Browsing by Subject "Transcription Factors"
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Item Biochemical and Functional Analysis of Members of the Myocardin Family During Cardiovascular Development(2006-12-20) Oh, Jiyeon; Olson, Eric N.The various stages of muscle development are characterized by distinct patterns of gene expression precisely controlled by combinatorial interaction between a large number of muscle-specific and ubiquitous transcription factors. Myocardin is a cardiac and smooth muscle-specific transcriptional coactivator of serum response factor (SRF) that forms a ternary complex with SRF on DNA and provides its strong transcriptional activation domain (TAD) to SRF. SRF has been shown to stimulate expression of smooth and cardiac muscle genes in association with GATA transcription factors, which play important roles in cardiac and smooth muscle development. I show that GATA transcription factors can either stimulate or suppress the transcriptional activity of myocardin, depending on the target gene. Modulation of myocardin activity by GATA4 is mediated by the physical interaction of myocardin with the DNA binding domain of GATA4 but does not require binding of GATA4 to DNA. The ability of GATA transcription factors to modulate myocardin activity provides a potential mechanism for fine tuning the expression of serum response factor target genes in a gene-specific manner. Two Myocardin Related Transcription Factors, referred to as MRTF-A and B, are expressed in numerous embryonic and adult tissues, implying their potential to modulate SRF target genes in a wide range of tissues. To determine the functions of MRTF-B in vivo, I generated MRTF-B mutant mice by targeted inactivation of the MRTF-B gene. I show that mice homozygous for an MRTF-B loss-of-function mutation die during mid-gestation from a spectrum of cardiovascular defects. These abnormalities are accompanied by a failure in differentiation of smooth muscle cells within the branchial arch arteries, which are derived from the neural crest. The phenotype of MRTF-B mutant mice is distinct from that of mice lacking myocardin and MRTF-A, revealing unique roles for these SRF coactivators in the development of different subsets of smooth muscle cells in vivo.Item Biochemical Characterization of Delta FosB(2006-12-19) Carle, Tiffany Lynn; Nestler, Eric J.; Phillips, Margaret A.; De Martino, George; Monteggia, LisaDeltaFosB, the truncated splice variant of FosB, is an important mediator of the long-term plasticity induced in brain by chronic exposure to many types of stimuli, such as repeated administration of drugs of abuse, stress, or compulsive running. Once induced, DeltaFosB persists in the brain for weeks or months following cessation of the chronic stimulus. In addition, DeltaFosB both activates and represses transcription. The biochemical basis of DeltaFosB's persistent expression and dual transcriptional regulation has remained unknown. Both the enhanced protein stability and transcriptional properties are unique to DeltaFosB, compared to FosB, and are critical for its role in neural plasticity. DeltaFosB lacks the C-terminal 101 amino acids of FosB as a result of alternative splicing. The purpose of this work is to biochemically characterize DeltaFosB relative to FosB, to determine how truncation of the FosB C-terminus directs its function. Here, I show that the FosB C-terminus contains two destabilizing elements that promote the degradation of FosB by both proteasome dependent and independent mechanisms. Pulse chase experiments of FosB C-terminal truncation mutants indicate that removal of these C-terminal degrons increases the FosB half-life ~5 fold and prevents its proteasome-mediated degradation and ubiquitylation, properties similar to FosB. These data indicate that alterative splicing specifically removes two destabilizing elements from FosB in order to generate a longer-lived transcription factor, DeltaFosB, in response to chronic perturbations to the brain. Truncation of the C-terminus from FosB also results in differing interaction partners for FosB and DeltaFosB that may contribute to the varying functions of each protein. Specifically, using co-immunoprecipitation assays both in vitro and in vivo, I determined that HDAC1 (histone deacetylase 1) is the preferential binding partner of DeltaFosB compared to FosB. These data suggest an intriguing hypothesis that DeltaFosB interactions with specific HATs and HDACs may be one mechanism by which DeltaFosB mediates both activating and repressive transcriptional activities. DeltaFosB is a unique transcription factor compared to its Fos family members. Truncation of the FosB C-terminal domain liberates DeltaFosB, enabling long-term protein stability and promoting specific interactions with protein partners that are critical for gene regulation important for neural plasticity.Item Building a Methodological Framework for Cell Fate Engineering(August 2021) Li, Boxun; Xu, Jian; Hon, Gary C.; Banaszynski, Laura; Cleaver, Ondine; Munshi, NikhilCell fate engineering has become an area of intense research in the last fifteen years. A useful framework of cell fate engineering should include three pillars: the discovery of new cell fate-reprogramming cocktails of factors, the evaluation of engineered cells, and the revelation of underlying molecular mechanisms. One major challenge has been the lack of a scalable screening approach in vitro for the performance of reprogramming cocktails. This limits the speed of discovering new cocktails that can efficiently reprogram diverse cell types. Such new cocktails are needed to unleash the full applicational potential of engineered cells in regenerative medicine, disease modeling, and drug discovery. Another challenge is that despite the advantages of in vivo reprogramming, such as more efficient and mature fate conversion, the underlying gene programs, and thereby the molecular mechanisms, have been