Browsing by Subject "Transcription"

Now showing 1 - 7 of 7
  • Thumbnail Image
    Item
    Chemistry and Biology of DNA-protein cross-links
    (2019-02) Ji, Shaofei
    DNA-protein cross-links (DPCs) are ubiquitous DNA lesions formed when proteins become covalently trapped on DNA strands upon exposure to various endogenous, environmental and chemotherapeutic agents. Because of their considerable size, DPCs interfere with the progression of replication and transcription machineries, potentially contributing to mutagenesis and carcinogenesis. However, unlike small DNA lesions of which the biological consequences and repair mechanisms have been well characterized, biological effects and repair mechanism of DPC lesions remain to be established. A significant challenge in the field is the structural diversity of DPC lesions and the scarcity of experimental mythologies to create site-specific DNA-protein conjugates. The main objective of this thesis was to synthesize model DPC and to investigate their biological consequences and repair mechanisms. In Chapter II, we discovered, characterized and quantified 5-formylcytosine(5fC) mediated DNA-histone conjugates in human cells. 5-Formylcytosine (5fC) is an endogenous DNA modification enzymatically generated in the genome as an oxidation product of 5-methyl-dC (5mC). While 5mC is known to be an epigenetic mark that controls the levels of gene expression, the biological functions of 5fC are incompletely understood. In this chapter, we discovered that 5fC bases in DNA readily form Schiff base conjugates with Lys side chains of nuclear proteins such as histones, forming covalent DNA-protein conjugates. Isotope dilution nanoLC-ESI-MS/MS methodology was employed to detect and quantify 5fC-lys conjugate in human cells. We hypothesize that reversible 5fC-histone cross-linking contributes to epigenetic signaling, transcriptional regulations and chromatin remodeling. After the discovery of 5fC-mediated DNA-histone crosslinks in mammalian cells, we investigated their effects on DNA replication in Chapter III. DNA substrates containing site-specific DPCs were subjected to in vitro translesion synthesis (TLS) in the presence of TLS DNA polymerases. We found that DPCs containing various full-length proteins conjugated to DNA via the C-5 position of cytosine completely blocked human DNA polymerases, while the corresponding lesions containing shorter peptides were bypassed by translesion synthesis (TLS) polymerases. These results are consistent with the proposed DPC repair pathway in the literature, in which full-length DPCs are subjected to proteolytic degradation to generate short DNA-peptide cross-links, which may serve as substrates for translesion synthesis. In addition, our steady-state kinetics analysis and mass- spectrometry-based sequencing and quantification revealed that the bypass of DNA- peptide cross-links by human TLS polymerases was highly error-prone, introducing significant amounts of C to T and deletion mutations. In Chapter IV, we investigated the effects of DPCs on transcription using two model DPCs where the proteins are conjugated to the C5 position of cytosine or the C7 position of 7-deazaguanine. The latter serves as a hydrolytically stable model of the N7- guanine lesions, which commonly form upon exposure to bis-electrophiles such antitumor agents. We found that full-length proteins cross-linked to either 5fC or 7-deazaguanine completely blocked T7 RNA polymerase, while relatively short peptide cross-links were bypassed, although with low efficiency. Interestingly, the two model DPCs exhibited completely different mutagenic patterns are revealed by PCR and mass spectrometry based assays. While the bypass of peptide cross-linked to 7-deaza-G by T7 RNA polymerases induced very small numbers of mutations, transcription past peptide lesions conjugated to C-5 of C induced significant amounts of C to T transcriptional mutations. In Chapter V, we investigated the effects of 5fC-mediated DNA-peptide/protein cross-links on transcription and its potential repair by nucleotide excision repair (NER) in living cells. To accomplish this goal, structurally defined DPCs were site-specifically incorporated into plasmid molecules, which was then transfected into wild type cells or cells deficient in NER. RT-PCR and LC-MS/MS based strategy was then employed to quantitatively study the effects of DPC lesions on efficiency and fidelity of transcription in mammalian cells. We found that the presence of peptide cross-links conjugated to C-5 of cytosine significantly inhibited DNA transcription in human embryonic kidney cells. However, in contrast to our in vitro results, no transcriptional mutagenesis was observed. In addition, we compared the transcription bypass efficiencies of DpC lesions in wild-type and NER-deficient cell-lines, and also conducted the in vitro NER assays using cell-free extracts from human HeLa cells. Collectively, our data suggested that 5fC-mediated DNA- peptide cross-links are poor NER substrates, requiring a different pathway for their repair. Recent studies suggested that the bulky DPCs in cells are proteolytically processed to shorter DNA-peptide cross-links before they can be tolerated by translation synthesis mechanism or removed by nucleotide excision repair. DPCs can block DNA replication, signaling for recruitment of specialized metalloprotease (Spartan). However, the mechanisms of protease-mediated DPC digestion in the absence of DNA replication are incompletely understood. In Chapter VI, we employed an immunoprecipitation(IP)-PCR methodology to demonstrate that DPCs present on non-replicating plasmids are rapidly ubiquitinylated in mammalian cells, which likely serves as a signal for the proteasome- mediated DPC processing or other ubiquitin-mediated pathways to facilitate the DPC repair.
