Browsing by Subject "Caenorhabditis elegans Proteins"
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Item C. Elegans OMA-1 and OMA-2 are Transcriptional and Translational Repressors of Germline Fate(2009-09-04) Ozkan, Tugba Guven; Lin, RueylingThe germline is a very specialized cell lineage for the proper transmission of genetic material through many generations, to ensure flawless perpetuation of the species and life cycles. The germline lineage is set aside as early as embryogenesis and kept quiescent until germ cells are needed for adult reproduction. During C. elegans germline development global transcription is repressed in specialized, mature diakinetic oocytes of the adult animal and transcription is reactivated as zygotic transcription in the 4-cell stage embryo but only in somatic blastomeres. Global transcription is kept repressed by PIE-1 in germline precursors beginning with 4-cell stage to protect germ lineage from inappropriate somatic differentiation pathways. During my graduate studies, I investigated the redundant roles for two CCCH type RNA binding zinc finger proteins OMA-1 and OMA-2 during C. elegans germline development and early embryogenesis. Previously, OMA proteins were shown to be required for oocyte maturation but they were not assigned any molecular functions. My thesis demonstrates transcriptional repression function of OMA proteins in newly fertilized embryos and translational repression functions during oogenesis. I showed that OMA-1/2 are redundantly required for global transcriptional repression before the onset of zygotic transcription in the 1-cell and 2-cell stages of C. elegans embryos by interacting with and sequestering in the cytoplasm TAF-4, a highly conserved essential basal transcription factor. Nuclear enrichment of TAF-4 requires interaction with another transcription factor TAF-12. OMA-1 competes with TAF-12 to interact with and change subcellular localization of TAF-4, in order to displace TAF-4 away from nuclei and prevent transcriptional initiation. I showed that interaction of OMA-1 and TAF-4 is regulated by MBK-2 phosphorylation at oocyte to embryo transition. My data suggest a model in which MBK-2 phosphorylated embryonic OMA-1 can change TAF-4 subcellular localization only in newly fertilized C. elegans embryos, not during oogenesis. When properly phosphorylated by MBK-2 kinase, ectopic OMA-1 is sufficient to repress transcription in later embryonic stages. Strikingly, reduction of oma-1/2 activities not only results in transcriptional derepression in newly fertilized embryos, but also in later germline blastomeres where wild type OMA-1 is normally absent. I show that OMA-1/2 indirectly repress global transcription in later germline blastomeres by preventing premature degradation of PIE-1 during germline development. OMA proteins protect PIE-1 and other CCCH RNA binding proteins from degradation by repressing zif-1 mRNA translation, the substrate specific binding partner for PIE-1 degradation. A zif-1 3'UTR reporter is repressed in the pachytene and proximal regions of the adult C. elegans germline, and expression of the reporter is activated in the 4-cell embryo only in anterior blastomeres, reciprocal to the PIE-1 expression pattern. I show that zif-1 3'UTR reporter is repressed in the proximal oocytes and in the pachytene region of the germline by OMA-1/2 and GLD-1 respectively. I further showed that zif-1 3'UTR reporter is kept repressed in germline blastomeres of the embryos by POS-1 and SPN-4 and its activation requires anterior cell fate determinants MEX-5/6 during embryogenesis. Contrary to the requirement for MBK-2 phosphorylated OMA-1/2 for embryonic transcriptional repression function, zif-1 3'UTR reporter repression by OMA proteins in the oocytes requires un/hypophosphorylated OMA proteins, the version of OMA-1/2 detected in the oocytes. In summary, my thesis shows that OMA-1/2 are dual function proteins redundantly required for germline development and maintenance of germline identity during oogenesis and embryonic development of C. elegans. OMA proteins are critical for the protection of CCCH type maternal proteins during oocyte development by preventing their premature proteasomal degradation through inhibiting translation of zif-1 mRNA. MBK-2 phosphorylation at the oocyte to embryo transition converts OMA proteins from oocyte translational repressors to embryonic transcriptional repressors. Phosphorylated OMA proteins can interact with TAF-4 in the newly fertilized C. elegans embryos and repress global transcription to prevent premature somatic differentiation during early stages of embryogenesis. OMA proteins protect germline identity at the level of both translational and transcriptional repression during the very critical time points of development to regulate a proper oocyte to embryonic transition.Item Identification and Characterization of EFL-3, a C. Elegans E2F Transcription Factor(2011-12-14) Yoshimoto, Jennifer Ann Winn; Avery, LeonThe development of an organism depends on cells receiving and executing their specific fates, though how this process is regulated remains largely unknown. Here, we