TSG101 is a tumor susceptibility gene, which exhibits many biological functions including the regulation of transcription, cell growth and differentation, protein trafficking and receptor recycling. Recent studies have revealed overexpression of TSG101 in human cancers of papillary thyroid carcinoma and breast cancer, whereas downregulation in the periphery of cancer tissue. These data indicated that the amount of TSG101 gene products might be relevant to tumor formation. Understanding the detail regulation of TSG101 gene expression might advance our knowledge on the processes of neoplastic conversion. In this regard, our lab have cloned and characterized upstream sequence of TSG101 transcription start site, and defined the region of -1~-436 as proximal promoter by luciferase reporter assay. Sequence analysis of this region using NSITE program on Softberry web site has revealed several transcription factor binding sites including MAZ, Sp1, ETF and ETS-1. Using EMSA and luciferase assay, we have demonstrated MAZ and Sp1 as major transcription factors regulate TSG101 proximal promoter. In this thesis, we advance our study to further explore the role of ETF and ETS-1 sites in regulation TSG101 promoter. We first cloned the region encompassing two ETS-1 sites in ¡V190~-1 region into pGL3-basic( -190/pGL3-basic ). The luciferase reporter assay of wildtype and mutant ¡V190/pGL3-basic plasmids further demonstrated important role of these ETS-1 sites. To explore detail contribution of the above mentioned transcription factor binding sites in regulation TSG101 promotor, we have made mutant reporter constructs containing different assortment of mutations in these binding sites, and assayed the effect of mutant on TSG101 promoter activity. The results showed that in cancer cell lines (COS-1, ARO and WRO) tested, Sp1, ETF, and ETS-1 binding sites are essential and they act co-operatively in regulating the activity of TSG101 proximal promoter. The results also indicated that Sp1 and ETS-1 were positive regulators, while ETF worked as a negative regulator.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0906105-174143 |
| Date | 06 September 2005 |
| Creators | Huang, man-yi |
| Contributors | Chung-lung Cho, Jiin-tsuey Cheng, Ming-hung Cho |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0906105-174143 |
| Rights | not_available, Copyright information available at source archive |
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