UT Southwestern Electronic Theses and Dissertations
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Browsing UT Southwestern Electronic Theses and Dissertations by Author "Abrams, John M."
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Item Apoptosis Determinants in Drosophila Melanogaster(2007-12-17) Chew, Su Kit; Abrams, John M.Apoptosis is a form of programmed cell death (PCD) that is governed by a core set of genes conserved across diverse metazoan phyla. Cells dying by apoptosis exhibit a characteristic series of morphological and biochemical changes that is also conserved. This form of PCD plays pivotal roles in homeostatic regulation of cell numbers, developmental sculpting of organs, damage and infection responses; conversely, its disregulation has profound implications in diseases such as cancers, immune disorders infertility and dystrophies. Common parallels in the regulation of the core apoptosis machinery have been elucidated in human and experimental model organisms, though many fundamental questions in our understanding of its regulation remain. A conserved node in the apoptosis pathway is the apoptosome, comprising the apical caspase and its adaptor protein. To understand the functions of this node, I generated a null allele of the apical caspase Dronc in the experimental model organism Drosophila melanogaster. Dronc is required for developmentally regulated apoptosis in multiple tissues during embryogenesis and larval development. Failure of apoptosis correlated with tissue hyperplasia. Notably, the removal of Dronc eliminated the cellular apopototic response to stresses in cells. In some of the stress contexts tested, Dronc depletion partially rescued cell viability to the same levels as pan-caspase inhibition by small peptide inhibitors, suggesting that Dronc functions map specifically to caspase activation and apoptosis. These and similar observations in its adaptor protein Dark point to the apoptosome as a key node for apoptosis in Drosophila. From these observations, I sought to use the induced apoptosis cellular response as a means to identify novel components and regulators in the apoptosis pathway. I optimized a cell culture system for high-throughput cell-based screening using RNA interference (RNAi) mediated gene silencing and a synthetic antagonist of inhibitors of apoptosis proteins (IAPs). From a genome-wide Drosophila RNAi library, I identified 42 potential genes required for apoptosis, of which I characterized 13 highly validated targets for their requirements in multiple stress contexts. One of these hits, Tango7, regulates pro-Dronc protein and represents an unprecedented point of apoptosis regulation. Collectively, my studies bolster the model for the crucial requirement of the apoptosome in apoptosis and identify new regulation entry-points into the apoptosis pathway.Item Bcl-2 Function in Drosophila(2007-12-17) Galindo, Kathleen A.; Abrams, John M.Bcl-2 family members are pivotal regulators of programmed cell death (PCD). In mammals, pro-apoptotic Bcl-2 family members initiate early apoptotic signals by causing the release of cytochrome c from the mitochondria, a step necessary for the initiation of the caspase cascade. Worms and flies do not show a requirement for cytochrome c during apoptosis, but both model systems express pro- and anti-apoptotic Bcl-2 family members. Drosophila encodes two Bcl-2 family members, Debcl (pro-apoptotic) and Buffy (anti-apoptotic). To understand the role of Debcl in Drosophila apoptosis, we produced an authentic null allele at the Debcl locus. Although gross development and lifespans were unaffected, we found that debcl was required for pruning cells in the developing central nervous system. debcl genetically interacted with the ced-4/Apaf-1counterpart, dark, but was not required for killing by RPR proteins. Surprisingly, in a model of caspaseindependent cell death, we found that heterologous killing by Murine Bax required debcl to exert its pro-apoptotic activity. DebclKO mutants were also significantly affected for mitochondrial density. Taken together, these findings suggest that evolutionary functions impacting mitochondrial properties represent ancient activities which preceded the evolution of these proteins as central regulators of PCD.Item Biochemical Analysis of Apoptosome Formation(2006-12-19) Kim, Hyun-Eui; Wang, Xiaodong; Abrams, John M.; Chen, Zhijian J.; Rosen, Michael K.Apoptosis is an active cell death program executed by proteases named caspases. One of the major caspase-activating pathways is initiated by mitochondria. Upon various apoptotic stimuli, the mitochondria releases cytochrome c into the cytosol where it binds to apoptotic protease activating factor 1 (Apaf-1). Then, the cytochrome c-bound Apaf-1 forms a heptameric complex named apoptosome. Apoptosome provides a platform to activate downstream caspases. The initiator caspase, procaspase-9 is recruited to apoptosome and gets activated to cleave downstream effector caspases. The series of this activation cascade is tightly regulated at each step. However, the role of cytochrome c and nucleotides during apoptosome formation is not clear. Also, how the apoptosome activity is stimulated by the positive regulator PHAP proteins is yet to be determined. Thus, my thesis work includes studies regarding these questions using biochemical analysis. I reconstituted apoptosome pathway using recombinant proteins in vitro. As a result, I discovered several biochemical steps during apoptosome formation that were previously unknown. And I identified a new mediator that positively regulates apoptosome formation. The new findings are, 1) Recombinant Apaf-1 obtained from insect cell expression system was already associated with dATP. 2) The Apaf-1-bound dATP got hydrolyzed upon cytochrome c binding. 