identify a mechanism by which a specific cell fate, apoptosis, is determined through the cooperative efforts of Hox and E2F proteins. E2F transcription factors are critical, conserved regulators of the cell cycle and apoptosis. However, little is known about the two most recently discovered mammalian E2Fs—-E2F7 and E2F8. In the nematode Caenorhabditis elegans, we identify a novel E2F7/8 homolog, EFL-3, and show that EFL-3 functions cooperatively with LIN-39, providing the first example in which these two major developmental pathways—-E2F and Hox—-are able to directly regulate the same target gene. Our studies demonstrate that LIN-39 and EFL-3 function in a cell type-specific context to regulate transcription of the egl-1 BH3 only cell death gene and determine cell fate during development.Item Identification of Ligands for the Orphan Nuclear Receptor DAF-12 That Govern Dauer Formation and Reproduction in C. Elegans(2006-08-15) Motola, Daniel Lewis; Mangelsdorf, David J.The orphan nuclear hormone receptor, DAF-12, plays a central role in the physiology of free-living nematode, C. elegans. DAF-12 is best known for its role in regulating dauer formation, a non-reproductive larval state entered in harsh environments and marked by developmental arrest, stress resistance, and extended life-span. Genetic screens for genes controlling dauer formation have identified conserved endocrine signaling pathways that converge on DAF-12 to influence the choice between dauer formation and reproductive development. Detailed genetic analysis of these signaling pathways suggests that they promote reproductive development in favorable environments by influencing the production of a ligand for DAF-12 by the cytochrome p450, DAF-9. Despite abundant evidence for hormonal control, the identity of the DAF-12 ligand has remained elusive. Using a cell-based ligand screening assay I initially identified a group of 3-keto-containing sterols that potently activated DAF-12 in a DAF-9-dependent manner. Subsequent analysis using a variety of techniques showed that DAF-9 catalyzes the non-stereo-selective addition of a carboxylic acid to the terminal side-chain methyl groups of 3-keto-sterols, producing 3-keto-cholestenoic acids. In collaboration with the laboratory of Dr. Eric Xu, we demonstrated that 3-keto-cholestenoic acids, referred to as dafachronic acids, directly bind to DAF-12 as bona fide ligands. In collaboration with Dr. Adam Antebi we found that these ligands also potently rescued the phenotypes resulting from mutations in daf-9 or its upstream activating genes. Dafachronic acids are also shown to be endogenous hormones, as they could be detected in DAF-12 activating lipid fractions from wild-type but not daf-9 null worms. Taken together, this work defines 3-keto-cholestenoic acids as the first hormonal ligands for an invertebrate orphan nuclear receptor and the first endogenous steroid hormones in nematodes. In addition, these findings demonstrate that steroid hormone control of reproduction is conserved from worms to humans. Finally, given the existence of DAF-12 homologs in parasitic nematodes, this work raises the possibility of targeting DAF-12 in parasitic nematodes as a means for controlling their growth.Item Nuclear Receptor Controls Nematode Metabolism And Development: Insight Into Man’s Nemesis, the Conqueror Worm(2011-02-01) Wang, Zhu; Mangelsdorf, David J.The nuclear receptor DAF-12 plays a central role in controlling the larval development of C. elegans. Activation of DAF-12 by its ligands called dafachronic acids (DAs) commits the nematode to development into reproductive adult, which will otherwise arrest at a diapause stage called dauer. But the molecular mechanisms remain unclear. Furthermore, whether the DAF-12 signaling pathway is conserved in other nematode species, especially parasitic ones, is also unknown. One aspect of my studies is to investigate the molecular mechanisms by which this DA-DAF-12 signaling pathway regulates the C. elegans development. By measuring a series of metabolic parameters, we demonstrated that DAF-12 activation markedly elevated aerobic utilization of fatty acids. In accordance with this, expression of a network of metabolic genes responsible for energetic catabolism of fatty acids was up-regulated as well. Importantly, inhibition of these metabolic genes abolished the reproductive growth stimulated by DAF-12. These results revealed a DAF-12-controlled metabolic network that coordinates energy metabolism and larval development in C. elegans. The other emphasis of my work is on the role of DAF-12 in parasitic nematodes. Our results showed that, as seen in C. elegans, DAF-12 activation also induced recovery from the infective L3 (iL3), which is the dauer larva of the parasites. Moreover, the metabolic genes controlled by C. elegans DAF-12 were identified in parasitic nematodes. These facts indicate that the DAF-12 signaling pathway is conserved in parasitic nematodes. Importantly, administration of DA dramatically reduced the formation of the pathogenic larvae that are mostly resistant to current anthelmintic drugs, indicating the unique therapeutic potential