largely unknown. This is in large part due to the difficulty of specifically isolating and analyzing reprogrammed cells, without contamination from their endogenous counterparts. To address these, in this thesis, I first develop Reprogram-seq, a method that screens thousands of transcription factor cocktails for their reprogramming performance by single-cell perturbation screens. Reprogram-seq found a cocktail of three factors that efficiently and functionally reprograms fibroblasts to epicardial-like cells. Thus, Reprogram-seq accelerates rational cell fate engineering. Next, I performed single-cell transcriptomic analysis of in vivo neurogenesis induced in astrocytes by a novel reprogramming factor, DLX2. This is enabled by a lineage tracer that highly specifically tracks all cells reprogrammed from astrocytes. My analysis reveals that DLX2 induces a neural stem cell-like behavior, transitioning from quiescence to activation, proliferation, and neurogenesis. Gene regulatory network analysis and mouse genetics identify and confirm key nodes mediating DLX2-dependent fate reprogramming. Therefore, this study dissects the gene programs of in vivo reprogramming with single-cell transcriptomics and paves the way for applying Reprogram-seq in vivo. Together, my thesis research has demonstrated that single-cell omic technologies accelerate the discovery of new reprogramming cocktails, streamline the transcriptional evaluation of engineered cells, and dissect gene programs that underlie reprogramming, contributing to all three pillars of the framework. I expect these methodologies to be generalizable to and useful for other cell fate engineering scenarios.Item Characterization of Receptor Protein Tyrosine Phosphatase Epsilon (PTPRE) Gene Promoter(2015-01-26) Isaacs, Thomas; Shukla, Abhay A.; Amatruda, JamesBACKGROUND: Receptor protein tyrosine phosphatase epsilon (PTPRE) is a receptor bound phosphatase that has been shown to be downregulated in Wilms' tumors compared to normal tissue, and could potentially be a target for future therapy. Our objective is to identify and characterize the promoter of the PTPRE gene and define the critical role of Wt1 transcription factor (commonly downregulated in Wilm's tumor) in PTPRE gene expression and in Wilms' tumor progression. METHODS: Our first step involved cloning and sequence analysis of the upstream region of the human PTPRE gene followed by PCR primer design and PCR amplification. The amplified fragment was then cloned into a promoterless reporter vector (pGl3 Basic) and transfected in Hek293 cells. Promoter DNA was used for deletion analysis where multiple PCRs were performed using a single forward primer and multiple reverse primers with nucleotides sequentially deleted from the 3' end. The different size PCR products were then cloned into pGL3 Basic vector DNA, transfected into HEK cells and had reporter assay (luciferase assay) performed to calculate fold change in PTPRE expression over promoterless vector. After the critical transcription factor binding motif was identified, PCR was performed to amplify full length promoter lacking 76 bases. The role of the deleted nucleotides was confirmed via luciferase assay. The sequence of deleted nucleotides was then analyzed for the presence of transcription factor binding motifs. Next, a predicted Wt1 transcription factor binding motif was mutated using site directed mutagenesis. Mutated fragment was cloned into pGl3Basic vector. Both wild type and mutated vector were transfected and luciferase assay was performed to confirm role of Wt1 binding motif for PTPRE promoter activity. Chromatin immunoprecipitation assay was then performed for further evidence. Promoter activity was also compared in two cells lines having differential expression of Wt1. Western blot and semi-quantitative PCR are used to confirm the expression levels of Wt1. RESULTS: Promoter deletion analysis confirmed that the Wt1 binding motif present at -16 position is critical for PTPRE expression and mutation of this site results in 95% loss in promoter activity in Hek293 cells. PTPRE promoter activity was shown to be high in Hek293 cells and low in Hela cells (high and low WT1 expression respectively), suggesting WT1 driving promoter activity. ChiP using WT1 antibody confirmed WT1 binding of the critical transcription factor binding motif. CONCLUSION: These results shed light on why PTPRE expression is lower in Wilms' tumors and reveals potential future targeted therapy.Item Characterization of β-glucuronidase for Enzyme Replacement Therapy in DYT6 Dystonia(2023-01-31) Lieu, Linh; Yim, Daron; Pappas, Samuel; Dauer, WilliamBACKGROUND: Dystonia is a debilitating disorder defined by sustained involuntary twisting movements. The current symptomatic treatments for dystonia offer only modest efficacy but numerous side effects. Dominantly inherited, loss of function mutations in the THAP1 transcription factor cause DYT6 dystonia (DYT-THAP1). THAP1 modulates the development of oligodendrocyte progenitor cells (OPC) by regulating the catabolism of glycosaminoglycans (GAGs), a crucial component of the extracellular matrix. The loss of THAP1 within OPCs directly reduces GAG-catabolic lysosomal enzyme β-glucuronidase (GusB) causing the accumulation of GAGs that inhibit their own maturation to myelinating cells. The result is severe dysmyelination during early CNS maturation and impaired neurodevelopment. Genetic overexpression of GusB rescues the maturation deficits and CNS myelination in THAP1 deficient mice raising the critical question of whether β-glucuronidase enzyme replacement could restore myelination in THAP1 null mice. OBJECTIVE: Characterization of β-glucuronidase for enzyme replacement therapy (ERT) in DYT6 dystonia models. METHOD: To establish a symptomatic model of DYT6 dystonia, we utilized a Cre/LoxP based Thap1 conditional knockout mouse model ("THAP1-NCKO") and a GusB transgenic mouse line ("GusB-TG"). We evaluated