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Molecular and Cellular Biology, May 2001, p. 3375-3386, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3375-3386.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Kinetics of p53 Binding to Promoter Sites In Vivo

Suzanne T. Szak,dagger Deborah Mays, and Jennifer A. Pietenpol*

Department of Biochemistry, Center in Molecular Toxicology, and The Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Received 17 November 2000/Returned for modification 29 January 2001/Accepted 9 February 2001

Downstream target genes of p53 are thought to mediate its tumor-suppressive activity, but it is unknown whether differential transactivation of these genes is regulated at the level of p53 binding to their promoters. To address this issue, p53 binding in vivo to consensus sites in the p21Waf1, MDM2, and PIG3 promoters was investigated in cells exposed to adriamycin (ADR) or ionizing radiation as well as in an inducible p53 cell line. p53-DNA complexes were cross-linked in vivo by treating the cells with formaldehyde and processed by chromatin immunoprecipitation-PCR. This methodology allowed for the analysis of relevant p53-DNA complexes by preventing redistribution of cellular components upon collection of cell extracts. Increased p53 binding to the p21Waf1, MDM2, and PIG3 promoters occurred within 2 h after p53 activation; however, significant increases in PIG3 transcription did not occur until 15 h after p53 binding. Gel shift analyses indicated that p53 had lower affinity for the consensus binding site in the PIG3 promoters compared to its consensus sites in the p21 and MDM2 genes, which suggests that additional factors may be required to stabilize the interaction of p53 with the PIG3 promoter. Further, acetylated p53 (Lys382) was found in chemically cross-linked complexes at all promoter sites examined after treatment of cells with ADR. In summary, the kinetics of p53 binding in vivo to target gene regulatory regions does not uniformly correlate with target gene mRNA expression for the p53 target genes examined. Our results suggest that target genes with low-affinity p53 binding sites may require additional events and will have delayed kinetics of induction compared to those with high-affinity binding sites.

* Corresponding author. Mailing address: 652 Medical Research Building II, The Vanderbilt Cancer Center, Nashville, TN 37232-6838. Phone: (615) 936-1512. Fax: (615) 936-1790. E-mail: pietenpol{at}toxicology.mc.vanderbilt.edu.

dagger Present address: National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD 20892.

Molecular and Cellular Biology, May 2001, p. 3375-3386, Vol. 21, No. 10
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.10.3375-3386.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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