UMD Theses and Dissertations
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Item COORDINATED TRAFFICKING OF HEME TRANSPORTERS BY CARGO SORTING COMPLEXES IS ESSENTIAL FOR ORGANISMAL HEME HOMEOSTASIS(2025) Dutt, Sohini; Hamza, Iqbal IH; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme, an iron-containing organic ring, is a vital cofactor responsible for diverse biologicalfunctions and is the major source of bioavailable iron in the human diet. As a hydrophobic and cytotoxic cofactor, heme must be transported in a highly controlled manner through membranes via specific intra- and inter-cellular pathways. However, the genes and pathways responsible for heme trafficking remain poorly understood. Unlike other metazoans, Caenorhabditis elegans cannot synthesize heme but requires heme for sustenance. Thus, C. elegans is an ideal animal model to identify heme trafficking pathways as it permits organismal heme homeostasis to be directly manipulated by controlling environmental heme. Heme is imported apically into the intestine by HRG-1-related permeases and exported basolaterally by MRP-5/ABCC5 to extra- intestinal tissues. Loss of mrp-5 causes embryonic lethality that can be suppressed by dietary heme supplementation raising the possibility that MRP-5-independent heme export pathways must exist. Here we show, by performing a forward genetic screen in mrp-5 null mutants, that loss of the vesicular cargo sorting Adaptor Protein complexes (AP-3) fully rescues mrp-5 lethality and restores heme homeostasis. Remarkably, intestinal heme accumulation due to mrp-5-deficiency causes a concomitant deficit in the lysosomal heme importer HRG-1 abundance and localization. Loss of both MRP-5 and AP-3 subunits resurrects HRG-1 levels and localization, thus underscoring the crucial role of HRG-1 in dictating mrp-5 mutant phenotypes. In the absence of MRP-5, heme is exported by SLC49A3 homolog, a previously uncharacterized transporter. Live- cell imaging reveals vesicular coalescence that facilitates heme transfer between the importers and exporters at the interface of lysosomal-related organelle. These results define a mechanistic model for metazoan heme trafficking and identifies SLC49A3 as a promising candidate for heme export in mammals.Item INVESTIGATING GENE REGULATORY ARCHITECTURES THAT DICTATE TRANSGENERATIONAL EPIGENETIC EFFECTS IN C. ELEGANS(2023) Chey , Mary Somontha; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The form and function of an organism rely on the recreation of similar gene expression patterns in every generation. The information for these expression patterns is stored in a single cell (e.g., zygote) in two forms – the genome sequence and the spatial arrangements of gene regulators. The interactions of regulators and the genome form intricate networks with different regulatory architectures. However, a change in the environment can impact gene expression by disrupting the physical and/or chemical properties of regulators or interactions without mutations in DNA sequence. Such epigenetic information can be transmitted across generations, but how long these effects can last is unclear. Here, we investigate the regulatory elements that promote transient or permanent epigenetic effects by analyzing the properties of a recombinant two-gene operon that expresses the fluorescent proteins mCherry and GFP and is susceptible to long-term RNA silencing in the nematode C. elegans. We reveal that 1) multiple mechanisms regulate transgenerational gene silencing and 2) the presence of the mCherry sequence can perturb RNA regulation within the germline to facilitate heritable epigenetic changes. Previous studies showed that the Argonaute protein HRDE-1 is required for the maintenance of silencing in the germline initiated by double-stranded (ds)RNAs and that poly-UG (pUG)-RNAs are key intermediates generated from the target mRNA. We found that loss of HRDE-1 can selectively rescue the expression of one cistron in a two-gene operon, suggesting that the two cistrons are not regulated by the same silencing pathway, but rather by a chromatin-independent mechanism that requires an unknown regulator. Surprisingly, we detected distinct populations of pUG-RNAs associated with expressed and silenced genes, suggesting that pUG-RNAs could potentially prime expressed genes for long-term silencing. Consistently, total RNA sequencing revealed trace amounts of anti-sense RNAs against mCherry and gfp that could trigger the production of pUG-RNAs. Examining the endogenous genes perturbed by the presence of mCherry suggests that long-term RNA silencing relies on the synergy between the sensing and processing of dsRNAs. Together, our results provide insights into the regulatory architectures and mechanisms of heritable gene silencing that occur without genetic mutations.Item Investigating Copper Acquisition And Delivery via Transporters and a Pharmacological Chaperone in Copper-Deficient Worms and Mice(2019) Yuan, Sai; Kim, Byung-Eun; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Copper (Cu) is a key micronutrient required for a variety of essential biochemical pathways. Systemic or tissue-specific Cu-deficiencies, caused by insufficient dietary Cu uptake or mutations in Cu transporting genes, result in impaired growth, neuropathy, ataxia, hypopigmentation, osteoporosis and