The REG1 Gene Product Is Required for Repression of INO1 and Other Inositol-Sensitive Upstream Activating Sequence-Containing Genes of Yeast

Corresponding author: Susan A. Henry, Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave., Pittsburgh, PA 15213., sh4b{at}andrew.cmu.edu (E-mail)

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

A search was conducted for suppressors of the inositol auxotrophic phenotype of the ino4-8 mutant of yeast. The ino4-8 mutation is a single base pair change that results in substitution of lysine for glutamic acid at position 79 in the bHLH domain of the yeast regulatory protein, Ino4p. Ino4p dimerizes with a second bHLH protein, Ino2p, to form a complex that binds to the promoter of the INO1 gene, activating transcription. Of 31 recessive suppressors of ino4-8 isolated, 29 proved to be alleles of a single locus, identified as REG1, which encodes a regulatory subunit of a protein phosphatase involved in the glucose response pathway. The suppressor mutation, sia1-1, identified as an allele of REG1, caused constitutive INO1 expression and was capable of suppressing the inositol auxotrophy of a second ino4 missense mutant, ino4-26, as well as ino2-419, a missense mutation of INO2. The suppressors analyzed were unable to suppress ino2 and ino4 null mutations, but the reg1 deletion mutation could suppress ino4-8. A deletion mutation in the OPI1 negative regulator was incapable of suppressing ino4-8. The relative roles of the OPI1 and REG1 gene products in control of INO1 expression are discussed.

IN the yeast Saccharomyces cerevisiae the products of the INO2 and INO4 regulatory genes are responsible for the transcriptional activation of a large number of structural genes encoding phospholipid biosynthetic enzymes. The structural genes subject to this regulation are repressed in response to the phospholipid precursors, inositol and choline. These enzymes are maximally derepressed in the absence of inositol and choline, partially repressed in the presence of inositol alone, and fully repressed when both inositol and choline are added to the growth medium (for review, see PALTAUF et al. 1992 ; HENRY and PATTON-VOGT 1998 ).

The INO2 and INO4 gene products both contain a basic helix-loop-helix (bHLH) domain (HOSHIZAKI et al. 1990 ; NIKOLOFF et al. 1992 ), which is characteristic of a family of proteins involved in transcriptional regulation and cell-type determination. The bHLH domain has been shown to be responsible for protein dimerization and DNA binding in a number of transcriptional regulatory proteins in mammalian cells. These include the mammalian oncogene cMyc (MURRE et al. 1989 ; DAVIS et al. 1990 ; VORONOVA and BALTIMORE 1990 ) and the upstream stimulatory factors (USF) that bind to the insulin response sequence of the fatty acid synthase (FAS) promoter (WANG and SUL 1995 , WANG and SUL 1997 ). A 10-bp repeated element (consensus sequence: 5' CATGTGAAAT 3'), first found in the promoter of INO1 gene (HIRSCH 1987 ), has been identified upstream of all the structural genes that are regulated in response to inositol and choline (PALTAUF et al. 1992 ; BACHHAWAT et al. 1995 ). This repeated element, the inositol-sensitive upstream activating sequence (UAS_INO), contains within it the CANNTG motif (i.e., 5' CATGTG 3') that has been shown to be the consensus-binding site for bHLH proteins (LASSAR et al. 1989 ; BLACKWELL and WEINTRAUB 1990 ; FISHER et al. 1991 ). Indeed, the UAS_INO core sequence, CATGTG, is identical to the E-box motif reported by WANG and SUL 1997 as the sequence required for USF binding and insulin regulation in the mammalian FAS promoter.

When a DNA fragment from the INO1 promoter that includes two copies of the 10-bp UAS_INO element is incubated with cell extracts prepared from wild-type cells, a protein-DNA complex is formed. This complex is absent when the extracts are derived from ino2 or ino4 mutant strains (LOPES and HENRY 1991 ). The INO2 and INO4 gene products have been demonstrated to bind directly to the UAS_INO site on the INO1 promoter (AMBROZIAK and HENRY 1994 ; NIKOLOFF and HENRY 1994 ; SCHWANK et al. 1995 ). The ino2 and ino4 mutants were originally isolated on the basis of an inositol auxotrophic phenotype (CULBERTSON and HENRY 1975 ; DONAHUE and HENRY 1981A ), which is due to their inability to derepress the INO1 gene, encoding inositol-1-phosphate synthase (DONAHUE and HENRY 1981B ; HIRSCH and HENRY 1986 ). Of 12 ino2 and ino4 loss-of-function mutants examined by DNA sequencing, 11 have mutations in the basic helix-loop-helix region (NIKOLOFF 1993 ; AMBROZIAK 1994 ; NIKOLOFF and HENRY 1994 ), suggesting the importance of the bHLH domain in the functioning of Ino2p and Ino4p. The ino4-8 allele contains a single amino acid change from glutamic acid to lysine in the loop region of the bHLH domain at the amino acid in position 79 in Ino4p, while ino4-26 has a single amino acid change from threonine to isoleucine at amino acid 42 in the basic region of the bHLH domain (AMBROZIAK 1994 ). The ino2-419 mutation contains lysine in place of arginine in the loop region at amino acid 273 (NIKOLOFF and HENRY 1994 ).

