Global quantitative analysis of genetic interactions provides a powerful approach for deciphering the roles of genes and mapping functional relationships amongst path-ways. Using colony size as a proxy for fitness, I developed a method for measuring ge-netic interactions from high-density arrays of yeast double mutants generated by synthet-ic genetic array (SGA) technology. I identified several experimental sources of systematic variation and developed normalization strategies to obtain accurate fitness measurements. I used this scoring method to map quantitative genetic interactions among 5.4 million yeast double mutants and generated the first functionally unbiased genetic interaction map of a eukaryotic cell. My map produced an unprecedented view of the cell in which genes of similar biological processes cluster together in coherent subsets and functionally interconnected bioprocesses map next to each other. We discovered several physiological and evolutionary gene features that are characteristic of genetic interaction hubs, and explored the relationship between genetic and protein-protein interaction networks. In particular, by comparing quantitative single and double mutant phenotypes, we identified specific cases of positive genetic interactions, termed genetic suppression, and constructed a global network of suppression interactions among protein complexes. I also demonstrated that an extensive and unbiased mapping of genetic interactions provides a key for interpreting chemical-genetic interactions and identifying drug targets. In addition, I used genome-wide SGA data to map profiles of genetic linkage along all sixteen yeast chromosomes. These linkage profiles recapitulated previously identified recombination patterns and uncovered an unexpected correlation between chromosome length and the extent of centromere-related recombination repression. These findings suggest a chromosome size-dependent mechanism for ensuring proper chromosome segregation and highlight the SGA methodology as a unique approach for systematic analysis of yeast meiotic recombination.

Global quantitative analysis of genetic interactions provides a powerful approach for deciphering the roles of genes and mapping functional relationships amongst path-ways. Using colony size as a proxy for fitness, I developed a method for measuring ge-netic interactions from high-density arrays of yeast double mutants generated by synthet-ic genetic array (SGA) technology. I identified several experimental sources of systematic variation and developed normalization strategies to obtain accurate fitness measurements. I used this scoring method to map quantitative genetic interactions among 5.4 million yeast double mutants and generated the first functionally unbiased genetic interaction map of a eukaryotic cell. My map produced an unprecedented view of the cell in which genes of similar biological processes cluster together in coherent subsets and functionally interconnected bioprocesses map next to each other. We discovered several physiological and evolutionary gene features that are characteristic of genetic interaction hubs, and explored the relationship between genetic and protein-protein interaction networks. In particular, by comparing quantitative single and double mutant phenotypes, we identified specific cases of positive genetic interactions, termed genetic suppression, and constructed a global network of suppression interactions among protein complexes. I also demonstrated that an extensive and unbiased mapping of genetic interactions provides a key for interpreting chemical-genetic interactions and identifying drug targets. In addition, I used genome-wide SGA data to map profiles of genetic linkage along all sixteen yeast chromosomes. These linkage profiles recapitulated previously identified recombination patterns and uncovered an unexpected correlation between chromosome length and the extent of centromere-related recombination repression. These findings suggest a chromosome size-dependent mechanism for ensuring proper chromosome segregation and highlight the SGA methodology as a unique approach for systematic analysis of yeast meiotic recombination.

xii, 86 p. : ill. A print copy of this thesis is available through the UO Libraries. Search the library catalog for the location and call number. / My dissertation has focused on identifying functions for non-essential genes in essential process, using the early C. elegans embryo as a model system. The fully sequenced C. elegans genome contains ∼19,800 protein coding genes of which about half have identifiable homologs in humans. Classical forward genetic mutagenesis screens, and more recently, genome-wide RNA interference (RNAi) screens has led to the identification of most essential genes in the genome. Analysis of the phenotypic data from mutants and RNAi screens shows that roughly 15% of the genes are essential and an additional 15% produce some other easily identifiable knockdown phenotype. This leaves about 70% of genes that have no functional information. Genetic modifier screening allows for the identification of roles for genes that do not produce a loss of function phenotype on their own but are able to modify the phenotype of a specific mutant. In my first chapter, I introduce approaches to identifying new gene functions and the usefulness V of C. elegans as a model system in this pursuit. In Chapter II, I describe a type of high-throughput genetic modifier screen that combines the sensitized genetic background of temperature-sensitive (ts) embryonic lethal mutants, and RNAi, to identify genes that either enhance or suppress embryonic lethality seen in the mutant background. I also summarize results from screening four ts mutants using this method. The following two chapters describe the identification and characterization of genetic modifier genes for two different ts embryonic-lethal mutants. Chapter III describes modifiers of rfl-1 , a conserved gene required for proper cytoskeletal regulation in the early C. elegans embryo. Chapter IV describes modifiers of lit-1 , also a conserved gene, that is required for regulation of Wnt signaling and cell fate specification in C. elegans . These findings reveal novel genetic interactions and provide functional information about many conserved but non-essential genes that have had no previous characterization. Conclusions are also made about the effectiveness of ts mutant/RNAi screening in the pursuit of identifying new gene functions. This dissertation contains co-authored material that has been previously published, and material that is currently in review, or is being prepared for publication. / Adviser: Bruce Bowerman

