Browsing by Author "Yao, Tingting, committee member"
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Item Open Access A new dawn for Aurora B regulation: shining light on multiple discrete populations of Aurora B kinase at centromeres and kinetochores(Colorado State University. Libraries, 2020) Broad, Amanda J., author; DeLuca, Jennifer G., advisor; Luger, Karolin, committee member; Markus, Steven, committee member; Yao, Tingting, committee member; Amberg, Greg, committee memberCell division is a fundamental biological process that is essential for all eukaryotes to divide the replicated genome with high fidelity into individual daughter cells. Improper segregation of replicated DNA results in chromosome instability, a characteristic that is deleterious to most cells. Critical to the proper segregation of mitotic chromosomes is attachment to spindle microtubules, which are dynamic cytoskeleton filaments that drive the movement of chromosomes during mitosis. A complex network of proteins, collectively called the kinetochore, mediates microtubule attachments to chromosomes. Kinetochores are recruited to individual chromosomes through a specialized heterochromatin domain known as the centromere. Centromeric heterochromatin is comprised of both canonical, H3-containing nucleosomes as well as nucleosomes that contain the histone H3 variant CENP-A. Centromeres serve as a central point of organization in mitotic cells, recruiting both structural and regulatory kinetochore proteins to chromosomes. This extensive protein/DNA network ensures the accurate segregation of chromosomes by regulation of proper kinetochore-microtubule attachments in mitosis. Kinetochore-microtubule interactions are regulated by Aurora B kinase, which phosphorylates outer kinetochore substrates to promote release of erroneous attachments. Although Aurora B kinase substrates at the kinetochore are defined, little is known about how Aurora B is recruited to and evicted from kinetochores, in early and late mitosis, respectively, to regulate these essential interactions. We set out to determine how Aurora B kinase is regulated during mitosis. We found that, contrary to the current model, Aurora B kinase and the Chromosomal Passenger Complex are recruited to distinct regions within the centromere and kinetochore. Furthermore, we found that accumulation of Aurora B kinase at centromeres is independent from Aurora B localization and activity at outer kinetochores. These results lead us to hypothesize a new model for Aurora B kinase regulation. In the direct recruitment model, a population of the kinase is recruited directly to kinetochores in early mitosis, then as mitosis progresses and kinetochore-microtubule attachments are stabilized, architectural changes within the kinetochore result in the eviction of outer-kinetochore localized Aurora B kinase and the stabilization of kinetochore-microtubule attachments.Item Open Access A pharmacokinetic investigation of chloroquine analogues in cancer autophagy modulation(Colorado State University. Libraries, 2020) Collins, Keagan P., author; Gustafson, Daniel, advisor; Prasad, Ashok, committee member; Yao, Tingting, committee member; Tham, Douglas, committee member; Thorburn, Andrew, committee memberHydroxychloroquine (HCQ) is currently being investigated for safety and efficacy as an autophagy inhibitor in Phase I/II cancer clinical trials. It is the only clinically-approved autophagy inhibitor for use in cancer clinical trials in the United States. HCQ is used in combination with other chemotherapeutics to augment their efficacy and has shown moderate success in treating patients with late stage cancers. While HCQ has a good safety index and shows promise as an addition to standard of care treatment regimens, it suffers from several critical pharmacologic shortcomings which we take steps to address herein. The primary issues with usage of HCQ addressed in this work are the metrics used to predict patient tumor concentration of the drug following various dosing regimens. HCQ pharmacokinetics (PK) are highly variable in patients, with no correlation between traditional plasma:tumor concentrations. The first step taken to address this problem is to characterize likely sources of interindividual variability in HCQ PK. To do this a physiologically-based pharmacokinetic (PBPK) model was developed to investigate absorption, distribution, metabolism, and toxicity (ADMET) factors relating to HCQ in a mathematical system representative of the human body. This model was developed based on physiological and biochemical parameters relevant to HCQ ADMET in mice and scaled to represent humans. The model was capable of simulating single and multiple dosing regimens in humans, that would be characteristic of a cancer clinical trial. PBPK modeling addressed variability that would be associated with the macrophysiologic scale, but intrinsic and extrinsic factors on the cellular scale needed to be further defined to strengthen the understand of HCQ PK. To investigate factors that affect cellular uptake and sub-compartment localization of HCQ, a base PK model of lysosomotropic agents like HCQ was applied. Model specific parameters were identified for a panel of four human breast cancer cell lines, and a majority of differences in cellular uptake of the drug could be attributed to differences in the relative lysosomal volume fraction of each cell line. The model was able to characterize HCQ PK under different extracellular pH conditions, and identified a positive-feedback loop, related to transcription factor EB (TFEB) activation. This feedback loop caused the cell to increase its lysosomal volume over time of exposure to HCQ, resulting in a continuous increase of HCQ concentration within the cell. Through sensitivity analysis of the model, acidic extracellular pH was identified as a critical limiting factor of HCQ uptake into cells – which is particularly important as the tumor microenvironment is physiologically acidic. HCQ concentrations in cells cultured in an acidic microenvironment are decreased up to 10-fold, which cannot be overcome without the aid of agents that neutralize this pH. Dimeric analogues of HCQ, Lys05 and DC661, have been reported to maintain potency in acidic conditions and so were investigated in a comparative context to HCQ. Lys05 and DC661 were found to behave similarly to HCQ pharmacokinetically – i.e. highly dependent on the lysosomal profile of the cell. These drugs exhibited similar kinetic uptake curves as HCQ, and also induced the lysosomal biogenesis PK feedback loop. Unlike HCQ, Lys05 and DC661 uptake was not completely inhibited by acidic extracellular pH, and they were able to maintain activity under these conditions. PK of these drugs was characterized in a murine model to investigate their potential as in vivo agents, suggesting they could maintain high concentrations for a longer duration than HCQ. Lys05 and DC661 share many pharmacologic similarities to HCQ, while not sharing significant shortcomings such as inactivity under acidic extracellular conditions suggesting they should be investigated for further application as next generation autophagy inhibitors.Item Open Access Binding of MBNL1 to CUG repeats slows 5'-to-3' RNA decay by XRN2 in a cell culture model of type I myotonic dystrophy(Colorado State University. Libraries, 2017) Zhang, Junzhen, author; Wilusz, Carol J., advisor; Wilusz, Jeffrey, advisor; Duval, Dawn, committee member; Di Pietro, Santiago, committee member; Yao, Tingting, committee memberType I myotonic dystrophy (DM1) is a multi-systemic inherited disease caused by expanded CTG repeats within the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene. The encoded CUG repeat-containing mRNAs are toxic to the cell and accumulate in nuclear foci, where they sequester cellular RNA-binding proteins such as the splicing factor Muscleblind-1 (MBNL1). This leads to widespread changes in gene expression. Currently, there is no treatment or cure for this disease. Targeting CUG repeat-containing mRNAs for degradation is a promising therapeutic avenue for myotonic dystrophy, but we know little about how and where these mutant mRNAs are naturally decayed. We established an inducible C2C12 mouse myoblast model to study decay of reporter mRNAs containing the DMPK 3' UTR with 0 (CUG0) or ~700 (CUG700) CUG repeats and showed that the CUG700 cell line exhibits characteristic accumulation of repeat-containing mRNA in nuclear foci. We utilized qRT-PCR and northern blotting to assess the pathway and rate of decay of these reporter mRNAs following depletion of mRNA decay factors by RNA interference. We have identified four factors that influence decay of the repeat-containing mRNA – the predominantly nuclear 5' 3' exonuclease XRN2, the nuclear exosome containing RRP6, the RNA-binding protein MBNL1, and the nonsense-mediated decay factor, UPF1. We have discovered that the 5' end of the repeat-containing transcript is primarily degraded in the nucleus by XRN2, while the 3' end is decayed by the nuclear exosome. Interestingly, we have shown for the first time that the ribonucleoprotein complex formed by the CUG repeats and MBNL1 proteins represents a barrier for XRN2-mediated decay. We suggest that this limitation in XRN2-mediated decay and the resulting delay in degradation of the repeats and 3' region may play a role in DM1 pathogenesis. Additionally, our results support previous studies suggesting that UPF1 plays a role in initiating the degradation of mutant DMPK transcripts. This work uncovers a new role for MBNL1 in DM1 and other CUG-repeat expansion diseases and identifies the nuclear enzymes involved in decay of the mutant DMPK mRNA. Our model has numerous applications for further dissecting the pathways and factors involved in removing toxic CUG-repeat mRNAs, as well as in identifying and optimizing therapeutics that enhance their turnover.Item Open Access Biogenic nanoparticles and their application in biological electron microscopy(Colorado State University. Libraries, 2018) Nemeth, Richard S., author; Ackerson, Christopher, advisor; Yao, Tingting, committee member; Bjostad, Louis, committee member; Peersen, Olve, committee memberInterest in nanomaterials has seen a dramatic increase over the past twenty years. In recent years many have turned toward proteins to aid in developing novel materials due to the mild reaction conditions, functionalization, and novel synthetic control of the resulting inorganic structures. Proteins have the ability to direct aggregation of inorganic nanostructures, while some enzymes are able to perform oxio/reductase activity to synthesize the materials as well. These two general properties are not always mutually exclusive and the dual function of certain proteins in nanoparticle synthesis is at the core of this work. Of all the applications for biogenic nanoparticles, generating tools for biological electron microscopy is one of the most appealing. The contrast issue, specifically with in vivo biological sample in the electron microscope has drastically limited the information obtainable by this method. An ideal biogenic nanoparticle would operate analogously to GFP in optical microscopy and contain the dual function characteristics stated above. More specifically it would have to fulfill three criteria: i) reduction of a metal precursor, ii) product size control, iii) product retention. To discover such a clonable contrast tag we must deepen our understanding of biogenic nanoparticle formation in tandem with discovering and developing novel dual function enzymes. This work encapsulates both aspects necessary for the development of a successful clonable nanoparticle for biological electron microscopy. Current biogenic synthetic methods produce nanomaterials with less desirable properties than their inorganic counterparts. Conducting fundamental research and establishing a set of rules and guidelines for biogenic methods will ultimately get us closer to mimicking the control nature has already developed. This dissertation contains 3 chapters. Chapter 2 focuses on the use of protein crystals as scaffolds for nanomaterial synthesis. Herein porous protein crystals were used to control the gold nanocluster seeded growth of gold nanorods in an attempt to help establish guidelines for biogenic nucleation controlled nanomaterial synthesis. High aspect gold nanorod products were generated from within the crystal pores. Subsequent dissolving of the crystals allowed for release of these rods from their template. The following two chapters focus on metalloid reductase nanoparticle synthesis in which we have discovered and characterized a novel selenophile bacteria. Through purification and mass spectrometry we found a glutathione reductase like enzyme to be responsible