the enzymatic activity and biodistribution of GusB in the CNS using biochemical and histochemical assays. RESULTS: We determined that THAP1-NCKO mice had lower GusB activity than their control counterparts. Interestingly, the activity of GusB is higher in adult (P60) mice compared to juvenile (P30) mice. Visualization of GusB activity showed that distribution of GusB was highest in white matter tracts. We showed that mice with THAP1- related deficits experienced a significant reduction in GusB activity within white matter tracts but not in other surveyed GusB positive brain areas. CONCLUSIONS: Age differentially affects CNS GusB enzymatic activity in a murine model of DYT6 dystonia. GusB enzyme exhibits a distinct biodistribution that varies regionally. White matter tracts experience more severe defects with THAP1 loss. Our results provide insights into the specific locations where GusB activity is deficient and highlight the importance of a "critical period" in which genetic insults have long lasting neurodevelopmental implications.Item Characterizing New Molecules and Mechanisms of Semaphorin/Plexin/MICAL Signaling(2013-07-16) Yoon, Jimok; Krämer, Helmut; Cobb, Melanie H.; Henkemeyer, Mark; Terman, Jonathan R.The mechanisms that regulate the cellular behaviors including morphology, motility, navigation, and connectivity that are critical for normal human health are still incompletely understood. These types of behaviors are regulated through the ability of guidance cues that are present outside of cells to exert precise effects on the cell’s internal actin cytoskeleton. To help characterize an underlying logic for these cellular changes and proper cellular function I have been characterizing how one of the largest families of extracellular guidance cues, members of the Semaphorin family of proteins, induces actin cytoskeletal changes. Interestingly, Semaphorins have been found to inhibit the movement of cells and their membranous processes but the molecules linking them to these specific behaviors have remained poorly understood. Therefore, during my graduate work, I began further characterizing a new family of proteins, the MICALs that were identified as cytoplasmic binding partners of the Semaphorin receptor Plexin. Combining Drosophila genetics with in vitro biochemical assays, my work revealed that Mical regulates actin organization both in vivo and in vitro and is a novel actin disassembly factor. These results provide a new basis for understanding how extracellular guidance cues regulate the actin cytoskeleton. I then went on to further explore Mical-mediated actin filament (F-actin) disassembly. In one line of investigation, my work revealed that Mical plays an antagonistic role to F-actin stabilizing/bundling proteins including fascin and espin in regulating the F-actin cytoskeleton in vivo. This work also indicated that Semaphorin/Plexin/Mical activity not only directly disassembles the F-actin cytoskeleton but also triggers other actin regulatory proteins to reorganize a more complex F-actin network, resulting in increased cellular plasticity. In another line of investigation, I found that the Abl non-receptor tyrosine kinase is a new Mical-interacting protein. My functional assays revealed that Abl and Semaphorin/Plexin/Mical work together to regulate F-actin arrangements and these interactions are conserved in many different contexts including bristle cell morphology, axon guidance, and cancer cell survival and invasion. Thus, I have found that MICAL family proteins are novel controllers of the actin cytoskeleton, functioning directly on F-actin and with other actin regulatory proteins to modulate diverse cellular behaviors.Item Chemical Genetic and Genomic Discovery of PARP-1-Dependent Mechansims of Transcriptional Regulation(2016-02-17) Gibson, Bryan Andrew; Rice, Luke M.; Rosen, Michael K.; Kraus, W. Lee; Li, BingPoly(ADP-ribose) polymerases, or PARPs, are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from NAD+ onto target proteins. These targets of post-translational modification as well as the genomic targets, or binding sites, to which the nuclear PARP family members localize reflects the molecular biology and cellular function of an individual PARP. Given that PARP proteins are implicated in the most devastating of human disease, including cancer, heart disease, stroke, and neuropathology, a deeper understanding of their proteomic and genomic targets may guide effective therapeutic intervention within this family of enzymes. Here, I report the development of new methodologies in an effort to identify the targets of PARPs. To identify the sites of ADP- ribsoylation, I've developed a simple and robust analog-sensitive approach for PARPs, which allows PARP-specific clickable ADP-ribosylation. Using this approach, I have mapped hundreds of protein targets and sites of ADP-ribosylation for PARPs 1, 2, and 3. I found that PARP-1 ADP-ribosylates and inhibits RNA-binding by NELF, a protein complex that regulates promoter-proximal pausing by RNA polymerase II. I have used this analog-sensitive approach to discover genomic sites of PARP-1-mediated ADP-ribosylation and their relationship to paused RNA Polymerase II. Furthermore, I have found that knockdown of PARP-1 resulted in the accumulation of paused RNA Polymerase II, implicating PARP-1 in RNA Polymerase II elongation through ADP-ribosylation and inhibition of NELF. PARP-1 is activated by both DNA lesions and nucleosomally wrapped DNA, the latter of which is a likely substrate for PARP-1 in its role as a regulator of transcription. In order to identify the nucleosomes that PARP-1 binds, I've developed an MNase ChIP-seq method using crosslinked cells called XL- MNase ChIP-seq. This technical advance has revealed that PARP-1 binds a nuclease-sensitive nucleosome that spans the "nucleosome free" region of regulatory elements of the genome, implicating PARP-1 as a modulator of genomic access at regulatory