The nucleoplasmin (NPM) family of acidic histone chaperones and the FK506-binding (FKBP) peptidyl proline isomerases are both linked to chromatin regulation. In vertebrates, NPM and FKBP domains are found on separate proteins. In fungi, NPM-like and FKBP domains are expressed as a single polypeptide in nucleoplasmin-like FKBP (NPL-FKBP) histone chaperones. Saccharomyces cerevisiae has two NPL-FKBPs: Fpr3 and Fpr4. These paralogs are 72% similar and are clearly derived from a common ancestral gene. This suggests that they may have redundant functions. Their retention over millions of years of evolution also implies that each must contribute non-redundantly to organism fitness. The redundant and separate biological functions of these chromatin regulators have not been studied. In this dissertation I take a systems biology approach to fill this knowledge gap. First, I refine the powerful synthetic genetic array (SGA) method of annotating gene-gene interactions, making it amenable for the analyses of paralogous genes. Using these ‘paralog-SGA’ screens I define distinct genetic interactions unique to either Fpr3 or Fpr4, shared genetic interactions common to both paralogs, and masked genetic interactions which are direct evidence for processes where these enzymes are functionally redundant. I provide transcriptomic evidence that Fpr3 and Fpr4 cooperate to regulate genes involved in polyphosphate metabolism and ribosome biogenesis. I identify an important role for Fpr4 at the 5’ ends of protein coding genes and the non-transcribed spacers of ribosomal DNA. Finally, I show that yeast lacking Fpr4 exhibit a genome instability phenotype at rDNA, implying that this histone chaperone regulates chromatin structure and DNA access at this locus. Collectively, these data demonstrate that Fpr3 and Fpr4 operate separately, cooperatively and redundantly to regulate a variety of chromatin environments. This work is the first comprehensive and comparative study of NPL-FKBP chaperones and as such represents a significant contribution to our understanding of their biological functions. / Graduate

Melanocytes, pigment-producing cells, derive from the neural crest (NC), a population of pluripotent cells that arise from the dorsal aspect of the neural tube during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The deletion of the transcription factor Ets1 in mice results in hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The goal of the present study was to establish the temporal requirement and role of Ets1 in murine melanocyte development. In the mouse, Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick cranial NC, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of melanocytes, enteric ganglia, and other NC derivatives. Using a combination of immunofluorescence and cell survival assays Ets1 was found to be required between embryonic days 10 and 11, when it regulates NC cell and melanocyte precursor (melanoblast) survival. Given the requirement of Ets1 for Sox10 expression in the chick cranial NC, a potential interaction between these genes was investigated. Using genetic crosses, a synergistic genetic interaction between Ets1 and Sox10 in melanocyte development was found. Since Sox10 is essential for enteric ganglia formation, the importance of Ets1 on gut innervation was also examined. In mice, Ets1 deletion led to decreased gut innervation, which was exacerbated by Sox10 heterozygosity. At the molecular level, Ets1 was found to activate a Sox10 enhancer critical for Sox10 expression in melanoblasts. Furthermore, mutating Ets1 at a site I characterized in the spontaneous variable spotting mouse pigmentation mutant, led to a 2-fold decrease in enhancer activation. Overexpression and knockdown of Ets1 did not affect Sox10 expression; nonetheless, Ets1 knockdown led to a 6-fold upregulation of the transcription factor Sox9, a gene required for melanocyte and chondrocyte development, but which impairs melanocyte development when its expression is prolonged. Together, these results suggest that Ets1 is required early during melanocyte development for NC cell and melanoblast survival, possibly acting upstream of Sox10. The transcription factor Ets1 may also act indirectly in melanocyte fate specification by repressing Sox9 expression, and consequently cartilage fate.

