Dual Regulation of Fzf1-Targeted Genes by Chemical Stresses and Nucleosome Occupancy in Saccharomyces cerevisiae

Saccharomyces cerevisiae Fzf1 contains five C2H2 zinc finger (ZF) domains and is a transcription activator that coordinates expression of DDI2, DDI3 (DDI2/3), SSU1, YHB1 and YNR064C in response to chemical stresses. The N-terminal three ZFs in Fzf1 are sufficient for sequence-specific binding to a consensus DNA sequence CS2 found in the promoters of all five genes, whereas functions of ZF4 and ZF5 remains unknown. This dissertation aimed to investigate Fzf1 functions and how it regulates downstream genes under stress conditions. Firstly, it was found that, in addition to previously reported cyanamide (CY) and methyl methanesulfonate (MMS) induction of DDI2/3 and nitric oxide (NO) induction of YHB1, all five Fzf1-regulated genes were induced by CY, MMS and NO, in an Fzf1-dependent manner, whereas sulfite, a substrate for the SSU1 product, repressed target gene expression. Secondly, amino acid substitutions in the ZF4 zinc coordinator residues or deletion of ZF4 resulted in sulfite resistance, elevated Fzf1 downstream gene expressions and loss of chemical induction except CY-induced DDI2/3, indicating that ZF4 represses Fzf1 activity. Thirdly, it was found that ZF5 serves as an activation domain not only within Fzf1 but also when fused to a heterologous Gal4 DNA-binding domain. Finally, previous studies led us to hypothesize that the nucleosome occupancy in the DDI2/3 promoters serves as a transcription repressor. To test this hypothesis, a yeast strain with endogenous histone H3/H4 genes replaced by GAL-promoter-driven H3/H4 was employed. Experimental depletion of H3/H4 resulted in approximately 30-fold increase in the DDI2/3 expression and CY induced DDI2/3 by another 20-fold in an FZF1-dependent manner. Meanwhile, other Fzf1-regulated genes differentially responded to chemical stresses upon histone depletion, which contributes to their variable levels of induction by tested chemicals. In summary, this study supports a regulatory mechanism by which Fzf1-ZF4 inhibits the ZF5 activity, and chemical stresses by CY, MMS and NO are sensed by Fzf1, resulting in relief of the ZF5 activity to induce downstream gene expressions. Hence, Fzf1-mediated transcription activation and detoxification is achieved by de-repression. In addition, DDI2/3 and possibly other genes under study are differentially regulated by their local nucleosome structures.