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Originally published In Press as doi:10.1074/jbc.M104130200 on May 30, 2001

J. Biol. Chem., Vol. 276, Issue 31, 29116-29125, August 3, 2001
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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*

George KoutsodontisDagger , Ioannis Tentes§, Paraskevi Papakosta, Aristidis Moustakas§, and Dimitris Kardassis||

From the Department of Basic Sciences, University of Crete Medical School and Institute of Molecular Biology and Biotechnology, Foundation of Research and Technology of Hellas, Heraklion GR-71110, Greece and the § Ludwig Institute for Cancer Research, Box 595, S-751 24 Uppsala, Sweden

In the present study we present evidence for the critical role of Sp1 in the mechanism of transactivation of the human cell cycle inhibitor p21WAF1/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 Sp1 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 Sp1 or related factors. Cotransfection of SL2 cells with p53 and Sp1 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 Sp1-binding site indicating functional cooperation between Sp1 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 Sp1 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 Sp1 is required for its physical interaction with p53. In conclusion, Sp1 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.

* This work was supported by a grant from the Human Frontier Science Program (to D. K. and A. M.) and funds from the Institute of Molecular Biology and Biotechnology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a fellowship from the "Maria Michael Manasaki Fund."

Permanent address: Democritus University of Thrace Medical School, Dept. of Biochemistry, Alexandroupolis GR-681 00, Greece.

|| To whom correspondence should be addressed: Dept. of Basic Sciences, University of Crete Medical School, Heraklion GR-71110, Crete, Greece. Tel.: 3081-394549; Fax: 3081-394530; E-mail: kardasis@ imbb.forth.gr.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.


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