This study examined the differential gene expression patterns between endothelial cells (EC) from 2D monolayer and EC from angiogenic capillary-like network in 3D matrices. Our microarray analysis comparing 3D to 2D EC cultures detected upregulation of 854 protein-coding genes and downregulation of 863 genes. We show that Notch signaling pathway is highly regulated in angiogenesis, induced by change in ECM dimension. Notch target genes Hey1, HeyL, Hes1 and Hes4 transcription factors were upregulated in 3D angiogenic EC, which were confirmed with qRT-PCR. Moreover, we are the first to report enrichment of FoxS1 transcription factor mRNA during angiogenesis in 3D ECM. Next, we asked whether epigenetic mechanisms partly mediate cis-trans response in angiogenesis. Our sodium bisulfite sequencing analyses did not indicate a role for DNA methylation in the expression of key Notch signaling components. However, our pilot studies indicate a potential role for lncRNAs in controlling EC phenotype in angiogenic response.

Increasing gene dosage provides a powerful means of probing gene function, as it tends to cause a gain-of-function effect due to increased gene activity. In the budding yeast, Saccharomyces cerevisiae, systematic gene overexpression studies have shown that in wild-type cells, overexpression of a small subset of genes results in an overt phenotype. However, examining the effects of gene overexpression in sensitized cells containing mutations in known genes is a powerful means for identifying functionally relevant genetic interactions. When a query mutant phenotype is rescued by gene overexpression, the genetic interaction is termed dosage suppression. I comprehensively investigated dosage suppression genetic interactions in yeast using three approaches. First, using one of two novel plasmid libraries cloned by two colleagues and myself, I systematically performed dosage suppression screens and identified over 130 novel dosage suppression genetic interactions for more than 25 essential yeast genes. The plasmid libraries, called the molecular barcoded yeast ORF (MoBY-ORF) 1.0 and 2.0, are designed to streamline dosage analysis by being compatible with high-throughput genomics technologies that can monitor plasmid representation, including barcode microarrays and next-generation sequencing methods. Second, I describe a detailed analysis of the novel dosage suppression interactions, as well as of literature-curated interactions, and show that the gene pairs exhibiting dosage suppression are often functionally related and can overlap with physical as well as negative genetic interactions. Third, I performed a systematic categorization of dosage suppression genetic interactions in yeast and show that the majority of the dosage suppression interactions can be assigned to one of four general mechanistic classifications. With this comprehensive analysis, I conclude that systematically identifying dosage suppression genetic interactions will allow for their integration into other genetic and physical interaction networks and should provide new insight into the global wiring diagram of the cell.

The ultimate purpose of this study was to investigate AHP2 protein function in Arabidopsis; AHP2 protein is known to form a heterodimer with MND1. But to do this an existing chromosome spreading protocol had to be modified to reproducibly provide large numbers of well preserved chromosomes and a multi-criteria meiotic staging method was developed to accurately identify chromosome spreads at specific prophase I substages. As well a technique for combined immuno-cytochemistry and fluorescent in situ hybridization (FISH) had to be developed. Coimmuno-localization of AHP2 and MND1 proteins, in wild type meiocytes, revealed synchronous temporal organization (signal initially peaks, for both, during zygotene) but spatially the AHP2 signal appeared exclusively as chromosome axis-associated foci whereas the MND1 signal was diffuse with some foci suggesting that MND1 may also be localizing to loop regions. This finding strongly suggests that MND1 also functions independently of AHP2 during meiosis. We coupled transmission electron microscopy (TEM) analysis of ultrathin sections of meiotic nuclei with light microscope (LM) analysis of chromosome spreads and demonstrated that ahp2 meiocytes fail to stabilize chromosome close alignment that normally occurs during zygotene. My method of combining 2-Bromo-5-deoxyUridine (BrdU) - determination of meiosis duration and the relative durations of each substage allowed us to calculate the absolute duration of each prophase I substage in wild type and revealed that early to mid-zygotene was prolonged in the ahp2 mutant. This finding was consistent with the ahp2 mutant’s overall lack of stabilized chromosome alignment. Scanning electron microscopy (SEM) showed that the Arabidopsis ahp2 short stamen filament was due to reduced cell elongation in filament parenchyma (epidermal) cells. Detection of illegitimate connections between chromosomes at anaphase I may trigger cell-to-cell signals that result in reduced epidermal cell elongation in the stamen filament. Finally, I report that the short arms of NOR-bearing chromosomes 2 and 4 homologously pair and synapse in the ahp2 mutant despite an overall lack of stabilized pairing. This NOR phenomenon has been observed in Drosophila but ours is the first report of the ability of NORs to locally induce pairing and synapsis in plants.

