Browsing by Department "Genetics and Genomics"
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Item Open Access A Genome-Wide RNAi Screen Identifies CDC-42 and GDI-1 as In Vivo Regulators of Invadopodia(2015) Lohmer, Lauren Renee LilleyBasement membrane (BM) is a sheet-like extracellular matrix that underlies most tissues and acts as a barrier to invading cells. Many cell types, including immune cells, cells migrating during development and morphogenesis, and metastatic cancer cells utilize F-actin-based structures called invadopodia to breach BM as they leave one tissue to enter another. Despite extensive study and interest in understanding invasion for its clinical importance, the molecular mechanisms regulating invadopodia and BM breach in vivo remain unclear. During uterine-vulval attachment in C. elegans, the specialized uterine anchor cell (AC) uses invadopodia to mediate breach of the underlying BM in order to contact the underlying vulval epithelium. The AC offers several advantages as a model, including experimental, visual, and genetic tractability, the presence of endogenous extracellular environment, and availability of the tissue targeted for invasion. In Chapter 2, I describe development of a novel technique for spatiotemporal-specific knockdown of proteins that will facilitate investigation of proteins, particularly those that are essential or required in other tissues, in the regulation of invadopodia and AC invasion. Using a sensitized genome-wide RNAi screen, classical genetics, and timelapse imaging of invadopodia at the AC-BM interface, Chapter 3 presents two in vivo invadopodia regulators that function by distinct mechanisms. This is the first in vivo evidence that the RhoGTPase CDC-42 regulates invadopodia formation through WSP-1. RabGTPase GDI-1 is a novel regulator of the unique membrane compartment required for invadopodia formation. CDC-42 and GDI-1 both function downstream of an unknown cue secreted by the cells targeted by the AC for invasion, illustrating that extracellular cues can play key roles in mediating cell invasion. The characterization of CDC-42 and GDI-1 as in vivo regulators of invadopodia is an important first step to understanding the mechanisms of this critical cellular process and we expect the AC will be an excellent model for future identification of novel regulators of BM breach.
Item Open Access A Genomic Definition of Centromeres in Complex Genomes(2011) Hayden, Karen ElizabethCentromeres, or sites of chromosomal spindle attachment during mitosis and meiosis, are non-randomly distributed in complex genomes and are largely associated with expansive, near-identical satellite DNA arrays. While the sequence basis of centromere identity remains a subject of considerable debate, one approach is to examine the genomic organization of satellite DNA arrays and their potential function. Current genome assembly and sequence annotation strategies, however, are dependent on robust sequence variation, and, as a result, these regions of near sequence identity remain absent from current genome reference sequences and thus are detached from explorations of centromere biology. This dissertation is designed as a foundational study for centromere genomics, providing the initial steps to characterize those sequences at endogenous centromeres, while further classifying `functional' sequences that directly interact with, or are capable of recruiting proteins involved in, centromere function. These studies build on and take advantage of the limited sequence variation in centromeric satellite DNA, providing the necessary genomic scope to promote biologically meaningful characterization of endogenous centromere sequences in both human and non-human genomes. As a result, this thesis demonstrates possible genomic standards for future studies in the emerging field of satellite biology, which is now positioned to address functional centromere sequence variation across evolutionary time.
Item Open Access A Novel Mechanism for Human Papillomavirus Mediated Tumorigenesis: Examining a Role for HPV E6 Protein in CYLD Mediated NF-kappaB Activation(2009) Shaw, CharlieHuman papillomavirus (HPV) infection of mucosal epithelium by `high-risk' HPV types has a prominent role in the development of anogenital intraepithelial neoplasias and carcinomas. Human epithelial cells transformed with the HPV E6 oncoprotein survive even under conditions that normally lead to cell apoptosis. This phenomenon has been attributed to HPV E6's ability to promote the degradation of the tumor suppressor protein p53. More recently, it has been demonstrated that HPV E6 contributes to activation of the NF-kB pathway. NF-kB is a transcription factor involved in the regulation of genes associated with cellular proliferation, apoptosis and inflammatory responses. In addition to p53 suppression, HPV E6 modulation of NF-kB activation presents another mechanism for HPV-driven tumorigenesis. However, it was not known how HPV E6 promotes NF-kB pathway activation. To address how HPV E6 leads to NF-kB activation, we identified an association between HPV E6 and the human cylindromatosis gene product (CYLD). CYLD is an endogenous inhibitor of canonical NF-kB activation. We showed that HPV E6 proteins could precipitate CYLD in vitro using a co-immunoprecipitation assay. Demonstrating that HPV E6 and CYLD proteins bind each other raised the possibility that this binding relationship would have a functional effect upon the NF-kB pathway by altering CYLD-mediated suppression of NF-kB activation. To identify HPV E6 functional relationship with CYLD and to determine how HPV E6 activates the NF-kB pathway, we transfected cells with either HPV E6 expression vectors containing the high-risk HPV type 16 E6 or the low-risk HPV type 11 E6 along with a CYLD expression vector. We showed HPV16 E6 expression in 293 cells blocked the ability of CYLD to inhibit CD40 ligand-stimulated NF-kB activation. Interestingly, HPV11 E6 was unable to inhibit CYLD mediated suppression of NF-kB in our system. CYLD had previously been shown to suppress NF-kB activation by removing stimulatory lysine 63-linked ubiquitin chains from TRAF2. We found CYLD expression in 293 cells leads to dose-dependent reduction in TRAF2 levels. This CYLD-mediated loss of TRAF2 is inhibited by co-expression of high-, but not low-risk E6 proteins. It was known that CYLD phosphorylation in vivo suppresses CYLD deubiquitination actions of canonical pathway proteins; we therefore tested the extent of CYLD phosphorylation when co-expressed with HPV E6, and discovered that CYLD phosphorylation was increased in the presence of HPV E6. This compilation of experiments suggests that with HPV16 E6 binding to CYLD, the E6 protein blocks CYLD-mediated TRAF2 loss and thereby TRAF2 is available to activate the canonical NF-κB pathway. Blocking HPV E6-mediated NF-kB activation may prove beneficial as a means for designing therapies that inhibit HPV-mediated tumorigenesis. The differences we detected between HPV11 E6 and HPV16 E6 are supported by other studies that showed E6 protein variations account for molecular and clinical differences among HPV infection outcomes. Similarly, there exist intratype E6 variations in HPV16. We obtained cervical specimens from patients with cytopathogenic changes consistent with the onset of cervical dysplasia infected with HPV16 E6 and the human immunodeficiency virus. We hypothesized the immunocompromised individual may harbor unique HPV16 E6 variants. Using PCR detection methods to amplify the HPV16 E6 DNA and sequencing technology, we identified that some of the samples indeed had nucleotide polymorphisms, resulting in amino acid sequence changes. However, the HPV E6 variants we detected were previously described, and fit know geographic HPV clades. Some of the HPV E6 variants we observed are suggested to be associated with progression to cervical cancer, but further evaluation is required.