for Se nanoparticle formation. A commercially available glutathione reductase from yeast was used for Se nanoparticle formation in vitro. This mechanism was characterized and the system was assessed for potential use as a clonable tag. The native enzyme was sequenced and isolated, followed by its own characterization. Our kinetic findings suggest this enzyme is the first documented metalloid reductase due to its specificity for selenium substrates. The enzymes transportability to foreign organisms demonstrates its potential use as a clonable contrast tag for electron microscopy.Item Open Access Characterization the role of telomeric RNA (TERRA) in telomeric DNA double-strand break (DSB) repair in human ALT (telomerase independent) cells(Colorado State University. Libraries, 2019) Alturki, Taghreed Mohammed, author; Bailey, Susan, advisor; Argueso, Lucas, committee member; Wiese, Claudia, committee member; Yao, Tingting, committee memberTelomeres are specialized nucleoprotein complexes that protect natural chromosomal termini from degradation and prevent their detection as of DNA damage. Therefore, telomeres play critical roles in maintaining genomic stability. Telomeres are composed of tandem arrays of conserved repetitive sequences (TTAGGG in vertebrates), bound by a suite of proteins collectively termed "shelterin". Shelterin proteins are essential for telomere length regulation and end-capping structure/function. Due to their repetitive nature and together with telomeres possessing an abundance of heterochromatic marks, telomeres have long been regarded as silenced, non-transcribed features of the genome. The relatively recent discovery of telomeric RNA (TElomere Repeat- containing RNA; TERRA) opened many new avenues of investigation. TERRA is a long, noncoding RNA (lncRNA) that serves a structural role at telomeres, as well as function in regulation of telomere length and telomerase activity, the specialized reverse transcriptase capable of elongating telomeres de novo. Further, TERRA participates in telomeric recombination in tumors that maintain telomere length in a telomerase independent fashion via the Alternative Lengthening of Telomeres (ALT) pathway. Emerging evidence also supports telomeric "DNA- TERRA hybrids" as indispensable for end protection and capping function; e.g., RNA interference mediated depletion of TERRA induced telomeric DNA damage responses (DDRs) and aberrations. Thus, TERRA participates in facilitating telomeric recombination and in preventing inappropriate telomeric DNA damage responses. We hypothesized that TERRA plays a critical role in the repair of telomeric DNA damage. To address the intriguing possibility that TERRA plays a role in the telomeric DNA damage response, we evaluated the colocalization of TERRA and γ-H2AX, a well-accepted marker of double-strand breaks (DSBs), at broken telomeres in human Osteosarcoma U2OS-ALT cells in different phases of the cell cycle. To test our hypothesis, we generated U2OS-ALT cells that stably expressed FUCCI green fluorescent signals to label cells in G2 phase. Telomeric DSBs were then induced in FUCCI-U2OS cells utilizing the ENT endonuclease fused to Telomere Repeat Factor 1 TRF1 (ENT-TRF1), and validated via colocalization of telomeres with γ-H2AX-FLAG. Forty-eight hours following transfection, FUCCI-U2OS cells were also treated with EdU to label cells in S phase. Cells negative for both FUCCI and EdU identified cells in G1 phase. Using this powerful strategy to distinguish cells in G1, S and G2 phases of the cell cycle, we showed that FUCCI-U2OS cells accumulate in G2 phase following transient transfection with ENT-TRF1. We validated that expression of ENT-TRF1 generates telomeric DSBs in U2OS-ALT cells through detection of telomere- γ-H2AX-FLAG colocalization events. Importantly, our data revealed that telomeric DSB induction triggers enrichment of TERRA in G2 phase. Taken together, these observations suggested that TERRA is increased in cells transfected with ENT-TRF1; i.e., in U2OS cells harboring telomere-specific DSBs. TERRA recruitment to telomeric DSB damage sites in G2 was validated by assessing co-localization between TERRA and ENT (FLAG). Similarly, TERRA recruitment to telomeric DSBs in G1/S was also evaluated. Futhermore, non-denaturing Telomere DNA FISH was employed to visualize G-rich and C-rich single-stranded (ss)telomeric DNA. Treatment of U2OS ENT transfected cells with Rnase A and Rnase H to remove TERRA, uncovered elevated levels of resected 5' C-rich (ss)telomeric DNA (complementary TERRA sequence), suggesting a potential role for TERRA in protecting resected telomeric DNA prior to cells entering G2 phase where Homologous Recombination (HR)-mediated elongation/repair would be possible. Consistent with published reports, telomeric DSBs were also significantly induced in cells transfected with TRF1-only (positive control). However, although TERRA co-localized with FLAG/broken telomeres, resected (ss)telomeric DNA was not detected upon removal of TERRA. Therefore, our results further support induction of telomeric DSBs with overexpression of TRF1, and additionally indicate that they are repaired via a different pathway than those induced by ENT, potentially alternative End Joining (alt-EJ), as previously proposed. In conclusion, our work revealed for the first time that the telomeric RNA TERRA, is enriched specifically at telomeric DNA DSB sites in U2OS (ALT) cells.Item Open Access Engineering and evolving helical proteins that improve in vivo stability and inhibit entry of Enfuvirtide-resistant HIV-1(Colorado State University. Libraries, 2019) Walker, Susanne N., author; Kennan, Alan, advisor; Yao, Tingting, committee member; McNally, Andrew, committee member; Paton, Robert, committee memberMethods for the stabilization of well-defined helical peptide drugs and basic research tools have received considerable attention in the last decade. Enfuvirtide is a 36-residue chemically synthesized helical peptide that targets the viral gp41 protein and inhibits viral membrane fusion. Enfuvirtide-resistant HIV, however, has been prolific, and this peptide therapy requires daily injection due to proteolytic degradation. In this dissertation I have developed a method for stabilizing helical peptide therapeutics termed helix-grafted display proteins. These consist of the HIV-1 gp41 C-peptide helix grafted onto Pleckstrin Homology domains. Some of these earlier protein biologics inhibit HIV-1 entry with modest and variable potencies (IC50 190 nM - >1 μM). After optimization of the scaffold and the helix, our designer peptide therapeutic potently inhibited HIV-1 entry in a live-virus assay (IC50 1.9-12.4 nM). Sequence optimization of solvent-exposed helical residues using yeast display as a screening method led to improved biologics with enhanced protein expression in Escherichia coli (E. coli, a common bio-expression host), with no appreciable change in viral membrane fusion suppression. Optimized proteins suppress the viral entry of a clinically-relevant double mutant of HIV-1 that is gp41 C-peptide sensitive and Enfuvirtide-resistant. Protein fusions engineered for serum-stability also potently inhibit HIV-1 entry.