The transactivation and transrepression functions of the simian virus 40 small t antigen

• Main Author: 彭瑞銘
• Other Authors: 王萬波
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/17926867621398470457

碩士 === 國立臺灣大學 === 微生物學研究所 === 88 === Previously our laboratory has found that simian virus 40 (SV40) small t antigen could transactivate the E2F-regulated genes by phosphorylating the retinoblastoma protein (pRB), and releasing E2F from the E2F-pRB complex. Our preliminary data suggest that this function of small t may to be related to its ability to interact with and inhibit protein phosphatase 2A (PP2A). To further confirm this, we asked whether a small t mutant, tC97S, which is defective in binding PP2A could still release E2F from E2F-pRB complex by electrophoretic mobility shift assay (EMSA). Our data indicate that while wild-type small t could efficiently release E2F from E2F-pRB complex, the tC97S mutant essentially lost this ability. We also studied whether the tC97S mutant could transactivate a synthetic promoter which contains only the E2F-binding sites. We found that in contrast to wild-type small t, which could efficiently transactivate this synthetic promoter, the tC97S mutant totally lost this ability. In consistent with our previous finding, which showed that small t could cooperate with SV40 large T to transactivate the E2F-controlled N-myc promoter, we also found that small t and large T could synergistically transactivate the synthetic E2F-site-containing promoter. Together these results suggest that small t could use this mechanism, i.e. transactivation of E2F-regulated genes, to help large T to transform cells. In the second part of this thesis, we wished to further demonstrate that small t can indeed interact with the transcriptional coactivator p300, and by doing so, small t obtains its ability to transrepress many promoters and enhancers. Previously our laboratory has found that small t could inhibit the trancriptional coactivator activity of p300. Mammlian two-hybrid assays also have suggested that small t may interact with p300. In this thesis, we further demonstrated that small t could interact with p300 by GST pull-down and immuno-coprecipitation assays. We also found that small t could inhibit p300 to coactivate AP-1 activity in CV-1P cells , but activate p300 to coactivate AP-1 activity in CV-1 cells. This result suggests that small t’s effect on p300 is cell-type specific. Since both E2F and p300 are important regulators in cell proliferation and differentiation, the understanding of regulation of these factors by small t should illuminate the roles of small t in modulating viral replication and cell transformation.