Sp1 Plays a Critical Role in the Transcriptional Activation of the Human Cyclin-dependent Kinase Inhibitor p21 Gene by the p53 Tumor Suppressor Protein

Abstract

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.

Multiplex enhancer-reporter assays uncover unsophisticated TP53 enhancer logic

“…This finding supports the clutch-like model, whereby transcription factors are able to displace nucleosomes, and are in competition with nucleosome binding. Finally, we find that TP53 mostly acts alone as a TF bound to its target enhancers, although previous studies had suggested cofactorship at the DNA level (Koutsodontis et al 2001;Thornborrow and Manfredi 2001). Note that non-sequence-specific cofactors (e.g., EP300) are likely to interact with TP53 at the protein level, independent of the DNA sequence, to recruit RNA polymerase and activate target gene transcription.…”

“…This suggests that TP53 mainly functions alone, without other regulatory factors cobinding at the DNA level. This is surprising as one of the proposed mechanisms for TP53 target specificity is through the recruitment of coregulatory transcription factors (Koutsodontis et al 2001;Thornborrow and Manfredi 2001).…”

“…Several elements of the TP53 binding site architecture have been proposed to contribute to the functional properties of the enhancer. These include variations of the spacer between two TP53 half-sites (Hoffman et al 2002;Godar et al 2008), or variations in binding sites for coregulatory factors (Koutsodontis et al 2001;Thornborrow and Manfredi 2001). However, these properties have not been evaluated on a global scale.…”

Modulation of the Cyclin-Dependent Kinase Inhibitor p21WAF1/Cip1 Gene by Zac1 through the Antagonistic Regulators p53 and Histone Deacetylase 1 in HeLa Cells

“…Because p21 is a transcriptional target of p53, it plays a crucial role in mediating growth arrest when cells are exposed to DNA-damaging agents such as doxorubicin and g-irradiation (16). Apart from p53, a variety of other factors including Sp1, p300/CBP, c-Jun, and E2F are known to activate p21 transcription (17)(18)(19)(20). Additionally, various mechanisms exist to regulate the levels of p21 protein in a cell including transcriptional regulation, epigenetic silencing, mRNA stability, and ubiquitin-dependent and ubiquitin-independent degradation of the protein (17,21,22).…”

ZNF424, a novel human KRAB/C2H2 zinc finger protein, suppresses NFAT and p21 pathway

“…The p21 gene, mainly regulated at the transcriptional level, plays a crucial role in mediating growth arrest when cells are exposed to DNA-damaging agents (7). Over-expression of p21 results in G1-, G2-, or S-phase arrest upon exposure to DNA-damaging agents (8,9). Whereas induction of p21 predominantly leads to cell cycle arrest, repression of p21 may have a variety of outcomes depending on the cellular context (10).…”