  • Thumbnail Image
    Item
    cis- and trans-acting transcriptional activators: characterization of single nucleotide polymorphisms and a novel two-component system of Staphylococcus aureus
    (2014-03) Hall, Jeffrey W.
    Staphylococcus aureus is a major opportunistic pathogen and a common cause of hospital- and community-acquired infections. Furthermore, infections of livestock animals by S. aureus results in billion dollar losses to agriculture producers annually. Over the last five decades antibiotic resistance has dramatically increased in S. aureus and highly pathogenic strains have emerged that threaten human and animal health. Characterization of highly pathogenic strains and novel transcriptional mechanisms and pathways is of utmost importance as it will provide a critical evolutionary understanding of the transcriptional changes that led to the emergence of successfully infectious S. aureus strains and may identify novel targets for antibacterial development. The overarching goal of research described in this thesis was to characterize and understand how novel cis- and trans-acting factors affect gene expression in S. aureus. To that end, the work and data presented investigate the effect of promoter based single nucleotide polymorphisms (SNPs) of the hla gene, encoding α-toxin, on gene transcription and gene product expression. The cis-acting SNPs increased the binding affinity of the promoter to the trans-acting transcription factor SarZ. Furthermore, the S. aureus RF122 strain had increased transcriptional expression of several positive regulators and decreased transcription of negative regulators of hla, which resulted in a dramatic increase in α-toxin expression and likely contributes to the increased mastitis pathogenesis of RF122. Additionally, the essentiality of the yhcSR two-component system was confirmed in the hospital-acquired methicillin resistant S. aureus WCUH29 strain. The YhcSR TCS was identified to transcriptionally activate the lacABCDE and opuCABC operons involved in cellular metabolism and osmoregulatory mechanisms, respectively. In an effort determine if a relationship existed between YhcSR and pathogenesis, studies revealed that the YhcSR TCS transcriptionally regulated, in a positive manner, the sspABC and crtOPQMN operons, encoding exported proteases and staphyloxanthin biosynthesis, which contribute to the survival of S. aureus in human blood. The data indicate that the YhcSR TCS system is an essential trans-acting global regulator in S. aureus.
  • Thumbnail Image
    Item
    Epigenetic regulation of killer immunoglobulin-like receptor Gene expression in developing human natural killer cells.
    (2010-05) Cichocki, Frank M.