Item Open Access A systems-level view of mammalian sex determination.(2010) Munger, Steven CarmenPathologies of sexual development are common in humans and reflect the precarious processes of sex determination and sexual differentiation. The gonad forms as a bipotential organ, and recent results from the Capel lab revealed that it is initially balanced between testis and ovarian fates by opposing and antagonistic signaling networks. In XY embryos, this balance is disrupted by the transient expression of the Y-linked gene, Sry, which activates genes that promote the testis pathway and oppose the ovarian pathway. While the roles of a few genes have been defined by mutation, current evidence suggests that the interactions of many genes and signaling pathways are involved in the establishment of sexual fate. For example, most cases of disorders of sexual development (DSDs) are unexplained by mutations in known sex determination genes. In addition, recent microarray studies in the mouse revealed that nearly half the transcriptome is expressed in the gonad at the time of sex determination (Embryonic day 11.5, or E11.5), and as many as 1,500 genes are expressed in a sexually dimorphic pattern at this early stage. Thus the sexual fate decision in the developing gonad likely depends on a complex network of interacting factors that converge on a critical threshold.
To begin to elucidate the transcription network topology underlying sex determination, we exploited two inbred mouse strains with well-characterized differences in sex reversal. The common inbred strain C57BL/6J (B6) is uniquely sensitive to XY male-to-female sex reversal in response to a number of genetic perturbations, while other strains, including 129S1/SvImJ (129S1) and DBA/2J (D2) are resistant to sex reversal. We hypothesized that these strain differences in gonad phenotype likely result from underlying expression differences in the gonad at the critical timepoint of E11.5. Using microarrays, we identified significant, reproducible differences in the transcriptome of the E11.5 XY gonad between B6 and 129S1 indicating that the reported sensitivity of B6 to sex reversal is consistent with a higher expression of a female-like transcriptome in B6 XY gonads. Surprisingly, a well-characterized master regulator of the testis pathway, Sox9, was found to be upregulated in the sensitive B6 background, which may serve as a compensatory mechanism to counteract the female-leaning transcriptome and activate the testis pathway in wild type B6 XY gonads.
We extended our expression analysis to a large set of F2 XY gonads from B6 and 129S1 intercrosses. From each pair of gonads, we analyzed the expression of 56 sex-associated genes by nanoliter-scale quantitative RT-PCR (qRT-PCR). The expression levels of most genes were highly variable across the F2 population, yet strong correlations among genes emerged. We employed a First-Order Conditional Independence (FOCI) algorithm to estimate the F2 coexpression network. From this unbiased analysis of XY expression data, we uncovered two subnetworks consisting of primarily male and female genes. Furthermore, we predicted roles for genes of unknown function based on their connectivity and position within the network.
To identify the genes responsible for these strain expression differences, we genotyped each F2 embryo at 128 single nucleotide polymorphisms (SNPs) located evenly throughout the 19 autosomes and X chromosome. We then employed linkage analysis to detect autosomal regions that control the expression of one or more of the 56 genes in the F2 population. These regions are termed expression quantitative trait loci, or eQTLs. We identified eQTLs for many sex-related genes, including Sry and Sox9, the key regulators of male sex determination. In addition, we identified multiple prominent trans-band eQTLs that controlled the expression of many genes. My work represents the first eQTL analysis of a developing vertebrate organ, the mouse gonad. This systems-level approach revealed the complex transcription architecture underlying sex determination, and provides a mechanistic explanation for sensitivity to sex reversal seen in some individuals.
Item Open Access A Tale of Two Proteins: Insights into the Haemophilus influenzae Hap and Hia Autotransporters(2011) Spahich, Nicole AnnNontypeable Haemophilus influenzae (NTHi) is a common commensal in the human nasopharynx that can cause localized respiratory tract diseases such as otitis media, bronchitis, and pneumonia. NTHi adheres to respiratory epithelial cells, a critical step in the process of colonization enabled by bacterial surface adhesive structures called adhesins. One group of NTHi adhesins are autotransporters, proteins that have an N-terminal signal sequence, a C-terminal β-barrel domain, and an internal passenger domain with effector function. The goal of this work was to increase our understanding of two NTHi autotransporters, Hap and Hia.
