Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / "December 2002." Typescript. Vita. Includes bibliographical references (leaves 179-198). Also issued on the Internet.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 133-141).

Groucho/transducin-like Enhancer of split (Gro/TLE) family proteins are corepressors found as part of multiple transcriptional complexes that play significant roles during many developmental processes, including neurogenesis. This thesis sought to characterize the molecular mechanisms underlying the biological activity of Gro/TLE1. More specifically, the aim was to clarify the contribution of different transcriptional cofactors, as well as phosphorylation events induced by cofactor binding, to Gro/TLE1 ability to inhibit neuronal differentiation from proliferating neural progenitor cells. / By characterizing specific point mutations within the C-terminal domain of Gro/TLE1, we were able to selectively impair binding of Gro/TLE1 to different classes of DNA-binding proteins and then assess the effect of those mutations on Gro/TLE1 anti-neurogenic function. These studies showed that the inhibition of cerebral cortex (cortical) neuron differentiation by Gro/TLE1 requires interaction with transcription factors that use short tetrapeptide sequences, WRP(W/Y), to recruit Gro/TLE1. In contrast, interactions with proteins that either interact with the C-terminal domain of Gro/TLE1 using a different type of binding sequence, termed engrailed homology 1 (Eh1) motif, or bind to the N-terminal part of the protein, are not required for Gro/TLE1 anti-neurogenic function. / Using a similar strategy based on mutation analysis, we characterized point mutations that block the hyperphosphorylation of Gro/TLE1 induced by transcription cofactor binding ("cofactor-activated phosphorylation") without impairing cofactor binding and transcriptional corepression ability. These mutations map at phosphorylatable serine residues, Ser-286, Ser-289, and Ser298. Mutation of those residues to alanine blocks/reduces both cofactor-activated phosphorylation and anti-neurogenic activity of Gro/TLE1, demonstrating that cofactor-activated phosphorylation is required for that function. Tandem mass spectroscopy analysis showed further that Ser-286 is phosphorylated. Taken together, these findings characterize the role of cofactor-activated phosphorylation and identify residues important for this mechanism. / Our studies also showed that homeodomain-interacting protein kinase 2 (HIPK2) mediates phosphorylation of Gro/TLE1 when the latter is complexed with transcriptional partners of the WRP(W/Y) motif family. However, HIPK2 is not involved in Gro/TLE1 cofactor-activated phosphorylation. Rather, HIPK2--mediated phosphorylation is antagonistic to the latter and decreases the ability of Gro/TLE1 to interact and repress transcription with WRP(W/Y) motif proteins. / Taken together, these results improve significantly our understanding of the mechanisms underlying the anti-neurogenic function of Gro/TLE1. This information provides new insight into the regulation of mammalian neuronal development and, possibly, other developmental processes controlled by Gro/TLE proteins.

Plasminogen activator inhibitor type 2 (PAI-2) is a serine protease inhibitor traditionally regarded as a regulator of fibrinolysis and extracellular matrix degradation. More recently, PAI-2 has been implicated in diverse processes such as keratinocyte differentiation, cell death and viral pathogenesis. Although PAI-2s limited pattern of expression in vivo generates significant interest in this molecule, little is known about the underlying mechanisms controlling its cell specific regulation. In this thesis, the function that the previously identified PAI-2 gene silencer (Antalis et al., 1996) plays in the regulation of PAI-2 gene expression was investigated. The PAI-2 upstream silencer element 1 (PAUSE-1) is located approximately 1800bp upstream of the PAI-2 transcription initiation site. By employing electrophoretic mobility shift assays and transient transfection assays with mutant PAUSE-1 sequences, the sequence that defines PAUSE-1 was identified as TCT N3 AGA N3 T4. This element was shown to bind a number of protein complexes of similar electrophoretic mobility from various cultured cell lines. Transient transfection assays with the cervical adenocarcinoma, HeLa S3 and the macrophage-like, U937 cell lines, showed that PAUSE-1 repressed transcription by approximately 2.5 fold when cloned into the SV40 promoter or the minimal PAI-2 promoter. Ultraviolet (UV)-crosslinking analyses determined that the PAUSE-1 binding protein (BP) was approximately 67kDa. Examination of several similar DNA promoter sequences, such as the human IFNb and insulin promoters, suggested that PAUSE-1 might be an example of a universal silencer with the consensus sequence TCT Nx AGA, where x=4. The PAUSE-1 sequence shows significant homology to the binding sequence of the transcriptional regulators Ski, Smad3 and Smad4. EMSAs incorporating anti-Ski, -Smad3 and -Smad4 antibodies suggested that each are members of the PAUSE-1 BP complex in HeLa S3 cells. The PAUSE-1 BP complex has been purified by employing DNA affinity chromatography using streptavidin labelled magnetic beads. Approximately nine PAUSE-1 associated proteins from HeLa S3 extracts were visualised. Amino-terminal protein sequencing identified the first eight amino acids of the PAUSE-1 BP as EIQQRAAQ. The PAUSE-1 BP fails to show significant sequence similarity to any known protein and therefore potentially represents a novel DNA binding protein.

