Browsing by Author "Hansen, Jeffrey, committee member"
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Item Open Access Investigation of transcriptional dynamics in the Caenorhabditis elegans intestine gene regulatory network(Colorado State University. Libraries, 2022) Williams, Robert Thomas Patton, author; Osborne Nishimura, Erin, advisor; Wilusz, Carol, committee member; Hansen, Jeffrey, committee member; Santangelo, Tom, committee memberELT-2 is the major transcription factor required for the activation of Caenorhabditis elegans intestinal development. ELT-2 expression initiates in embryos to promote development and persists after hatching through larval and adult stages. Though the sites of ELT-2 binding have been defined and the transcriptional changes that result from ELT-2 depletion described, the intestine-specific transcriptome profile over developmental time has not been characterized, in part because of the difficulty in isolating intestine from other tissues. To address this knowledge gap, we used Fluorescence Activated Cell Sorting (FACS) to enrich intestine cells and performed RNA-seq analysis at distinct developmental stages. By linking the transcriptome profiles to previous ELT-2 studies, we were able to gain new insight into the role of ELT-2 in the intestinal regulatory network throughout development. Correlation of ELT-2 binding to the intestine transcriptome data, revealed that only 33% of intestine-enriched genes were direct targets of ELT-2 binding in embryos, but that number increased to 75% by the L3 stage. This suggests additional transcription factors may promote intestine-specific transcription early in development. Consistent with this possibility, half of the ELT-2 direct target genes were not transcriptionally dependent on ELT-2 for appropriate expression (i.e. their expression was not impacted following ELT-2 depletion) implying that other factors may compensate in the absence of ELT-2. Among direct target genes that were affected by ELT-2 depletion, equal proportions were over and under-expressed thus ELT-2 can both activate and repress direct target genes. Both activated and repressed sets of ELT-2 target genes were enriched for defense response genes reinforcing recent findings demonstrating ELT-2 participating in mediating the immune response upon pathogen exposure. Fluorescent reporter assays demonstrated that expression of two direct targets of ELT-2 cebp-1 and ets-4 are indeed repressed by ELT-2. Moreover, we observed that ELT-2 repressed its own promoter in a negative feedback loop that regulates elt-2 gene expression. Together, our findings illustrate that ELT-2 contributes directly to roughly 20 – 50% of intestine-specific gene expression, that ELT-2 exerts both positive and negative regulatory control on its direct targets, and that our overall picture of the intestinal regulatory network is incomplete with more intestine specific transcription factors and mechanisms remaining to be discovered.Item Open Access Live-cell imaging uncovers the relationship between histone acetylation, RNA polymerase II phosphorylation, transcription, and chromatin dynamics(Colorado State University. Libraries, 2023) Saxton, Matthew Neeley, author; Stasevich, Timothy J., advisor; Nishimura, Erin O., committee member; Hansen, Jeffrey, committee member; Krapf, Diego, committee memberLiving cells are capable of turning a one dimensional strand of nucleic acids into a functional polypeptide. A host of steps and factors are involved in the process of transcription and translation, and understanding each of them is necessary for comprehending and characterizing life. While new technologies and assays have expanded our understanding of eukaryotic transcription, there is still much to be learned. In particular, single-molecule microscopy provides a powerful and versatile platform for studying the genesis of RNA with unparalleled spatiotemporal resolution (Chapter 1). First, we characterize the timing, kinetics, and occupancy of phosphorylated RNA polymerase II (RNAP2) using a single-copy HIV-1 reporter system. This work provides strong evidence for clusters of phosphorylated, initiating RNAP2 which is spatially separated from bursty, downstream RNA synthesis. It is found that RNAP2-Ser5-phosphorylation (Ser5ph) precedes RNA output by ~1 minute, and RNAP2 arrives at the locus in a phosphorylated state (Chapter 2). Then, we examine the spatial correlation between H3K27 acetylation and Ser5ph in living cells on the course of minutes to hours. Contrary to expectations based upon ChIP data, we find that the two signals are in fact spatially separated. This argues for a functional separation between transcriptional poising and initiation, likely aiding bursty behavior. Next, the dynamics