regions across the genome. The utility of these new methodologies is evident in their use in the discovery of new biological roles for PARP-1 in transcriptional regulation.Item Chemical Inhibition of RNA Viruses Reveals REDD1 as a Host Defense Factor(2013-08-07) Mata, Miguel Angel; Roth, Michael G.; White, Michael A.; Lum, Lawrence; Fontoura, BeatrizInfluenza (flu) is a contagious infectious respiratory illness. The flu can cause from mild to life-threatening illness. The current therapeutic intervention strategies to prevent or treat influenza infection are not sufficient in the event that a pathogenic virus strains reaches pandemic proportions. Therefore, the development of anti-influenza therapeutic modalities is critical to respond to a future influenza pandemic. In this study, a chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reversed NS1-mediated inhibition of host gene expression. A counterscreen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus (VSV). The mechanism of action occurs through activation of REDD1 expression and concomitant inhibition of mammalian target of rapamycin complex 1 (mTORC1) via TSC1–TSC2 complex. The antiviral activity of naphthalimides was abolished in REDD1−/− cells. Inhibition of REDD1 expression by viruses resulted in activation of the mTORC1 pathway. REDD1-/- cells prematurely upregulated viral proteins via mTORC1 activation and were permissive to virus replication. In contrast, cells conditionally expressing high concentrations of REDD1 downregulated the amount of viral protein. Whole animal studies revealed REDD1-/- mice are highly susceptible to virus infection. Influenza infection of REDD1-/- mice results in decreased TLR7 and MHC class II expression by dendritic cells and macrophages. In addition, excessive inflammatory cell infiltration in the lungs of REDD1-/- infected mice was observed. Preliminary evidence suggests a potential defect in NF-κB signaling upon influenza virus infection in REDD1 deficient mice. Thus, REDD1 is a new host defense factor, and chemical activation of REDD1 expression represents a potent antiviral intervention strategy. Our studies also reveal passage immortalization of REDD1-/- MEFs require loss of the type I IFN response pathway as these cells are unable to induce the expression of interferon genes and interferon inducible genes when challenged with synthetic dsRNA. In contrast, primary or SV40 large T antigen transformed REDD1-/- MEFs activate a type I IFN response when exposed to synthetic dsRNA.Item Chemical Intervention of Influenza Virus mRNA Nuclear Export(2020-07-15) Esparza, Matthew Aaron; Shay, Jerry W.; Fontoura, Beatriz; Grinnell, Frederick; Minna, John D.Influenza A viruses are human pathogens with limited therapeutic options, making it crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. We show that influenza virus utilizes nuclear speckles to promote post-transcriptional splicing of its M1 mRNA. We assign previously unknown roles for the viral NS1 protein and cellular factors to an intranuclear trafficking pathway that targets the viral M1 mRNA to nuclear speckles, mediates splicing at these nuclear bodies, and exports the spliced M2 mRNA from the nucleus. In addition, gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway, inhibits M mRNA nuclear export without significantly altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that inhibits nuclear export of a subset of viral and cellular mRNAs via the mRNA export factor UAP56 without altering bulk cellular mRNA nuclear export. These findings underscore specific nuclear export requirements for viral mRNA nuclear export. This RNA export inhibitor also impaired replication of diverse influenza virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.Item Composition, Assembly, and Dynamics of PML Nuclear Bodies(2021-05-01T05:00:00.000Z) Rice, Allyson M.; Jaqaman, Khuloud; Banaszynski, Laura; Yu, Hongtao; Rosen, Michael K.Biomolecular condensates concentrate biomolecules into two- or three-dimensional foci that lack surrounding membranes and can scale in diameter from tens of nanometers to several microns in cells. Promyelocytic leukemia nuclear bodies (PML NBs) are a biomolecular condensate conserved across mammalian, avian and reptilian species. They are primarily composed of the PML protein, and over two hundred additional proteins have reported associations with PML NBs. While the precise function of PML NBs has remained ambiguous, they have been implicated in diverse cellular pathways, from viral response to epigenetics. Previous studies surrounding PML NBs in cells have been largely qualitative, and the physical mechanisms underlying PML NB assembly, dynamics and composition remain largely unknown. In this thesis, I have used quantitative imaging methods in live cells to elucidate properties of PML NBs in their physiological environment. I identified the role of a PML post translational modification, addition of the small ubiquitin-like modifier (SUMO) at lysine 65, in balancing the composition with the dynamics of PML NBs. Additionally, I discuss PML NB behaviors throughout the cell cycle, investigate client recruitment into PML NBs, and evaluate the contribution of ordered domains in PML in PML NB formation. Finally, I analyzed different models for PML NB assembly and propose future lines of study that could uncover their assembly mechanism. Collectively, my work sheds light on a natural condensate and illustrates the complexity of condensate regulation in vivo.Item Controlling Gene Expression with Synthetic Molecules(2006-08-11) Alluri, Prasanna G.; Kodadek, Thomas J.Aberrant gene expression patterns have been implicated in several pathological states. Synthetic molecules capable of functionally mimicking native transcription factors and regulating gene expression in a specific and predictable manner may represent a new paradigm in drug development. Native transcription factors are minimally composed