3) The hydrolyzed nucleotide on Apaf-1 needed to be exchanged with dATP/ATP to form an active apoptosome. 4) CAS is a mediator of PHAP proteins. PHAPI and CAS enhanced the nucleotide exchange on Apaf-1 to stimulate apoptosome formation.Item Chemical Disruption of Wnt Signaling and Telomere Length Maintenance(2015-01-28) Kulak, Ozlem; White, Michael A.; Minna, John D.; Abrams, John M.; Lum, LawrenceA nearly universal feature of colorectal cancer (CRC) incidents is the presence of genetic alterations that promote deviant activation of the TCF/LEF transcriptional regulators. The TCF/LEF proteins control the transcriptional output of intercellular signaling mediated by the Wnt family of secreted ligands. Several chemical screening efforts devoted to disrupting deviant TCF/LEF activity have converged on two vulnerabilities in the Wnt pathway -- the poly-ADP-ribose polymerases, Tankyrase 1 and 2 (Tnks1 and 2) that control the threshold response levels to Wnt ligands, and the Wnt acyltransferase Porcupine that provides an essential fatty acyl adduct to all nineteen Wnt ligands. My thesis focuses on the chemical biology of one of these strategies -- the Tnks enzymes -- with the goal of understanding the strengths and limitations of drugging the Tnks proteins for achieving therapeutic goals in regenerative medicine and cancer. Given the previously assigned role of Tnks enzymes in telomere maintenance, I have also devoted considerable effort to understanding the cell biological effects of disrupting Tnks activity on telomere integrity. Finally, I mined a high-confidence collection of Wnt pathway inhibitors with previously unidentified mechanisms of action to identify novel small molecules that directly target the TCF/LEF transcriptional apparatus. This effort netted a chemical approach for disabling deviant transcriptional activity in CRC that is distinct from the one afforded by Tnks and Porcn inhibitors. Taken together, my thesis establishes a chemical toolkit for interrogating the inner workings of Wnt-mediated signaling and also reveals new avenues for disabling deviant Wnt responses in cancer and normal Wnt responses in tissue engineering.Item Communal Cell Death and P53 Mediated Transcriptional Control in Drosophila Melanogaster(2011-08-10) Link, Nichole Lynn; Abrams, John M.Apoptosis is essential for all metazoan development. The key component that functions in apoptosis, the apoptosome, is a molecular machine that initiates caspase activation and is conserved throughout the animal kingdom. Drosophila strains that are mutated for genes encoding the apoptosome show pronounced defects in programmed cell death (PCD). Using a characteristic phenotype associated with mosaic animals, we conducted a screen in Drosophila to discover new regulators or effectors of the apoptosome. Using this model, we also discovered a unique communal form of cell death where large regions of epithelial cells are eliminated within minutes. We also produced 'saturation tile' arrays by digital optical chemistry for an unbiased sampling of transcriptional activity in the Drosophila genome. We found that the scope of unannotated transcriptional activity is extensive and widespread. A dominant population of noncanonical transcripts was stress-responsive and required p53, a master regulator of conventional stress-responsive target genes in vertebrates and invertebrates. This prompted us to examine stimulus dependent activity surrounding a single p53 enhancer in our tiled region. Through genetic analyses, we showed that this enhancer coordinates stimulus dependent induction of multiple genes spanning over 300kb throughout the Reaper region. Surprisingly, this same enhancer regulated a gene positioned across the centromere at distances over 20Mb and also controlled at least one gene mapping to a different chromosome. Chromosome conformation capture analyses placed this enhancer in close proximity to these distant targets in vivo through specific DNA looping and these interactions were influenced by p53. Therefore, a single p53 enhancer is necessary and sufficient for long range, multigenic regulation in cis and in trans.Item Fibulin-5 Promotes Pancreatic Tumor Growth through Inhibition of Integrin-induced ROS: Insights into Tumor-Matrix Signaling(2016-07-18) Topalovski, Mary; Terada, Lance; Cobb, Melanie H.; Abrams, John M.; Brekken, Rolf A.Elevated oxidative stress is an aberration seen in many solid tumors, and exploiting this biochemical difference has the potential to enhance the efficacy of anti-cancer agents. Homeostasis of reactive oxygen species (ROS) is important for normal cell function, but excessive production of ROS can result in cellular toxicity and therefore ROS levels must be balanced finely. Here, we highlight the