Browsing by Author "Stargell, Laurie, committee member"
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Item Open Access A yeast-based assay system for the study of environmentally induced copy number variation(Colorado State University. Libraries, 2012) Stanton, Jacquelyn Lee, author; Argueso, Juan Lucas, advisor; Legare, Marie, committee member; Stargell, Laurie, committee memberMultiple studies have shown that in different individuals, specific genomic segments can occur at a variable copy number relative to the reference human genome. Chromosomal rearrangements resulting in Copy Number Variations (CNVs) have long been recognized as contributing factors in carcinogenesis, and more recently in Autism Spectrum Disorders. The molecular mechanisms underlying the formation of CNVs are not completely understood. The goal of this research project was to complete the development of an assay system to study CNV, and to validate it as a tool to investigate the relationship between environmental exposures and CNV formation. We have optimized a CNV detection assay using the budding yeast Saccharomyces cerevisiae as an experimental model system. This CNV reporter contains two yeast genes, SFA1 and CUP1 that confer gene dosage-dependent tolerance to formaldehyde and copper, respectively. This system enables the detection of rare clones containing an amplification of the chromosomal segment containing the reporter by selection in media containing high levels of formaldehyde and copper, allowing the estimation of the rate of CNV formation. Results obtained in diploid cells under basal growth conditions (un-induced / un-exposed) showed that most spontaneous CNV events detected in our system were mediated through non-allelic homologous recombination (NAHR) between dispersed repetitive DNA sequences, mainly Ty1 and Ty2 retrotransposable elements and their LTRs. Another set of repeats involved in NAHR included conserved gene family. Single copy sequences and microhomology motifs were detected in our dataset, but were exceedingly rare. The most abundant classes of CNVs observed involved segmental duplications and non-reciprocal translocations. In order to characterize the effect of environmental factors on CNV, cells were exposed to relatively low doses of three different known mutagens: Hydroxyurea, Methyl Methanesulfonate, and Camptothecin. These exposures resulted in an increase of the CNV rate ranging from 3 to 17 fold over the un-induced cultures. The spectra of chromosomal rearrangements induced by these exposures was analyzed, revealing that not only did exposures result in more chromosomal breaks but often a higher frequency of resulting segmental copies, and allowing further understanding of the CNV mechanisms associated with these exposures.Item Open Access Assessing histone H2A.Z and the H2A tails in chromatin structure(Colorado State University. Libraries, 2018) Seidel, Erik, author; Hansen, Jeffrey, advisor; Stargell, Laurie, committee member; Bailey, Susan, committee memberDeoxyribose nucleic acid (DNA) is a negatively charged macromolecule that encodes life's genetic material. In organisms, it is bound to net positively charged histone proteins in specific fashions and then compacts with magnesium and calcium to form domains and then chromosomes, which occupy territories in the nucleus during interphase. The mechanism of this compaction has been debated and studied for decades, and the employment of specific protein structures in molding chromatin morphology is still under review. This thesis adds to this story by testing how higher order chromatin structure is influenced by a histone H2A variant, H2A.Z, and the combined effect that the so-called histone H2A N and C terminal tails, when contrasted to arrays involving wildtype canonical H2A. An in vitro model system of nucleosomal arrays consisting of sea urchin derived 5S ribosomal DNA and recombinant mammalian histone proteins was used. Both the H2A.Z and H2A tailless arrays required increased magnesium to oligomerize into possibly domain-like structures. The H2A.Z protein produced similarly accessible structures as the fully accessible wildtype control as learned through a micrococcal nuclease digestion method designed for these chromatin structures. The deletion of the H2A N and C terminal tails produced oligomers with slightly less accessible linker DNA than its wildtype control according to the micrococcal nuclease digestion. Furthermore, the H2A.Z, H2A double tailless, and H2A wildtype oligomers were globular in shape. When subjected to fluorescence recovery after photobleaching (FRAP), the oligomers involving H2A.Z agreed with the current literature describing its presence in euchromatin and heterochromatin, and its mobility correlated with that of a more mobile and possibly more open structural agent. Taken together, the H2A and H2A.Z proteins are influential in determining and providing variability to the overall chromatin structure that is vital to DNA's role in biology.Item Open Access Biochemical, biophysical and functional characterization of histone chaperones(Colorado State University. Libraries, 2014) Zhang, Ling, author; Luger, Karolin, advisor; Krapf, Diego, committee member; Nyborg, Jennifer, committee member; van Orden, Alan, committee member; Stargell, Laurie, committee memberNucleosomes, the basic repeating unit of chromatin, are highly dynamic. Nucleosome dynamics allow for various cellular activities such as replication, recombination, transcription and DNA repair, while maintaining a high degree of DNA compaction. Each nucleosome is composed of 147 bp DNA wrapping around a histone octamer. Histone chaperones interact with histones and regulate nucleosome assembly and disassembly in the absence of ATP. To understand how nucleosome dynamics are regulated, it is essential to characterize the functions of histone chaperones. The first project of my doctoral research focused on the comparison of different nucleosome assembly proteins employing various biochemical and molecular approaches. Nucleosome assembly proteins (Nap) are a large family of histone chaperones, including Nap1 and Vps75 in Saccharomyces cerevisiae, and Nap1 (also Nap1L1), Nap1L2-6 (Nap1-like 2-6, with Nap1L4 being Nap2) and Set in metazoans. The functional differences of nucleosome assembly proteins