Saccharomyces cerevisiae PIP2 mediating oleic acid induction and peroxisome proliferation is regulated by Adr1p and Pip2p-Oaf1p.

Saccharomyces cerevisiae genes involved in fatty acid degradation contain in their promoters oleate response elements (OREs) and type 1 upstream activation sequences (UAS1s) that bind Pip2p-Oaf1p and Adr1p, respectively. The promoter of the PIP2 gene was found to contain a potential UAS1 that consists of a tandem array of CYCCRR half-sites in an overlapping arrangement with a previously characterized ORE. Electrophoretic mobility shift analysis demonstrated that Adr1p bound to UAS1PIP2, and Northern analysis in combination with a lacZ reporter gene confirmed that Adr1p influenced the transcription of PIP2. Immunoprecipitation showed that, in adr1delta mutant cells grown on oleic acid, Pip2p was less abundant compared with the corresponding wild-type. In addition, the amount of Pip2p-Oaf1p that bound to a target ORE in vitro was reduced in mutant extracts compared with the wild-type. Transcription of the oleic acid-inducible genes SPS19 and CTA1, which rely on both Pip2p-Oaf1p and Adr1p for their regulation, was reduced in adr1delta mutant cells. However, by ectopically restoring levels of Pip2p in adr1delta cells grown on oleic acid medium, transcription of both genes increased 2-fold compared with the control. This partial suppression of the adr1delta mutant phenotype was additionally manifested by moderate utilization of oleic acid. Hence, both the expression as well as the action of the two transcription factors, Adr1p and Pip2p-Oaf1p, are interconnected, which allows for an elaborate control of fatty acid-inducible genes.

In addition to containing an ORE, the promoters of several fatty acid-responsive genes also have a type 1 upstream activation sequence (UAS1) that acts as target for the Adr1p transcription factor (11). The UAS1 consensus sequence consists of CYCCR(A/T/G)N 4 -36 (T/A/C)YGGRG (12). Adr1p has been identified previously as a regulator of the glucose-repressible alcohol dehydrogenase gene ADH2 (13,14) and was additionally implicated in the growth of yeast cells on oleic acid medium (11,(15)(16)(17). Adr1p was subsequently shown to regulate directly the transcription of the ORE-dependent genes CTA1, SPS19, and POX1 (15,18,19).
Investigations into the expression of SPS19 and POX1 have exposed a strict adherence to regulation by both Adr1p and Pip2p-Oaf1p. The fact that Pox1p represents peroxisomal acyl-CoA oxidase, which is the first and the rate-limiting enzyme of the ␤-oxidation spiral, elucidated the reason for the requirement for Adr1p during the growth of yeast cells on fatty acids (Ref. 19, and references therein). The promoters of these two genes exhibit an ORE/UAS1 overlap, which is postulated to be significant for transcriptional up-regulation. Studies on this type of overlap in the CTA1 promoter revealed a synergy between the oleic acidspecific Pip2p-Oaf1p transcription factor and the more general sensor of less favored carbon sources, Adr1p (19).
Several mechanisms ensure that Adr1p initiates transcription of its target genes only in the presence of non-fermentable carbon sources. With respect to ORE-regulated genes, chromatin immunoprecipitations revealed that Adr1p binds to the UAS1 of CTA1 and POT1/FOX3 in vivo but does so only in the absence of glucose (20). Pip2p-Oaf1p is also tightly regulated by multiple layers of control. Although glucose inhibition is a feature common to both partners of this transcription factor, activation of the constitutively expressed Oaf1p depends on fatty acids (21), whereas PIP2 transcription is thought to be primarily regulated in response to its own abundance through an autoregulatory loop based on an ORE in the promoter of the PIP2 gene (21).
A potential ORE/UAS1 overlap has since been identified in the promoter of PIP2 (19), which raises the issue of the relationship between Adr1p and Pip2p-Oaf1p with respect to bulk gene transcription in cells grown on oleic acid. Here, the role of Adr1p in regulating PIP2 was investigated, and the results are discussed in terms of a transcriptional program that culminates in the induction of genes involved in fatty acid breakdown.

