Snf2 is acetylated by Gcn5 and deacetylated by Hst2 and Rpd3. (A) TAP-purified proteins from Snf6-TAP-tagged strain (Snf6-TAP) or untagged strain (nontagged control) were resolved with SDS-PAGE and analyzed by immunoblotting with acetyl lysine (α-AcK) and Snf2 (α-Snf2) antibodies. (B) Immunoprecipitated SWI/SNF from either wild-type or mutant cells was analyzed by immunoblotting with α-AcK and α-Snf2. (C) SWI/SNF complexes from wild type or gcn5Δ were resolved with SDS-PAGE and analyzed by silver staining and Western blot using α-AcK. (D) SWI/SNF was immunoprecipitated with Flag agarose from wild type or a series of HDAC deletion mutants. Immunoprecipitated complexes were analyzed with acetylated Snf2 antibody (α-AcSnf2) and α-Snf2.

Gcn5 acetylates two lysine residues located between the AT hook domains of Snf2, both in vivo and in vitro. (A) Schematic diagram of Snf2 fragments: Snf2N (1–1383 amino acids), Snf2C (1384–1703 amino acids), AT hook (1384–1546 amino acids), and bromodomain (1547–1703 amino acids). GST-Snf2 fragments acetylated by Ada2 complexes were subjected to SDS-PAGE and visualized by autoradiograph. (B) Wild-type and mutant AT hook proteins acetylated by Ada2 complexes in vitro were subjected to SDS-PAGE and visualized by autoradiograph (³H). (C) SWI/SNF was immunoprecipitated with Flag agarose from wild type or mutants expressing K1493R, K1497R, or K1493R and K1497R (RR) Snf2 under native promoter. Immunoprecipitated complexes were analyzed by immunoblotting using α-AcK and Flag antibody (α-Flag).

Acetylation of Snf2 facilitates intramolecular interactions between the bromodomain and the two acetyl lysine residues. (A) Acetylated or unacetylated Snf2C proteins were tested for binding to unmodified (H3) or acetylated histone H3 (AcH3) peptides. (B) Acetylated or unacetylated Snf2C proteins were tested for binding to unmodified (H4) or acetylated histone H4 (AcH4) peptides. (C) GST-AT2 (1466–1546 amino acids) purified from E. coli was acetylated using Ada2 complexes with or without acetyl CoA. AT2 protein that was treated with or without acetyl CoA was repurified using glutathione sepharose. Acetylation of AT2 proteins was confirmed by Western blot. (D) Coimmunoprecipitation assay was performed using His-tagged bromodomain of Snf2 and either unacetylated GST-AT2 (AT2) or acetylated GST-AT2 (AcAT2). (E) GST-bromodomain of Snf2 was incubated with biotinylated AcH3 peptide or biotinylated H3 peptide in the presence of excess Snf2 peptides acetylated at K1493 (1493Ac), K1497 (1497Ac), or K1493 and K1497 (1493Ac, 1497Ac), or the absence of Snf2 peptide. Bound proteins were visualized by Coomassie staining.

Acetylation site mutant SWI/SNF is enriched at the gene promoters. (A) Wild-type or RR mutant cells grown in dextrose (2%) media were shifted to low-dextrose media (0.05%). Cells were harvested at indicated time points. Cross-linking of wild-type (Snf2-Flag; black line) and mutant (RR Snf2-Flag; red line) Snf2 to the SUC2 promoter was analyzed by ChIP using anti-Flag antibody. The level of Snf2 occupancy at the SUC2 promoter was normalized to the level of Snf2 signal at the control TELVIR locus. (B) Occupancy of wild-type Snf2 (black line) and RR mutant Snf2 (red line) at the SUC2 promoter was analyzed by ChIP assay in the gcn5Δ background. (C,D) Wild type (black line), RR mutant (red line), and gcn5Δ (blue line) grown in synthetic dextrose (SD) media were shifted to SD media without phosphate. Cells were harvested at indicated time points. Occupancy of SWI/SNF at PHO5 and PHO8 promoters was analyzed by ChIP using Flag antibody. (E) The model illustrating an in vivo role for Snf2 acetylation by Gcn5.