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1.
J Biol Chem ; 276(31): 29116-25, 2001 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-11384995

RESUMO

In the present study we present evidence for the critical role of Sp1 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 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.


Assuntos
Ciclinas/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Carcinoma Hepatocelular , Linhagem Celular , Clonagem Molecular , Inibidor de Quinase Dependente de Ciclina p21 , Drosophila melanogaster , Inibidores Enzimáticos , Escherichia coli , Genes Reporter , Genes p53 , Humanos , Queratinócitos , Neoplasias Hepáticas , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Sondas de Oligonucleotídeos , Osteossarcoma , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Fator de Transcrição Sp1/genética , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética
2.
J Biol Chem ; 274(41): 29572-81, 1999 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-10506225

RESUMO

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.


Assuntos
Ciclinas/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteínas de Saccharomyces cerevisiae , Fator de Transcrição Sp1/metabolismo , Transativadores/farmacologia , Animais , Sequência de Bases , Inibidor de Quinase Dependente de Ciclina p21 , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Proteínas Fúngicas/genética , Regulação da Expressão Gênica , Genes Reporter , Humanos , Ligação Proteica , Proteínas Recombinantes de Fusão , Fatores de Transcrição/genética , Células Tumorais Cultivadas
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