relationship between the extracellular matrix and ROS production by reporting a novel function of the matricellular protein Fibulin-5 (Fbln5). We found that Fbln5 is abundantly expressed in mouse and human pancreatic cancer compared to normal pancreas. By employing genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDA), we showed that mutation of the integrin-binding domain of Fbln5 led to decreased tumor growth, increased survival, and enhanced chemoresponse to standard PDA therapies. Through mechanistic investigations, we found that improved survival was due to increased levels of oxidative stress in Fbln5 mutant tumors. Furthermore, loss of the Fbln5-integrin interaction augmented fibronectin (FN) signaling, driving integrin-induced ROS production in a 5-lipooxygenase-dependent manner. These data indicate that Fbln5 promotes PDA progression by functioning as a molecular rheostat that modulates cell-ECM interactions to reduce ROS production and thus tip the balance in favor of tumor cell survival and treatment-refractory disease. The latter part of this thesis is focused on the underlying mechanism that leads to upregulation of Fbln5 in PDA. The deposition of ECM is a defining feature of PDA where ECM signaling can promote cancer cell survival and epithelial plasticity programs. ECM-mediated signaling is governed by expression of the ECM proteins, the presence of cell surface receptors and the expression and activity of matricellular proteins that function as extracellular adaptors to reduce ECM-cell interaction. As stated above, Fbln5 is a matricellular protein that blocks FN-integrin interaction and thus directly limits ECM-driven ROS production and supports PDA progression. Compared to normal pancreatic tissue, Fbln5 is expressed abundantly in the stroma of PDA; however, the mechanisms underlying the stimulation of Fbln5 expression in PDA are undefined. Using in vitro and in vivo approaches, we report that hypoxia triggers Fbln5 expression in a transforming growth factor β (TGF-β)- and PI3K-dependent manner. Pharmacologic inhibition of TGF-β receptor (TGF-βR), PI3K, or protein kinase B (AKT) was found to block hypoxia-induced Fbln5 expression in mouse embryonic fibroblasts and 3T3 fibroblasts. Moreover, tumor-associated fibroblasts from mouse PDA were also responsive to TGF-βR and PI3K/Akt inhibition with regard to suppression of Fbln5. In genetically engineered mouse models of PDA, therapy-induced hypoxia elevated Fbln5 expression while pharmacologic inhibition of TGF-β signaling reduced Fbln5 expression. These findings offer insight into the signaling axis that induces Fbln5 expression in PDA and a potential strategy to block its production.Item GCNA: Guardian of the Genome(2020-05-01T05:00:00.000Z) Goldstein, Courtney DaVee; Abrams, John M.; Buszczak, Michael; Brekken, Rolf A.; Olson, Eric N.The propagation of species depends on the ability of germ cells to protect their genome in the face of numerous exogenous and endogenous threats. While germ cells employ a number of know repair pathways, specialized mechanisms that ensure high-fidelity replication, chromosome segregation, and repair of germ cell genomes remain incompletely understood. Here, we identify Germ cell nuclear acidic peptidase (GCNA) as a conserved regulator of genome stability in flies, worms, zebrafish and human germ cell tumors. GCNA contains an acidic intrinsically disordered region (IDR) and a protease-like SprT domain. In addition to chromosomal instability and replication stress, Gcna mutants accumulate DNA-protein crosslinks (DPCs). GCNA acts in parallel with a second SprT domain protein Spartan. Structural analysis reveals that while the SprT domain is needed to limit meiotic and replicative damage, much of GCNA's function maps to its IDR. This work shows GCNA protects germ cells from various sources of damage, providing novel insights into conserved mechanisms that promote genome integrity across generations.Item Genetic and Biochemical Analyses of the Necessity for Caspase Activation by the CED4-Domain Proteins, APAF-1 and DARK(2003-04-03) Oliver, George Reinhold; Abrams, John M.Activation of caspase proteases by Ced4 domain proteins is a critical step in the induction of programmed cell death, or apoptosis. Understanding of the genetic and biochemical regulation of the mammalian Ced-4 gene, Apaf-1, may be crucial to the understanding of autoimmune diseases, neurodegenerative disorders and cancer progression. Located in chromosomal band 12q22, Apaf-1 is in a locus frequently deleted in Male Germ Cell Tumors (GCT's). Though not homozygously inactivated in these tumors, Apaf-1 mediated caspase activation is impaired in GCT cell lines and may be a frequent event in other cancer types. Analysis of human genomic DNA facilitated the discovery of the homologous gene DARK, the Drosophila Apaf-1 Related Killer. Hypomorphic alleles of DARK cause developmental disruption, including wing defects, body wall defects, supernumerary bristles, male sterility and an enlarged nervous system. Mutation of DARK potently suppresses the apoptotic function of the genes reaper, grim, and hid. Recombinant Grim protein was shown to antagonize IAP-mediated caspase inhibition in vitro. Peptides corresponding to the conserved Amino-termini of the reaper, grim