Epigenetic studies hold the promise of addressing some of the fundamental questions of human biology including development, cell differentiation, and the aetiological mechanisms of complex disease. Over the last years, several new large scale high throughput technologies have been developed to allow genome wide profiling of epigenetic signals such as DNA methylation and histone modifications. Two of such technologies were developed in our laboratory enabling a genome wide microarray based profiling of DNA methylation signatures and a high throughput method for the site specific interrogation of the density of methylated cytosine. Using these techniques, we identified a DNA methylation difference in the 3’UTR of the DLX1 gene with potentially functional implications to discordance in risk taking behavior in a single pair of MZ twins. We modeled a power analysis on the effect size of the detected difference and determined that approximately 6~25 discordant twin pairs will be adequate to yield 80% power across the entire 12 K CpG island microarray platform using our epigenomic microarray profiling technique. We performed a DNA methylome analysis of MZ twins in white blood cells (WBC), buccal epithelial cells, and gut (rectum) biopsies (N=57 pairs in total) using 12K CpG island microarrays providing the basis for the first annotation of epigenetic metastability of ~6,000 unique genomic regions in MZ twins. We performed a classical twin study on DNA methylation differences in WBC and buccal epithelial cells from 39 pairs of MZ twins to 40 pairs of DZ twins. DZ co-twins exhibited significantly higher epigenetic difference compared to the MZ co-twins in buccal cells (p=1.2x10-294). While such higher epigenetic discordance in DZ twins can result from DNA sequence differences, our in silico SNP analyses and comparison of methylomes in inbred vs. outbred mice favour the hypothesis that this is due to epigenomic differences in the zygotes. This study suggests that molecular mechanisms of heritability may not be limited to DNA sequence differences.

Behaviour genetics deals with complex phenotypes which respond flexibly to environments animals experience. Change of phenotype in response to environment is phenotypic plasticity. A central question is how genes influence plasticity. I study plasticity and gene by environment interactions (GEI) relating to behaviours, metabolic, and genomic phenotypes of adults of the fruit fly Drosophila melanogaster. Chapters 1-3 study cuticular hydrocarbon (CH) levels of male flies. Chapter 1 shows male CH levels respond to time of day and light. Methods are developed to reduce high variability of CH. I show variation in CH parallels activity of two classes of CH synthesis hormones. Analysis of rate of variation gives estimates of turnover rates of CH and the metabolic cost of signaling. Chapter 2 studies mixed groups of genetically different flies, “hosts” and “visitors”. GEI of CH are found with both abiotic factors and with social mix. Social mix results in GEI as strong as abiotic factors. Indirect Genetic Effects (IGE) theory is used to show frequency-dependent IGE interactions. Chapter 3 shows that males in mixed social environments reduce expression of clock and CH synthesis genes, resulting in different signals. Females mate more often with males in a mixed group than with single-genotype males. Plasticity in male gene expression in response to social environment leads to different signals, mating levels, and potentially different fitness. Chapter 4 deals with behaviour, metabolite, and genomic phenotypes in flies differing in foraging gene alleles, as the food environment is changed. Strong GEI is found, structured by food type, chemical class of metabolite, and gene metabolic roles. A concept called “relative nutrient sensitivity” suggests an interaction between foraging and the insulin signaling pathway. I demonstrate epistasis between for and insulin with quantitative genetic methods and bioinformatics. These results lead to the conclusion that GEI are common in many fly phenotypes in response to well studied environments such as food and less studied ones such as social group. Some implications of this for maintenance of genetic variance are discussed.