are thus interesting to explore. We show that Nap1, Nap2 and Set bind to histones with similar and high affinities, but Nap2 and Set do not disassemble non-nucleosomal DNA-histone complexes as efficiently as Nap1. Also, nucleosome assembly proteins do not display discrepancies for histone variants or different DNA sequences. In the second project, we identified Spn1 as a novel histone chaperone and look into new functions of Spn1 on the regulation of chromatin structural states. Spn1 was identified as a transcription regulator that regulates post-recruitment of RNA polymerase II in yeast. We demonstrated that Spn1 is a H3/H4 histone chaperone, a novel finding that was not observed previously. Spn1 also interacts with Nap1, and forms ternary complexes with Nap1 and histones. We also show that Spn1 has chromatin assembly activity and N- and C- terminal domains of Spn1 are required for its histone chaperone properties. At the same time, we had an interesting observation that Spn1 potentially has topoisomerase/nuclease activity, which is dependent on magnesium ions. This activity of Spn1 can also help answer questions raised by in vivo assays related to Spn1, including its correlation with telomere length, the heat sensitivity in the reduction of function yeast strains, and the elongated lifespan in the Spn1ΔNΔC strain. Our studies on the functional comparison of nucleosome assembly proteins revealed their distinct roles in the regulation of nucleosome dynamics. Our findings on the histone chaperone functions and nuclease/topoisomerase activities disclosed new roles of Spn1 in chromatin regulation, by regulating histone-DNA interaction and also maintenance of DNA integrity.Item Open Access Complex regulation of BpeEF-OprC mediated drug efflux in Burkholderia pseudomallei(Colorado State University. Libraries, 2016) Rhodes, Katherine, author; Belisle, John, advisor; Schweizer, Herbert, advisor; Dow, Steven, committee member; Stargell, Laurie, committee memberBurkholderia pseudomallei (Bp) is a Gram-negative bacillus and the etiologic agent of melioidosis, a multifaceted syndrome causing high mortality in tropical regions of the world. The bacteria is classified as a Tier-1 Select Agent due to the seriousness of infection, low infectious dose, lack of effective vaccine, and difficulty of treatment. Bp’s many acquired and intrinsic antimicrobial resistance determinants make the study of these factors vital to improving the efficacy of bi-phasic treatment currently used to treat melioidosis. This study examines one factor in particular: the BpeEF-OprC efflux pump, a member of the resistance-nodulation and cell division family of efflux proteins, and capable of extruding both trimethoprim and sulfamethoxazole. A combination of these compounds (co-trimoxazole) is the first line of eradication phase therapy, making BpeEF-OprC the most clinically important efflux pump encoded by Bp. In spite of this, little is understood of the regulation of bpeEF-oprC, other than it is controlled in part by two LysR family proteins, BpeS, and BpeT. We hypothesized that these regulatory proteins 1) exert their action(s) by interacting with bpeEF-oprC at a specific site within the bpeT-llpE-bpeEF-oprC intergenic region, 2) are capable of influencing transcription of additional operons, and 3) that mutations to these proteins altered ability to form multimers, thereby influencing their function as observed by increased co-trimoxazole resistance and bpeF transcript levels. In Aim I of the study, we identified the cis regulatory regions by which these proteins interact within the bpeT-llpE-bpeE intergenic region using a combination of 5’ deletion assays, S1 nuclease protection, fluorescent-linked oligo extension and electrophoretic mobility assays. With this information we were able to locate bpeT transcriptional start sites and promoter regions as well as binding sites for both BpeT and BpeS. In Aim II, we examined the function of BpeT and BpeS as trans regulatory factors of BpeEF-OprC through mutation and deletion of both genes in part I, and as global regulatory factors in part II. Through overexpression and qRT-PCR or MIC analysis of wild type and mutant forms of both genes, we observed that while BpeT is a direct transcriptional activator of bpeEF-oprC, BpeS is not. Additionally, mutation position in BpeS seems to play a role in the expression phenotype of bpeEF-oprC. However, these mutations do not influence the ability of BpeS or BpeT to form multimers, as we observed no change between wild type and mutant protein oligomer formation through low-pressure gel chromatography and native gel electrophoresis. These same mutations also appeared to have no deleterious effect on the ability of the protein to bind their consensus region within the IR. Additionally, the loss of both genes did not interrupt the ability of bpeEF-oprC to be induced by substrates of BpeEF-OprC, suggesting an additional regulatory factor is at play. In Part II, RNA sequencing analysis and confirmation of select transcriptionally altered operons by RT-qPCR revealed that BpeS might influence expression of the Bsa Type 3 Secretion System (T3SS), while BpeT seems only to target bpeEF-oprC. This may have implication in the pathogenesis of Bp, and must be confirmed in in-vivo cell models using Select Agent excluded strain Bp82 in order to solidify the link between efflux and T3SS during infection. Ultimately, more work is needed to identify the missing regulatory factors in play during expression of bpeEF-oprC, understand how mutations to BpeT and BpeS alter their function, and confirm the relevance of a putative link between co-regulation of efflux and virulence during Bp infection.Item Open Access Factor dependent archaeal transcription termination(Colorado State University. Libraries, 2017) Walker, Julie, author; Santangelo, Thomas J., advisor; Montgomery, Tai, committee member; Stargell, Laurie, committee member; Yao, Tingting, committee memberRNA polymerase activity is regulated by nascent RNA sequences, DNA template sequences and conserved transcription factors. Transcription factors regulate the activities of RNA polymerase (RNAP) at each stage of the transcription cycle: initiation, elongation, and termination. Many basal transcription factors with common ancestry are employed in eukaryotic