    The immune system is our primary defense against infection and disease. Immune cells need to recognize and efficiently destroy invasive pathogens while, at the same time, exercising tolerance towards normal cells and tissues within the body. Because pathogenic organisms are constantly evolving to evade detection, the immune system must employ multiple recognition strategies to keep pace. Natural killer (NK) cells have evolved a self versus non-self recognition strategy known as “missing self” that is based upon the recognition of self major histocompatibility complex (MHC) molecules by stochastically expressed inhibitory receptors on the surface of NK cells. When MHC expression is downregulated by a virus or cellular transformation event, the dampening signals that balance against NK cell activation are lost due to a lack of inhibitory receptor engagement. This lack of inhibitory signaling, along with the engagement of activating receptors, leads to the elimination of the distressed cell through targeted NK cell-mediated cytotoxicity. The work presented in this manuscript focuses on the transcriptional regulation of a critically important family of human NK cell inhibitory receptors known as killer immunoglobulin-like receptors (KIR). The KIR genes are present within the leukocyte receptor complex on chromosome 19 and are expressed in a variegated, clonally restricted pattern on fully differentiated NK cells. How this pattern of gene expression is regulated during NK cell development is not well understood despite the demonstrated clinical relevance of KIR during hematopoietic cell transplantation to treat patients with leukemia, the influence of the KIR repertoire on the progression of HIV to AIDS, and the importance of KIR during pregnancy. Progress in the elucidation of how KIR genes are regulated has been slow due to the complexity of the KIR locus and the lack of KIR genes in mice, which are much more amenable to genetic manipulation. We have shown that the 5’ upstream regulatory region of each KIR gene contains a previously uncharacterized distal promoter with a functional c-Myc binding site. Stimulation of primary peripheral blood NK cells with IL-15 induces c-Myc binding at the distal promoter, which acts to promote KIR transcription. We also found that the overexpression of c-Myc protein in the NK92 cell line, which lacks surface KIR due to dense methylation of CpG dinucleotides proximal to the transcriptional start site, causes de novo surface KIR expression. Taken together, these results suggest that IL-15 directly promotes KIR transcription by inducing the binding of c-Myc to the distal promoter. We hypothesize that the recruitment of c-Myc and the initiation of active transcription from the distal promoter may also be key steps in the removal of repressive epigenetic marks within KIR promoters during human NK cell development to allow for stable gene expression. In addition to identifying a novel distal promoter, our group has found that the conventional proximal KIR promoter exhibits bi-directional transcriptional activity, meaning that transcription can initiate in either the sense or antisense orientation. We observed a strong inverse correlation between the expression of KIR antisense transcripts and receptor expression on the cell surface, leading to the hypothesis that antisense transcripts directly participate in RNA-mediated transcriptional repression of individual KIR genes. We found that over-expressing full-length antisense transcripts during NK cell development led to an approximately 70% reduction in KIR expression compared to controls. Furthermore, we determined that full-length antisense transcripts are processed into a 28 base RNA with biochemical properties similar to those attributed to members of the PIWI family of small RNAs. We also demonstrate that the 28 base sequence is necessary for antisense transcript-mediated repression of KIR gene expression. This work establishes a direct association between KIR antisense transcription and the initiation of DNA methylation within the KIR promoter. Further elucidation of the mechanisms that regulate KIR expression during NK cell development may provide a basis for new strategies in the design of NK cell-based therapies
  • Thumbnail Image
    Item
    Influence of Poly(ethylene oxide)-b-Poly(propylene oxide) Block Copolymers and Poly(ethylene oxide) Homopolymer on Cellular Function
    (2022-12) Crabtree, Adelyn
    Poly(ethylene oxide) (PEO) and poloxamers, a class of poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers are widely used for biological applications. In the 1990s, these polymers, specifically Poloxamer 188 (P188, 8.4 kDa, 80 wt% EO), were found to provide therapeutic effects through membrane stabilization. However, the complete mechanism by which the polymer interacts with the membrane to provide stabilization is unclear. To better understand the mechanisms behind the polymer-membrane interaction, this work aims to investigate (i) the role of different architectures, including the inverted (PPO-PEO-PPO) architecture, (ii) the effect of polymer dose, and (iii) the effects of polymer treatment and/or osmotic stress on transcription.Previous work in this field has shown that protective copolymers have a hydrophobic block to engage the molecule with the membrane and a hydrophilic block to solubilize the molecule in aqueous solution and stabilize the interaction with the membrane. To further expound on this proposed mechanism, an inverted poloxamer analog to P188 (PPO15-PEO200-PPO15) and a systematic variation of PPO-PEO diblocks were synthesized and tested in vitro to determine if increasing the number of hydrophobic ends would increase protection against enzyme leakage in osmotically stressed murine myoblasts. Through titrating the polymer dose, small, yet significant, differences showed enhancing the hydrophobicity through one or more hydrophobic ends, with possible addition of a non-polar end group, did increase the molecule’s potency. The titration of polymer dose also elucidated a concentration threshold where all the polymers tested reduced enzyme leakage by 50% compared to the untreated control between a dose of 0.8-4 µM. Through collaborative efforts, this trend was further seen in poly(butylene oxide)-PEO and bottlebrush poloxamers with a slightly larger threshold of 0.3-4 µM. Although enzyme leakage is a good metric to measure membrane permeability, the definition of cell health should be defined more broadly. To determine if the P188 or PEO181 (8 kDa) have an effect on the biochemical response of healthy or osmotically stressed murine myoblasts, RNA sequencing was used to quantify the transcriptome. Differential gene expression analysis showed that short-term exposure to 14 µM of P188 or PEO significantly changed expression of genes compared to the unstressed, untreated control. Subjecting the myoblasts to osmotic stress also impacted the transcriptome; however, the addition of polymer treatment to stressed cells did not result in significant differential expression compared to stress alone, which is consistent with a protective mechanism based on the physical polymer-membrane interaction.
