FIG. 7.

Histone removal is reversible and requires the continuous presence of the stimulus. (A) Schematic of the experiment to test the effect of stimulus withdrawal on histone loss from the IL-2 promoter. The times of stimulation (P/I), withdrawal (W/D), and harvest are shown. (B) EL-4 T cells were stimulated with P/I (stippled lines) for the times indicated. In parallel samples, the stimulus was removed after 4 h, and cells were harvested 2 h and 20 h later (solid lines). These samples were all subjected to ChIP assays with antibodies against acetylated H3K9 (AcK9) and the C terminus of H3 (H3C), and the immunoprecipitated DNA was amplified with IL-2 primer sets B and F. Data are presented as ratios of immunoprecipitated sample to total input normalized to unstimulated cell samples, which were given a value of 1. (C) Schematic of the experiment to test the effect of stimulus withdrawal and restimulation on histone loss at the IL-2 promoter. The times of stimulation (P/I), stimulus withdrawal (W/D), restimulation (P/I), and harvest are shown. (D) EL-4 cells were stimulated with P/I for 4 h, and the stimulus was withdrawn for a further 2 or 16 h and then restimulated with P/I for 30 min and 2 h. In parallel, untreated cells were stimulated for 30 min and 2 h with P/I. The amount of acetylated H3 (AcH3) and total H3 were measured by ChIP assays using IL-2 primer set B. Data are presented as ratios of immunoprecipitated sample to total input normalized to unstimulated cell samples, which were given a value of 1.