Hap is a monomeric autotransporter that mediates adherence to epithelial cells and extracellular matrix (ECM) proteins. Hap also self-associates with protein on neighboring bacteria, resulting in bacterial aggregation and microcolony formation. The Hap passenger domain contains the regions responsible for adhesive activity. To define the molecular mechanism of Hap adhesive activity, we crystallized the Hap passenger domain. Characterization of the crystal structure revealed an N-terminal globular domain and a more ordered, prism-like C-terminal domain. Interestingly, Hap crystallized as a multimer, suggesting that Hap-Hap interactions occurred in the passenger domain. Progressive deletions of the β-loops that comprise the C-terminal region disrupted Hap-Hap interactions and led to a defect in bacterial settling. To further support that the C-terminal domain was responsible for Hap-Hap interactions,
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we purified the wild type and truncated passenger domains and conjugated the proteins to latex beads. By light microscopy we visualized bead aggregation when the wild type passenger domain was conjugated to the beads, but not when the truncated passenger domain was conjugated. These results show that the C-terminal portion of the Hap passenger domain is responsible for Hap-Hap interactions leading to multimerization. Hap multimerization could be important in microcolony formation that leads to biofilm formation in vivo.
The ECM binding domain in located in the final 511 amino acids of the Hap passenger domain. To pin-point the region of the ECM protein fibronectin that is recognized by Hap, we spotted small fragments of fibronectin onto nitrocellulose membranes and incubated the membrane with purified Hap passenger domain. Far Western analysis using Hap antibody revealed that the smallest fibronectin region necessary for binding was comprised of the first two type III repeats, FNIII(1-2). To define the regions of Hap responsible for interaction with fibronectin, we mutated motifs in the Hap passenger domain that are important for fibronectin binding in other bacterial proteins. Based on assessment by ELISA, many of the mutations located between amino acids 525-725 caused reduced bacterial binding to fibronectin. However, no mutation totally ablated binding, suggesting that a larger Hap region is involved in fibronectin binding.
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In an additional study, we identified a relationship between Hap levels in the outer membrane and the expression of lipopolysaccharide (LPS) biosynthesis enzymes. Through Western and qPCR analysis, we found that mutation of the rfaF, pgmB, lgtC, kfiC, orfE, rfbP, lsgB and lsgD genes involved in the synthesis of LPS oligosaccharide core in H. influenzae strain Rd/HapS243A resulted in loss of Hap in the bacterial outer membrane and a decrease in hap transcript. In contrast, the same mutations had no effect on outer membrane localization of H. influenzae P5 and IgA1 protease or levels of the p5 or iga1 transcripts, suggesting a Hap-specific effect. Elimination of the HtrA periplasmic protease resulted in a return of Hap to the outer membrane and restoration of wild type levels of hap transcript. We speculate that the lack of certain LPS biosynthesis enzymes causes Hap to mislocalize and accumulate in the periplasm, where it is degraded by HtrA. This degradation then leads to a decrease in hap transcript. lgtC is one of several phase variable LPS biosynthesis genes. Using an antibody against the epitope formed in part by the lgtC gene product, we identified lgtC phase-off bacteria by Western analysis of colony blots. Consistent with our previous observations, in lgtC phase off bacteria Hap was absent from the outer membrane and hap transcript was reduced. By analyzing a lgtC/lic2A double mutant, we found that Hap localization in the outer membrane and hap transcript levels were not related to LPS size but instead to the functions of the LPS synthesis enzymes themselves. This relationship could be beneficial to bacteria in vivo as a way to regulate Hap expression.
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Early models suggested that autotransporters do not require accessory factors for folding and OM insertion. However, mounting recent evidence has suggested that the Bam complex is required for OM localization of most β-barrel proteins, including autotransporters. We studied the role of the Bam complex in OM localization of the trimeric autotransporter Hia. We expressed Hia in E. coli strains with mutations in the Bam complex and found that BamA and BamD were needed for Hia localization, while BamB, BamC, and BamE were not necessary. In further studies, we mutated the C-terminus of Hia and found that the final and third-to-last amino acids were the most important for outer membrane localization.
In summary, this work provides insights into the regulation and adhesive activity of Hap and the outer membrane localization of Hia. We have learned important details about these factors that shed light on aspects of H. influenzae disease and could lead to new antimicrobial therapies.
Item Embargo AAV Delivery of CFH Constructs for Complement Regulation in the Murine Eye(2022) Grigsby, DanielAge-related macular degeneration (AMD), a major cause of irreversible vision loss in elderly populations, has been associated genetically with the complement system, and especially with complement factor H (CFH). However, it remains unknown how CFH affects risk of developing AMD. Humans additionally express a truncated splice variant of CFH, Factor H-like 1 (FHL-1). The retinal pigmented epithelium (RPE) is separated from circulation by Bruch’s membrane (BrM), and it has been reported that CFH is unable to diffuse through BrM, while FHL-1 can. Therefore, it is uncertain whether FHL-1 is responsible for most complement regulation in BrM and on RPE cells, or if local production of CFH is more important. We examined this question in Cfh-/- animals through the use of AAVs, allowing expression of both FHL-1 or truncated versions of CFH retaining the C-terminal end of protein, 18tCFH and 12tCFH, from either local sources following subretinal injection or from systemic sources following tail vein injection. While all constructs showed regulation of the complement system using Western blots in circulation, only 18tCFH, the most complete of the constructs, showed definitive regulation of the complement system following subretinal injection. Additionally, there was more evidence of complement regulation following expression locally in the eye, as opposed to circulation. These results suggest that the C-terminus of the protein is essential for complement regulation in the eye, and that local delivery of the protein is the better delivery route. These findings are important for the design of future gene therapies for complement regulation in AMD patients.
