Browsing by Subject "Gene Expression Regulation"
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Item Biochemical Characterization Of Delta FosB(2006-12-19) Carle, Tiffany Lynn; Phillips, MegDeltaFosB, 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 Characterization of Drosophila SCAP: Analysis of Mutants and Evidence for a Retention Factor(2011-08-26T17:34:23Z) Ozdemir, Cafer; Rawson, Robert B.The SREBP pathway is one of the major regulators of lipid homeostasis and it is highly conserved among metazoans. SREBP is a transcription factor whose precursor is an endoplasmic reticulum (ER) transmembrane protein. In order to be activated it must travel to the Golgi apparatus via interaction with an escort protein, Scap. Scap, in turn can interact with components of the coatamer protein complex II (COPII) when lipid levels fall. In the Golgi, SREBP is cleaved sequentially by two proteases, S1P and S2P. By contrast to mammalian cells, which cannot survive without S2P or Scap, flies lacking Scap or S2P can activate SREBP. These mutants survive owing to non-canonical mechanisms of SREBP activation. Scap has a intrinsic tendency to travel to Golgi. In vertebrates, the ER retention factor, Insig, anchors the Scap:SREBP complex to the ER membrane when de novo lipid synthesis is not required. In Drosophila dSREBP pathway there is no Insig orthologues. However, our data suggest that there should be an analogous component that retains dScap in the ER. In order to discover the putative retention factor and other modifiers of the dSREBP, I set up a high through-put genome-wide screen. Employing luciferase as reporter, knocking-down each gene in genome through RNA interference will reveal the genes that modulate the activity of dSREBP.Item Controlling Gene Expression With Synthetic Molecules(2006-08-11) Alluri, Prasanna G.; Kodadek, ThomasAberrant 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 Design and Development of Artificial Zinc Finger Transcription Factors and Zinc Finger Nucleases to the HTERT Locus(2011-02-01T19:36:40Z) Wilson, Kimberly Anne; Porteus, MatthewThe 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 The Function and Mechanism of RNA Interference in Neurospora(2009-01-14) Lee, Heng-Chi; Liu, YiRNA interference (RNAi) is a conserved gene silencing mechanism important for various biological processes, including developmental timing, genome defense, and heterochromatin formation. RNAi is triggered by double stranded RNA (dsRNA), which is processed by Dicer to siRNA. siRNA is loaded onto RNA-induced silencing complex (RISC), in which an Argonaute family protein, guided by a siRNA, mediates the cleavage of homologous RNAs. In the filamentous fungus Neurospora, we show that dsRNA not only trigger RNAi, it also transcriptionally activates several key components of RNAi pathway, including qde-2 (an Argonaute) and dcl-2 (a Dicer). A genome wide identification of dsRNA activated genes suggests that RNAi is part of a broad ancient host-defense response against viral and transposon infections. Our research on qde-2 regulation also suggests a role of RNAi during DNA damage. We show that DNA damage induces qde-2 expression, and the purification of QDE-2 bound RNAs identifies a novel class of small RNAs named qiRNAs. qiRNAs are averaged 21 nt in length and are mostly derived from ribosomal DNA (rDNA) locus. Importantly, qiRNA biogenesis requires RNAi components and RNAi mutants exhibit increased sensitivity to DNA damage, suggesting a role for qiRNAs during DNA repair. Further analysis suggests that the qiRNA contributes to the DNA damage checkpoints by inhibiting protein translation after DNA damage. To trigger RNAi against transgenes, it has been proposed that transgene- specific aberrant RNA (aRNA) is made and converted into dsRNA by RNA dependent RNA polymerase (RdRP). How aRNA is produced and specifically recognized by RdRP is not known. We show that QDE-1, a RdRP is also the DNA-dependent RNA polymerase (DdRP) that produces aRNA from ssDNA. QDE-1 is recruited to ssDNA by Replication Protein A (RPA) and QDE-3 (an RecQ helicase), both of them are also essential for aRNA production. Moreover, QDE-1 can produce dsRNA from ssDNA, a process facilitated by RPA. Our results provide a molecular mechanism of aRNA production in RNAi pathway.Item The Functional Roles of Steroidogenic Factor 1 in the Ventromedial Nucleus