c-Jun transactivates the promoter of the human p21WAF1/Cip1 gene by acting as a superactivator of the ubiquitous transcription factor Spl
c-Jun transactivates the promoter of the human p21WAF1/Cip1 gene by acting as a superactivator of the ubiquitous transcription factor Spl
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DOI：
10.1074/jbc.274.41.29572
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发表时间：
1999-10-08
影响因子：
4.8
通讯作者：
Moustakas, A
中科院分区：
文献类型：
--
作者：
Kardassis, D;Papakosta, P;Moustakas, A
细胞周期抑制蛋白p21(WAF1/Cip1) （p21）是p53依赖性生长控制机制和p53非依赖性终末分化途径的关键下游效应物。我们最近报道了转化生长因子-p通路特异性的Smad3和Smad4蛋白通过一个短的近端区域反激活人类p21启动子，该区域包含普遍存在的转录因子Spl的多个结合位点。在本研究中，我们发现sp1占据的启动子区域介导c-Jun和相关蛋白JunB、JunD和ATF-2对p21启动子的反激活。通过凝胶电泳迁移位移测定，我们发现该区域不包含c-Jun的结合位点。根据DNA结合数据，c-Jun在sp1缺陷果蝇来源的SL2细胞中过表达时，无法反激活p21启动子。c-Jun和Sp1在这些细胞中的共表达导致了该启动子的强协同反激活。此外，在HepG2细胞中，一个由6个串联高亲和力sp1结合位点与CAT基因融合组成的嵌合启动子被过表达的c-Jun反激活。以上数据表明c-Jun和Sp1之间存在功能上的合作。利用细菌中表达的GST-Sp1杂交蛋白和体外转录翻译的c-Sun，在体外证明了这两个因子之间的物理相互作用。c-Jun介导Sp1相互作用的区域位于亮氨酸拉链结构域的基本区域内。在体内，c-Jun和Sp1之间的功能相互作用通过基于gal4的转激活实验得到证实。在HepG2细胞中，过表达的c-Jun仅在与GAL4-(Sp1-(83-778)融合蛋白共表达时才会反激活一个由5个串联gal1结合位点组成的嵌合启动子。通过同样的实验，我们发现Sp1的B结构域富含谷氨酰胺的片段（Bc，氨基酸424-542）对于c- jun诱导的p21启动子的转激活是足够的。总之，我们的数据支持c-Jun对Sp1的超激活机制，这是基于这两个转录因子在人类p21上以及可能的其他Sp1依赖启动子之间的物理和功能相互作用。
The cell cycle inhibitor protein p21(WAF1/Cip1) (p21) is a critical downstream effector in p53-dependent mechanisms of growth control and p53-independent pathways of terminal differentiation. We have recently reported that the transforming growth factor-p pathway-specific Smad3 and Smad4 proteins transactivate the human p21 promoter via a short proximal region, which contains multiple binding sites for the ubiquitous transcription factor Spl. In the present study we show that the Sp1-occupied promoter region mediates transactivation of the p21 promoter by c-Jun and the related proteins JunB, JunD, and ATF-2. By using gel electrophoretic mobility shift assays we show that this region does not contain a binding site for c-Jun. In accordance with the DNA binding data, c-Jun was unable to transactivate the p21 promoter when overexpressed in the Sp1-deficient Drosophila-derived SL2 cells. Coexpression of c-Jun and Sp1 in these cells resulted in a strong synergistic transactivation of this promoter. In addition, a chimeric promoter consisting of six tandem high affinity Sp1-binding sites fused with the CAT gene was transactivated by overexpressed c-Jun in HepG2 cells. The above data propose functional cooperation between c-Jun and Sp1. Physical interactions between the two factors were demonstrated in vitro by using GST-Sp1 hybrid proteins expressed in bacteria and in vitro transcribed-translated c-Sun. The region of c-Jun mediating interaction with Sp1 was mapped within the basic region leucine zipper domain. In vivo, functional interactions between c-Jun and Sp1 were demonstrated using a GAL4-based transactivation assay. Overexpressed c-Jun transactivated a chimeric promoter consisting of five tandem GAL1-binding sites only when coexpressed with GAL4-(Sp1-(83-778) fusion proteins in HepG2 cells. By utilizing the same assay, we found that the glutamine-rich segment of the B domain of Sp1 (Bc, amino acids 424-542) was sufficient: for c-Jun-induced transactivation of the p21 promoter. In conclusion, our data support a mechanism of superactivation of Sp1 by c-Jun, which is based on physical and functional interactions between these two transcription factors on the human p21 and possibly other Sp1-dependent promoters.