of single chromatin-incorporated nucleosomes in the context of H3K27 acetylation and transcription initiation is determined with super-resolution single-molecule imaging. The physical movement of chromatin inside of H3K27ac and RNAP2-Ser5ph enriched regions is found to be significantly different, despite both marks being traditionally associated with transcriptionally active chromatin. (Chapter 3). Much of this work utilizes bead-loading in order to introduce proteins and DNA into living cells. A simple, effective, and cheap procedure, bead-loading is a highly effective and versatile technique that is generally underutilized. To facilitate communication of this process, a detailed protocol is included (Chapter 4). While this culmination of work furthers our understanding of cellular genetic expression and eukaryotic transcription, it also introduces many new questions that are promising areas of study. Fortunately, the combination of imaging technology and knowledge developed here provides promising new fronts for studying transcription in living cells (Chapter 5).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 Selective halogenation of pyridines and diazines via unconventional intermediates(Colorado State University. Libraries, 2022) Levy, Jeffrey N., author; McNally, Andrew, advisor; Bandar, Jeffrey, committee member; Neilson, Jamie, committee member; Hansen, Jeffrey, committee memberPyridines and diazines are prevalent in pharmaceuticals, agrochemicals, ligands, and other organic materials, and it's vital that synthetic chemists can selectively functionalize these heterocycles. We have shown that heterocyclic phosphonium salts and Zincke imine intermediates can be used to regioselectively functionalize pyridine rings. This dissertation describes the development of these strategies with an emphasis on new approaches to selectively halogenate pyridines, which we view as a long-standing challenge in organic chemistry. Chapter One introduces the importance of pyridines and diazines, as well as established methods and limitations in halogenating these azines. Chapter Two provides an overview of the synthesis and reactivity of heterocyclic phosphonium salts, and then describes a new strategy to access 4-halogenated pyridines via these reagents. Chapter Three examines further developments of heterocyclic phosphonium salts, including as how they can be used to selectively add amines and fluoroalkyl substituents to pyridines. Chapter Four provides an overview of pyridine ring-opening reactions and then shows how this approach can be applied to selectively halogenate the 3-position of pyridines. Chapter Five describes how modifications to the ring-opening strategy can be used to change halogenation site-selectivity. This chapter also shows that the ring-opened intermediates can be used to form isotopically labeled pyridines and aniline derivatives.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 Spn1, a multifunctional player in the chromatin context(Colorado State University. Libraries, 2016) Li, Sha, author; Stargell, Laurie, advisor; Argueso, J. Lucas, committee member; Hansen, Jeffrey, committee member; Luger, Karolin, committee member; Yao, Tingting, committee memberSpn1 was initially identified as a transcription factor that copurified with Spt6. Spn1 functions in transcription initiation and elongation, mRNA processing and export, histone modification, as well as in heterochromatic silencing. Our recent study demonstrated that Spn1 could bind histones and assemble nucleosomes in vitro. Therefore, Spn1 is a new member of the histone chaperone family. Here we found that Spt6 regulates Spn1-nucleosome interaction and conversely, Spn1 regulates Spt6-H2A-H2B interaction. Co-regulation between Spn1 and Spt6 enables them to be independent histone chaperones in nucleosome assembly. In addition, abrogation of Spn1-Spt6 interaction does not generate cryptic transcripts at certain genes. Furthermore, we identified a new interaction between Spn1 and the histone chaperone Nap1. Spn1, Nap1 and histones can form a large complex. We also found Spt6 could compete Nap1 for Spn1 binding, therefore disrupting Spn1-Nap1 interaction and releasing Nap1. In sum, Spn1 plays a multifunctional role in the chromatin context via dynamic interactions with its binding partners.Item Open Access The application of new methodology to complex molecule synthesis: studies toward the synthesis of pordamacrine A and liphagal(Colorado State University. Libraries, 2015) Seizert, Curtis A., author; Kennan, Alan, advisor; Ferreira, Eric, committee member; Chen, Eugene, committee member; Prieto, Amy, committee member; Hansen, Jeffrey, committee memberThe coevolution of organic synthesis and methodology has contributed greatly to the