anemia-like symptoms in mammals. How organisms regulate Cu homeostasis at the systemic levels in response to Cu deficiencies remain elusive. In this study, we use Caenorhabditis elegans (C. elegans), a genetically tractable, multi-tissue metazoan to explore Cu homeostasis and investigate these unknowns. The high-affinity Cu transporters encoded by CTR family genes are required for dietary Cu uptake and maintaining systemic Cu balance from yeast to mammals. However, little is known about Cu acquisition mechanisms in C. elegans. We identified ten CTR ortholog genes in C. elegans; of these, chca-1 was functionally characterized. Cu availability regulates transcription of chca-1 in both the intestine and hypodermis, and chca-1 is essential for normal growth, and reproduction in the worm. Additionally, altered Cu balance caused by the loss of CHCA-1 results in defects in Cu-responsive avoidance behavior. Identification of this CTR-like gene in C. elegans, which appears to be essential for normal Cu homeostasis in the worm, illustrates the importance of Cu delivery via CHCA-1 for normal metazoan development and behavioral phenotypes. In addition, we show that a Cu-binding pharmacological chaperone, elesclomol (ES), fully restores the developmental defects and Cu deficiencies in chca-1-depleted worms, as well as the lethality in worms lacking cua-1 expression (Cu exporter ATP7A ortholog), suggesting ES is able to efficiently deliver Cu from dietary sources to peripheral tissues through the intestine in C. elegans. Our study was further expanded to mammalian models such as cardiac-specific Ctr1-depleted (Ctr1hrt/hrt) mice. We found that ES administration fully restores the postnatal lethality, developmental defects and cardiac hypertrophy found in Ctr1hrt/hrt mice, as well as rescuing the secondary systemic Cu homeostasis responses, including aberrant ATP7A protein levels in the liver and intestine. Moreover, ES shows the potential ability to transport Cu across the blood-brain-barrier in in vitro studies. These results illustrate the capability of ES to rescue systemic Cu deficiency in worms and mice, independent of the presence of functional Cu transporters, and shed light on the therapeutic usage of ES in Cu-deficient human diseases.Item Mobile RNA reveal differential requirements for gene silencing in C. elegans at single-cell resolution(2018) Ravikumar, Snusha; Jose, Antony M; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Delivery of double-stranded RNA (dsRNA) into animals can silence genes of matching sequence in diverse cell types through mechanisms that have been collectively called RNA interference. In the worm C. elegans, such organism-wide silencing relies on the transport of dsRNA to most cells and requires amplification mechanisms for effective gene silencing. Amplification of silencing signals is accomplished by two tissue-specific RNA-dependent RNA polymerases - EGO-1 in the germline and RRF-1 in somatic cells. Here, we reveal instances of RRF-1-independent silencing in somatic cells, which are dictated by three variables. First, when the same intestinal target gene was silenced using ingested, intestinal, or neuronal dsRNA, only silencing by mobile RNAs derived from neuronal dsRNA was independent of RRF-1. Second, when the same source of mobile RNA was used to silence a target sequence in different genomic contexts, the requirement for RRF-1 could change. Third, measurement of silencing by mobile RNAs at single-cell resolution revealed cell-to-cell and animal-to-animal variation in the requirement for RRF-1. Therefore, the requirements for gene silencing can vary based on the source of dsRNA, the target context, and even the particular cell examined, suggesting that each C. elegans animal is a functional mosaic with respect to RNA interferenceItem PERPETUATION OF NON-GENETIC CHANGES AT A TRANSGENE LOCUS IN C. ELEGANS(2018) Devanapally, Sindhuja; Jose, Antony M; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Development of a multicellular organism from a single cell or from a few cells in every generation relies on the reproducible expression pattern of genes. At the beginning of every generation in every organism, at least a single progenitor cell is necessary to produce the organism. This cell is subjected to reprogramming mechanisms that erase epigenetic information transmitted from the previous generation and as it develops, the organism goes through experiences that affect gene expression. Despite this, developmental processes give rise to nearly the same organism in the next generation, suggesting that the components in the cells are similarly regulated in every generation. How a complex organism can develop and reproduce its gene expression pattern using only the information present within the progenitor cell is not understood. Here, we describe an engineered genetic locus in the nematode worm C. elegans that shows robust transgenerational expression like many loci in the genome but, unlike other tested loci, can be uniquely susceptible to transgenerational silencing by one of two distinct processes. This locus could be silenced by double-stranded RNA (dsRNA) transported from neurons and could also be silenced when inherited solely from the male parent in a genetic cross. Each process could initiate transgenerational silencing within the