Browsing by Author "Kim, Tae Hoon"
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Item Beyond Nitrogen Limitation – Novel Mechanisms Regulating Glutamine Synthetase Expression in Escherichia coli and a Possible Alternative Pathway of Glutamine Synthesis(May 2023) Urs, Karthik; Reitzer, Lawrence J.; Conlon, Ronan; Palmer, Kelli; Spiro, Stephen; Kim, Tae HoonThe expression of glnA (ammonia-assimilating glutamine synthetase) is high for uropathogenic E. coli grown in urine. Because glnA is part of an operon that codes for regulators of the nitrogen-regulated (Ntr) response, high glnA expression has been interpreted to suggest nitrogen limitation, which is unexpected because of the high urinary ammonia concentration which should suppress glnA expression. We present evidence that glnA expression does not result from nitrogen limitation. First, in the presence of ammonia, urea induced expression of glnA from the cAMP receptor protein (Crp)- dependent glnAp1 promoter, which circumvents control from the nitrogen-regulated glnAp2 promoter. This urea effect on glnA expression has not been previously described. Second, the most abundant amino acids in urine inhibited GS activity, based on reversal of the inhibition by glutamate and glutamine, and increased glnA expression. The relevance of these inhibitory amino acids in natural environments has not been previously demonstrated. Third, urea and the inhibitory amino acids did not induce other Ntr genes, i.e., high glnA expression can be independent of other Ntr genes. Finally, the urea- dependent induction did not result in GlnA synthesis because of a previously undescribed translational control. We conclude that glnA expression in urea-containing environments does not imply growth rate-limiting nitrogen restriction and is consistent with rapid growth of uropathogenic E. coli. ΔglnA mutants are glutamine auxotrophs, however, UTI89ΔglnA mutants, were unexpectedly able to grow in a synthetic urine medium. This phenotype was conditional and required the presence of both glutamate and ammonia, the substrates for glutamine synthetase. Additionally, overexpression of proA, which is part of the proline biosynthesis pathway, whose product catalyzes the formation of glutamate-5-semialdehyde, improved growth. In contrast, an increase in proC expression, which directs pyrroline-5-carboxylate, the cyclized form of glutamate-5-semialdehyde to proline, impaired growth. We describe a possible alternate route of glutamine production in these mutants involving the enzymes of proline synthesis and the substrates, glutamate and ammonia, via the intermediate glutamate-5-semialdehyde. The pathway may be facilitated by a putative secondary activity of the ProA enzyme – the reduction of a proposed imine intermediate to ultimately form glutamine. Members of the B2 clade of E. coli exhibit high glnA transcript levels in nitrogen-rich glucose-tryptone medium. This increased expression did not translate to increased protein production and enzyme activity as evidenced by low translation levels and glutamine synthetase activity in these strains. Transcriptomic analysis revealed an inverse correlation between phoB, a phosphate-dependent transcriptional regulator, and glnA expression. Consistent with this, overexpression of phoB, reduced glnA transcription levels. The effect was not a complete repression of glnA transcription. Additionally, translational expression appeared to be stabilized upon phoB overexpression. Our findings suggest a novel mechanism of glnA regulation at both the transcriptional and translational levels that involves PhoB operating either directly or indirectly, and possibly in combination with other unidentified factors.Item Boundary Associated Long Noncoding RNA Mediates Long-Range Chromosomal Interactions(Public Library of Science, 2015-08-24) Nwigwe, I. J.; Kim, Yoon Jung; Wacker, D. A.; Kim, Tae Hoon; Chadwick, B. P.; Kim, Yoon Jung; Kim, Tae HoonCCCTC binding factor (CTCF) is involved in organizing chromosomes into mega base-sized, topologically associated domains (TADs) along with other factors that define sub-TAD organization. CTCF-Cohesin interactions have been shown to be critical for transcription insulation activity as it stabilizes long-range interactions to promote proper gene expression. Previous studies suggest that heterochromatin boundary activity of CTCF may be independent of Cohesin, and there may be additional mechanisms for defining topological domains. Here, we show that a boundary site we