Transcriptional Repression of the Plasminogen Activator Inhibitor Type 2 Gene

Ogbourne, Steven (2000). Transcriptional Repression of the Plasminogen Activator Inhibitor Type 2 Gene PhD Thesis, Medicine, University of Queensland.

Attached Files (Some files may be inaccessible until you login with your UQ eSpace credentials)
Name Description MIMEType Size Downloads
n01front.pdf n01front.pdf application/pdf 45.09KB 1
n02chapter1.pdf n02chapter1.pdf application/pdf 203.65KB 0
n03chapter2.pdf n03chapter2.pdf application/pdf 79.08KB 0
n04chapter3.pdf n04chapter3.pdf application/pdf 440.60KB 0
n05chapter4.pdf n05chapter4.pdf application/pdf 170.02KB 0
n06chapter5.pdf n06chapter5.pdf application/pdf 105.52KB 0
n07chapter6.pdf n07chapter6.pdf application/pdf 78.81KB 0
n08appendix.pdf n08appendix.pdf application/pdf 10.68KB 0
n09bibliography.pdf n09bibliography.pdf application/pdf 139.91KB 0
Author Ogbourne, Steven
Thesis Title Transcriptional Repression of the Plasminogen Activator Inhibitor Type 2 Gene
School, Centre or Institute Medicine
Institution University of Queensland
Publication date 2000
Thesis type PhD Thesis
Supervisor Dr. Toni Antalis
Abstract/Summary 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.
Keyword transcription

Citation counts: Google Scholar Search Google Scholar
Access Statistics: 203 Abstract Views, 1 File Downloads  -  Detailed Statistics
Created: Fri, 21 Nov 2008, 20:51:25 EST