  • Thumbnail Image
    Item
    Role of transcriptional activation unit 5 (TAU5) in mediating transcriptional activity of androgen receptor splice variants
    (2012-12) Mutha, Sarita Kumari
    The standard treatment for advanced prostate cancer is chemical castration, which inhibits the activity of the androgen receptor (AR). Eventually, prostate cancer reemerges with a castration-resistant phenotype (CRPC) but still depends on AR signaling. One mechanism of AR activity in CRPC is the synthesis of AR splice variants, which lack the ligand binding domain. These splice variants function as constitutively active transcription factors that promote expression of endogenous AR target genes and support androgen independent prostate cancer cell growth. Previous work has shown transcriptional activation unit 5 (TAU5) is necessary for ligand independent activity of the full length AR in low or no androgen conditions and that this activation is mediated by the WHTLF motif. The purpose of this study was to determine whether the TAU5 region was also important for regulating the constitutive activity of truncated AR variants. We generated deletion mutants of the AR variants and tested transcriptional activity by luciferase assay. We found that that the constitutive, ligand independent transcriptional activity of truncated AR variants was dependent on TAU5 for transcription. Surprisingly, we found that AR variants did not require WHTLF to mediate this ligand independent activity. We attempted to narrow down the region that may be important for variant transcriptional activity and found that deletion of amino acids 420-490 resulted in lower activity compared to AR 1/2/3 CE3. However, testing smaller regions of 420-490 did not elucidate a specific motif. These findings highlight TAU5 as a key AR domain through which AR activity could be inhibited for treatment of CRPC.
  • Thumbnail Image
    Item
    Strain dependent variations in iron metabolism of Mycobacterium avium subsp. paratuberculosis
    (2010-06) Janagama, Harish Kumar
    Johne’s disease is a major animal health problem of ruminant species worldwide and imposes significant economic losses to the industry. Our ability to culture the causative agent--Mycobacterium avium subsp. paratuberculosis (MAP)--and therefore its rapid diagnosis and our understanding of its virulence is limited. MAP is difficult to culture because of its unusually strict iron requirements. For optimal growth in laboratory media, MAP requires a siderophore (mycobactin) supplementation that makes MAP fastidious, often requiring eight to sixteen weeks to produce colonies in culture – a major hurdle in timely diagnosis and therefore implementation of optimal control measures. Understanding iron regulatory networks in the pathogen in vitro is therefore of great importance. Several microbiological and genotyping studies and clinical observations suggest that Johne’s in certain hosts such as sheep, goats, deer, and bison is caused by a distinct set of strains that show a relatively high degree of host preference. At least two microbiologically distinct types of MAP have been recognized. A less readily cultivable type is the common, but not invariable, cause of paratuberculosis in sheep (type I), while another readily cultivable type is the most common cause of the disease in cattle (type II). In addition, since the MAP genome sequence was published in 2005, very little research has focused on iron physiology and its contribution to metabolic networks of this fastidious organism. Based on these observations, I hypothesize that iron dependent gene regulation is different between type I and type II MAP strains. Iron dependent Regulator (IdeR), a transcription factor, is an essential gene in MAP and differentially controls the expression of genes involved in iron physiology in the two strain types of MAP. We identified polymorphisms in the IdeR open reading frame (ORF) and the promoters of putative IdeR regulated genes between the type I and type II strains of MAP. Structure-function association studies revealed repression of an iron storage gene, bfrA in the presence of iron by type I MAP strain alone. In contrast, bfrA was upregulated in the presence of iron in type II MAP strain. This leads us to propose that type I MAP strains may