growth of both fields. This has been enabled by the invention of new methods during the prosecution of a synthesis in order to solve an unforeseen problem as well as by the novel application of independently developed methods to complex synthetic settings. Our own studies have encompassed both of these strategies, and we present their results herein. Our initial efforts consisted of synthetic studies towards the complex hexacyclic alkaloid pordamacrine A. This molecule presented many difficulties, and we were forced develop and employ new methods in its synthesis. Ultimately, these studies were stymied by the difficulty of forming the central carbocyclic ring system of this molecule. Among the methods used in the synthesis of pordamacrine A was a variant of a previously reported boron promoted Ireland-Claisen rearrangement. This rearrangement has been reported in very few papers in the literature, and many details of the reaction were undisclosed at the outset of ourstudies. We report here our investigations of the scope and stereochemical features of this rearrangement. Finally, methods based on the use of Pt carbenoids have formed a central element in our group's research focus. We apply here the use of this intermediate to the synthesis of liphagal, a complex tetracyclic compound. Our explorations of Pt-catalyzed cycloaddition reactions based on Pt carbenoids in this study have shed valuable light on the scope of this method. Though our studies culminated in a formal synthesis of an epimer of the natural product, we expect that future work towards liphagal will be able to use this methodology to make the correct diastereomer of liphagal, potentially in enantioenriched form.Item Open Access The chromatin binding factor Spn1 contributes to genome instability in Saccharomyces cerevisiae(Colorado State University. Libraries, 2018) Thurston, Alison K., author; Stargell, Laurie, advisor; Bailey, Susan, committee member; DeLuca, Jennifer, committee member; Hansen, Jeffrey, committee member; Luger, Karolin, committee memberMaintaining the genetic information is the most important role of a cell. Alteration to the DNA sequence is generally thought of as harmful, as it is linked with many forms of cancer and hereditary diseases. Contrarily, some level of genome instability (mutations, deletions, amplifications) is beneficial to an organism by allowing for adaptation to stress and survival. Thus, the maintenance of a "healthy level" of genome stability/instability is a highly regulated process. In addition to directly processing the DNA, the cell can regulate genome stability through chromatin architecture. The accessibility of DNA for cellular machinery, damaging agents and spontaneous recombination events is limited by level of chromatin compaction. Remodeling of the chromatin for transcription, repair and replication occurs through the actions of ATP remodelers, histone chaperones, and histone modifiers. These complexes work together to create access for DNA processing and to restore the chromatin to its pre-processed state. As such, many of the chromatin architecture factors have been implicated in genome stability. In this study, we have examined the role of the yeast protein Spn1 in maintaining the genome. Spn1 is an essential and conserved transcription elongation factor and chromatin binding factor. As anticipated, we observed that Spn1 contributes to the maintenance of the genome. Unexpectedly, our data revealed that Spn1 contributes to promoting genome instability. Investigation into a unique growth phenotype in which cells expressing a mutant form of Spn1 displayed resistance to the damaging agent, methyl methanesulfonate revealed Spn1 influences pathway selection during DNA damage tolerance. DNA damage tolerance is utilized during replication and G2 to bypass lesions, which could permanently stall replication machinery. This pathway congruently promotes and prevents genome instability. We theorize that these outcomes are due to the ability of Spn1 to influence chromatin structure throughout the cell cycle.Item Open Access The discovery of novel proteins regulating melanosome biogenesis and function(Colorado State University. Libraries, 2022) Detry, Anna, author; Di Pietro, Santiago, advisor; Hansen, Jeffrey, committee member; Hoerndli, Frederic, committee memberMelanosomes are lysosomal related organelles found in cells which are responsible for making pigment such as skin melanocytes. They are membrane bound organelles that form from the endosomal pathway and have specific proteins and enzymes which allow them to perform the function of melanin production. The process of melanosome biogenesis involves the melanosomes developing through four stages that are classified by electron microscopy