The ubiquitin ligase Highwire is responsible for cell-autonomously promoting synapse formation in the Drosophila Giant Fiber system. highwire mutants show defects in synaptic function and extra branching at the axon terminal, corresponding to transient branching that occur in the course of giant synapse formation during metamorphosis. The MAP kinase pathway, including Wallenda and JNK/Basket, plus the transcription factor Jun, act to suppress synaptic function and axon pruning in a dosage sensitive manner, suggesting different molecular mechanisms downstream of the MAP kinase pathway govern function and pruning. A novel role for Highwire is revealed, regulating the giant fiber axon’s ability to respond to external cues regulated by Fos. When expression of the transcription factor Fos is disrupted in the post-synaptic TTMn or surrounding midline glia of highwire mutants, the giant fiber axons show a marked increase in axon overgrowth and midline crossing. However, synaptic function is rescued by the cell nonautonomous manipulation of Fos, indicating distinct mechanisms downstream of Highwire regulating synaptic function and axon morphology. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Sox proteins all contain a single ~70 amino acid High Mobility Group (HMG) DNA-binding domain with strong homology to that of Sry, the mammalian testisdetermining factor. In Drosophila melanogaster, there are four closely related members of the B group, Dichaete (D), Sox Neuro (Sox N), Sox 21a, and Sox 21b that each exhibit ~90% sequence identity within the HMG domain.The previous study has shown that Dichaete plays a major role in embryonic nervous system development and is expressed in several clusters of neurons in the brain, including intermingled olfactory LNs and central-complex neurons strongly expressed in local interneuron of the olfactory system. However, little is known about the possible expression and functions of the related group B Sox genes in the larval and adult brain. In particular, it is unclear if Sox N may function along with Dichaete in controlling the development or physiology of the adult olfactory system. Our data suggests Sox N potential role in the elaboration of the olfactory circuit formation. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2017. / FAU Electronic Theses and Dissertations Collection

Transcriptional regulation is an important area of research due to the fact that it leads to gene expression. Transcription factors associated with the regulation can either be activators or repressors of target genes, acting directly or with the aid of other factors. A majority of transcriptional repressors are zinc finger proteins (ZFPs) which bind to specific DNA sequences. The Snail/Gfi (SNAG) domain family, with members such as Slug, Smuc, Snail, and Scratch, are transcriptional repressors shown to play a role in various diseases such as cancer. The SNAG transcription factors contain a conserved SNAG repression domain and DNA binding domain zinc fingers. The specific DNA sequences to which each SNAG-ZFP binds, as well as a general consensus -TGCACCTGTCCGA, have been determined. Also, putative protein-protein interactions in which the Slug domain participates has been identified via binding assays. All these results contribute to better understanding of SNAG-ZFP functions. / by Cindy Chung-Yue Chiang. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

The classic guidance molecules, Netrin and its receptor Frazzled (Fra), dictate the strength of synaptic connections in the giant fiber system (GFS) of Drosophila melanogaster by regulating gap junction localization in the pre-synaptic terminal. In Netrin mutant animals the synaptic coupling between a giant interneuron and the jump motor neuron was weakened. Dye-coupling between these two neurons was severely compromised or absent. These mutants exhibited anatomically and physiologically defective synapses between the giant fiber (GF) and tergotrochanteral motor neuron (TTMn). In cases where Netrin mutants displayed apparently normal synaptic anatomy, half of the specimens exhibited physiologically defective synapses. Dye-coupling between the giant fiber and the motor neuron was reduced or eliminated, suggesting that gap junctions were disrupted in the Netrin mutants. When we examined the gap junctions with antibodies to Shaking-B Innexin (ShakB), they were significantly decreased or absent in the pre-synaptic terminal of the mutant GF. This data is the first to show that Netrin and Frazzled regulate placement of gap junctions pre-synaptically at a central synapse. In the Drosophila Giant Fiber System, we demonstrate a mechanism that ensures the monoinnervation of two homologous motor neurons by two homologous interneurons. In a scenario where both interneurons could synapse with both motor neuron targets, each interneuron exclusively synapsed with only one target and the circuit functions at normal physiological levels. This innervation pattern depended on the ratio of netrin-to-frazzled expression. When Netrin was over expressed in the system, shifting the ratio in favor of Netrin, both interneurons synapsed with both target motor neurons and physiological function was reduced. This resulted in the polyinnervationof a single target. In contrast, when Frazzled was over expressed in the system, one interneuron innervated both targets and excluded the remaining interneuron from making any synaptic contact. This resulted in a single interneuron mono-innervating both motor neurons and physiological function was mutant. The orphaned interneuron made no synaptic contact with either motor neuron target. Physiological function was only normal when the Netrin-Frazzled ratio was at endogenous levels and each GF monoinnervated one motor neuron. When we examined the gap junctions at this synapse in experimental animals, there was a significant reduction of gap junction hemichannels in the presynaptic terminal of axons that deviated from normal innervation patterns. While the synapse dyecoupled, the reduction in gap junction hemichannels reduced function in the circuit. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2013.

