubulin and TRPM2 during LPS-induced macrophage activation and taxol-induced microtubule stabilization. We propose that the stabilization of microtubules in activated macrophages enhances the interaction of tubulin with TRPM2 resulting in the gating and/or localization of the channel resulting in a contribution to increased intracellular calcium and downstream production of chemokines.

TTumor hypoxia (poor oxygenation) adversely affects patient prognosis by promoting therapeutic resistance and an aggressive tumor phenotype. We aimed to understand how urokinase plasminogen activator receptor (uPAR), a cysteine-rich protein implicated in the malignant phenotype and poor patient prognosis, matures in hypoxia. We hypothesized that secretion of uPAR during hypoxia is conferred by a superior ability to form disulfide bonds without oxygen. A model and assay was established to monitor the oxygen-dependency of suPAR (a soluble secreted isoform of uPAR) folding and secretion. We found that suPAR maturation involves disulfide formation and N-linked glycosylation in normoxia. In anoxia, suPAR disulfide formation was impaired, but suPAR was nevertheless secreted. We propose that suPAR has low dependency on disulfide formation for efficient secretion in comparison to other disulfide-containing proteins. Mechanisms supporting protein expression during hypoxia may potentially be targeted to mitigate the adverse effects of tumor hypoxia and ultimately improve cancer therapy.

The yeast Saccharomyces cerevisiae is a prevalent system for studying gene regulation because of the ease of experimental methods and the simplicity of its gene structure. Here, I describe my work that aims to identify the sequences and factors responsible for demarcating genes within the genome sequence. With comparative genomics and RNA-Seq, we are quite adept at identifying gene structure. However, the cell does not have access to this kind of information. Instead, it uses the specificities of DNA- and RNA-binding proteins to read and interpret the sequence of the genome; it is this process that I have studied in my thesis. In the first chapter, I describe my work collecting yeast transcription factor specificities. I evaluated these specificities using available confirmatory data to determine which one best represents the transcription factor; this gave me a high-confidence description of what DNA sequences yeast transcription factors recognize. Next, I look for over- and under-represented DNA words within and surrounding gene structures and attempt to explain these in terms of the specificities of known factors or other known biological phenomena. I found that the sequences in the 5' and 3' gene ends are very similar and can often be explained by similar phenomena. I also provide evidence that several factors may be involved in regulating transcription in non-canonical ways. In the final chapter, I describe my efforts to build a model that uses my collection of transcription factor specificities as well as DNA structural features to identify gene structure as we think the cell would. This model is comprised of two classifiers that identify mRNA initiation and termination sites, and these are used to provide evidence to a hidden Markov model that predicts gene structure. I test that the predicted determinants of promoter structure are sufficient to initiate transcription, and that the transcription arising from randomly-generated DNA is correctly predicted. Overall, my work demonstrates that the sequence elements demarcating yeast genes are relatively simple in nature, which has implications for how transcription is regulated and how genes evolve.

Tyrosine kinases (TK) are important for cardiac function, but their downstream targets in the adult heart have yet to be established. The ShcA docking protein binds specific phosphotyrosine (pTyr) sites on activated TKs through its N-terminal PTB and C-terminal SH2 domains and stimulates downstream pathways through motifs such as pTyr sites in its central CH1 region. To explore the role of this TK scaffold in the adult heart, we generated a myocardial-specific knockout of murine ShcA (ShcA CKO). Such mice developed a dilated cardiomyopathy phenotype involving impaired systolic function with enhanced cardiomyocyte contractility. This uncoupling of global heart and intrinsic myocyte functions was associated with altered perimysial collagen and extracellular matrix complicance properties, suggesting disruption of mechanical coupling. In vivo dissection of ShcA signaling properties revealed that selective inactivation of the PTB domain in the myocardium had effects resembling those seen in ShcA CKO mice, while disruption of the SH2 domain caused a less severe cardiac phenotype. Downstream signaling through the CH1 pTyr sites was dispensable for baseline cardiac function, but necessary to prevent adverse remodeling after hemodynamic overload. Therefore, ShcA mediates pTyr signaling in the adult heart through multiple distinct signaling elements that control myocardial functions and response to stresses.

Tyrosine kinases (TK) are important for cardiac function, but their downstream targets in the adult heart have yet to be established. The ShcA docking protein binds specific phosphotyrosine (pTyr) sites on activated TKs through its N-terminal PTB and C-terminal SH2 domains and stimulates downstream pathways through motifs such as pTyr sites in its central CH1 region. To explore the role of this TK scaffold in the adult heart, we generated a myocardial-specific knockout of murine ShcA (ShcA CKO). Such mice developed a dilated cardiomyopathy phenotype involving impaired systolic function with enhanced cardiomyocyte contractility. This uncoupling of global heart and intrinsic myocyte functions was associated with altered perimysial collagen and extracellular matrix complicance properties, suggesting disruption of mechanical coupling. In vivo dissection of ShcA signaling properties revealed that selective inactivation of the PTB domain in the myocardium had effects resembling those seen in ShcA CKO mice, while disruption of the SH2 domain caused a less severe cardiac phenotype. Downstream signaling through the CH1 pTyr sites was dispensable for baseline cardiac function, but necessary to prevent adverse remodeling after hemodynamic overload. Therefore, ShcA mediates pTyr signaling in the adult heart through multiple distinct signaling elements that control myocardial functions and response to stresses.

