The results of this study show that Cyc8p and, to a lesser extent, Tup1p are required for high-level activation of Gcn4p target genes in vivo. Deletion of CYC8 confers sensitivity to SM and impairs transcriptional induction of two Gcn4p-dependent reporters (UASGCRE-CYC1-lacZ and HIS3-GUS) and authentic Gcn4p target genes (ARG1, ARG4, HIS4, and TRP3). ChIP analysis revealed that cyc8Δ cells exhibited diminished recruitment of SAGA and Srb mediator to the UAS, reduced promoter occupancy of TBP and Pol II, and decreased Pol II association with the 3′ ends of the ORFs at ARG1 and ARG4. The tup1Δ mutant showed similar defects in coactivator recruitment, PIC assembly, and transcriptional activation of authentic target genes but was less sensitive to SM and did not significantly impair induction of the reporter genes. Thus, it appears that Cyc8p is more broadly required than Tup1p for transcriptional activation by Gcn4p.
It is possible that all of the defects in transcriptional activation of Gcn4p-dependent promoters observed with cyc8
Δ and tup1
Δ cells can be attributed to the decreased Gcn4p binding to the UAS elements at its target genes in these mutants. This decrease in UAS occupancy by Gcn4p cannot be attributed to reduced expression of GCN4
mRNA or decreased steady-state levels of Gcn4p protein in cyc8
Δ or tup1
Δ cells. No other mutations in subunits of SAGA, SWI/SNF, Srb mediator, or RSC that impair transcriptional activation by Gcn4p have been found to reduce Gcn4p binding at ARG1
). Thus, reduced Gcn4p promoter occupancy in cells containing WT levels of Gcn4p is a unique phenotype of cyc8
Δ and tup1
One way to account for the role of Cyc8p and Tup1p in Gcn4p binding to UAS elements is to propose that they are required for efficient nuclear import of Gcn4p. For example, expression of one or more factors involved in nuclear import of Gcn4p could be reduced in cyc8
Δ and tup1
Δ cells. It was shown previously that a functional green fluorescent protein (GFP)-Gcn4 fusion protein is localized primarily in the nucleus independently of amino acid abundance (37
). Thus, nuclear localization of Gcn4p is probably not regulated by amino acid levels. We have compared levels of localization of this GFP-Gcn4p fusion for cyc8
Δ versus WT cells and found only a small decrease in the percentage of cells (from 95% to 79%) containing GFP-Gcn4p that was localized exclusively in the nucleus in cyc8
δ cells (see Fig. S2 in the supplemental material). Thus, we believe that Cyc8p/Tup1p has no significant role in nuclear localization of Gcn4p.
A direct role for Cyc8p/Tup1p in promoting UAS binding by Gcn4p is suggested by our finding that both proteins are recruited by Gcn4p to its target genes in vivo. This raises the possibility that the Cyc8p/Tup1p complex modifies the chromatin structure of the UAS to increase accessibility of Gcn4p to its binding sites, perhaps by positioning nucleosomes in an organized array (5
) or by recruiting histone deacetylases to reduce histone acetylation (1
). Another possibility is that Cyc8p/Tup1p impedes degradation of UAS-bound Gcn4p. This idea is prompted by the finding that Srb10p, a component of the mediator, phosphorylates the Gcn4p activation domain and targets the protein for rapid degradation by the proteosome (2
). Hence, recruitment of the mediator by Gcn4p could accelerate the degradation of UAS-bound Gcn4p without affecting the turnover of unbound Gcn4p (2
). According to this last model, Cyc8p/Tup1p recruited to the UAS by Gcn4p would decrease the rate of Srb10p-mediated degradation of UAS-bound Gcn4p. To account for the fact that total steady-state levels of Gcn4p are not reduced by cyc8
Δ, it would be necessary to stipulate that UAS-bound Gcn4p represents only a small fraction of the total cellular pool of the protein and that Gcn4p degraded at the UAS in cyc8
Δ cells is quickly replenished by new synthesis.
Whatever the mechanism involved, it seems that Cyc8p plays a more important role than Tup1p in promoting Gcn4p binding to UAS elements. Thus, in addition to the less severe activation defects in tup1Δ cells versus cyc8Δ cells, we found that overexpressing Gcn4p can alleviate the Gcn4p binding defect in tup1Δ cells but not in cyc8Δ cells. Presumably, Gcn4p binding to the UAS can be driven to nearly WT levels in tup1Δ cells by increasing the cellular concentration of Gcn4p, but the impediment to UAS binding is too great to overcome by mass action in cyc8Δ cells.