of DAF-12 ligands to treat nematode parasitic diseases. To understand the pharmacology of targeting DAF-12, we solved the 3-demenstional structure of DAF-12 in a parasitic nematode called Stronglyloides stercoralis that infects human. These results reveal the molecular basis for DAF-12 ligand binding and identify DAF-12 and its downstream metabolic genes as unique therapeutic targets in parasitic nematodes. Based on this, we have discovered several small molecules that activate Stronglyloides stercoralis DAF-12 and these molecules may provide lead compounds for developing novel anthelmintic drugs.Item Phosphatase Regulation of Mechanical Stress and Aging in C. elegans(2020-08-01T05:00:00.000Z) Egge, Nathan Chandler; Goldsmith, Elizabeth J.; Douglas, Peter; Mendell, Joshua T.; Terman, Jonathan R.; Joachimiak, LukaszStress and aging embody two related processes driving cellular dysfunction. In either case, environmental stimuli and genetically encoded regulatory mechanisms affect cellular homeostasis and influence adaptation. Phosphatases represent master regulators of stress signaling and modulate cellular responses and fate during stress and aging. Yet, physiologically relevant mechanisms by which these phosphatases are regulated or orchestrate stress response and lifespan are incompletely understood. Herein, I present two scenarios, mechanical trauma and intestinal aging, both of which involve regulation by phosphatases. Mechanical stimuli initiate adaptive signal transduction pathways, but exceeding cell tolerance for physical stress results in degeneration and death via unclear mechanisms. In the nematode C. elegans, I developed a model to study cellular degeneration in response to mechanical stress caused by blunt force trauma. I identified a dual-specificity MAPK phosphatase, VHP-1, as a stress-inducible modulator of neurodegeneration. VHP-1 regulates the transcriptional response to mechanical stress and itself is dually regulated by its target, KGB-1. KGB-1 both activates VHP-1 via a negative feedback loop and represses via inhibition of a deubiquitinase, MATH-33, affecting proteasomal degradation. Thus, I describe an uncharacterized stress response pathway in C. elegans and identify transcriptional and post-translational components comprising a feedback loop on Jun kinase and phosphatase activity. Like stress, aging challenges cell tolerances, instigating death upon inadequacy of homeostatic regulation. Intestinal cells form a vital barrier separating environment from organism. Age impairs intercellular interactions and the cells' capacity to tightly associate within tissues and form an effective barrier necessary for normal systemic function. In particular, the actin cytoskeleton represents a key determinant in maintaining tissue architecture; how age disrupts the actin cytoskeleton, and, in turn, promotes mortality remains unclear. Herein, I show that phosphorylation of ACT-5 compromises C. elegans intestinal barrier integrity and accelerates pathogenesis. Age-related loss of the heat shock transcription factor, HSF-1, disrupts the Jun kinase/Protein Phosphatase I equilibrium, increasing ACT-5 phosphorylation within a troponin-binding site. Phosphorylated ACT-5 accelerates decay of the intestinal terminal web and impairs cell junctions. Therefore, age-associated dysregulation of phosphatase/kinase activity contributes to intestinal dysmorphogenesis and organism death.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 Starvation-Signaling in the Nematode Caenorhabditis Elegans Using Regulator of G-Proteins GPB-2(2013-08-30) Pollok, Robert Harding; Galindo, Rene; Avery, Leon; Abrams, John M.; Castrillon, Diego H.During starvation, C. elegans adjust their behavior in order to survive. Using the starvation-sensitive gpb-2(ad541) loss-of-function mutant, components in a starvation-signaling pathway were identified. The goals of the studies presented here were to identify neurons that propagate a starvation signal, and to identify genes that regulate fat storage in the gut during starvation. Starvation in gpb-2(ad541) worms is lethal, and this lethality can be induced by arecoline, an acetylcholine receptor antagonist. Starvation sensitivity in gpb-2(ad541) worms is inhibited by atropine, an acetylcholine receptor antagonist. Previous work suggests that cholinergic signaling propagates a starvation signal in the pharynx of the worm, and the MC neurons are responsible for sending that signal. By ablating the MC neurons in newly hatched L1 worms, I aimed to prevent starvation-induced lethality due to the gpb-2(ad541) background. Several genes have also been identified to act downstream of gpb-2 in the regulation of fat in the gut. Both flp-20 loss-of-function and mgl-2 loss-of-function mutations rescue the starvation-induced lethality of gpb-2(ad541), while introduction of a gcy-28 loss-of-function mutation restores lethality. When fat was assayed using Oil Red O, it was found that GCY-28, a receptor-type guanylate cyclase, is necessary to maintain fat levels during starvation. GCY-28 is expressed in various head neurons and throughout the gut, and GCY-28 may play a