Abstract
The p53 tumor suppressor protein regulates the transcription of regulatory genes involved in cell cycle arrest and apoptosis. We have reported previously that inducible expression of the p53 gene leads to the cell cycle arrest both at G(1) and G(2)/M in association with induction of p21 and reduction of mitotic cyclins (cyclin A and B) and cdc2 mRNA. In this study, we investigated the mechanism by which p53 regulates transcription of the cdc2 gene. Transient transfection analysis showed that wild type p53 represses whereas various dominant negative mutants of p53 increase cdc2 transcription. The cdc2 promoter activity is not repressed in cells transfected with a transactivation mutant, p53(22/23). An adenovirus oncoprotein, E1B-55K inhibits the p53-mediated repression of the cdc2 promoter, while E1B-19K does not. Since the cdc2 promoter does not contain a TATA sequence, we performed deletion and point mutation analyses and identified the inverted CCAAT sequence located at -76 as a cis-acting element for the p53-mediated regulation. We found that a specific DNA-protein complex is formed at the CCAAT sequence and that this complex contains the NF-Y transcription factor. Consistently, a dominant negative mutant of the NF-YA subunit, NF-YAm29, decreases the cdc2 promoter, and p53 does not further decrease the promoter activity in the presence of NF-YAm29. These results suggest that p53 negatively regulates cdc2 transcription and that the NF-Y transcription factor is required for the p53-mediated regulation.
Highlights
Inactivation of p53 tumor suppressor gene occurs in over half of all human tumors, implying that loss of this gene represents a fundamentally important step in genomic instability and susceptibility to malignant transformation [1, 2]
In the absence of tetracycline in the medium, the expression of dCCAATmt-Chloramphenicol Acetyltransferase (CAT) was decreased only about 1.3- fold, while pCCAATmt-CAT was decreased by 7.7-fold, which is a level similar to that of the wild type cdc2 promoter (Fig. 3). These results suggest that the distal CCAAT sequence located at Ϫ76 is necessary for regulation of the cdc2 promoter by p53
The antibodies against A or B subunit of NF-Y led to the formation of a supershifted band at the expense of band a, while the antibodies against the C/EBP isoforms did not give rise to such supershifted bands. These results indicate that the complex formed at the distal CCAAT sequence, corresponding to band a, contains the heterotrimeric transcription factor, NF-Y
Summary
Inactivation of p53 tumor suppressor gene occurs in over half of all human tumors, implying that loss of this gene represents a fundamentally important step in genomic instability and susceptibility to malignant transformation [1, 2]. Repression of hsp70 transcription by p53 is mediated by an interaction between p53 and CCAAT-binding protein (CBF) a transcription activator of the hsp70 promoter [19]. Negative Regulation of cdc2 Transcription by p53—To examine the effect of p53 on cdc2 transcription, we transfected the cdc2 promoter-CAT reporter construct (pcdc2-CAT) into HepG2 cells with the plasmid carrying either the wild type p53 or its dominant negative mutant form, p53273.
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