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. 2005 Jul;25(13):5626-38.
doi: 10.1128/MCB.25.13.5626-5638.2005.

Simultaneous recruitment of coactivators by Gcn4p stimulates multiple steps of transcription in vivo

Chhabi K Govind  1 , Sungpil Yoon, Hongfang Qiu, Sudha Govind, Alan G Hinnebusch
Affiliations

Simultaneous recruitment of coactivators by Gcn4p stimulates multiple steps of transcription in vivo

Chhabi K Govind et al. Mol Cell Biol. 2005 Jul.

Abstract

Transcriptional activation by Gcn4p is dependent on the coactivators SWI/SNF, SAGA, and Srb Mediator, which are recruited by Gcn4p and stimulate assembly of the pre-initiation complex (PIC) at the ARG1 promoter in vivo. We show that recruitment of all three coactivators is nearly simultaneous with binding of Gcn4p at ARG1 and is followed quickly by PIC formation and elongation by RNA polymerase II (Pol II) through the open reading frame. Despite the simultaneous recruitment of coactivators, rapid recruitment of SWI/SNF depends on the histone acetyltransferase (HAT) subunit of SAGA (Gcn5p), a non-HAT function of SAGA, and on Mediator. SAGA recruitment in turn is strongly stimulated by Mediator and the RSC complex. Recruitment of Mediator, by contrast, occurs independently of the other coactivators at ARG1. We confirm the roles of Mediator and SAGA in TATA binding protein (TBP) recruitment and demonstrate that all four coactivators under study enhance Pol II recruitment or promoter clearance following TBP binding. We also present evidence that SWI/SNF and SAGA stimulate transcription elongation downstream from the promoter. These functions can be limited to discrete time intervals, providing evidence for multiple stages in the induction process. Our findings reveal a program of coactivator recruitment and PIC assembly that distinguishes Gcn4p from other yeast activators studied thus far.