In the developing CNS, presynaptic neurons often have exuberant overgrowth and form excess (and overlapping) postsynaptic connections. Importantly, these excess connections are refined during circuit maturation so that only the appropriate connections remain. This synaptic rearrangement phenomenon has been studied extensively in vertebrates but many of those models involve complex neuronal circuits with multiple presynaptic inputs and postsynaptic outputs. Using a simple escape circuit in Drosophila melanogaster (the giant fiber circuit), we developed tools that enabled us to study the molecular development of this circuit; which consists of a bilaterally symmetrical pair of presynaptic interneurons and postsynaptic motorneurons. In the adult circuit, each presynaptic interneuron (giant fiber) forms a single connection with the ipsilateral, postsynaptic motorneuron (TTMn). Using new tools that we developed we labeled both giant fibers throughout their development and saw that these neurons overgrew their targets and formed overlapping connections. As the circuit matured, giant fibers pruned their terminals and refined their connectivity such that only a single postsynaptic connection remained with the ipsilateral target. Furthermore, if we ablated one of the two giant fibers during development in wildtype animals, the remaining giant fiber often retained excess connections with the contralateral target that persisted into adulthood. After demonstrating that the giant fiber circuit was suitable to study synaptic rearrangement, we investigated two proteins that might mediate this process. First, we were able to prevent giant fibers from refining their connectivity by knocking out highwire, a ubiquitin ligase that prevented pruning. Second, we investigated whether overexpressing Netrin (or Frazzled), part of a canonical axon guidance system, would affect the refinement of giant fiber connectivity. We found that overexpressing Netrin (or Frazzled) pre- & postsynaptically resulted in some giant fibers forming or retaining excess connections, while exclusively presynaptic (or postsynaptic) expression of either protein had no effect. We further showed that by simultaneously reducing (Slit-Robo) midline repulsion and elevating Netrin (or Frazzled) pre- & postsynaptically, we significantly enhanced the proportion of giant fibers that formed excess connections. Our findings suggest that Netrin-Frazzled and Slit-Robo signaling play a significant role in refining synaptic circuits and shaping giant fiber circuit connectivity. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Genes identified by SAGE may serve as potential prognostic marker or therapeutic target. 14-3-3 sigma is a putative tumor suppressor and can be induced in response to DNA damage following irradiation, leading to cell cycle arrest in G2/M in human cancer cells. Our SAGE results revealed that 14-3-3 sigma expression is significantly downregulated in C666-1 cells. The study of 72 primary NPCs showed that an increased expression of 14-3-3 sigma was associated with a poorer clinical outcome in terms of shorter overall survival (OS; p=0.0297) and shorter disease free survival (DFS; p=0.042) using univariate analysis. Hence, 14-3-3 sigma may be used as an independent prognostic marker for NPC patients. / In conclusion, a NPC transcription profile has been successfully generated and several candidate NPC-associated genes have been identified by Serial Analysis of Gene Expression (SAGE) and NPC transcriptome map. These novel findings lead to better understanding of the molecular basis of NPC development and provide a foundation for discovery of new therapeutic strategies. / Nasopharyngeal carcinoma (NPC) is one of the most prevalent cancers among Southern Chinese. To better understand the genetic basis of this disease, Serial Analysis of Gene Expression (SAGE) was performed to investigate the transcriptional profiles of an EBV-positive NPC cell line (C666-1) and a normal NP outgrowth (NP4). A total of 102,059 SAGE tags were extracted in both libraries and 250 genes with 10-fold or more differential expression were found in NPC cells compared to normal NP cells. Eleven differentially expressed genes identified by SAGE were selected for confirmation using real time RT-PCR. The transcripts for 5 of the 11 genes, CD 74, Transcriptional intermediary factor 1, Ferritin 1, Claudin 4, and fatty acid synthase were overexpressed in NPC cells. Conversely, the remaining transcripts including Keratin 17, Keratin 5, S100 calcium-binding A2, Cystatin A, 14-3-3 sigma and Caveolin 1 were underexpressed in NPC cells. The aberrant expression of CD74, Claudin 4, Fatty acid synthase, 14-3-3 sigma, Caveolin 1 were further validated by immunohistochemistry on 20 NPC patients. / On the other hand, fatty acid synthase (FASN), a key enzyme for de novo lipogenesis, is a putative therapeutic target in treating NPC. Immunohistochemical studies showed upregulation of FASN in 20.8% (15/72) of the NPC cases compared with the adjacent normal NP epithelium. In addition, FASN expression also had prognostic significance in predicting the outcome of patients after radiotherapy, as high levels of FASN expression were associated with worse overall survival (OS, p=0.032) and disease free survival (DFS, p=0.002) in NPC patients. FASN inhibitors, such as C75 which inhibit cell growth via cell cycle arrest in G2/M phase, are potential chemotherapeutic agents in treating NPC. / The genome-wide quantitative analysis of gene expression by SAGE with matched chromosomal positions enables the construction of a transcriptome map of NPC. A total of 8 and 29 overexpressed and underexpressed gene clusters were observed, respectively. Some novel regions that have never been illustrated in previous reports such as amplification regions at 2p11.2-p25.1, 2q33-q37, 9q22-q34, 17p11.2-p13.2 and deletion regions at 1p12-p31.2, 1q25-q42.12, 2q21.3-q33, 8p21.1-p22, 9q33-q34.3, 10q23.3-q26.3, 12p13, 16p13, 17q23.2-q25, 19p13, 19q12-q13.2, 20p11-p13, 22q13, Xp11.2-p11.4, and Xq26-q28 were also identified. A candidate tumor suppressor gene named MEG3 has been found within an underexpressed region at 14q32.2 in the NPC transcriptome map. Our FISH analysis revealed that chromosome loss at 14q32 is associated with hypermethylation of MEG3 promoter region in 9/13 (75%) of NPC patients. Loss of imprinting is the major mechanism that governs the MEG3 expression. Moreover, transient transfection of one of the MEG3 isoforms (accession no. AF119863) could obviously inhibit cell colony formation of NPC cells. Taken together, MEG3 gene on chromosome 14q32.2 might act as a tumor suppressor in NPC. / Chan, Yat Yee. / "March 2008." / Adviser: Lo Kwok Wai. / Source: Dissertation Abstracts International, Volume: 70-03, Section: B, page: 1605. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (p. 196-225). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

This thesis describes the development of an systems biology method to study transcriptional programs that are activated during early and late phases of cell-fusion mediated reprogramming, as well as an implementation of systems-level analysis using reverse-engineered regulatory networks to study CNS disorders like Alcohol Addiction, and neurodegenerative disorders like Alzheimer's Disease (AD), and Parkinson's Disease (PD). The results will show an unprecedented view into the mechanisms underlying complex processes and diseases, and will demonstrate the predictive power of these methodologies that extended far beyond their original contexts.

