The transcription factor p53: not a repressor, solely an activator

Abstract

The predominant function of the tumor suppressor p53 is transcriptional regulation. It is generally accepted that p53-dependent transcriptional activation occurs by binding to a specific recognition site in promoters of target genes. Additionally, several models for p53-dependent transcriptional repression have been postulated. Here, we evaluate these models based on a computational meta-analysis of genome-wide data. Surprisingly, several major models of p53-dependent gene regulation are implausible. Meta-analysis of large-scale data is unable to confirm reports on directly repressed p53 target genes and falsifies models of direct repression. This notion is supported by experimental re-analysis of representative genes reported as directly repressed by p53. Therefore, p53 is not a direct repressor of transcription, but solely activates its target genes. Moreover, models based on interference of p53 with activating transcription factors as well as models based on the function of ncRNAs are also not supported by the meta-analysis. As an alternative to models of direct repression, the meta-analysis leads to the conclusion that p53 represses transcription indirectly by activation of the p53-p21-DREAM/RB pathway.

Keywords: CDE, cell cycle-dependent element; CDKN1A; CHR, cell cycle genes homology region; ChIP, chromatin immunoprecipitation; DREAM complex; DREAM, DP, RB-like, E2F4, and MuvB complex; E2F/RB complex; HPV, human papilloma virus; NF-Y, Nuclear factor Y; cdk, cyclin-dependent kinase; genome-wide meta-analysis; p53.

Figure 1.

Solely genes activated by p53 are found enriched for p53 binding. A regulation score, named Expression Score, ranging from −6 to +6 was assigned to 19,736 known protein-coding genes from 6 genome-wide p53-dependent gene expression analyses.^, (A) All ChIP-peaks from 6 genome-wide p53 binding studies, that were identified in at least 2 studies, were allocated to the nearest gene.^, Out of the 19,736 genes, 13.4% were assigned at least one such p53 ChIP-peak. The percentage of genes with a p53 ChIP-peak in a specific Expression Score group is displayed by the black line. The blue line indicates a theoretical uniform distribution of ChIP-peak-containing genes across the 13 Expression Score groups. (B) The percentage of default p53 targets (Table S2) in each Expression Score group is given by the black line. The theoretical uniform distribution of default p53 targets (n = 171 or 0.8% of 19,736 genes) across the 13 Expression Score groups is indicated by the blue line.

Figure 2.

Experimental validation of data from the meta-analysis. (A) p53 protein binding to reported p53 response regions in untreated anddoxorubicin-treated HCT116 cells was tested by ChIP. A fragment of the GAPDHS promoter served as a negative control while CDKN1A and MDM2 served as positive controls. (B) mRNA expression in HCT116 cells treated with doxorubicin or nutlin3a for 24 h. Cells treated with DMSO served as a control. The log₂ fold-expression from doxorubicin- or nutlin3a-treated cells compared to DMSO control cells is displayed as. GAPDH, L7, and U6 served as negative controls, while CDKN1A, MDM2, and PPM1D were employed as positive controls. Significance of expression was tested against U6 expression levels using paired Student's t-test. Experiments were performed with 2 biological replicates and 2 technical replicates each (n = 4). *P ≤ 0.05; **P ≤ 0.01;***P ≤ 0.001.

Figure 3.

Indirect repression through p53-p21-DREAM or -RB/E2F pathways. (A) The percentage of genes bound by DREAM in proximity to their transcriptional start site (TSS) in each Expression Score group is displayed. The theoretical uniform distribution across the 13 Expression Score groups of genes bound by DREAM is indicated by the blue line (3.5% of 19,736 genes). (B) Displayed for each Expression Score group is the percentage of genes bound by RB in proximity to their TSS. The blue line indicates a theoretical uniform distribution of genes bound by RB (4.1% of 19,736 genes) across the 13 Expression Score groups. (C) The percentage of genes bound by p130 in proximity to their TSS is shown for each Expression Score group. A theoretical uniform distribution of genes bound by p130 (15.2% of 19,736 genes) across the 13 Expression Score groups is indicated. (D) Compilation of targets displayed in (A-C). The blue line indicates a theoretical uniform distribution of genes bound by DREAM, p130, or RB (16.1% of 19,736 genes) across the 13 Expression Score groups. The red area marks the fraction of genes bound by DREAM, p130, or RB in Expression Score groups −6, −5 and −4 (76.7 % of 399 genes).

Figure 4.

Genes repressed by p53 are enriched for CHRs which bind DREAM and E2F sites which recruit RB/E2F complexes. (A) The percentage of genes possessing a phylogenetically conserved CHR element in proximity to their TSS in each Expression Score group is displayed. The theoretical uniform distribution across the 13 Expression Score groups of genes with a phylogenetically conserved CHR element is indicated by the blue line (12.1% of 19,736 genes). (B) The percentage of genes harboring a phylogenetically conserved E2F site in proximity to their TSS in each Expression Score group is displayed. The theoretical uniform distribution across the 13 Expression Score groups of genes possessing a phylogenetically conserved E2F sites is indicated by the blue line (8.2% of 19,736 genes). (C) The percentage of genes with a phylogenetically conserved TATA-box in proximity to their TSS in each Expression Score group is displayed. The theoretical uniform distribution across the 13 Expression Score groups of genes holding a phylogenetically conserved TATA-box is indicated by the blue line (5.9% of 19,736 genes).

Figure 5.

CCAAT- and GC-boxes do not mediate repression by p53 independent of DREAM, p130, or RB. (A) The percentage of genes harboring a phylogenetically conserved CCAAT-box in proximity to their TSS in each Expression Score group is displayed. The theoretical uniform distribution across the 13 Expression Score groups of genes with a phylogenetically conserved CCAAT-box is indicated by the blue line (15.9% of 19,736 genes). (B) The percentage of genes holding a phylogenetically conserved GC-box (Sp1 site) in proximity to their TSS in each Expression Score group is displayed. The theoretical uniform distribution across the 13 Expression Score groups of genes possessing a phylogenetically conserved GC-box (Sp1 site) is indicated by the blue line (31.1% of 19,736 genes). (C) All genes bound by DREAM, p130, or RB (n = 3,189) are removed from the total set of 19,736 genes for further analyses. (D) The percentage of genes harboring a phylogenetically conserved CCAAT-box in proximity to their TSS in each Expression Score group is displayed. The theoretical uniform distribution across the 13 Expression Score groups of genes with a phylogenetically conserved CCAAT-box is indicated by the blue line (13.3% of 16,547 genes). (E) The percentage of genes possessing a phylogenetically conserved GC-box (Sp1 site) in proximity to their TSS in each Expression Score group is displayed. The theoretical uniform distribution across the 13 Expression Score groups of genes holding a phylogenetically conserved GC-box (Sp1 site) is indicated by the blue line (29.0% of 16,547 genes).

Figure 6.

The tumor suppressor p53 is not a direct repressor of transcription, it solely activates its target genes upon binding to DNA. In order to activate transcription, the p53 tetramer binds to the p53 RE of its target gene. The transcription factor p53 acts as repressor by activation of the p53-p21-DREAM/RB pathway ultimately leading to indirect p53-dependent transcriptional repression.