appearance. The different melanosome stages have amyloid fibrils formed by proteolytically processed PMEL protein, different amounts of melanin, and different melanosomal proteins. In addition to melanosome biogenesis, another key factor in proper melanin formation and pigmentation is the melanosome luminal pH. The melanin producing enzyme tyrosinase is a pH dependent enzyme. When melanosomes are more acidic, tyrosinase is less functional, leading to less melanin production and a hypopigmentation phenotype. The Di Pietro lab and others have shown that the Two Pore Channel Two (TPC2) is a key regulator of melanosome pH, as well as a regulator of melanosome size and localizes to melanosome membranes. A proximity-dependent biotin identification experiment was preformed using TPC2 and eight potential melanosome proteins were identified. Each of these candidate proteins were knocked down in a human melanoma cell line using small interfering RNA and studied for a potential pigmentation phenotype. Tetraspanin10, phospholipase D1, myosin heavy chain 9, and myosin heavy chain 10 all showed a hypopigmentation phenotype. Two independent tetraspanin 10 knockout cell lines were generated using CRISPR-Cas9 which reproduced the hypopigmentation phenotype. In addition, the phenotype was rescued by re-expressing tetraspanin 10 in the knockout cells and overexpressing tetraspanin 10 in wild type cells showed a hyperpigmentation phenotype. This shows that tetraspanin 10 is involved in the pigmentation process. CD63 is another tetraspanin known to play vital roles in melanosome biogenesis and based on the minimal information aviable on tetraspanin 10, it can be hypothesized as being involved in PMEL processing. The discovery that tetraspanin 10 is involved in skin pigmentation will lead to better understanding of the pigmentation process and pigmentation related diseases.Item Open Access The role of Ferredoxin 3 in hydrogen metabolism in the hyperthermophilic archaeon Thermococcus kodakarensis(Colorado State University. Libraries, 2022) Stettler, Meghan, author; Santangelo, Thomas, advisor; Hansen, Jeffrey, committee member; Peers, Graham, committee memberLife faces innumerable challenges to cellular maintenance and reproduction, including access to sufficient energy. As such, all domains of life ubiquitously utilize energetically conservative mechanisms to maximize energy gains from the environment. Use of proteinaceous electron carriers, like ferredoxins, allows cells to harness energy from catabolic reactions that would otherwise be lost to the system as entropy or enthalpy. The hyperthermophilic, anaerobic archaeon Thermococcus kodakarensis is of particular interest as a target for bioengineering to maximize total energy gains, as it natively produces hydrogen gas resulting from terminal electron transport through a Membrane Bound Hydrogenase. T. kodakarensis encodes for three physiologically distinct ferredoxins. Prior to this thesis, only the sequence and molecular weight of the T. kodakarensis ferredoxins were known. Efforts in this thesis laid the groundwork for the biophysical characterization of each ferredoxin isoform via protein-film voltammetry and x-ray crystallography by the development of a reliable recombinant expression and purification scheme. Preliminary biophysical assay trials resulted in a Ferredoxin 1 crystal capable of diffracting to 1.1 Ångstroms, and midpoint reduction potentials for Ferredoxin 1 and Ferredoxin 3 confirming the predicted redox center geometry, demonstrating the efficacy of the developed protein expression and purification scheme for producing high-quality samples. Further investigation into the activity of the ferredoxins resulted in the generation of T. kodakarensis strains encoding for a tether protein between Ferredoxin 3 and its presumed sole electron acceptor Membrane Bound Hydrogenase at two respective locations. The parent strain includes a deletion of Ferredoxin 3, resulting in a deficient phenotype during sulfur-independent growth. The tethered strains of T. kodakarensis demonstrates a full recovery of sulfur-independent growth. Additionally, western-blotting revealed retention of the tethered protein in-vivo, and headspace measurements demonstrated restoration of hydrogen gas production compared to the parent deletion strain, and a reduction in total hydrogen gas output per cell compared to the lab parent strain. These findings implicate the importance of Ferredoxin 3 in hydrogen metabolism in T. kodakarensis and indicate Ferredoxin 3 as a potential target for bioengineering. Furthermore, this thesis is the foundation for further characterization of the T. kodakarensis ferredoxins as proteinaceous electron carriers with potential applications outside of this model organism.