An important hallmark of cancer is the inactivation of tumor suppressor genes. The most common genetic alteration in cancer is the mutation of the TP53 gene occurring in about half of all cancers, but very little progress has been made on how to therapeutically target the signaling defects in these cancers. Additionally, the PTEN tumor suppressor is mutated in a wide variety of cancer types, and its expression is often lost in the absence of mutation. PTEN is a haploinsufficient tumor suppressor that exhibits dose-dependent effects in cells. In the context where PTEN is lost or downregulated, PI3K signaling and downstream signaling through AKT is overactive, leading to an increase in cell growth and proliferation, among other effects. Acting as both a protein and lipid phosphatase, loss of PTEN also affects the PI3K-independent signaling of PTEN, and results in an increase of migration and invasion phenotypes. Importantly, PTEN transcript level is the key determinant for PTEN protein expression, and downregulation of PTEN is part of a poor-prognosis gene expression signature in breast cancer. Downregulation of tumor suppressor gene expression represents a reversible change that is often sufficient to drive tumorigenesis. However, our understanding of the broad molecular mechanisms by which the expression of these tumor suppressors is lost remains limited, but is required to develop effective therapeutic strategies to target malignancies driven by tumor suppressor loss. In Chapter 2, we characterize the problem of transcriptional downregulation of PTEN in breast cancer. We investigate the expression of PTEN in various normal and tumor cells at both the transcript and protein level. We identify various model systems that we believe are suitable to model normal PTEN expression and the PTEN downregulation that mimics what is observed in tumors. We employ a sophisticated approach that couples RNA-sequencing with Nanostring nCounter analysis in order to obtain a detailed and thorough transcriptional profile of the PTEN and pseudogene PTENP1 genomic loci, as well as expression of the poor-prognosis gene signature associated with PTEN downregulation. In this study, we obtained an understanding of the changes in the PTEN transcriptional profile that occur in the progression from normal to cancer, and we believe this approach could be applied to other key tumor suppressor genes. In Chapter 3, we discovered that basally expressed p53 maintains expression of thirteen well-validated tumor suppressors. p53 is expressed at low levels under normal, low-stress conditions, and is expressed at much higher levels under enhanced stress, leading to the activation of stress-response genes. We begin the study by highlighting an association between TP53 mutation and downregulation of PTEN expression. Upon performing chromatin immunoprecipitation coupled with next generation sequencing for p53 under normal, low-stress conditions, we found that p53 binds in the vicinity of thirteen tumor suppressor genes, including PTEN. Basally expressed p53 binds to classic consensus binding sites in enhancers and promoters of target tumor suppressors to maintain their expression at baseline. CRISPR/Cas9-mediated knockout of the endogenous basal p53 binding site upstream of PTEN led to a decrease in PTEN expression and an increase in tumorigenic phenotypes. Given that mutation of TP53 leads to tumorigenesis in mice, but loss of p53 stress-response targets or loss of the ability of p53 to activate these stress-response targets does not lead to spontaneous tumorigenesis, it is likely that these tumor suppressor targets of basal p53 contribute to p53-mediated tumor suppression. In Chapter 4, we identified yet another mechanism by which transcriptional repression of PTEN occurs in triple-negative breast cancer (TNBC) through polycomb repressive complex 2 (PRC2)-mediated repression of the PTEN promoter and upstream regulatory region. Previous research has shown that mutated NOTCH1 represses PTEN through the HES-1 transcription factor in acute myeloid leukemia (AML), and that NOTCH translocations are frequent in TNBC and are sufficient for transformation in vitro. We discovered that NOTCH1 and NOTCH2 mutations and translocations correlate with PTEN downregulation by immunohistochemistry in a cohort of TNBC cases. The TNBC cell line exhibiting PRC2-mediated repression of PTEN also harbors a SEC22B-NOTCH2 translocation that creates a gene product resembling the NOTCH2 intracellular domain. The NOTCH target HES-1 co-localizes on the PTEN promoter with EZH2 (the lysine methyltransferase involved in PRC2-mediated transcriptional repression), and knockdown of NOTCH2 in this cell line led to decreased expression of EZH2, and restoration of PTEN expression at the transcript and protein level. We also demonstrated that EZH2 inhibitors, HDAC inhibitors, and DNA hypomethylating agents robustly restore PTEN transcript levels. Taken together, these results elucidate another mechanism by which PTEN is transcriptionally repressed in the highly aggressive and poor-prognosis TNBC subtype of breast cancer that may be applicable to other cancer types. The results also suggest that this repression is reversible by pharmacological approaches, highlighting a promising therapeutic avenue. Taken together, the studies presented in this thesis begin to unravel the complex mechanisms of transcriptional repression of tumor suppressor genes in cancer. As is the case with PTEN and p53, multiple regulatory mechanisms can influence expression in combination or in a context-dependent manner. The loss of expression of tumor suppressor genes is one of the key hallmarks of cancer, yet very few of the therapeutic approaches used in the clinic today aim to restore tumor suppressor expression. Our results demonstrate proof of concept that restoration of tumor suppressor expression is a plausible and promising therapeutic approach for many different types of cancer, but requires a detailed understanding of the underlying molecular mechanisms of transcriptional regulation.

