p53-dependent gene repression through p21 is mediated by recruitment of E2F4 repression complexes

Abstract

The p53 tumor suppressor protein is a major sensor of cellular stresses, and upon stabilization, activates or represses many genes that control cell fate decisions. While the mechanism of p53-mediated transactivation is well established, several mechanisms have been proposed for p53-mediated repression. Here, we demonstrate that the cyclin-dependent kinase inhibitor p21 is both necessary and sufficient for the downregulation of known p53-repression targets, including survivin, CDC25C, and CDC25B in response to p53 induction. These same targets are similarly repressed in response to p16 overexpression, implicating the involvement of the shared downstream retinoblastoma (RB)-E2F pathway. We further show that in response to either p53 or p21 induction, E2F4 complexes are specifically recruited onto the promoters of these p53-repression targets. Moreover, abrogation of E2F4 recruitment via the inactivation of RB pocket proteins, but not by RB loss of function alone, prevents the repression of these genes. Finally, our results indicate that E2F4 promoter occupancy is globally associated with p53-repression targets, but not with p53 activation targets, implicating E2F4 complexes as effectors of p21-dependent p53-mediated repression.

Figure 1

p21 is required for the downregulation of p53-repression targets. HCT116 WT, HCT116 p53−/−, and HCT116 p21−/− cells were treated with 0.5 μM doxorubicin (Doxo), 20 μM nutlin-3, or vehicle control for 24 h. (a) Cell-cycle analysis. The propidium iodine intensities and the percentages of cells in each cell-cycle phase are indicated. (b, c) Quantitative RT–PCR showing the expression of (b) p53 upregulated target genes and (c) p53-repression target genes. Error bars indicate s.d. of representative experiments performed in triplicate.

Figure 2

Inhibition of p21 abrogates the ability of p53 to repress its target genes. p53 was induced in EJp53 cells stably expressing either shGFP or shp21 by reducing the tet concentration from 1.0 to 0.02 μg/ml for 24 h. (a) Cell-cycle analysis. The propidium iodine intensities and the percentages of cells in each cell-cycle phase are indicated. (b, c) Quantitative RT–PCR (b) and immunoblots (c) showing the expression of p53 and its upregulated target genes p21 and MDM2. (d) Quantitative RT–PCR of p53 downregulated genes. Error bars indicate s.d. of representative experiments performed in triplicate.

Figure 3

p21 in the absence of p53 is sufficient for the downregulation of p53-repression targets. p53 and p21 were induced in EJp53 and EJp21 cells, respectively, by the complete removal of tet for the indicated time points. (a) Cell-cycle analysis. The propidium iodine intensities and the percentages of cells in each cell-cycle phase are indicated. (b, c) Quantitative RT–PCR (b) and immunoblots (c) showing the expression of p53 and its upregulated target genes p21 and MDM2. (d) Quantitative RT–PCR of p53-repression target genes. Error bars indicate s.d. of representative experiments performed in triplicate.

Figure 4

p16 is sufficient for the downregulation of p53-repression targets. p16 was induced in EJp16 cells by the complete removal of tet for the indicated time points. (a) Cell-cycle analysis. The propidium iodine intensities and the percentages of cells in each cell-cycle phase are indicated. (b, c) Quantitative RT–PCR showing the expression of (b) p16 and (c) p53-repression target genes. Error bars indicate s.d. of representative experiments performed in triplicate.

Figure 5

Regulation of known E2F target genes by p53, p21, and p16. (a) Quantitative RT–PCR of E2F target genes in EJp53, EJp21, and EJp16 cells. p53, p21, and p16 were induced in their respective cells by the complete removal of tet for 24 h. (b) Quantitative RT–PCR showing the expression of E2F target genes in HCT116 WT, HCT116 p53−/−, and HCT116 p21−/− cells treated with 0.5 μM doxorubicin (Doxo), 20 μM nutlin-3, or vehicle control for 24 h. Error bars indicate s.d. of representative experiments performed in triplicate.

Figure 6

E2F4, but not E2F1 or E2F7, is recruited to p53-repression target promoters in response to p53 or p21 induction. ChIP analysis showing E2F1, E2F4, and E2F7 promoter occupancy on p53 repression and E2F target promoters in the presence and absence of (a) p53 in EJp53 and (b) p21 in EJp21 cells. p53 and p21 were induced in their respective cells by the complete removal of tet for 72 h. AChR was used as a negative control. Target sequences were detected by quantitative RT–PCR of eluted DNA. The promoter occupancy is shown as the percent of input DNA. Error bars indicate s.d. of representative experiments performed in triplicate.

Figure 7

The p53-repression target ARF is not regulated by p21 or E2F4 repressive complexes. (a) Quantitative RT–PCR showing the expression of ARF in EJp53, EJp21, and EJp16 cells. p53, p21, and p16 were induced in their respective cells by the complete removal of tet for the indicated time points. (b) Quantitative RT–PCR showing the expression of ARF in EJp53 cells stably expressing shGFP or shp21. p53 was induced in by reducing the tet concentration from 1.0 to 0.02 μg/ml. (c, d) ChIP analysis of the indicated genes showing the ratio of E2F4 to E2F1 promoter occupancy in (c) EJp53 cells induced for p53 for 72 h and (d) EJp21 cells induced for p21 for 24 and 48 h. Error bars indicate s.d. of representative experiments performed in triplicate.

Figure 8

E2F4 promoter recruitment by RB family members is essential for p53-mediated gene repression. p21 was induced in EJp21 cells expressing either HPV E7 or vector control by the removal of tet. (a, b) Quantitative RT–PCR of p21 (a) and immunoblot of p21 and HPV E7 levels (b). (c) ChIP analysis showing E2F4 promoter occupancy on p53-repression target promoters. AChR was used as a negative control. Target sequences were detected by quantitative RT–PCR of eluted DNA. The promoter occupancy is shown as the percent of input DNA. (d) Quantitative RT–PCR of p53-repression target genes. Error bars indicate s.d. of representative experiments performed in triplicate.

Figure 9

Global correlation between p53-mediated gene repression and E2F4 promoter occupancy. (a) Heatmap showing the correlation between the E2F4 ChIP-seq peak score and the p53 microarray expression levels. Row Z-score was performed on the log2-transformed values for each gene. (b) E2F4 ChIP-seq peak scores were divided into low (<4.5), medium (4.5–7.5), high (7.5–20), and very high (>20) binding groups. The percentage of p53 upregulated and downregulated genes, normalized for total gene number, that fall into each of these categories is shown in the form of a bar graph.