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 Factors and mechanisms of archaeal transcription termination and DNA repair(Colorado State University. Libraries, 2022) Marshall, Craig, author; Santangelo, Thomas J., advisor; Peersen, Olve, committee member; Wilusz, Carol, committee member; Yao, Tingting, committee memberRNA synthesis by RNA polymerase (RNAP) is an essential process and must be properly regulated both temporally and spatially to ensure cellular health in dynamic environments. Regulation of RNA synthesis in response to internal and environmental stimuli is typically achieved through interactions with RNAP at all stages of the transcription cycle- initiation, elongation, and termination. While studies of transcription initiation and elongation have identified multiple regulatory transcription factors and defined mechanisms, only a handful of protein factors able to terminate transcription have yet been described, and the general mechanism of transcription termination is still highly debated. We previously identified the first two factors capable of terminating transcription elongation complexes (TECs) in Archaea from the genetically tractable Thermococcus kodakarensis, and use both factors as models to explore the molecular mechanisms involved in collapse of the TEC. The Factor that terminates transcription in Archaea (FttA), a close homolog of the human CPSF subunit CPSF73, is completely conserved throughout Archaea, and appears to act analogously to the bacterial termination factor Rho, terminating transcription after the uncoupling of transcription and translation at the end of protein coding genes. We employed a novel genetic screen to verify the role of FttA in the polar repression of transcription, a phenomenon specific to regulation of genes contained within operons in prokaryotes. Eta, a euryarchaeal-specific superfamily 2 (SF2) helicase, appears to terminate transcription in a more specialized context, potentially terminating transcription of TECs arrested at sites of DNA damage while concurrently recruiting appropriate DNA repair enzymes, akin to the bacterial termination factor Mfd. A structure-function study of Eta employing select mutations derived from a crystallographic structure was conducted to elucidate the Eta-TEC contacts and various activities of Eta required for Eta-mediated termination. Further, many efforts were directed at establishing a role of Eta as an archaeal transcription-repair coupling factor (TRCF), and while this was not achieved, a state-of-the-art next-generation sequencing based approach to monitor nucleotide excision repair (NER) and the sub pathway transcription-coupled repair (TCR) genome-wide was developed and verified in E.coli. The work in this dissertation adds valuable insight to multiple fields of research. First, exploration into the mechanism of Eta-mediated transcription termination reveals a potential shared susceptibility of core RNAP subunits to transcription termination while elucidating activities of SF2 helicases- enzymes which are ubiquitously distributed in multiple essential cellular pathways. Second, our genetic screen identifies FttA as the archaeal polarity factor, shedding light on functions of an ancestral factor indispensable in mammalian transcription termination pathways. Establishment of the novel RADAR-seq/RNA-seq measurement of NER genome-wide will likely prove instrumental in future studies of archaeal DNA repair, and potentially presents a new paradigm in research of eukaryotic-like NER by use of Archaea as a advantageous model organism.Item Open Access Genomics and transcriptomics of the molting gland (Y-organ) in the blackback land crab, Gecarcinus lateralis(Colorado State University. Libraries, 2016) Martin, Lindsay, author; Mykles, Donald L., advisor; Garrity, Deborah M., committee member; Yao, Tingting, committee memberMolting is required for growth and development in crustaceans. In the blackback land crab Gecarcinus lateralis, molting is stimulated by ecdysteroids, hormones produced in the Y-organ (YO). Throughout the molting cycle, the YO demonstrates phenotypic plasticity. The phenotypic plasticity is correlated with the stages of the molt cycle, during which YO ecdysteroid production varies. During intermolt, the longest stage of the molt cycle, the circulating ecdysteroid titers are low and molting is suppressed. In preparation for molting, the YO increases ecdysteroid production during premolt. Circulating ecdysteroids continue to rise, dropping right before the ecdysis and remaining low in the subsequent postmolt period. During the molt cycle, the YO's sensitivity to inhibitory cues also varies, which contributes to ecdysteroid fluctuations. To better understand how changes in gene expression modulate the YO's phenotypic plasticity, a YO transcriptome from five molt stages was generated. Using over 5.6 million reads from Illumina, 229,278 contigs were assembled to comprise the reference transcriptome. By comparing expression levels of the transcripts between the molt stages, 13,189 unique differentially expressed contigs were identified in G. lateralis. Based on differential expression, insect hormone biosynthesis and oxidative phosphorylation pathways were enriched, validating the YO transcriptome identity. Using GO enrichment, MAP kinase was identified as a possible candidate gene for regulating YO ecdysteroid synthesis. To complement and validate the transcriptome, claw muscle genomic DNA was sequenced and assembled using 2.6 million reads. 