and hid genes could compete for a binding site also used by the mammalian anti-IAP protein SMAC to block IAP-caspase interaction. Despite these peptides' failure to allow for reconstitution of caspase activation in vitro, genetic inactivation of IAP's leads to significant activation of caspases in vivo. DARK plays a critical role in caspase activation in vivo, and mutations of DARK suppress several genetic measures of cell death due to IAP inactivation. These studies show DARK to be an important apoptosis gene in the fly and necessary for caspase amplification and apoptotic initiation in certain cell death pathways. Further understanding of the regulation of cell death in the genetically tractable Drosophila model may help shed light on the regulation of apoptosis in human cells and disease states as well.Item Genetics of Stress-Induced Responses in Drosophila(2006-08-11) Akdemir, Fatih; Abrams, John M.Apoptosis is a highly conserved process responsible for elimination of cells during normal development and after cellular damage. Apical caspases that initiate caspase cascades are stimulated upon interaction with adaptor molecules. The Drosophila adaptor protein Dark, a homolog of nematode Ced-4 and mammalian Apaf-1, regulates the apical caspase Dronc, through interactions involving respective caspase recruitment domains (CARD). Dronc is the only caspase in the fly genome with a caspase recruitment domain. Here I pursue functional characterization of dronc and dark animals especially to find out whether they are required for all programmed cell death and whether they have distinct functions. dronc mutants have extensive hyperplasia of hematopoietic tissues and adult structures lacking dronc are disrupted for fine patterning. In diverse models of metabolic injury, dronc- cells are completely insensitive to induction of cell killing. I also show the generation and functional characterization of dark null mutant animals. Using in vivo and ex vivo assays, I demonstrate a global apoptogenic requirement for dark and show that a required focus of dark- organismal lethality maps to the central nervous system. Finally I show functional similarities of dronc and dark null mutants by a diverse set of experiments. Together these findings illustrate broad requirements for Dark and Dronc in adaptive responses during stress-induced apoptosis and in normal cell death. Treatment of cells with DNA-damaging agents upregulates the transcription of many genes, many of which are not functionally characterized. In a series of independent studies I characterize an ionizing radiation (IR)-induced gene, CG17836, designated as xrp1. Xrp1 is robustly responsive to IR, and it is a nuclear protein with DNA-binding activity as inferred from domain structure. I have characterized two different loss-of-function mutants of xrp1. In a loss-of-heterozygosity (LOH) assay xrp1 mutant animals display higher genomic instability than wild types after IR challenge. Even though xrp1 is not required for apoptosis or cell cycle arrest after IR treatment in animals, surprisingly, its overexpression in cell culture prevents cell proliferation. Thus, Xrp1 might maintain genomic stability by modulating cell cycle checkpoints upon IR exposure.Item Illuminating the P53 Regulatory Network in Genetic Models(2011-02-01) Lu, Wan-Jin; Abrams, John M.The tumor suppressor gene p53 is mutated in more than 50% of human cancers, and functions as a central component of stress response machinery that mediates a wide variety of downstream responses. Interestingly, the evolutionary appearance of p53 preceded its role in tumor suppression, suggesting that there may be unappreciated functions for this protein. In order to examine physiologic functions of p53 in vivo, a green fluorescent protein (GFP) reporter was designed to follow the activation of this regulatory network in a genetic model, Drosophila melanogaster. By following the reporter during Drosophila development, physiological activation of the p53 regulatory network in the female germ line was discovered. It is provoked by the first enzymatic step for meiotic recombination and conserved in both flies and mice. The functional relevance of the p53 activities in the germ line was shown by the meiotic recombination frequency and genetic interactions with a meiotic effector gene, Rad54. Additionally, genotoxic stress selectively activates p53 in germ line stem cells and promotes regeneration of fertility after IR. Activation of p53 was also found in uncontrolled growth of germ cells by blocked differentiation, and surprisingly by overexpression of oncogenic protein in the germ line. Together, my thesis work indicate that the need for controlling growth by the p53 regulatory network is an evolutionary conserved feature, which may serve as a selective pressure to preserve this network. Future studies on the mechanisms of p53 actvities during meiosis and in response to oncogene activation could provide novel insights on its cancer-related functions.Item In Vivo Genome-Wide Analyses of the Drosophila p53 Transcriptional Network(2017-07-27) Kurtz, Paula S.; Fontoura, Beatriz; Abrams, John M.; Kraus, W. Lee; Krämer, Helmutp53 is the most commonly mutated gene in human cancers. Despite decades of p53 studies we do not fully understand