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

The activation of transcription initiation in eukaryotes involves the binding of a sequence-specific activator protein to an enhancer, or upstream activation/regulatory sequence (UAS/URS), followed by activator-dependent recruitment of the general transcription machinery. Because packaging of promoter DNA in nucleosomes impedes assembly of the preinitiation complex (PIC), activators must recruit chromatin-modifying complexes to the promoter. These include coactivator complexes with histone acetyltransferase (HAT) activity, such as the Gcn5p-containing SAGA complex of Saccharomyces cerevisiae, and ATP-dependent remodeling enzymes, such as SWI/SNF and RSC. Nucleosome remodeling by SWI/SNF has also been implicated in removing a barrier to transcription elongation by RNA polymerase II (Pol II) (8). The recruitment of TATA binding protein (TBP) and Pol II is facilitated by coactivators which serve as adaptors between the activator and these general transcription factors (GTFs). For example, SAGA complex contains subunits that interact with TBP and has been shown to mediate TBP recruitment by various activators (2, 22, 30). Similarly, the Mediator complex interacts with Pol II and has been shown to stimulate the recruitment of both Pol II and TBP (21, 25, 26, 30). The recruitment of one coactivator may stimulate the recruitment of another, and this interdependence can be reflected in a sequential order of coactivator recruitment to the promoter. Such staged recruitment was shown for the HO gene where SWI/SNF recruitment is necessary for, and temporally precedes, recruitment of SAGA by the activator Swi5p to the URS2 element (9). SWI/SNF is also required for Mediator recruitment to HO, even though these two coactivators are recruited simultaneously to URS1, whereas Mediator recruitment is dispensable for SWI/SNF recruitment to this promoter (4). At the GAL1 gene of yeast, by contrast, SAGA recruitment by Gal4p precedes that of Mediator (6). Although SWI/SNF is recruited to GAL1 as rapidly as SAGA, and independently of SAGA function, strong SWI/SNF recruitment requires Mediator and downstream steps in PIC assembly (19, 24). Thus, the temporal order of coactivator recruitment at GAL1 does not seem to reflect an obligate sequence of coactivator functions. The requirement for SWI/SNF function as a prerequisite for SAGA recruitment by Swi5p at HO appears to be restricted to late mitosis and applies even to Gal4p- and Gcn4p-regulated promoters in this phase of the cell cycle, most likely reflecting a highly condensed state of promoter chromatin (19). In fact, both SAGA and Mediator functions are required during interphase for wild-type steady-state recruitment of SWI/SNF by Gcn4p at the target genes ARG1 and SNZ1 (43). Thus, the degree of interdependency among coactivators in recruitment can vary from one yeast activator to the next, and even for the same activator depending on the chromatin structure of the promoter. A mechanism to explain SAGA-SWI/SNF interdependency has been proposed wherein histone acetylation by Gcn5p enhances recruitment of SWI/SNF and of SAGA itself via the bromodomains in the Snf2p and Gcn5p subunits, respectively. This mechanism was demonstrated in vitro using reconstituted nucleosome arrays, and genetic data indicated a requirement for the Snf2p bromodomain in SWI/SNF recruitment to the SUC2 promoter in vivo (15). However, a different result was obtained in another study where the Snf2p bromodomain was dispensable for wild-type SWI/SNF recruitment to SUC2, even though mutations in the HAT subunits of SAGA (Gcn5p) and NuA4 (Esa1p) decreased SWI/SNF recruitment in the early phase of SUC2 induction (13). Similarly, a deletion of GCN5 did not reduce steady-state recruitment of SWI/SNF by Gcn4p (38, 43), although it impaired chromatin remodeling by the recruited SWI/SNF complex (38). However, it has not been determined whether the kinetics of SWI/SNF (or SAGA) recruitment by Gcn4p is influenced by the Gcn5p HAT function in vivo. A key objective of this study was to determine whether Gcn4p mounts a staged recruitment of SAGA, SWI/SNF, and Mediator, followed by PIC assembly, in which earlier events are prerequisites for later steps in the process. To that end, we analyzed the kinetics of Gcn4p binding, coactivator recruitment, binding of TBP and Pol II to the promoter (PIC assembly), and the appearance of Pol II at the 3′ end of the open reading frame (ORF) during induction of the ARG1 gene in wild-type cells and in various coactivator mutants. Our results indicate that transcriptional activation by Gcn4p is a rapid process commencing with the immediate occupancy of the ARG1 UAS by Gcn4p on amino acid starvation, followed quickly by nearly simultaneous recruitment of SAGA, SWI/SNF, and Mediator. These coactivators, together with RSC, coordinate the rapid recruitment of TBP and Pol II to the promoter and elongation through the ORF by Pol II without any detectable delay in promoter clearance. Despite the nearly simultaneous recruitment of coactivators, our mutant analyses reveal extensive interdependencies among the coactivators for their rapid recruitment by Gcn4p. In particular, we provide in vivo evidence that the SAGA HAT subunit is required for wild-type kinetics of SWI/SNF recruitment and that RSC function is needed for optimal SAGA recruitment. We also show that Mediator is strongly required for activator recruitment of both SAGA and SWI/SNF throughout the entire course of induction. Our kinetic analyses of PIC formation in coactivator mutants show that TBP recruitment per se is not sufficient for wild-type promoter occupancy by Pol II, suggesting that all four coactivators enhance Pol II recruitment or promoter clearance after binding of TBP to the promoter. We also uncovered functions for SWI/SNF and SAGA in transcription elongation downstream of the promoter. Together, our results provide a detailed picture of the activation mechanism for Gcn4p that differs significantly from that described for other activators, and they extend the range of functions stimulated by the participating coactivators in vivo.