EXPERIMENTAL PROCEDURES
Strains, Plasmids, and Oligonucleotides-The Escherichia coli strain DH10B was used for all plasmid amplifications and isolations as well as for producing recombinant Adr1p-LacZ from plasmid pQC-A229Z (22). References for the S. cerevisiae strains used are listed in Table I, and  references for the plasmids and oligonucleotides are in Table II. Strains yAG1250 through yAG1253 were generated by transforming BJ1991pip2⌬ or the corresponding adr1⌬ cells expressing SPS19-lacZ from pAG454 (yAG485 and yAG1045) with either YCplac22 or pTPI1-PIP2HA3, respectively. For expressing PIP2 ectopically, cells were transformed with plasmid pTPI1-PIP2. Construction of GA1-8C-based strains MF14 and MF14adr1⌬ or BJ1991pip2⌬ and BJ1991pip2⌬oaf1⌬ has been described. The deletion of the PIP2 gene in strain MF14 was performed previously. Strain BJ1991adr1⌬ was a gift from Dr. H. F. Tabak's laboratory. Strains 500 -16 (adr1-1), 16ϫADR1, ADR1, and ADR1-5 c were kindly provided by the laboratory of Dr. E. T. Young.
Plasmid Constructions-Double-stranded oligonucleotides representing the Adr1p elements in the promoter of PIP2 were ligated to the matching SalI site in the plasmid vector pBluescript® SKϩ (Stratagene, La Jolla, CA), resulting in plasmid pLW82. The nucleotides of all cloned inserts were confirmed by automatic sequencing. Plasmid pHPR184 was created by exchanging the ClaI-SalI fragment of pHPR183 with that of pYIMPIP2-HA3, followed by an exchange of the NotI-demarcated hemagglutinin A epitope cassette with a similarly delineated cassette containing the sequence for 9ϫ Myc.
Media and Growth Conditions-Production of Adr1p-LacZ in E. coli cells was performed as described (22). For RNA isolations, cultures of the wild-type strain MF14 or the otherwise isogenic mutants devoid of Adr1p or Pip2p were shifted to YP medium (1% w/v yeast extract, 2% w/v peptone) containing the indicated carbon sources and grown for a further 16 h (1). For ␤-galactosidase measurements, cells were induced in oleic acid medium as follows. Late exponential phase cells from overnight pre-cultures consisting of YP medium and 5% (w/v) D-glucose were transferred to 100-ml conical flasks with 50 ml of YP medium containing both 0.2% (w/v) oleic acid (Merck) and 0.02% (w/v) Tween 80 (Sigma-Aldrich) that were adjusted to pH 7.0 with NaOH, 0.05% D-glucose, and 75 g/ml ampicillin to an absorbance of A 600 ϭ 0.2. Cultures were grown at 30°C with vigorous aeration for the periods indicated.
For immunoblotting, BJ1991 wild-type cells and otherwise isogenic adr1⌬ and pip2⌬oaf1⌬ mutants were grown in liquid YP medium containing 2% D-glucose and shifted to YP media supplemented with either 4% D-glucose, 2% (v/v) ethanol, or pH-adjusted 0.2% oleic acid and 0.02% Tween 80. Cultures were aerated vigorously for 8 h in the case of cells grown on glucose (to A 600 Ͻ 1.0) and for 20 h in the cases of growth on ethanol or oleic acid. For assays using culture drops, cells were grown in liquid rich glucose YPD medium (YP and 2% D-glucose) overnight to late exponential phase, diluted serially, and applied to solid YPD medium solidified with 2% (w/v) agar. For qualitative estimates of ␤-galactosidase expression levels on solid YPD medium, 50 l of 4% (w/v) 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal) in di-methyl formamide was added to the plates. Oleic acid plates contained a 0.67% (w/v) yeast nitrogen base with amino acids added, 0.1% yeast extract, 0.5% (w/v) potassium phosphate buffer at pH 6.0, 2% agar, autoclaved with 0.5% Tween 80 and 0.125% oleic acid. Plates were prepared by pouring a thin layer at 55°C. These were used to assess fatty acid breakdown by clear zone formation.