Ng Lui. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 243-257). / Abstracts in English and Chinese. / Abstract --- p.i_iii / Abstract (Chinese version) --- p.iv-v / Acknowledgements --- p.vi / Table of Contents --- p.vii-xvii / List of Abbreviations --- p.xviii-xx / List of Figures --- p.xxi-xxvi / List of Tables --- p.xxvii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Peroxisome Proliferators (PPs) --- p.1 / Chapter 1.2 --- Peroxisome proliferator-activated receptors (PPARs) --- p.3 / Chapter 1.2.1 --- What are PPARs? --- p.3 / Chapter 1.2.2 --- PPAR isoforms --- p.3 / Chapter 1.2.2.1 --- PPARp/δ --- p.3 / Chapter 1.2.2.2 --- PPARγ --- p.4 / Chapter 1.2.2.3 --- PPARα --- p.5 / Chapter 1.2.3 --- PPARα target genes --- p.5 / Chapter 1.2.3.1 --- Transcriptional regulation --- p.5 / Chapter 1.2.3.2 --- PPRE --- p.6 / Chapter 1.2.4 --- Physiological roles --- p.9 / Chapter 1.2.4.1 --- Lipid metabolism --- p.9 / Chapter 1.2.4.1.1 --- Cellular fatty acid uptake and fatty acid activation --- p.9 / Chapter 1.2.4.1.2 --- Intracellular fatty acid transport --- p.11 / Chapter 1.2.4.1.3 --- Mitochondrial fatty acid uptake --- p.12 / Chapter 1.2.4.1.4 --- Mitochondrial fatty-acid P-oxidation / Chapter 1.2.4.1.5 --- Peroxisomal fatty acid uptake --- p.13 / Chapter 1.2.4.1.6 --- Peroxisomal fatty acid oxidation --- p.13 / Chapter 1.2.4.1.7 --- Micorsomal co-hydroxylation of fatty acids --- p.14 / Chapter 1.2.4.1.8 --- Ketogenesis --- p.15 / Chapter 1.2.4.1.9 --- Bile acid metabolism --- p.15 / Chapter 1.2.4.1.10 --- Lipoprotein metabolism --- p.17 / Chapter 1.2.4.1.11 --- Hepatic lipogenesis --- p.18 / Chapter 1.2.4.2 --- Glucose metabolism --- p.19 / Chapter 1.2.4.2.1 --- Glycogenolysis --- p.19 / Chapter 1.2.4.2.2 --- Glycolysis --- p.20 / Chapter 1.2.4.2.3 --- Gluconeogenesis --- p.20 / Chapter 1.2.4.3 --- Urea cycle --- p.21 / Chapter 1.2.4.4 --- Biotransformation --- p.22 / Chapter 1.2.4.5 --- Inflammation --- p.23 / Chapter 1.2.4.6 --- Acute phase response --- p.23 / Chapter 1.2.5 --- Toxicological roles --- p.24 / Chapter 1.2.5.1 --- PPs induce hepatocarcinoma formation through PPARα --- p.24 / Chapter 1.2.5.2 --- Mechanism of PPARa-mediated PP-induced hepatocarcinoma --- p.25 / Chapter 1.2.5.2.1 --- Oxidative stress --- p.25 / Chapter 1.2.5.2.2 --- Hepatocellular proliferation and inhibition of apoptosis --- p.26 / Chapter 1.3 --- Discovery of novel PPARα target genes --- p.27 / Chapter 1.3.1 --- Peroxisome proliferator and starvation-induced gene (PPSIG) --- p.28 / Chapter 1.3.1.1 --- PPSIG is a putative PPARa target gene --- p.28 / Chapter 1.3.1.2 --- Examination of PPSIG FDD fragment cDNA sequence --- p.28 / Chapter 1.4 --- Objectives --- p.32 / Chapter Chapter 2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Cloning of the full-length mouse PPSIG cDNA --- p.38 / Chapter 2.1.1 --- Rapid amplification of cDNA ends (RACE) --- p.38 / Chapter 2.1.1.1 --- Total RNA extraction --- p.38 / Chapter 2.1.1.1.1 --- Materials --- p.38 / Chapter 2.1.1.1.2 --- Methods --- p.38 / Chapter 2.1.1.2 --- Primers design --- p.39 / Chapter 2.1.1.3 --- 5' and 3' cDNA ends amplification --- p.42 / Chapter 2.1.1.3.1 --- Materials --- p.42 / Chapter 2.1.1.3.2 --- Methods --- p.42 / Chapter 2.1.2 --- Subcloning of 5' and 3'RACED products --- p.45 / Chapter 2.1.2.1 --- Ligation and transformation --- p.45 / Chapter 2.1.2.1.1 --- Materials --- p.45 / Chapter 2.1.2.1.2 --- Methods --- p.46 / Chapter 2.1.2.2 --- Screening of the recombinants --- p.48 / Chapter 2.1.2.2.1 --- PhenoI:chloroform test --- p.48 / Chapter 2.1.2.2.1.1 --- Materials --- p.48 / Chapter 2.1.2.2.1.2 --- Methods --- p.48 / Chapter 2.1.2.2.2 --- Restriction enzyme digestion --- p.48 / Chapter 2.1.2.2.2.1 --- Materials --- p.48 / Chapter 2.1.2.2.2.2 --- Methods --- p.49 / Chapter 2.1.3 --- DNA sequencing of the 5'and 3'RACED subclones --- p.49 / Chapter 2.1.4 --- Northern blot analysis using PPSIG 5' and 3' RACED cDNA as probes --- p.52 / Chapter 2.1.4.1 --- RNA sample preparation --- p.52 / Chapter 2.1.4.1.1 --- Materials --- p.52 / Chapter 2.1.4.1.2 --- Methods --- p.52 / Chapter 2.1.4.2 --- Formaldehyde-agarose gel electrophoresis and blotting of RNA --- p.52 / Chapter 2.1.4.2.1 --- Materials --- p.52 / Chapter 2.1.4.2.2 --- Methods --- p.53 / Chapter 2.1.4.3 --- Probe preparation --- p.55 / Chapter 2.1.4.3.1 --- DIG labeling of RNA probe from 5'RACED PPSIG cDN A subclone 5'#32 --- p.55 / Chapter 2.1.4.3.1.1 --- Materials --- p.55 / Chapter 2.1.4.3.1.2 --- Methods --- p.55 / Chapter 2.1.4.3.2 --- PCR DIG labeling of 3´ة RACED PPSIG cDNA subclone 3' #12 --- p.56 / Chapter 2.1.4.3.2.1 --- Materials --- p.56 / Chapter 2.1.4.3.2.2 --- Methods --- p.57 / Chapter 2.1.4.4 --- Hybridization --- p.57 / Chapter 2.1.4.4.1 --- Materials --- p.57 / Chapter 2.1.4.4.2 --- Methods --- p.57 / Chapter 2.1.4.5 --- Post-hybridization washing and colour development --- p.59 / Chapter 2.1.4.5.1 --- Materials --- p.59 / Chapter 2.1.4.5.2 --- Methods --- p.59 / Chapter 2.2 --- Cloning of the PPSIG genomic DNA --- p.61 / Chapter 2.2.1 --- Screening of bacterial artificial chromosome (BAC) clones --- p.61 / Chapter 2.2.1.1 --- Screening of a mouse genomic library --- p.61 / Chapter 2.2.1.2 --- "Purification of BAC DNA by solution I, II,III" --- p.61 / Chapter 2.2.1.2.1 --- Materials --- p.61 / Chapter 2.2.1.2.2 --- Methods --- p.61 / Chapter 2.2.2 --- Confirmation of PPSIG genomic BAC clones --- p.64 / Chapter 2.2.2.1 --- Genomic Southern blot analysis --- p.64 / Chapter 2.2.2.1.1 --- Agarose gel electrophoresis and blotting of BAC DNA --- p.64 / Chapter 2.2.2.1.1.1 --- Materials --- p.64 / Chapter 2.2.2.1.1.2 --- Methods --- p.64 / Chapter 2.2.2.1.2 --- DIG labeling of DNA probe by random priming --- p.65 / Chapter 2.2.2.1.2.1 --- Materials --- p.65 / Chapter 2.2.2.1.2.2 --- Methods --- p.65 / Chapter 2.2.2.1.3 --- Hybridization --- p.66 / Chapter 2.2.2.1.4 --- Post-hybridization washing and colour development --- p.66 / Chapter 2.2.2.2 --- EcoR I digestion --- p.67 / Chapter 2.2.2.2.1 --- Materials --- p.67 / Chapter 2.2.2.2.2 --- Methods --- p.67 / Chapter 2.2.2.3 --- Large scale preparation of BAC DNA --- p.67 / Chapter 2.2.2.3.1 --- Materials --- p.67 / Chapter 2.2.2.3.2 --- Methods --- p.68 / Chapter 2.2.3 --- Determination of PPSIG genomic sequences --- p.68 / Chapter 2.2.3.1 --- Primers design --- p.68 / Chapter 2.2.3.2 --- PCR --- p.73 / Chapter 2.2.3.2.1 --- Materials --- p.73 / Chapter 2.2.3.2.2 --- Methods --- p.73 / Chapter 2.2.3.3 --- Subcloning of the PPSIG genomic fragments --- p.73 / Chapter 2.2.3.3.1 --- Ligation and transformation --- p.73 / Chapter 2.2.3.3.2 --- PCR screening --- p.74 / Chapter 2.2.3.3.2.1 --- Materials --- p.74 / Chapter 2.2.3.3.2.2 --- Methods --- p.74 / Chapter 2.2.3.4 --- DNA sequencing --- p.75 / Chapter 2.3 --- Cloning of PPSIG-promoter reporter constructs --- p.75 / Chapter 2.3.1 --- Amplification of PPSIG 5'-flanking fragment by PCR --- p.75 / Chapter 2.3.1.1 --- Materials --- p.75 / Chapter 2.3.1.2 --- Methods --- p.75 / Chapter 2.3.2 --- Preparation of pGL3-Basic vector DNA --- p.81 / Chapter 2.3.2.1 --- Materials --- p.81 / Chapter 2.3.2.2 --- Methods --- p.81 / Chapter 2.3.3 --- Ligation and transformation --- p.84 / Chapter 2.3.3.1 --- Materials --- p.84 / Chapter 2.3.3.2 --- Methods --- p.84 / Chapter 2.3.4 --- Screening and confirmation of recombinants --- p.85 / Chapter 2.3.4.1 --- Materials --- p.85 / Chapter 2.3.4.2 --- Methods --- p.85 / Chapter 2.4 --- Cloning of PPSIG 5'-deletion promoter constructs --- p.87 / Chapter 