and archaeal systems that directly bind to RNAP and influence intramolecular movements of RNAP and modulate DNA or RNA interactions. We describe and employ a flexible methodology to directly probe and quantify the binding of transcription factors to the archaeal RNAP in vivo. We demonstrate that binding of the conserved and essential archaeal transcription factor TFE to the archaeal RNAP is directed, in part, by interactions with the RpoE subunit of RNAP. As the surfaces involved are conserved in many eukaryotic and archaeal systems, the identified TFE-RNAP interactions are likely conserved in archaeal-eukaryal systems and represent an important point of contact that can influence the efficiency of transcription initiation. While many studies in archaea have focused on elucidating the mechanism of transcription initiation and elongation, studies on termination were slower to emerge. Transcription factors promoting initiation and elongation have been characterized in each Domain but transcription termination factors have only been identified in bacteria and eukarya. Here we characterize the first archaeal termination factor (termed Eta) capable of disrupting the transcription elongation complex, detail the rate of and requirements for Eta-mediated transcription termination and describe a role for Eta in transcription termination in vivo. Eta-mediated transcription termination is energy-dependent, requires upstream DNA sequences and disrupts transcription elongation complexes to release the nascent RNA to solution. Deletion of TK0566 (encoding Eta) is possible, but results in slow growth and renders cells sensitive to DNA damaging agents. Structure-function studies reveal that the N-terminal domain of Eta is not necessary for Eta-mediated termination in vitro, but Thermococcus kodakarensis cells lacking the N-terminal domain exhibit slow growth compared to parental strains. We report the first crystal structure of Eta that will undoubtedly lead to further structure-function analyses. The results obtained argue that the mechanisms employed by termination factors in archaea, eukarya, and bacteria to disrupt the transcription elongation complex may be conserved and that Eta stimulates release of stalled or arrested transcription elongation complexes.Item Open Access Functional characterization of nucleosome assembly proteins(Colorado State University. Libraries, 2021) Krzizike, Daniel, author; Luger, Karolin, advisor; Kennan, Alan, committee member; Nyborg, Jennifer, committee member; Stargell, Laurie, committee member; Woody, Robert, committee memberThe amount of DNA found within the human body will span from the earth to the sun ~50 times. With the DNA providing the genetic blueprint of all living things, it needs to be packaged in a way that allows accessibility. The first step in this packaging involves nucleosomes, large macromolecular complexes made up of histone proteins and DNA. Nucleosomes must remain dynamic as they are constantly assembled and disassembled for processes such as DNA replication, repair, and transcription. Both assembly and disassembly occur in a specific stepwise manner orchestrated by multiple proteins employed by the cell. Specifically, histone chaperones have been implicated in almost every aspect of nucleosome dynamics such as shuttling histones into the nucleus, histone storage, and both nucleosome assembly and disassembly in an ATP-independent manner. While the structures of many histone chaperones have been determined, the mechanism of how they regulate nucleosome dynamics is still largely unknown. I investigated the mechanism of the nucleosome assembly protein family (Nap family) through several biochemical approaches. The Nap family of proteins are implicated in histone homeostasis through interactions with core histones, histone variants, and linker histones. They are conserved among all eukaryotes from yeast to humans. Members of the Nap family contain a conserved core region flanked by highly disordered N- and C-terminal tails varying in length and charge between species. Using yNap1, we investigated how these tails impact the overall function in regards to histone binding, histone selectivity among core histones and histone variants, and in mediating histone-DNA interactions. We found that the tails are critical for overall function, with the charge of the tails being crucial in regulation. We also investigated Vps75, another member of the Nap family. Similar to Nap1, Vps75 binds core histones, but also stimulates the acetylation activity of Rtt109, a histone acetyltransferase. In light of a recent debate regarding the stoichiometry with which these Nap members bind their histone cargo, we characterized the Vps75-histone interaction using core histones H2A-H2B and H3-H4. Comparing Vps75 with yeast Nap1, we found that the mechanism of histone binding is not conserved among these Nap family members. Further expanding on Vps75, we investigated the interaction with Rtt109 in both the presence and absence of H3-H4. We discovered dimeric Vps75 is capable of binding either one histone tetramer or two units of Rtt109 with the ternary complex consisting of only one unit of Rtt109 and one H3-H4 tetramer. While characterizing Nap family members I became very familiar with Analytical Ultracentrifugation (AUC). AUC is a powerful in-solution technique that provides first-principle hydrodynamic information to determine size, shape, and molecular interactions, making it ideal for the characterization of proteins, DNA, and the interactions among them. As our lab traditionally used AUC to obtain van Holde-Weischet plots, an excellent graphical representation of homogeneity or heterogeneity, we incorporated new analysis techniques for improved accuracy in molecular mass and gross shape determination. Using the added-on fluorescence detection system, we obtained a level of sensitivity and selectivity that was otherwise not possible. Using the powerful method of analytical ultracentrifugation combined with fluorescent studies, we provide insight into the regulation mechanism of Nap family members along with establishing a framework to study other macromolecular complexes.Item Open Access Integrating discrete stochastic models with single-cell and single-molecule experiments(Colorado State University. Libraries, 