Gender	Male
E-mail	james.manfredi@mssm.edu
Education and Training	Ph.D., Albert Einstein College of Medicine
	M.S., Albert Einstein College of Medicine

Education and Training	Ph.D., Albert Einstein College of Medicine
	M.S., Albert Einstein College of Medicine

The two binding sites for p53 in the human p21 promoter are representative of two distinct classes
Two response elements for the tumor suppressor p53 have been identified in the promoter of the gene encoding the cyclin-dependent kinase inhibitor p21. Binding of a monoclonal antibody directed against the carboxyl terminus of p53 selectively enhanced binding by p53 to the upstream (5' site) without affecting the affinity of p53 for the downstream (3' site). Mutational analysis showed that a single base change can cause one site to behave similarly to the other site. These sites in the p21 promoter represent two distinct classes of p53 response elements found in a variety of genes. The mAb 421-enhanced class was shown to include elements from the gadd45, mdm2, and cyclin G genes as well as the box A element from the IGF-BP3 gene, whereas the mAb 421-inhibited class included bax, the 3' element from the p21 promoter, a novel element from the cdc25C gene, and the box B element from the IGF-BP3 gene. These results demonstrate the existence of two classes of p53 binding sites in the human genome and that the binding of p53 to these two classes of sites can be differentially regulated by binding of mAb 421. This novel example of the regulation of binding site selection by a transcription factor suggests a possible mechanism for selectivity in target gene activation by the p53 protein.

Three high mobility group proteins, HMG-1, HMG-2, and HMG-I, all enhanced the DNA binding ability of p53 to both the 5' and 3' sites and partially restored the ability of the antibody to enhance binding of p53 to the 3' site in vitro. Although the closely related HMG-2 and the unrelated HMG-I had similar effects on the DNA binding abilities of p53 in vitro, only HMG-1 was capable of stimulating the transcriptional activity of p53 in cells. These results demonstrate that, in spite of their close sequence similarity, HMG-1 and HMG-2 are functionally distinct in their regulation of p53.

To examine further the role of the C-terminal region in the differential binding of p53 to these two elements, truncated forms of p53 were produced either by proteolytic digestion of the full-length protein or by recombinant expression of a defined deletion mutant. While full-length p53 bound comparably to both sites, fragments of p53 lacking the carboxyl terminus showed enhanced binding to the p21 5' site without substantially affecting binding to the p21 3' site. Such assays can be used to classify other p53 response elements, confirming that each of the sites in the p21 promoter is indeed representative of a distinct subset of p53 binding sites. These results demonstrate that the C-terminal region of p53 selectively negatively regulates the binding of p53 to elements such as the p21 5' site and, consistent with previous studies, suggest that modification of the C-terminus is required to achieve high affinity binding to these sites in cells. Although such C-terminal modification can explain how p53 transcriptional activates via an element like the p21 5' site, a distinct mechanism is clearly necessary for p53-dependent activation via elements such as the p21 3' site.

Tang HY, Zhao K, Pizzolato JF, Fonarev M, Manfredi JJ, Langer JC. Constitutive expression of the cyclin-dependent kinase inhibitor p21 is regulated by the tumor suppressor protein p53. J Biol Chem 1998 Oct 30; 273(44): 29156-63.

Resnick-Silverman L, St. Clair S, Maurer M, Zhao K, Manfredi JJ. Identification of a novel class of genomic DNA binding sites for the tumor suppressor protein p53 suggests a mechanism for the regulation of the seque. Genes Dev 1998 July 15; 12(14): 2102-7.

Thornborrow EC, Manfredi JJ. ) One mechanism for cell-type specific regulation of the bax promoter by the tumor suppressor p53 is dictated by the p53 response element. J Biol Chem 1999 Nov 19; 274(47): 33747-56.