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We describe a genome-wide characterization of mRNA transcript levels in yeast grown on the fatty acid oleate, determined using Serial Analysis of Gene Expression (SAGE). Comparison of this SAGE library with that reported for glucose grown cells revealed the dramatic adaptive response of yeast to a change in carbon source. A major fraction (>20%) of the 15,000 mRNA molecules in a yeast cell comprised differentially expressed transcripts, which were derived from only 2% of the total number of approximately 6300 yeast genes. Most of the mRNAs that were differentially expressed code for enzymes or for other proteins participating in metabolism (e.g., metabolite transporters). In oleate-grown cells, this was exemplified by the huge increase of mRNAs encoding the peroxisomal beta-oxidation enzymes required for degradation of fatty acids. The data provide evidence for the existence of redox shuttles across organellar membranes that involve peroxisomal, cytoplasmic, and mitochondrial enzymes. We also analyzed the mRNA profile of a mutant strain with deletions of the PIP2 and OAF1 genes, encoding transcription factors required for induction of genes encoding peroxisomal proteins. Induction of genes under the immediate control of these factors was abolished; other genes were up-regulated, indicating an adaptive response to the changed metabolism imposed by the genetic impairment. We describe a statistical method for analysis of data obtained by SAGE.

In the yeast Saccharomyces cerevisiae, two transcriptional activators belonging to the Zn2Cys6 protein family, Pip2p and Oaf1p, are involved in fatty-acid-dependent induction of genes encoding peroxisomal proteins. This induction is mediated via an upstream activation sequence called the oleate-response element (ORE). DNA-bandshift experiments with ORE probes and epitope-tagged proteins showed that two binary complexes occurred: in wild-type cells the major complex consisted of a Pip2p x Oaf1p heterodimer, but in cells in which Oaf1p was overexpressed an Oaf1p homodimer was also observed. The genes encoding Oaf1p and Pip2p were controlled in different ways. The OAF1 gene was constitutively expressed, while the PIP2 gene was induced upon growth on oleate, giving rise to positive autoregulatory control. We have shown that the Pip2p x Oaf1p heterodimer is responsible for the strong expression of the genes encoding peroxisomal proteins upon growth on oleate. Pip2p and Oaf1p form an example of a heterodimere of yeast Zn2Cys6 zinc-finger proteins binding to DNA.

In Saccharomyces cerevisiae, peroxisomes are the exclusive site for the degradation of fatty acids. Upon growth with the fatty acid oleic acid as sole carbon source, not only are the enzymes of beta-oxidation and catalase A induced, but also the peroxisomal compartment as a whole increases in volume and the number of organelles per cell rises. We previously identified a cis-acting DNA sequence [oleate response element (ORE)] involved in induction of genes encoding peroxisomal proteins. The aim of our investigation was to test whether a single mechanism acting via the ORE coordinates the events necessary for the proliferation of an entire organelle. Here we report the cloning and characterization of the oleate-specific transcriptional activator protein Pip2p (pip: peroxisome induction pathway). Pip2p contains a typical Zn(2)-Cys(6) cluster domain and binds to OREs. A pip2 deletion strain is impaired in growth on oleate as sole carbon source and the induction of beta-oxidation enzymes is abolished. Moreover, only a few, small peroxisomes per cell can be detected. These results indicate that fatty acids activate Pip2p, which in turn activates the transcription of genes encoding beta-oxidation components and acts as the crucial activator of peroxisomes.

In Saccharomyces cerevisiae induction of the FOX3 gene, encoding peroxisomal 3-oxoacyl-CoA thiolase, by growth on oleate as sole carbon source, is exerted via the cis-acting DNA element designated oleate response element (ORE) (Einerhand et al. (1991) Eur. J. Biochem. 200, 113-122). The transcription factor(s) binding to this upstream activation site (UAS) are still unknown, however. Induction of another peroxisomal enzyme, citrate synthase (CIT2) is dependent on the products of two genes called RTG1 and RTG2 (Liao and Butow (1993) Cell 72, 61-71). In the present study we have investigated whether RTG1 controls other genes coding for peroxisomal proteins, and whether such control takes place via the ORE. A number of genes coding for a variety of peroxisomal proteins such as: thiolase and catalase (peroxisomal matrix proteins), PAS3p (a peroxisomal membrane protein) and PAS10p (a protein involved in the import of peroxisomal proteins) were studied in their response to RTG1. Although the RTG1 and 2 products proved to be required for the increase in number and volume of peroxisomes upon induction by oleate, the single promoter output of the chosen set of genes remained practically unchanged in a rtg1 mutant strain. In addition gel retardation experiments indicated that RTG1 does not bind to the ORE. The behavior of genes coding for the various proteins also varied during repression, derepression and induction, indicating that probably a number of proteins are involved in tuning the output of each gene to cellular demand.

Expression of the FOX3 gene, which encodes yeast peroxisomal 3-oxoacyl-coenzyme A thiolase, can be induced by oleate and repressed by glucose. Previously, we have shown that induction was mediated by an oleate response element. Just upstream of this element a negatively acting control region that mediated glucose repression was found. In order to study this negative control region, we carried out DNA-binding assays and analyzed phenotypes of mutations in this region and in the trans-acting factor CAR80, which is identical to UME6. DNA-binding assays showed that two multifunctional yeast proteins, ABF1 and RP-A, interacted with the negative control element independently of the transcriptional activity of the FOX3 gene. ABF1 and RP-A, the latter being identical to BUF, were able to bind to DNA independently of one another but also simultaneously. The phenotypes of mutations in either DNA-binding sites of ABF1, RP-A, or both, which affected the DNA binding of these factors in vitro, indicated that these sites and the proteins that interact with them participate in glucose repression. The involvement of the RP-A site in glucose repression was further supported by our observation that the CAR80 gene product, which is required for repression mediated by the RP-A site, was essential for maintenance of glucose repression. In addition to the RP-A site in the FOX3 promoter, similar sequences were observed in other genes involved in peroxisomal function. RP-A proved to bind to all of these sequences, albeit with various affinities. From these results it is concluded that the ABF1 and RP-A sites are being required in concert to mediate glucose repression of the FOX3 gene. In addition, coordinated regulation of expression of genes involved in peroxisomal function in response to glucose is mediated by proteins associated with the RP-A site, probably RP-A and CAR80.