Figure 3.

Reversal of targeted histone deacetylation upon dissociation of TetR–Ume6 from the HIS3 promoter. Cross-linked chromatin from TetR–Ume6-expressing YKT2 cells (contain a nonactivated HIS3 promoter) treated with Dox for 0–15 min or 4 h was immunoprecipitated with antibodies to acetylated histones H3 and H4 or the HA epitope for measuring TetR–Ume6 occupancy, and analyzed by quantitative PCR. (A) Histone acetylation at the HIS3 promoter was analyzed as described in Figure 1. (B) Histone acetylation at the nucleosome +2 region within the HIS3 ORF was determined using the PGK1 promoter as an internal control. After normalizing each IP signal to the input signal, the HIS3 results were divided by those of the control locus, and expressed relative to the 4-hour-treated sample. (C) TetR–Ume6 binding at the HIS3 promoter was determined using the PGK1 promoter as an internal control. After normalizing each IP signal to the input signal, the specific binding of TetR–Ume6 at HIS3 was determined by dividing the HIS3 results by the control PGK1 results. (D,E) Kinetics of changes in histone acetylation and TetR–Ume6 dissociation from the promoter throughout the time course. At each time-point, the change in IP value from the time 0 value was determined, and expressed relative to the change at 4 h set as 100%.