2.4.1 --- Deletion of target fragments by restriction enzyme digestion --- p.87 / Chapter 2.4.1.1 --- Materials --- p.87 / Chapter 2.4.1.2 --- Methods --- p.88 / Chapter 2.4.2 --- Ligation and transformation --- p.90 / Chapter 2.4.2.1 --- Materials --- p.90 / Chapter 2.4.2.2 --- Methods --- p.90 / Chapter 2.4.3 --- Screening and confirmation of recombinants --- p.91 / Chapter 2.5 --- Cloning of PPSIG-PPRE reporter constructs --- p.91 / Chapter 2.5.1 --- Amplification of PPSIG-PPRE fragments --- p.91 / Chapter 2.5.1.1 --- Materials --- p.91 / Chapter 2.5.1.2 --- Methods --- p.93 / Chapter 2.5.2 --- Preparation of pGL3-Basic vector DNA --- p.96 / Chapter 2.5.2.1 --- Materials --- p.96 / Chapter 2.5.2.2 --- Methods --- p.96 / Chapter 2.5.3 --- Ligation and transformation --- p.97 / Chapter 2.5.3.1 --- Materials --- p.97 / Chapter 2.5.3.2 --- Methods --- p.97 / Chapter 2.5.4 --- Screening and confirmation of recombinants --- p.97 / Chapter 2.6 --- Cloning of PPSIG-PPRE deletion construct --- p.101 / Chapter 2.6.1 --- Deletion of PPRE fragment by Stu I/Xho I digestion --- p.101 / Chapter 2.6.1.1 --- Materials --- p.101 / Chapter 2.6.1.2 --- Methods --- p.101 / Chapter 2.6.2 --- "Ligation, transformation, screening and confirmation of recombinants" --- p.103 / Chapter 2.7 --- Construction of PPSIG-PPRE-deletion and PPSIG- PPRE-mutation constructs by site-directed mutagenesis --- p.105 / Chapter 2.7.1 --- Primers design --- p.105 / Chapter 2.7.2 --- Amplification of the left and right halves of the PPRE-deletion and PPRE-mutation constructs by PCR --- p.109 / Chapter 2.7.2.1 --- Materials --- p.109 / Chapter 2.7.2.2 --- Methods --- p.109 / Chapter 2.7.3 --- "Ligation, Dpn I digestion and transformation" --- p.110 / Chapter 2.7.3.1 --- Materials --- p.110 / Chapter 2.7.3.2 --- Methods --- p.110 / Chapter 2.7.4 --- Screening and confirmation of recombinants --- p.111 / Chapter 2.7.4.1 --- Materials --- p.111 / Chapter 2.7.4.2 --- Methods --- p.111 / Chapter 2.8 --- Cloning of mouse malonyl-CoA decarboxylase (MCD) and rat acyl-CoA binding protein (ACBP) PPRE reporter constructs --- p.112 / Chapter 2.8.1 --- Preparation of mouse and rat genomic DNA --- p.112 / Chapter 2.8.1.1 --- Materials --- p.112 / Chapter 2.8.1.2 --- Methods --- p.113 / Chapter 2.8.2 --- Amplification of MCD and ACBP PPRE fragments by PCR --- p.113 / Chapter 2.8.2.1 --- Materials --- p.113 / Chapter 2.8.2.2 --- Methods --- p.114 / Chapter 2.8.3 --- Ligation and transformation --- p.117 / Chapter 2.8.4 --- Screening and confirmation of recombinants --- p.117 / Chapter 2.9 --- Cloning of mPPARα and mRXRα expression plasmids --- p.119 / Chapter 2.9.1 --- RT-PCR of mouse PPARα and RXRa cDNAs --- p.119 / Chapter 2.9.1.1 --- Materials --- p.119 / Chapter 2.9.1.2 --- Methods --- p.119 / Chapter 2.9.2 --- Preparation of pSG5 vector DNA --- p.123 / Chapter 2.9.2.1 --- Materials --- p.123 / Chapter 2.9.2.2 --- Methods --- p.123 / Chapter 2.9.3 --- Ligation and transformation --- p.125 / Chapter 2.9.3.1 --- Materials --- p.125 / Chapter 2.9.3.2 --- Methods --- p.125 / Chapter 2.9.4 --- Screening and confirmation of recombinants --- p.125 / Chapter 2.9.4.1 --- Materials --- p.125 / Chapter 2.9.4.2 --- Methods --- p.126 / Chapter 2.10 --- Transient transfection and reporter assays --- p.128 / Chapter 2.10.1 --- Cell culture and transient transfection --- p.128 / Chapter 2.10.1.1 --- Materials --- p.128 / Chapter 2.10.1.2 --- Methods --- p.128 / Chapter 2.10.2 --- Assay for reporter construct luciferase activity --- p.131 / Chapter 2.10.2.1 --- Materials --- p.131 / Chapter 2.10.2.2 --- Methods --- p.131 / Chapter 2.11 --- Electrophoretic mobility-shift assay (EMSA) --- p.133 / Chapter 2.11.1 --- In vitro transcription/translation --- p.133 / Chapter 