Transcription activation is an important step in gene regulation, and much remains to be understood about the mechanism of activation. Here, we used the yeast acidic activator Gcn4 to investigate the targets of its tandem activation domains and to determine the functional consequences of these activator-target interactions. Our results show a surprising overlap in activation domain function where both Gcn4 activation domains interact with a common set of coactivator targets. Another unexpected finding is the mechanism of Gcn4-Gal11 interaction where both Gcn4 activation domains contact the same three conserved Gal11 activator-binding domains with relatively low affinity. These three activator-binding domains additively contribute to Gal11 function and Mediator recruitment in vivo (Fig. ).
FIG. 10. Summary of Gcn4 activation domain (AD) and Gal11-Gcn4 interactions. (A) Cross-linking targets of the tandem Gcn4 activation domains in PICs. (B) Three Gal11 activator-binding domains bind both Gcn4 activation domains. Gray boxes 1 to 5 indicate conserved (more ...)
We used site-specific cross-linking in PICs to identify Taf12, Gal11, Tra1, and Sin4 as targets of the Gcn4 N-terminal activation domain in both nonchromatin and chromatin-assembled promoters. With the exception of Sin4, the coactivator subunits Taf12, Gal11, and Tra1 are also cross-linking targets of two other acidic activation domains: the Gcn4 central activation domain and the Gal4 activation domain (19
). Thus, unlike p53, in which the two individual activation domains may target distinct factors, the tandem Gcn4 activation domains primarily target the same three coactivator subunits, which may be general targets of acidic activators.
We performed a series of experiments to test the functional importance of these Gcn4-coactivator interactions. Gcn4 cross-links to Tra1 in the context of two distinct coactivator complexes, SAGA and NuA4, and it has previously been shown that Gcn4-Tra1 interaction is important for transcription activation via interaction with SAGA (7
). Here, we showed that the NuA4 complex, containing the histone acetyltransferase subunit Esa1, is an important chromatin-specific coactivator for Gcn4 function. However, we were surprised to find that the activator-binding domain of Taf12 was not required for Gcn4 activity and was not obviously important for the expression of any tested yeast gene under starvation conditions. It is possible that activator-Taf12 interaction is redundant with other activator-target interactions or that it is simply not functionally important. In contrast to these two extremes, Gcn4-regulated genes show a broad spectrum of dependence on Gal11, ranging from 10-fold dependence to complete Gal11 independence. We speculate that modest or absent Gal11 dependence at some genes is, at least in part, due to other transcription factors that bind to these gene regulatory regions. For example, at the Gal11-independent genes, Gcn4 may work in concert with other factors that make contacts with alternative Mediator subunits, and these other contacts likely compensate for the loss of Gcn4-Gal11 contact. Alternatively, Mediator may be recruited to these Gal11-independent genes by cooperative interactions with other coactivators that are recruited by different pathways.
We found that the two Gcn4 activation domains cooperate in a gene-specific fashion, which was surprising since the two activation domains contact three identical coactivator subunits. At three Gcn4-dependent genes examined, we found that the two activation domains can synergize or function additively or less than additively to promote transcription. Although the basis for this behavior is unknown, it may be due to variable coactivator requirements at these three genes. A better understanding of the reasons for this variable cooperation will help explain how tandem activation domains function in a wide variety of eukaryotic transcription factors.
Three independent experimental approaches mapped the Gal11 domains that specifically bind the two Gcn4 activation domains. These three Gal11 activator-binding domains bound each Gcn4 activation domain with low micromolar affinity. Two of these domains bound either activator with approximately equal affinity, while Gal11 residues 277 to 368 bound with 6-fold higher affinity to the Gcn4 N-terminal activation domain. At least two of these activator-binding domains have previously been suggested to bind the combined Gcn4 activation domains (31
). Also, the Gal11 activator-binding domain corresponding to conserved region 2 has been reported to bind the activation domain of glucocorticoid receptor Tau, which normally interacts with a glutamine-rich domain in mammalian steroid receptor coactivator 1 (34
Recently, Jedidi et al. (31
) suggested a key functional interaction between Gcn4 and the Gal11 KIX domain based on pulldown assays, in vivo
functional tests, and a nuclear magnetic resonance binding experiment. However, our results from several lines of experiments do not support specific binding of the KIX domain to the Gcn4 activation domains. Particularly revealing are FP experiments showing very weak interaction of Gcn4 with the Gal11 KIX domain that did not saturate at concentrations of KIX polypeptide near 200 μM. This weak interaction is not specific for the activation function of Gcn4 since it is not reduced by a Gcn4 mutation lacking transcription activation function.
Measurement of transcription and Mediator recruitment in vivo showed the functional importance of the activator-binding domains and the KIX domain. Although the KIX domain contributes to Gal11 function, we believe it does not function via direct interaction with Gcn4. Perhaps it interacts with other gene-specific regulators at the promoters tested here, with another Gal11 domain, or with other coactivators or general transcription factors, contributing to the stability of PIC and/or Mediator recruitment.
The three Gcn4 activator-binding domains we identified are unrelated to each other, and sequence analysis did not detect similarity to any known protein fold, although all are predicted to be alpha helical (40
). However, one common feature of these activator-binding domains, as well as those found in the Swi/Snf subunits Swi1 and Snf5 (51
), is that they are located in close proximity to nearby polyglutamine or polyglutamine/asparagine repeats. Since these glutamine-rich repeats are expected to be flexible (1
), they may function to enhance accessibility or exposure of the activator-binding domains in the context of large coactivator complexes.
It was surprising that the Gal11 activator-binding domains acted additively to promote transcription by Gcn4 since we found that a polypeptide containing two or three activator-binding domains bound with significantly higher affinity to Gcn4 than did the single activator-binding domains. This shows that activator-target affinity does not strictly correlate with function and that multiple weak low-micromolar interactions are sufficient to recruit Gal11 and Mediator to gene regulatory regions. Perhaps the relatively weak affinity sufficient for activation is due to the ability of the activation domains to interact with multiple coactivators, which in turn cooperatively bind to the promoter. In other words, recruitment of multiple factors by protein-protein interactions of modest affinity is an effective strategy for assembly of the PIC since many of these recruited factors cooperatively interact with each other and with DNA. The cooperative interactions among coactivators, general transcription factors, and DNA likely circumvent the need for high-affinity, high-specificity protein-protein interactions that are important for the specific interaction of individual molecules and small protein complexes.
In summary, although activator-target contacts may be conserved at many promoters, the relative importance of these contacts varies in a promoter-specific manner. It will be important for future studies to examine specific classes of genes to determine the basis for these mechanistic differences. These differences likely play an important role in regulating the response of different genes to specific activators and signaling pathways and provide a mechanism for a single activator to regulate transcription in a gene-specific fashion.