Figure 2: p53 activation promotes the binding of transcriptional repressor E2F4 at the Fancd2 gene.

(a) p21 is required for the downregulation of Fancd2. mRNAs from untreated or Nutlin-treated p21^−/− MEFs were quantified. Results from three independent experiments. (b) Increased E2F4 binding at the Fancd2 promoter upon p53 activation. A map surrounding the Fancd2 transcription start site (TSS) is shown on the left (white box: UTR (Ex1: exon 1); lollipops: putative E2F4-binding sites according to ref. 70 (Supplementary Fig. 3); arrows: ChIP PCR primers), and ChIP data on the right. ChIP assay for E2F4 binding was performed in Nutlin-treated p53^−/− MEFs, and untreated or Nutlin-treated WT MEFs, with an antibody against E2F4 or rabbit IgG as a negative control. Immunoprecipitates were quantified using real-time PCR, fold enrichment was normalized to data over an irrelevant region, then E2F4 binding at Fancd2 in untreated WT cells was given a value of 1. Data from two independent ChIP experiments, each quantified in triplicates. (c) The p53-dependent regulation of Fancd2 occurs via a CDE/CHR motif. CDE/CHR motifs are required for gene repression by an E2F4-containing DREAM complex³². These motifs consist of a 6-bp long GC-rich CDE site (bound by E2F4) located 4-bp upstream of a 6-bp long AT-rich CHR site. On top, CDE/CHR motifs regulating the expression of five mouse genes are presented, as well as a putative CDE/CHR motif 23–38-bp downstream of the mouse Fancd2 TSS, and its mutated counterpart (with mutations in the CDE). Below, a 2-kb fragment centred around the Fancd2 TSS, containing a WT or mutant CDE/CHR, was cloned upstream a Luciferase gene and transfected into NIH-3T3 cells, treated or not with Nutlin, then Luciferase activity was measured after 24 h. Although the cell cycle kinetics of cells transfected with either plasmid were identical (Supplementary Fig. 4), Nutlin led to decreased luciferase activity only with the construct containing a WT CDE/CHR motif. Mutation of the putative CDE site increased Luciferase basal expression, and abrogated the effect of Nutlin. Results from three independent experiments. In all figures, means+s.e.m. are shown; ***P≤0.001, *P≤0.05, NS, not significant by Student’s t-test.