## 3 Results
### Calcineurin Affects the Expression of Fungal-Specific Zinc-Cluster Transcriptional Factors  
[PERSON] et al. (2012) reported that the \(crz1\Delta\) mutant is not sensitive to cationic stress, but \(cna1\Delta\) is, suggesting that other transcription factors may be post-translationally modified by calcineurin in response to cationic stress, or calcineurin may directly or indirectly induce one or more transcription factors at the transcriptional level in \(C\). _neofurans_[14]. Therefore, it is reasonable to propose that, since calcineurin is a signaling complex, the disabling of its signaling pathway may have consequences for the induction of transcription factor genes. We directly searched for differential \(CRZ1\) expression in the transcriptomes generated in this work, but \(CRZ1\) expression levels were unaffected by the presence of NaCl in the wild type, and no differential expression was detected for \(CRZ1\) in \(cna1\Delta\) or \(cnb1\Delta\) under this condition, suggesting that \(CRZ1\) does not respond directly to cationic stress. However, a comparison of the wild-type and \(gpp2\Delta\) strains highlighted the category (GO:0006355) related to the regulation of transcription, where 14 transcription factors were induced in the wild type and repressed in the \(GPP2\) deletion mutant in the presence of NaCl. Of the 14 transcription factors, 11 belong to the Zinc-Cluster class of proteins, which are fungal-specific. The other three are BZip (CNAG_00871), GATA (CNAG_01883), and Fork-Head (CNAG_05535) transcription factors. The analysis was expanded to the \(cna1\Delta\) and \(cnb1\Delta\) strains, showing that all three mutants failed to induce most of the 14 putative transcription factors (heat map in Figure 8A). In the \(gpp2\Delta\) strain, only two Zinc-Cluster proteins continued to be induced compared to the wild type during cationic stress, but to a lesser extent than in the wild type (CNAG_01977 and CNAG_05333). In the calcineurin mutants, only the GATA factor and one BZip continued to be induced. All other transcription factors were either not induced or repressed. In this case, the most repressed were CNAG_05333, CNAG_03132, CNAG_00841, and CNAG_07545. Out of 14 DEGs analyzed in the \(gpp2\Delta\) strain, 7 transcription factors appeared repressed, all Zinc-Cluster transcription factors, except for the Fork-Head (Figure 8A). Finally, an important hallmark of the transcription factors identified in this study is that five of them are involved in the stress response and especially in fatty acid utilization and lipid accumulation in _S. cerevisiae_ (CNAG_06252, CNAG_05479, CNAG_07545, CNAG_04774, and CNAG_06097). The most strongly induced gene under this condition (CNAG_05333) was validated by quantitative PCR (qPCR) under cationic stress in the wild-type strain, confirming the expression pattern observed in RNA-seq (Figure 8B) and the calcineurin-dependent expression in Figure 8C. These observations allowed us to suggest that (i) cationic stress induces a set of Zinc-Cluster transcription factors in a calcineurin-dependent manner, and (ii) several of these transcription factors seem to be related to lipid metabolism in other organisms. Figure 7: **(A)** A heat map of DEGs categorized under oxidative stress response (GO:0006979) and glutathione metabolism (GO:0006749); **(B)** the growth phenotypes of wild-type (H99) and _cnb1A_, _cna1A_, _met3A_, and _gpp2A_ mutant strains cultured in YEPD medium containing NaCl, KCl (1 M each), or LiCl (0.09M) and supplemented with glutathione (10 mM). Figure 8: (**A**) A heat map of the DEGs categorized under transcription regulation (GO:0006355) in the wild type and mutants exposed to cationic stress. On the right side is the manual annotation based on the Saccharomyces genome database (SGD) and FungiDB.