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Figures

FIG. 1.
FIG. 1.
ChIP analysis of the kinetics of Gcn4p binding and recruitment of coactivators, TBP, and Pol II to ARG1 on induction of Gcn4p by SM. GCN4 ADA2-myc strain HQY392 was cultured at 25°C in SC medium and treated with SM at 0.5 μg/ml. Aliquots of cells were cross-linked with formaldehyde at the indicated times and processed for ChIP analysis of factor binding to ARG1 using primers to amplify the UAS- or TATA-containing sequences, or ORF sequences, at ARG1 indicated in the schematic shown in panel A. Coding sequences of the POL1 gene were amplified to control for nonspecific immunoprecipitation. Chromatin fragments were immunoprecipitated with the antibodies shown to the right of each panel, and the DNA was extracted from the immunoprecipitates (IP) and from 5% of the corresponding input chromatin (Inp) samples. A 1,000-fold dilution of the Inp and the undiluted IP DNA samples were PCR amplified in the presence of [33P]dATP, and the PCR products were resolved by polyacrylamide gel electrophoresis and visualized by autoradiography. In parallel, gcn4Δ ADA2-myc strain HQY503 was treated with SM for 120 min and handled identically (last lane). The PCR-amplified fragments were quantified with a phosphorimager, and the ratios of the ARG1 signals to the POL1 signals in the IP samples were normalized for the corresponding ratios for the Inp samples to yield the “relative % IP ARG1/POL1” for each sample.
FIG. 2.
FIG. 2.
Kinetic ChIP analysis suggests sequential binding of Gcn4p, coactivators, and TBP/Pol II at the ARG1 promoter on induction of Gcn4p. The results of replicate ChIP experiments conducted as exactly as described in Fig. 1 were quantified and the mean “relative % IP ARG1/POL1” values with standard errors were plotted as a function of time for each factor, indicated along the y axes. Note that different y axis scales were employed to normalize the peak heights for the different curves.
FIG. 3.
FIG. 3.
Coactivator mutations do not substantially alter the kinetics or extent of Gcn4p binding at the ARG1 UAS. Kinetic ChIP analysis was conducted as described for Fig. 1 using the wild-type (WT) and mutant strains with the relevant genotypes listed in the inset of each graph. Antibodies against Gcn4p were employed for the ChIP assays using primers to measure its binding to the ARG1 UAS.
FIG. 4.
FIG. 4.
Coactivator mutations alter the kinetics and extent of SWI/SNF and SAGA recruitment by Gcn4p at the ARG1 UAS. Kinetic ChIP analysis was conducted as described in Fig. 1 using the wild-type (WT) and mutant strains with the relevant genotypes listed in the inset of each graph. Antibodies against myc-tagged Ada2p (A and B), Snf6p (C and D), or Gal11p (E and F) were employed for the ChIP assays using primers to measure binding of these factors to the ARG1 UAS. In panel A, myc-Ada2p binding in WT and snf2Δ cells is indicated by the scale along the left-hand y axis, while the results for gcn5Δ refer to the scale on the right-hand y axis.
FIG. 5.
FIG. 5.
Coactivator mutations alter the kinetics and extent of TBP and Pol II occupancy of the promoter and of Pol II occupancy in the ORF at ARG1. Kinetic ChIP analysis was conducted as described in Fig. 1 using the wild-type (WT) and mutant strains with the relevant genotypes listed in the inset of each graph. Antibodies against TBP (A, D, and G) or Pol II subunit Rpb1p (B, C, E, F, H, and I) were employed for the ChIP assays using the appropriate primers to measure binding of TBP and Pol II to the promoter (A, B, D, E, G, and H) or Pol II occupancy in the ORF (C, F, and I). In panel D, TBP binding in WT and ada1Δ cells is indicated by the scale along the left-hand y axis, while the results for gcn5Δ refer to the scale on the righthand y-axis.
FIG. 6.
FIG. 6.
A model for simultaneous interdependent recruitment of SAGA, SWI/SNF, and Mediator by Gcn4p and multiple functions of these coactivators in PIC assembly and elongation. Transcriptional activation is divided into the two stages that were temporally resolved in wild-type cells. The top two panels depict the first stage in transcriptional activation involving binding of Gcn4p to the UAS and the nearly simultaneous recruitment of SAGA, SWI/SNF, and Mediator. Even though binding of Gcn4p and recruitment of SAGA and SWI/SNF commence immediately on induction of Gcn4p synthesis, binding of Gcn4p is depicted as the first step because mutational inactivation of these coactivators (or of Mediator) does not affect Gcn4p binding to the UAS. Although recruitment of RSC is also depicted here, the kinetics of its recruitment have not been determined. Interdependencies among the coactivators are summarized using arrows color coded for the stimulatory factor. The bottom three panels depict the second stage in activation involving TBP recruitment, Pol II recruitment or promoter clearance downstream of TBP binding, and elongation. While these three steps were not temporally resolved, genetic analysis reveals that Pol II recruitment is dependent on TBP binding to the TATA element and that the coactivators independently stimulate these reactions in the manner depicted by the color-coded arrows. See text for further details.
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References

    1. Akhtar, A., G. Faye, and D. L. Bentley. 1996. Distinct activated and non-activated RNA polymerase II complexes in yeast. EMBO J. 15:4654-4664. - PMC - PubMed
    1. Bhaumik, S. R., and M. R. Green. 2001. SAGA is an essential in vivo target of the yeast acidic activator Gal4p. Genes Dev. 15:1935-1945. - PMC - PubMed
    1. Bhaumik, S. R., T. Raha, D. P. Aiello, and M. R. Green. 2004. In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer. Genes Dev. 18:333-343. - PMC - PubMed
    1. Bhoite, L. T., Y. Yu, and D. J. Stillman. 2001. The Swi5 activator recruits the Mediator complex to the HO promoter without RNA polymerase II. Genes Dev. 15:2457-2469. - PMC - PubMed
    1. Blau, J., H. Xiao, S. McCracken, P. O'Hare, J. Greenblatt, and D. Bentley. 1996. Three functional classes of transcriptional activation domain. Mol. Cell. Biol. 16:2044-2055. - PMC - PubMed

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