c-Jun transactivates the promoter of the human p21(WAF1/Cip1) gene by acting as a superactivator of the ubiquitous transcription factor Sp1.
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-beta 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 Sp1. 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-Jun. 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 GAL4-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.
Kardassis
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Department of Basic Sciences, University of Crete Medical School, Institute of Molecular Biology, Foundation of Research and Technology of Hellas, Heraklion GR-71110, Crete, Greece. kardasis@imbb.forth.gr
Papakosta
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P
Pardali
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K
Moustakas
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A
D000818:Animals; D001483:Base Sequence; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004330:Drosophila / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D015534:Trans-Activators / Q000494:pharmacology*; D014157:Transcription Factors / Q000235:genetics; D014407:Tumor Cells, Cultured
C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D016329:Sp1 Transcription Factor; D015534:Trans-Activators; D014157:Transcription Factors