375,152 scaffolds ≥ 500 bp were built, with an N50 of 1,841 bp. Using k-mer frequencies, the genome size was estimated to be 3.07 Gb, similar to mammalian vertebrates. The median gene size of G. lateralis was approximated to be 6,300 bp; the disparity between the median estimate and the N50 prohibited further computational analysis. Genome scaffolds were sufficient in length for manual comparison. Alignment of the transcriptome and genome sequences of the Rheb gene showed 100% nucleotide alignment in the open reading frame, and extended the sequence by 7.7 fold, including the identification of four introns. The sequence comparison validated both genome and transcriptome assemblies and extended the gene sequence. Next-generation sequencing provided us with a global perspective of molecular variations within the YO throughout the molt cycle. We hypothesize variations in gene expression regulate YO phenotypic plasticity by varying ecdysteroid production. YO transitions throughout molting are essential for regulation. YO activation and commitment, both corresponding to increased ecdysteroids, are required to induce ecdysis. YO repression, during which circulating ecdysteroid titers are low, is needed to prevent precocious molting. Identifying changes in gene expression and key regulatory elements correlating with variations in YO phenotype will increase our understanding of molt cycle regulation, which is critical for crustacean development, growth, and repair.Item Embargo Identification of the TPC2 interactome reveals TSPAN10 and OCA7 as key players in the biogenesis of melanosomes(Colorado State University. Libraries, 2023) Beyers, Wyatt, author; Di Pietro, Santiago, advisor; Amberg, Gregory, committee member; Santangelo, Thomas, committee member; Yao, Tingting, committee memberMany specialized cell types gain their function through the generation of specialized organelles that make or store cell-specific biomolecules. A group of specialized organelles are called Lysosome Related Organelles (LROs) because they are derived from Golgi and endolysosomal compartments and their biogenesis depends on trafficking pathways and machinery shared with lysosomes, many have protein contents partially overlapping with lysosomes, and typically have low pH during stages of their maturation. One well-studied model LRO is the melanosome, the organelle in melanocytes and retinal pigment epithelial cells responsible for melanin pigment production in the eyes, hair, and skin, and defects in melanosome function lead to pigmentation diseases such as oculocutaneous albinism. Melanosome biogenesis is a complex process requiring ubiquitous membrane trafficking machinery to be repurposed for the differentiation of melanosomes from other endosomal compartments and specific delivery of melanosome synthesizing enzymes, Tyrosinase and Tyrosinase Related Proteins 1 and 2. Furthermore, correct melanosome maturation requires remodeling of the melanosome membrane, recycling of membrane trafficking machinery, generation of intraluminal amyloid fibrils with the correct structure for melanin packaging, tight pH control, as well as coordinated influx of copper, zinc, tyrosine, and cysteine for melanin synthesis. These processes require the temporospatial coordination of at least 100 known proteins, and probably dozens more remain undiscovered. In this dissertation, I present the discovery of new proteins involved in the biogenesis of melanosomes. Proximity biotinylation by promiscuous biotin ligase enzymes followed by biotin pulldown and mass spectrometry has emerged as a powerful technique for the identification of protein-protein interactions, protein complex determination, and identification of organelle membrane proteomes. I utilized the melanosome localized cation channel, TPC2, genetically fused with the BioID2 biotin ligase, to identify proteins in proximity to TPC2 at the cytosolic surface of melanosome membranes of MNT1 melanoma cells. Through mass spectrometry analysis of biotinylated proteins enriched through Streptactin pulldown, a TPC2 proximity interactome was identified comprising over 200 proteins. Subsequent fluorescence confocal microscopy analysis confirmed several proteins, including PLD1, SV2A, TSPAN10, and OCA7/C10orf11/LRMDA all colocalize highly with TPC2-EGFP, confirming they are new melanosome proteins. In follow-up functional studies, TSPAN10 and OCA7 were confirmed to be involved in pigmentation, with severe melanin depletion in TSPAN10 or OCA7 knockout MNT1 cells. TSPAN10 and OCA7 both influence the processing of the PMEL protein, which is required for correct melanosome ultrastructure and for melanin packaging. Further investigation of TSPAN10 revealed it functions with the pigmentation associated metalloproteinase, ADAM10, and is required for ADAM10 expression and localization to endosomal compartments. On the other hand, OCA7 was found to work with the melanosome localized Rab proteins, Rab32 and Rab38, and regulates the pH of melanosomes. Thus, the newly defined TPC2 interactome in melanocytes was proven as a valuable dataset that robustly identifies new melanosome proteins. Chapter 1 of this dissertation provides a broad overview of membrane trafficking pathways, as well as a synopsis of the specific proteins and pathways involved in melanosome biogenesis and homeostasis. Chapter 2 investigates the TPC2 interactome in MNT1 cells, and it characterizes TSPAN10 as a new player in melanosome biogenesis. Finally, Chapter 3 provides a characterization of the OCA7 protein associated with oculocutaneous albinism type 7 and investigates OCA7 function using a newly generated OCA7 knockout cell model.Item Open Access In vitro and in vivo characterization of RAD51AP1 in homologous recombination DNA repair(Colorado State University. Libraries, 2020) Pires, Elena, author; Wiese, Claudia, advisor; Argueso, Lucas, committee member; Thamm, Douglas, committee member; Yao, Tingting, committee memberCancer embodies a large group of diseases that is responsible for illness and deaths in millions of people annually around the world. Many tumors arise due to accumulated, unrepaired damage and alterations to genes, from endogenous or exogenous sources of DNA damage. Among the DNA lesions associated with cancer, DNA double-strand breaks (DSBs) are considered the most dangerous and require coordinated and conserved machinery to prevent unfavorable consequences, such as apoptosis and cancer-causing mutations. One