Item Open Access Acute and Intergenerational Nutrient Responses in Caenorhabditis elegans(2017) Hibshman, Jonathan DavidNearly all animals live in environments with fluctuations in nutrient availability. The ability to sense and respond to these changes is essential for survival. Nutrition impacts physiology immediately, but can also have long-lasting effects across generations. The nematode Caenorhabditis elegans is particularly well-adapted to thrive in conditions of variable food availability. Here we find that starvation responses in C. elegans are largely independent of the larval stage at which worms experience starvation. Starvation in worms results in shrinkage, delayed growth upon recovery, and ultimately death. In order to adapt to starvation, metabolism is dramatically altered. At a gross level, this can be seen in a reduction of mitochondrial genomes and a more fragmented network of mitochondria.
Insulin-like signaling is a key cell signaling pathway controlling nutrient responses. We interrogate the role of insulin-like signaling in regulation of the acute starvation response. We show that daf-16/FoxO restructures carbohydrate metabolism by driving carbon flux through the glyoxylate shunt and gluconeogenesis and into synthesis of trehalose, a disaccharide of glucose. Trehalose is a well-known stress protectant, capable of preserving membrane organization and protein structure during abiotic stress. Metabolomic, genetic, and pharmacological analyses confirm increased trehalose synthesis and further show that trehalose not only supports survival as a stress protectant, but also serves as a glycolytic input. Further, we provide evidence that metabolic cycling between trehalose and glucose is necessary for this dual function of trehalose. This work demonstrates that daf-16/FoxO promotes starvation resistance by shifting carbon metabolism to drive trehalose synthesis, which in turn supports survival by providing an energy source and acting as a stress protectant.
In addition to acute changes in response to the nutrient environment, effects can persist intergenerationally. Maternal effects of environmental conditions produce intergenerational phenotypic plasticity. Adaptive value of these effects depends on appropriate anticipation of environmental conditions in the next generation, and mismatch between conditions may contribute to disease. However, regulation of intergenerational plasticity is poorly understood. Dietary restriction (DR) delays aging but maternal effects have not been investigated. We demonstrate maternal effects of DR in the roundworm C. elegans. Worms cultured in DR produce fewer but larger progeny. Nutrient availability is assessed in late larvae and young adults, rather than affecting a set point in young larvae, and maternal age independently affects progeny size. Reduced signaling through the insulin-like receptor daf-2/InsR in the maternal soma causes constitutively large progeny, and its effector daf-16/FoxO is required for this effect. nhr-49/Hnf4, pha-4/FoxA, and skn-1/Nrf also regulate progeny-size plasticity. Genetic analysis suggests that insulin-like signaling controls progeny size in part through regulation of nhr-49/Hnf4, and that pha-4/FoxA and skn-1/Nrf function in parallel to insulin-like signaling and nhr-49/Hnf4. Furthermore, progeny of DR worms are buffered from adverse consequences of early-larval starvation, growing faster and producing more off- spring than progeny of worms fed ad libitum. These results suggest a fitness advantage when mothers and their progeny experience nutrient stress, compared to an environmental mismatch where only progeny are stressed. This work reveals maternal provisioning as an organismal response to DR, demonstrates potentially adaptive intergenerational phenotypic plasticity, and identifies conserved pathways mediating these effects.
Item Open Access An evolutionary genomics approach towards understanding Plasmodium vivax in central Africa(2022) Gartner, ValerieIncreased attention has recently been placed on understanding the natural variation of the malaria parasite Plasmodium vivax across the globe, as in 2020 alone, P. vivax caused an estimated 4.5 million malaria cases and lead to over 600,000 deaths around the world. P. vivax infections in central Africa have been of particular interest, as humans in Sub-Saharan Africa frequently possess a P. vivax resistance allele known as the Duffy-negative phenotype that is believed to prevent infection in these individuals. However, new reports of asymptomatic and symptomatic infections in Duffy-negative individuals in Africa raise the possibility that P. vivax is evolving to evade host resistance.Whole genome sequencing has become more common as a means of understanding the population diversity of P. vivax. However, there is still a scarcity of information about P. vivax in central Africa. In this dissertation, I analyze whole genome sequencing data from a new P. vivax sample collected from the Democratic Republic of the Congo in central Africa. By studying P. vivax from central Africa, we can begin to understand the evolutionary history of the pathogen in this part of the world as it relates to the global context of this pathogen. I also investigate the relationship of P. vivax in the DRC with a potential animal reservoir of a closely related species, P. vivax-like, in non-human primates in this region. Due to the scarcity of P. vivax samples in central Africa, I also investigated methods with which to best make use of whole genome sequencing data, particularly in generating phylogenetic trees. While many studies of P. vivax genetic diversity employ whole genome variation data in order to study evolutionary relationships of P. vivax populations, in this dissertation I make use of the P. vivax apicoplast, a non-photosynthetic plastid organelle genome. The apicoplast genome is five times longer than the mitochondrial genome and does not undergo recombination, making it a valuable locus for studying P. vivax evolutionary history using phylogenetic trees.
Item Open Access Analysis of the Drosophila Sugar Receptor Genes(2009) Slone, Jesse DavidGustation, also known as taste perception, is critical for the survival of most animal species. The fruit fly Drosophila melanogaster employs 68 different gustatory receptors (GRs) for the detection of sugars, bitter or toxic compounds, and pheromones. However, with a few notable exceptions, the functions of most GRs involved in feeding are unknown. Our research has focused on a cluster of highly-related Drosophila Grs, known as the Gr64 family, that have been shown to be critical for the perception of multiple sugars. Furthermore, we have demonstrated that another gene related to the Gr64 genes, Gr61a, is a sugar receptor that is narrowly tuned to a subset of pyranose sugars and may (along with the Gr64 genes) be indispensable for early fly development.