Enzyme Assays, Protein Extract Preparation, Immunoprecipitation, and Immunoblotting-␤-Galactosidase activities were assayed in soluble protein extracts prepared by breaking cells with glass beads (26) and expressed as nanomole of O-nitrophenyl-␤-D-galactopyranoside hydrolyzed per minute and milligram of protein. Catalase measurements were performed as described (27). Unless stated otherwise, values reported here are the average of three experiments Ϯ S.D. Whole-cell extracts were prepared according to a published protocol (28). Preparation of soluble protein extracts and immunoprecipitation of a Myctagged protein were performed essentially as described (29). Briefly, soluble protein extracts obtained from yeast cells (2 g wet weight) were incubated with Dynabeads goat anti-mouse IgG (Dynal Biotech, Oslo, Norway) coated with anti-Myc (9E10) antibodies. Following triple washing, the beads were boiled in 50 l of 1ϫ SDS sample buffer. The anti-Kar2p antibody was described previously (30). The polyclonal rabbit antibody against Cta1p was a gift from Dr. H. F. Tabak, and the monoclonal antibody 9E10 (c-myc) was obtained from Berkeley Antibody, Richmond, CA. Immunoreactive complexes were visualized using anti-rabbit or anti-mouse IgG-coupled horseradish peroxidase in combination with the ECL™ system from Amersham Biosciences.

UAS1 PIP2 Binds Adr1p in Vitro-
The PIP2 promoter contains a potential UAS1 that could be important for the expression of the corresponding gene (Fig. 1A). The nucleotide sequence of UAS1 PIP2 resembles that in the promoter of the GUT1 gene (37), which similarly consists of two single Adr1p-binding half-sites ordered as a tandem repeat (38). To examine whether UAS1 PIP2 could interact with Adr1p, EMSAs were performed on a fragment representing this promoter element as well as on the analogous sequence in the CTA1 promoter, UAS1 CTA1 . Containing a single copy of ADR1 23 ADR1-5 C7 Containing one copy of the ADR1-5 C allele 23 8) yAG485 4 Expressing SPS19-lacZ from pAG454 25, 39 9) yAG1045 6 Expressing SPS19-lacZ from pAG454 18 yAG1250 8 Harboring YCplac22 This study yAG1251 8 Carrying pTPI1-PIP2HA3 This study yAG1252 9 Harboring YCplac22 This study yAG1253 9 Carrying pTPI1-PIP2HA3 This study a The number in superscript following the strain's designation refers to its parental genotype, e.g. MF14 wild type 1 was derived from 1) GA1-8C.
Incubation of labeled UAS1 CTA1 with recombinant Adr1p-LacZ formed a high molecular weight complex (Fig. 1B, lane 2) that was specific to Adr1p elements, because the addition of excessive amounts of UAS1 CTA1 (Fig. 1B, lane 3) or the corresponding element in the ADH2 promoter (18), UAS1 ADH2 (Fig.  1B, lane 4), caused the intensity of the signal due to this complex to diminish. The results also showed that the intensity of the Adr1p signal could be reduced following the addition of excess DNA representing UAS1 PIP2 (Fig. 1B, lane 5), although this reduction was less pronounced compared with those associated with UAS1 CTA1 and UAS1 ADH2 . On the other hand, excess PIP2 ORE, which flanks the half-site of UAS1 PIP2 (Fig.  1A), did not weaken the signal's intensity (Fig. 1B, lane 6). UAS1 PIP2 could also be shown to be targeted directly by Adr1p (Fig. 1B, lanes 7-12). The top two bands seen in Fig. 1B, lane 2 (marked with arrows and asterisks) were reasoned to not represent genuine complexes because they could be competed by a nonspecific competitor DNA that failed to reduce the signal of the predominant complex (Fig. 1B, lane 6). Hence, EMSAs performed using a recombinant Adr1p-LacZ fusion protein revealed that UAS1 PIP2 was able to recruit the transcription factor in vitro.
Adr1p Influences PIP2 Transcription on Oleic Acid-Northern analysis was performed to determine whether Adr1p actually affected PIP2 transcription ( Fig. 2A). The results demonstrated that levels of PIP2 transcripts were higher in wild-type cells grown on oleic acid medium compared with the situation on ethanol ( Fig. 2A; top pair of panels), albeit the PIP2 signal was weaker compared with those corresponding to the two peroxisomal enzyme genes, POT1/FOX3 and CTA1. Nevertheless, the Northern blot could expose a reduction in the transcription of PIP2 by the adr1⌬ mutant under oleic acid medium conditions ( Fig. 2A, top right hand panel) even when considering the moderately unequal loading seen using ACT1 (encoding actin), which served as a control gene that is not affected by Adr1p ( Fig. 2A, bottom pair of panels). Hence, the reduced PIP2 signal resembled the situation with the two Adr1p-dependent positive control genes (11,17) seen in the middle panels.