of two domains, a DNA-binding domain (DBD) and an activation domain (AD). Several synthetic DBDs capable of recognizing DNA in a sequence specific manner have been reported in the literature. Furthermore, studies have demonstrated that coupling of these synthetic DBDs to peptides that are capable of acting as activation domains results in chimeric molecules that are capable of activating target gene expression. Since peptides and other biomolecules generally have poor cell-membrane permeability and are prone to rapid enzymatic inactivation inside cells, it is highly desirable to develop artificial molecules that are capable of mimicking native ADs. Towards this goal, a comprehensive methodology for the synthesis, screening and characterization of large peptoid libraries has been developed. Peptoids are a new class of peptidomimetic compounds that are resistant to proteolytic cleavage and are relatively simple and cheap to synthesize. One of the combinatorial libraries was screened against CBP (CREB-binding protein), an important transcriptional coactivator, and three novel, low micromolar affinity ligands were isolated. A cellbased reporter gene assay was employed to assess the cell permeability and transcription activation potential of the synthetic ligands in live mammalian cells. The assay consists of transfecting into HeLa cells a luciferase reporter gene harboring Gal4 binding sites and a construct in which the ligand binding domain of the Glucocorticoid receptor has been fused to Gal4 DBD. The cells are treated with the CBP-binding peptoids that have been chemically coupled to a dexamethasone derivative. Among the three peptoids tested, one of the molecules as a steroid conjugate, has been found to activate the transcription of a reporter gene nearly 1000-fold suggesting that it may be acting as an activation domain surrogate. The mechanistic aspects of the observed transcriptional activity of the peptoid-steroid conjugate remain to be elucidated.Item Defining Genes and Circuits Affecting Naïve and Experience-Dependent Alcohol Preference in Drosophila melanogaster(2015-07-22) Ojelade, Shamsideen Adeniyi; Krämer, Helmut; Terman, Jonathan R.; Self, David W.; Rothenfluh, AdrianDespite alcohol being one of the most used and abused drugs in the world, the molecular mechanisms underlying alcohol abuse disorders remain largely unknown. In this dissertation, I utilized the model system Drosophila melanogaster to identify genes and circuits affecting ethanol-induced behaviors. From an unbiased genetic screen, I identified a novel gene that affects ethanol consumption in both flies and humans. Ras suppressor 1 (Rsu1) is required in the adult Drosophila nervous system for normal sensitivity to ethanol-induced sedation, and acts upstream of Rac1 and downstream of integrin to regulate the actin cytoskeleton. In a two bottle choice assay called the capillary feeding (Café) assay, loss of Rsu1 causes immediate heightened alcohol preference compared to wild type's initial naïve aversion. In contrast, flies specifically lacking Rsu1 in the mushroom bodies show normal initial aversion to alcohol, but then fail to acquire ethanol preference like normal flies do. Our data show that not only is Rsu1 required for normal alcohol responses, it suggests that different anatomical brain structures in flies control distinct alcohol behavioral responses. In humans, we find that polymorphisms in RSU1 are associated with brain activation in the ventral striatum during reward anticipation in adolescents and alcohol consumption in both adolescents and adults. Together, these data suggest a conserved role for integrin/Rsu1/Rac1/actin signaling in modulating reward-related phenotypes, including ethanol consumption in flies and humans. Using a modified Café paradigm, we investigated whether dopamine plays a role in both the aversive and experience-dependent properties of alcohol. I show that distinct subsets of DA neurons innervating the Fan-shaped body (FSB) and Mushroom body (MB) mediate naïve alcohol aversion (NAA) and experience-dependent alcohol preference (EDAP) respectively in flies. Furthermore, Rac1-dependent actin alteration in these anatomical structures (FSB and MB) also mirror dopaminergic-induced neuronal activity in these circuits suggesting that dopamine functions upstream of Rac1-signaling to affect alcohol preference in flies. Taken together, my dissertation suggests a conserved role for dopamine and the integrin/Rsu1/Rac1/Cofilin/Actin signaling pathway in modulating drug-induced behavioral plasticity across phyla, and highlights Drosophila as an effective model for integrative translational research.Item Defining the Constellation of RNA Elements That Associate with Bacillus Subtilis HFQ(2013-01-17) Dambach, Michael David; Winkler, Wade C.Bacteria utilize a wide variety of genetic regulatory strategies in order to sense and respond to various environmental fluctuations in nutrient availability, temperature, salinity, and oxygen among others. As such, bacterial species have evolved highly coordinated and tightly regulated systems as a means of efficiently responding to potentially deleterious changes in environmental conditions. Traditionally DNA binding transcriptions factors were thought to be the primary means by which the cell executes a selective genetic response. However, the advent of microarray and next generation sequencing platforms, coupled with the wealth of sequenced genomes and powerful bioinformatics have revealed that RNA mediated post transcriptional gene regulation is wide spread in bacterial species and may in fact rival protein based regulatory systems in scope and breadth. RNA mediated post transcriptional gene regulation is broadly divided into two categories-those in which the RNA element is transcribed with the mRNA it regulates (cis-acting regulatory RNAs) or those which are transcribed independently from the gene that they regulate (trans-acting regulatory