The budding yeast Saccharomyces cerevisiae is the most well characterized model organism for systematic analysis of fundamental eukaryotic processes. Approximately 19% of S. cerevisiae genes are considered essential. Essential genes tend to be more highly conserved from yeast to humans when compared to nonessential genes. The set of essential yeast genes spans diverse biological processes and while the primary role of most essential yeast genes has been characterized, the full breadth of function associated with essential genes has not been examined, due, at least in part, to the lack of adequate genetic reagents for their conditional and systematic perturbations. To systematically study yeast essential gene functions using synthetic genetic array analysis and to complement the current yeast deletion collection, I constructed a collection of temperature-sensitive yeast mutants consisting of 795 ts strains, covering 501 (~45%) of the 1,101 essential yeast genes, with ~30% of the genes represented by multiple alleles. This is the largest collection of isogenic ts yeast mutants constructed to date. I confirmed the correct integration of over 99% of the ts alleles using PCR-based strategy and the identity of the ts allele by complementation of the ts phenotype with its cognate plasmid. The ts mutant collection was characterized by high-resolution profiling of the temperature sensitivity of each ts strain, distribution analysis of gene ontology molecular function and biological process, and comparison of ts allele strains to the strains carrying Tet-repressible alleles of essential genes. The results demonstrated that the ts collection is a powerful reagent for the systematic study of yeast essential gene functions and provides a valuable resource to complement the current yeast deletion collection. I validated and demonstrated the usefulness of the ts collection in a number of different ways. First, I carried out detailed temperature profiling of each mutant strain using liquid growth assays and found that ts mutants that define particular biological pathways often show highly similar profiles. Second, I showed that the ts mutant array can be used to screen compounds for suppression of growth defects and thus is useful for exploration of chemical-genetic interactions. Third, I demonstrated that the ts collection represents a key reagent set for genetic interaction analysis because essential genes tend to be highly connected hubs on the global genetic network. Fourth, I further validated the ts array as a key resource for quantitative phenotypic analysis by using a high-content screening protocol to score six different fluorescent markers, diagnostic for different subcellular compartments or structures, in hundreds of different mutants. Quantification of the marker behaviour at the single-cell level enabled integration of this data set to generate a morphological profile for each ts mutant to reveal both known and previously unappreciated functions for essential genes, including roles for cohesion and condensin genes in spindle disassembly.

Many current efforts in cancer research focus on understanding the molecular mechanisms driving oncogenesis and to advance molecular diagnostics and targeted therapeutics. The MYC oncoprotein is estimated to be deregulated in over 50% of human cancers, and its deregulation is often associated with aggressive disease and poor patient outcomes. While the ability of MYC to promote cellular transformation is well established, a better understanding of the mechanisms promoting MYC-mediated tumorigenesis is essential. While MYC has been shown to undergo a number of post-translational modifications (PTMs), our current understanding of biological significance of these modifications is largly limited to two phosphorylation sites located in the N-terminal domain of the protein. Our work, therefore, aimed to further our understanding of how PTMs regulate MYC-dependent transformation. To this end we have identified and characterized three novel human cell line models of MYC-dependent transformation: MCF10A, SH-EP Tet21/N-Myc, and LF1/TERT/LT/ST cells. Using a combination of these novel models and classic systems, we have evaluated point mutants of MYC at key serine/threonine and lysine residues for their ability to influence MYC-dependent transformation. Using a six lysine to arginine substitution mutant, we have identified and chacterized six C-terminal lysines to be important for the negative regulation of MYC activity. We have additionally demonstrated for the first time that MYC can undergo SUMOylation at one of the lysines in this region. We further completed a functional and transcriptional characterization of MYC phosphorylation mutants. We have assigned biological significance to previously identified phosphorylation sites through the characterization of two mutants that have increased transformation potential over wild-type MYC. Expression array analysis identified gene expression changes both common to deregulated MYC and unique to the different gain-of-function phosphorylation mutants. Combined, this work has advanced our understanding of several of the mechanisms that may regulate MYC-induced transformation.

Myc is an oncogene that is commonly deregulated in human cancers. Mechanistic studies reveal that Myc is a transcription factor that interacts with a protein partner called Max. Heterodimerization and the formation of the Myc:Max complex enables Myc:Max to bind to the E-box and subsequently regulate the activation and repression of Myc target genes. Since regulation of its target genes are essential for Myc to drive transformation, the Myc and Max interaction has been targeted in mouse model studies to determine whether the oncogenic activity of Myc can be inhibited. Surprisingly, these studies reveal that targeting Myc is not only possible but a powerful way to suppress tumour growth. Since a better understanding of how Myc carries out its biological functions makes the possibility of targeting Myc a reality, it is essential to investigate and study the mechanisms of how Myc promotes tumourigenesis. In the first part of this thesis, we investigate the idea that the N-terminal end of Myc may be post-translationally modified and this modification may dictate Myc activities. Indeed we report here that the N-terminal end of Myc can be ubiquitylated as well as acetylated, and that the loss of these modifications results in a decrease in Myc activities. Furthermore, we characterize Mdm2 as a potential E3 ubiquitin ligase that may ubiquitylate the N-terminal end of Myc. In the second part of this thesis, we investigate CUL7 as a novel oncogene that may inhibit Myc-potentiated apoptosis and cooperate with Myc in transformation. Indeed, CUL7 is a novel p53 interacting protein that inhibits Myc potentiated apoptosis through the inhibition of p53. We have also characterized CUL7 to be overexpressed in primary human lung cancers, and a higher level of CUL7 expression associates with short-term survival of lung cancer patients. Through the better understanding of the enzymes that post-translationally modify the N-terminal end of Myc and proteins, such as CUL7, that can cooperate with Myc to drive tumourigenesis, we may begin to devise ways to target and control deregulated Myc in cancer cells.