2019) Fox, Zachary R., author; Munsky, Brian, advisor; Stargell, Laurie, committee member; Wilson, Jesse, committee member; Prasad, Ashok, committee memberModern biological experiments can capture the behaviors of single biomolecules within single cells. Much like Robert Brown looking at pollen grains in water, experimentalists have noticed that individual cells that are genetically identical behave seemingly randomly in the way they carry out their most basic functions. The field of stochastic single-cell biology has been focused developing mathematical and computational tools to understand how cells try to buffer or even make use of such fluctuations, and the technologies to measure such fluctuations has vastly improved in recent years. This dissertation is focused on developing new methods to analyze modern single-cell and single-molecule biological data with discrete stochastic models of the underlying processes, such as stochastic gene expression and single-mRNA translation. The methods developed here emphasize a strong link between model and experiment to help understand, design, and eventually control biological systems at the single-cell level.Item Open Access Integrating p300 functions in HTLV-1 transcription initiation(Colorado State University. Libraries, 2014) Luebben, Whitney R., author; Nyborg, Jennifer K., advisor; Stargell, Laurie, committee member; Laybourn, Paul, committee member; Prenni, Jessica, committee member; Quackenbush, Sandra, committee memberThe HTLV-1 provirus overcomes a repressive chromatin environment for efficient transcription of its genome. This is accomplished by the robust recruitment of the coactivator protein, p300, to the viral enhancer sites through interactions with DNA bound pCREB and the viral transactivating protein, Tax. Recruitment of p300 to the HTLV-1 promoter results in histone acetylation and nucleosome depletion from the promoter region in the presence of the histone chaperone, Nap1. To study the histone acetylation requirements for Nap1-dependent nucleosome disassembly, we utilized immobilized in vitro assembled chromatin templates containing site specific K→R mutations within the N-terminal tails of the histones. Through these studies, we identified histone H3, lysine 14 as the functionally relevant acetylation site for Nap1-dependent nucleosome disassembly. Additionally, we found a significant correlation between nucleosome disassembly from the HTLV-1 promoter and acetylation-dependent transcription activation. These studies suggest that nucleosome disassembly is a prerequisite for transcription activation, as nucleosome disassembly creates a nucleosome free region within the HTLV-1 promoter, allowing for the subsequent recruitment of Pol II and general transcription machinery for activation of transcription. The identification of a single and specific acetyl-lysine residue led us to the hypothesis that the p300 acetyl-lysine binding domain (bromodomain) was involved in HTLV-1 transcription activation through recognition of H3K14ac. To test this hypothesis, we utilized a p300 bromodomain deletion mutant and a CBP/p300 specific bromodomain inhibitor, (SGC-CBP30), to investigate the involvement of the p300 bromodomain in HTLV-1 transcription activation. Importantly, we found that the p300 bromodomain is not involved in the initial recruitment of the coactivator to the chromatin template as previously proposed, rather the bromodomain functions after recruitment to the promoter and following acetylation of the histone tails. These findings are consistent with a role for the p300 bromodomain in nucleosome disassembly and uncover a novel function for the bromodomain in gene activation.Item Open Access Investigation of the molecular mechanisms and therapeutic potential of oncogene-induced kinetochore-microtubule defects(Colorado State University. Libraries, 2015) Herman, Jacob A., author; DeLuca, Jenniver G., advisor; Bamburg, James, committee member; Stargell, Laurie, committee member; Nickoloff, Jac, committee memberKinetochores, large protein structures assembled on centromeric DNA during mitosis, bind to microtubules of the mitotic spindle to orchestrate and power chromosome movements. Deregulation of kinetochore-microtubule (kinetochore-MT) attachments has been implicated in driving chromosome instability and cancer evolution; however, the nature and source of kinetochore-MT attachment defects in cancer cells remain largely unknown. Here, we identify kinetochore-MT attachments, and their regulation by Aurora B kinase (ABK) as key targets for selective therapeutic intervention in glioblastoma and other cancers. We observe that accessory regulators of ABK and kinetochore-microtubule attachment stability are compromised in some cancers and fundamentally alter kinetochore signaling. First we identify RAS/MAPK oncogenic transformation as sufficient to induce these defects through an enzymatic cascade targeting the kinetochore. We then identify BUBR1 kinetochore recruitment and kinetochore-associated PP2A activity as cancer-essential activities, which are required for some cancers to form robust physical interactions between kinetochores and MTs. We also verify previous findings that many cancers are characterized by chromosome segregation errors arising from merotelic kinetochore-microtubule attachments (a single kinetochore bound to microtubules emanating from both spindle poles). We attribute the cause of these errors to be a decrease in MT dynamics independent of the physical attachments status. Finally we characterize a novel kinetochore component, BUGZ, which serves as a molecular chaperone for BUB3 and thus indirectly stimulates ABK activity. We find that BUGZ binds to BUB3 through a conserved GLEBS domain, and this interaction is required for BUB3 kinetochore localization. BUGZ depletion decreases ABK activity resulting in lethal chromosome alignment defects in glioblastoma cells and genetically transformed cells. Together these findings further elucidate the molecular mechanism by which kinetochore-MT attachments are regulated and importantly, how this mechanism is perturbed upon transformation. These results will make the design and application of novel anti-cancer drugs with reduced side effects possible because the specifically target cancer populations.Item Open Access Nucleosomal array