Development of genome-wide reagents has allowed systematic analysis of gene function. The experimental accessibility of budding yeast makes it a test-bed for technology development and application of new functional genomic tools and resources that pave the way for comparable efforts in higher eukaryotes. In this Thesis, I describe a two-color GFP-RFP reporter system I developed to assess the consequences of genetic perturbations on a promoter of interest. The dual-reporter system is compatible with the synthetic genetic array methodology, an approach that enables marked genetic elements to be introduced into arrays of yeast mutants via an automated procedure. I use this approach to probe cell cycle-regulation of histone gene transcription by introducing an HTA1 promoter-GFP reporter gene construct into an ordered array of ~4500 yeast deletion mutants. I scored defects in reporter gene expression for each mutant, generating a quantitative analysis of histone promoter activity. The results of my screen motivated a number of follow-up experiments, including chromatin immunoprecipitation, transcript profiling and genome-wide analysis of nucleosome positions, which revealed a previously unappreciated pathway that specifies regions of repressed chromatin in a cell cycle-sensitive manner. A novel aspect of this pathway is that it involves histone chaperones and a chromatin boundary element. Specifically, we discovered that the histone chaperone Rtt106 works with two other chaperones, Asf1 and the HIR complex, to create a repressive chromatin structure at histone promoters which is bound by the protein Yta7. It was clear from previous work that Asf1 and HIR repress transcription at HTA1 and that HIR localizes to and functions through a specific element in histone promoters. However, there was no previous data demonstrating a role for Rtt106 in cell cycle-dependent gene transcription. In sum, I describe a new genomic screen that I used to discover a novel pathway regulating cell cycle-dependent transcription. While I examined histone gene expression as proof-of-principle, my screening system could be applied to virtually any pathway for which a suitable reporter can be devised. I anticipate this methodology will enable yeast researchers to collect quantitative data on hundreds of gene expression pathways.

Increasing gene dosage provides a powerful means of probing gene function, as it tends to cause a gain-of-function effect due to increased gene activity. In the budding yeast, Saccharomyces cerevisiae, systematic gene overexpression studies have shown that in wild-type cells, overexpression of a small subset of genes results in an overt phenotype. However, examining the effects of gene overexpression in sensitized cells containing mutations in known genes is a powerful means for identifying functionally relevant genetic interactions. When a query mutant phenotype is rescued by gene overexpression, the genetic interaction is termed dosage suppression. I comprehensively investigated dosage suppression genetic interactions in yeast using three approaches. First, using one of two novel plasmid libraries cloned by two colleagues and myself, I systematically performed dosage suppression screens and identified over 130 novel dosage suppression genetic interactions for more than 25 essential yeast genes. The plasmid libraries, called the molecular barcoded yeast ORF (MoBY-ORF) 1.0 and 2.0, are designed to streamline dosage analysis by being compatible with high-throughput genomics technologies that can monitor plasmid representation, including barcode microarrays and next-generation sequencing methods. Second, I describe a detailed analysis of the novel dosage suppression interactions, as well as of literature-curated interactions, and show that the gene pairs exhibiting dosage suppression are often functionally related and can overlap with physical as well as negative genetic interactions. Third, I performed a systematic categorization of dosage suppression genetic interactions in yeast and show that the majority of the dosage suppression interactions can be assigned to one of four general mechanistic classifications. With this comprehensive analysis, I conclude that systematically identifying dosage suppression genetic interactions will allow for their integration into other genetic and physical interaction networks and should provide new insight into the global wiring diagram of the cell.

The ultimate purpose of this study was to investigate AHP2 protein function in Arabidopsis; AHP2 protein is known to form a heterodimer with MND1. But to do this an existing chromosome spreading protocol had to be modified to reproducibly provide large numbers of well preserved chromosomes and a multi-criteria meiotic staging method was developed to accurately identify chromosome spreads at specific prophase I substages. As well a technique for combined immuno-cytochemistry and fluorescent in situ hybridization (FISH) had to be developed. Coimmuno-localization of AHP2 and MND1 proteins, in wild type meiocytes, revealed synchronous temporal organization (signal initially peaks, for both, during zygotene) but spatially the AHP2 signal appeared exclusively as chromosome axis-associated foci whereas the MND1 signal was diffuse with some foci suggesting that MND1 may also be localizing to loop regions. This finding strongly suggests that MND1 also functions independently of AHP2 during meiosis. We coupled transmission electron microscopy (TEM) analysis of ultrathin sections of meiotic nuclei with light microscope (LM) analysis of chromosome spreads and demonstrated that ahp2 meiocytes fail to stabilize chromosome close alignment that normally occurs during zygotene. My method of combining 2-Bromo-5-deoxyUridine (BrdU) - determination of meiosis duration and the relative durations of each substage allowed us to calculate the absolute duration of each prophase I substage in wild type and revealed that early to mid-zygotene was prolonged in the ahp2 mutant. This finding was consistent with the ahp2 mutant’s overall lack of stabilized chromosome alignment. Scanning electron microscopy (SEM) showed that the Arabidopsis ahp2 short stamen filament was due to reduced cell elongation in filament parenchyma (epidermal) cells. Detection of illegitimate connections between chromosomes at anaphase I may trigger cell-to-cell signals that result in reduced epidermal cell elongation in the stamen filament. Finally, I report that the short arms of NOR-bearing chromosomes 2 and 4 homologously pair and synapse in the ahp2 mutant despite an overall lack of stabilized pairing. This NOR phenomenon has been observed in Drosophila but ours is the first report of the ability of NORs to locally induce pairing and synapsis in plants.