previously identified known as CTCF binding site 5 (CBS5) from the homeotic gene cluster A (HOXA) locus exhibits robust promoter activity. This promoter activity from the CBS5 boundary element generates a long noncoding RNA that we designate as boundary associated long noncoding RNA-1 (blncRNA1). Functional characterization of this RNA suggests that the transcript stabilizes long-range interactions at the HOXA locus and promotes proper expression of HOXA genes. Additionally, our functional analysis also shows that this RNA is not needed in the stabilization of CTCF-Cohesin interactions however CTCF-Cohesin interactions are critical in the transcription of blncRNA1. Thus, the CTCF-associated boundary element, CBS5, employs both Cohesin and noncoding RNA to establish and maintain topologically associated domains at the HOXA locus.Item Chronic IL-1 Exposure Drives Prostate Cancer Progression(May 2023) Dahl, Haley C 03/03/1993-; Delk, Nikki; Kolodrubetz, Michael; Palmer, Kelli; Kim, Tae Hoon; Winkler, Duane D.Prostate cancer (PCa) is the second most common cause of cancer-related deaths among American men. Androgen Receptor (AR) transcriptional activity is required for PCa tumor growth. Androgens regulate normal prostate tissue growth and differentiation via androgen receptor (AR) activation. Due to the role of androgens in prostate cancer, androgen-deprivation therapy (ADT), either through chemical or surgical castration or the use of anti-androgens, has become the standard therapy. However, ~10-20% of PCa patients will develop treatment resistance, referred to as castration-resistant PCa (CRPC). One mechanism of CRPC is the loss of dependence on AR for cell growth and survival. As such, over 84% of CRPC patients will develop incurable, lethal bone metastasis. Thus, it is important to uncover the mechanisms that drive CPRC. IL-1 is elevated in PCa patient tissue and serum and is associated with disease progression and metastasis. IL-1 is clinically relevant, but the role of IL-1 in CRPC development is not fully elucidated. Chronic inflammation is a known hallmark of cancer initiation and progression. Therefore, we exposed the cancer cells to IL-1 for several months to make the chronic IL-1 sublines, LNas1 and LNbs1. The chronic IL-1 sublines restore AR and AR activity but evolve AR independence and acquire a constitutive p62-KEAP1 interaction. p62 is a multi-domain, multifunctional pro-survival protein that mediates autophagic turnover of damaged proteins and organelles, promotes NF-κB inflammatory signaling and induces NRF2 antioxidant signaling through its binding to and sequestering of KEAP1 from NRF2. Despite constitutive p62- KEAP1 binding, the chronic IL-1 sublines only show elevated NRF2 signaling in the NRF2 target genes, HMOX1 and GCLC. Furthermore, the chronic IL-1 sublines evolve insensitivity to IL-1 extracellular signaling and, thus, do not activate NF-κB nor NRF2 signaling. Thus, the regulation and function of the constitutive p62-KEAP1 interaction in the chronic IL-1 sublines is novel. To investigate the regulation and function of the constitutive p62-KEAP1 interaction in PCa cells chronically exposed to IL-1, I dissected p62-KEAP1 regulation and function under the oxidative stress-inducing stimulus, androgen deprivation. Under androgen deplete conditions, there is attenuation of HMOX1 and overexpression of GCLC in the chronic IL-1 sublines. This suggests that there is active but aberrant NRF2 signaling that may allow these cells to be primed to withstand oxidative stress. Furthermore, knockdown of KEAP1 results in upregulation of HMOX1 suggesting that KEAP1 negatively regulates HMOX1. Both HMOX1 and GCLC function as an antioxidant to attenuate iron-induced lipid ROS and thus regulate iron- dependent cell death known as ferroptosis. Based on what we have found, we hypothesize that the sublines activate a novel pathway that primes them to withstand ferroptosis-induced cell death.Item Heme Sequestration as an Effective Strategy for the Suppression of Tumor Growth(2021-08-01T05:00:00.000Z) Wang, Tianyuan; Zhang, Li; Lumata, Lloyd; Kim, Tae Hoon; Spiro, Stephen; Xuan, ZhenyuHeme is an essential prosthetic group in proteins and enzymes involved in oxygen utilization and metabolism. Heme also plays versatile and fascinating roles in regulating fundamental biological processes ranging from aerobic respiration to drug metabolism. Increasing experimental and epidemiological data have also shown that altered heme homeostasis accelerates the development