role in regulating how gut cells store fat.Item Study of Oocyte-To-Embryo Transition Regulators, OMA-1 and OMA-2 in C. Elegans(2007-05-22) Nishi, Yuichi; Lin, RueylingA non-dividing, developmentally dormant oocyte is transformed into a rapidly dividing, differentiating embryo during a short period termed oocyte-to-embryo transition. Oocyte-to-embryo transition encompasses oocyte maturation and fertilization and is characterized by both cell cycle and developmental events. Understanding the mechanisms underlying oocyte-to-embryo transition is a fundamental goal for developmental biology and reproductive medicine. However, our current understanding of the transition is very limited. Two CCCH Tis-11 type zinc finger proteins of C. elegans, OMA-1 and OMA-2 are expressed exclusively in oocytes and 1-cell embryos, and are rapidly degraded at the first mitosis. Previous studies suggested that oma-1 and oma-2 are redundantly required for oocyte maturation, and the degradation of OMA-1/2 proteins at the end of the 1-cell stage is essential for embryogenesis. However, their roles in the 1-cell embryo, and the mechanism of the OMA-1/2 degradation were elusive. In addition, the molecular functions of OMA-1/2 proteins were unknown. In this study, I investigated the mechanism controlling OMA-1/2 degradation as well as the molecular and genetic functions of OMA-1/2. I showed that two proline directed kinases, MBK-2/DYRK2 and GSK-3 directly and likely sequentially phosphorylate OMA-1/2 to mark them for degradation at the end of the 1-cell stage. My data further suggest that SCF and/or ECS E3 ubiquitin ligase and the proteasome are likely responsible for the execution of OMA-1/2 degradation. Secondly, I characterized the molecular and genetic functions of OMA-1/2 in oocytes and 1-cell stage embryos. My data suggest that OMA-1/2 regulate multiple processes. These processes include transcription and translation. At the 1-cell stage, transcription is inactive. My data suggest that OMA-1/2 render 1-cell embryos transcriptionally quiescence by preventing the nuclear localization of a general transcription factor, TAF-4. OMA-1/2 (oma-1/2) interact with translational regulators, MEX-3 and SPN-4 physically, and other translation factors, puf-3/5/8, and cpb-3 genetically, suggesting that OMA-1/2 also regulate translation in oocytes and 1-cell embryos. In summary, my study revealed that phosphorylation and ubiquitination events regulate the degradation of OMA-1/2 proteins, and provided insights into functions of OMA-1/2 during oocyte-to-embryo transition.Item Using C. Elegans as Model Organism to Study the Mode of Action of a Natural Toxin, Psymberin(2011-12-15) Wu, Cheng-Yang; Roth, Michael G.Psymberin is an extremely potent cytotoxin isolated from the marine sponges Psammocinia and Ircinia ramose. Several cancer cell lines are sensitive to psymberin, including breast, melanoma and colon cancer cell lines. Psymberin is the only member of the pederin natural product family that contains a dihydroisocoumarin side chain. The cytotoxicities of psymberin in various human tumor cell lines are between sub-nanomolar to nanomolar IC50. Like pederin, the first member of this natural product family, psymberin and mycalamide A inhibit translation in vivo and in vitro. This inhibition by psymberin is 40 to 100 fold more potent than cycloheximide, which inhibits >90% translation at 100 micromolar in vivo. In a SAR study, both the cytotoxicity of psymberin and psymberin-induced translation inhibition were attenuated by substituting the psymberin side chain with the pederin side chain. However, the attenuation of cytotoxicity was relatively greater than of translation. The stereo configuration and both side chains of psymberin are required for both inhibition of translation and cytotoxicity. The result of the SAR study suggests that additional bioactivity is contained in psymberin. Psymberin is at best a poor substrate for small molecule pumps in the cell. Two separate forward genetics screens in C. elegans isolated seven independent psymberin-resistant mutants. In each the mutation was a C361T point mutation in the rpl-41 gene that changes Pro65 to Leu65 in the protein coding sequence. The psymberin-resistant mutant strain DA2312 is resistant to psymberin only. This mutation did not appear to cause weaker binding of psymberin to the ribosome, but must allow translation to continue with the toxin bound. There are additional modes of actions of psymberin compared to mycalamide A. The endogenous protein level of LC3, an autophagy marker, is decreased faster with psymberin treatment than mycalamide A. In HT-29 cells, psymberin is capable of synergizing TNFa-induced necrotic cell death more efficiently than mycalamide A. The results from SAR study and from study of the psymberin-specific mutation in C. elegans suggest that psymberin may induce fast cell death through multiple pathways, including translation inhibition, apoptosis and necrosis. The structural uniqueness of psymberin has functional consequences suggesting that the mode of action of psymberin on the ribosome is different from other members of the pederin family.