PMC

Genes associated with cell cycle and mitosis are enriched among targets of the p53-p21-DREAM-CDE/CHR pathway. Venn diagram displaying the overlap in the groups of genes found as repressed by p53, bound by DREAM, and containing a conserved CHR element. Top 10 GO terms enriched among the 210 genes displayed in Table and the other group overlaps as identified using the DAVID Functional Annotation tool (Supplementary Table S3).

p53 does not bind to promoters of genes harboring CHR sites. Protein binding to promoters of the indicated genes was tested by ChIP in (A) HCT116 wild-type cells, (B) HCT116 p21^−/− and (C) HFF cells either left untreated or treated with doxorubicin for 24 h (HFF) or 48 h (HCT116) followed by real-time PCR. The CDKN1A promoter served as a positive control for p53 binding; the GAPDHS promoter which does not bind p53 was used as a negative control. One representative experiment with three technical replicates (n = 3) is displayed.

p53 induces p21-dependent binding of DREAM to genes with CHR sites. Protein binding to CHR-containing promoters in untreated or 48 h doxorubicin-treated HCT116 (A) wild-type or (B) p21^−/− cells was tested by ChIP followed by real-time PCR. Protein binding to the GAPDHS promoter served as negative control. One representative experiment with three technical replicates (n = 3) is displayed. Significance was tested using the paired Student's t-test; n.s. not significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. (C) Flow cytometry of cells used in Figure ; cells were stained with propidium iodide (PI).

Repression of non-canonical CHR-containing genes by p53 requires p21 and is conserved between mouse and human. The log₂-fold change of mRNA expression from treated compared to untreated (A) NIH3T3, (B) HCT116 wild-type, (C) HCT116 p21^−/− and (D) HFF cells is displayed. Cells were treated with doxorubicin, nutlin-3a or 5-FU for 24 h. Untreated cells and cells treated with DMSO served as controls. Normalization was carried out against measurements from untreated cells. GAPDH, L7 and U6 served as negative controls for p53 response, while CDKN1A and MDM2 were employed as positive controls. (A–C) Experiments were performed with two biological replicates and three technical replicates each (n = 6). (D) Experiments were performed with three technical replicates (n = 3). Significance of changes in expression levels was tested against GAPDH expression levels using the unpaired Student's t-test; n.s. not significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.