how p53 suppresses tumors. Similar to human p53, the Drosophila counterpart is a transcription factor that can respond to genotoxic stress and promote adaptive responses at the cellular level. Our lab has leveraged the powerful genetics of Drosophila to study p53 functions in vivo. In the context of the developing fly, p53 robustly activates important apoptotic genes in response to DNA damage to promote cell death. In the embryo model, we discovered an important p53 enhancer that forms chromatin contacts through long genomic distances and enables p53 to activate various genes. How p53 programs are adapted in different cellular contexts is poorly understood. In my dissertation work I examined two layers of p53 regulation, long-range enhancer looping and p53 DNA occupancy. To further examine enhancer looping, I exploited the established embryo model and the well characterized p53 reaper enhancer. At the single cell resolution, I demonstrated that the p53 enhancer can contact multiple targets simultaneously; however these multigenic complexes appear in low frequency. I also have preliminary genome-wide data suggesting in embryos this p53 enhancer contacts additional p53 targets. In addition, through genome-scale analyses I dissected novel p53 programs in a postmitotic model (the Drosophila head). Interestingly, postmitotic p53 programs are distinct from networks described in developing cells. I found that the canonical p53 apoptotic program is unresponsive in Drosophila heads, establishing this system as an ideal in vivo model to study alternate functions of p53. To determine how p53 differential programs are specified, I tested two distinct mechanisms for tissue specific target activation, p53 enhancer looping and DNA binding. Interestingly, I observed no change in enhancer looping to cell death targets in heads. However, I did detect loss of p53 enhancer binding. Lastly, I integrated genome-wide analyses of p53 DNA occupancy and transcriptional control in embryos and heads. Interestingly, I found that at the genome-scale p53 binding landscapes poorly correlate with nearby transcriptional effects, indicating that p53 enhancers could be generally acting through long distances.Item Investigating the Role of DIS3L2 in Perlman Syndrome and Wilms Tumor(2018-06-29) Hunter, Ryan Wayne; Amatruda, James F.; Mendell, Joshua T.; Abrams, John M.; Conrad, NicholasWilms tumor, while the most common pediatric kidney tumor, has a poorly understood etiology. Several recent studies have uncovered a role for loss of let-7 in its pathogenesis. One crucial mechanism through which let-7 expression is controlled is via the activity of the RNA-binding protein LIN28, which binds the precursor of let-7 and mediates the addition of a series of uridines to the 3′ end. This oligouridylation marks pre-let-7 for degradation by the exoribonuclease DIS3L2. Loss-of-function mutations in DIS3L2 lead to the Perlman congenital overgrowth syndrome, characterized by high neonatal mortality and, interestingly, a strong predisposition to Wilms tumor. Furthermore, DIS3L2 has been found to be deleted or mutated in some cases of sporadic Wilms tumor. The importance of let-7 in Wilms tumorigenesis and a purported role for DIS3L2 in the LIN28-let-7 pathway have led to speculation that aberrant let-7 expression underlies Wilms tumor susceptibility in DIS3L2-deficient contexts. It is still unclear, though, how loss of DIS3L2 could lead to altered let-7 levels, as the uridylated pre-let-7 species that DIS3L2 degrades is believed to be a dead-end product that cannot be further processed by DICER into mature let-7. Thus far, regulation of mature let-7 levels by DIS3L2 has only been examined in a limited number of cell lines. To determine whether DIS3L2 regulation of let-7 differs in a broader set of cell types, we used genome-editing to knockout DIS3L2 in a wide-ranging panel of cell lines with differing levels of LIN28 expression. Consistent with prior reports, loss of DIS3L2 had no effect on mature let-7 expression in these cell lines. However, it remained possible that the regulation of let-7 by DIS3L2 differed in cell populations relevant to Wilms tumor pathogenesis in vivo. Thus, to examine DIS3L2 function in these contexts, we used CRISPR-Cas9 to generate mouse lines harboring either Dis3l2-null alleles or mutations commonly observed in Perlman syndrome. Interestingly, Dis3l2 mutants recapitulated some aspects of Perlman syndrome, including neonatal mortality and genitourinary abnormalities, but not overgrowth or Wilms tumor. Moreover, the phenotype that results from the most common Perlman mutation is the same as that seen in the Dis3l2 loss-of-function mouse model, suggesting that the DIS3L2 mutations reported in Perlman syndrome are indeed loss-of-function. Finally, we examined the molecular function of DIS3L2 in isolated primary nephron progenitor cells (NPCs), a likely cell of origin of Wilms tumors, from Dis3l2-null and wild-type embryos. Consistent with our findings in the cell lines, loss of DIS3L2 in NPCs does not affect let-7 expression or activity, yet leads to upregulation of Igf2, which is reported to be overexpressed in 70-80% of Wilms tumors. Therefore, Igf2 poses an attractive candidate for both overgrowth and oncogenesis associated with DIS3L2 