Induction of p53-dependent Activation of the Human Proliferating Cell Nuclear Antigen Gene in Chromatin by Ionizing Radiation

Abstract

A human fibroblast cell line with conditional p53 expression displayed a p53-dependent increase in both the protein and mRNA levels of proliferating cell nuclear antigen (PCNA) after exposure to ionizing radiation (IR). The combination of p53 induction and IR cooperated to activate a transiently expressed human PCNA promoter-reporter gene via a p53-responsive element. Chromatin immunoprecipitation assays with antibodies specific for p53 or p300/CREB-binding protein revealed specific p53-dependent enrichment of PCNA promoter sequences in immunoprecipitates of sheared chromatin prepared from irradiated cells. Maximal and specific association of acetylated histone H4 with the PCNA promoter also depended on p53 induction and exposure to IR. These data demonstrate p53 binding to a target site in the PCNA promoter, recruitment of p300/CREB-binding protein, and localized acetylation of histone H4 in an IR-dependent manner. These molecular events are likely to play a role in mediating activation of PCNA gene expression by p53 during the cellular response to DNA damage. The analyses indicate that the combination of p53 induction and IR activate the PCNA gene via mechanisms similar to that of p21/wild-type p53-activated factor but to a lesser extent. This differential regulation of PCNA and p21/wild-type p53-activated factor may establish the proper ratio of the two proteins to coordinate DNA repair with cell cycle arrest.Cellular responses to DNA damage bear upon tumorigenesis and the development of anti-cancer therapies. The p53 tumor suppressor protein integrates many of the cellular responses to DNA damage (for recent reviews, see Refs. 1 and 2). p53 possesses sequence-specific DNA binding activity and primarily functions as a transcription factor that binds to degenerate response elements in a variety of target genes, including p21/ WAF1, 1 mouse double minute-2, and Bax (3). Activation of p53target genes leads to either growth arrest and subsequent DNA repair or apoptosis depending on a number of variables including the extent of DNA damage (4). DNA-bound p53 recruits transcriptional co-activators that render localized remodeling of the chromatin structure to favor promoter access by the transcriptional machinery (5-7). To date, the best characterized p53 transcriptional co-activator is p300/CBP, which possesses intrinsic histone acetyltransferase activity and interacts with other proteins possessing histone acetyltransferase activity (8, 9). C-terminal acetylation of p53 by p300/CBP along with phosphorylation in the N terminus converts inert p53 to a transcriptionally active form (10, 11). p53-mediated transcriptional activation of the p21/WAF1 promoter correlates with p53 binding and p300/CBP-mediated acetylation of promoter-associated histones (12)(13)(14). The essential role of the TRRAP transcriptional co-activator in potentiating activation of the mouse double minute-2 promoter by p53 suggests that p53-associated transcriptional co-activators possess promoter selectivity (15). The PCNA gene encodes a highly co...

Estrogen Receptor-α Binds p53 Tumor Suppressor Protein Directly and Represses Its Function

“…S3B). A similar pattern (data not shown) of p53 and ER␣ binding was observed on the p53-response element of the PCNA gene, another transcriptional target of p53 (20). Immunoblotting analysis showed that the ER␣ expression was unaffected, and the p53 level, as expected, was increased by irradiation (Fig.…”

Functional Analysis of p53 Binding under Differential Stresses

“…As expected, the ChIP-chip screen identified some known p53 target genes: glutathione peroxidase 1 (GPX1) (74), PCNA (72), jagged 2 (JAG2) (70), and c-myc (MYC) (30). GPX1, PCNA, and MYC have p53 response elements that have been described previously (72,74,78). The promoters for both GPX1 and PCNA have p53REs within the CGIs identified from the array.…”

“…The promoters for both GPX1 and PCNA have p53REs within the CGIs identified from the array. Interestingly, three CGIs have been mapped to the PCNA locus (6 kb upstream, 500 bp upstream, and 6 kb downstream) (28), but only one was enriched for p53 binding (2.11-fold enrichment) and contains a previously reported p53RE at Ϫ278 bp (72). The MYC locus has several predicted p53REs located throughout the exon and intron structure of the human gene (78).…”

Changes in Whole Blood Gene Expression after Computed Tomography in Children: A Pilot Study

“…We observed dose-dependent increases in PCNA expression with increasing CT dose (mGy cm) and dose-dependent increases in TP53 and FLT3LG expression with increasing effective CT dose (mSv), which are in accordance with in vitro studies reporting increases in gene expression or protein concentrations after IR exposure. 45,46 Next, we examined correlations between the gene expression levels and IR history. In our subjects, regression analyses revealed that previous exposure to IR (either through prior CT scans or X-rays) was dose dependently related to MDM2 levels.…”