RNAs). In general cis-acting RNA elements are embedded within a 5' UTR of a gene that they regulate and may or may not require a protein cofactor to execute genetic regulation. Whereas, trans-acting regulatory RNAs, also known as sRNAs, function via base pairing with their target mRNA and this usually requires the protein chaperone Hfq. Hfq mediated gene regulation is poorly understood in Gram-positive organism, thus I undertook studies of this protein in the model Gram-positive organism Bacillus subtilis. I used co-immunoprecipitation and deep-sequencing to define the suite of RNA elements that associate with this regulatory protein. In addition I performed global transcriptomic studies on an Hfq deletion mutant in order to identify genes that are regulated via Hfq. These studies identified sRNAs that may be involved with sporulation. This led me to analyze the transcriptomic profile of Bacillus subtilis spores in an attempt to identify new sRNA regulators.Item The Design and Development of Artificial Zinc Finger Transcription Factors and Zinc Finger Nucleases to the hTERT Locus(2011-02-01) Wilson, Kimberly Anne; Porteus, Matthew H.The ability to direct hTERT expression through genetic control or tunable regulatory factors would advance our understanding of the transcriptional regulation of hTERT, and also potentially produce new strategies for addressing telomerase-associated disease. In this work, we describe the engineering of artificial zinc finger transcription factors (ZFTFs) and zinc finger nucleases (ZFNs) to target sequences at the hTERT promoter. We first explored expansions to the repertoire of sites that can be targeted by ZFNs and modifications of ZFN architecture to accommodate such sites. A ZFN is made of a zinc-finger DNA binding domain (ZFP) linked to the FokI nuclease domain by a short amino acid “inter-domain linker”. The general sequence motif of a ZFN target is 5’-(ZFN site1)-(6 bp spacer)-(ZFN site2)-3’ and each half-site is 5’-GNNGNNGNN-3’. Variations of this motif come in the forms of variable spacer lengths, extra basepairs in-between triplets, and the inclusion of non-GNN triplets. To explore these types of target sites, we created ZFN variants that contained different inter-domain linkers, lengthened inter-finger linkers, and DNA binding domains created through hybridizing the modular assembly and OPEN methodologies. We show that through altering ZFN architecture, target sites with 5-7-bp spacers and those with ANN, CNN, and TNN triplets can be efficiently recognized and cut by ZFNs. We then generated new ZFPs to five ZFN target sites with 5- or 6-bp spacers in the hTERT locus based on those findings and made ZFTFs by linking the ZFPs to the VP16 transcriptional activation domain. We were able to identify several active ZFTFs that demonstrate a dose-dependent response. The same ZFPs were also converted into ZFNs and screened in combinatorial pairs in cell-based single-strand annealing assays and gene targeting assays. These screening strategies have pinpointed several ZFN pairs that may be useful in genomic editing of the hTERT locus. Our findings provide guidelines for modifying ZFP architecture to a wider array of potential target sites for use in developing ZFTFs and ZFNs at the hTERT promoter, which may be applicable towards inheritable, telomerase-based diseases and answering basic science questions about hTERT transcriptional regulation.Item Downregulation of the Cytosolic Iron-Sulfur Assembly Pathway in Cancer by an E3 Ubiquitin Ligase(2017-06-19) Weon, Jenny Linda; Tu, Benjamin; Potts, Patrick Ryan; Minna, John D.; Liu, YiIron-sulfur (Fe-S) clusters are considered to be one of the oldest cofactors utilized by proteins and are essential for life from bacteria to mammals. Multiple processes in the cell require Fe-S cofactors, such as electron transfer in mitochondrial respiration, enzymatic reactions, and as structural components in DNA repair enzymes. We describe here the first post-translational mechanism to regulate Fe-S assembly and delivery through the ubiquitination and degradation of a key cytosolic iron-sulfur cluster assembly (CIA) pathway component by a MAGE-RING ligase (MRL). The MAGE protein family consists of ~40 members in humans that function in complex with E3 ubiquitin ligases to enhance ubiquitination activity, alter E3 subcellular localization, and/or specify E3 targets. Using biochemical and cellular approaches we have discovered that the MAGE-F1-NSE1 ligase disrupts Fe-S cluster delivery through ubiquitination and degradation of the CIA pathway protein MMS19. MMS19 is a substrate specifying, late-acting component of the CIA pathway that facilitates Fe-S transfer from the multi-component cascade of assembly proteins to specific recipient apoproteins. Notably, many MMS19 targets are enzymes involved in DNA repair. We found that MAGE-F1 directs the E3 ligase NSE1 to target MMS19 for ubiquitination and degradation. Knockdown of MAGE-F1 stabilized MMS19 and overexpression of MAGE-F1 decreased MMS19 levels without affecting MMS19 mRNA levels. We further confirmed MAGE-F1 inhibits Fe-S incorporation into known MMS19-dependent Fe-S proteins, such as FANCJ, POLD1, RTEL1, XPD, and DPYD, but not MMS19-independent Fe-S proteins, such as PPAT. Loss of Fe-S incorporation leads to decreased DNA repair capacity of cells, exemplified by decreased homologous recombination rates and altered sensitivity to DNA damaging agents. Importantly, numerous cancer types harbor copy-number amplification of MAGE-F1, including lung squamous carcinoma and head and neck squamous carcinoma. Consistent with MAGE-F1 inhibitory activity on Fe-S incorporation into key DNA repair enzymes, MAGE-F1-amplified tumors bear a significantly greater mutational burden than non-MAGE-F1-amplified cancers and the expression of MAGE-F1-NSE1 correlates with poor patient prognosis. In summary, we provide the first evidence for post-translational regulatory control