Diss. (sammanfattning) Stockholm : Stockholms universitet, 2007. / Härtill 3 uppsatser.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p. 165-182.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 77-92).

No description available.

Pantoea stewartii subsp. stewartii is the causative agent of Stewart's wilt disease in maize plants. The bacteria are injected into the plant by corn flea beetles during feeding. They colonize the xylem and overproduce a capsular exopolysaccharide (EPS) at high cell densities. The production of EPS is regulated by an EsaI/EsaR quorum sensing mechanism, homologous to the LuxI/R system. Although activation of the EPS encoding genes by EsaR occurs after it complexes to the AHL (3-oxo-C6-HSL), unlike the LuxI/R system, this activation occurs by a different mechanism. At low cell densities, dimerized EsaR acts as a repressor. At a high cell population, derepression of the EPS genes occurs via an unknown mechanism once the AHL complexes to EsaR. Hence, a random mutagenesis genetic approach to isolate EsaR* variants that are immune to the effects of AHL has been utilized. Error-prone PCR and site-directed mutagenesis were used to generate desired mutants, which were subsequently screened for their ability to repress transcription in the presence of AHL. Several individual amino acids playing a critical role in the AHL-insensitive phenotype have been identified and mapped onto a homology model of EsaR. A separate study attempted to localize the dimerization region and analyze the stability of the N-terminal domain of EsaR. Truncations of EsaR at amino acids 169 and 178, without and with the extended linker region respectively, were generated using PCR. Dimerization assays similar to those by Choi and Greenberg in 1991 were performed but proved to be unsuccessful. However, the N-terminal domain is stable as determined by western blotting, which may facilitate its future structural analysis. Together, these efforts have contributed to the molecular understanding of AHL-dependent derepression of EsaR. / Master of Science

The LuxR protein family is a class of quorum-sensing regulated bacterial transcription factors that alter gene expression as a function of ligand detection. This coincides with a high population density and/or a low rate of signal ligand diffusion. The majority of LuxR proteins are activated only in the presence of the signal ligand, an acyl-homoserine lactone (AHL). EsaR, from the corn pathogen Pantoea stewartii, represents a subset of LuxR homologues that are active in the absence of AHL and deactivated by its presence. The mechanism by which EsaR responds to AHL in a manner opposite to that of the majority of LuxR homologues remains elusive. Unlike the majority of LuxR homologues, which require AHL for purification, EsaR can be purified and biochemically investigated in the absence and presence of AHL. This work sought to answer questions regarding the structure-function relationship of the LuxR homologue, EsaR. Fluorescence anisotropy was used to determine the relative DNA-binding affinity of wild type EsaR and three AHL-independent EsaR variants in the presence and absence of AHL. This enabled for quantitative analysis of the relative binding affinities of these AHL-independent variants for the EsaR binding site, the esa box. The results demonstrate that one AHL-independent EsaR variant has a slightly higher affinity for the esa box in the presence, rather than the absence of AHL. The affinity of the other two for the DNA is not impacted by AHL, potentially due to an inability to transduce the signal of ligand detection to the DNA binding domain. Constructs containing only the EsaR N-terminal domain (NTD) were also developed. These constructs circumvented solubility issues associated with the full-length protein, allowing for additional biochemical analysis. It was determined that the EsaR NTD alone is sufficient for multimerization and ligand binding. Additionally, preliminary X-ray crystallography efforts have established some of the early parameters required to solve the crystal structure of the EsaR ligand binding domain in both the presence and absence of AHL. If pursued, these structures would be the first solved of a LuxR homologue ligand binding domain in both the presence and absence of the native AHL, potentially demonstrating the conformational change that occurs as a result of ligand binding. Collectively, these findings have established some of the groundwork required to resolve the question of what sort of conformational changes occur in EsaR as a result of ligand binding. / Master of Science