Significance of the amino terminus of Sp1 in gene regulation

• Main Authors: Chih-Chia Lu, 盧志嘉
• Other Authors: Jin-Ding Huang
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/82775905931469683226

碩士 === 國立成功大學 === 藥理學研究所 === 92 === 　　Sp1 is a ubiquitous transcription factor that regulates a variety of mammalian genes. Previous studies utilized a truncated Sp1 cDNA lacking the first 82 amino acids of the Sp1 coding sequence (696 amino acids). It has been reported that transcriptional activation of the rat CYP2D5 gene involves synergism between Sp1 and C/EBPb (MCB 14:1383, 1994). We found that the full-length (778 amino acids) Sp1 was less active than Sp1 (696) in transactivating CYP2D5 promoter. We therefore examined the role of this amino terminus of Sp1 in gene regulation. 　　Using Drosophila cells that lack endogenous Sp1 activity, we showed that Sp1 (696) was more potent than Sp1 (778) in transactivation of CYP2D5 promoter. Similar result was observed in rat Mrp3 promoter. However, the amino terminus of Sp1 significantly interfered synergistic effect between Sp1 and C/EBPb in CYP2D5 promoter, but moderately affect the synergistic effect between Sp1 and C/EBP�� in Mrp3 promoter. The amino terminus of Sp1 prevent Sp1, C/EBPb, or CBP/p300 synergistic transactivation of CYP2D5 promoter. We further construct truncated Sp1 cDNA lacking the first 28; 46; 64 amino acids of the Sp1 coding sequence (Sp1-N1; Sp1-N2; Sp1-N3 respectively). We observed that these deletion constructs were almost equal to Sp1 (696) in transactivation of CYP2D5 promoter in Drosophila SL2 cells. By using SUMOplot�� prediction software, it contains one SUMO targeted motifs with high probability within the first 28 a.a. Mutational analyses of this site enhance the activity of the CYP2D5 promoter about two to three folds, but the activity of Mrp3 promoter was no obvious increase. Coexpression of Sp1 mutants with C/EBP�� enhance the activity of Mrp3 promoter about two to three folds than Sp1 (778) with C/EBP��. We found that Sp1 mutants with higher protein expression and equal DNA binding in Western blotting and DAPA assay, and the ability of of these mutants translocation into nuclear is not altered. Taken together, it suggests that sumoylation at the amino terminus of Sp1 regulated its transcriptional activity.