Regulation of PIP2 was also investigated in response to excess levels of Adr1p (Fig. 2B). Multiple ADR1 copies have been reported previously to result in increased transcription of certain genes whose promoters contain a UAS1, including ADH2, CTA1, and SPS19 (15,18,24). Northern analysis was performed on immobilized RNA extracted from haploid 16ϫADR1 cells containing multiple copies of the ADR1 gene (24) as well as from isogenic ADR1 cells containing a single wild-type ADR1 gene copy, adr1-1 cells with an adr1-1 mutation, or ADR1-5 C cells expressing a partially constitutive ADR1 (23). The results showed that PIP2 transcription was elevated in oleic acid-grown cells harboring 16 ADR1 copies compared with the other cell types (Fig. 2B). In addition, a reduction in transcript accumulation could also be seen in the adr1-1 mutants (Fig. 2B), which was in agreement with the corresponding adr1⌬ lane in Fig. 2A. Signal intensity notwithstanding, the RNA profile of PIP2 under these oleic acid-medium conditions was essentially identical to that of POT1/ FOX3 and CTA1 (16,17). Hence, PIP2 transcription appeared to rely on Adr1p.
To confirm the dependence of PIP2 on Adr1p, transcription was studied using a PIP2-lacZ reporter construct (1). Levels of ␤-galactosidase activity were analyzed at various time points following the shifting of cells from low glucose-to oleic acidcontaining media. The results demonstrated that PIP2-lacZ expression in the adr1⌬ strain reached ϳ50% of the wild-type level at all time points measured (Table III). Only minimal ␤-galactosidase activity was detected in the corresponding pip2⌬ mutant.
The influence of Adr1p on the transcription of PIP2 was also investigated under non-inducing conditions using the aforementioned lacZ reporter gene (1). Serial 10-fold dilutions of wild-type and adr1⌬ cells harboring the PIP2-lacZ reporter gene were applied to YPD plates. The chromogenic substrate X-Gal was added to the surface of the medium to assess the expression of the reporter construct. As expected, Adr1p was not critical for the onset of growth on rich-glucose medium, as verified by comparing colony sizes (Fig. 3). The results demonstrated that expression of ␤-galactosidase was reduced in the mutant strain because, unlike the wild-type strain, which turned light blue, mutant colonies remained white (Fig. 3). This experiment also revealed the importance of Adr1p for TRP1-marked centromeric vector 54 This study PIP2 transcription in the absence of a fatty acid substrate, i.e. in cells sensing the depletion of glucose. Adr1p Is Critical for Pip2p Expression on Oleic Acid-To further substantiate the apparent requirement for Adr1p to express Pip2p, protein levels were monitored using a plasmidborne gene that encodes a Myc-tagged Pip2p expressed from the native promoter. This tag did not interfere with the function of Pip2p, because Pip2p-Myc was as efficient as the untagged protein in complementing the growth phenotype of a pip2⌬ strain (not shown). Immunoprecipitation was performed on soluble protein extracts from wild-type, adr1⌬, or pip2⌬oaf1⌬ cells harboring this centromeric plasmid. The results demonstrated that, under oleic acid-medium conditions, Adr1p was important for obtaining wild-type levels of Pip2p (Fig. 4, top panel). The pattern of Cta1p signals (Fig. 4, middle panel) concurred with those of previous experiments using immunoblotting as well as catalase assays, which demonstrated a higher level of dependence on Pip2p-Oaf1p than on Adr1p for expression under these conditions (19). Kar2p served as an internal loading control (Fig. 4, bottom lane), whereas the control lane on the far left of Fig. 4 consisted of an extract from a wild-type strain expressing an untagged Pip2p.
Pip2p-Oaf1p Complexes Formed on OREs Are Less Abundant in Cells Devoid of Adr1p-The above data indicated that, in the absence of Adr1p, Pip2p expression was compromised. It followed, therefore, that the amount of Pip2p-Oaf1p binding to target OREs might also be affected in the adr1⌬ mutant. Hence, EMSA was performed on an ORE obtained from the promoter of the POT1/FOX3 gene, which, unlike the corresponding element in the PIP2 promoter, does not overlap its neighboring UAS1 (Fig. 5A). Formation of the Pip2p-Oaf1p complex was monitored using soluble protein extracts derived from BJ1991 wild-type, adr1⌬, or pip2⌬oaf1⌬ strains that were incubated with the labeled ORE fragment.
