Figure 2
miR-27a is a transcriptional target of p53 273H. (a) Schematic illustration of miR-27a promoter and the positions of PCR amplicons used in ChIP assays. (b) ChIP analysis showing p53 273H binding to the promoter region of miR-27a. Input and immunoprecipitated DNA (by an antibody against p53) from H1299-Tet-On-p53 273H cells after treatment with doxycycline for 24 h were amplified by quantitative PCR to examine abundance of the indicated amplicons. An isotope-matched anti-IgG (anti-immunoglobulin G) antibody was also used as a negative control. (c) ChIP analysis showing p53 273H but not wild-type p53 binding to the −3056 to −2832 region of miR-27a promoter. Input and immunoprecipitated DNA (by an antibody against p53) from H1299-Tet-On-p53, H1299-Tet-On-p53 273H, or control cells after treatment with doxycycline for 24 h were amplified by PCR to detect abundance of the indicated amplicons. Amplification for GAPDH was used as a negative control. (d) MDA-MB-468 cells stably expressing either control or p53-specific shRNA were subjected to ChIP assays using anti-p53 antibody or an isotope-matched control IgG. ChIP products were amplified by PCR to determine abundance of the indicated amplicons. (e) A schematic illustration of pGL-3-basic-based reporter construct used in luciferase assays to examine the transcriptional activity of the −3556 to −2675 region in response to wild-type or mutant p53 expression. (f) The increasing amounts of constructs encoding either wild-type or mutant p53 were cotransfected into H1299 cells together with pGL3-miR-27a and Renilla luciferase plasmids. Twenty-four hours after transfection, reporter activity was measured by luciferase assays and plotted after normalizing with respect to Renilla luciferase activity. The data are represented as mean±S.D. of three independent experiments. ***P<0.001