and progression of common diseases, including various cancers, diabetes, vascular diseases, and Alzheimer's disease. The effects of heme on the pathogenesis of these diseases may be mediated via its action on various cellular signaling and regulatory proteins, as well as its function in cellular bioenergetics, specifically, oxidative phosphorylation (OXPHOS). Elevated heme levels in cancer cells intensify OXPHOS, leading to higher ATP generation and fueling tumorigenic functions. In contrast, lowered heme levels in neurons may reduce OXPHOS, leading to defects in bioenergetics and causing neurological deficits. Additionally, heme has been shown to modulate the activities of diverse cellular proteins influencing disease pathogenesis. These include tumor suppressor P53 protein, progesterone receptor membrane component 1 protein PGRMC1, cystathionine-βsynthase CBS, and the nuclear receptor subfamily member Rev-Erbα. Here, we generated small heme-sequestering proteins (HeSPs) based on bacterial hemophores. These HeSPs contain neutral mutations in the heme-binding pocket of hemophores and hybrid sequences from hemophores of different bacteria. We showed that HeSPs bound to heme and effectively extracted heme from hemoglobin. They strongly inhibited heme uptake and cell proliferation and induced apoptosis in non-small lung cancer (NSCLC) cells, while their effects on non-tumorigenic cell lines representing normal lung cells were not significant. HeSPs strongly suppressed the growth of human NSCLC tumor xenografts in mice. HeSPs decreased oxygen consumption rates and ATP levels in tumor cells isolated from treated mice, while they did not affect liver and blood cell functions. Immunohistochemistry revealed that HeSPs reduced the levels of key enzymes and transporters involved in heme synthesis and uptake, as well as the uptake and metabolism of the main fuels for cancer cells, glucose and glutamine. Further, we found that HeSPs reduced the levels of angiogenic and vascular markers, as well as vessel density in tumor tissues. Together, these results demonstrate that HeSPs act via multiple mechanisms, including the inhibition of oxidative phosphorylation, to suppress tumor growth and progression. Evidently, heme sequestration can be a powerful strategy for suppressing lung tumors and likely drug-resistant tumors that rely on oxidative phosphorylation for survival.Item Histone Deacetylases Positively Regulate Transcription Through the Elongation Machinery(Elsevier B.V., 2015-11-17) Greer, Celeste B.; Tanaka, Yoshiaki; Kim, Yoon Jung; Xie, Peng; Zhang, Michael Q.; Park, In-Hyun; Kim, Tae Hoon; 0000 0001 1707 1372 (Zhang, MQ); Kim, Yoon Jung; Xie, Peng; Zhang, Michael Q.; Park, In-HyunTranscription elongation regulates the expression of many genes, including oncogenes. Histone deacetylase (HDAC) inhibitors (HDACIs) block elongation, suggesting that HDACs are involved in gene activation. To understand this, we analyzed nascent transcription and elongation factor binding genome-wide after perturbation of elongation with small molecule inhibitors. We found that HDACI-mediated repression requires heat shock protein 90 (HSP90) activity. HDACIs promote the association of RNA polymerase II (RNAP2) and negative elongation factor (NELF), a complex stabilized by HSP90, at the same genomic sites. Additionally, HDACIs redistribute bromodomain-containing protein 4 (BRD4), a key elongation factor involved in enhancer activity. BRD4 binds to newly acetylated sites, and its occupancy at promoters and enhancers is reduced. Furthermore, HDACIs reduce enhancer activity, as measured by enhancer RNA production. Therefore, HDACs are required for limiting acetylation in gene bodies and intergenic regions. This facilitates the binding of elongation factors to properly acetylated promoters and enhancers for efficient elongation.; Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.Item Identification of a Novel Myc Associated Oncogenic Enhancer RNA(2020-07-15) Cao, Lian; Kim, Tae HoonAmplification or elevated expression of the Myc oncogene is a major mechanism associated with malignant tumor progression and poor prognosis in many cancers, including breast cancer. Enhancers play dominant roles in driving the overexpression of oncogenes. Long noncoding RNA known as enhancer RNA (eRNA) produced from enhancers can mediate the function of enhancers. Although many eRNA-dependent regulatory functions have been demonstrated, direct association of eRNA, with its immediate effectors and