As a complementary approach to our behavioral analysis, we have examined the expression pattern of the Drosophila sugar receptors using knock-in driver alleles created by homologous recombination. As expected, most of these drivers have shown strong expression in various taste tissues. Intriguingly, some of these knock-in alleles also show expression in the maxillary palp and antenna, tissues previously thought to be involved only in olfaction. These expression patterns raise interesting questions about the true range of function of these chemosensory receptors and whether or not they might be involved in olfaction as well as gustation.
Item Open Access Ancestry-based Methods for Characterizing the Evolutionary History of Admixed Populations(2022) Hamid, ImanAdmixture occurs when previously isolated populations come together to form a new population with genetic ancestry from those sources. Admixture is ubiquitous across the tree of life, including humans, and is often associated with migration and exposure to new environments and selective pressures. Admixed populations provide a unique opportunity to study adaptation on short timescales by introducing beneficial alleles at high frequency. However, admixed populations are often excluded from genomic studies due to lack of applicable methodology. Instead of relying on classical methods confounded by the process of admixture itself, we can detect changes in patterns of genetics ancestry that are informative about selection in admixed populations and at the short timescales often relevant for post-admixture selection. However, we lack theoretical expectations and methods to detect and characterize ancestry-based genomic signals indicative of post-admixture selection and adaptation. Common ancestry outlier approaches discard information about the surrounding genomic context and are prone to false positives due to drift and demography. Here, I present three studies which leverage patterns of genetic ancestry to investigate the evolutionary history of admixed populations. First, I develop a suite of ancestry-based summary statistics and computational methods to detect post-admixture adaptation, and demonstrate their application in a case study of human adaptation to malaria. In particular, these summary statistics incorporate patterns of ancestry beyond the site under selection, such as the length of contiguous ancestry tracts surrounding the locus, and are informative about the strength and timing of selection in admixed populations. I observe one of the strongest signals of recent selection in humans at the malaria protective Duffy-null allele, and show that this mode of strong single-locus selection over 20 generations has impacted genome-wide patterns of ancestry. Next, I move beyond summary statistics to develop a deep learning strategy for localizing regions of the genome under selection. This method takes images of chromosomes painted by ancestry as input to avoid the loss of information and bias that can occur when relying on user-defined summary statistics. I demonstrate this approach on simulated admixture scenarios and find that the method successfully localizes variants under selection 95% percent of the time, outperforms the common ancestry outlier approach, and is robust to demographic misspecification. Lastly, I present the first Illyrian genome sequences available from the Iron Age in a study of the ancestry and genetic relationships of five neonates buried in Korčula, Croatia. I find genetic support for classifying these individuals as Illyrian, and show that patterns of ancestry and genetic variation are consistent with their geographic location between Italy and the mainland Balkans. In the combined work presented here, I advance our ability to study the evolutionary history of admixed populations, which has implications for our understanding of phenotypic variation, disease risk, and conservation genetics across many study systems. Further, these methods tailored to the mosaic ancestry of admixed populations is a step towards expanding the diversity of populations, especially humans, who benefit from discoveries and advancement in genomic research.
Item Open Access Aneuploidy Tolerance in a Polyploid Organ(2016) Schoenfelder, Kevin PaulEndopolyploid cells (hereafter - polyploid cells), which contain whole genome duplications in an otherwise diploid organism, play vital roles in development and physiology of diverse organs such as our heart and liver. Polyploidy is also observed with high frequency in many tumors, and division of such cells frequently creates aneuploidy (chromosomal imbalances), a hallmark of cancer. Despite its frequent occurrence and association with aneuploidy, little is known about the specific role that polyploidy plays in diverse contexts. Using a new model tissue, the Drosophila rectal papilla, we sought to uncover connections between polyploidy and aneuploidy during organ development. Our lab previously discovered that the papillar cells of the Drosophila hindgut undergo developmentally programmed polyploid cell divisions, and that these polyploid cell divisions are highly error-prone. Time-lapse studies of polyploid mitosis revealed that the papillar cells undergo a high percentage of tripolar anaphase, which causes extreme aneuploidy. Despite this massive chromosome imbalance, we found the tripolar daughter cells are viable and support normal organ development and function, suggesting acquiring extra genome sets enables a cell to tolerate the genomic alterations incurred by aneuploidy. We further extended these findings by seeking mechanisms by which the papillar cells tolerated this resultant aneuploidy.
Item Embargo Aptamers as Reversible Sorting Ligands in Dual FACS and MACS: Antisense and Nuclease-Mediated Approaches(2023) Requena, MartinFluorescence Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS) are two essential tools for cell separation in research and medicine. Antibodies, the gold standard in both of these methods, are effective ligands for cell-surface biomarkers, but their irreversible binding precludes a wide variety of downstream medical and experimental applications. Aptamers – nucleic acid ligands with a defined three-dimensional structure that enables them to bind a molecular target with a high degree of specificity – offer a viable alternative for this particular obstacle because their RNA- or DNA-based chemistry enables their removal from cellular targets. In these studies, we present examples of successful sorting of cells and removal of the targeting aptamers with MACS and FACS using both the previously-published antisense-based method of post-sorting aptamer removal and a more general approach using nuclease-based digestion of targeting aptamers on the cell surface after cell isolation. We believe this work can be used in a number of potential post-sorting applications where targeting ligands or attached magnetic or fluorescent moieties could interfere with experimental or clinical results.