condensation: new insights into an old "tail"(Colorado State University. Libraries, 2011) Sorensen, Troy C., author; Hansen, Jeffrey C., advisor; Stargell, Laurie, committee member; Luger, Karolin, committee member; Bailey, Susan, committee memberThe DNA present within the nucleus of each human somatic cell, when extended end to end, would span a distance of about one meter. The first level of compaction critical to fitting the entire genome into the nucleus is the nucleosome, consisting of 147 base pairs of DNA wrapped 1.7 times around an octameric structure composed of the four core histones H2A, H2B, H3 and H4. Nucleosomes separated by up to 80 base pairs of linker DNA called nucleosomal arrays compact the DNA further through short range intra-array and long range inter-array contacts that generate different levels of higher order condensed structures. This dissertation investigates the involvement of the core histone "tail" domains as well as the influence of the H3 centromeric variant CENP-A in nucleosomal array condensation events. In vitro, 12-mer nucleosomal arrays condense intra- and inter-molecularly through nucleosome-nucleosome interactions driven primarily by the core histone tail. This dissertation details the contributions and the molecular determinants of the histone tail domains to the condensation processes. Importantly, we found that the H3 and H4 tail domains were the largest contributors to array condensation. The mode of action used by the H4 tail domain in intra- and intermolecular condensation centered on the following determinants: 1) position of the H4 tail, 2) amino acid composition, 3) positive charge density and 4) tail domain length. Importantly, the primary sequence of the H4 tail was found to not be an important molecular determinant. To date no study has been performed to determine short-range compaction between "bulk" H3 containing and H3 centromeric specific variant, CENP-A chromatin. 12-mer nucleosomal arrays containing either H3 or CENP-A histones were reconstituted and tested for their ability to fold intra-molecularly. Major finding include that CENP-A containing nucleosomal arrays assemble in the same stepwise manner as conical arrays and were always more compact than H3 containing arrays at every salt concentration tested. The increased compaction was found to be in part due to a lysine to arginine mutation at position 49 of CENP-A.Item Open Access Reexamining the role of linker histones beyond 30 nm fibers in a complex chromatin environment(Colorado State University. Libraries, 2024) Kuerzi, Amanda, author; Hansen, Jeff, advisor; Stargell, Laurie, committee member; Stasevich, Tim, committee member; Mykles, Donald, committee memberEukaryotic cells store DNA in the cell nucleus in the form of chromatin. Chromatin is composed of nearly equal parts proteins and DNA. It is both highly compacted and organized into discrete domains within the nucleus. However, the manner in which chromatin is compacted, and domains are organized, remains elusive. The primary players in chromatin compaction are core histones, which bind DNA to form the nucleosome and the basis for 10 nm fibers. Linker histones also play an important role in chromatin compaction. Previous work showed that linker histones are important for the formation of 30 nm structures. 30 nm structures were long held to be folding intermediates for repressive chromatin domains. However, there is little evidence for these structures in most eukaryotic cell types. Instead, chromatin appears to be composed of an interdigitated 10 nm fibers in both repressive and accessible chromatin types. The role of linker histones in 10 nm fibers is not well characterized. Previous work showed that linker histones stabilized 30 nm structures, rendering them inaccessible to binding by additional proteins. In the following, we investigate the behavior of linker histones in an interdigitated 10 nm fiber environment. We use an in vitro model called "condensates" to mimic the formation of 200 nm chromatin domains. We find that linker histones stabilize these condensates by cross-linking chromatin fibers. Importantly, we show that the presence of linker histones does not preclude binding by additional proteins. Linker histones readily bind condensates in ratios above an expected one linker histone per nucleosome. Additional binding by linker histones suggests that 10 nm fibers provide a complex environment in which linker histones dynamically interact with both nucleosomes and linker DNA.Item Open Access Regulation of Halloween genes and ecdysteroid responsive genes in molting gland of Blackback land crab Gecarcinus lateralis by molt-inhibiting hormone, mTOR and TGFβ signaling pathways pathways(Colorado State University. Libraries, 2019) Benrabaa, Samiha Abd Al Salem, author; Mykles, Donald, advisor; Garrity, Deborah, committee member; Kanatous, Shane, committee member; Stargell, Laurie, committee memberMolting is necessary for growth and development in all arthropods. Halloween genes are expressed in the Y-organs (YO) and encodes cytochrome P450 enzymes. These enzymes catalyze the synthesis of ecdysteroid hormones that regulate the molt cycle. This hormone binds to ecdysteroid receptor to activate a cascade of ecdysteroid-responsive genes that effect tissue responses to hormone. We used P. leptodactylus Halloween gene and insect ecdysteroid-responsive gene sequences to extract and characterize the land crab orthologs in the YO transcriptome. This resulted in identification and characterization of eight ecdysteroidogenic genes that encode phantom, disembodied, spook, shadow, Cyp18a1, neverland, NADK and ALAS and nine ecdysteroid-responsive genes that encoded EcR, RXR, broad complex, E75, E74, Hormone receptor 4, Hormone receptor 3, forkhead box transcription factor (FOXO) and Fushi tarazu factor-1. Sequences were validated by end-point PCR and Sanger sequencing. We used phylogenetic analysis to infer evolutionary relationships among contig sequences and ortholog of Halloween genes and ecdysteroid-responsive genes in other species. The results showed the contig sequences clustered with their corresponding orthologous genes. Tissue distributions for spook and phantom showed significantly higher mRNA levels in the YO compared to other tissues. By contrast, the mRNA levels of