To identify additional factors involved in regulation of phospholipid biosynthesis and to acquire more information about the regulatory network, we conducted a screen for suppressors of inositol auxotrophy in an ino4-8 strain. We report here the isolation of suppressor mutations which proved to be allelic to the REG1 locus. The mutations identified in this screen also suppressed ino4-26 and ino2-419. However, these suppressor mutations do not suppress null alleles of either INO4 or INO2, while a reg1 deletion mutation can suppress the ino4-8 mutation. reg1 mutants have been identified in numerous previous genetic screens (MATSUMOTO et al. 1983 ; NEIGEBORN and CARLSON 1987 ; TUNG et al. 1992 ; NAIK et al. 1997 ) designed to identify regulatory loci controlling such diverse functions as glucose repression, RNA processing, and proteinase B expression. reg1 mutants also exhibit growth and glycogen storage defects (FREDERICK and TATCHELL 1996 ). Here we demonstrate that the REG1 gene product is also involved in the control of the coordinately regulated genes of phospholipid biosynthesis.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, medium, and growth conditions:
The genotypes and sources of strains used in this study are listed in Table 1. The following growth media were used: YEPD (1% yeast extract, 2% Bactopeptone, 2% glucose); complete synthetic medium [2% glucose, 0.67% Difco Yeast Nitrogen Base without vitamins, vitamin mix, supplements (amino acids, uracil, and adenine); GREENBERG et al. 1982A , GREENBERG et al. 1982B ], minimal synthetic medium (same as complete synthetic medium but lacking supplements); drop-out medium (same as complete synthetic medium but lacking a single supplement); sporulation medium (0.1% yeast extract, 0.05% glucose, 1% KAc). Solid medium (plates) contained 2.5% agar in addition to the above ingredients. Inositol-free (I^-) plates contained complete synthetic medium with no inositol supplement; inositol-supplemented (I⁺) plates contained complete synthetic medium with 75 µM inositol. For liquid growth studies, derepressing medium (D) was defined as complete synthetic medium containing 10 µM inositol. This level of inositol has been shown to allow partial derepression of the INO1 gene, while still supporting growth of inositol auxotrophs such as ino4 mutants (HIRSCH and HENRY 1986 ). Repressing (R) medium contained 75 µM inositol. Yeast cultures were grown at 30°.

To test for sensitivity to 2-deoxyglucose (2-DG), yeast strains were incubated on plates containing 2% peptone, 1% yeast extract, 2% sucrose, 200 µg of 2-deoxy-D-glucose and 1 µg/ml antimycin A to stimulate anaerobic conditions. Control plates lacked 2-DG.

Mutagenesis and isolation of suppressor mutants:
Strains SH405 (MAT ino4-8) and SH406 (MATa ino4-8) were mutagenized with ethyl methanesulfonate (EMS) as previously described (CULBERTSON and HENRY 1975 ) and screened for cells that were able to grow in the absence of inositol (I^- plates). Cells were harvested at stationary phase from 5-ml cultures grown on YEPD medium, washed twice with 10 ml of phosphate-glucose solution (0.2 M Na₂HPO₄, 2% glucose, pH 8.0) and resuspended in 9.7 ml of phosphate-glucose buffer. Cells were mutagenized by adding 0.3 ml of EMS and incubating at 30° for 30 min for strain SH405 (31% survival) and 20 min for strain SH406 (37% survival). The EMS was inactivated as previously described (CULBERTSON and HENRY 1975 ) and appropriate dilutions were made into liquid YEPD medium and spread onto YEPD plates to achieve a density of about 200 viable cells per plate. Approximately 40,000 colonies were screened by replicating to I^- and I⁺ plates.

Yeast genetic manipulations:
Genetic techniques such as mating, sporulation, and tetrad dissection were carried out using standard methods (SHERMAN et al. 1978 ). The potential suppressors were colony-purified and the growth phenotype on I^- plates was reconfirmed. Candidates were then crossed to the ino4-8 strain of the opposite mating type (i.e., SH405 or SH406), and diploids were scored on I^- plates to determine whether the suppressor mutation was dominant or recessive. Diploids having the parental phenotype of inositol auxotrophy indicated the presence of a recessive suppressor while diploids having the suppressor phenotype growth on I^- medium indicated a dominant suppressor.