Item Open Access Basement Membrane Dynamics During Anchor Cell Invasion(2015) Morrissey, Meghan AnnBasement membranes are a dense, sheet-like form of extracellular matrix that underlie epithelia and endothelia, and surround muscle, fat and Schwann cells. Basement membranes separate tissues and protect them from mechanical stresses. Although traditionally thought of as a static support structure, a growing body of evidence suggests that dynamic basement membrane deposition and modification instruct cell behavior and morphogenetic processes. In this thesis, I discuss how changes to basement membrane affect anchor cell (AC) invasion during C. elegans uterine vulval attachment. During AC invasion, the uterine AC breaches two juxtaposed basement membranes to contact the underlying vulval epithelium. Using live-cell imaging, genetics, molecular biology and electron microscopy I identify three modifications to the BM that affect AC invasion. In Chapter 2, I describe a system for linking juxtaposed basement membranes to stably align or connect adjacent tissues. This adhesion system promotes rapid AC invasion and also regulates a more long-term connection between the uterine tissue and the hypodermal seam cell in the adult worm. Chapter 3 elucidates how the BM component SPARC promotes cell invasion. As SPARC overexpression is correlated with cancer metastasis, this aims to understand how SPARC overexpression promote invasion in a pathological situation. In Chapter 4, I discuss preliminary data showing that the AC actively secretes laminin into the basement membrane targeted for invasion. I outline how future studies could elucidate the mechanism by which AC-derived laminin might promote cell invasion. Finally, Chapter 5 discusses conclusions and future directions for these studies.
Item Open Access Causes and consequences of microbial symbioses; insights from comparative genomics of plant associated bacterial-fungal interactions(2017) Uehling, Jessie UehlingSymbioses have shaped our modern world, providing for the air we breathe; for the plant and animal diversity we celebrate; and for the functioning of ecosystems from the tops of mountains to the ocean floor. Here I study symbiosis using fungal bacterial interactions as a model for understanding symbiotic dynamics. In this dissertation I present interpretations of experimental data about fungal bacterial interactions that lend insight into dynamics of symbiotic establishment and consequences of long-term endosymbiosis. More specifically, I examine the interactions of a plant-associated zygomycete, Mortierella elongata, and its interactions with several Betaproteobacteria in the Burkholderiales. I used genome sequencing, comparative genomics, physiological assays, and time-lapse microfluidic videography to ask the following questions; How are bacterial fungal symbioses initiated? How do bacteria and fungi communicate? What resources do these microbes share? Are long-term symbioses essential for one or both partners? What are the impacts of removing long-term endosymbionts for fungal host physiology? What are the effects long-term fungal endosymbiosis on bacterial genome content?
In chapter 1 I present lessons learned from genome sequencing of fungus Mortierella elongata and its primary resident endosymbiont, Mycoavidus cysteinexigens. I tested the hypothesis that genome reduction is a commonality of eukaryotic endosymbionts, and that characteristic genes and pathways are impacted by gene loss and inactivation in endosymbionts. I found that compared to its free-living relatives, M. cysteinexigens has a highly reduced genome and has lost genes coding for the biosynthesis of amino acids and intermediates of glycolysis, among other metabolic pathways. I describe a method for clearing fungi of endosymbionts using antibiotics. I report comparative physiological data for the cleared and uncleared strains and draw conclusions about the nature of their interactions based on the behavior of the fungal host lacking the endosymbiont. I tested the hypothesis that sharing of fungal fatty acids underpins this symbiosis, as suggested by the genome sequences of both microbes. I found that when cleared of endosymbionts, M. elongata grows more rapidly and accumulates fatty acids that are likely used by M. cysteinexigens when present.
In chapter 2 I investigate the transcriptional control of fungal-endosymbiont phenotypes. I continued working with the cleared and uncleared strains developed in chapter 1 and quantified transcript abundance in each isolate. I assigned functions to differentially expressed genes by identifying homologues in the fungal genetic model organism Saccharomyces cerevisiae. I layered on transcriptional data to the patterns that emerged from comparative analyses in chapter 1to better understand fungal response to endosymbiosis. I showed that differential expression of conserved genes underpin the increases in growth and altered metabolism in M. elongata when cleared of M. cysteinexigens. I found that endosymbiont presence is associated with toggling of metabolic programs that result in resources more or less bioavailable to M. cysteinexigens based on metabolic capability predicted by genome annotation. I found that genes with homologues in mating pheromone perception pathways are differentially regulated in cleared isolates of M. elongata, and that this aspect of clearing is shared by other isolates of M. elongata when cleared of their bacterial endosymbionts.
In chapter 3 I examine dynamics of pre-symbiotic signaling events between fungi and bacteria using Mortierella elongata and a free-living bacterium, Burkholderia BT03. Using microbial growth assays and a suite of conditioned medias I showed that growth stimulation is mutual for fungi and bacteria, and that signaling leading up to symbiotic phenotypes involves multiple bi-directional signal exchanges. I designed and used a microfluidic platform along with plate based and liquid culture systems to compare fungal growth rates in response to conditioned medias. By extrapolating rates from microbial growth assays including M. elongata, Burkholderia BT03 and related microbes, I inferred directionality, order, conditionality, specificity, and nature of signal exchange leading to microbial growth stimulation in this system.
As a whole this thesis explores how comparative microbial genomics and phenotypic assays can provide mechanistic insight into symbiotic establishment and the effects of long-term symbioses. The results presented here provide novel insights into biotic and abiotic factors dictating symbiotic establishment. Second, they suggest long-term endosymbionts of eukaryotic cells experience convergent gene loss. Lastly they emphasize that long-term endosymbionts strongly impact host metabolism, and that host-microbe metabolic intertwining is a commonality of many symbioses. The use of a systems biology approach to generate comparative genomics data on multiple levels that enable insight into the consequences of fungal bacterial symbioses is a novel contribution for the field.