of Fe-S cluster assembly and a novel mechanism by which a broad spectrum of DNA repair enzymes can be regulated and lead to genomic instability in cancer.Item Dynamics of Cell Fate Decision Making Between Sporulation and Competence in Bacillus Subtilis(2013-01-02) Kuchina, Anna 1985-; Altschuler, Steven J.; Alto, Neal; Graff, Jonathan M.; Süel, Gürol M.During multipotent differentiation cells must reliably make a cell fate decision under a variety of conditions, yet remain sensitive to changes in extracellular environment. It is unclear how the cells reconcile these seemingly contradictory requirements. To complicate the issue, the cells often face a decision between multiple fates mediated by the respective differentiation programs which could become active at once. How cells make a specific cell fate choice when presented with several possibilities is a fundamental, yet poorly resolved question. To study cell-fate decision-making dynamics, I utilized the soil bacterium Bacillus subtilis which under stress can either become competent for DNA uptake or undergo sporulation. The master regulator of sporulation is the transcription factor Spo0A. Single cell measurements of Spo0A dynamics along with activities of stage-specific sporulation reporters Spo0F, SpoIIE and SpoIIR revealed the reversible and noisy progression of sporulation up until the final irreversible decision point. Mathematical modeling suggested that such strategy might be advantageous for coping with unpredictable environment. The alternative cell fate of competence is controlled by the transcription factor ComK. Using time-lapse fluorescence microscopy, I quantitatively measured the activities of Spo0A and ComK, along with other cross-regulatory genes, simultaneously in single B. subtilis cells. I found that, surprisingly, sporulation and competence progressed independently in the same cell without cross-regulation up to the final decision point. This finding was confirmed by the discovery of cells in a conflicted state that progressed to sporulation despite the expression of ComK. Measurements of gene expression dynamics in these cells revealed key differences in the relative timing of differentiation programs. To investigate the importance of relative timing, I altered it by engineering artificial cross-regulatory links between the sporulation and competence genetic circuits. Results favor a simple model for cellular decision-making that does not require intricate cross-regulation prior to the decision. Rather, cell fate choice appears to be the outcome of a "molecular race" between independently progressing differentiation programs. This temporal competition mechanism provides a simple, yet efficient way to generate mutually exclusive cell fates. Investigation of the benefits and limitations of such strategy opens a promising venue for future studies.Item The Effects of Chromatin Remodeling and Pseudokinase Activity on Liver Pathophysiology(August 2021) Moore, Austin Bradley; Agathocleous, Michalis; Hobbs, Helen H.; Hoshida, Yujin; Zhu, HaoThe liver exhibits a remarkable capability to regenerate itself in the face of injury; however, in the setting of sustained damage, this capability can be overwhelmed and eventually lead to chronic liver diseases such as non-alcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. Although these processes are complex and not completely understood, specific genetic and epigenetic factors that drive aspects of this pathophysiology can shed further light on both how these diseases develop and on how normal, healthy regeneration differs from liver disease. In this body of work, we show that loss of Arid1a, a DNA-binding component of the SWI/SNF chromatin remodeling complex, shifts hepatocyte metabolism to promote lipid accumulation in a manner similar to that seen in non-alcoholic fatty liver disease. We further explore the dynamics of the SWI/SNF complex by examining a mutually exclusive homolog of Arid1a, Arid1b, and provide evidence to suggest that its role is to stabilize the hepatocyte SWI/SNF complex in the absence of Arid1a seen in regeneration and hepatocellular carcinoma. Finally, we harness the power of in vivo CRISPR screening within the liver to identify the pseudokinase STK31 as a positive regulator of hepatocyte proliferation and liver oncogenesis. Our findings underscore the important role that chromatin remodeling has in enforcing hepatocyte identity and functionality as well as allowing for plasticity. Additionally, our work with STK31 highlights the power of in vivo screening within the liver to identify new potential therapeutic targets not only for hepatocellular carcinoma, but other tumor types as well.Item Epigenetic Regulation of Oligodendrocyte Development and Regeneration in the Central Nervous System(2016-10-26) He, Danyang; Kraus, W. Lee; Lu, Q. Richard; Johnson, Jane E.; Olson, Eric N.Oligodendrocytes (OLs) produce myelin sheaths that electrically insulate axons and promote rapid propagation of action potentials in the CNS. The onset and timing of CNS myelination and remyelination requires precise coordination between epigenetic programming and transcriptional regulation. In this thesis, I present my findings on two epigenetic regulatory complexes Chd7/Sox10 and lncOL1/Suz12 in CNS myelination and remyelination. First, we show that chromatin remodeler Chd7 is required for proper onset of CNS myelination and remyelination. Genome-occupancy analyses, coupled with transcriptome profiling, reveal that Chd7 interacts with Sox10 and targets the enhancers of key myelinogenic genes, and identify novel Chd7 targets including bone formation regulators Osterix/Sp7 and Creb3l2, which are also critical for oligodendrocyte maturation. Thus, Chd7 coordinates with Sox10 to regulate the initiation of myelinogenesis and acts as a molecular nexus of regulatory networks that account for the development of a seemingly diverse array of lineages including