Williams-Beuren Syndrome (WBS) is an autosomal dominant neurodevelopmental disorder caused by hemizygous deletion of a 1.5 Mb region on chromosome 7q11.23. Symptoms are numerous and include behavioural and cognitive components. One of the deleted genes, GTF2IRD1, a putative transcription factor, has been implicated in the neurological features of WBS by studying patients with atypical deletions of 7q11.23. Gtf2ird1-targeted mice have features consistent with the WBS phenotype, namely reduced innate fear and increased sociability. To identify neural targets of GTF2IRD1, microarray analyses were performed comparing gene expression in whole brains of Gtf2ird1-/- and wildtype (WT) mice at embryonic day 15.5 and at birth. Overall, the changes in gene expression in the mutant mice were not striking, with most falling in the range of 0.3 to 2 fold. qRT-PCR was used to verify the expression levels of candidate genes and examination of verified genes revealed that most were located on chromosome 5, within 50 Mb of Gtf2ird1. Expression of these candidate genes in Gtf2ird1-/- mice was found to be the same as in WT 129S1/SvImJ mice, indicating the differences were the result of flanking chromosomal material from the, 129-derived, R1 ES cells from which the Gtf2ird1-/- mice were generated, and that expression differences were unrelated to Gtf2ird1 dosage. Further analysis found that while many genes showed decreased expression using primers targeting the 3’ UTR, expression of upstream exons was not affected. Transcripts using alternative polyadenylation sites were identified using 3’ RACE, and qRT-PCR showed that expression of different 3’ UTR isoforms can occur in a strain specific manner. Expression analysis of previously identified GTF2IRD1 targets also failed to demonstrate an in vivo effect. In summary, I was unable to find any in vivo neuronal targets of this putative transcription factor, despite its robust expression in the developing rodent brain.

Cell migration and extension is essential for development. The ability of a cell or cell extension to reach its target is dependent on spatial cues and receptors that translate positional information into directed plasma membrane extension. For example, the UNC-40/DCC receptor is required to direct circumferential migrations towards the source of the ligand, UNC-6/Netrin, expressed at the ventral midline. To better understand the process of cell extension, I used a specialized process called muscle arms as a model system. In C. elegans, body wall muscles (BWMs) extend membrane projections called muscle arms to the nearest nerve cord at the midline. These muscle arms harbor the postsynaptic element of the neuromuscular junction and extend in a stereotypical and regulated manner. In a screen for muscle arm development defective (Madd) mutants, I isolated madd-2, a novel component of the UNC-40 pathway. MADD-2 is a C-1 TRIM protein and functions cell-autonomously to direct numerous muscle and axon extensions to the ventral midline of worms. In a striking analogy, mutations in a human homologue of MADD-2, MID1, cause numerous ventral midline defects that culminate as Opitz Syndrome. How MID1 regulates midline development is unclear. MADD-2 enhances UNC-40 pathway activity by facilitating the physical interaction between UNC-40 and the downstream Rho-GEF, UNC-73. It is possible that MID1 may mediate the function of a DCC-like pathway at the ventral midline of humans. This work provides the first indication that C1-TRIM proteins may have a conserved biological role of regulating midline-oriented development events and may provide key insights into the role of MID1 in the pathogenesis of Opitz syndrome.