Theses and Dissertations

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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 member
Deoxyribose 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.
Open Access
Aurora A kinase phosphorylates serine 62 on Hec1 to affect mitotic kinetochore microtubule interactions
(Colorado State University. Libraries, 2024) Sparrow, Sarah, author; DeLuca, Jennifer, advisor; Markus, Steven, committee member; Bailey, Susan, committee member
The Hec1 protein plays an important role in ensuring successful chromosome segregation during cell division. Its 80 amino acid, unstructured, "tail" region is critical for kinetochore-microtubule attachment regulation, which is mediated through Aurora kinase phosphorylation. At least nine phosphorylation target sites within this domain have been identified, including the recently confirmed target site, serine 62 (S62). However, the functional significance of phosphorylation of this residue remains elusive. Here, we selectively target Aurora A and Aurora B kinase protein activities using the inhibitors MLN8054 and ZM447439, respectively, and study their effects on the dynamics of serine 62 phosphorylation in the Hec1 tail. Utilizing immunofluorescence, we demonstrated that inhibition of Aurora A kinase activity leads to a significant reduction in phosphorylation levels at serine 62. Additionally, using phospho-null mutants, we studied the effect of serine 62 phosphorylation on the creation of stable, tension-generating kinetochore-microtubule attachments by measuring the distance between sister kinetochores. Our findings reveal that alterations in serine 62 phosphorylation status result in subtle changes in interkinetochore distances showcasing the functional relevance of this phosphorylation event in regulating kinetochore-microtubule attachments. Furthermore, under conditions of nocodazole-induced mitotic arrest, we observe a marked decrease in phosphorylation at serine 62 suggesting a microtubule dependent regulation of this phosphorylation. These findings provide evidence supporting the role of Aurora A kinase in phosphorylating serine 62 of the Hec1 tail and shed light on the regulation of this critical post-translational modification during mitosis.
Open Access
Investigating mitotic vulnerabilities that arise upon oncogenic cell transformation
(Colorado State University. Libraries, 2020) Shirnekhi, Hazheen K., author; DeLuca, Jennifer G., advisor; Markus, Steven, committee member; Nishimura, Erin, committee member; Bailey, Susan, committee member
During mitosis, cells must accurately divide their duplicated chromosomes into two new daughter cells. This process is highly regulated and much of this regulation is centered around kinetochores. Kinetochores are large proteinaceous structures built upon centromeric heterochromatin that must form stable, load-bearing attachments to microtubules (MTs) emanating from the spindle poles. Failure to undergo high-fidelity cell division can result in aneuploidy and even progress to continued mis-segregation in a phenomenon known as chromosome instability (CIN). As aneuploidy results from defective pathways in mitosis, it is important to characterize the changes cancer cells exhibit in their mitotic machinery, with the goal of identifying targets for therapeutic potential. Here, we utilize a human papillomavirus cell culture model system to determine how expression of E6 or E7, two viral transforming proteins, influences mitosis. We find that E6-expressing cells exhibit a weakened spindle assembly checkpoint (SAC) and an increased incidence of pole-associated chromosomes. This combination of mitotic errors allows some of these cells to exit mitosis in the presence of improper kinetochore-MT attachments, leading to aneuploid daughter cells. Defective mitotic processes in cancer cells provide a means of differentiating them from healthy cells, which may be important in developing new effective cancer therapeutics. Through two independent cancer lethality screens, the mitotic proteins BuGZ and BubR1 were identified as essential for Glioblastoma Multiforme cancer cell survival but dispensable for healthy neural cell survival. We characterize the important chaperone-like role BuGZ plays in mitosis to examine its apparent dispensability in healthy cells. BuGZ aids in the kinetochore loading of Bub3, which in turn is needed for the kinetochore loading of proteins with important roles in kinetochore-MT attachment and in spindle assembly checkpoint signaling. BubR1's cancer lethality has also been previously described in Glioblastoma cells. BubR1 is needed to recruit the PP2A phosphatase to kinetochores, where it stabilizes kinetochore-microtubule attachments. We identify the HEC1 tail as a substrate for the BubR1-recruited population of PP2A, and we demonstrate that kinetochore-microtubule attachment defects in BubR1 depleted cells can be rescued with a phospho-null HEC1 mutant. This work identifies important changes in the mitotic machinery of transformed cells, providing potential pathways to target for therapeutics that may apply to many different cancers.
