Multiple mechanistically distinct functions of SAGA at the PHO5 promoter

Abstract

Our previous studies have shown that the rate of chromatin remodeling and consequently the rate of PHO5 activation are strongly decreased in the absence of Gcn5 histone acetyltransferase activity. Using chromatin immunoprecipitation, we demonstrate that SAGA is physically recruited to the PHO5 promoter. Recruitment is dependent on the specific activator Pho4 and occurs only under inducing conditions. Spt3, another subunit of SAGA, also plays a role in PHO5 activation but has a function that is completely different from that of Gcn5. An SPT3 deletion severely compromises the PHO5 promoter and reduces the extent of transcriptional activation by diminishing the binding of the TATA binding protein to the promoter without, however, affecting the rate or the extent of chromatin remodeling. A gcn5 spt3 double mutant shows a synthetic phenotype almost as severe as that observed for an spt7 or spt20 mutant. The latter two mutations are known to prevent the assembly of the complex and consequently lead to the loss of all SAGA functions. The absence of the Ada2 subunit causes a strong delay in chromatin remodeling and promoter activation that closely resembles the delay observed in the absence of Gcn5. A deletion of only the Ada2 SANT domain has exactly the same effect, strongly suggesting that Ada2 controls Gcn5 activity by virtue of its SANT domain. Finally, the Gcn5 bromodomain also contributes to but is not essential for Gcn5 function at the PHO5 promoter. Taken together, the results provide a detailed and differentiated description of the role of SAGA as a coactivator at the PHO5 promoter.

FIG. 1.

Pho4-mediated SAGA recruitment to the PHO5 promoter. Strain 8136, which contains 18-myc-tagged Ada2 and a wild-type PHO4 allele, and strain 8136Δpho4 were grown logarithmically in YPDA (+Pi) or phosphate starved for 4 h (−Pi). Acid phosphatase levels in strain 8136 increased from 22 U (+Pi) to 172 U (−Pi) under these conditions. For the chromatin immunoprecipitation analysis, chromatin was fixed by formaldehyde treatment and Ada2-myc was precipitated with anti-myc monoclonal antibody 9E11. The amounts of coimmunoprecipitated DNA determined by quantitative PCR were normalized to the respective input DNA and are shown in arbitrary units. The locations of the PCR fragments used for chromatin immunoprecipitation analysis of the PHO5 locus are shown above the nucleosomal organization of the repressed PHO5 promoter. Stable nucleosomes (solid circles), unstable nucleosomes (open circles) UASp1 (black box), UASp2 (grey box), and the TATA box (T) are indicated. As a further control, the PGK1 promoter was included in the analysis. Error bars show SDs.

FIG. 2.

Disintegration of the SAGA complex by an SPT7 or SPT20 deletion abolishes PHO5 induction. The time course of PHO5 induction in wild-type (wt), gcn5, spt7, and spt20 strains was monitored by measuring acid phosphate activity at the indicated times after transfer of cells from phosphate-containing medium to phosphate-free medium.

FIG. 3.

Deletion of SAGA subunit Spt3 affects PHO5 activation in a manner distinct from that of Gcn5. The time course of PHO5 induction in wild-type (wt), spt3, and spt7 strains (A) or in gcn5, spt3, spt3 gcn5, and spt7 strains (B) was monitored by measuring acid phosphatase activity at the indicated times after transfer of cells from phosphate-containing medium to phosphate-free medium.

FIG. 4.

The rate of chromatin remodeling at the PHO5 promoter is not affected by the absence of Spt3. Nuclei isolated from a wild-type (wt) strain, an spt3 strain, a gcn5 strain, and an spt3 gcn5 strain after 4 h of induction in phosphate-free medium were treated for 30 min with 50 U (lanes 1, 3, 5, and 7) or 200 U (lanes 2, 4, 6, and 8) of ClaI. In order 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 (3). In all instances, the ratio of the lower band to the upper band reflects the relative accessibility of the ClaI site. The position of the ClaI site is shown above the nucleosomal organization of the repressed PHO5 promoter (see the legend to Fig. 1 for details).

FIG. 5.

Spt3 strongly increases TBP recruitment to the activated PHO5 promoter. Strains 8136 (wild type) and 8136Δspt3 were phosphate starved for up to 6 h. TBP occupancy was determined by chromatin immunoprecipitation analysis with an anti-TBP antibody. The amounts of coimmunoprecipitated DNA determined by quantitative PCR were normalized to the respective input DNA and are shown in arbitrary units. The locations of the PCR fragments used for chromatin immunoprecipitation analysis of the PHO5 locus are shown with respect to the nucleosomal structure of the repressed PHO5 promoter (3) (see the legend to Fig. 1 for details). As a control, the PGK1 promoter was included in the analysis. Error bars show SDs.

FIG. 6.

Deletion of the Ada2 subunit or its SANT domain causes a strong delay in PHO5 promoter activation. The kinetics of PHO5 induction in wild-type (wt), ada2, ada2Δ97-106 (ada2ΔSANT), and gcn5 strains were monitored by measuring acid phosphate activity at the indicated times after transfer of cells from phosphate-containing medium to phosphate-free medium.

FIG. 7.

The rate of chromatin remodeling at the PHO5 promoter is strongly decreased in ada2 and ada2Δ97-106 strains. Nuclei isolated from ADA2 (wild-type [wt]), ada2, and ada2Δ97-106 (ada2ΔSANT) strains after 4 h of induction in phosphate-free medium were analyzed for chromatin remodeling by the ClaI accessibility assay as described in the legend to Fig. 4. ClaI concentrations in lanes 1 to 6 correspond to those used in lanes 1 to 6 of Fig. 4.

FIG. 8.

The Gcn5 bromodomain contributes to Gcn5 function at the PHO5 promoter. The kinetics of PHO5 induction in a wild-type (wt) strain, a gcn5 strain, and strains in which the Gcn5 bromodomain had been either deleted (gcn5Δbromo) or mutated (gcn5bromo-mut) (as described in Materials and Methods) were monitored by measuring acid phosphatase activity at the indicated times after transfer of cells from phosphate-containing medium to phosphate-free medium.