The results demonstrated that the signal due to the Pip2p-Oaf1p complex was appropriately missing from the lane containing the control pip2⌬oaf1⌬ extract (Fig. 5B, lane 4). In addition, this signal, using extracts obtained from oleic acidgrown cells lacking Adr1p (lane 3), appeared to be less intense compared with that using wild-type cells extracts (lane 2). This situation seemed to be exacerbated using cells grown to a late exponential phase on glucose or ethanol (Fig. 5C, lanes 3 and  6). Hence, in accordance with abnormally low levels of Pip2p in FIG. 1. Adr1p binds UAS1 PIP2 in vitro. A, arrangement of an ORE and a potential UAS1 in the PIP2 promoter. The terminal 3Ј G in the PIP2 sequence is situated 121 bp 5Ј to the ATG start site. B, EMSA of UAS1 PIP2 . Labeled UAS1 CTA1 or UAS1 PIP2 DNA (indicated below the gels) was incubated with recombinant Adr1p-LacZ, and free and bound DNA fragments were resolved on a 5% (w/v) polyacrylamide gel. Arrows with asterisks indicate unidentified complexes. The effect of adding a 25-fold excess of unlabeled UAS1 PIP2 on the signal due to the Adr1pdependent complex was compared with that of UAS1 CTA1 and UAS1 ADH2 as well as the nonspecific competitor PIP2 ORE. Competitor DNA representing elements in the promoters of CTA1, ADH2, and PIP2 were generated by annealing oligonucleotide pairs CTA1ADR1-F/R, ADH2ADR1-F/R, PIP2ADR1-F/R, and PIP2ORE1/2 (Table II).
FIG. 2. The effect of manipulating the number of ADR1 gene copies on PIP2 transcription. RNA was extracted from cells that had been induced on oleic acid and immobilized on filters that were probed with labeled DNA fragments containing the genes PIP2, POT1/FOX3, CTA1, or ACT1. A, comparison of transcript levels between MF14derived adr1⌬ cells and the corresponding wild-type or pip2⌬ strains. B, influence of 16 copies of the wild-type ADR1 gene or of the ADR1-5 C mutant allele on the transcriptional activation of PIP2. The strains used were isogenic to strain 500 -16; ADR1 contains a single ADR1 copy, adr1-1 is mutated in ADR1, 16ϫADR1 has 16 copies of ADR1, and ADR1-5 c harbors a constitutively active ADR1 allele. oleic acid-grown adr1⌬ cells (Fig. 4), the amount of transcription factor that could bind OREs in vitro was appropriately reduced.
Uncoupling PIP2 Transcription from Adr1p Control Partially Suppresses the adr1⌬ Mutant Phenotype on Oleic Acid-Overexpression in wild-type cells of just Pip2p (without also overproducing Oaf1p) does not lead to an increase in ORE-dependent gene transcription (8,21). To determine the effect of uncoupling PIP2 transcription from the control of Adr1p on the expression of SPS19 and CTA1, PIP2 was placed behind the constitutive TPI1 promoter in plasmid pTPI1-PIP2HA3 (Table  II) that was used to transform the adr1⌬ or pip2⌬ mutant cells that additionally harbored an SPS19-LacZ reporter gene.
The result of propagating cells on oleic acid medium for 18 h demonstrated that SPS19-lacZ reporter gene activities were 2.5-fold greater in adr1⌬ mutant cells ectopically expressing Pip2p compared with those producing Pip2p exclusively from the native locus (Table IV). This effect was also manifested on catalase activity, which had increased 2-fold in the former cell type. Complementation with the TPI1-driven PIP2 was also monitored in a pip2⌬ mutant, and, as anticipated, this demonstrated a 28-fold increase in SPS19-lacZ expression as well as a 3.6-fold increase in catalase activity (Table IV). Hence, it was reasoned that levels of SPS19-lacZ and CTA1 expression were elevated in the adr1⌬ mutant because TPI1-PIP2 gave rise to sufficient amounts of Pip2p. However, due to Adr1p also acting directly at the SPS19 and CTA1 promoters, the observed effects were only partial.