It is interesting that high-level UAS occupancy of Gcn4p is dependent on Cyc8p and Tup1p, while at the same time, Gcn4p recruits Cyc8p and Tup1p. Thus, Gcn4p and Cyc8p/Tup1p are interdependent for high-level binding at ARG1 and ARG4. Although the reduction in UAS occupancy by Gcn4p in cyc8Δ cells is substantial, it is not complete (e.g., Fig. ). Indeed, the SM sensitivity and reductions in reporter and target gene transcription are less severe in cyc8Δ cells than in gcn4Δ cells. Hence, it seems that Gcn4p can bind to a UAS element lacking associated Cyc8p, albeit with reduced efficiency. By subsequently recruiting Cyc8p/Tup1p, Gcn4p may achieve a higher level of UAS occupancy, thereby creating a positive-feedback loop (Fig. ). We cannot rule out the possibility, however, that Cyc8p/Tup1p binds at ARG1 independently of Gcn4p at a level below the detection limit of the ChIP assay and then promotes binding of Gcn4p to the unoccupied UAS. Once bound to the UAS, Gcn4p would then recruit Cyc8p/Tup1p to the higher levels observed under inducing conditions with our ChIP assays.
FIG. 8. Model for interdependent binding of activator Gcn4p and Cyc8p/Tup1p at ARG1 and ARG4 in vivo. Cyc8p/Tup1p functions to stimulate Gcn4p binding to the UAS elements at ARG1and ARG4. Interestingly, Gcn4p recruits Cyc8p/Tup1p to the promoter and ORF regions (more ...)
We have done several experiments to determine whether Gcn4p recruits Cyc8p/Tup1p to target genes by direct binding to these cofactors but have uncovered no evidence to support this possibility. First, in cells where myc-tagged Gcn4p was induced by SM, we observed no coimmunoprecipitation of Tup1p with myc-Gcn4p from cell extracts, whereas Tup1p coimmunoprecipitated with myc-Cyc8p under the same conditions (see Fig. S3 in the supplemental material). We also failed to observe a specific interaction of GST-Gcn4p with myc-Tup1p or myc-Cyc8p in yeast WCEs under conditions where robust binding of GST-Gcn4p to the mediator was readily detected (see Fig. S3 in the supplemental material). We presume that Gcn4p recruits Cyc8p/Tup1p to the UAS and ORF of its target genes in a manner dependent on other coactivators or histone modifications, and future experiments will address the mechanism of this indirect recruitment, particularly as it applies to the coding sequences.
Most previous studies of Cyc8p and Tup1p have underscored their functions as corepressors (42
), but the complex can also function in activation. As noted above, Cyc8p/Tup1p remains bound at promoters regulated by Sko1p (38
) and at GAL1
) under inducing conditions and participates in recruitment of coactivators to overcome its own repressing functions. In the Cyc8p-dependent induction of CIT2
by Rtg3p (4
), it is unknown whether Cyc8p stimulates UAS occupancy by Rtg3p or provides a coactivator function for UAS-bound Rtg3p. The exact step in activation of CYC1
by Hap1p that is dependent on Tup1p/Cyc8p is also unknown (52
). Regarding Cyc8p-dependent activation of FRE2
by Aft1p, Cyc8p binds in vitro to the DNA binding domain of Aft1p, and Aft1p binding at FRE2
was apparently reduced by a small amount in cyc8
Δ cells. Nevertheless, activation by a LexA-Aft1p fusion from LexA binding sites was strongly Cyc8p dependent, pointing to a role for Cyc8p in the activation function of Aft1p rather than in Aft1p promoter binding (13
). Thus, the prominent role of Cyc8p in stimulating Gcn4p binding to the UAS elements at ARG1
described here appears to be unique among the yeast activators studied thus far.
We obtained one indication that Cyc8p/Tup1p also enhances the ability of Gcn4p to recruit coactivators. In strains overexpressing Gcn4p from the h.c. GCN4-HA plasmid, there was essentially the same level of Gcn4p binding to the ARG1 UAS in WT and tup1Δ cells (Fig. ); however, the tup1Δ cells showed a relatively lower level of SAGA and mediator recruitment to the UAS and also less myc-Rpb3p recruitment to the promoter (Fig. ). Hence, Cyc8p/Tup1p may play a dual role at Gcn4p target genes, increasing the efficiency of coactivator recruitment by Gcn4p in addition to stimulating Gcn4p binding to the UAS.