Volume 1, Issue 4, Pages 495-505 (March 1998) Absence of Gcn5 HAT Activity Defines a Novel State in the Opening of Chromatin at the PHO5 Promoter in Yeast  Philip D Gregory, Andrea Schmid, Maasoumeh Zavari, Lin Lui, Shelley L Berger, Wolfram Hörz  Molecular Cell  Volume 1, Issue 4, Pages 495-505 (March 1998) DOI: 10.1016/S1097-2765(00)80050-7

Figure 1 Effect of a GCN5 Deletion on Activity of the PHO5 Promoter Acid phosphatase activity is shown under (A) repressing (high Pi) and (B) activating (no Pi) conditions. The relevant genotypes of the strains are indicated. YS18 (wt), YS5189 (gcn5), YS30 (pho85), YS5309 (pho85/gcn5), YS31 (pho80), and YS5319 (pho80/gcn5). Molecular Cell 1998 1, 495-505DOI: (10.1016/S1097-2765(00)80050-7)

Figure 2 Gcn5 Dependence of PHO5 Promoter Variants The results for the activation of lacZ reporter plasmids in the presence or absence of phosphate for the wild-type (YS18) and gcn5 (YS5189) strains are shown. Values in (A) were determined in the presence or absence of phosphate and in (B) before (−galactose) and after (+galactose) induction in phosphate-containing media. The particular promoter constructs employed are indicated. Molecular Cell 1998 1, 495-505DOI: (10.1016/S1097-2765(00)80050-7)

Figure 3 Deletion of GCN5 Defines a Novel Chromatin Structure at the PHO5 Promoter (A) Nuclei isolated from the three strains, YS18, YS5319, and YS31, (relevant genotypes indicated) grown in the presence of phosphate were treated with 0.25 (lanes 1, 5 and 15), 0.5 (lanes 2,6 and 14), 1 (lanes 3,7 and 13), and 2 (lanes 4, 8 and 12) U/ml DNaseI for 20 min at 37°C. DNA was isolated, digested with ApaI, analyzed on a 1.5% agarose gel, blotted, and hybridized with probe D (Almer et al. 1986). Double digests of purified genomic DNA with ApaI/BstEII, ApaI/ClaI, and ApaI/BamHI shown in lanes 9, 10, and 11, respectively, serve as markers. The schematic on the left shows the positions of the restriction sites used for generating marker fragments together with the nucleosomes and is drawn approximately to scale. (B) The chromatin organization of the repressed PHO5 promoter, including the TATA box (T) and the positions of the Pho4-binding sites UASp1 and UASp2. Also shown are probe D and the location of the ApaI site used for indirect endlabeling. The nucleosomes removed under wild-type conditions are shown as open circles. Molecular Cell 1998 1, 495-505DOI: (10.1016/S1097-2765(00)80050-7)

Figure 4 Effect of Phosphate Starvation on the Chromatin Structure of the PHO5 Promoter in a gcn5/pho80 Background Nuclei isolated from the YS5319 (pho80/gcn5) and YS31 (pho80) strains grown in the presence or absence of phosphate indicated were treated with 0.25 (lanes 1, 5, and 15), 0.5 (lanes 2, 6, and 14), 1 (lanes 3, 7, and 13), and 2 (lanes 4, 8 and 12) U/ml DNaseI for 20 min at 37°C. DNA was analyzed as in Figure 3. Molecular Cell 1998 1, 495-505DOI: (10.1016/S1097-2765(00)80050-7)

Figure 5 Effect of a GCN5 Deletion on the Accessibility of Restriction Sites at the PHO5 Promoter Nuclei isolated from YS31 (GCN5/pho80), which is referred to as “wt”, and YS5319 (gcn5/pho80), referred to as “gcn5” and grown in the presence of phosphate, were treated for 30 min at 37°C with 50 U (left) or 200 U (right) of the restriction enzyme indicated. To monitor the extent of cleavage, DNA was isolated, cleaved with HaeIII, analyzed on a 1.5% agarose gel, blotted, and hybridized with probe D (see Figure 3). The positions of the restriction sites with respect to the nucleosomal organization of the repressed PHO5 promoter are shown schematically underneath. In all cases, the appearance of the lower band represents accessibility of the corresponding site. The nucleosomes that are perturbed during normal activation are shown as open circles. Molecular Cell 1998 1, 495-505DOI: (10.1016/S1097-2765(00)80050-7)

Figure 6 The Effect of Gcn5 HAT Domain Mutants on the Activation of the PHO5 Promoter (A) Diagram of yeast Gcn5, indicating the domains of the protein and position and protein sequence of conserved region I (Brownell et al. 1996). The substitution mutants, named according to the group of amino acids mutated to alanine, are shown in brackets. (B) The histone acetylation activities of the Gcn5 HAT domain mutants tested are shown as a percentage of wild type (Wang et al. 1998; and this study). The PHO5 promoter activity of these mutants in the gcn5/pho80 (YS5319) background is shown in units of acid phosphatase activity in the presence or absence of phosphate. Molecular Cell 1998 1, 495-505DOI: (10.1016/S1097-2765(00)80050-7)

Figure 7 Mutation of Specific Residues Critical for Gcn5 HAT Activity Is Sufficient To Generate the Novel Chromatin Organization of the PHO5 Promoter (A) Nuclei isolated from the PKM, YIA, and KQL (see Figure 6) Gcn5 HAT mutant strains in a gcn5/pho80 background, grown in the presence of phosphate, were treated with 0.25 (lanes 1, 4, and 12), 0.5 (lanes 2, 5, and 11), and 1 (lanes 3, 6, and 10) U/ml DNaseI for 30 min at 37°C. DNA was analyzed as in Figure 3. (B) Nuclei isolated from YS30 (GCN5/pho80) denoted “wt,” YS5319 (gcn5/pho80) denoted “Δgcn5,” and the PKM, YIA, and KQL Gcn5 HAT mutant strains grown in the presence of phosphate were treated for 30 min at 37°C with 50 U (left) or 200 U (right) of ClaI. DNA was analyzed as in Figure 5. Molecular Cell 1998 1, 495-505DOI: (10.1016/S1097-2765(00)80050-7)