This paper provides a thorough illustration of the gene, GATA activator and inducer specificities among NCR responses. Earlier reports have given some possible explanations to these observations. First, the promoter architecture, the number and orientation of GATA sites likely influence the way how an NCR-sensitive gene responds to a particular stimulus 34–36. It has been recognized that in some cases, auxiliary promoter sequences were shown to contribute to transcriptional activation 37, although this was never as efficient as additional GATA sites 38. Second, we cannot neglect the impact of the two other GATA factors on NCR-sensitive transcription, the Dal80 and Gzf3 repressors 39–42. Under limiting nitrogen conditions, DAL80 is highly induced 40, and Gzf3 has been detected at NCR-sensitive promoters 43. Given the elevated sequence conservation among the four yeast GATA factors, with 100% conservation of the residues involved in contacts with DNA 44, it is very likely that the repressors do bind the exact same DNA sequences in vivo and may interfere with the GATA activators, in a gene-specific manner. Third, we have previously shown that Gln3 and Gat1 localizations are controlled by two different regulatory pathways, with Gln3 localization predominantly responding to intracellular nitrogen levels, and Gat1 localization mainly responding to rapamycin 45, suggesting that the different stimuli do not borrow the same signalization cascades. Differences of GATA activator requirements for the transcriptional responses to different activation signals can therefore be anticipated.
This paper also provides a deep characterization of the role of the SAGA complex controlling DAL5 and GDH2 expression, together with the GATA activators. In wild type cells, elevated binding of both GATA activators and SAGA ensure maximal activation of DAL5 upon rapamycin treatment. The HAT module is dispensable here. In the absence of Ada1, DAL5 expression is strongly reduced despite the presence of Gln3 at the promoter, probably because SAGA is not recruited in this CORE mutant. Interestingly, the absence of SAGA prevents Gat1 recruitment at the DAL5 promoter. Low expression of DAL5 is also observed in cells lacking Gat1 or Gln3, in which recruitment of the other GATA activator to the DAL5 promoter is largely impaired, as well as the SAGA complex. DAL5 activation is nevertheless significant in all three single mutants, though, and this could be attributed to a binding of the GATA activators and/or SAGA subunits so weak that it escaped our detection. Since DAL5 expression is abolished in the double gln3Δ ada1Δ and gat1Δ ada1Δ mutants, we propose that SAGA is indeed recruited even though we could not detect it in the gln3Δ and gat1Δ single mutants. Since Gln3 binding can occur without SAGA, which is not the case for Gat1 binding, we propose that SAGA could be a better coactivator for Gat1-dependent transcriptional activation. Accordingly, Gln3 could be co-recruited with Gat1, leading to SAGA recruitment, allowing chromatin remodelling, thereby increasing Gat1 binding and subsequent transcription. Interdependence for binding suggest the existence of synergistic interferences between the different partners at the DAL5 promoter upon rapamycin treatment.
As far as GDH2 is concerned, the conclusions are clearly different. GDH2 expression is maximal in wild type cells treated with methionine sulfoximine, consistent with maximal GATA factor and SAGA subunit binding. Interestingly, this expression does not require Gat1, even though the activator is detected at the GDH2 promoter at elevated levels. Consistently, Gln3 and SAGA still bound at the GDH2 promoter in gat1Δ cells, ensuring elevated GDH2 transcription levels. The activation levels were even higher in gat1Δ mutant cells, and this could be due to Gat1 being negatively interfering with Gln3 transcription activation, more potent. Alternatively, increased GDH2 expression in gat1Δ cells could be explained by the decreased presence of the Gat1-dependent Dal80 negative regulator. In the absence of Gln3, nor Gat1 nor SAGA could be recruited at the GDH2 promoter, consistent with the lack of activation in gln3Δ mutant cells. Finally, in ada1Δ cells, binding of the GATA factors was only weakly altered, with much lower activation in the mutant cells, suggesting that SAGA is not mainly needed for GATA factor recruitment but most strongly for transcription activation. In sum, Msx-induced Gln3 binding at the GDH2 promoter can occur on its own, allowing for concomitant Gat1 and SAGA recruitment. Gat1 recruitment is ineffective for GDH2 activation whereas that of SAGA is crucial.
In this work, we have demonstrated an absolute interdependence for rapamycin-induced GATA activator binding at the DAL5 promoter. This has not always been the case. Our previous reports have shown that rapamycin-induced Gat1 binding at the DAL5 promoter could occur in the absence of Gln3, in both TB 14 and FY 15 genetic backgrounds. In these experiments, however, cells were grown with glutamine as the nitrogen source whereas they were grown with ammonia in the present work. Such a difference is striking and suggests that much more complexity is to be expected in the context of nitrogen source-specific regulations of NCR-sensitive genes.
Our work has demonstrated that SAGA subunits are recruited to NCR-sensitive promoters upon derepression. The SAGA CORE module is structurally made of a histone octamer-like fold and an adjacent submodule (Taf5, 6 and Spt2032). The octamer-like domain is made of three pairs of subunits (Taf6-Taf9, Taf10-Spt7 and Taf12-Ada1), each pair contributing to two histone folds, plus Spt3, which brings two more histone folds on its own 46. In our experiments, the strongest phenotypes with respect to NCR-sensitive transcription in SAGA mutants have been observed in the ada1Δ and spt7Δ mutant strains. This observation is consistent with the fact that Ada1 and Spt7 belong to the CORE of SAGA and their absence has previously been shown to fully impair SAGA complex integrity 47−49. Consistently, Ada1 and Spt7 recruitment at NCR-sensitive genes was fully interdependent. In addition, our observation that Spt7 ChIP levels were systematically twice higher than those of Ada1 could indicate a better accessibility of the former. Indeed, from the published structure of SAGA, Ada1 could be less accessible, interfacing the CORE module with the large Tra1 subunit and the DUB module, whereas Spt7’s position appears more peripheral 46.
Interestingly, the HAT module was fully dispensable for NCR-sensitive gene transcription, whereas the DUB module was only mildly required, for rapamycin-induced DAL5 expression only. This could indicate that SAGA modules fulfil complementary activities at NCR-sensitive promoters, and altering only one of these activities may not alter its capacity to mediate transcriptional activation. More surprisingly, the reduced impact, on NCR-sensitive transcription, of the joint deletion of SPT3 and SPT8, supposedly altering TBP recruitment suggests that other coactivator complexes may help for its recruitment, or that GATA activators can perform this function themselves.
Our results illustrate the complexity and interactions taking place at NCR-sensitive promoters, enabling fine-tuned responses to environmental changes. Further work will be required to decipher all mechanisms involved in gene- and stimulus-specific transcriptional responses of NCR-sensitive genes.