Interdependent recruitment of SAGA and Srb mediator by transcriptional activator Gcn4p - PubMed (original) (raw)
Interdependent recruitment of SAGA and Srb mediator by transcriptional activator Gcn4p
Hongfang Qiu et al. Mol Cell Biol. 2005 May.
Abstract
Transcriptional activation by Gcn4p is enhanced by the coactivators SWI/SNF, SAGA, and Srb mediator, which stimulate recruitment of TATA binding protein (TBP) and polymerase II to target promoters. We show that wild-type recruitment of SAGA by Gcn4p is dependent on mediator but independent of SWI/SNF function at three different promoters. Recruitment of mediator is also independent of SWI/SNF but is enhanced by SAGA at a subset of Gcn4p target genes. Recruitment of all three coactivators to ARG1 is independent of the TATA element and preinitiation complex formation, whereas efficient recruitment of the general transcription factors requires the TATA box. We propose an activation pathway involving interdependent recruitment of SAGA and Srb mediator to the upstream activation sequence, enabling SWI/SNF recruitment and the binding of TBP and other general factors to the promoter. We also found that high-level recruitment of Tra1p and other SAGA subunits is independent of the Ada2p/Ada3p/Gcn5p histone acetyltransferase module but requires Spt3p in addition to subunits required for SAGA integrity. Thus, while Tra1p can bind directly to Gcn4p in vitro, it requires other SAGA subunits for efficient recruitment in vivo.
Figures
FIG. 1.
Gcn4p recruits myc-Spt7p, myc-Ada2p, myc-Srb6p, and myc-Gal11p, dependent on hydrophobic residues in the activation domain. (A) SPT7-myc gcn4Δ and ADA2-myc gcn4Δ strains bearing empty vector (gcn4Δ) and SPT7-myc GCN4 and ADA2-myc GCN4 strains carrying high-copy-number GCN4-HA plasmid pHQ1239 were cultured in SC medium lacking Ile and Val and treated with sulfometuron for 2 h to induce Gcn4p synthesis by starvation for Ile/Val and then subjected to ChIP analysis with myc antibodies. DNA was extracted from the immunoprecipitates (IP), and 5% of the input (Inp) samples and a 1,000-fold dilution of the Inp and the undiluted IP DNA samples were PCR amplified using primers specific for the _POL1_ORF, _ARG1_UAS, _SNZ1_UAS, or ARG4 promoter (UAS and TATA sequence), in the presence of [33P]dATP. The PCR products were resolved by polyacrylamide gel electrophoresis and visualized by autoradiography. (B) The SPT7-myc gcn4Δ and ADA2-myc gcn4Δ strains described above, along with SRB6-myc gcn4Δ and GAL11-myc gcn4Δ strains, bearing empty vector (gcn4Δ), high-copy-number GCN4 plasmid pHQ1303 (GCN4), or high-copy-number gcn4-14Ala plasmid pHQ1304 (gcn4-14Ala), were subjected to ChIP analysis as above, and the results were quantified with a phosphorimager. The ratios of the _ARG1_UAS signals to the POL1 signals in the IP samples were normalized for the corresponding ratios for the Inp samples, and the resulting values measured for the GCN4 strain were normalized to the corresponding values obtained for the gcn4Δ strain to produce the “ratio %IP(GCN4/gcn4Δ)” values plotted in the histograms for each protein.
FIG. 2.
Deletion of Spt3p or subunits required for SAGA integrity, but not Gcn5p, impairs recruitment of SAGA by Gcn4p. (A) ChIP analysis of a gcn4Δ SPT7-myc strain (gcn4Δ) and GCN4 SPT7-myc strains containing WT SAGA subunits or the indicated SAGA subunit deletions and harboring high-copy-number GCN4-HA plasmid pHQ1239 was carried out as described in Fig. 1 using anti-myc antibodies. (B) Same as panel A except that ADA2-myc strains were analyzed. (C) Same as panel A except that TRA1-FL strains were analyzed and anti-Flag M2 antibodies were used. The ratio %IP (GCN4/gcn4Δ) values as defined in Fig. 1 were calculated for the _ARG1_UAS, _SNZ1_UAS, and ARG4 probes, and the average results obtained from two or more independent cultures and two or more PCR amplifications for each culture were plotted in the histograms with standard errors shown as error bars. The numbers under the histograms, corresponding to percentages of the WT Gcn4p-dependent binding of myc-Spt7p, myc-Ada2p, or FL-Tra1p, were calculated by subtracting unity from all ratio %IP (GCN4/gcn4Δ) values for each mutant and expressing the result as a percentage of the corresponding WT value.
FIG. 3.
Western analysis of tagged SAGA or mediator subunits in coactivator mutants. (A-D) myc- or Flag-tagged strains used in Fig. 2 or 5 or the untagged parent strain BY4741 (panel C) was grown under the same conditions used for ChIP analysis, and WCEs were prepared and subjected to Western blot analysis using anti-myc, anti-Flag M2, or anti-Gcd6p antibodies, the last serving as loading control. The Western signals obtained using the ECL chemiluminescence kit (Amersham) were quantified by video densitometry using NIH image software, and the ratios of myc-Spt7p, myc-Ada2p, myc-Srb6p, or myc-Gal11p to Gcd6p signals are listed for each mutant relative to the WT strain between the two blots.
FIG. 4.
Coimmunoprecipitation analysis of SAGA integrity in coactivator mutants. WCEs from the appropriate yeast strains were immunoprecipitated with monoclonal c-myc or Flag M2 antibodies. The immune complexes were collected, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subjected to Western analysis to detect the proteins listed on the left of each panel or with anti-myc or anti-Flag M2 antibodies to detect the myc- or Flag-tagged proteins. I, 10% of the input WCEs; P, 50% of the pellet fraction from the immunoprecipitates; S, 10% of the supernatant fractions.
FIG. 5.
Interdependent recruitment of SAGA and Srb mediator by Gcn4p. (A) ChIP analysis of a gcn4Δ SPT7-myc strain carrying empty vector (gcn4Δ) or GCN4 SPT7-myc strains containing no coactivator mutations (WT) or the indicated deletions of Srb mediator subunits, or swi2Δ, and harboring high-copy-number GCN4-HA plasmid pHQ1239, conducted as described in Fig. 1 and 2. (B) Same as panel A except that ADA2-myc strains were employed. (C-D) Same as panels A and B, except that SRB6-myc and GAL11-myc strains were employed, containing no coactivator mutations (WT) or the indicated deletions of SAGA subunits or swi2Δ.
FIG. 6.
Deletion of the ARG1 TATA does not affect recruitment of SAGA, Srb mediator, and SWI/SNF by Gcn4p. Transformants of gcn4Δ strains carrying empty vector (gcn4Δ) or high-copy-number GCN4-HA plasmid pHQ1239 (WT) and of gcn4Δ arg1-ΔTATA strains carrying pHQ1239 (ΔTATA), harboring SRB6-myc, SPT7-myc, or SWI2-myc, were subjected to ChIP analysis as described in Fig. 1 and 2. (Data for TBP-myc and RPB3-myc shown in the histogram were published previously [53] and are provided here only for comparison.)
FIG. 7.
TBP binding to TATA box is a prerequisite for recruitment of GTFs by Gcn4p. gcn4Δ, WT, and arg1-ΔTATA strains containing chromosomal TOA1-myc, SUA7-myc, TFA1-myc, TFG2-myc, or KIN28-myc alleles were subjected to ChIP analysis as described in Fig. 1 and 2. (Data for TBP-myc and RPB3-myc in the histogram were published previously [53].)
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