Medulloblastoma is the most common pediatric brain tumour, yet many of the underlying genetic and epigenetic factors have yet to be discovered. After a genome wide screen of a large cohort of primary medulloblastomas, we discovered that many of the genes within the cell adhesion family are affected by either copy number loss and/or decreased expression unexplained by copy number change. This led us to believe that both genetic and epigenetic factors were affecting this gene family. Through methylation-specific PCR, RT-PCR and high-throughput methylation status analysis, we have concluded that promoter CpG methylation plays a role in the expression of the PCDH10 protein in both medulloblastoma cell lines and primary tumours. Through functional validation with a stable cell line re-expressing PCDH10, I show that cell cycle and proliferation remain unchanged but migration is decreased in cells with PCDH10 re-expression. This suggests that PCDH10 has characteristics of a tumour suppressor in medulloblastoma.

Mucopolysaccharidoses result from genetic mutations in lysosomal enzymes required for degradation of glycosaminoglycans. The deficiency in any of eight lysosomal enzymes needed to degrade heparan sulfate leads to an accumulation of both non-degraded and partially degraded polysaccharides within the lysosomes of many tissues. Interestingly, six of these deficient enzymes can be treated by a relatively new approach – substrate reduction therapy (SRT), which aims to reduce the synthesis of the substrate for the deficient enzyme being targeted. I developed a cell-based high throughput screen assay for the identification of compounds that decrease the expression of the first modifying enzyme in HS biosynthesis, N-deacetylase/N-sulfotransferase 1, by inhibiting the transcription of its mRNA. From the high throughput screen, I identified several compounds, with a previous history of use in humans, which significantly decreased the endogenous NDST1 expression and therefore, could be considered as potential SRT agents for up to six Mucopolysaccharidoses.

Mucopolysaccharidoses result from genetic mutations in lysosomal enzymes required for degradation of glycosaminoglycans. The deficiency in any of eight lysosomal enzymes needed to degrade heparan sulfate leads to an accumulation of both non-degraded and partially degraded polysaccharides within the lysosomes of many tissues. Interestingly, six of these deficient enzymes can be treated by a relatively new approach – substrate reduction therapy (SRT), which aims to reduce the synthesis of the substrate for the deficient enzyme being targeted. I developed a cell-based high throughput screen assay for the identification of compounds that decrease the expression of the first modifying enzyme in HS biosynthesis, N-deacetylase/N-sulfotransferase 1, by inhibiting the transcription of its mRNA. From the high throughput screen, I identified several compounds, with a previous history of use in humans, which significantly decreased the endogenous NDST1 expression and therefore, could be considered as potential SRT agents for up to six Mucopolysaccharidoses.

Programmed cell death (PCD or apoptosis) is an evolutionarily conserved, genetically controlled suicide mechanism for cells, which when deregulated, can lead to developmental defects, cancers and degenerative diseases. In C. elegans, DNA damage induces germ cell death by signaling through cep-1/p53 ultimately leading to the activation of the CED-3/caspase. It has been hypothesized that the major regulatory events controlling cell death occur by cell autonomous mechanisms, that is within the dying cell. In support of this, genetic studies in C. elegans have shown that the core apoptosis pathway genes ced-4/APAF1 and ced-3/caspase are required in cells fated to die. However, it is not known whether the upstream signals that activate apoptosis function in a cell autonomous manner. Here I show that two genes, kri-1, an ortholog of KRIT1/CCM1 that is mutated in the human neurovascular disease cerebral cavernous malformations (CCMs) and daf-2, an insulin-like receptor, are required to activate DNA damage-dependent cell death independently of cep-1/p53. Interestingly, I found that both genes can regulate cell death in a non-autonomous manner, revealing a novel role for non-dying cells in eliciting death in response to DNA damage.