NADK, ALAS, and all ecdysteroid-responsive genes were not higher in the YO than those in other tissues. These data show that the YO is the primary source of ecdysteroid production and that the YO can respond to ecdysteroid, suggesting a feedback regulation on ecdysteroid synthesis and secretion. qPCR was used to quantify gene expression of Halloween and ecdysteroid-responsive gene expression in the YO of Gecarcinus lateralis induced to molt by multiple limb autotomy (MLA) or eyestalk ablation (ESA). ESA decreased mRNA levels of Gl-Phm, Gl-E75 and Gl-RXR at 3 days post-ESA. Gl-Dib and shadow increased at 3 days post ESA and decreased at 7 and 14 days post-ESA. Gl-Cyp18a1, Gl-BR-C, Gl-NADK and Gl-ALAS mRNA were higher at Day 0 and 1 post-ESA and lower at Day14 post ESA. Gl-HR3, Gl-HR4, and Gl-E74 were expressed at low levels. MLA lowered mRNA levels of Halloween genes, Gl-Nev, and Gl-E75, except Gl-dib, at premolt and postmolt stages. Gl-Dib, Gl-NADK, Gl-ALAS, and Gl-BR-C mRNA levels were not affected by molt stage. Gl-EcR, Gl-HR4 and Gl-HR3 mRNA levels were highest in premolt and lowest in postmolt. Gl- FOXO mRNA levels were highest in premolt and lowest in intermolt. These data suggest that molting has an indirect effect on the regulation of Halloween genes and that molting directly regulates HR3, HR4, RXR and FOXO to increase ecdysteroid synthetic capacity of the YO in premolt animals. The presence of EcR/RXR and ecdysteroid-responsive genes suggest that elevated ecdysteroid represses the YO at the end of premolt. TGFβ/activin signaling mediates the transition of the YO from the activated to the committed state, as SB431542 blocks this transition. G. lateralis were eyestalk-ablated to induce molting and injected with vehicle (DMSO) or SB431542 at Day 0. In controls, ESA increased hemolymph ecdysteroid titers at 3, 7 and 14 days post-ESA. There were significant increases in the mRNA levels of Gl-Nvd at 7 and 14 days post-ESA and other Halloween genes (Gl-Spo, Gl-Phm, Gl-Dib, and Gl-Sad), as well as Gl-CYP18a1, Gl-ALAS, Gl-NADK, Gl-BR-C,Gl- FOXO, Gl-EcR, and Gl-RXR, at 14 days post-ESA. SB431542 reduced hemolymph ecdysteroid titers at 7 and 14 days post-ESA compared to control animals, but titers were no different from controls at 1, 3, and 5 days post-ESA, indicating that SB431542 had no effect on YO activation. SB431542 blocked the increases in mRNA levels of Gl-Nvd, Gl-Spo, Gl-Phm, Gl-Dib, Gl-Sad, Gl-CYP18a1, Gl-ALAS, Gl-NADK, Gl-BR-C, Gl-EcR, and Gl-RXR by ESA. SB431542 had no effect on mRNA levels of the ecdysteroid-responsive genes Gl-HR3 Gl-HR4, Gl-E74, Gl-E75 and Gl-FTZ-F1. These data suggest that an Activin-like TGFβ factor stimulates YO ecdysteroidogenesis in the committed YO by up-regulating Halloween, Gl-BR-C, and Gl-FOXO genes.Item Open Access Regulation of transcription by ubiquitin and the INO80 chromatin remodeling complex(Colorado State University. Libraries, 2015) Ndoja, Ada, author; Yao, Tingting, advisor; Cohen, Robert, advisor; Stargell, Laurie, committee member; Hansen, Jeffrey, committee member; Wilusz, Carol, committee memberTranscription in eukaryotes is a crucial process that is tightly regulated in order to maintain cellular homeostasis and offer optimal responses to environmental changes. Transcriptional activators (TAs) regulate this process by orchestrating time and locus-specific assembly of complex transcription machinery. Thus, the abundance, localization and activity of TAs are all subject to tight control. One way in which TAs are controlled is by the covalent attachment of the conserved protein ubiquitin. Ubiquitination of TAs has been reported to affect transcription via proteolytic and non-proteolytic routes, yet the function of the ubiquitin signal in the non-proteolytic process is poorly understood. Through studies of a series of synthetic and natural activators in yeast and mammalian cells, we found that mono-ubiquitinated TAs cannot stably interact with DNA to promote transcription. We have identified the AAA+ ATPase Cdc48 and its cofactors as the Ub receptor responsible for extracting mono-ubiquitinated activators from chromatin, independently of proteolysis. These findings describe a novel mechanism by which gene repression can be maintained without destroying the activator. Upon appropriate stimulus, deubiquitinating enzymes can readily reverse mono- or oligo-ubiquitination of the activator and transcription can initiate without the requirement for new protein synthesis. This mechanism may facilitate rapid switching between “on” and “off” states of transcription and may serve as a general strategy to prevent spurious transcription in the “off” state. Compaction of DNA into chromatin imparts further layers of complexity to the control of eukaryotic gene expression. Cooperation between chromatin remodelers, histone variants, and histone post-translational modifications (PTMs) offers diverse regulatory options in DNA metabolic processes, including transcription and DNA repair. The human INO80 chromatin-remodeling complex (hINO80) has been shown to facilitate transcription by promoting an open chromatin environment at promoter regions. How and whether hINO80 directly promotes an open chromatin environment is not yet understood. In an effort to elucidate how hINO80 regulates transcription, we have characterized the nucleosome sliding activity of hINO80 and examined how histone variant H2A.Z and histone PTMs modulate its activity in vitro. Our results suggest that nucleosomes containing H2A.Z or the H3 acetylation mimic, K56Q, are mobilized by hINO80 with faster kinetics compared to canonical unmodified nucleosomes, and their effects are additive. In contrast, ubiquitination of H2A or H2B does not affect the sliding activity of hINO80. Nucleosomes containing both H2A.Z and H3-K56Ac are enriched at promoter regions and DNA damage sites in mammalian cells. These nucleosomes have been shown to exhibit rapid turnover kinetics in vivo. Our studies provide biochemical evidence that hINO80 participates in transcription and DNA repair processes by ATP-dependent mobilization of H2A.Z/H3-K56Ac-containing nucleosomes. Future studies will be required to elucidate how nucleosome mobilization in vitro relates to chromatin dynamics in vivo.Item Open Access RNA replication by poliovirus RNA-dependent RNA polymerase: