c-Jun transactivates the promoter of the human p21(WAF1/Cip1) gene by acting as a superactivator of the ubiquitous transcription factor Spl

The cell cycle inhibitor protein p21(WAF1/Cip1) (p21) is a critical downstr eam effector in p53-dependent mechanisms of growth control and p53-independ ent pathways of terminal differentiation. We have recently reported that th e transforming growth factor-p pathway-specific Smad3 and Smad4 proteins tr ansactivate the human p21 promoter via a short proximal region, which conta ins multiple binding sites for the ubiquitous transcription factor Spl. In the present study we show that the Sp1-occupied promoter region mediates tr ansactivation of the p21 promoter by c-Jun and the related proteins JunB, J unD, 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 w ith the DNA binding data, c-Jun was unable to transactivate the p21 promote r when overexpressed in the Sp1-deficient Drosophila-derived SL2 cells. Coe xpression of c-Jun and Sp1 in these cells resulted in a strong synergistic transactivation of this promoter. In addition, a chimeric promoter consisti ng of six tandem high affinity Sp1-binding sites fused with the CAT gene wa s transactivated by overexpressed c-Jun in HepG2 cells. The above data prop ose functional cooperation between c-Jun and Sp1. Physical interactions bet ween the two factors were demonstrated in vitro by using GST-Sp1 hybrid pro teins 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-Ju n and Sp1 were demonstrated using a GAL4-based transactivation assay. Overe xpressed c-Jun transactivated a chimeric promoter consisting of five tandem GAL1-binding sites only when coexpressed with GAL4-(Sp1-(83-778) fusion pr oteins in HepG2 cells. By utilizing the same assay, we found that the gluta mine-rich segment of the B domain of Sp1 (Bc, amino acids 424-542) was suff icient: for c-Jun-induced transactivation of the p21 promoter. In conclusio n, our data support a mechanism of superactivation of Sp1 by c-Jun, which i s based on physical and functional interactions between these two transcrip tion factors on the human p21 and possibly other Sp1-dependent promoters.