of the Hypothalamus(2009-06-17) Kim, Ki Woo; Parker, Keith L.Steroidogenic factor 1 (SF-1) is a nuclear receptor that plays important roles in the hypothalamic-pituitary-steroidogenic organ axis. Global knockout studies in mice revealed the essential in vivo roles of SF-1 in the ventromedial hypothalamic nucleus (VMH), adrenal glands, and gonads. One limitation of global SF-1 knockout mice is their early postnatal death from adrenocortical insufficiency. To overcome limitations of the global knockout mice and to delineate the roles of SF-1 in the brain, we used Cre/loxP recombination technology to genetically ablate SF-1 specifically in the central nervous system (CNS). Mice with CNS-specific knockout of SF-1 mediated by nestin-Cre showed increased anxiety-like behavior, revealing a crucial role of SF-1 in a complex behavioral phenotype. Our studies with CNS-specific SF-1 KO mice also defined roles of SF-1 in regulating the VMH expression of target genes implicated in anxiety and energy homeostasis. Therefore, present work will focus on the functional roles of SF-1 in the VMH linked to anxiety and energy homeostasis.Item HIF-2: Standing Guard at the Crossroads of Stress and Aging(2009-06-15) Dioum, El Hadji Mamadou; Garcia, Joseph A.The capacity of mammalian organisms to cope with hypoxic or ischemic stress is in part mediated by stress-induced transcription factors. Hypoxia-induced mediators include transcription factors, such as the α (alpha) subunit of Hypoxia inducible factors (HIF-1alpha and HIF-2 alpha). HIF-1 alpha and HIF-2 alpha have similar structural organization, and after forming an obligate heterodimer with the common partner ARNT/HIF-1 alpha, bind to the same recognition element located in target gene promoter or enhancer regions. However, despite these similarities, HIF-1 alpha and HIF-2 alpha regulate distinct target genes. In previous studies from the Garcia laboratory using mouse knockout studies, we demonstrated the importance of HIF-2 alpha in the in vivo regulation of genes involved in the cellular response to hypoxic and oxidative stress. These genes include Erythropoietin (epo), vascular endothelial cell growth factor (Vegf), superoxide dismutase 2 (Sod2) and other genes encoding major antioxidant enzymes (AOE). Novel roles for HIF-2 alpha have been found not only in hematopoiesis, but also in the control of reactive oxygen species and mitochondrial homeostasis. The molecular mechanism by which HIF-2 alpha selectively regulates its target genes remains an exciting area of research. In the first part of my thesis, I identified a novel molecular mechanism regulating activity of the enhancer region in the Epo gene. First, by using bioinformatics to perform an unbiased sequence comparison of several mammalian 3 prime Epo enhancer region, we identified a previously unrecognized evolutionary conserved region. Second, we determined the functional significance of these conserved sequences using transient transfection and mutation analyses in cell culture studies and determined that these sequences contribute to HIF-2 alpha selectivity. Finally, using a candidate factor strategy, we determined that members of the early growth response (Egr) transcription factor family bind to these elements and act synergistically with HIF-2 alpha to augment Epo gene expression. In the second part of my thesis, we demonstrate that the redox-sensing, NAD+ dependent deacetylase enzyme Sirtuin 1, also known as Sirt1 or silent mating type information regulator 2 (Sir2) homolog 1, selectively stimulates HIF-2 alpha signaling during hypoxia. In lower organisms and cell culture models, the FoxO family of transcription factor regulates the transcription of SOD2 and other major AOE. During oxidative stress, Sirt1 modulates FoxO transcriptional activity, promoting the protective cellular response to oxidative stress. We hypothesized that Sirt1 would be activated by redox changes induced by hypoxia and that activated Sirt1 would in turn modulate HIF-2 signaling. We determined that HIF-2 alpha signaling is indeed increased by Sirt1 in transfection assays. Sirt1/HIF-2 alpha signaling does not involve previously described oxygen-dependent HIF-2 alpha modifications. Sirt1 augmentation of HIF-2 alpha transcriptional activity involves direct binding to and deacetylation of HIF-2 alpha. In cultured cells and in mice models, interventions that