effects of residue 5 on elongation complex stability and processivity(Colorado State University. Libraries, 2011) Hobdey, Sarah Elizabeth, author; Peersen, Olve B., advisor; Stargell, Laurie, committee member; Chen, Chaoping, committee member; Wilusz, Carol, committee memberThe poliovirus (PV) RNA-dependent RNA polymerase (RdRP) is a small, single-subunit, enzyme that is responsible for the replication of the viral genome. PV genome replication is much more involved than just repetitively incorporating a single nucleotide into an oligonucleotide primer. The polymerase must first go through multiple steps of initiation before processive replication can happen. During primer-dependent initiation, the polymerase must bind the primer/template RNA substrate and undergo a conformational change to form a stable RNA-polymerase complex. After the stable RNA-polymerase complex is formed the polymerase undergoes a second conformational change associated with the addition of the first nucleotide to the primer. It is only after these few steps of initiation that the polymerase can begin processive elongation. The work presented in this dissertation addresses a structure-function relationship related to initiation and processivity of the PV RdRP. Specifically, my work shows that the PV RdRP residue 5 is involved in forming and maintaining the stable, elongation-competent complex. Also, the complex stability can be modulated by downstream RNA interactions or by the number of nucleotides that are incorporated to form the stable complex. Lastly, my data demonstrate evidence of an RNA rearrangement during elongation complex formation and show that the maintenance of a stable elongation complex is required for processive RNA replication, which is required for virus replication. To end, these data elucidate a more complete understanding of the structure-function relationships of the viral RNA polymerase and could eventually facilitate in the design of specific polymerase inhibitors.Item Open Access Site-specific function of Endonuclease G and CPS6 to enable vertebrate function in an invertebrate model(Colorado State University. Libraries, 2021) Czarny, Ryan Scott, author; Ho, P. Shing, advisor; Stargell, Laurie, committee member; Hansen, Jeffrey, committee member; Argueso, Lucas, committee memberThe role of mitochondrial localized Endonuclease G (EndoG) remains relatively elusive. Studies have shown that EndoG has implications in mitochondrial DNA copy number, nuclear DNA cleavage during apoptosis, and oncogenesis; however, the mechanisms and pathways have yet to be determined. Our initial work investigates the nuclease activity of EndoG as well as its binding preference for duplex DNA and Holliday Junctions. It appears that EndoG and its C. elegans homolog, cps6, have slightly different functions in their in vivo systems, which has led us to query the structural modifications between the proteins. EndoG has been shown to have a preference for the 5-hydroxymethylcytosine (5hmC) epigenetic marker, an interesting feature due to the fact that invertebrate systems do not contain 5hmC in their epigenome. A key difference in the homologs arises in their DNA binding domain. The invertebrate model (cps6) contains two additional amino acids within this region that potentially allow for an alpha helix not seen in the vertebrate model to form. This alpha helix repositions a conserved cysteine in a way that it is pointed away from the active site in cps6, which could have consequences with regards to function. Our work investigates the addition/removal of this helix from the vertebrate and inveterate system to elucidate its role. In conjunction with the primary DNA binding site, there is a second site next to and orthogonal to the first. The vertebrate system contains multiple positively charged residues positioned to interact with the DNA backbone while the invertebrate contains two prolines that seem to be responsible for repositioning charges away from the site. We investigate the role of this secondary binding site as well as the importance of the invertebrate prolines. Overall, we propose a model to determine the role of EndoG in vivo utilizing the suite of protein mutations characterized herewithin.Item Open Access The effects of the histone chaperone and histone modifications on nucleosome structure(Colorado State University. Libraries, 2016) Wang, Tao, author; Luger, Karolin, advisor; Stargell, Laurie, committee member; Yao, Tingting, committee member; Williams, Robert M., committee memberThe nucleosome, composed of 147-bp DNA and a histone octamer, is the basic unit of chromatin in eukaryotes, which is considered as a barrier for all DNA dependent processes. Understanding how nucleosome structure is regulated provides new insights into pivotal cellular processes. Histone modifications and histone chaperones have potential roles in the regulation of nucleosome structure. Here, I investigated the role of FACT in regulating nucleosome structure. FACT (FAcilitate Chromatin Transcription) is a conserved histone chaperone that is essential for gene transcription elongation. Our biochemical data show that FACT is not only a H2A-H2B chaperone, but also a H3-H4 chaperone. By binding H3-H4, FACT facilitates tetrasome assembly. In the presence of H2A-H2B, FACT facilitates H2A-H2B deposition onto tetrasomes and hexasomes, and thus promotes nucleosome assembly. FACT is also able to tether partial nucleosome components, composed of a histone hexamer and DNA, and results in forming an unstable complex. Interaction with H2A-H2B is essential for FACT binding to tetrasomal H3-H4. In order to hold a histone hexamer, FACT also stabilizes dimer-tetramer interaction. Previous study shows that H2BK120ub facilitates FACT function in gene transcription with the help of other transcription factors. Here, we show that H2AK119ub and H2BK120ub have no effects on FACT-(H2A-H2B) interaction and FACT assembly activity. The role of select histone modifications in nucleosome structure was also determined in this dissertation. Histone modifications selected in this work are located at the entry-exit region of nucleosomal DNA. By using biochemical approaches, we find that H3Y41E (mimic phosphorylation) and H3R45E (mimic phosphorylation) affect the shape of nucleosome by facilitating nucleosomal 