loss.Item Nuclease-Mediated Targeted Gene Insertion at the Adenosine Deaminase Locus in Primary Cells(2013-07-24) Checketts, Joshua Allen; Albanesi, Joseph P.; Porteus, Matthew H.; Burma, Sandeep; Abrams, John M.; Sternweis, Paul C.Gene therapy is the ability to correct diseases at the DNA level and has long been a goal of science and medicine. The earliest gene therapy clinical trial was for a patient with severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency. Initial trials looked promising and the technique was extended to other forms of primary immunodeficiency. Unfortunately, some of the patients enrolled in these trials using retroviral vectors to carry replacement genes resulted in insertional oncogenesis. To avoid the insertional oncogenesis caused by random integration into the genome, we postulated that targeted insertion of the gene of interest through homologous recombination would prove to be a safer alternative to random viral insertion of a gene. To this end, we developed several pairs of TAL effector nucleases (TALENs) designed to target exon 1 of ADA. These TALENs function as dimers, and each pair creates a different targeted double strand break near the start site of the ADA gene. The most effective pair induces a DNA double strand break immediately preceding the ADA start codon. Targeted activity of these TALENs was measured through determining the percent of alleles that undergo mutagenic non-homologous end joining upon exposure to the TALENs, with up to 14% of alleles undergoing such mutations. In order to stimulate gene targeting at the ADA locus in human cells, these TALENs were nucleofected into the cells as plasmid DNA, along with a donor plasmid that contains the DNA to be inserted flanked by 800bp arms of homology to the cut site. These TALENs were able to stimulate site-specific integration of the desired fragment at rates of up to 10% in human cell lines. Successful targeted gene insertion was verified through maintained fluorescence, western blots, and sequencing of the targeted alleles through PCR amplification. We demonstrated the ability to enrich for targeted cells through the expression of a selectable marker within the DNA cassette integrated at the ADA locus. In addition to the editing of cell lines, we showed successful stimulation of gene targeting in patient-derived fibroblasts in 1.5% of cells. We demonstrated the feasibility of using the ADA locus as a safe harbor through the targeted insertion of three therapeutically interesting genes. Finally, we demonstrated the successful targeted gene insertion in human CD34+ in up to 0.5% of cells treated. The successful targeting of human CD34+ is especially relevant, as these cells will need to undergo gene targeting in order to be therapeutically relevant as a curative therapy for SCID due to ADA deficiency.Item On Cholesterol Transport Between Membranes(August 2021) Trinh, Michael Nguyen; Mendell, Joshua T.; Chen, Zhijian J.; Abrams, John M.; Brown, Michael S.; Goldstein, Joseph L.The studies described in this dissertation focus on investigation of the pathways for transport of cholesterol from one organelle to another in animal cells. Cells have evolved elaborate transport mechanisms to assure an optimum cholesterol content within their membranes. Dysregulation of cholesterol transport causes common diseases, including atherosclerosis. The major source of cellular cholesterol comes from Low Density Lipoprotein (LDL). When plasma membranes are low in cholesterol, cells produce LDL receptors which bind LDL and mediate its uptake by endocytosis and its delivery to lysosomes. Within lysosomes the cholesteryl esters of LDL are hydrolyzed. The free cholesterol binds to a soluble lysosomal protein called Niemann Pick C2 (NPC2) which delivers it to a membrane-embedded protein called NPC1 which inserts the cholesterol into the lysosome membrane. From there the cholesterol moves to the plasma membrane (PM) through a pathway that is unknown. When the PM becomes saturated with cholesterol, any excess is transported to the endoplasmic reticulum (ER) to repress production of LDL receptors and to be stored in lipid droplets. The work described here 1) showed that triazole antifungal drugs inhibit lysosomal cholesterol export by binding to the membrane domain of NPC1 2) used itraconazole to solve the crystal structure of NPC1 at 3.3Å, 3) revealed that cholesterol is transported out of lysosomes through interactions between two or more NPC1 molecules, and 4) utilized CRISPR-Cas9 whole-genome knockout screens to identify all the genes involved in the transport and uptake of LDL cholesterol. From these screens in the latter study, we discovered that a specific phospholipid, phosphatidylserine (PS), is required for PM-to-ER cholesterol transport. These studies provide supporting evidence towards a vision of one-way directional transport of LDL-derived cholesterol from lysosomes to the PM to the ER.Item P53 Genes Act to Restrain Mobile Elements(2015-11-24) Wylie, Annika Dawn; Amatruda, James F.; Abrams, John M.; Hobbs, Helen H.; Morrison, Sean J.Oncogenic stress provokes tumor suppression by p53 but the extent to which this regulatory axis is conserved remains unknown. Using a biosensor to visualize p53 action, we find that Drosophila p53 