Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21WAF1/Cip1 gene by the p53 tumor suppressor protein
Sp1 plays a critical role in the transcriptional activation of the human cyclin-dependent kinase inhibitor p21WAF1/Cip1 gene by the p53 tumor suppressor protein
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DOI：
10.1074/jbc.m104130200
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发表时间：
2001-08-03
影响因子：
4.8
通讯作者：
Kardassis, D
中科院分区：
文献类型：
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作者：
Koutsodontis, G;Tentes, I;Kardassis, D
在本研究中，我们提供了Spl在肿瘤抑制因子p53蛋白反激活人类细胞周期抑制剂p21(WAF1/Cip1) （p21）基因启动子的机制中的关键作用的证据。我们发现，在肝癌HepG2细胞中，p21启动子的远端p53结合位点可作为同源或异源启动子的增强子。在转染实验中，p53在表达Spl但具有突变p53形式的HaCaT细胞中反激活了p21启动子。相反，在缺乏内源性Spl或相关因子的果蝇胚胎源性Schneider's SL2细胞中，p53不能反激活p21启动子。p53和Spl共同转染SL2细胞导致p21启动子的协同反激活。在SL2细胞和HaCaT细胞中，由于p53结合位点或-82/-77 Spl结合位点的突变，协同反激活大大降低，这表明Spl和p53在p21启动子的反激活中存在功能合作。协同反活化也会因p53反活化结构域的突变而降低。通过谷胱甘肽s -转移酶下拉和共免疫沉淀法确定了Spl和p53蛋白之间的物理相互作用。通过使用缺失突变体，我们发现Spl的DNA结合域是其与p53物理相互作用所必需的。综上所述，Spl通过p21基因激活，在p53控制的DNA修复、细胞生长、分化和凋亡等重要生物学过程中发挥关键作用。
In the present study we present evidence for the critical role of Spl in the mechanism of transactivation of the human cell cycle inhibitor p21(WAF1/Cip1) (p21) gene promoter by the tumor suppressor p53 protein. We found that the distal p53-binding site of the p21 promoter acts as an enhancer on the homologous or heterologous promoters in hepatoma HepG2 cells. In transfection experiments, p53 transactivated the p21 promoter in HaCaT cells that express Spl but have a mutated p53 form. In contrast, p53 could not transactivate the p21 promoter in the Drosophila embryo-derived Schneider's SL2 cells that lack endogenous Spl or related factors. Cotransfection of SL2 cells with p53 and Spl resulted in a synergistic transactivation of the p21 promoter. Synergistic transactivation was greatly decreased in SL2 cells and HaCaT cells by mutations in either the p53-binding site or in the -82/-77 Spl-binding site indicating functional cooperation between Spl and p53 in the transactivation of the p21 promoter. Synergistic transactivation was also decreased by mutations in the transactivation domain of p53. Physical interactions between Spl and p53 proteins were established by glutathione S-transferase pull-down and coimmunoprecipitation assays. By using deletion mutants we found that the DNA binding domain of Spl is required for its physical interaction with p53. In conclusion, Spl must play a critical role in regulating important biological processes controlled by p53 via p21 gene activation such as DNA repair, cell growth, differentiation, and apoptosis.