As mentioned previously, cells devoid of Adr1p fail to degrade fatty acids (15) because they do not express Pox1p, which is necessary for ␤-oxidation to occur (19). To examine whether ectopic expression of Pip2p could also re-establish fatty acid breakdown in adr1⌬ cells, the formation of clear zones was monitored on solid oleic acid medium. This medium also contained Tween 80, which acted to disperse the fatty acids but was also a poor carbon source, and, therefore, mutant cells could grow to some extent on such plates, but the transparent zones in the opaque medium around regions of cell growth indicated utilization of the fatty acid substrate (39). Three independent adr1⌬ mutant strains expressing a functional  3. Expression of a PIP2-lacZ reporter gene on glucose medium. Episomal plasmid pPIP2LacZ (Table II) was introduced into BJ1991 wild-type cells or otherwise isogenic mutants devoid of Adr1p. Serially diluted cultures were applied to solid rich glucose YPD medium to which was added 50 l of 4% (w/v) X-Gal. Ura minus medium was used to verify the presence of the URA3-marked plasmid loaded with the gene fusion, and Leu minus medium was used to corroborate the adr1⌬::LEU2 disruption.
FIG. 4. Immunoprecipitation of Pip2p-Myc. Equal amounts of soluble protein extracts prepared from oleic acid-induced BJ1991 wildtype, adr1⌬, and pip2⌬oaf1⌬ cells were analyzed for the presence of Pip2p-Myc, Cta1p, and Kar2p by immunoblotting. Prior to the determination of the amounts of Pip2p-Myc, samples were subjected to immunoprecipitation with anti-Myc antibodies. All strains used were transformed with pHPR184 except the wild-type control strain, which expressed an untagged Pip2p from plasmid pHPR183.

FIG. 5. EMSA of Pip2p-Oaf1p/ORE complexes in cells lacking
Adr1p. A, the arrangement of the UAS1 and ORE elements in the POT1/FOX3 promoter. The terminal 3Ј G is 118 bp 5Ј to the ATG start site. B and C, labeled POT1/FOX3 ORE was mixed with soluble protein extracts from BJ1991 wild-type, adr1⌬, or pip2⌬oaf1⌬ strains grown on the indicated carbon sources and resolved on a 5% (w/v) polyacrylamide gel. The DNA representing POT1/FOX3 ORE was generated by annealing the oligonucleotide pair FOX3ORE1/2 (Table II).
Pip2p from a pTPI1-PIPHA3 construct were compared with a mutant harboring the YCplac22 plasmid vector for formation of clear zones. As seen in Fig. 6A, all three transformants overexpressing Pip2p could give rise to narrow zones of clearing that were absent from the region surrounding the vector-containing adr1⌬ strain. Expression of the construct in the pip2⌬ mutant gave rise to more pronounced clear zones in the medium, as could be expected from self-complementation (Fig.  6B). Hence, restoration of Pip2p levels in the adr1⌬ mutant using the TPI1-driven construct probably caused POX1 and possibly other Adr1p-regulated genes to be expressed in amounts sufficient for moderate levels of fatty acid degradation.

DISCUSSION
Here we have shown that the inducible transcription factor Pip2p-Oaf1p, which is specific for up-regulating genes involved in fatty acid breakdown, is directly governed by the more general sensor of less-favored carbon sources, Adr1p. This novel regulatory route is postulated to proceed through the binding of Adr1p to the PIP2 promoter at a deviant UAS1. UAS1 PIP2 is only the second example, after UAS1 GUT1 (38), of a tandem array of UAS1 half-sites capable of binding Adr1p in vitro. Consistent with Adr1p binding to the PIP2 promoter, the respective levels of PIP2 transcript, gene product, and in vitro binding potential to OREs were found to be reduced in the adr1⌬ mutant compared with the situation in wild-type cells grown under oleic acid medium conditions. This is the first demonstration of an Adr1p interaction preceding the step that depends on Pip2p-Oaf1p for oleic acid induction and peroxisome proliferation.
The present data allowed us to conclude that Adr1p-dependent control over Pip2p affects ORE-dependent gene regulation. EMSA performed on cells lacking Adr1p demonstrated that such cells gave rise to a reduced Pip2p-Oaf1p signal. This indicated that Adr1p influenced the amount of the transcription factor being expressed, or alternatively, it modulated the factor's binding activity, e.g. by loss of synergy. Because the fragment used in these EMSAs consisted of an ORE in isolation, and immunoprecipitation revealed that less Pip2p was produced in cells devoid of Adr1p, we argue that the reason for the importance of Adr1p lies most probably in maintaining normal levels of Pip2p and not in increasing the binding efficiency of the transcription factor that subsequently forms with Oaf1p.