2.11.1.1 --- Materials --- p.133 / Chapter 2.11.1.2 --- Methods --- p.133 / Chapter 2.11.2 --- Preparation of AML-12 nuclear extract --- p.134 / Chapter 2.11.3 --- Preparation of DIG-labeled PPSIG-PPRE oligonucleotides --- p.136 / Chapter 2.11.3.1 --- Oligonucleotides design --- p.136 / Chapter 2.11.3.2 --- Annealing of single-stranded oligonucleotides to form double- stranded oligonucleotides --- p.136 / Chapter 2.11.3.2.1 --- Materials --- p.136 / Chapter 2.11.3.2.2 --- Methods --- p.138 / Chapter 2.11.3.3 --- 3' end labeling of the double-stranded oligonucleotides --- p.138 / Chapter 2.11.3.3.1 --- Materials --- p.138 / Chapter 2.11.3.3.2 --- Methods --- p.138 / Chapter 2.11.3.4 --- Testing the labeling efficiency of the double-stranded oligonucleoides --- p.139 / Chapter 2.11.3.4.1 --- Materials --- p.139 / Chapter 2.11.3.4.2 --- Methods --- p.139 / Chapter 2.11.4 --- Preparation of unlabeled oligonucleotides as competitors --- p.140 / Chapter 2.11.5 --- Binding reactions --- p.142 / Chapter 2.11.5.1 --- Perform with in vitro transcribed/translated proteins --- p.142 / Chapter 2.11.5.1.1 --- Materials --- p.142 / Chapter 2.11.5.1.2 --- Methods --- p.142 / Chapter 2.11.5.2 --- Perform with AML-12 nuclear extracts --- p.144 / Chapter 2.11.5.2.1 --- Materials --- p.144 / Chapter 2.11.5.2.2 --- Methods --- p.144 / Chapter 2.11.6 --- Detection of shift-up pattern --- p.145 / Chapter 2.11.6.1 --- Materials --- p.145 / Chapter 2.11.6.2 --- Methods --- p.145 / Chapter 2.12 --- Statistical analysis --- p.146 / Chapter Chapter 3 --- Results --- p.147 / Chapter 3.1 --- PPSIG cDNA sequence analysis --- p.147 / Chapter 3.1.1 --- Cloning of PPSIG full-length cDNA sequence --- p.147 / Chapter 3.1.2 --- Northern blot analysis of PPSIG --- p.160 / Chapter 3.1.3 --- "Comparison of PPSIG, Riken cDNA 0610039N19 and all-trans-13'14-dihydroretinol saturase cDNA sequences" --- p.163 / Chapter 3.2 --- PPSIG genomic sequence analysis --- p.166 / Chapter 3.2.1 --- Screening of the PPSIG BAC clone --- p.166 / Chapter 3.2.2 --- Cloning of PPSIG genomic fragments --- p.167 / Chapter 3.2.3 --- Examination of PPSIG genomic organization --- p.170 / Chapter 3.2.3.1 --- "Comparison of PPSIG, Riken cDNA 0610039N19 and all-trans-13'14-dihydroretinol saturase genomic sequence" --- p.177 / Chapter 3.3 --- Characterization of the 5'-flanking region of PPSIG --- p.184 / Chapter 3.4 --- Identification of a functional PPRE in the intron 1 of PPSIG gene --- p.201 / Chapter 3.5 --- Gel shift analysis of PPARa/RXRa heterodimer to PPSIG-PPRE --- p.222 / Chapter Chapter 4 --- Discussion --- p.234 / Chapter Chapter 5 --- Future studies --- p.241 / References --- p.243 / Appendix A Seating plan of transfection experiments (24-wells) / Chapter A1 --- Transfection experiment to study PPSIG-promoter reporter constructs --- p.258 / Chapter A2 --- Transfection experiment to study the PPSIG- promoter deletion constructs --- p.259 / Chapter A3 --- Transfection experiment to study the PPSIG-PPRE reporter constructs --- p.260 / Chapter A4 --- Transfection experiment to study PPSIG-PPRE- deletion and PPSIG-PPRE-mutation constructs --- p.262 / Appendix B Alignment result of RACE clone DNAs --- p.265 / Chapter B1 --- Alignment result of 5´ة#7 --- p.265 / Chapter B2 --- Alignment result of 5'#11 --- p.267 / Chapter B3 --- Alignment result of 5'#12 --- p.269 / Chapter B4 --- Alignment result of 5´ة#16 --- p.271 / Chapter B5 --- Alignment result of 5´ة#20 --- p.274 / Chapter B6 --- Alignment result of 5´ة#31 --- p.276 / Chapter B7 --- Alignment result of 5´ة#32 --- p.278 / Chapter B8 --- Consensus sequence of each 5'RACED clone --- p.280 / Chapter B9 --- Alignment result of all 5'RACE clones consensus sequence --- p.287 / Chapter B10 --- Alignment result of 3´ة#2 --- p.290 / Chapter B11 --- Alignment result of 3´ة#3 --- p.291 / Chapter B12 --- Alignment result of 3´ة#14 --- p.292 / Chapter B13 --- Alignment result of 3´ة#5 --- p.293 / Chapter B14 --- Alignment result of 3´ة#6 --- p.294 / Chapter B15 --- Alignment result of 3´ة#8 --- p.295 / Chapter B16 --- Alignment result of 3´ة#10 --- p.297 / Chapter B17 --- Alignment result of 3´ة#11 --- p.298 / Chapter B18 --- Alignment result of 3´ة#12 --- p.299 / Chapter B19 --- Alignment result of 3´ة#16 --- p.301 / Chapter B20 --- Alignment result of 3´ة#22 --- p.302 / Chapter B21 --- Alignment result of 3´ة#25 --- p.303 / Chapter B22 --- Consensus sequence of each 3'RACED clone --- p.305 / Chapter B23 --- Alignment result of all 3' RACE clones consensus sequence --- p.310 / Appendix C DNA sequencing and alignment result of PPSIG genomic fragments --- p.312 / Chapter C1 --- Exon 1 to exon 2 --- p.312 / Chapter C2 --- Exon 2 to exon 3 --- p.315 / Chapter C3 --- Exon 3 to exon 4 --- p.316 / Chapter C4 --- Exon 4 to exon 5 --- p.318 / Chapter C5 --- Exon 5 to exon 6 --- p.319 / Chapter C6 --- Exon 6 to exon 7 --- p.321 / Chapter C7 --- Exon 7 to exon 8 --- p.322 / Chapter C8 --- Exon 8 to exon 9 --- p.323 / Chapter C9 --- Exon 9 to exon 10 --- p.324 / Chapter C10 --- Exon 10 to exon 11 --- p.325 / Chapter C11 --- Exon 11 to downstream --- p.326 / Chapter C12 --- Consensus sequence of each BAC genomic DNA fragment --- p.328 / Chapter C13 --- The alignment result of all the PPSIG genomic sequence --- p.335 / Appendix D DNA sequencing and alignment result of constructs --- p.347 / Chapter D1 --- "pGL3-PPSIG (-2936/+119), pGL3-PPSIG (-1534/+119), pGL3-PPSIG (-879/+119) and pGL3- PPSIG (-375/+119) reporter constructs DNA sequencing and alignment result" --- p.347 / Chapter D2 --- pSG5-PPARa expression plasmid DNA sequencing and alignment result --- p.351 / Chapter D3 --- pSG5-RXRa expression plasmid DNA sequencing and alignment result --- p.353 / Chapter D4 --- pGL3-MCD reporter constructs DNA sequencing and alignment result --- p.355 / Chapter D5 --- pGL3-PPSIG (-229/+435) reporter construct DNA sequencing and alignment result --- p.356 / Chapter D6 --- pGL3-PPSIG (+94/+435) and pGL3-PPSIG (+94/+190) reporter constructs DNA sequencing and alignment result --- p.357 / Chapter D7 --- pGL3-PPSIG (-229/+3031) reporter construct DNA sequencing and alignment result --- p.358 / Chapter D8 --- pGL3-PPSIG (+94/+3031) reporter construct DNA sequencing and alignment result --- p.360 / Chapter D9 --- pGL3-ACBP reporter construct DNA sequencing and alignment result --- p.362 / Chapter D10 --- PPSIG-PPRE-deletion and PPSIG-PPRE-mutation constructs DNA sequencing and alignment result --- p.363

There are approximately 20,000 genes in the human genome. Around 2% of these genes code for transcriptional repressors known as KRAB-ZFPs. It is already known that Zinc-Finger Proteins contain two main functional domains at either end of the polypeptide. In today's database, you will find a KRAB (Kruppell-associated Box) domain at one end and a tandem array of Zinc-finger repeats at the other end. The carboxyl terminal tandem Zinc-finger repeats function as sequence-specific DNA-binding domains. The amino terminal KRAB domain serves as a repressor domain, which will recruit a co-repressor termed KAP-1 (KRAB Associated Protein-1). Located in between these two domains is a region of uncharacterized DNA referred to as the "Linker Region". This thesis will explore the DNA-binding domains of 6 known KRAB-ZFPs, as well as utilize the linker regions to derive an evolutionary history for this superfamily. / by Alain Chamoun. / Thesis (M.S.)--Florida Atlantic University, 2010. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2010. Mode of access: World Wide Web.