is selectively active in gonadal stem cells after exposure to stressors that destabilize the genome. Similar p53 activity occurred in hyperplastic growths that were triggered either by the RasV12 oncoprotein or by failed differentiation programs. In a model of transient sterility, p53 was required for the recovery of fertility after stress, and entry into the cell cycle was delayed in p53- stem cells. Together, these observations establish that the stem cell compartment of the Drosophila germline is selectively licensed for stress-induced activation of the p53 regulatory network. Furthermore, the findings uncover ancestral links between p53 and aberrant proliferation that are independent of DNA breaks and predate evolution of the ARF/Mdm2 axis. While exploring the role of p53 in this context, we made a series of observations that justify a comprehensive examination of the relationship between p53 and transposon biology. Using Drosophila, zebrafish, and mouse models, we found that p53 functions to restrict the activity of retrotransposons. Furthermore, Drosophila p53 genetically interacted with components of the piRNA pathway and, in complementation studies, normal human p53 alleles restrained these mobile elements, but mutant p53 alleles from cancer patients could not. Consistent with these results, we also found patterns of unrestrained retrotransposons in p53-driven human cancers. Together, these observations indicate that ancestral functions of p53 operate through conserved mechanisms to suppress retrotransposons. Furthermore, since human p53 mutants are disabled for this activity, our findings raise the possibility that p53 mitigates oncogenic disease, in part, by restricting retrotransposon mobility.Item p53 in a Genetic Model: Illuminating Adaptive Radiation Responses(2005-05-03) Sogame, Naoko; Abrams, John M.When cells are challenged by genotoxic stress, the tumor suppressor protein p53 promotes adaptive responses, including cell cycle arrest, DNA repair, or apoptosis. How these distinct fates are specified through an action of a single protein is not known. To study its functions in vivo we produced a targeted mutation at the Drosophila p53 (Dmp53) locus. I show that Dmp53 is required for damage-induced apoptosis but not for cell cycle arrest. Dmp53 function is also required for damage-induced transcription of two tightly linked cell death activators, reaper and sickle. When challenged by ionizing radiation, Dmp53 mutants exhibit radiosensitivity and genomic instability, indicating in our model, apoptosis is important for maintenance of genomic stability in response to ionizing radiation. I also examined a global transcriptional change in response to ionizing radiation in the absence of Dmp53. Only 35 genes were constantly radiation responsive in wild type animals and Dmp53 was required for induction of a vast majority of the genes. The Radiation Induced p53 Dependent (RIPD) genes include genes implicated in apoptosis and DNA repair as well as genes with unknown functions. The functional significance of RIPD genes for the activation of apoptosis was tested using RNAi. Thus far, I uncovered ribonucleotide reductase large subunit (RnrL) as a novel Dmp53 target that is necessary for induction of caspase activation. Taken together, my study supports the notion that core ancestral functions of the p53 gene family are intimately coupled to cell death and possibly DNA repair as an adaptive response.Item Post-Transcriptional Regulation of Maternal mRNA Shapes Early C. Elegans Embryogenesis(2014-03-17) Burleson, Marieke Oldenbroek; Buszczak, Michael; Abrams, John M.; Cleaver, Ondine; Hobbs, Helen H.Much of early embryogenesis is controlled through complex networks comprised of maternally provided factors. Oocytes are packed with protein and RNA that are ready to spring into action after fertilization to guide early embryonic development. The regulation of maternally provided factors is therefore critical and is a fundamental goal of developmental biology. During my studies, I investigated how two maternally provided mRNAs, zif-1 and mom-2, are regulated post-transcriptionally through their 3’ untranslated region (3’ UTR) to ensure proper spatio-temporal protein expression. I discovered that seven RNA binding proteins bind directly to the zif-1 3’ UTR in a combinatorial fashion thereby ensuring that zif-1 is only translated in somatic blastomeres, beginning at the four cell stage embryo. Interestingly a similar set of RNA binding proteins (nine total) regulate the spatio-temporal expression of mom-2 in a similar fashion despite the fact that mom-2 has a reciprocal expression pattern when compared to zif-1. My studies on zif-1 and mom-2 regulation indicate that a “code” is embedded within the 3’ UTR of mRNAs to mediate translational regulation. The precise combination of RNA binding proteins present in a particular cell at a particular time, each with the intrinsic capability of binding to regulatory sequences contained in this “code”, determines when and where mRNAs get translated. I also investigated mechanisms by which maternal mRNAs get degraded. Zygotic transcription activation is often linked to maternal mRNA degradation, which I showed to be the case in C. elegans embryos. Specifically, I discovered a gene