The sets of recessive suppressors isolated in strains of opposite mating types were crossed with each other, and the resulting diploids were tested on I^- plates to estimate the number of complementation groups. Representatives of each complementation group were crossed to the ino4-8 parental strain and the diploids were sporulated and the tetrads, dissected. In many cases, this first cross yielded low sporulation efficiency and/or poor spore viability. A second backcross to the ino4-8 parental strain, using spore colonies retrieved from the dissection of the first cross of the primary suppressor-bearing strains to an ino4-8 strain, often resulted in higher sporulation efficiency and better spore viability. Tetrads from the second cross were scored on I^- plates to determine the segregation of the suppressor phenotype.

Test for Opi^- phenotype:
The test for the inositol excretion (overproduction of inositol, Opi^-) phenotype has been described in detail elsewhere (GREENBERG et al. 1982B ; SWEDE et al. 1992 ). Briefly, strains to be tested were spotted or replicated onto I^- medium, allowed to grow for 2 days, and then sprayed with an indicator strain, which is an inositol auxotroph. Growth of a halo of the indicator strain around a colony indicated inositol excretion (Opi^- phenotype). An opi1 mutant strain (SH308) and wild type (W303) were used as positive (Opi^-) and negative (Opi⁺) controls, respectively.

Construction of reg1::URA3:
An ~4.2-kb EcoRI-XbaI fragment from plasmid pUCsrn1::URA3, kindly provided by A. K. Hopper (TUNG et al. 1992 ), was transformed into the diploid strain SH701 and allowed to integrate into the yeast genome by homologous recombination. Southern analysis of four independent Ura⁺ transformants confirmed the integration of this fragment into the REG1 locus. Sporulation and subsequent tetrad analysis of one of these transformants confirmed 2:2 segregation for Ura⁺:Ura^- and cosegregation of reg1 phenotypes with the Ura⁺ phenotype. Strains harboring the disruption allele were found to be viable but grew more slowly than their isogenic wild-type counterparts, as previously reported (TUNG et al. 1992 ; FREDERICK and TATCHELL 1996 ). Spore colonies from this cross were also tested for the Opi^- phenotype, as described above, and all reg1 segregants were found to be Opi^-.

ß-Galactosidase assays:
A single copy of an INO1-lacZ gene fusion stably integrated at the URA3 locus was introduced into strains of interest (LOPES and HENRY 1991 ). To assay ß-galactosidase, cells containing the gene fusion were grown under partially derepressing (with 10 µM inositol) or repressing (with 75 µM inositol) conditions (HIRSCH and HENRY 1986 ). The use of completely inositol-free medium for the derepressing condition was not possible because the parental ino4-8 strains cannot grow in the absence of inositol. Cells were harvested at midlogarithmic phase. Cell extracts were prepared and ß-galactosidase assays were performed as described by LOPES and HENRY 1991 , except that reaction aliquots were removed at 5, 10, and 15 min. ß-Galactosidase units are defined as (OD₄₂₀/min/mg total protein) x 1000.

RNA isolation and analysis:
For RNA isolation, yeast cells were grown in repressing (75 µM inositol) and derepressing (10 µM inositol) medium to midlog phase. Cells from a 10-ml culture were harvested and washed once with 5 ml of RE buffer (100 mM LiCl, 100 mM Tris-HCl, pH 7.5, 1 mM EDTA) and suspended in 0.4 ml RE buffer. This solution was transferred to a fresh tube containing ~2/3 volume of ice-cold glass beads and vortexed four times, 1 min each, being placed on ice between pulses. Proteins were removed by sequential extractions with 0.3 ml equilibrated phenol, 0.3 ml phenol/CHCl₃/isoamyl alcohol (50:49:1), and 0.3 ml CHCl₃. RNA was precipitated at -20° overnight and suspended in 0.1 ml diethyl pyrocarbonate-treated water.

Northern analysis was done by electrophoresis of 20-µg samples of RNA loaded onto 1% agarose-6% formaldehyde/1x MOPS gels and run overnight in 1x MOPS. Gels were electroblotted to Nytran Plus membrane in 1x TAE at 4° for 30 min at 10 V, followed by an additional 1 hr 30 min at 40 V.

