FIG 9.

Identification of hormone-dependent BRG1-mediated DNA breaks within chromatin MMTV. Transient DNA breaks were detected using a modified reiterative primer extension technique. (A) Schematic of the proximal portion of the MMTV promoter indicating nucleosomal location, restriction endonuclease cleavage sites, transcriptional factor binding regions, and positions of oligonucleotides used for primer extension. (B) SW-13/MMTV cells expressing GR and BRG1 or ΔHSA mutant protein were treated with 100 nM Dex for 0, 10, 15, or 30 min prior to isolation of nuclei and DNA. Purified genomic DNA was digested to completion with AvaII or HaeIII and analyzed for DNA breaks using iterative primer extension with ³²P-labeled primers specific for the upper or lower strand of MMTV. (C) Merbarone (Mer) treatment attenuates hormone-induced transcriptional activation of chromatin MMTV. U2OS/UL3 cells containing integrated copies of MMTV reporter or SW-13/MMTV cells expressing GR and BRG1 were incubated for 5 h with 100 μM Mer at the indicated concentrations, followed by treatment with Dex. Posttreatment, cells were lysed and assayed for luciferase activity. Relative luciferase units (RLU) values were normalized to total protein measured and reported as the average from three independent experiments. Error bars are standard deviations (n = 3). (D) Inhibition of TOP2β activity blocks hormone-dependent BRG1-mediated DNA break formation. SW-13/MMTV cells expressing GR and BRG1 were treated with Mer and/or Dex. Following treatment, purified genomic DNA was digested with AvaII or HaeIII and analyzed for DNA break formation as described above. Extension products were resolved on 7% denaturing polyacrylamide gels and exposed to a phosphorimager screen. DNA breaks were identified using ImageQuant peak-finder analysis software. (E) Hormone-stimulated recruitment of GR, BRG1, Ku70, or TOP2β to chromatin MMTV is not affected by treatment with Mer. SW-13/MMTV cells expressing GR and BRG1 were treated with Mer or vehicle (DMSO) for 5 h prior to addition of Dex or ethanol (EtOH) for 15 min or 1 h. Data are reported as the average from three independent experiments. (F) In vivo restriction enzyme accessibility assay to determine chromatin architecture at MMTV-NucB upon treatment with Mer and hormone (Dex). Nuclei purified from cells expressing GR and BRG1 were pretreated with Mer (5 h) and then subjected to treatment with Dex (15 min) followed by digestion with SstI (in vivo) restriction endonuclease. Purified products were further digested to completion with HaeIII (in vitro) and used as the template for iterative primer extension with an MMTV-specific primer. Extension products were resolved by 6% denaturing PAGE.