The identification and characterization of novel upstream regulatory elements within the murinep53 promoter

p53 mRNA levels are tightly regulated during the cell cycle with its transcription being induced prior to DNA synthesis. However, the mechanism controlling this regulation is not well defined. Through characterizing an additional 1000bp of upstream DNA sequences of the murine p53 gene, we identified new positive and negative regulatory elements. Furthermore, we found a trans-acting factor(s) that binds within a positive cis-acting element (-972/-953) in a manner indicative of cell cycle regulation. When Swiss3T3 cells are arrested by serum depletion p53 mRNA levels decrease and binding of this regulatory factor(s) to the promoter is reduced. Upon serum stimulation p53 mRNA levels increase prior to the cells entering S phase. When the factor(s) are experimentally sequestered from the promoter or when the regulatory element is deleted from the promoter overall p53 promoter activity is reduced. There is no further reduction in p53 promoter activity upon serum depletion and the kinetics of induction is delayed by approximately 5 hours. These findings indicate that a factor(s) binding within the -972/-953 regulatory element on the p53 promoter is important for the tight regulation of p53 mRNA expression in response to mitogen stimulation. One of the factors binding within the -972/-953 regulatory element has been identified as C/EBPbeta, a CCAAT/enhancer binding protein critical for normal growth and differentiation of various cell types that exists in three different isoforms. Characterizing of C/EBPbeta has indicated that C/EBPbeta-2 is the most likely candidate of the three C/EBPbeta isoforms binding the p53 promoter and regulating p53 promoter activity during the cell cycle.