Recruitment of Snf1, SAGA, and cognate histone modifications to the INO1 promoter in vivo. (A) PCR analysis of ChIP with anti-Snf1 antibody (Snf1) on the INO1 promoter. Left panel, PCR results of ChIP-assay-performed WT cells under repressing (−, 10 μM inositol) or inducing conditions (+, inositol starvation). Inp=Input DNA and Ip=immunoprecipitated DNA by anti-Snf1. ACT1 was the internal control. See Materials and methods for quantitation methods. Right panel, PCR results of ChIP assay performed from WT compared to snf1Δ cells. Titration of input DNA (Inp) and immunoprecipitated DNA (Ip) by anti-Snf1 antibody are as indicated. Intergenic region on chromosome V (IntV) was the internal background control. (B) Time-course ChIP analysis of protein complexes and histone modifications on the INO1 promoter during induction. Cells were grown under repressing conditions to 0.8 OD₆₀₀ (time 0) and then in inducing conditions at the time points indicated. Upper panel: Antibodies were against Snf1 (Snf1c), pS10, Ada2 (SAGA), and acK14. Lower panel: Quantitation of time course and statistical evaluation of multiple ChIP and PCR reactions. IntV was used as a control for ChIP.

Cooperation of histone H3 phosphorylation and acetylation in galactose utilization and GAL1 transcriptional regulation in vivo. (A) Growth phenotype of enzyme deletions and histone substitutions. Five-fold serial dilutions of strains with the genotypes indicated on the left were spotted on glucose (2% glucose) and galactose (2% galactose) plates. Phenotypes were scored after 3-day growth at 30°C (top panels) and at 39°C (lower panels). (B) S1 nuclease RNA protection assay of enzyme deletions and histone substitutions. GAL1 transcription from WT, deletion strains (snf1, gal83, gal4, gcn5) and histone substitution strains (S10A, K14A, and S10AK14A) under repressing (−, 2% glucose) and inducing conditions (+, 2% galactose; upper panel). The induced level of GAL1 transcription in each strain was normalized to internal tRNA level and then calculated as percentage of WT level. (C) ChIP analysis of histone modifications on the GAL1 promoter during galactose induction time course. Cells were collected every 60 min over a 5 h time course. Antibodies were against Snf1 (Snf1c), pS10, Spt3-myc (SAGA), and acK14. Primer pairs were located in the GAL1 upstream activating sequence (GAL1-UAS) or the core promoter of GAL1 (GAL1-TATA).

Interaction of Snf1c with transcriptional activators in vitro. (A) Subunit composition of the Snf1c. Purification scheme for the Snf1c from a strain bearing Snf1-FLAG is described in Materials and methods. Purified Snf1c was separated by 4–12% gradient SDS–PAGE and visualized by Coomassie blue staining. Subunits identified by mass spectrometry are indicated, and molecular mass markers (kDa) at left. The bands at 40 and 33 kDa were Sip2 and likely to be degradation products. (B) Description of Ino2 and Ino4 constructs. The diagrams show GST fusion proteins containing full-length Ino2 (Ino2-FL: aa 1–304), acidic AD of Ino2 (Ino2-AD: aa 1–100), deletion of first AD (Ino2-AD1Δ, aa 1–27), deletion of second AD (Ino2-AD2Δ, aa 28–100), or deletion of both (Ino2-AD1/2Δ, aa 1–100). Ino4 (aa 1–151) was purified via a 6His-tag at its N-terminus. The DNA binding and interaction domains (basic helix–loop–helix) of Ino2 (aa 213–304) and Ino4 are indicated. (C) GST pulldown assay of Ino2 or Gal4 activator interaction with Snf1c. Fusion proteins as indicated and GST alone were tested for Snf1c binding. Top panel: Input and bound Snf1c were analyzed on 10% SDS–PAGE and immunoblotted using anti-Snf1antibody. Input represents 50% of amount in binding reaction. Bottom panel: Relative amounts of recombinant fusion proteins used in the assay were visualized by Ponceau S staining on the same membrane.

Role of Ino2/Ino4 activator in recruitment of Snf1 and SAGA complexes at the INO1 promoter in vivo. (A, C) S1 nuclease assay of INO1 gene expression in ino2, ino4, and ino2 AD deletion strains. RNA was prepared from indicated deletion strains or ino2D bearing plasmid-borne full-length or deleted Ino2, from repressing conditions (0 time) and inducing conditions (inositol starvation). Plasmid-born Ino2-FL (panel C) conferred 81% of transcription compared to endogenous Ino2 (panel A). (B, D) ChIP analysis of Snf1c, SAGA, and cognate histone modifications of complexes dependant on Ino2 and Ino4 activators at INO1. Same as above, except that Spt3-Myc was used to examine SAGA recruitment.

Comparative interaction of SAGA and Snf1c with Ino2/Ino4 and Gal4 activators in vitro. (A, B) GST pulldown between Ino2, Gal4, or Gcn4 ADs, and Snf1c or SAGA. See Figure 3 legend for details. Anti-Ada3 antibody was used to visualize SAGA. In panel B, prior to GST pulldown, 6His-tagged Ino4 protein was incubated with GST-Ino2-FL in high salt buffer (350 mM NaCl) for 30 min at 4°C.

Effect of loss of Snf1 or pSer10 on recruitment of SAGA complex and acK14 to GAL1 and INO1 promoters. (A, B) ChIP assays of snf1Δ or S10A mutant effect on recruitment of SAGA or Snf1c and cognate histone modifications at GAL1 (A, B) and INO1 (B).

Effect of loss of Snf1 or S10A substitution on recruitment of TBP to INO1 or GAL1 promoters. ChIP assay of TBP and H3 trimethyl Lys4 recruitment. Cells were collected before (A) or after 3 h induction (A, B). In panel B, WT, snf1D, or S10A mutant cells were used. Primer pairs were located in TATA regions.

Model for the establishment of pS10, acK14, and recruitment of TBP. The upper panel shows Snf1c recruitment by activators for both INO1 and GAL1 genes to establish pSer10. The middle panels show two putative mechanisms for establishment of acLys14: at INO1 pS10 sets up acK14 via SAGA recruitment, whereas at GAL1 the activator recruits SAGA. Lower panel, TBP recruitment is mediated by pSer10 at both genes.