| Summary: | 碩士 === 國立成功大學 === 臨床藥學研究所 === 91 === The promoter specificity factor Sp1 is required for efficient transcriptional activation of many cellular and viral genes. Our previous studies indicated that there are five Sp1 binding site in the rat Mrp3 promoter region, and the third and the fourth Sp1 binding sites in —157~+19 region of rat Mrp3 promoter play important role in the gene transcription. Our recent data revealed that there is a C/EBP protein binding site in front of the third Sp1 binding site, and Sp1 has synergistic effect with C/EBPδin rat Mrp3 transcription. However, the mechanism is unclear. In this study, we studied how Sp1 mutants affect the interaction in the rat Mrp3 promoter. We designed a series of Sp1 variants, and transfected them into Drosophila Schneider cell line 2 (SL2 cell), which is lack of Sp1 protein. Then, We observed the difference of rat Mrp3 promoter activity to detect if these Sp1 variants could influence gene regulation.
In overexpression studies, we found that H701G of Sp1 variants could significantly diminish rat Mrp3 promoter activity by 50%, almost completely inhibited Sp1 activity. T419E, T476E, S713D and P718I increased promoter activity . They locate at glutamine-rich regions of B domain and D domain of Sp1. E65K, S353A, W457S, A499Y and R590E located at A, B and C domain of Sp1 protein could down-regulate the synergestic effect of Sp1 and C/EBPδ. In the other topic associated with Sp1 variants and c-Jun in epidermal growth factor-induced gene expression of human 12(S)-lipoxygenase, the result evidences that E65K, T419E, W457S and H701G of Sp1 variants with EGF treatment could decrease 12(S)-lipoxygenase promoter activity, and A499Y, R590E and P718I could increase the activity. Although the results of these two studies are not the same, it may be due to the different transcription mechanism of two cell lines. The studies also suggest that C/EBPδ and c-jun may interact with Sp1 differently.
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