GR physically interacts with the
promoter in the absence of ligand. A. Schematic of walking ChIP primers covering ~2800 bp upstream and ~500 bp downstream of the transcription start site of Ch25h. The thick black line indicates the Ch25h promoter. The arrow and +1 indicate the location of the transcription start site. The numbers above indicate the bp location on Ch25h. The boxes below indicate the locations of the ChIP primers, and the region amplified by each primer set is indicated by a line between the boxes. The numbers above and below these lines indicate the designated “region” of the Ch25h promoter covered by each primer set. B. Chromatin was prepared from EPH-4 non-malignant mouse mammary cells 24 hours after treatment with either ethanol vehicle (-HC) or 1 μg/mL HC (+HC) in serum-free medium. ChIP analysis was carried out with the indicated antibodies and PCR primers amplifying the region between -447 to -219 bp of the Ch25h promoter. Ab, antibody; HA, hemagglutinin; H3, acetylated histone H3.