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In addition to its well-characterized function as a sequence specific transcriptional activator, there is growing evidence that the p53 tumor suppressor protein is also a sequence-specific transcriptional repressor. The concept that a transcription factor can exist as both an activator and a repressor of transcription is not new. In fact, it is the rare transcription factor that can perform only one of these functions. The initial challenges for individuals studying the repression function of p53 have been met; that is, a set of genes whose expression is decreased following p53 induction has been identified. These include the genes encoding alpha-fetoprotein, bcl-2, cyclin B, cdc2, cdc25, Map4, Mdr1, presenilin-1, and survivin, as well as others. The promoters for many of these genes have been cloned, and p53 has been found to bind to sites within these promoters using assays that measure binding in vivo, such as chromatin immunoprecipitation. The p53 binding sites in these promoters have been identified, and in many cases the mechanism whereby p53 represses transcription, for example by promoter occlusion or by recruitment of histone deacetylases, has been elucidated. The current challenge is to create mutant forms of p53 that are capable of repressing transcription but not activating it (or vice versa), such that the contribution of this activity to tumor suppression by p53 can be effectively delineated. [ABSTRACT FROM AUTHOR]

Differential display is a reverse-transcription polymerase chain reaction (RT-PCR) technique that was introduced in 1992 by Liang and Pardee (1). In this technique, messenger RNA 3′ termini are amplified using an anchored oligo-dT primer and a series of arbitrary 13-mers; a single round of cDNA synthesis is followed by PCR amplification in the presence of radiolabeled nucleotide, resulting in the amplification of subsets of mRNAs expressed in the cell. This complex PCR product is resolved on a denaturing polyacrylamide gel; following autoradiography, gene expression is detected as a ladder of cDNAs that differ in size by a single nucleotide. Theoretically, the use of different sets of primers from both directions in discrete RT-PCR reactions allows for the examination of the majority of expressed genes within the cell. This chapter will focus first on a brief review of the use of differential display to identify p53 target genes, as well as secondary tumor suppressor genes important for tumor progression. This will be followed by an overview of the methodology of differential display, along with recent advances in this technique; a more comprehensive analysis can be found in Methods in Molecular Biology, Volume 85: Differential Display Methods and Protocols, edited by Liang and Pardee (2). An outline of the methodology involved in differential display is provided in Fig. 1, and individual steps are described below; typically these steps follow the protocol described by Liang and Pardee (1,2), but in some instances changes have been made and noted. [ABSTRACT FROM AUTHOR]

There is substantial evidence in the literature that, in addition to functioning as an activator of transcription, the p53 tumor suppressor protein can also function as a sequence-specific transcriptional repressor of a separate set of genes. However, elucidation of the mechanism whereby p53 functions as a transcriptional repressor has been obscured by the use of artificial assays to measure this activity; these assays include transient transfection analyses, where both p53 and target promoters are overexpressed. This chapter describes alternative approaches for the definition of sequence elements that mediate transcriptional repression by p53. These include the McKay (immunobinding) assay, which measures the in vitro binding of large fragments of DNA, as well as chromatin immunoprecipitations (ChIPs), which measure in vivo binding. The use of such assays should better define the mechanism of transcriptional repression by p53 and should aid in the elucidation of the contribution of this activity to p53-dependent growth arrest and programmed cell death (apoptosis). [ABSTRACT FROM AUTHOR]