Identification and Characterization of the Fzf1-mediated Response to Nitrosative Stress in Saccharomyces Cerevisiae

The mechanisms by which microorganisms sense and detoxify nitric oxide (NO & middot;) are of particular interest due to the central role this molecule plays in innate immunity. I used DNA microarrays to investigate the genome-wide transcriptional response to exogenously supplied NO & middot; in the model organism S. cerevisiae. Exposure to NO & middot; generating compounds resulted in both a general stress response as well as a specific NO & middot;detoxification response. This was characterized by the induction of a small set of genes, including the yeast flavohemoglobin YHBI, SSUI, and three additional uncharacterized open reading frames. The YHB1 gene encodes a NO & middot; dioxygenase gene that converts NO & middot; to nitrate. Induction of YHB1 in response to NO & middot; is consistent with a specific NO & middot;detoxification mechanism. Transcriptional induction of SSU1, which encodes a putative sulfite transporter, has previously been shown to require the zinc finger transcription factor Fzf1p. I discovered that deletion of Fzf1p eliminated the nitrosative stress-specific transcriptional response; while overexpression of Fzf1p recapitulated this response in the absence of exogenously supplied NO & middot;. In addition to discovering the response and determining a necessary transcription factor, I used a combination of bioinformatic, phylogenetic, and experimental approaches to discover a cis-acting sequence unique to the promoter regions of Fzf1p-dependent, NO & middot;-responsive genes. This sequence was found to be sufficient to activate reporter gene activity in an NO & middot;- and Fzf1p-dependent manner. I also verified that the RNA transcript level increases seen by array led to increases in the Yhb1p and Ssu1p protein levels using both Western blotting and flow cytometry. My results suggest that the presence of NO & middot; or NO & middot; derivatives activate Fzf1p, which leads to a physiologically relevant response that protects the cell from NO & middot;-mediated stress. My results have already led to the characterization of a similar response in the pathogenic fungi C. albicans.