crucial DNA repair pathway for mending DSBs and maintaining genome integrity is homologous recombination (HR) DNA repair. This relatively error-free mechanism employs the RAD51 recombinase and involves the joining of homologous DNA strands to restore lost DNA sequence information at the damage site. RAD51-Associated Protein 1 (RAD51AP1) is a key protein that interacts with RAD51 and stimulates its activities during HR. Nonetheless, there are knowledge gaps in understanding how this HR player functions mechanistically and in vivo for protection against DNA damage. To test our overarching hypothesis that disrupted RAD51AP1 inhibits cellular and organismal protection against spontaneous or induced DNA damage, we assessed the biochemical and biological functions of RAD51AP1 through three main avenues of study: its role in the context of chromatin, the effects of its post-translational modifications in cells, and the penalties of its loss in an animal system. This dissertation describes findings from these pursuits that have not been previously characterized and offers new insights into RAD51AP1's functions in vitro and in vivo. At the start of this dissertation, our first objective was to define key attributes of RAD51AP1 in the HR reaction by further characterizing the DNA binding properties of recombinant human RAD51AP1. Using the electrophoretic mobility shift assay, we found that RAD51AP1 avidly associates with both naked and chromatinized double-stranded (ds)DNA. Deletional and mutational analyses were used to further define the chromatin-binding region in RAD51AP1, which occurs within its C-terminal DNA binding domain. Two post-translational modification (PTM) sites, which undergo phosphorylation at S277 and S282 (in isoform 2) and lie within its C-terminal DNA binding region, were also evaluated and showed decreased affinity to chromatinized dsDNA. These results unveil a novel RAD51AP1 interaction with chromatin DNA. Next, we further assessed these PTMs in regard to their impacts on RAD51AP1 function and HR capability in cells facing spontaneous or induced DNA damage. Using RAD51AP1 KO cells expressing phosphorylation mimic (S2D) or non-phosphorylatable (S2A) mutants, we found that S2D expressing cells behaved similarly to wild-type expressing cells. Notably, S2A expressing cells were significantly compromised in their growth, cell survival, cell cycle progression, and HR kinetics. The results of these studies provide an important role for PTMs that affect RAD51AP1's functions during HR. To examine the role of RAD51AP1 in providing protection against DNA damage in an animal system, we utilized a recently available mouse knockout model to evaluate the impacts of Rad51ap1 deletion from spontaneous DNA damage. Given the role of RAD51AP1 in meiotic HR and its high expression in murine testes, we specifically monitored fertility ratios, spermatogenesis in testes cross sections, and meiosis via synaptonemal complex formation. We found that Rad51ap1 heterozygous mice do not breed in a Mendelian pattern. Furthermore, while synaptonemal complex formation was not impaired in Rad51ap1 KO mice, advanced stages of spermatogenesis were impacted, suggestive of a biological role for RAD51AP1 in maintaining the fidelity of this process. Collectively, the results of these studies characterizing the in vitro and in vivo roles of RAD51AP1 provide new insights into this important HR player. For the first time, we reveal a new association between RAD51AP1 and chromatinized dsDNA and propose a model integrating this interaction within the HR reaction, when homology search and hetero-duplex formation after presynaptic filament formation occurs. Additionally, previously uncharacterized PTMs were assessed functionally in cells, and we unveil that the lack of these PTMs negatively impacts cells against spontaneous and induced DNA damage. Lastly, our studies on the biological effects of Rad51ap1 loss in a recently available Rad51ap1 KO mouse describe a novel role of Rad51ap1/RAD51AP1 during late spermatogenesis in an animal system for the first time. Ultimately, by understanding the mechanisms and biology of this important HR protein, this knowledge can guide the optimization of treatments for cancers that exploit DNA repair factors as well as help us comprehend how this factor protects against DNA damage in mammals.Item Open Access Quantifying ubiquitin dynamics(Colorado State University. Libraries, 2019) Bollinger, Sarah A., author; Cohen, Robert E., advisor; Peersen, Olve, committee member; Yao, Tingting, committee member; Prenni, Jessica, committee member; Alan, Kennan, committee memberUbiquitin (Ub) is a small protein that is frequently attached to other proteins as a post-translational modification (PTM) to elicit a new function, cellular localization, or otherwise modulate the activity of the substrate protein. Ub addition and removal serves as a signal for proteasome degradation, regulation of cell division, gene expression, membrane and protein trafficking and signaling in a multitude of stress response mechanisms. Defects in ubiquitination or deubiquitination have been linked to cancer onset and progression, muscle dystrophies, and disorders in inflammation and immunity; these findings further highlight the critical processes regulated by Ub. Due to its high demand, cellular Ub levels are highly regulated, such that the abundance of free Ub is above a threshold enabling new ubiquitination events, a critical part of normal cell function and survival. Due to the high demand on the cellular free Ub pool to supply substrate for thousands of ubiquitination reactions, it is tightly regulated in many ways. Our knowledge of Ub homeostasis has not advanced, likely due to the lack of accurate, sensitive methods for pool quantitation that can be performed routinely. Here, a method is presented that utilizes a high affinity free Ub binding protein to quantify cellular pools of Ub after a series of treatment protocols. The methods can be performed within a day and are amenable to high throughput applications. Using these methods, the Ub pool distributions of cells under conditions such as proteasome inhibitor and heat stress were assessed. However, this assay will only report the steady-state concentration of Ub in each pool; it provides no information about the rate of movement through them. The rates of competing ubiquitination and