The SLIK and SAGA complexes generate similar patterns of acetylation on histone H3 from nucleosomes. HAT assays were performed with nucleosome substrates incubated together with purified SLIK or SAGA complexes. Radiolabeled histone H3 was subsequently purified and subjected to microsequence analysis followed by direct determination of radioactivity at each position in the H3 sequence. The counts per minute for each cycle are plotted against the amino acid detected for that cycle.

Rtg2 is a unique component of SLIK. (A) Rtg2 associates with the Spt7 protein in vivo. Extracts prepared from strains expressing TAP-tagged Rtg2 or Spt8 or untagged control proteins were incubated with calmodulin resin. Inputs and protein eluted from the resin were subjected to Western blotting with antibodies directed against protein A or Spt7. (B) Size-exclusion chromatography of the Rtg2-TAP and Spt8-TAP complexes isolated from the calmodulin resin. Western blotting of Superose 6 fractions was performed with antibodies against protein A or Ada3. (C) Western blots of Rtg2-TAP and Spt8-TAP fractions eluted from Mini Q columns. Fractions from the Superose 6 column were bound to Mini Q columns, and fractions eluted were assayed by Western blotting with protein A antibodies. The relative positions of the SAGA and SLIK complexes are indicated.

Genetic phenotypes of SLIK and SAGA mutant yeast. (A) Wild-type (BY4742) and spt7Δ, rtg2Δ, spt8Δ, and rtg2Δspt8Δ derivative yeast strains were assayed for their ability to grow on SC medium containing dextrose, acetate, glycerol, or glycerol plus ethanol as carbon sources. (B) Synthetic phenotypes generated in SAGA and SLIK mutant yeast. Wild-type (BY4742), spt7Δ, rtg2Δ, spt8Δ, and rtg2Δ spt8Δ yeast strains were assayed for their ability to grow on SC medium containing 2% dextrose or raffinose carbon sources or in medium lacking inositol.

Identification of the SLIK complex. (A) Schematic representation of the isolation of SLIK. Complexes were partially purified from whole-cell extracts prepared from yeast strain CY396. (B) Fluorogram of HAT assays and Western blot analyses performed with SLIK and SAGA complexes. Fractions from the Mono Q columns were incubated with nucleosomes and assayed for HAT activity or assayed by Western blotting with the indicated antisera. The position of histone H3 is indicated, as determined by Coomassie blue staining of the same gel.

Identification of the components of SLIK. (A) Schematic representation of the chromatographic purification of SLIK, as determined by HAT assays and Western blotting. (B) HAT assay and Western blotting of peak SLIK fractions eluted from the final Mini Q column. Fluorogram and Western blotting analyses indicate that SLIK predominantly elutes in a single protein peak. (C) Silver stain gel of the purified SLIK complex in comparison to SAGA. The peak SLIK fraction from the final Mini Q column was analyzed by silver staining of proteins separated on an SDS-4 to 20% polyacrylamide gel. For comparison a highly purified SAGA complex is shown. The components identified by mass spectrometry are grouped according to their different classes of transcriptional regulators, namely, Spts, Adas, TAF_IIs, or others. The position of a protein which likely represents TAF_II25 is also shown. Candidate protein bands which appear unique to SLIK are marked with asterisks. Numbers at left of panel C are molecular masses in kilodaltons.

Effect of deletion of RTG2 on the SLIK and SAGA complexes. (A) HAT assay and Western blot analysis of SLIK and SAGA complexes prepared from wild-type (SFY526) or rtg2Δ strains. Mono Q fractions of partially purified SLIK and SAGA were subjected to nucleosomal HAT assay and Western blotting with Ada2 antiserum. (B) SLIK fractions 27 to 31 eluted from the Mono Q column in panel A were separated on a Superose 6 column. Shown is a fluorogram of a nucleosomal HAT assay performed with Superose 6 fractions from wild-type and rtg2Δ strains. (C) Northern blot analysis of CIT2 expression from wild-type (WT, BY4742), spt7Δ, rtg2Δ, and spt8Δ isogenic strains grown in SC medium containing 2% dextrose (D) or 2% acetate (A) as a carbon source. ACT1 (encoding actin) is shown as a loading control. The relative fold increase in CIT2 RNA levels normalized to control RNA for each strain is shown, as estimated by densitometry.

Recruitment of SAGA and SLIK complexes to specific promoters is coincident with acetylation and gene activation. (A) Western blot prepared from yeast extracts expressing GST, GST-Spt8, or GST-Rtg2 proteins following binding to glutathione-Sepharose. Anti-GST antiserum was used for immunoblotting. (B) The expression of the CIT2 gene and the control ACT1 gene from the indicated yeast strains grown in SC medium containing dextrose (D) or acetate (A) as carbon sources was monitored by Northern blotting. The presence of histone H3 acetylation was measured through PCR-amplified CIT2 promoter DNA from chromatin immunoprecipitated with anti-acetylated lysine 9/14 H3 antiserum. The binding of GST, GST-Rtg2, or GST-Spt8 on the CIT2 promoter was monitored by PCR amplification of DNA from chromatin precipitated with glutathione-Sepharose affinity resin. PCR amplifications from input and precipitated (bound) DNA are shown. (C) Similar assays were performed to study the induction, acetylation, and promoter binding mediated by SLIK and SAGA at the GAL10 locus with the indicated yeast strains grown in SC medium containing dextrose (D) or galactose (G).