termed vet-5 that is first transcribed in the somatic blastomeres of the four-cell embryo and is sufficient to degrade at least several maternal mRNAs when provided exogenously as dsRNA. vet-5 maps to a highly repetitive locus and has been shown to be a target of siRNA production. Consistent with vet-5 derived siRNA production I found that the siRNA pathway is, at least partly, required for the degradation of maternal mRNAs and that removing components of the siRNA pathway affects vet-5 expression. Therefore, I hypothesize that siRNAs could be produced from the vet-5 locus that target maternally provided mRNAs for degradation.Item Reductive Carboxylation Is a Novel Pathway of Glutamine Metabolism That Supports the Growth of Tumor Cells with Metabolic Defects(2013-10-23) Mullen, Andrew Robbins; Burgess, Shawn C.; Abrams, John M.; Pearson, Gray W.; DeBerardinis, Ralph J.In growing cancer cells, oxidative metabolism of glucose and glutamine in the mitochondria provide precursors needed for de novo synthesis of proteins, nucleic acids and lipids. Yet, a subset tumors harbor genetic mutations in the electron transport chain or tricarboxylic acid cycle that disable normal oxidative mitochondrial function. Importantly, it has been unknown how these cells generate the biosynthetic precursors required for growth. To address this, I used models of mitochondrial dysfunction in isogenic cancer cell lines and studied their metabolism using a combination of Gas Chromatography- Mass Spectrometry and Nuclear Magnetic Resonance spectroscopy. In all cases, mitochondrial dysfunction stimulated a novel pathway of glutamine metabolism, characterized by reversal of the canonical tricarboxylic acid cycle, termed reductive carboxylation; providing a plausible mechanism for how cancer cells with mitochondrial defects generate biosynthetic precursors required for growth. To gain mechanistic insight into how this unusual pathway was regulated I carried out a targeted metabolomics analysis in our isogenic tumor cell models. This led to the striking discovery that cells engaged in the reductive carboxylation pathway also operate an additional metabolic pathway that, at first glance, would appear to be superfluous and inefficient. Functional characterization of this second pathway revealed, however, that its activity was necessary for the optimal function of the reductive carboxylation pathway. In summary, this work has given us insights into how cancer cells are able to grow in the context of defective mitochondria. Additionally, this has exposed a potential Achilles ’ heel that might be used to selectively kill tumors which rely on this pathway for growth.Item The Role of Autophagy in Early Development and Tumor Suppression Using a Zebrafish Model System(2013-06-25) Lee, Eunmyong; Brugarolas, James B.; Abrams, John M.; Cleaver, Ondine; Levine, Beth; Amatruda, James F.Autophagy is an evolutionarily conserved lysosomal degradation pathway which involves the sequestration of cytoplasmic components into a double membraned structure called the autophagosome. By using genetically manipulated autophagy-deficient models, important roles for autophagy in development and tumorigenesis have been suggested. Genetic analyses indicate that autophagy is essential for eukaryotic differentiation and development. However, little is known about whether autophagy contributes to morphogenesis during embryonic development. To address this question, the role of autophagy in early development was examined using zebrafish, a model system for studying vertebrate tissue and organ morphogenesis. Active autophagy was observed in multiple tissues during early embryonic development, as evidenced by the presence of autophagosomes in electron microscope images or GFP-LC3 puncta in autophagy reporter fish line Tg(cmvItem The Role of HNF4A in Germ Cell Tumor Development(2017-08-10) Pierce, Joshua Logan; Burma, Sandeep; Amatruda, James F.; Abrams, John M.; Castrillon, Diego H.Yolk sac tumor is a histological category of germ cell tumors, which represent the most frequent malignancy in young men. Little is known about the molecular mechanisms responsible for the aberrant differentiation and oncogenic potential of yolk sac tumors. Multiple recurrent chromosomal copy number variations are the primary genetic lesions discovered in yolk sac tumors. Frequent gains of chromosome 20q13 provide clues as to the identity of important YST driver genes. We have determined that the Hepatocyte Nuclear Factor 4 alpha locus, which resides in this region, is frequently amplified in YSTs. Overexpression of HNF4A in an undifferentiated GCT line is sufficient to drive extraembryonic endodermal gene programs. This endodermal program subsequently utilizes endogenous WNT signaling to grow. Additionally, isoform specific manipulation of HNF4A revealed differential oncogenic potential amongst HNF4A isoforms. This isoform specific manipulation was involved in modulation of the WNT pathway which has previously been identified as active in YSTs. These results allowed us to uncover HNF4A isoform specific differences in histological samples from YST patients. Our findings reveal a possible new drug target in YST treatment and reveal an interesting isoform dependent mechanism for understanding tumor development.