oligodendrocytes and osteoblasts, pointing to the hitherto previously uncharacterized Chd7 functions in white matter pathogenesis in CHARGE syndrome. To understand the role of lncRNAs in CNS myelination, we establish dynamic expression profiles of lncRNAs at different stages of oligodendrocyte development and uncover a cohort of stage-specific oligodendrocyte-restricted lncRNAs including a conserved chromatinassociated lncOL1. Genetic inactivation of lncOL1 causes defects in CNS myelination and remyelination following injury. Functional analyses illustrate that lncOL1 interacts with Suz12, a component of PRC2, to promote oligodendrocyte maturation in part through Suz12-mediated repression of a differentiation inhibitory network that maintains the precursor state. Collectively, these studies show that epigenetic circuitry between lncRNAs and transcription factors with chromatin-modifying complexes play roles in balancing inhibitory and activating gene program, allowing the timely CNS myelination and myelin repair.Item Examining the Role of PRDM13 in Dorsal Interneuron Specification(2016-07-29) Uruena, Ana Cristina; Wu, Jiang I.; Johnson, Jane E.; MacDonald, Raymond J.; Morrison, Sean J.PTF1A is a transcription factor transiently expressed as neural progenitor cells become post-mitotic and begin to express neuronal specific genes. PTF1A specifies these cells to become GABAergic (inhibitory) neurons while suppressing the glutamatergic (excitatory) program. A fundamental principle in bipotential fate decisions is the necessity to repress gene programs in the alternative fate. Our lab identified PRDM13, a zinc finger transcription factor and direct downstream target of PTF1A that may serve this function in the inhibitory/excitatory neuron fate choice. Overexpression of PRDM13 in chick neural tube shows it represses markers of the excitatory neuronal lineage. To explore PRDM13 function in vivo and expand these findings to regions outside the neural tube, a Prdm13GFP_KI and Prdm13ΔZF mutant mouse strains was generated and are null for PRDM13 expression. These mice die neonatally and at E10.5 show an increase in the dorsal neural tube excitatory neuron population at the expense of the inhibitory neurons. These phenotypes recapitulate that seen in Ptf1a null mice. These models have revealed additional insights into the function of PRDM13 in the developing spinal cord. First, PRDM13 negatively regulates Ptf1a providing a mechanism for downregulating PTF1A as development progresses. Second, in contrast to the phenotype seen with Ptf1a mutants, late stage mutant embryos show only a partial loss of the inhibitory interneuron population, possibly due to the higher levels of PTF1A in these mutants. Finally, ChIP-Seq and RNA-Seq analysis of heterozygote vs homozygote Prdm13 mutants revealed a novel function of PRDM13 to keep neuronal subtype specification genes for the ventral neural tube suppressed in the dorsal region. These mouse models has placed PRDM13 in a pivotal role in the specification of neuronal subtypes in the spinal cord, a function that will likely extend to the retina and cerebellum where PRDM13 is also present.Item FHL2 Inhibits Calcineurin and Represses Pathological Cardiac Hypertrophy(2010-11-02) Hojayev, Berdymammet; Hill, Joseph A.Stress-induced cardiac hypertrophy is a hallmark feature of pathological remodeling which, left unchecked, predisposes hearts to arrhythmia and failure. FHL2 is a member of the four-and-a-half LIM domain (FHL) family of proteins expressed predominantly in the heart. Targeted disruption of FHL2 leads to an exaggerated response to beta-agonist (isoproterenol)-induced cardiac hypertrophy. Isoproterenol-induced hypertrophy relies on activation of the calcineurin-NFAT pathway, and inhibition of calcineurin is sufficient to block growth in response to isoproterenol. I also observed that FHL2 is up-regulated in mouse hearts after isoproterenol treatment. Based on this, we hypothesized that FHL2 negatively regulates the calcineurin-NFAT pathway and consequently, the hypertrophic growth response. To determine whether calcineurin signaling is enhanced in the absence of FHL2, wild type (WT) and FHL2 knockout (FHL2-/-) mice were treated with isoproterenol (32 mg/kg/day). We observed a significant increase in isoproterenol-induced expression of the NFAT target genes RCAN1.4 and BNP in FHL2-/- hearts as compared to WT. To determine whether the effect of FHL2 on the abundance of NFAT target gene transcripts was mediated by calcineurin-NFAT-dependent transcription, HEK 293 cells were transfected with luciferase reporter constructs containing the NFAT-driven promoters of either RCAN1 or IL-2. Consistent with the in vivo data, knockdown of FHL2 message using siRNA led to increases in both RCAN1 and IL-2 promoter activities elicited by constitutively active calcineurin or the calcium ionophore, ionomycin. Importantly, activation of the RCAN1 promoter by ionomycin, in control and FHL2 knockdown cells, was abolished by the calcineurin inhibitor cyclosporin A, confirming the calcineurin dependence of the response. Over-expression of FHL2 in HEK 293 cells inhibited the activation of both NFAT reporters triggered by either constitutively active calcineurin or ionomycin. Furthermore, neonatal rat ventricular myocytes over-expressing FHL2 exhibited reduced hypertrophic growth in response to constitutively active calcineurin (measured by cell cross-sectional area and fetal gene expression). Finally, immunostaining of adult cardiomyocytes revealed co-localization of FHL2 and calcineurin predominantly at the sarcomere, and activation of calcineurin by endothelin-1 treatment resulted in interaction between FHL2 and calcineurin as demonstrated by coimmunoprecipitation. These observations demonstrate that FHL2 represses calcineurin-NFAT signaling and thereby suppresses hypertrophic cardiac growth at least in part by interacting with calcineurin and inhibiting its activation.