Fig. 5.

The p53R248Q and p53R273C mutants increase histone acetylation and p53 bind to HER2 promoter distal region. Chromatin immunoprecipitation (ChIP) assays were carried out in p53R248Q, p53R273C and empty vector stably transfected Saos-2 cells as described in the Material and Methods. Antibodies against acetylated histone H3, H4, p53 protein and IgG as non-relevant antibody were used for the immunoprecipitation. a Shows a graphic representation of the regions encompassed by the primers used for ChIP assay. Precipitated DNA was amplified with primers described in Additional file 2: Table S2. HER2d (b) -4000 bp; HER2e (c) -4500 bp, and HER2f (d) up-steam the HER2 promoter. Relative amounts of acetylated H3 and H4 histones, as well as p53 interaction with HER2 distal promoter regions were determined by quantitative PCR (qPCR). Normalized data were calculated relative to each primer set (b-d), comparing p52R248Q or p53R273C vs IgG ChIP 1.0% input sample. The results of Fold Enrichment are reported as the mean ± SEM of three independent experiments. Statistical analysis was performed with t-student test by comparing the results obtained for the Fold Enrichment in Saos-2 cells transfected with p53R48Q or p53R273C against empty vector transfected cells: *p < 0.05