Genome-wide expression patterns in Saccharomyces cerevisiae: comparison of drug treatments and genetic alterations affecting biosynthesis of ergosterol

Abstract

Enzymes in the ergosterol-biosynthetic pathway are the targets of a number of antifungal agents including azoles, allylamines, and morpholines. In order to understand the response of Saccharomyces cerevisiae to perturbations in the ergosterol pathway, genome-wide transcript profiles following exposure to a number of antifungal agents targeting ergosterol biosynthesis (clotrimazole, fluconazole, itraconazole, ketoconazole, voriconazole, terbinafine, and amorolfine) were obtained. These profiles were compared to the transcript profiles of strains containing deletions of one of the late-stage ergosterol genes: ERG2, ERG5, or ERG6. A total of 234 genes were identified as responsive, including the majority of genes from the ergosterol pathway. Expression of several responsive genes, including ERG25, YER067W, and YNL300W, was also monitored by PCR over time following exposure to ketoconazole. The kinetics of transcriptional response support the conditions selected for the microarray experiment. In addition to ergosterol-biosynthetic genes, 36 mitochondrial genes and a number of other genes with roles related to ergosterol function were responsive, as were a number of genes responsive to oxidative stress. Transcriptional changes related to heme biosynthesis were observed in cells treated with chemical agents, suggesting an additional effect of exposure to these compounds. The expression profile in response to a novel imidazole, PNU-144248E, was also determined. The concordance of responsive genes suggests that this compound has the same mode of action as other azoles. Thus, genome-wide transcript profiles can be used to predict the mode of action of a chemical agent as well as to characterize expression changes in response to perturbation of a metabolic pathway.

FIG. 1

Treatments used in the study and their relationships to ergosterol biosynthesis. Gene names are as listed in reference 9). Genes in deletion strains are boldfaced and boxed. Arrows point to the sites of action of the antifungal agents. The structure of PNU-144248E is shown under the azoles.

FIG. 2

Genes involved in the biosynthesis of ergosterol and membrane components. Boldfaced genes were responsive in the study; boldface italics indicate genes with decreased transcript levels. Superscripts indicate the number of treatments to which the gene responded. The first number in each superscript is the number of genetic perturbations (out of a total of three) which elicited a response, and the second number is the number of drug treatments (out of a total of eight) which elicited a response. Lists of genes were obtained from the YPD (18) and reference .

FIG. 3

Genes involved in the biosynthesis and utilization of heme. Boldface, italics, and superscripts are as described for Fig. 2. Lists of genes were obtained from the YPD (18).

FIG. 4

Comparison of expression measurements by RT-PCR and microarrays. Each data point represents the measurement of a given gene by each method under a specific treatment, as indicated by the label. Squares, ERG25; circles, YER067W; solid symbols, untreated control. The vertical axis is the change from the TEF1 level; larger numbers signify more cycles needed and thus less starting material in the sample. The horizontal axis indicates the microarray intensity in ADI units, normalized as described in Materials and Methods; ADI units are proportional to transcript levels.

FIG. 5

Expression time course following exposure to ketoconazole. (A) Culture OD at the times sampled (solid bars, untreated culture; heavily shaded bars, 4 μM ketoconazole; open bars, 8 μM ketoconazole; light shaded bars, 16 μM ketoconazole) and change in TEF1 level as culture goes into saturation (line, average of three measurements of untreated culture in panels B, C, and D; error bars, standard errors). (B through D) Responses of ERG25, YER067W, and YNL300W transcripts (solid symbols) and an internal TEF1 control (open symbols). Culture was left untreated (circles) or treated with ketoconazole at 4 μM (triangles), 8 μM (squares), or 16 μM (diamonds).