Prehybridization and hybridization conditions were as described in HIRSCH and HENRY 1986 . RNA probes for hybridization were enzymatically synthesized from the following plasmids described in HUDAK et al. 1994 : pAB309 (TCM1); pMH203 (OPI3); pJH301 (INO1); pAB103 (CHO1); pTG109 (CHO2). The TCM1 RNA, whose expression is unaffected by the availability of inositol, was used as a loading control. The results were visualized by autoradiography and quantified by a FUJIX BAS2000 phosphoimager using MacBAS version 2.4 software.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of mutants that suppress the inositol auxotrophy of the ino4-8 mutation:
From the original screening of 40,000 colonies, 200 suppressor candidates were isolated after two rounds of testing on I^- plates as described in MATERIALS AND METHODS. Strains with respiratory deficient phenotypes were eliminated from the collection. Of the 200 original putative suppressor-bearing strains, ~70% appeared to be due to recessive mutations while ~30% appeared to harbor a dominant mutation. The mutations conferring the strongest suppressor phenotypes (showing growth within two days after replicating to I^- plates) were all dominant. Preliminary genetic analysis suggested that most of these strains carried primary reversions of the ino4-8 allele. The remaining recessive suppressors were subjected to further analysis.

Complementation and segregation analysis:
The strains carrying recessive suppressors were classified according to growth on I^- plates. Approximately 31 putative suppressor-bearing strains with stronger growth phenotypes (showing definite growth within 3–4 days after replicating to I^- plates) were selected for further analysis. Because the mutants had been isolated in two strains of opposite mating type, it was possible to conduct an initial complementation analysis crossing the mutant collections of opposite mating types against each other. The recessive mutants were found to fall into three complementation groups with one complementation group (sia1) containing 29 members exhibiting the stronger growth phenotypes. The other two groups had only a single representative each and only one of these (sia2) was subjected to further characterization. The suppressor mutations representing the sia1 and sia2 (for suppressor of inositol auxotrophy) complementation groups were subjected to further genetic analysis.

Two suppressor-bearing strains from the larger (sia1) complementation group and the single sia2 strain were backcrossed to the ino4-8 parent strain of the opposite mating type (i.e., SH405 or SH406; Table 1). Twenty-three tetrads with four surviving spores were recovered from the crosses of the two representatives from the sia1 complementation group and all exhibited 2:2 segregation of the growth phenotype on I^- plates. Nineteen tetrads with four surviving spores were tested from crosses involving suppressors of the single sia2 representative, and all showed 2:2 segregation for growth on I^- plates, indicative of a mutation in a single gene.

sia1 and sia2 are not linked to the INO4 locus or to each other:
Strains carrying the sia1-1 or the sia2-1 mutation (i.e., ino4-8 sia1-1 or ino4-8 sia2-1) were crossed to wild-type strains SH155 or SH224 (see Table 1 for full genotypes). In 34 tetrads with four surviving spores recovered from crosses of an ino4-8 sia1-1 strain to wild type, 22 showed 3⁺:1^- segregation for inositol auxotrophy (i.e., Ino⁺:Ino^-), 10 exhibited 2⁺:2^- segregation and 2 segregated 4⁺:0^- (Table 2A). In the 17 tetrads with four surviving spores from crosses involving ino4-8 sia2-1 strains to wild-type strains, 14 tetrads segregated 3⁺:1^- for inositol auxotrophy, 3 segregated 2⁺:2^-, and no 4⁺:0^- tetrads were recovered. In these crosses, the 4⁺:0^- segregation represents the parental ditype category, 3⁺:1^- reflects a tetratype ascus, and 2⁺:2^- segregation is expected for nonparental ditype asci. However, the proportion of 4⁺:0^- asci was lower than the expected ratio of 1 in 6. Explanations include the possibility that the suppressor phenotype might not be fully penetrant (i.e., some ino4-8 sia1-1 or ino4-8 sia2 segregants may score as Ino^-), or spores of the ino4-8 sia1-1 and ino4 sia2-1 genotypes may not germinate as well as other genotypes. Since only tetrads with four surviving spores were analyzed, a lower viability for the ino4-8 sia1-1 (or sia2-1) genotype would lead to a reduced percentage of 4⁺:0^- tetrads among the tetrads analyzed. Consistent with either of these explanations, an excess of 2⁺:2^- tetrads was observed, compared to 4⁺:0^- tetrads, in reciprocal crosses (i.e., crosses of strains of the ino4-8 SIA1 or ino4-8 SIA2 genotypes to strains of the INO4 sia1-1 or INO4 sia2-1 genotypes, respectively; data not shown). However, the excess of 2⁺:2^- vs. 4⁺:0^- tetrads and the high proportion of 3⁺:1^- and 2⁺:2^- asci shown in Table 2A indicate that it is unlikely that either sia1 or the sia2 is closely linked to the INO4 gene.