The roundworm Caenorhabditis elegans possesses a single, conserved phosphatidylinositol 3-kinase (PI3K) signaling pathway that regulates somatic developmental decisions and lifespan through the Insulin-like receptor tyrosine kinase (RTK) DAF-2, the class I PI3K AGE-1 and the 3-phosphoinositide-dependent protein kinase 1 (PDK1) homologue PDK-1. This pathway ultimately controls the action of Akt homologues on the forkhead transcription factor DAF-16. The C. elegans Akt orthologue akt-1 also negatively regulates the DNA damage-dependent apoptosis of worm germ cells by indirectly interfering with activation of the key transcription factor CEP-1, the sole homologue of p53 in the worm. Because upstream regulation by RTK/PI3K signaling is known to couple with downstream Akt kinase activity, I hypothesized that the worm daf-2/age-1/pdk-1 pathway would function upstream of akt-1/Akt in response to DNA damage. Surprisingly, this was not the case: daf-2/InsR and pdk-1/PDK1 do not function upstream of akt-1/Akt and instead promote DNA damage-induced germ cell apoptosis independently of CEP-1/p53 by regulating the B cell lymphoma (Bcl2) homologue CED-9 and the Apoptotic protease-activating factor 1 (Apaf1)-like adapter protein CED-4, respectively. Furthermore, PDK-1/PDK1 promotes germ cell apoptosis by a mechanism that does not include changes in the subcellular localization or absolute levels of CED-4/Apaf1, but does require the presence of CEP-1/p53. Therefore, daf-2/RTK, pdk-1/PDK1, and cep-1/p53 co-operate from independent pathways to drive germ cell death. The separation of worm Akt function from canonical RTK/PI3K regulation is consistent with the ability of AKT-1 to function without major changes in phosphorylation at threonine 350, a site homologous to Thr308 in mammals. Since this modification is an essential step in the activation of Akt proteins by PDK1, it is likely that damage-dependent germline activity of AKT-1 is controlled by a novel mechanism that does not involve phosphorylation by PDK-1 on key regulatory sites. These data argue that C. elegans re-arranges single homologous components of a signalling pathway to respond to different stimuli in vivo. Finally, I present data identifying the C. elegans ataxia and telangectasia and Rad3-related (ATR) kinase homologue ATL-1 as a potential target of AKT-1. Collectively, my work has uncovered a novel DNA damage-dependent pathway that allows AKT-1 to control CEP-1/p53-dependent apoptosis.

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.

The fundamental repeating unit of all eukaryotic chromatin is the 147bp DNA:histone complex known as the nucleosome. Genome-wide studies have demonstrated that nucleosomes are organized with the 5’ promoter being nucleosome depleted and the transcribed region is occupied by a periodic array of positioned nucleosomes. While this organization is well described, the determinants, particularly trans-acting factors that contribute to this architecture are only partly described with gene expression, however, while the connection between chromatin and the various facets of gene expression regulation, especially in evolution, is apparent the detailed mechanisms remain to be described. In this thesis, I describe 1) The role of nucleosomes in gene expression evolution in closely related yeast species 2) The role of trans-acting factors (particularly transcription factors and co-factors) in determining the nucleosome depleted region of promoters and 3) The role of trans-acting factors in nucleosome spacing within genes.

Genomic evolution is a continuous process that involves the accumulation of neutral and adaptive variation within DNA sequences. Duplication, a mechanism that introduces new genetic material into a genome, is thought to be the primary source of new genes that have arisen during vertebrate evolution. This hypothesis, popularized by Susumu Ohno in 1970, has transformed the field of evolutionary biology. Consequently, many evolutionary studies have concentrated on identifying examples of gene duplication and assessing their impact on the evolution of genomes. This thesis presents the identification and analysis of three examples of gene duplication involved in shaping mammalian genomes. Through these analyses, I investigate the fate of duplicated genes and discuss the potential impact of duplication on genomic evolution. The fates depicted within these studies range from the pseudogenization of recent gene duplications to the preservation of ancient duplications for over 100 million years in multiple mammalian genomes. The consequences of these fates include neofunctionalization, subfunctionalization, and gene relocation. In additional, the analyses in this thesis demonstrate different rates and directions of evolution following gene duplication. Some duplicated genes are shown to diverge gradually over time throughout mammalian evolution, while others exhibit an accelerated evolutionary rate within a specific lineage. In other rare cases, divergence is impeded such that duplicated genes evolve in synchronization, under a process known as concerted evolution. This can lead to examples showing mosaic evolution, where both divergent and concerted evolutionary signatures are observed within a single duplicated gene. Through the analyses presented in this thesis, I illustrate some of the different evolutionary histories that result from gene duplication and examine the variety of forces that influence the evolution of duplicated genes. These studies examine the role of duplication in mammalian evolution and represent a significant contribution to the growing body of knowledge in the field of evolutionary biology.

Programmed cell death (PCD or apoptosis) is an evolutionarily conserved, genetically controlled suicide mechanism for cells, which when deregulated, can lead to developmental defects, cancers and degenerative diseases. In C. elegans, DNA damage induces germ cell death by signaling through cep-1/p53 ultimately leading to the activation of the CED-3/caspase. It has been hypothesized that the major regulatory events controlling cell death occur by cell autonomous mechanisms, that is within the dying cell. In support of this, genetic studies in C. elegans have shown that the core apoptosis pathway genes ced-4/APAF1 and ced-3/caspase are required in cells fated to die. However, it is not known whether the upstream signals that activate apoptosis function in a cell autonomous manner. Here I show that two genes, kri-1, an ortholog of KRIT1/CCM1 that is mutated in the human neurovascular disease cerebral cavernous malformations (CCMs) and daf-2, an insulin-like receptor, are required to activate DNA damage-dependent cell death independently of cep-1/p53. Interestingly, I found that both genes can regulate cell death in a non-autonomous manner, revealing a novel role for non-dying cells in eliciting death in response to DNA damage.

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.