Open Access
Multiple domains in the NDC80 complex are required for generating and regulating kinetochore-microtubule attachments in mitosis
(Colorado State University. Libraries, 2012) Sundin, Lynsie, author; DeLuca, Jennifer G., advisor; Bamburg, James, committee member; Curthoys, Norman, committee member; Bailey, Susan, committee member
The goal of mitosis is to accurately segregate chromosomes into two new daughter cells. It is critical that this process occurs appropriately because the consequences of chromosome nondisjunction or missegregation are severe, most notably birth defects and cancer. Kinetochores are built at the centromeric region of mitotic chromosomes and serve several functions during mitosis. First, the kinetochore is the physical scaffold at which microtubule binding sites are built. Second, kinetochores regulate the strength of the attachments to microtubules to ensure proper chromosome movements. Finally, the kinetochore is the origin of a soluble 'wait anaphase' signal that prevents premature entry into anaphase. Together these functions culminate with chromosome alignment at the spindle equator of a cell, ultimately resulting in accurate chromosome segregation in anaphase. While the kinetochore can be considered the director of kinetochore-microtubule attachment, microtubules drive the process of cell division by providing the force behind chromosome movements. The mechanism of kinetochore-microtubule attachment remains elusive as kinetochores must generate and maintain connections to microtubules that are constantly polymerizing and depolymerzing. Extensive studies into this process have revealed that the KMN (KNL1 complex, MIS12 complex, and NDC80 complex) network, a supercomplex of proteins at the outer kinetochore, comprises the core microtubule binding site in cells. As part of this network the NDC80 complex has been an attractive candidate as an essential part of the microtubule binding machinery. Here we have used a combination of in vivo, in vitro, and in silico methods to characterize three discrete domains of the NDC80 complex that each contribute to the process of kinetochore-microtubule attachment in distinct ways. Our data have elucidated some of the molecular details of how kinetochore-microtubule attachments are both generated and regulated. We show that the Hec1 CH domain is absolutely required for kinetochore-microtubule attachment. Our data suggest that the Hec1 CH domain makes direct contacts with microtubules, while the CH domain of Nuf2 does not, indicating functionally distinct roles for these protein domains in mitosis. We characterize the Hec1 loop domain, demonstrating that it is required for stable kinetochore-microtubule attachments and mitotic progression. Our data suggest that the Hec1 loop domain is required to recruit accessory proteins to the kinetochore during mitosis. Furthermore, we show that kinetochore-microtubule attachment strength is highly sensitive to small changes in Hec1 tail phosphorylation. Finally we also demonstrate that incremental phosphorylation of the Hec1 tail domain is a primary mechanism of regulating kinetochore-microtubule attachment strength. Together our data highlight the diverse functions of a single kinetochore component and implicate the NDC80 complex as the principle site for direct binding to microtubules and as a site of regulation for these attachments.
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 member
The 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.
Open Access
Parsing PARP: the enzymatic and biophysical characterization of poly (ADP-Ribose) polymerases I and II
(Colorado State University. Libraries, 2015) Hepler, Maggie R. D., author; Luger, Karolin, advisor; Bailey, Susan, committee member; Yao, TingTing, committee member
The ADP-ribosyl transferase (ART) family is a prominent group of at least seventeen enzymes comprised of mono (ADP-ribose) transferases (MARTs) and poly (ADP-ribose) polymerases (PARPs). Each family member contains a conserved PARP signature motif in the catalytic domain. Enzymatically active proteins, in the presence of co-factor NAD+, catalyze individual or multiple ADP-ribose groups onto themselves or other proteins in automodification and heteromodification, respectively. The act of ADP-ribosylation implicates the ART family in a multitude of cellular processes including, but not limited to, transcription, apoptosis, DNA damage, metabolism, and inflammation. The founding member of the ART family is PARP-1, a first responder to DNA damage and regulator of active gene expression. In its inactive state and as a chromatin architectural protein, PARP-1 tightly binds chromatin, thereby regulating cellular activities, signifying the importance of PARP-1 and chromatin interaction. Importantly, PARP-1 must be activated and automodified in order to bind histones and gain nucleosome assembly function. Structurally similar and in many ways thought to be functionally redundant, PARP-2 is also thought to primarily function in the DNA damage response. PARP-2 has a non-canonical DNA binding domain, and therefore it is able to recognize different types of DNA structures in comparison to PARP-1, which