regulators, has not been explored and little is known about how eRNA might work directly to affect gene expression. In our efforts to discover potential oncogenic eRNAs, we identified a novel eRNA that drives Myc expression in breast cancer cells that we named MYC EN-1. We find that MYC EN-1 is stabilized selectively in cancer cells via possible circularization and that reduction of MYC EN-1 decreases Myc transcription, protein expression, and cell viability. The transcriptome profile after knocking down of MYC EN-1 and Myc mRNA, respectively, reveals that both eRNA and mRNA share a large portion of similar transcriptomic changes. Biochemical characterization of MYC-EN-1 reveals that it is intimately associated with both Myc pre-mRNA and mature mRNA as it is synthesized and processed from its genomic locus. This physical interaction between MYC-EN-1 and Myc mRNA is achieved through direct interaction by direct base pairing. Our study has uncovered a broad role of oncogenic eRNAs with ability to promote target mRNA transcription.Item Long Genes Linked to Autism Spectrum Disorders Harbor Broad Enhancer-Like Chromatin Domains(Cold Spring Harbor Laboratory Press) Zhao, Y. -T; Kwon, D. Y.; Johnson, B. S.; Fasolino, M.; Lamonica, J. M.; Kim, Yoon Jung; Zhao, B. S.; He, C.; Vahedi, G.; Kim, Tae Hoon; Zhou, Z.; Kim, Yoon Jung; Kim, Tae HoonGenetic variants associated with autism spectrum disorders (ASDs) are enriched in genes encoding synaptic proteins and chromatin regulators. Although the role of synaptic proteins in ASDs is widely studied, the mechanism by which chromatin regulators contribute to ASD risk remains poorly understood. Upon profiling and analyzing the transcriptional and epigenomic features of genes expressed in the cortex, we uncovered a unique set of long genes that contain broad enhancer-like chromatin domains (BELDs) spanning across their entire gene bodies. Analyses of these BELD genes show that they are highly transcribed with frequent RNA polymerase II (Pol II) initiation and low Pol II pausing, and they exhibit frequent chromatin-chromatin interactions within their gene bodies. These BELD features are conserved from rodents to humans, are enriched in genes involved in synaptic function, and appear post-natally concomitant with synapse development. Importantly, we find that BELD genes are highly implicated in neurodevelopmental disorders, particularly ASDs, and that their expression is preferentially down-regulated in individuals with idiopathic autism. Finally, we find that the transcription of BELD genes is particularly sensitive to alternations in ASD-associated chromatin regulators. These findings suggest that the epigenomic regulation of BELD genes is important for post-natal cortical development and lend support to a model by which mutations in chromatin regulators causally contribute to ASDs by preferentially impairing BELD gene transcription.Item A Novel Virus-Inducible Enhancer of the Interferon-β Gene with Tightly Linked Promoter and Enhancer Activities(2014-10-27) Banerjee, A. Raja; Kim, Yoon Jung; Kim, Tae Hoon; Kim, Yoon Jung; Kim, Tae HoonLong-range enhancers of transcription are a key component of the genomic regulatory architecture. Recent studies have identified bi-directionally transcribed RNAs emanating from these enhancers known as eRNAs. However, it remains unclear how tightly coupled eRNA production is with enhancer activity. Through our systematic search for long-range elements that interact with the interferon-beta gene, a model system for studying inducible transcription, we have identified a novel enhancer, which we have named L2 that regulates the expression of interferon-beta. We have demonstrated its virus-inducible enhancer activity by analyzing epigenomic profiles, transcription factor association, nascent RNA production and activity in reporter assays. This enhancer exhibits intimately linked virus-inducible enhancer and bidirectional promoter activity that is largely dependent on a conserved Interferon Stimulated Response Element and robustly generates virus inducible eRNAs. Notably, its enhancer and promoter activities are fully retained in reporter assays even upon a complete elimination of its associated eRNA sequences. Finally, we show that L2 regulates IFNB1 expression by siRNA knockdown of eRNAs, and the deletion of L2 in a BAC transfection assay. Thus, L2 is a novel enhancer that regulates IFNB1 and whose eRNAs exert significant activity in vivo that is distinct from those activities recapitulated