Synergistic transcriptional activation of the MCK promoter by p53: tetramers link separated DNA response elements by DNA looping.

Abstract

The WAF1, Cyclin G and muscle creatine kinase (MCK) genes, all contain multiple copies of the consensus p53-binding element within their regulatory regions. We examined the role of these elements in transactivation of the muscle creatine kinase (MCK) gene by p53. The MCK promoter possesses distal (-3182 to -3133) and proximal (-177 to -81) p53-binding elements within which residues -3182 to -3151 (distal) and -176 to -149 (proximal) show homology to the consensus p53-binding site. Using promoter deletion studies, we find that both proximal and distal elements are required for high level, synergistic transcriptional activation in vivo. Electron microscopy indicates that p53-p53 interactions link proximal and distal p53-binding elements and cause looping out of intervening DNA, suggesting that this DNA sequence may be dispensable for synergy. This idea was confirmed by progressive deletion of the DNA between p53-binding elements. Synergism persisted with spacing reduced to only 150 bp. Tetramerization-deficient p53 mutants were defective for transcriptional activation but still capable of synergy. Our results provide evidence for a model by which high level transcriptional activation of promoters with multiple p53 response elements is achieved.