Item Open Access Cell Fate Specification and the Regulation of RNA-dependent DNA Methylation in the Arabidopsis Root Meristem(2016) Valdes, ManuelThe Arabidopsis root apical meristem (RAM) is a complex tissue capable of generating all the cell types that ultimately make up the root. The work presented in this thesis takes advantage of the versatility of high-throughput sequencing to address two independent questions about the root meristem. Although a lot of information is known regarding the cell fate decisions that occur at the RAM, cortex specification and differentiation remain poorly understood. In the first part of this thesis, I used an ethylmethanesulfonate (EMS) mutagenized marker line to perform a forward genetics screen. The goal of this screen was to identify novel genes involved in the specification and differentiation of the cortex tissue. Mapping analysis from the results obtained in this screen revealed a new allele of BRASSINOSTEROID4 with abnormal marker expression in the cortex tissue. Although this allele proved to be non-cortex specific, this project highlights new technology that allows mapping of EMS-generated mutations without the need to map-cross or back-cross. In the second part of this thesis, using fluorescence activated cell sorting (FACS) coupled with high throughput sequencing, my collaborators and I generated single-base resolution whole genome DNA methylomes, mRNA transcriptomes, and smallRNA transcriptomes for six different populations of cell types in the Arabidopsis root meristem. We were able to discover that the columella is hypermethylated in the CHH context within transposable elements. This hypermethylation is accompanied by upregulation of the RNA-dependent DNA methylation pathway (RdDM), including higher levels of 24-nt silencing RNAs (siRNAs). In summary, our studies demonstrate the versatility of high-throughput sequencing as a method for identifying single mutations or to perform complex comparative genomic analyses.
Item Open Access Cell Polarity Establishment in the Budding Yeast Saccharomyces Cerevisiae(2009) Howell, AudreyEstablishing an axis of cell polarity is central to cell motility, tissue morphogenesis, and cell proliferation. A highly conserved group of polarity regulators is responsible for organizing a wide variety of polarized morphologies. One of the most widely expressed polarity regulators is the Rho-type GTPase Cdc42. In response to cell cycle cues the budding yeast Saccharomyces cerevisiae polarizes Cdc42p to a discrete site on the cell periphery. GTP-Cdc42p recruits a number of effectors that aid in the organization of a polarized actin cytoskeleton. The polarized actin cytoskeleton acts as tracks to facilitate the delivery of the secretory vesicles that will grow the bud, an essential process for an organism that proliferates by budding. We have employed treatment with the actin depolymerizing drugs Latrunculin A and B as well as high-speed timelapse microscopy of fluorescently labeled polarity proteins to characterize the assembly of the incipient bud site.
Often, ensuring that only a single axis of polarity is established is as important as generating asymmetry in the cell. Even in the absence of positional cues dictating the direction of polarization, many cells are still able to self-organize and establish one, and only one, polarity axis through a process termed symmetry breaking. Symmetry breaking is thought to employ positive feedback to amplify stochastic fluctuations in protein concentration into a larger asymmetry. To test whether singularity could be guaranteed by the amplification mechanism we re-wired yeast to employ a synthetic positive feedback mechanism. The re-wired cells could establish polarity, however they occasionally made two buds simultaneously, suggesting that singularity is guaranteed by the amplification mechanism.
Item Open Access Cerebral Cavernous Malformations: From Two-Hit Mechanism to Developing a Targeted Therapy(2013) McDonald, David AndrewCerebral cavernous malformations (CCMs) are multicavernous vascular lesions affecting the central nervous system. Affected individuals have a lifetime risk of recurrent headaches, focal neurological deficits, seizures, and intracerebral hemorrhage leading to stroke. Patients tend to fall into two classes: familial cases with a known family history and multiple lesions, and; sporadic cases with no family history and single lesions. This epidemiological pattern suggests a two-hit mutational mechanism for CCM. While somatic mutations have been identified in lesions from familial patients, it is unknown if sporadic cases follow the same genetic mechanism. Using a next-generation sequencing strategy, I have identified somatic mutations from sporadic CCM lesions in the three known CCM genes, including one lesion bearing two independent mutations in CCM1. These data support a two-hit mutation mechanism in CCM for sporadic patients.
The mechanism of CCM pathogenesis (how mutations in one of the three CCM genes causes lesions to form and develop) is currently unknown. We developed mouse models that recapitulate the human disease. We have further shown that inhibition of Rho Kinase decreases the number of late-stage, multicavernous lesions. This is the first potential therapeutic strategy to specifically treat CCM, and suggests that the RhoA pathway is a central player in CCM pathogenesis.
Item Open Access Characterization of Drosophila Ctr1a: New Roles for Ctr1 Proteins and Copper in Physiology and Cell Signaling Pathways(2008-10-21) Turski, Michelle LynnCopper is an essential trace element required by all aerobic organisms as a co-factor for enzymes involved in normal growth, development and physiology. Ctr1 proteins are members of a highly conserved family of copper importers responsible for copper uptake across the plasma membrane. Mice lacking Ctr1 die during embryogenesis from widespread developmental defects, demonstrating the need for adequate copper acquisition in the development of metazoan organisms via as yet uncharacterized mechanisms. The early lethality of the Ctr1 knockout mouse has made it difficult to study the functions of copper and Ctr1 proteins in metazoan development and physiology. Drosophila melanogaster, a genetically tractable system expresses three Ctr1 genes, Ctr1A, Ctr1B and Ctr1C, and may help to further understand the roles of copper and Ctr1 proteins in metazoan development and physiology. Described here is the characterization of Drosophila Ctr1A.
