Analysis of promoter activity of TSG101 tumor susceptibility gene 101

• Main Authors: Shih-Fang Hsu, 許世芳
• Other Authors: Jiin-Tsuey Cheng
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/18922155964236790727

博士 === 國立中山大學 === 生物科學系研究所 === 101 === Tumor susceptibility gene, TSG101, participates in regulation of cellular functions such as gene transcription, vesicular trafficking, cell growth and differentiation. The steady-state level of TSG101 protein is kept in a narrow range. Deprivation or overexpression of TSG101 leads to neoplastic in mouse model. The purpose of this dissertation is to define the upstream promoter region of TSG101 and investigates the role of putative transcription factor binding sites reside in this promoter region. The results of promoter activity reporter assay indicated that TSG101 proximal promoter reside -1〜-436 region, Which contains binding site sequences for Sp1, MAZ , ETF and ETS transcription factors. It exhibits as a TATA-Less housekeeping type gene promoter. DNA methylation led to inhibition of TSG101 promoter reporter activity, while treatment with AzaC, a DNA methylation inhibitor, resulted in a dose-dependent elevation of TSG101 protein. The findings indicate that TSG101 promoter could be regulated by promoter DNA methylation. In addition, up-stream -1280〜-1757 region could function as an enhancer that orientation-independently increases TSG101 promoter activity in COS-1 transformed cells and thyroid carcinoma ARO and WRO cells. Interestingly, this region did not behave as an enhancer in primary rat thyroid FRTL cell. These results indicate TSG101 promoter could be regulated in normal and neoplastic thyroid cells, which corresponds to previous reports that TSG101 overexpression in thyroid and breast carcinoma specimens. We further demonstrated in vitro binding of Sp1 and MAZ transcription factors with electrophoretic mobility-shift assy (EMSA) assay by using probs with corresponding binding sequences. Experiments using mutant reporter constructs created by site-directed mutagenesis also demonstrated that both Sp1 and MAZ binding sites are essential for TSG101 promoter activity in transformed or primary thyroid cells. Whereas, reporter construct encompassing further upstream (-1～-2647) region showed differential promoter activities in transformed and primary thyroid cells. In these two type of cells, Sp1 binding site exhibits positive role in activation TSG101 promoter. However, MAZ played a positive role in transformed cell, but negative role in primary thyroid cells. Taken together, our results implied that in transformed cells, Sp1 and MAZ binding sites and their bound Sp1 and MAZ transcription factors together articipate in recruiting activators bound on enhancer to further stimulat TSG101 promoter activity. Whereas, in primary thyroid cells, MAZ binding site and presumably its bound MAZ transcription factor might function to recruit negative transcription regulator; hence, negatively regulate TSG101 promoter activity.