The sia1-bearing strains were also crossed to sia2 strains (Table 2A). Although a relatively small number of tetrads with four spores surviving were recovered (13 in two crosses), the high proportion of 3⁺:1^- and 2⁺:2^- tetrads indicated that sia1 and sia2 mutations are not closely linked. No unusual growth phenotype was observed for sia1 sia2 strains, which were viable and resembled the sia1 and sia2 single mutants in ability to suppress ino4-8. In contrast, a cross involving two sia1 alleles (i.e., ino4-8 sia1-1 with ino4-8 sia1-2) produced 21 4⁺:0^- tetrads and only 1 tetrad exhibiting a 3⁺:1^- segregation pattern (which could be due to reversion of ino4-8 or a gene conversion; Table 2A).

Both the sia1-1 and the sia2-1 mutations can suppress a second ino4 missense allele, but neither can suppress ino4:
Strains SH407 and SH408 carrying the ino4-26 allele, a missense mutation in the basic region of the bHLH domain at amino acid position 42 (AMBROZIAK 1994 ), were crossed to strains SH368 and SH372 carrying the sia1-1 or the sia2-1 mutation, respectively, in the INO4 genetic background (Table 2B). The high proportions of 3⁺:1^- and 4⁺:0^- tetrads recovered from these crosses indicate that the sia1-1 and the sia2-2 mutations can both suppress the inositol auxotrophy of the ino4-26 strain. In contrast, crosses of sia1-1- or sia2-1-bearing strains SH369 and SH376 to strain SH309 carrying an ino4 deletion mutation (ino4) produced only tetrads exhibiting a 2⁺:2^- segregation for inositol auxotrophy (Table 2B), indicating that neither sia1-1 nor sia2-1 can suppress the ino4 allele.

The sia1 and sia2 mutations can suppress an ino2 missense mutation, but not an ino2 allele:
Strains harboring sia1-1 or sia2-1 were crossed to a strain carrying the ino2-419 allele (a missense mutation in the loop region of the bHLH domain at amino acid 273; NIKOLOFF and HENRY 1994 ). A high proportion of tetrads exhibiting 3⁺:1^- and 4⁺:0^- ratios was observed, suggesting that neither sia1-1 nor sia2-1 is linked to INO2, but both are capable of suppressing the ino2-419 allele. Strains bearing the sia1-1 or the sia2-2 mutation were also crossed to a strain carrying an ino2 null allele. The observed 2⁺:2^- segregation for inositol auxotrophy (Table 2C) indicates that neither sia1-1 nor sia2-1 can suppress the ino2 mutation.

The growth characteristics of suppressor strains:
Growth of strains carrying the sia1-1 (Figure 1) and sia2-1 (data not shown) mutations was compared to growth of ino4-8 and wild-type strains in medium containing 10 µM inositol (derepressing, D) and 75 µM inositol (repressing, R). As expected, the suppressor-bearing strain SH352 (ino4-8 sia1-1) grew more rapidly and reached a higher optical density in D medium containing 10 µM inositol than did SH406 (ino4-8 SIA1 SIA2; Figure 1). However, the ino4-8 sia1-1 strain did not grow as rapidly as wild type or sia1-1 INO4 (SH368) in D medium (Figure 1). Similarly, the sia2-1 ino4-8 strain grew more rapidly than the ino4-8 (SH406) strain, but not as rapidly as wild type or SH374 (INO4 sia2-1) in D medium containing 10 µM inositol (data not shown). Strain SH374 (INO4 sia2-1) exhibited a growth rate comparable to wild type in either D or R medium (data not shown). However, SH368 (sia1-1 INO4) grew slightly more slowly and reached a lower optical density than wild type (SH155; Figure 1) in medium containing either 10 µM or 75 µM inositol.

View larger version (16K): In this window In a new window Download PPT slide   Figure 1. Growth of sia1-1 (SH368) and sia1-1 ino4-8 (SH352) strains compared to wild-type (SH155) and ino4-8 (SH406) strains in the presence and absence of inositol. Strains were inoculated from overnight cultures into synthetic complete medium with 75 µM inositol (R; ) and 10 µM inositol (D; ), as described in the MATERIALS AND METHODS. Growth at 30° was monitored by optical density using a Klett-Summerson spectrophotometer. The doubling times of each strain in each type of medium are shown in parentheses on the figure next to the medium designation (R or D).

