In this study, we initiated a characterization of the coactivator requirements of the Zap1 transcription factor. A genetic screen was used to identify coactivator complex subunits required for optimal expression of one Zap1 target gene, YOR387C
, in low zinc. A similar approach was used previously to identify coactivators used by the Gcn4 transcription factor (Swanson et al., 2003
). Among the approximately 60 coactivator mutants that we analyzed, 10 were found to have reduced YOR387C
expression below an arbitrary threshold value of 50%. The identity of those mutants implicated SWI/SNF and SAGA complexes as being particularly important for Zap1-induced expression. In addition, given the very poor growth of the med15Δ
mutant in low zinc, Mediator complex was also implicated. Other complexes that we identified that may play lesser roles on Zap1-regulated genes include CCR4-NOT, THO-TREX, and Paf1 complexes. These complexes play various roles in transcription initiation, transcription elongation, RNA export, and RNA degradation (Collart, 2003
; Jaehning, 2010
; Rondon et al., 2010
We focused our attention on the SWI/SNF, SAGA, and Mediator complexes because of the strong effect mutations affecting those complexes had on YOR387C expression. It should be noted, however, that our genetic screen was not exhaustive and many more coactivator mutants await analysis. In addition, other coactivator mutants that we found to reduce YOR387C expression but not below our threshold value may also affect coactivator complexes that are recruited by Zap1 and contribute significantly to transcription. Thus, while we have identified coactivator complexes important for Zap1 function, other unrecognized coactivators may be involved as well.
Alternative approaches to identifying coactivators recruited by a given transcription factor include chromatin immunoprecipitation coupled with mass spectrometry to identify interacting proteins. While this approach has been used successfully by others, we believe that the genetic approach we have used has a significant advantage. Our approach can identify coactivator complexes that are not only recruited to a promoter but must also play significant functional roles in the initiation process. While the genetic approach does not specifically identify coactivators that interact directly with Zap1, it does highlight those factors that are especially important for gene expression whether they are directly or indirectly recruited by the Zap1 activation domains.
The initial goal of our genetic screen was to identify those factors required for AD1 function. This was possible because YOR387C transcription is entirely dependent on AD1 and AD2 does not activate this promoter. Thus, mutations found to decrease YOR387C-lacZ expression are likely to affect AD1-mediated activation. Once identified, we could then test the role of these factors on other Zap1-regulated promoters. By analyzing the DPP1 and ZRT1 promoters, we found that the effects of the coactivator mutants were similar. These results suggest that at least for the YOR387C, DPP1, and ZRT1 promoters, Zap1-mediated activation has similar coactivator requirements. Because DPP1 and ZRT1 can be activated by either AD1 or AD2, we could also compare the effects of these mutations on either AD1 or AD2 function. We found that the effects of these mutations on these two activation domains were also similar. These results suggest that AD1 and AD2 have similar coactivator requirements. This hypothesis was supported by chromatin immunoprecipitation experiments showing that AD1 and AD2 can both recruit SWI/SNF, SAGA, and Mediator to a Zap1 target promoter.
We began this study with the hypothesis that, in zinc-replete cells, zinc binding to ligand residues within and flanking AD1 and AD2 blocks coactivator recruitment (Bird et al., 2003
; Herbig et al., 2005
). This was confirmed when we examined the zinc and Zap1 dependence of coactivator recruitment. Assembly of coactivators on Zap1 target promoters was also found to be highly interdependent with SAGA recruitment requiring SWI/SNF activity and vice versa. Optimal recruitment of Mediator complex was also dependent on both SWI/SNF and SAGA function. From our results, we cannot assess the order of recruitment as has been done previously with other promoters that can be quickly activated. This is not possible for Zap1 target promoters because it takes several hours to induce these promoters as a cell transitions from a zinc-replete to a zinc-limited state. On other promoters where these experiments have been done, it was found that assembly is a highly ordered process (Biddick and Young, 2009
). On the HO promoter, for example, Mediator is recruited by the Swi5 activator following SWI/SNF and SAGA entry (Cosma, 2002
). In contrast, Mediator is recruited by Gal4 to the GAL1
promoter after SAGA but before SWI/SNF (Bryant and Ptashne, 2003
; Lemieux and Gaudreau, 2004
). Zap1-mediated induction differs from that of Gal4 where SWI/SNF and SAGA are recruited by the activator independently of each other. Thus, our findings reflect the heterogeneity of coactivator interactions as observed in other coactivator recruitment studies.
If AD1 and AD2 require the same coactivators, as our data suggest, why are some promoters responsive to AD2 and other promoters are not responsive? As described above, our knowledge of what coactivators are required for function of AD1 and AD2 is still incomplete and there may be some key differences in the specific complexes recruited by these two domains. Continued analysis of coactivator recruitment by AD1 and AD2 may identify AD-specific coactivators. Our results do support a second hypothesis that is AD2 is a weaker-activation domain and may therefore be incapable of activating those genes that require a strong activation domain due to, for example, especially repressive nucleosome positioning. Promoters that are better poised for activation would then be responsive to either a strong activation domain (AD1) or a weaker domain like AD2. We showed previously that while AD1 is sufficient to activate transcription of the ZRT1
gene under normal conditions, it was not sufficient to activate transcription at 37°C where AD2 was also required (Frey and Eide, 2011
). We suggested that AD2 may therefore be needed to aid AD1 when zinc deficiency is combined with other stresses such as heat stress. Now that we know some of the coactivators recruited by Zap1, we can further explore the novel roles of AD1 and AD2 in Zap1-mediated transcription.