Figure 3. Inactivated FANCD2 promotes the expression of ΔNp63 via a 1.2 kb DNA fragment downstream of the P2 promoter.

(A) Schematic representation of 1 kb and 1.2 kb DNA fragments up or downstream of the P2 promoter. (B) Inactivated FANCD2 enhances the 1.2 kb reporter activity. Both the 1.0 kb and 1.2 kb DNA fragments (Fig. 3A) were individually cloned into the upstream of pGL-3-promoter- reporter, named 1 kb or 1.2 kb reporters. Cells showed a higher reporter activity when the 1.2kb reporter was co-transfected with K561R FANCD2 cDNA-containing plasmid. The relative reporter activity was plotted upon photon counts as we did previously. Cells carrying the 1.0kb reporter along with either wt or mtFANCD2 cDNA did not show a noticeable difference in the reporter activity as compared to the 1.0 kb reporter alone (not shown). The results shown are a representative of five independent experiments performed each time in triplicate, and error bars indicate the standard deviation. The transfection efficiency of the reporter assay for pEGFP-Flag-wtFANCD2 or -mtFANCD2 was measured via western blotting analysis with antibodies against GFP (the pEGFP vector produces polycistronic mRNAs encoding non-fusion GFP protein) and Flag-fused FANCD2 protein. (C) The inactivated FANCD2 associates more strongly with the 1.2 kb DNA fragment. Both HCT116 and U2OS sets of stably-transfected cell pairs carrying an intact or impaired FA pathway, respectively (Supplementary Figures 1 and 3 right panel) were used to perform FANCD2 ChIP analysis using primers to bracket DNA fragments 1 kb up or 1.2 kb downstream of the P2 promoter (Figure 3A). (The relative folds were calculated upon the band density measured by NIH image J program with the corresponding bands generated from control cells as “1”.)