Differential requirement of SAGA components for recruitment of TATA-box-binding protein to promoters in vivo

Abstract

The multisubunit Saccharomyces cerevisiae SAGA (Spt-Ada-Gcn5-acetyltransferase) complex is required to activate transcription of a subset of RNA polymerase II-dependent genes. However, the contribution of each SAGA component to transcription activation is relatively unknown. Here, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation assay, we have systematically analyzed the role of SAGA components in the recruitment of TATA-box binding protein (TBP) to SAGA-dependent promoters. We show that recruitment of TBP is diminished at a number of SAGA-dependent promoters in ada1delta, spt7delta, and spt20delta null mutants, consistent with previous biochemical data suggesting that these components maintain the integrity of the SAGA complex. We also find that Spt3p is generally required for TBP binding to SAGA-dependent promoters, consistent with biochemical and genetic experiments, suggesting that Spt3p interacts with and recruits TBP to the core promoter. By contrast, Spt8p, which has been proposed to be required for the interaction between Spt3p and TBP, is required for TBP binding at only a subset of SAGA-dependent promoters. Ada2p and Ada3p are both required for TBP recruitment to Gcn5p-dependent promoters, supporting previous biochemical data that Ada2p and Ada3p are required for the histone acetyltransferase activity of Gcn5p. Finally, our results suggest that TBP-associated-factor components of SAGA are differentially required for TBP binding to SAGA-dependent promoters. In summary, we show that SAGA-dependent promoters require different combinations of SAGA components for TBP recruitment, revealing a complex combinatorial network for transcription activation in vivo.

FIG. 1.

Identification of a set of promoters that require SAGA for transcription. Total cellular RNA was prepared from either a wild-type (WT) or spt20Δ strain, and transcription from the indicated promoter was monitored by primer extension. The level of transcription in the spt20Δ strain relative to that of the wild type is indicated (%WT).

FIG. 2.

General requirement of Ada1p, Spt7p, and Spt20p for recruitment of TBP to SAGA-dependent promoters. (A) Wild-type (WT), ada1Δ, spt7Δ, and spt20Δ strains were first grown in glucose-containing medium (YPD) and then shifted to galactose-containing medium (YPG) 5 h prior to treatment with formaldehyde. Formaldehyde-based in vivo cross-linking and ChIP were carried out as previously described (28). Immunoprecipitation assays were performed with polyclonal antibodies against TAF10, TAF12, or TBP. TAF10 and TAF12 are representative SAGA components used to monitor recruitment of the SAGA complex. Primer pairs located in the GAL1 UAS or core promoter were used for PCR analysis of the input and immunoprecipitated (IP) DNA samples. All PCRs were carried out in the linear range of DNA amplification, as indicated by the arrow in the curve shown in the top right panel. The percentage of DNA immunoprecipitated relative to that of the wild type (%WT) is indicated. Background levels in the immunoprecipitation assay are shown by using an irrelevant DNA sequence (GAL4 ORF [open reading frame]) and an irrelevant antibody control (TAF1 is a specific component of the TFIID complex, which is not associated with the core promoters of the genes analyzed) which immunoprecipitated less than 0.1% of DNA. (B) All strains were grown in YPD to an OD₆₀₀ of 1.0 prior to formaldehyde treatment. Analysis of TBP binding to the core promoters of ADH1, VTC3, BDF2, and PHO84 was performed as described for panel A. The background signal obtained with the irrelevant anti-TAF1 antibody control is shown on the right.

FIG. 3.

General requirement of Spt3p but not Spt8p in recruitment of TBP to SAGA-dependent promoters. Wild-type (WT), spt3Δ, and spt8Δ deletion mutants were grown as described in the legend to Fig. 2 prior to formaldehyde treatment. Primer pairs located in the core promoters of the GAL1, ADH1, VTC3, BDF2, and PHO84 genes were used for PCR analysis of the input and immunoprecipitated (IP) DNA samples.

FIG. 4.

Gcn5p, Ada2p, and Ada3p are required for TBP recruitment to a common set of SAGA-dependent promoters. Wild-type (WT), gcn5Δ, ada2Δ, and ada3Δ strains were grown as described in the legend to Fig. 2 prior to formaldehyde treatment. Primer pairs located in the core promoters of the GAL1, ADH1, VTC3, BDF2, and PHO84 genes were used for PCR analysis of the input and immunoprecipitated (IP) DNA samples.

FIG. 5.

Differential requirement of SAGA TAFs for recruitment of TBP to SAGA-dependent promoters. Yeast strains harboring temperature-sensitive mutations in TAF9, TAF6, and TAF12 were first grown at 23°C to an OD₆₀₀ of 0.8 and then transferred to 37°C for 1 h prior to treatment with formaldehyde. Primer pairs located in the core promoters of the GAL1, ADH1, VTC3, BDF2, and PHO84 genes were used for PCR analysis of the input and immunoprecipitated (IP) DNA samples.