FIG. 9.

Effect of TRA1 deletions on HAT subunit recruitment, histone acetylation levels, and HAT module integrity. (A to C) Strains were grown as described for Fig. 4 and analyzed by ChIP at the indicated genes. Shown are the ChIP results obtained with Esa1-6HA and H4K8Ac (A) or Gcn5-13Myc and H3K9Ac (B and C). Histone ChIP results are shown as the ratio of acetylated to total H3 or H4 relative to the wild-type value, set at 1.0. HAT ChIP results are shown relative to the wild-type value, set at 1.0. UI, not induced. Brackets below the TRA1 mutant labels indicate the domain(s) disrupted by the deletion. P/Fc, junction between PI3K C-terminal lobe and FATc domains. (D) Relative mRNA levels from a gcn5Δ strain grown at 30°C. (E) Relative mRNA levels from an esa1 temperature-sensitive mutant strain grown at 30°C or shifted to 37°C for the indicated times before gene induction. Experiments were performed in biological duplicate, and errors bars represent standard deviations. (F and G) Western analysis of immunoprecipitated Flag-Tra1 (F) and Gcn5-13myc (G) complexes. Amounts of the immunoprecipitate (IP) loaded were normalized to load equivalent amounts of Tra1 and Gcn5, respectively. Purified complexes were analyzed by Western blotting using the following antibodies: Flag, Myc, Ada2, and Ada3. (H) Histone H3 HAT activity of Flag-Tra1 purified complexes. Equivalent amounts of Flag-Tra1 purified complexes were incubated with free histone H3 and analyzed by Western blotting using antibodies against H3K9Ac. Coomassie blue staining was used as a loading control to assess total H3 levels in each reaction mixture. Relative HAT activity levels (HA) represent the percentages of the H3K9Ac acetylation levels relative to wild-type Flag-Tra1.