could suggest a unique role for PARP-2 in repair. PARP-2 has not been extensively studied in a chromatin or gene regulation context due to this assumed redundancy. Given the pronounced functional changes in PARP-1 upon automodification, it is important to better understand what exactly triggers its enzymatic activity. Similarly, due to the functional redundancy of PARP-2, insight into activators of its enzymatic activity could indicate specificity and selectivity for the protein. However, determining the details of nuclear components that activate PARP-1 and PARP-2 are limited by the availability of a reliable quantitative and kinetic assay, as well as by the availability of defined substrates. These limitations hinder the separation of potent, and thus biologically relevant, activators from weak or non-specific activators. Utilizing a fluorescence based enzyme assay adapted for this system, kinetic parameters of PARP-1 and PARP-2 allosteric activators are reported here. As proof of principle and to test the reliability of the enzymatic assay, PARP-1 and PARP-2 activity was first tested with nucleic acids and other previously reported activators, such as nucleosomes and histones. Next, potentially novel activators were tested. Notably, PARP-1 is activated in the presence of its enzymatic product, PAR, indicating a mechanism by which PARP-1 could spread at sites of DNA damage and active gene expression. PARP-2 exhibits unique activation and specificity different from that of PARP-1 through its enzymatic preference for RNA. Further, PARP-1 remains the prominent chromatin related PARP due to the weak interaction, both activity and affinity, of chromatin with PARP-2. However, while PARP-1 and PARP-2 can act individually, affinity and activity studies demonstrate a PARP-1 and PARP-2 complex suggesting that these proteins can act sequentially and simultaneously with one another during a PAR-mediated recruitment and signaling cascade. Overall, these data indicate novel functions and mechanisms for PARP-1 and PARP-2 within the nucleus as critical responders to DNA damage and gene regulation.
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 member
Proper 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.
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 member
The 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.
Open Access
The biophysical, biochemical and structural characterization of Poly(ADP-ribose) Polymerase-1 (PARP-1) and its complexes with DNA-damage models and chromatin substrates
(Colorado State University. Libraries, 2013) Clark, Nicholas James, author; Luger, Karolin, advisor; Bailey, Susan, committee member; DeLuca, Jennifer, committee member; Hansen, Jeffrey C., committee member; Woody, Robert, committee member
Eukaryotic DNA is highly dynamic and must be compacted and organized with the help of cellular machines, proteins, into 'heterochromatin' state. At its basic level, chromatin is comprised of spool-like structures of protein complexes termed histones, which bind and organize DNA into larger fibrous structures. Cellular processes like transcription and DNA-damage repair require that chromatin be at least partially stripped of its protein components, which in turn allows for complete accessibility by DNA-repair or transcription machinery. A number of protein factors contribute to chromatin structure regulation. Poly(ADP-ribose) Polymerase-1 (PARP-1) is one of these proteins that exists in all eukaryotic organisms except for yeast. In its inactive form, it compacts chromatin, but performs its chromatin-opening function by covalently modifying itself and other nuclear proteins with long polymers of ADP-ribose in response to DNA damage. Thus, it also serves as a first responder to many types of DNA damage. The highly anionic polymers serve to disrupt protein-DNA interactions and thus allow for the creation of a temporary euchromatin structure. Contained herein are investigations aimed at addressing key questions regarding key differences between the interactions of PARP-1 and chromatin and its DNA-damage substrates. Our experiments show that human PARP-1 interacts with and is enzymatically activated to a similar level by a variety of different DNA substrates. In terms of chromatin, it appears that PARP-1 fails to interact with nucleosomes that do not have linker DNA. PARP-1 most effectively interacts with chromatin by simultaneously binding two DNA strands through contacts made by its two N-terminal Zn-finger domains. Small-Angle X-ray (SAXS) and Neutron Scattering (SANS) and molecular dynamics (MD) experiments were combined with biophysical and biochemical studies to better describe the structural effects of DNA binding on PARP-1. The average solution structure of PARP-1 indicates that the enzyme is a monomeric, non-spherical, elongated molecule with a radius of gyration (Rg) of ~55Å. The DNA-bound form of PARP-1 is also monomeric and binding DNA causes the molecule to become more elongated with an average Rg of ~80Å.
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 member
Maintaining 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.

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