The link between the genotype and the phenotype of an organism is immensely complex. Despite this it can, to a great extent, be captured using models that assume that gene variants combine their effects in an additive manner. This thesis explores aspects of genetics that cannot be fully captured using such additive models. Using experimental data from three different model organisms, I study two phenomena that fall outside of the additive paradigm: genetic interactions and genetic variance heterogeneity. Using the model plant Arabidopsis thaliana, we show how important biological insights can be reached by exploring loci that display genetic variance heterogeneity. In the first study, this approach identified alleles in the gene CMT2 associated with the climate at sampling locations, suggesting a role in climate adaption. These alleles affected the genome wide methylation pattern, and a complete knock down of this gene increased the plants heat tolerance. In the second study, we demonstrate how the observed genetic variance heterogeneity was the result of the partial linkage of many functional alleles near the gene MOT1, all contributing to Molybdenum levels in the leaves. Further, we explore genetic interactions using data from dogs and budding yeast (Saccharomyces cerevisiae). In the dog population, two interacting loci were associated with fructosamine levels, a biomarker used to monitor blood glucose. One of the loci displayed the pattern of a selective sweep in some of the studied breeds, suggesting that the interaction is important for the phenotypic breed-differences. In a cross between two strains of yeast, with the advantage of large population size and nearly equal allele frequencies, we identified large epistatic networks. The networks were largely centered on a number of hub-loci and altogether involved hundreds of genetic interactions. Most network hubs had the ability to either suppress or uncover the phenotypic effects of other loci. Many multi-locus allele combinations resulted in phenotypes that deviated significantly from the expectations, had the loci acted in an additive manner. Critically, this thesis demonstrates that non-additive genetic mechanisms often need to be considered in order to fully understand the genetics of complex traits.

Septins are a highly conserved family of eukaryotic proteins having significant homology within and among species. In the budding yeast, Saccharomyces cerevisiae, a septin-based hierarchy of proteins is required to localize chitin in the bud neck prior to septum formation. However, this process has not been clarified in a filamentous, conidiogenous fungus capable of yeast growth, such as Wangiella dermatitidis, a polymorphic agent of human phaeohyphomycosis. Prior studies of this melanized mold showed that some chitin synthase mutants (wdchsΔ) have defects in yeast septum formation, suggesting that the septins of W. dermatitidis might functionally associate with some of its chitin synthases (WdChsp). To test this hypothesis, four vegetative septin homologs of S. cerevisiae were cloned from W. dermatitidis and designated WdCDC3, WdCDC10, WdCDC11, and WdCDC12. Of the four, only WdCDC3 functionally complemented completely a strain of S. cerevisiae with a ts mutation in the corresponding gene, although WdCDC12 did so partially. Functional characterizations by mutagenesis of the four W. dermatitidis septin genes revealed that resulting mutants (wdcdc[delta]) each had unique defects in yeast growth and morphology, indicating that each septin carried out a distinct function. Furthermore, when a wdcdc10[delta] mutation was introduced into five different wdchs[delta] strains, weak genetic interactions were detected between WdCDC10 and WdCHS3 and WdCHS4, and a strong interaction between and WdCHS5. Cytological studies showed that WdChs5p was mislocalized in some septin mutants, including wdcdc10[delta]. These results confirmed that in W. dermatitidis septins are important for proper cellular morphogenesis, cytokinesis, and especially septum formation through associations with some chitin synthases. / text