INO1 gene expression in sia1 and sia2 strains:
Strains containing a single copy of an INO1-lacZ gene fusion at the URA3 locus were used to assay INO1 gene expression (Table 3). As in the growth experiments described above, medium containing a low amount of inositol (10 µM), which allows partial derepression of INO1, was used as the D growth condition and 75 µM inositol was used as the fully R growth condition (DONAHUE and HENRY 1981B ; HIRSCH and HENRY 1986 ). The wild-type strain (SH155) expressed ~155 units of activity in derepressing D medium (Table 3). In fully repressing R medium, the wild-type strain expressed <20 units of ß-galactosidase. Such repression of the INO1 reporter construct in response to high levels of inositol is consistent with previous reports (LOPES and HENRY 1991 ). Also consistent with previous reports (HIRSCH and HENRY 1986 ; HOSHIZAKI et al. 1990 ), the ino4-8 parental strain (SH406) exhibited no detectable ß-galactosidase activity under either D or R growth condition. In contrast to the ino4-8 parental strain, strains SH352 and SH357 (carrying the sia1-1 or the sia2-1 suppressor, respectively, in an ino4-8 genetic background) expressed measurable ß-galactosidase from the INO1 lacZ fusion in D medium (Table 3). The level of activity in D medium [~29 units in the case of SH352 (ino4-8 sia1-1) and 35 units in the case of SH357 (ino4-8 sia2-1)] was lower than the level observed in wild type (SH155), but higher than in the parental strain (SH406, ino4-8 SIA1 SIA2).

Expression of derepressed levels of ß-galactosidase from the INO1-lacZ reporter construct was observed in R medium in the sia1-1 INO4 strain (SH368). Constitutive expression, but at a lower level, was observed in the sia1-1 ino4-8 genetic background (strain SH352). Thus, the sia1-1 mutation results in constitutive expression of the INO1-lacZ reporter construct in both genetic backgrounds (i.e., ino4-8 or INO4). The sia1-1 mutant strains were tested for the Opi^- phenotype, but none of these strains was Opi^-.

In contrast to the constitutive pattern of INO1 expression observed in sia1-1-bearing strains, expression of the INO1-lacZ gene fusion was repressed in R medium in sia2-1-bearing strains (SH357 and SH374). Levels of ß-galactosidase activity observed in both D and R medium in the SH357 strain (ino4-8 sia2-1) were lower than the levels observed in SH374 (INO4 sia2-1; Table 3).

Expression of phospholipid biosynthetic structural genes in sia1-1 and sia2-1 strains:
The INO1, CHO1, CHO2, and OPI3 genes are coordinately regulated in response to inositol (PALTAUF et al. 1992 ). An autoradiogram of a Northern blot showing the expression of these transcripts in the wild type, sia1-1 INO4, and sia2-1 INO4 strains is shown in Figure 2. Under the derepressing growth condition employed in this study (10 µM inositol; D medium), the wild-type strain exhibited 4-fold derepression of INO1 compared to levels observed in cells grown under repressing conditions (R). This level of derepression is comparable to previous reports of INO1 expression in cells grown in the presence of 10 µM inositol, whereas 10-fold or greater derepression is typically seen in cells grown in completely inositol-free medium (HIRSCH and HENRY 1986 ). In D medium containing 10 µM inositol, the sia2-1 cells exhibited approximately 9-fold derepression of INO1, a much greater degree of derepression than observed in the wild-type strain (Figure 2). The sia2-1 strain also had a copy of the INO1-lacZ reporter construct integrated at the URA3 locus, and the transcript from this construct was regulated in a pattern comparable to the native INO1 transcript (Figure 2).

View larger version (58K): In this window In a new window Download PPT slide   Figure 2. Northern analysis of transcripts of genes, INO1, OPI3, CHO2, and CHO1, encoding enzymes subject to repression by inositol in wild-type, sia1-1 INO4, sia2 INO4, and reg1 INO4 strains. DR denotes partially derepressing growth condition (10 µM inositol), as described in MATERIALS AND METHODS. R denotes fully repressing growth condition (75 µM inositol). Ribosomal protein gene TCM1 is included as a control for RNA loading. Lanes from left to right: Lane 1, SH706 (reg1), 75 µM inositol; lane 2, SH706 (reg1), 10 µM inositol; lane 3, W303 (wild type), 75 µM inositol; lane 4, W303 (wild type), 10 µM inositol; lane 5, SH368 (sia1-1 INO4), 75 µM inositol; lane 6, SH368 (sia1-1 INO4), 10 µM inositol; lane 7, SH376 (sia2-1 INO4), 75 µM inositol; lane 8, SH376 (sia2-1 INO4), 10 µM inositol. Quantification of the Northern blot is depicted below the blot. The numbers were obtained by normalizing to the signal obtained with the TCM1 probe and are expressed as a proportion of the amount of the specific RNA present in wild-type cells grown under derepressing (D) growth conditions (i.e., the amount of each RNA in wild type normalized to TCM1 is set at 1.0 for each of the four probes). The repression ratio (D/R) was obtained by dividing the level of each transcript expressed in D medium by the level observed in R medium.