germline that lasted for >25 generations. The two processes depended on distinct mechanisms to initiate silencing – while neuronal dsRNA required the conserved dsRNA importer SID-1 and the Argonaute RDE-1, mating-induced silencing required the Piwi-interacting Argonaute PRG-1. Both processes engaged the same germline Argonaute HRDE-1 to maintain heritable silencing suggesting that both processes trigger silencing independently but converge on the same pathway for maintenance. No other locus that was tested showed such indefinite silencing by either mechanism, suggesting that most loci are resistant to changes in gene expression. Thus, the discovery of a locus that is susceptible to transgenerational change provides us with the first instance of how a single change at a gene sequence can be used to explain evolution of gene regulation.Item MRP5/ABCC5, A CONSERVED ABC TRANSPORTER, REGULATES METAZOAN HEME HOMEOSTASIS(2014) Korolnek, Tamara; Hamza, Iqbal; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Hemes are metalloporphyrins used by nearly all organisms as cofactors for proteins involved in respiration, binding and sensing gases, and as catalysts for various reactions. Despite extensive knowledge about heme biosynthesis and catabolism, the pathways for transporting heme between cells and within cells remain poorly understood. C. elegans serves as a unique animal model for uncovering these pathways, as it is unable to synthesize its own heme and depends on the uptake of dietary heme for growth and reproduction. Functional RNAi screens implicated mrp-5 as a potential heme transporter in C. elegans. This gene encodes a membrane-bound ABC transporter that localizes to the basolateral intestinal membrane and is required for worm growth and reproduction. Depletion of mrp-5 activates heme deprivation signals within the worm, protects worms from toxicity associated with a toxic heme analog, and results in worms accumulating the fluorescent heme analog, zinc mesoporphyrin IX, in intestinal cells. Taken together, these results indicate a defect in heme export from the intestine when MRP-5 activity is lost. Functional assays in yeast support the hypothesis that MRP-5 is capable of exporting heme across cell membranes, and that this function is conserved in the human ortholog. Knockdown of mrp5 in zebrafish embryos results in developmental defects and decreased blood formation, indicating that this transporter likely regulates heme homeostasis in vertebrates. Loss of Mrp5 in mammalian cells leads to decreased heme transport into the secretory pathway as measured by activity of a Golgi-targeted heme-dependent enzyme. Furthermore, macrophages from mice lacking Mrp5 are unable to activate a number of cellular responses when undergoing erythrophagocytosis, the process whereby the heme-iron in senescent red bloods is recycled. Altogether, our results implicate MRP-5 as a key heme transporter in C. elegans, and point to an evolutionarily conserved role for MRP5 proteins in regulating heme homeostasis.Item Genetic Characterization of Toxic Resistant Mutants in C. elegans(2010) Walston, Jonathan Dean; Hamza, Iqbal; Animal Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heme is an essential cofactor that plays a key role in diverse biological processes. Free heme, however, is hydrophobic and toxic to cellular macromolecules. C. elegans lack the heme biosynthetic pathway, and therefore contains a highly regulated trafficking network to redistribute heme throughout the worm. A forward genetic screen in C. elegans identified thirteen mutants which grow at toxic concentrations of heme in axenic liquid media. These mutants, termed them for Toxic HEMe resistant, belong to five complementation groups of which IQ7280, IQ7310, IQ8280 and IQ9110 strains were characterized. them mutants exhibit abnormal responses to heme analogs, mating defects, and growth on heme-deficient bacteria. Pyrosequencing analysis mapped IQ7310, IQ8280, and IQ7280 to a common genetic interval on chromosome I and IQ9110 to chromosome V. Solexa deep sequencing identified mutations in novel genes which may play an essential role in organismal heme homeostasis.Item ISOLATION AND CHARACTERIZATION OF CAENORHABDITIS BRIGGSAE TRA MUTANTS.(2005-12-12) Kelleher, Danielle Fay; Haag, Eric S; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In order to aid in the reconstruction of the Caenorhabditis briggsae sex determination pathway, for the purposes of studying the convergent evolution of hermaphroditism and mating system change in nematodes, Cb-tra mutants have been isolated through forward mutagenesis. Based on phenotype, genetic linkage, molecular linkage, and in some cases sequence analysis, the C. briggsae homologs of tra-1, tra-2, and possibly tra-3 have been identified. Upon further characterization, the function of the tra genes during sex determination between C. elegans and C. briggsae appears to be largely conserved. However, notable differences, with respect to the role of tra-1 and potentially the role of tra-3, have been observed between these two species. In addition, intra-species suppression of the putative tra-3 mutant (AF16 background) by genomic variants in the wild-type strain HK104 suggests that molecular interactions underlying sex determination are changing between these two C. briggsae populations.