Transcriptional regulation of the budding yeast DDI2 gene in response to environmental stresses

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DDI2 and DDI3 are two duplicated budding yeast DNA-damage inducible (DDI) genes identified through our microarray analysis. During the current granting period, we demonstrated that DDI2 encodes a HD-domain-containing cyanamide (CY) hydrotase, and determined the recombinant Ddi2 protein structure and its detailed mechanism of reaction. More interestingly, the DDI2/3 gene was induced by CY by up to 2,000-fold, and deletion of both DDI2 and DDI3 genes sensitizes cells to CY and MMS. Through a genome-wide genetic screen, we have identified a transcriptional factor Fzf1 that is absolutely required for the induction of DDI2 by CY and MMS. However, how DDI2 is induced by CY and MMS remains largely unknown. This proposed research is to characterize molecular mechanisms by which budding yeast DDI2 is induced in response to CY and MMS, and to investigate the stress responsive network mediated by Fzf1. (1) Define cis-acting element(s) required for the DDI2 . A DDI2 promoter-lacZ fusion construct is able to support full induction by CY and MMS. We will make DDI2 promoter deletions and subsequently point mutations in this construct and test their effects on the lacZ reporter expression to define the critical cis-acting element. This sequence will be used as a probe to perform electrophoresis mobility shift assay (EMSA) in vivo and in vitro to see if it is the Fzf1-binding site. If other cis-acting elements are also defined through the above analysis, we will search for their cognate transcriptional regulators by bioinformatics analysis and yeast one-hybrid screen. (2) Investigate how cells sense CY-induced stress. Upon CY or MMS treatment, the Fzf1 level was not significantly altered, nor was it modified by means like phosphorylation or ubiquitination. Based on our preliminary analysis, we are entertaining three alternative but non-exclusive hypotheses: (i) Certain amino acid residues in Fzf1 itself are modified by treatment with CY or MMS, which turns it into an active form; (ii) a histone subunit is modified by treatment with CY or MMS that alters nucleosome structure, allowing access of Fzf1 to the DDI2 promoter; and (iii) an unknown co-factor is modified by treatment with CY or MMS that recruits Fzf1 to the DDI2 promoter. (3) Structure and functions of Fzf1 in coordinating multiple chemical stress response pathways. Fzf1 appears to coordinate cellular response pathways to several chemical stresses. To further address how Fzf1 coordinately regulates these pathways, we will purify the Fzf1 protein and determine its crystal structure bound to the consensus DNA sequence and functional domains. Meanwhile, we will assess global transcriptional regulatory network by Fzf1 through RNA-seq analysis. The clustered genes will be further characterized to see if they are coordinately regulated in response to several chemical stresses including CY, MMS, sulphite and nitrogen oxide