Figure 3.

Histone H3K37A mutants exhibit a rapamycin-induced cell-cycle defect. (A) H3K37A mutants have defects in S-phase after rapamycin treatment. H3WT and histone mutants were grown asynchronously to log phase before mock treating or treating with 25 nM rapamycin for 1.5 hours and 4 hours. Cells were then stained with SYTOX Green and analyzed by flow cytometry. The percentages of cells in each phase are presented and are listed in Additional file 3. (B)tco89Δ results in a complete absence of S-phase cells after rapamycin treatment. The experiment in (A) was repeated using wild-type (BY4741) and tco89Δ. The percentages of cells in each phase are listed in Additional file 4. (C) Both H3K37 charge and acetylation mutations rescue the cell-cycle phenotype. Experiment as (A), except H3WT, H3K37A, H3K37R, and H3K37Q mutants were analyzed. Additional file 5 lists the percentages of each cell-cycle phase. (D) Strains from (C) were spotted to YPD control plates or YPD plates containing 25 nM rapamycin and incubated at 30°C for four days. (E,F) H3K37A and tco89Δ mutants exhibit sensitivity to transient rapamycin treatment. The indicated strains were grown to log phase before spotting equal numbers of cells to control YPD plates. The remaining cultures were treated with 25 nM (sub-inhibitory) or 200 nM (inhibitory) rapamycin for 5.5 hours (E) or 24 hours (F). Cells were washed, 5-fold serially diluted, spotted to YPD plates, and incubated at 30°C for 3 days. Rap, rapamycin; WT, wild-type.