Nucleosome turnover in the absence of TBP. See also Supplemental Figure S7. (A) Cells from a TBP-anchor-away strain expressing a galactose-inducible HA-tagged version of histone H3 (H3HA) from a single copy plasmid were grown overnight in raffinose before being arrested in G1 with alpha factor. Galactose was then added directly to the medium at T0 to induce expression of H3HA. Glucose was added 10 min later (T10) to suppress further expression of H3HA, or rapamycin was added to deplete TBP from the nucleus. Culture aliquots were removed at these and subsequent time points and directly processed for Western blot and ChIP analyses. H3HA and TBPFRB protein levels in whole-cell extracts prepared before cross-linking were assessed using anti-HA and anti-TBP antibodies. G6PDH served as a loading control. Note that the left and right panels are from the same gel exposure for each protein and are directly comparable. (B) TBP promoter occupancy at the highly transcribed CDC19 and ADH1 genes and at the silent STE3 gene was measured at the indicated time points after galactose addition, as in Figure 2. The results are expressed relative to the T0 value for CDC19, which was set to 100. (C) H3HA incorporation at the same promoters and time points after addition of glucose or rapamycin (red curves) to deplete TBP. The values are expressed as percentage of input DNA recovered.