The OPI3, CHO1, and CHO2 transcripts, in general, exhibit much less dramatic repression ratios than INO1 and are not fully repressed in medium containing inositol unless choline is present in addition to inositol (BAILIS et al. 1987 ; GAYNOR et al. 1991 ). In the present study, no choline was added to the R growth medium and, consistent with previous reports, we observed that the OPI3, CHO1, and CHO2 transcripts showed much less repression than the INO1 transcript under these conditions in both wild-type and sia2-1 cells. Compared to the wild-type strain, the sia2-1 strain showed elevated derepression of all of these transcripts in D medium. The sia1-1 strain exhibited no significant repression of any of the transcripts tested under these conditions (Figure 2).

Analysis of INO1-lacZ expression in diploid strains:
The coregulated structural genes of phospholipid biosynthesis are also constitutively expressed in opi1 (HIRSCH and HENRY 1986 ), sin3 (HUDAK et al. 1994 ; SLEKAR and HENRY 1995 ), and dep1 (LAMPING et al. 1995 ) mutant strains. sia1-1-bearing strains were mated to strains carrying opi1, sin3, and dep1 mutations, and ß-galactosidase activity was assayed in the resulting diploids under both derepressing and repressing conditions (Table 4). The diploids all exhibited wild-type regulation in response to growth in R medium. Thus, the sia1-1 mutation complements the opi1, dep1, and sin3 mutations and is unlikely to be an allele of any of these loci. A plasmid containing the OPI1 gene was also transformed into sia1-1 strain and failed to complement the sia1 suppressor phenotype (data not shown).

Cloning of an sia1 complementing clone:
Strain SH700 (sia1-1 ino4-8) was transformed with a CEN-based genomic library and transformants were tested for their ability to grow in the absence of inositol. Because the sia1-1 mutation is recessive, we reasoned that the presence of the wild-type copy of this gene in a ino4-8 sia1-1 strain would render it auxotrophic for inositol. Therefore, we looked for those transformants that had lost their ability to grow in the absence of inositol upon transformation. Among 3600 transformants tested, only one showed the expected phenotype. Sequencing analysis of a portion of the genomic fragment containing the complementing activity (pMR1034), followed by a search of the Saccharomyces Genome Database, showed that this fragment maps to coordinates 494,689–504,734 on the right arm of chromosome IV (inserts numbered as in Saccharomyces Genome Database). This fragment contains five different open reading frames (YDR027–YDR031) of which only one corresponded to a previously characterized gene, REG1/HEX2/SRN1 (NEIGEBORN and CARLSON 1987 ; TUNG et al. 1992 ; NAIK et al. 1997 ). Subsequent subcloning of this ~10-kb fragment showed that the only complementing subclones were those that carried a full-length copy of the REG1 gene. Additional REG1 clones, kindly provided by M. Johnston and K. M. Arndt, further confirmed this observation. Thus, it appeared likely that the SIA1 gene was, in fact, REG1.

To explore this possibility further, we constructed a reg1 mutant as described in MATERIALS AND METHODS and examined its growth and other characteristics. The reg1 mutant exhibited a longer lag period than wild type or sia1-1 when inoculated into fresh medium. However, once it reached logarithmic phase, its growth was no more impaired than the growth of sia1-1 (which is shown in Figure 1). As previously described (FREDERICK and TATCHELL 1996 ), and similar to the sia1-1 mutant, the reg1 mutant grew slightly more slowly than wild type under all growth conditions employed in this study. The doubling time of the reg1 mutant in R medium containing 75 µM inositol was ~2.5 hr compared to approximately 3 hr for sia1-1 and ~2 hr for wild type. Unlike the sia1-1 INO4 mutant strain, however, the reg1 INO4 strain had a weak inositol excretion (Opi^-) phenotype. The reg1 strain also exhibited elevated constitutive expression of INO1, comparable to the expression pattern seen in sia1-1 cells (Figure 2).

Among 28 full four-spore tetrads recovered from a cross of SH703 (ino4-8) to SH703 (reg1), 17 exhibited 3⁺:1^- segregation of inositol auxotrophy, 4 showed 2⁺:2^- segregation, and 7 tetrads exhibited 4⁺:0^- segregation (Table 2D). Furthermore, all reg1 ino4-8 segregants grew in the absence of inositol. Thus, the null allele, reg1::URA3, has the same ability to suppress ino4-8 as does sia1-1 (Table 2A).

