FIG 2.

Hormone-dependent recruitment of Ku to chromatin MMTV and endogenous promoters requires the HSA domain of BRG1. Recruitment of Ku70/86 to stably integrated MMTV or endogenous PLZF or HSD11B2 promoters requires the N-terminal HSA domain of BRG1, as demonstrated by chromatin immunoprecipitation analysis. (A) Western blot analysis using anti-BRG1 or -GR antibodies in order to monitor protein expression of transfected plasmids, with stably expressed GR serving as protein loading control. (B to E) Purified immunoprecipitated DNA fragments from treated SW-13/MMTV cells expressing BRG1, K798R, or ΔHSA proteins were analyzed by real-time PCR using primer sets covering the MMTV promoter nucleosome B region (B) and the 3′ coding region of the integrated luciferase reporter (C) or gene-specific primers targeting the endogenous promoters of HSD11B2 (D) and PLZF (E). Cross-linked DNA-protein complexes were isolated using antibodies specific for GR, BRG1, Ku70, or Ku86 protein. Normal IgG was used as a control for nonspecific interactions. Quantitative analysis was performed, with results displayed as percentage of ChIP input. Values are shown as mean ± standard deviation from 3 biological replicates.