deubiquitination or degradation reactions determine the steady-state level of every Ub-protein conjugate; however, measurement of the rate of Ub movement these conjugates remains a challenge. Thus, the relative contributions of conjugation and disassembly rates in cellular responses to different signals are rarely known. Moreover, even though the concentration of a particular Ub-protein conjugate may appear unchanged, the flux of Ub through that conjugate might change dramatically. To address these deficits in our understanding of ubiquitination, we have developed a method to label Ub and follow its movement through conjugation pathways that we call SILOW or Stable Isotope Labeling with ¹⁸O-Water. Our method is applicable to both yeast and mammalian cells, does not perturb cellular physiology in any way and can be used with conventional proteomics methods. SILOW permits rapid changes in Ub flux to be evaluated over short times across hundreds of sites within the human cell proteome to reveal the intracellular dynamics of Ub-conjugation in specific Ub-Ub linkages of polyUb compared with Ub-protein linkages of histones.Item Open Access SARS-CoV-2 viral RNA biology and its impact on the infected cell(Colorado State University. Libraries, 2023) Altina, Noelia H., author; Wilusz, Jeffrey, advisor; Argueso, Lucas, committee member; Geiss, Brian, committee member; Perera, Rushika, committee member; Yao, Tingting, committee memberThe fine-tuning of the replication and transcription of RNA viruses often requires the interaction of viral RNAs with cellular RNA binding proteins. This project addresses fundamental knowledge gaps on the molecular mechanisms that underlie SARS-CoV-2 gene expression, regulation, and viral RNA-host interactions. After infection, SARS-CoV-2 generates a large set of sub-genomic mRNAs, each containing an identical ~70 base 'leader' region in their 5'UTR (from position 14 to position 75 in RefSeq NC_045512) and a 229 base region at the 3'UTR (from position 29,675 to position 29,903 in RefSeq NC_045512) generated by discontinuous transcription. The accumulation of a considerable amount of these leader/3'UTR regions during the infection represents a possible sink for cellular RNA binding proteins. We demonstrated that PTBP1, a cellular protein involved in the regulation of alternative splicing, binds to the SARS-CoV-2 leader region. SARS-CoV-2 infection critically impacted the splicing of several cellular pre-mRNAs that are normally regulated by PTBP1. Mechanistically, we suggest that SARS-CoV-2 sequesters and influences the re-localization of shuttling splicing factors like PTBP1 from the nucleus to the cytoplasm resulting in significant effects on the host cell splicing machinery leading to changes in cellular mRNA splicing patterns during SARS-CoV-2 infection. Given the current extensive interest in epigenetic methylations of both cellular and viral RNAs, our study also explored the role of post-transcriptional RNA modifications on viral mRNAs. We demonstrated that SARS-CoV-2 can usurp the cellular enzyme, namely PCIF1, to place the m6Am modification at the cap proximal position in its mRNAs. This double methylation is usually found on all host mRNAs that initiate with an adenosine residue, and thus SARS-CoV-2 likely installs this modification on its mRNAs to avoid host immune recognition. Interestingly, we also discovered that capping and m6Am modification are tightly regulated throughout the infection. The highest levels of these 5' end RNA modifications were observed at 12 hours post infection, correlating with the full establishment of viral gene expression in infected cells. These findings indicate that 5' end modification of SARS-CoV-2 transcripts is not simply a default process but rather undergoes unanticipated regulation throughout the infection. Collectively, the data presented provide not only new insights into the complex interactions that SARS-CoV-2 has with the RNA biology of the cell during infection, but also identify attractive potential targets for developing novel anti-coronavirus drugs to treat future emerging coronavirus diseases.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 Structural and functional insight into kinetochore protein CENP-N and its interaction with CENP-A nucleosome(Colorado State University. Libraries, 2018) Zhou, Keda, author; Luger, Karolin, advisor; Yao, Tingting, committee member; Deluca, Jennifer, committee member; Bailey, Susan, committee memberProper chromosome segregation during mitosis is one of the most important processes to ensure genome integrity. During this process, the microtubules are captured by a multi-unit complex called kinetochore. The kinetochore is assembled specifically at centromere through recognizing nucleosomes containing the histone H3 variant CENP-A. CENP-N and CENP-C are the only two kinetochore proteins that specifically recognize CENP-A nucleosomes. There are about 1 in 25 nucleosomes that contain CENP-A at the centromere. Therefore, how these two proteins 'ignore' the abundant H3 nucleosomes to interact selectively with a handful of centromeric CENP-A nucleosomes has important implications for genome stability during cell division. To obtain deep insight into the mechanism behind this, I solved the structure of CENP-A nucleosome in complex with CENP-N by single particle cryo electron microscopy (cryo-EM) at 4 Å. Through charge and space complementarity, the unique "RG" loop on CENP-A is decoded by CENP-N. CENP-N also engages in extensive interactions with a long segment of the distorted nucleosomal DNA double helix. These interactions were validated in vitro and in vivo.The DNA ends of CENP-A nucleosome which are disordered in the crystal structure are mostly visible in the cryo-EM structure when it is in complex with CENP-N. By micrococcal nuclease digestion assay, the CENP-A nucleosome DNA ends are shown to be less flexible when CENP-N is presented in solution, which is consistent with structural study. Since CENP-N does not interact with DNA ends directly, the less dynamics on the DNA ends indicate a more stable nucleosome. By quantitative electrophoretic mobility shift assay (EMSA) and electron microscopy, the stabilizing effect of CENP-N on CENP-A nucleosome was confirmed in vitro. However, this effect was not significant in vivo, which indicates that the CENP-A nucleosome stability in vivo is determined by multiple