decrease or increase Sirt1 activity affect expression of the HIF-2 alpha target gene epo accordingly. Thus, Sirt1 is a molecular switch that promotes HIF-2 signaling during hypoxia and likely other environmental stresses.Item Paxillin is a Novel Regulator of Xenopus Oocyte Maturation(2010-05-14) Young, Melissa Rasar; Hammer, Stephen R.Oocyte maturation is triggered by steroids in a transcription-independent fashion that involves an unusual positive feedback loop whereby MOS (a germ cell specific Raf) activates MEK1, which in turn activates ERK2. ERK2 then acts back on MOS to enhance its expression resulting in amplification of the kinase signaling cascade. To date, little is known regarding other factors that regulate this powerful feedback kinase cascade. Here we present the scaffolding molecule, Paxillin, as a newly recognized essential regulator of meiosis in Xenopus leavis oocytes. Reduction of Paxillin expression using RNA interference and antisense oligonucleotides completely abrogates steroid-triggered meiotic resumption. Detailed signaling studies reveal that Paxillin is acting early in the kinase cascade, as it is required for accumulation of MOS protein and complete activation of downstream kinase signaling in response to steroids. Surprisingly, full Paxillin activity also requires serine phosphorylation by a kinase downstream of MOS and MEK1, possibly ERK2. Together, these data suggest that Paxillin is an important regulator of the positive feedback effects of MEK/ERK signaling on MOS protein expression. The ability of Paxillin to function as a MAPK scaffold was analyzed, revealing Paxillin can interact with MOS in mammalian cells. Furthermore, the ability of Paxillin to regulate activity of proteins important for translation, specifically polyadenylation binding proteins, is briefly explored. In all, these experiments reveal a novel and critical function for Paxillin in meiosis, and support the notion that Paxillin may be general modulator of MAPK signaling and/or mRNA translation by polyadenylation binding proteins.Item Silencing Transcription: Promoter-Targeted Oligonucleotides Bind Chromosomal DNA Inside Cells(2009-01-08) Beane, Randall L.; Corey, DavidAberrant gene expression can lead to multiple disease-states that can be difficult or impossible to treat using traditional small-molecule medications. An alternative approach to treating such diseases is oligonucleotide-based therapeutics, which are theoretically capable of treating or curing genetic diseases, infections, and abnormalities. Oligonucleotide-based molecules targeted to DNA are referred to as antigene agents. These molecules can silence or activate gene transcription of alleles and have many potential medical applications. However, the growth of antigene technologies has been slow despite broad therapeutic potential and unique molecular applications. Through the development of chemical modifications, oligonucleotide-based molecules are actively being improved and refined. Chemical modifications can alter the cellular uptake, toxicity, biodistribution, and plasma retention of oligonucleotides. My research goal was to further the field of synthetic antigene oligonucleotides. To do this, I targeted endogenous genes in human cancer cell lines with chemically-modified oligonucleotides, including MOEs, PNAs, ENAs, and LNAs. I established that LNAs were robust antigene agents capable of inhibiting transcription under multiple conditions. Specifically, I established that mixed-base antigene agents physically associate with the hPR-B promoter and decrease the occupancy of RNA polymerase II on the hAR and hPR genes inside human cells. Furthermore, my research indicates that antigene LNAs function in an orientation-dependent manner and that functional LNAs must target the template strand of DNA to have appreciable potency. This body of work comprised the first extensive study of a mixed-base antigene oligonucleotide in multiple human cell lines and provides the first evidence that mixed-base antigene agents can physically associate with chromosomal DNA and inhibit transcription of endogenous mammalian genes inside human cells. Collectively, my data suggest that antigene LNAs are a potent and general strategy for silencing gene expression, and that antigene LNAs also have potential therapeutic applications and possible utility in modern functional genomics.