Overview of nutrient refeeding effects on histone acetylation in SP yeast cells. (A) Outline of culture/refeeding protocol. Time 0 corresponds to 8 days in culture, before nutrient addition. (B) Culture medium glucose concentration (solid lines), and cell viability (dashed lines), plotted as a function of time after refeeding. Viability is normalized to 100% live cell recovery at time 0. (C) Immunoblot analysis of H4 acetylation (H4ac antibody) and bulk H4 expression (loading control) at the indicated time points after nutrient refeeding. (D) Immunoblot analysis of H4 acetylation and bulk H4 expression at the indicated time points after refeeding with 2 or 10% glucose. (E) Immunoblot analysis of histone acetylation using residue- and acetylation-specific anti-H4 antibodies, and an antibody raised against an H3 K9ac K14ac tail peptide. *Indicates an unknown cross-reacting species. (F) Immunoblot analysis of acetylated and bulk H4 in the soluble and DNA-associated pools of histones obtained from cells before and after (4 h) glucose refeeding.

Identification of the KATs required for glucose induction of H3/H4 acetylation in SP. (A) Expression of Esa1-TAP in unfed and glucose-fed (1 h) SP cells monitored by anti-CBP immunoblotting. Equivalent loading is confirmed by the intensity of the background band (*). (B) Immunoblotting analysis of H4 acetylation in wild-type and two ESA1 temperature-sensitive strains. Cells were unfed or glucose-fed, at either the permissive (26°C) or restrictive (40°C) temperature. (C) Immunoblotting analysis of H4 acetylation in wild-type and HAT1 and SAS2 null mutants. Glucose-fed cells were harvested at 1 h. (D) Immunoblotting analysis of H3 acetylation in wild-type and GCN5, HPA2 and SAS3 null mutants. Glucose-fed cells were harvested at 1 h. Glucose was added to 2% weight/volume for all experiments.

Identification of H3 and H4 KAT complexes required for glucose induction of H3/H4 acetylation in SP. (A) Expression of HA-tagged Epl1 and epl1(1–485) monitored by anti-HA immunoblotting. In (A–D), glucose-fed cells were harvested at 1 h. (B) Immunoblotting analysis of H4 acetylation in wild-type and epl1(1–485) cells. (C) Immunoblotting analysis of H3 acetylation in wild-type cells compared to null mutants lacking the genes encoding Gcn5, Spt7 (specific for SAGA/SLIK) and Ahc1 (a specific subunit of ADA). (D) Immunoblotting analysis of H3 acetylation in wild-type and null mutant strains lacking the genes encoding Spt8 (a specific subunit of SAGA) and Rtg2 (a specific subunit of SLIK). (E) Immunoblotting analysis of H3 acetylation in wild-type and a TRA1 temperature sensitive strain. Cells were incubated for 30 min at the restrictive temperature (40°C), then harvested after another 1 h at the same temperature in the presence or absence of added glucose. (F) Immunoblotting analysis of H3 acetylation in wild-type and mutant strains lacking CHD1 in a TRA1 or tra1-2 background. The chd1Δ single mutant was fed at room temperature and harvested at 1 h. The double mutant was fed after 30 min at the restrictive temperature (40°C) and harvested 1 h later. In (C–F), ‘H3 Kac’ denotes acetylated H3 detected using an anti-acetyl lysine antibody.

Metabolite profiles in unfed and glucose-fed SP cells. (A) Relationship of glycolysis to acetyl-CoA production for use by nuclear KATs. (B) ¹H-NMR spectra of a glucose reference sample (top panel) and samples of metabolites isolated from SP cells (bottom panel; only glucose peaks are shown). (C) Targeted quantitative profiling of cellular metabolite levels by ¹H-NMR, before and after glucose refeeding. Identical volumes were analyzed from samples prepared from the same number of cells and resuspended in the same final volume. The results are the average of two independent experiments. (D) Cellular level of acetate in glucose-fed cells relative to the level in unfed cells (average of three independent experiments ± SD). Acetate was measured by an enzymatic assay.

Metabolic regulation of global histone acetylation by glucose. (A) Expression of TAP-tagged Acs1 and Acs2 in unfed and fed cells monitored by anti-CBP immunoblotting. Glucose-fed cells were harvested at 1 h. (B) Immunoblotting analysis of H3 K9 and H4 acetylation in wild-type and ACS1/ACS2 mutant strains. Cells were unfed or glucose-fed (1 h), at either the permissive (26°C) or restrictive (40°C) temperature. (C) Immunoblotting analysis of H4 acetylation in wild-type and acs1Δ acs2-Ts1 mutant strains shifted from room temperature to 16°C at the time of glucose refeeding. (D) Immunoblotting analysis of H4 acetylation in wild-type cells fed 2% glucose, 2% 2-deoxy-D-glucose (2DG), or a mixture of both sugars at 1% each. (E) Immunoblotting analysis of H4 acetylation in wild-type and a PYK1 (CDC19) temperature sensitive mutant strain. Cells were unfed or glucose-fed (1 h), at either the permissive (26°C) or restrictive (40°C) temperature.