factors. Besides the change on DNA ends of CENP-A nucleosome, the orientation of H4 N-terminal tail is altered due to its interaction with CENP-N, with important implications for the multiple biological processes involving the H4 N-terminal tail, especially with respect to the formation of chromatin higher order structure The structural and functional studies in this thesis shed light on how CENP-N ensures that the kinetochore assembles specifically at the centromere.Item Open Access Structural insights into chromatin assembly factor 1 and nucleosome assembly mechanism(Colorado State University. Libraries, 2018) Gu, Yajie, author; Luger, Karolin, advisor; Bailey, Susan, committee member; Peersen, Olve, committee member; Yao, Tingting, committee memberThe eukaryotic genome is highly packed with histones to form chromatin. The basic building unit of chromatin is the nucleosome, which consists of a histone octamer core, wrapped by 147 base pairs of DNA. The nature of the nucleosome structure presents a formidable barrier for DNA-related processes, especially for DNA replication. Therefore, the chromatin will undergo dramatic dynamics during replication, involving disassembly of old nucleosomes and distribution of both new and old histones to form nucleosomes onto both daughter DNA strands. These nucleosome dynamics suggest a challenge for the maintenance of histone density and epigenetic inheritance in the wake of DNA replication. Chromatin Assembly Factor-1 (CAF-1) is a conserved histone chaperone that directly interacts with the replication machinery via the polymerase processivity factor PCNA, and is involved in assembling nucleosomes behind the DNA replication fork. CAF-1 is essential for multicellular eukaryotes, while deletion of CAF-1 in yeast is not lethal, but results in increased sensitivity to DNA damage and aberrant telomeric silencing. Despite the significance of this histone chaperone, the structural organization of this complex remains largely unknown, and thus the mechanism underlying CAF-1-mediated nucleosome assembly is elusive. In this study, we identified the key peptides involved in CAF-1 subunit assembly by performing HDX-MS analysis followed by site-directed mutagenesis studies, which were confirmed by yeast genetic studies. This structural information allows us to further characterize functional domains within CAF-1, and provides unprecedented details for future structural studies using crystallization and/or cryo-EM. This work also shows how histones H3-H4 are bound by CAF-1, and how this histone binding regulates the nucleosome assembly activity by CAF-1. We also show that DNA is acting as a bridge to bring two histone-bound CAF-1 together, thereby promoting (H3-H4)2 tetramer formation as well as the tetramer hand-off between CAF-1 and DNA, resulting in the formation of tetrasome ((H3-H4)2 tetramer wrapped with DNA), the initial step for nucleosome assembly. Overall, this study provides a mechanistic explanation for efficient nucleosome assembly by CAF-1 following DNA replication, and highlights a direct nucleosome assembly mechanism by a histone chaperone for the first time. Moreover, the concerted mechanism of CAF-1-mediated nucleosome assembly suggests that two H3-H4 dimers are brought together right before the (H3-H4)2 tetramer deposition onto DNA, shedding light on the future directions of epigenetic maintenance regulation during replication.Item Open Access TATA binding protein dynamics within the cellular chromatin landscape(Colorado State University. Libraries, 2013) Yearling, Marie N., author; Stargell, Laurie A., advisor; Luger, Karolin, committee member; Nyborg, Jennifer K., committee member; Yao, Tingting, committee member; Slayden, Richard A., committee memberRNA polymerase II (RNAPII) is a twelve subunit enzyme that catalyzes messenger (mRNA) in eukaryotic organisms. A number of essential transcription factors associate with RNAPII to form the pre-initiation complex (PIC) at gene promoter regions. TATA binding protein (TBP) is one member of the transcription machinery indispensable for transcription. At some genes, the formation of the PIC correlates strongly with the transcription output (Ptashne, 2005). These genes have a low occupancy of TBP and other PIC components prior to activation. Upon activation, these factors assemble onto the promoter and transcriptional output increases. Genes that become active upon PIC formation are termed recruitment regulated because their transcription is regulated at the level of recruitment of the PIC to the promoter. While recruitment of the PIC is required for transcription, in many cases promoter-occupancy is not correlated with transcription output. Post-recruitment gene regulation has been conserved across evolution from prokaryotes to humans (Choy et al., 1997; Guenther et al., 2007). At these genes, TBP and RNAPII and other transcription-related factors occupy the promoter region regardless of whether transcription is occurring. Upon gene activation, the occupancy increases only slightly when compared to the increase in transcript level. These genes are described as being poised. At poised genes, these transcription proteins constitutively occupy the promoter region, but it is unknown if the promoter interaction is stable or dynamic. One principal objective of my work was to investigate TBP-promoter dynamics at the poised CYC1 gene in yeast. Due to the genetic and biochemical amenability of the yeast system, studies of the transition of poised CYC1 gene to the active form have provided key insights into the sophisticated molecular requirements involved in this post-recruitment process. To describe the dynamics of the transcription complex bound at the CYC1 promoter I developed a TBP exchange assay. The results suggest that the TBP within the RNAPII transcription complex exists in a relatively stable configuration at the poised gene prior to activation. Upon induction, TBP-promoter dynamics increased at the CYC1 gene promoter. Rapid exchange during activated transcription was also observed at other genes, including at recruitment regulated gene promoters. Overall, we found rapid TBP-promoter exchange to be associated with active transcription. From my findings I propose a model where frequently clearing the promoter offers a functional advantage to support activated transcription.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'.