'DNA breathing'.Item Open Access The impact of insulin like growth factor 2 mRNA binding protein 1 (IGF2BP1) in human and canine osteosarcoma(Colorado State University. Libraries, 2019) Alyami, Nouf Mahdi, author; Duval, Dawn, advisor; Wilusz, Jeffrey, committee member; Argueso, Lucas, committee member; Stargell, Laurie, committee memberOsteosarcoma (OS) is a malignant bone tumor that afflicts over 10,000 dogs. Most dogs and approximately 30-40% of children with OS succumb to metastatic disease. We identified elevated insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1) as one of the biomarkers of poor prognosis in canine OS. IGF2BP1 is an oncofetal protein that regulates mRNA subcellular localization, nuclear export, stability, and translation. IGF2BP1 controls the expression of oncogene targets and correlates with poor outcome in a variety of human cancers. Using microarray analysis, we identified elevated insulin-like growth factor II mRNA binding protein 1 (IGF2BP1) expression as a biomarker of poor prognosis in canine osteosarcoma. Also, our preliminary data show that IGF2BP1 knockdown (shRNA) in a human OS cell line increased sensitivity to doxorubicin by ≥ tenfold compared to control. Significant reductions in cellular migration, invasion, proliferation, and tumor growth in nude mice were also observed (p < 0.05). The current research explores mechanisms for increased IGF2BP1 expression in panels of human and canine osteosarcoma cell lines. Gene amplification, hypomethylation, increased transcription, and alterations in microRNA (miRNA) regulation directly or through 3'UTR shortening have all been hypothesized by many studies as mechanisms to increase IGF2BP1 expression in cancer. We evaluated the expression and alternative polyadenylation of IGF2BP1 using RT-qPCR and western blot analysis in human and dog osteosarcoma cell lines. We assessed transcriptional activation of IGF2BP1 using luciferase reporters containing promoter sequences from the human and canine IGF2BP1 genes. To detect genomic amplification and methylation, we used qPCR to assess gene copy numbers and treatment with the DNA methylase inhibitor, 5-Azacytidine, to explore activation of gene expression through hypomethylation. Using qPCR analysis, we observed genomic amplification in 35% of canine tumors and cell lines and correlated amplification with IGF2BP1 transcript expression (p = 0.0006, Pearson r = 0.88). We observed no genomic amplification in human cell lines. Significant loss of 3'UTR regulatory sequences was found in 20% of canine cell lines (p < 0.05). The promoter analysis showed that most regulatory elements were located within ~580bp from the translational start site in both species. Using pathway-focused luciferase reporter assays, we identified activation of the following factors: MYC, NF-Kappa B, AP-1, and TCF4: β-catenin. Thus, our data show that multiple mechanisms can contribute to elevated IGF2BP1 expression, and these results can be used to develop new treatment strategies that target elevated IGF2BP1 or regulatory mechanisms. Using the McKinley canine OS cell line, we generated and validated stable overexpression of IGF2BP1 (IGF2BP1-pLVX-Puro, Clontech). The stable OS cell line pool and individual clones with a corresponding empty vector control were analyzed and tested for migration, invasion, proliferation, and resistance to standard chemotherapeutic agents. We analyzed migration and invasion using a scratch wound assay and measured cellular proliferation as a surface confluence for 90 hours on an IncuCyte Zoom. We also assessed the clones' sensitivity to doxorubicin over 48 hours using a bioreductive resazurin-based fluorometric assay. We assessed changes in transcript expression in response to IGF2BP1 from isolated total RNA analyzed on Affymetrix Canine 1.0ST microarrays (University of Colorado Cancer Center Genomic and Microarray Shared Resource). The overexpressing IGF2BP1 clones had increased resistance to doxorubicin compared to the control, and the IC50 levels correlated with IGF2BP1 mRNA levels (p < 0.05, r2 = 0.89). For cellular proliferation, we found that only the IGF2BP1-expressing pool, that represents random insertion of the plasmid without selecting isolated clones, exhibited a significantly higher rate of proliferation relative to the empty vector control (p < 0.05). However, one of the highest expressing IGF2BP1 isolated clones had significantly greater cellular mobility and invasion than this pool, and both the pool and isolated clone had significantly higher rates of migration and invasion that cells transfected with the empty plasmid (p < 0.05). Microarray analysis of control and over¬expressing cells was used to detect global changes in gene expression and to identify potential targets of IGF2BP1. Differentially expressed genes were cross ¬referenced to the RNA¬ Binding Protein Immunoprecipitation database, published by Conway et al. (2016) using human stem cells, to identify direct mRNA targets bound by IGF2BP1. We identified 162 genes that were differentially expressed between control and overexpressing cells (FC ≥2, FDR< 0.05), and 13 of those genes have been previously reported to bind IGF2BP1 directly. Pathway analysis of these 13 genes identified enrichment for genes involved in the regulation of cell adhesion, migration, and the extracellular matrix. Altered expression and IGF2BP1 binding of a subset of these transcripts were confirmed using RNA immunoprecipitation and RT-qPCR. Our data suggest that IGF2BP1 plays a significant role in human and canine osteosarcoma. This study revealed the functional relevance of IGF2BP1 and identified it as a biomarker for aggressiveness in osteosarcoma. With this knowledge, new treatment strategies can be developed that target IGF2BP1 or it is signaling pathways for osteosarcoma, or any cancer that expresses high levels of IGF2BP1. This treatment may have a high impact on the cell's ability to metastasize.Item Open Access Transcriptome and elemental analysis of the selenium hyperaccumulator Stanleya pinnata and non-accumulator Stanleya elata(Colorado State University. Libraries, 2015) Wang, Jiameng, author; Pilon-Smits, Elizabeth, advisor; Sloan, Daniel, committee member; Stargell, Laurie, committee memberTo view the abstract, please see the full text of the document.