Figure 4. Transcriptional regulation of LKB1 by p53.

A) Schematic representation of the putative p53 binding sites in upstream sequence of LKB1 and the DNA fragments cloned into the upstream of the firefly luciferase reporter gene (LUC). Each reporter construct was transiently transfected into ECC-1 cells and the cells were harvested after 24 h for the dual luciferase assay. The promoter activity of each construct was measured using the firefly luciferase activity that was first normalized to Renilla activity and then calculated as fold induction relative to that of the empty pGL3 Basic plasmid. B) The effect of p53 knockdown and overexpression on the promoter activity of LKB1. After transfection with p53 siRNA or infection with retrovirus encoding full-length p53 (p53-VC) for 6 h, ECC-1 cells were then transfected with LKB1(−164/−1) reporter construct and subjected to the dual luciferase assay. C) The sequence of the p53-binding site in LKB1 genomic sequence is shown and compared to the p53 consensus binding. R, G or A; W, A or T; Y, C or T. The sequence of the mutant version of the p53-binding site is also shown. ECC-1 cells were transduced with p53-VC or control retroviral particles (VC) for 6 h and then transfected with LKB1(−164/−1) (Wt–LKB1) or mutated construct (MutLKB1). D) Chromatin immunoprecipitation assay was performed to determine the association of p53 with the LKB1 promoter. Normal rabbit IgG was included as the negative control (IgG). The recovered chromatin was subjected to quantitative PCR analysis using primers shown in the diagram. PCR amplification of the p53 site in the p21 promoter was included as positive control. Results are given as fold enrichment relative to IgG.