In vivo evidence for binding of p53 to consensus binding sites in the p21 and GADD45 genes in response to ionizing radiation

Abstract

The tumor suppressor protein p53 has a transcriptional activation activity thought to mediate its biologic function including G 1 arrest and perhaps apoptosis. To learn more about p53's transactivator function in vivo, we performed genomic footprinting experiments examining p53-DNA interactions in the regulatory regions of the p53-regulated genes p21, GADD45, and MDM2. Using ionizing radiation to induce DNA damage in human ML-1 myeloblastic leukemia cells, the promoter and intronic regions of these genes containing p53-consensus binding sites were examined for in vivo footprints. There was a uniform and sustained expression of p53 protein as well as a strong induction of p21, GADD45, and MDM2 mRNA following irradiation. At the two p53 consensus binding sites in the p21 promoter, reduced DNaseI cleavage was observed in irradiated cells beginning 1 to 2 h after irradiation, being most pronounced after 2 h and diminishing after 8 h. A partial in vivo footprint was also observed in the third intron of the GADD45 gene beginning 2 h after irradiation. No in vivo footprints were seen at the two p53 binding sites in the MDM2 gene. Our study provides direct evidence that the DNA damage-induced activity of p53 is mediated by its consensus DNA binding sites in the p21 and GADD45 genes. We suggest that the transient nature and relative instability of p53-DNA interactions in vivo may make the p53 protein more accessible to a rapid turnover pathway which might be impaired under conditions when the protein is stably bound to DNA.

p53 binds to a constitutively nucleosome free region of the mdm2 gene

“…Recently one study examining the in vivo binding ability of p53 has been reported (Chin et al, 1997). However in this study the authors were unable to detect any p53 dependent protection changes at the mdm2 gene.…”

“…This suggests that p53 may be tethered to the complex via association with other DNA bound proteins. In a recent study the relative instability of p53-DNA interactions in vivo has been noted (Chin et al, 1997). In fact the authors were unable to explain their complete lack of an in vivo footprint at the mdm2 gene.…”

“…Studies on the ability of p53 to bind to DNA have been carried out predominantly in vitro, but little is known about the ability of the protein to bind to speci®c target sites in vivo (Chin et al, 1997). The region of the mdm2 gene with which p53 interacts was originally identi®ed by co-immunoprecipitation of a speci®c DNA element as well as by demonstrating a minimal sequence able to confer p53 speci®c transactivation of a reporter construct (Juven et al, 1993;Wu et al, 1993).…”

The specific activation of gadd45 following UVB radiation requires the POU family gene product N-oct3 in human melanoma cells

“…Most studies related to gadd45 expression in response to DNA damage have used genotoxic agents such as ionizing radiation (Carrier et al, 1994;Chin et al, 1997;Graunke et al, 1999;Papathanasiou et al, 1991;Zhan et al, 1993Zhan et al, , 1994, UVC irradiation Takahashi et al, 2001;Zhan et al, 1993Zhan et al, , 1998, camptothecin (Takahashi et al, 2001), or methyl methane sulphonate (Hollander et al, 1993;Zhan et al, 1998). Until now, studies of gadd45-responsiveness to DNA damage in human melanoma cell lines have used ionizing radiation or UVC irradiation as stressors (Bae et al, 1996;Haapajarvi et al, 1999).…”

Transition from In Situ to Invasive Testicular Germ Cell Neoplasia is Associated with the Loss of p21 and Gain of mdm-2 Expression

“…One such protein is p21 (8,9), which is actively transcribed in the presence of p53 protein bound to a site in the p21 promoter region (10,11). The transcription and subsequent translation of p21 leads to cell cycle arrest through p21's inhibitory interactions with the cyclins/cyclin-dependent kinases.…”