Positive and negative regulation of squalene synthase (ERG9), an ergosterol biosynthetic gene, in Saccharomyces cerevisiae

To identify regulatory cis-elements in the proximal promoter of the yeast E RG9 squalene synthase gene, promoter deletion analysis was performed. This approach identified two regulatory elements, one an upstream repressing cis -element (URS), and the other an upstream activating cis-element (UAS). Ele ctromobility shift assays (EMSAs) demonstrated that distinct proteins bind each element. Genetic screens were performed to identify yeast mutants that altered expression of ERG9 promoter-reporter gene fusions. Three non-ergos terol biosynthetic pathway genes were identified. A mutation in TPO1(YLL028 W) led to a 5.5-fold increase in ERG9 expression while mutations in YER064C and SLK19 (YOR195W) led to a 3.1- and 5.6-fold decrease, respectively. Del etion analysis of these genes demonstrated that TPO1 and SLK19 specifically regulated ERG9 expression when tested with several different promoter-repo rter gene fusions. Additionally, EMSAs demonstrated that extracts derived f rom the TPO1 deletion strain was unable to shift the repressing cis-element while protein extracts from the SLK19 deletion strain had a reduced shift of the activating cis-element. Furthermore, these two mutants showed quanti tative differences in sterols and antifungal drug susceptibilities consiste nt with their role in regulating ERG9 expression. (C) 2001 Elsevier Science B.V. All rights reserved.