Adr1p-independent expression of PIP2 in adr1⌬ cells was sufficient to partially release these mutants from their oleic acid induction deficiency. Analysis of the expression of two genes encoding peroxisomal enzymes, Cta1p and Sps19p, showed that, as a result of the uncoupling of PIP2 from Adr1p control, their respective levels increased 2-fold, reaching between about a third (Sps19p-LacZ) to half (Cta1p) of the poten-tial of the wild-type expression levels, as measured by the self-complemented BJ1991pip2⌬ strain. Moreover, the capability of this strain to degrade fatty acids improved significantly. Hence, it was reasoned that the expression of additional OREregulated genes that are critical for ␤-oxidation, e.g. POX1, was also likely to be positively affected by such uncoupling. The extent of the transcriptional cascade operating through Adr1p and Pip2p-Oaf1p is very significant for metabolism because, by regulating Pip2p-Oaf1p, Adr1p could influence at least 24 additional genes involved in diverse functions, including those encoding peroxisomal proteins but also a mitochondrial protein as well as proteins of unknown function (2,40,41).
Cells shifted directly from fresh, rich glucose medium to that  supplemented with fatty acids as the sole carbon source without first undergoing derepression fail to achieve the metabolic switch in a timely manner and do not undergo efficient oleic acid induction. However, the molecular mechanism ensuring the integrity of this link between the two phases of derepression and induction has remained hitherto unclear. Adr1p might turn out to be important for maintaining normal levels of Pip2p in non-induced cells in which Pip2p-Oaf1p is transcriptionally inactive. For example, a PIP2-lacZ reporter gene failed to turn blue in cells devoid of Adr1p that were grown on glucose plates containing X-gal. In addition, adr1⌬ mutants grown to late exponential phase on glucose or ethanol media gave rise to weaker signals representing Pip2p-Oaf1p binding to OREs than did wild-type cells. Although non-induced wild-type cells contain only trace amounts of Pip2p, it is attractive to speculate that these quantities nevertheless support a faster response in derepressed cells whose growth medium was supplemented with oleic acid. There exists some evidence that Adr1p is additionally dependent on a signal emerging from the mitochondria. A temperaturesensitive mutation in the RML2 gene encoding a mitochondrial ribosomal protein disables the induction of peroxisomal catalase (42). Interestingly, this deficiency could be alleviated by overexpressing Adr1p (42). However, the fact that transcription of Adr1pdependent POX1 and POT1/FOX3 was not affected by this mutation argued against a communication pathway appearing to course from the mitochondria to the peroxisomes via the nucleus. Nevertheless, such a retrograde pathway is clearly at work in respiratory-deficient [rho 0 ] cells in which several ORE-regulated genes are induced despite the absence of fatty acids from the growth medium (43). A subsequent microarray-based study using cells devoid of Adr1p underscored the fact that, of the 43 genes that are responsive to changes in mitochondrial function, no less than 17 are regulated by Adr1p (44). Hence, it would be interesting to determine the extent of the sharing of transcription factors between the metabolic events triggered by oleic acid and respiratory deficiency. It is worth noting that, in such Adr1p-less cells, at least two ORE-regulated genes, DCI1 (2,45,46) and IDP3 (47,48), are down-regulated (44) despite lacking obvious UAS1s in their promoters (18). It is not entirely impossible that this is due to a regulatory circuitry based on Adr1p controlling Pip2p abundance at their OREs.
In addition to acting on PIP2, Adr1p also regulates a number of ORE-regulated genes directly by binding to UAS1 elements that are situated in the vicinity of OREs. Experimental evidence for such a dual control exercised by Pip2p-Oaf1p and Adr1p was demonstrated previously for the promoters of CTA1, SPS19, POX1, and POT1/FOX3 (15,18,19,20). This type of combinatorial control seems to be a more general feature of Adr1p, as it also acts in concert with the transcription factor Cat8p at promoters of genes encoding gluconeogenic and glyoxylate-cycle enzymes so as to allow their expression in glucose-exhausted media (49 -51). Our demonstration of PIP2 being regulated by Adr1p extends this concept of combinatorial control in that Adr1p acts not only together with Pip2p-Oaf1p under fatty acid medium conditions but also in the step preceding the oleic acid induction cascade.