The evolution of antibiotic resistance is shaped by interactions between genes, the chemical environment, and an antibiotic's mechanism of action. This thesis explores these interactions with experiments, theory, and analysis, seeking a mechanistic understanding of how different interactions between genes and drugs can enhance or constrain the evolution of antibiotic resistance. Chapter 1 investigates the effects of the chemical decay of an antibiotic. Tetracycline resistant and sensitive bacteria were grown competitively in the presence of tetracycline and its decay products. Antibiotic decay did not only remove selection for resistance, but long-lived decay products favored tetracycline sensitivity by inducing costly drug efflux pumps in the resistant strain. Selection against resistance by antibiotic-related compounds may contribute to the coexistence of drug-sensitive and resistant bacteria in nature. Chapter 2 investigates how genetic interactions can favor particular combinations of resistance-conferring mutations. All possible combinations of a set of trimethoprim resistance-conferring mutations in the drug's target gene were constructed and phenotyped. Incompatibilities between mutations arose in a high-order, not pairwise, manner. One mutation was found to induce this ruggedness and create a multi-peaked adaptive landscape. Chapters 1 and 2 observed that non-optimal expression of a drug resistance gene or a drug's target could compromise antibiotic resistance. Chapter 3 broadly characterizes non-optimal gene expression under antibiotic treatment, using a functional genetic screen to identify over one hundred pathways to antibiotic resistance through positive and negative changes in gene expression. Genes with the potential to confer antibiotic resistance were found to often go unused during antibiotic stress. The optimization of gene expression for drug-free growth was found to cause non-optimal expression under drug treatment, creating a situation where regulatory mutations can confer resistance by correcting errors in gene expression. Chapter 4 investigates whether it is beneficial to up-regulate the genes encoding antibiotic targets when they are inhibited. Drug target genes were quantitatively over-expressed, and drug resistance was found to not always increase, but alternatively to remain unchanged or even decrease. These diverse effects were explained by simple models that consider toxicity arising from gene over-expression, and mechanisms of drug action in which drugs induce harmful enzymatic reactions.

This work presents the investigation of fungal AdoMet-dependent methyltransferases. The first part of the dissertation focuses on two distinct methyltransferases with previously unknown functions in the budding yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans. To characterize these enzymes I used a combinatorial approach that exploits contemporary high-throughput techniques available in yeast (chemical genetics, expression, lipid profiling and genetic interaction analysis) combined with rigorous biological follow-up. First, I showed that S. cerevisiae CRG1 (ScCRG1) is a small molecule methyltransferase that methylates cytotoxic drug cantharidin and is important for maintaining lipid homeostasis and actin cytoskeleton integrity in response to small-molecule cantharidin in the baker’s yeast. Similarly to ScCRG1, orf19.633 in the human fungal pathogen C. albicans (CaCRG1) methylates cantharidin and is important for GlcCer biosynthesis. I also demonstrated that CaCrg1 is a ceramide- and PIP-binding methyltransferase involved in Candida’s morphogenesis, membrane trafficking and fungal virulence. Together, the analysis of two genes in yeast illuminated the important roles of the novel small molecule methyltransferases in coupling drug response to lipid biosynthesis and fungal virulence. In the second part of my dissertation, I present the systematic characterization of the genetic architecture of the yeast methyltransferome by examining fitness of double-deletion methyltransferase mutants in standard and under environmental stress conditions. This analysis allowed me to describe specific properties of the methyltransferome network and to uncover functional relationships among methyltransferases inspiring multiple hypotheses and expanding the current knowledge of this family of enzymes.

This work presents the investigation of fungal AdoMet-dependent methyltransferases. The first part of the dissertation focuses on two distinct methyltransferases with previously unknown functions in the budding yeast Saccharomyces cerevisiae and the human fungal pathogen Candida albicans. To characterize these enzymes I used a combinatorial approach that exploits contemporary high-throughput techniques available in yeast (chemical genetics, expression, lipid profiling and genetic interaction analysis) combined with rigorous biological follow-up. First, I showed that S. cerevisiae CRG1 (ScCRG1) is a small molecule methyltransferase that methylates cytotoxic drug cantharidin and is important for maintaining lipid homeostasis and actin cytoskeleton integrity in response to small-molecule cantharidin in the baker’s yeast. Similarly to ScCRG1, orf19.633 in the human fungal pathogen C. albicans (CaCRG1) methylates cantharidin and is important for GlcCer biosynthesis. I also demonstrated that CaCrg1 is a ceramide- and PIP-binding methyltransferase involved in Candida’s morphogenesis, membrane trafficking and fungal virulence. Together, the analysis of two genes in yeast illuminated the important roles of the novel small molecule methyltransferases in coupling drug response to lipid biosynthesis and fungal virulence. In the second part of my dissertation, I present the systematic characterization of the genetic architecture of the yeast methyltransferome by examining fitness of double-deletion methyltransferase mutants in standard and under environmental stress conditions. This analysis allowed me to describe specific properties of the methyltransferome network and to uncover functional relationships among methyltransferases inspiring multiple hypotheses and expanding the current knowledge of this family of enzymes.