experience iron toxicity when excess iron is provided in the medium. The rationale is that excess free iron is detrimental to the cells and must be stored in bacterioferritins, a feature that type I strains lack. Transcriptional and proteomic profiling of these MAP strains under iron-replete or –deplete conditions revealed that iron-sparing response to iron limitation was unique to the type II strain as evidenced by repression of non-essential iron utilization enzymes (aconitase and succinate dehydrogenase) and upregulation of proteins of essential function (iron transport, [Fe-S] cluster biogenesis and cell division). Under iron-replete conditions, type II MAP alone increased expression of BfrA (bacterioferritin) and MhuD (mycobacterial heme utilization, degrader) protein, which is intricately involved in iron recycling. These findings further supported the contention that type I MAP strains are metabolically inept under iron-replete conditions. The intracellular lifestyle of MAP in the intestines and lymph nodes of natural infection revealed that MAP deployed genes involved in maintaining iron homeostasis under iron stress in the tissues of infected animals. There was a clear dichotomy in in vitro infected macrophages and natural infection in the expression profiles of both iron acquisition genes and other virulence factors involved in MAP survival inside the host. In summary, our studies revealed that IdeR of type II strain regulates mycobactin synthesis and iron storage genes, similar to the function of IdeR in M. tuberculosis (MTB), while the type I strain is deficient in iron storage function. Given our inability to delete ideR, it appears that this is an essential gene (as in MTB) for MAP survival. MAP IdeR regulon studies led us to define a novel operon carrying genes encoding a potential secretory apparatus (ESX-3/type VII secretory system). Functional analysis of the iron-induced proteome also identified novel ESAT-6 (early secreted antigenic target) family of proteins belonging to ESX-5, which have been identified as major virulence factors in MTB. We also established that, type I MAP strains are more sensitive to fluctuations of environmental iron due to defective regulation of bfrA and may grow better under lower iron levels in the culture media. Taken together, our studies suggest that MAP employs a sophisticated repertoire of proteins that are inter-connected and function in response to environmental stress.
  • Thumbnail Image
    Item
    Transcriptional regulation of transcription Factor 8
    (2010-05) Broege, Aaron M.
    Zinc-Finger E-box binding protein (ZEB) 1 (Zfhx1a, AREB6, delta-EF1), encoded by the gene tcf8, is a transcription factor that binds to E-box sequences in regulatory regions of its target genes to either repress or activate transcription. ZEB1 induces the epithelial to mesenchymal transition (EMT) during development and cancer progression. The most prominent phenotype of tcf8 null mice is abnormal skeletal development, and ZEB1 inhibits differentiation of mesenchymal precursors into chondrocytes and osteoblasts; however, little is known about the transcriptional regulation of tcf8 throughout these processes. The transcription factor Runx2 is a master regulator of skeletal formation, making it a likely candidate to regulate expression of tcf8 in this tissue. Seven putative Runx binding sites are present in the tcf8 proximal promoter, and we hypothesized that Runx2 regulates tcf8 activity by binding to one or more of these sites. Reporter based assays indicate that Runx2 either represses or activates transcription of tcf8 in a cell type dependent manner and that this activity is mediated through a region between 164 and 112 relative to the translation start site. Furthermore, Runx2 was found to cooperate synergistically with the transcription factor LEF 1 to activate transcription of tcf8 in Cos7 cells. Given that LEF 1 mediates the effects of the canonical Wnt signaling pathway, the effect of the Wnt3a on tcf8 expression was examined in the C2C12 mesenchymal precursor cell line, where an induction of endogenous tcf8 was observed. These data provide the first evidence for regulation of tcf8 by Runx2 and suggest that Runx2 may interact with LEF 1 to mediate the effects of Wnt signaling on expression of tcf8.