in the luciferase reporter assays.Item Probing Dynamic Cellular Properties Using Genome Editing and Systems Biology(2021-12-01T06:00:00.000Z) Nowak, Chance Michael; Bleris, Leonidas; Lee, Mark; Palmer, Kelli; Kim, Tae Hoon; Campbell, ZacharyGenome editing has revolutionized not only the future of biological research, but also holds the promise of being a powerful therapeutic for genetic diseases. When considering the multitude of genetic regulations that contribute to various biological processes and their individual contributions that permit diseased cellular states, especially in instances where more than a single genetic aberration is attributed to the diseased phenotype, it is crucial to consider the interconnectivities of gene regulators and their individual contributions to cell health. Biological network maps that reveal the relation of gene products to one another can provide insight into the biological properties they govern. A biological network map consists of nodes (gene products) connected by edges that are dictated by the nature of the interaction between the two nodes. Nodal ablation (i.e., knocking out a gene to render it non-functional) has been crucial in understanding diseased states. However, this type of mutational analysis essentially disregards the impact that individual edges have on the network as a whole. The goal of my dissertation work was to utilize the genome editing tool Cas9 to disrupt the p53-miR-34a network in an edge-specific manner in order to demonstrate not only the complexity of these networks, but to also underscore the importance that individual edges have on the tumor suppressor phenotype. To this end, I, along with a team of researchers, developed a genetic screen using Cas9-bearing lentiviral vectors to disrupt 93 miR-34a binding sites within the 3’ untranslated region (UTR) of 71 genes impactful to cell survival under apoptotic conditions. I quantified the degree of apoptosis in two colorectal cancer cell lines that differ in functional p53 status, and that each harbored miR-34a binding site mutations within the pro-survival gene Bcl-2 3’UTR, demonstrating the importance of the miR-34a-Bcl-2 edge on apoptotic progression. Concurrently, I investigated the phenomenon of cell cycle desynchronization by tracking the DNA distribution of a population of cells starting from a synchronized state until asynchrony with flow cytometry analysis. In doing so, I utilized statistical tools to quantify the degree of desynchronization that does not rely on individual cell cycle phase labeling. Additionally, with the help of my peers, tested and validated a mathematical model the capitulates experimental observations. I explored the sensitivity of the model to changes in its parameters to reveal that cell cycle variability within the population is a main contributor to cell cycle desynchronization. Furthermore, I tested this model prediction by treating cells with lipopolysaccharide to enhance cellular noise, resulting in a greater variability of cell cycle duration, which was also shown to increase the rate of cell cycle desynchronization. Taken together, my research provides insight into the importance individual edges have to biological networks and their resulting phenotypes, as well as the underlying sources of cell population heterogeneity and its contribution to cell cycle variability.Item Synthesis and Characterization of Metal-organic Frameworks for Potential Uses in Cancer Therapy(2021-12-01T06:00:00.000Z) Vizuet Mata, Juan Pablo; Balkus Jr., Kenneth J.; Kim, Tae Hoon; Ferraris, John P.; Novak, Bruce M.; Smaldone, Ronald A.Metal-organic frameworks (MOFs) are crystalline materials, characterized for their high surface areas and defined pore architectures. These materials, first synthesized and characterized in the 1990s, have seen an increase interest due to their inherent properties. As a result of their hybrid nature, MOFs can be used in a wide range of applications, from catalysis and gas storage, to drug delivery and cancer therapy. While commonly using transition metals as building blocks, using lanthanide as their metal centers further increases the range of MOF applications. Using holmium in these materials could potentially create an improved cancer therapy method by delivering both a radiation source and a radiosensitizer to the cancer sites. This work in particular focuses on the synthesis and characterization of several frameworks, with the focus of using these materials for cancer therapy applications.