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Synergistic transcriptional activation of the MCK promoter by p53: tetramers link separated DNA response elements by
P Jackson1, I Mastrangelo, M Reed
1Oncology Research Centre, Prince of Wales Hospital, Randwick, NSW, Australia.
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.
Insights
The tumor suppressor p53 protein activates muscle creatine kinase (MCK) gene transcription. Multiple p53-binding sites on the MCK gene work together to boost gene activation.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The tumor suppressor protein p53 plays a critical role in regulating gene expression.
- Several p53 target genes, including WAF1, Cyclin G, and muscle creatine kinase (MCK), possess multiple p53-binding elements in their regulatory regions.
Purpose of the Study:
- To investigate the functional significance of multiple p53-binding elements in the transactivation of the muscle creatine kinase (MCK) gene.
- To elucidate the mechanism by which p53 mediates synergistic transcriptional activation through these elements.
Main Methods:
- Promoter deletion studies were employed to analyze the MCK gene's regulatory regions.
- Electron microscopy was used to visualize the interaction of p53-binding elements.
- Analysis of tetramerization-deficient p53 mutants assessed their role in transcriptional activation and synergy.
Main Results:
- Both proximal and distal p53-binding elements in the MCK promoter are essential for high-level, synergistic transcriptional activation in vivo.
- p53-p53 interactions facilitate looping of the DNA between binding sites, indicating intervening DNA may not be crucial for synergy.
- Synergistic activation was maintained even with reduced spacing (150 bp) between elements.
- Tetramerization-deficient p53 mutants showed impaired activation but retained the ability to induce synergy.
Conclusions:
- High-level transcriptional activation of genes with multiple p53 response elements is achieved through a mechanism involving synergistic interaction of p53-binding sites.
- The spatial arrangement and DNA sequence between p53-binding elements are less critical than the elements themselves for synergistic activation.