Mechanisms of differential activation of target gene promoters by p53 hinge domain mutants with impaired apoptotic function

Suppression of tumor cell growth by p53 results from the activation of both apoptosis and cell cycle arrest functions that have been shown to be separ able activities of p53. We report here that some mutants in the p53 hinge d omain, a short linker between the DNA binding and tetramerization domains, differentially activated the promoters of p53 target genes and possessed an impaired apoptotic function. Our results indicate that the hinge domain ma y play an important role in differentially regulating p53 cell cycle arrest and apoptotic functions. However, the mechanisms by which p53 hinge domain mutants differentially activate its target genes, e.g. p21(WAF1/CIP1) and Bax, remain unknown. To investigate the possible mechanisms, recombinant p2 1(WAF1/CIP1) and Bax promoters were constructed, resulting in rearrangement of the existing p53 binding sites within a given promoter or actually swap ping p53 binding sites between the two promoters. Our results suggest that multiple mechanisms of differential transactivation occur, depending on the molecular nature of the relevant hinge domain mutant, such as the possibil ity that dual separate DNA binding sites in the p21(WAF1/CIP1) promoter are responsible for the selective transactivation activity of p53 hinge domain mutant del300-327, which has a large deletion in the hinge domain. Lack of ideal p53 binding sites in the Bax promoter results in less potent activat ion than that seen with the p21(WAF1/CIP1) promoter when it is transactivat ed by hinge domain point mutant mutR306P or short deletion mutant del300-30 8 proteins. How the single mutation or the short deletion affect the confor mation of p53 and consequently the transactivation of the Bax promoter will require further investigation of the relevant p53 protein: DNA-binding dom ain by NMR and x-ray crystallographic techniques.