Figure 4

ΔNp63 isotypes bind the ATM promoter in vivo and stimulate ATM transcription. (A) The indicated genes (1 μg) were transfected into Saos2 cells. Cells were harvested after 24 hrs, total RNA was isolated and analyzed for ATM gene expression by real time RT-PCR. ATM gene expression was normalized to β-actin, and the data is represented as fold-change over empty vector control. (B) ΔNp63α and E2F-1 bind the endogenous ATM promoter in vivo. Protein-DNA complexes in cycling HaCat cells were crosslinked and analysed by chromatin immunoprecipitation using anti-p63 or anti-E2F-1 antibodies or no antibody control. Crosslinks were reversed and purified DNA was quantified by real-time PCR using ATM primers. Relative amounts of INPUT DNA were 1/10 for ATM. (C) ΔNp63 isoforms stimulate ATM -LUC reporter activity. H1299 were co-transfected with 1 μg ATM-LUC, 0.2 μg pRL-CMV plasmids, and 1 μg empty pCDNA3.1, p53, E2F1 plasmids, or p63 plasmids expressing ΔNα, ΔNγ, TAα or TAγ isoforms. Cells were harvested after 24 hrs and luciferase activity was analysed by Dual-Luciferase reporter assay kit (Promega). Data is normalised to the empty vector control. (D) The ATM promoter is more sensitive to E2F-1 than ΔNp63α. H1299 cells were transfected with 10ng, 100ng or 1000ng E2F-1 or p63 expression plasmids, and reporter plasmids, and harvested as described above. Specific ATM reporter activity was determined as described previously (C). (E)-(F) ΔNp63α and E2F-1 bind an exogenous ATM promoter in vivo. H1299 cells were transfected the ATM-LUC reporter plasmid, and co-transfected with ΔNp63α (E) or E2F-1 (F) expression plasmids Protein-DNA complexes were crosslinked after 24 hrs, and ChIP analysis was done using anti-p63 or anti-E2F-1 antibodies. Crosslinks were reversed and purified DNA was quantified by real-time PCR using ATM primers. Relative amounts of INPUT DNA were 1/100.