Localization studies using an affinity purified anti-Ctr1A peptide antibody show Ctr1A is predominantly expressed at the plasma membrane in whole embryos and in larval tissues. Ctr1A is an essential gene in Drosophila as loss-of-function mutants, generated by imprecise p-element excision arrest at early larval stages of development. Inductively coupled plasma mass spectroscopy (ICP-MS) demonstrated that whole body copper levels are reduced in Ctr1A mutants and consequently, a number of copper-dependent enzyme deficiencies were detected by in vitro enzyme and cell biological assays. Ctr1A maternal and zygotic mutants have a more severe developmental phenotype and also showed reductions in heart rate, which could be partially rescued by dietary copper supplementation. Heart-specific Ctr1A knockdown flies were subsequently examined for heart rate defects using optical coherence tomography (OCT) and while they did have reduced heart rate measurements, heart contractility was compromised. While investigating tissue-specific requirements for Ctr1A in the development of Drosophila, a genetic interaction between Ctr1A and Ras was observed. Genetic experiments in Drosophila and cell culture experiments in both Drosophila and mammalian cell lines demonstrate a conserved role for Ctr1 proteins and copper as positive modulators of Ras/MAPK pathway signaling. Immunoblot analysis shows that signal transduction is intact until the point at which MEK1/2 phosphorylates ERK1/2. MEK2 protein levels are reduced in copper deficient cells, while MEK1 is able to bind copper-chelated beads, suggesting that these two proteins may be copper-binding proteins. In summary, this work demonstrates that Ctr1A is an essential gene in Drosophila and through characterization studies of Ctr1A, has uncovered conserved roles for Ctr1 proteins and copper in physiological processes and in an important signaling pathway that controls a number of fundamental biological processes.
Item Open Access Characterizing Genetic Drivers of Lymphoma through High-Throughput Sequencing(2016) Zhang, JennyThe advent of next-generation sequencing, now nearing a decade in age, has enabled, among other capabilities, measurement of genome-wide sequence features at unprecedented scale and resolution.
In this dissertation, I describe work to understand the genetic underpinnings of non-Hodgkin’s lymphoma through exploration of the epigenetics of its cell of origin, initial characterization and interpretation of driver mutations, and finally, a larger-scale, population-level study that incorporates mutation interpretation with clinical outcome.
In the first research chapter, I describe genomic characteristics of lymphomas through the lens of their cells of origin. Just as many other cancers, such as breast cancer or lung cancer, are categorized based on their cell of origin, lymphoma subtypes can be examined through the context of their normal B Cells of origin, Naïve, Germinal Center, and post-Germinal Center. By applying integrative analysis of the epigenetics of normal B Cells of origin through chromatin-immunoprecipitation sequencing, we find that differences in normal B Cell subtypes are reflected in the mutational landscapes of the cancers that arise from them, namely Mantle Cell, Burkitt, and Diffuse Large B-Cell Lymphoma.
In the next research chapter, I describe our first endeavor into understanding the genetic heterogeneity of Diffuse Large B Cell Lymphoma, the most common form of non-Hodgkin’s lymphoma, which affects 100,000 patients in the world. Through whole-genome sequencing of 1 case as well as whole-exome sequencing of 94 cases, we characterize the most recurrent genetic features of DLBCL and lay the groundwork for a larger study.
In the last research chapter, I describe work to characterize and interpret the whole exomes of 1001 cases of DLBCL in the largest single-cancer study to date. This highly-powered study enabled sub-gene, gene-level, and gene-network level understanding of driver mutations within DLBCL. Moreover, matched genomic and clinical data enabled the connection of these driver mutations to clinical features such as treatment response or overall survival. As sequencing costs continue to drop, whole-exome sequencing will become a routine clinical assay, and another diagnostic dimension in addition to existing methods such as histology. However, to unlock the full utility of sequencing data, we must be able to interpret it. This study undertakes a first step in developing the understanding necessary to uncover the genomic signals of DLBCL hidden within its exomes. However, beyond the scope of this one disease, the experimental and analytical methods can be readily applied to other cancer sequencing studies.
Thus, this dissertation leverages next-generation sequencing analysis to understand the genetic underpinnings of lymphoma, both by examining its normal cells of origin as well as through a large-scale study to sensitively identify recurrently mutated genes and their relationship to clinical outcome.
Item Open Access Characterizing the Relationship Between Cell-Cycle Progression and a Transcriptional Oscillator(2013) Bristow, Sara LynnThe cell division cycle is the process in which the entirety of a cell's contents is duplicated completely and then equally segregated into two identical daughter cells. The order of the steps in the cell cycle must be followed with fidelity to guarantee two viable cells. Understanding the regulatory mechanisms that control cell-cycle events remains to be a fundamental question in cell biology. In this dissertation, I explore the mechanisms that coordinate and regulate cell-cycle progression in the budding yeast, Saccharomyces cerevisiae.
Cell-cycle events have been shown to be triggered by oscillations in the activity of cyclin dependent kinases (CDKs) when bound to cyclins. However, several studies have shown that some cell-cycle events, such as periodic transcription, can continue in the absence of CDK activity. How are periodic transcription and other cell-cycle events coupled to each other during a wild-type cell cycle? Currently, two models of cell-cycle regulation have been proposed. One model hypothesizes that oscillations in CDK activity controls the timing of cell-cycle events, including periodic transcription. The second model proposes that a transcription factor (TF) network oscillator controls the timing of cell-cycle events, via proper timing of gene expression, including cyclins. By measuring global gene expression dynamics in cells with persistent CDK activity, I show that periodic transcription continues. This result fits with the second model of cell-cycle regulation. Further, I show that during a wild-type cell cycle, checkpoints are responsible for arresting the bulk of periodic transcription. This finding adds a new layer of regulation to the second model, providing a mechanism that coordinates cell-cycle events with a TF network oscillator. Taken together, these data provide further insight into the regulation of the cell cycle.