Abstract
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.
Highlights
P21WAF1/Cip1 (p21) modulates cyclin-dependent kinase activity resulting in cell growth arrest or progression [1, 2]
We have recently reported that the transforming growth factor- 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
Transactivation of the Human p21WAF1 Promoter by Jun Proteins Is Mediated by the Proximal Ϫ122 to Ϫ64 Region—The proximal region of the human p21 promoter extending between positions Ϫ122 and Ϫ64 is GC-rich and contains five sequences that resemble or match exactly the recognition sequence of the ubiquitous transcription factor Sp1 (5Ј-GGGCGG-3Ј, Fig. 1A, double underline). One of these Sp1 sites was shown previously to be required for the stimulation of the p21 promoter by TGF- in HaCaT keratinocytes [21], whereas at least one of these Sp1 sites was shown to mediate stimulation of the same promoter by phorbol esters during U937 cell differentiation [18]. Since both TGF- and phorbol ester signal transduction pathways are mediated, at least in part, by AP1 proteins (Jun and Activating transcription factor 2 (ATF-2)), we investigated the role of different AP1 family members in human p21 gene regulation
Summary
P21WAF1/Cip1 (p21) modulates cyclin-dependent kinase activity resulting in cell growth arrest or progression [1, 2]. We have recently reported that the transforming growth factor- 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.
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