A reg1 ino4-8 strain (SH707) was crossed with a sia1-1 ino4-8 strain (SH352) and the diploid was found to be inositol prototrophic, indicating that the sia1-1 and reg1 mutations do not complement. A second diploid was constructed by crossing SH368 (sia1-1 INO4) with SH706 (reg1 INO4). Both diploids were analyzed for ß-galactosidase expression from the INO1-lacZ reporter gene (Table 4). In both cases, consistent with failure of sia1-1 and reg1 to complement, ß-galactosidase expression was constitutive (Table 4). However, the level of expression of the reporter construct was lower in the strain that was homozygous for ino4-8 (i.e., SH352 x SH707). This diploid sporulated poorly. Therefore, a diploid heterozygous for ino4-8 was produced by crossing strains SH368 (sia1-1) and SH707 (reg1::URA3 ino4-8). This strain was sporulated and dissected. Among 23 tetrads recovered from this cross, all showed 4⁺:0^- segregation for growth on inositol-free plates (Table 2D). In addition, all spore colonies were tested for resistance to 2-DG (LOBO and MAITRA 1977 ), a phenotype associated with reg1 mutants (TU and CARLSON 1995 ). The parental strains, sia1-1 and reg1, and all progeny from the cross exhibited 2-DG resistance. In all 23 tetrads, 2-DG resistance segregated 4^R:0^S resistant:sensitive. Thus, reg1 and sia1-1 are allelic and we have renamed sia1-1 as reg1-600.

The opi1 mutation cannot suppress ino4-8:
The observation that a REG1 null allele is able to suppress the ino4-8 growth phenotype indicates that the mechanism of suppression does not involve specific contact between the REG1 gene product and the mutant ino4-8 gene product. This caused us to question whether the absence of a negative regulator in the ino4-8 genetic background was sufficient to confer suppression. To determine whether mutations in the negative regulator encoded by the OPI1 gene would have a similar effect, a strain carrying the opi1 mutation (SH308) was crossed to strain SH703 (ino4-8). In all 38 tetrads (Table 2D), from which four surviving spores were recovered, 2⁺:2^- segregation for inositol auxotrophy was observed, indicating that opi1 cannot suppress ino4-8.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

INO1 expression is regulated not only in response to the availability of inositol in the growth medium, but also in response to growth phase (LAMPING et al. 1995 ; GRIAC et al. 1996 ; JIRANEK et al. 1998 ), ongoing phosphatidylcholine biosynthesis (GRIAC et al. 1996 ), and turnover (PATTON-VOGT et al. 1997 ; SREENIVAS et al. 1998 ). Specific regulatory genes shown to affect INO1 expression and regulation include INO2, INO4, and OPI1 (PALTAUF et al. 1992 ). Under derepressing conditions (absence of inositol), opi1 mutants excrete inositol (Opi^- phenotype) due to overexpression of the INO1 gene product, inositol-1-phosphate synthase (GREENBERG et al. 1982B ). opi1 mutants also fail to repress the INO1 gene in response to the presence of inositol or stationary growth phase signals (HIRSCH and HENRY 1986 ; WHITE et al. 1991 ; JIRANEK et al. 1998 ). The INO2 and INO4 loci encode transcriptional activators of the bHLH class which form a heterodimer that binds to the repeated element UAS_INO found in the INO1 promoter (AMBROZIAK and HENRY 1994 ; NIKOLOFF and HENRY 1994 ; BACHHAWAT et al. 1995 ). Deletion of either the INO2 or the INO4 locus causes inositol auxotrophy (Ino^- phenotype) which is not alleviated by mutations at the OPI1 locus (GRAVES 1996 ). However, the precise function of the OPI1 gene product (Opi1p) is not yet established and Opi1p does not appear to bind to DNA directly (GRAVES 1996 ).

Mutations at a large number of other loci produce Ino^- and Opi^- phenotypes, indicating defects in INO1 expression and/or regulation (reviewed in HENRY and PATTON-VOGT 1998 ). For example, mutations in RNA polymerase II (SCAFE et al. 1990A , SCAFE et al. 1990B , SCAFE et al. 1990C ), the TATA binding protein (ARNDT et al. 1995 ), and the global regulatory factors SWI1/ADR6, SWI2/SNF2, and SWI3 (PETERSON and HERSKOWITZ 1992 ; PETERSON et al. 1994 ; PETERSON and TAMKUN 1995 ) all cause inositol auxotrophy. In contrast, mutations in the SIN3 (HUDAK et al. 1994 ; SLEKAR and HENRY 1995 ) and UME6 (JACKSON and LOPES 1996 ) regulatory factors result in Opi^- phenotypes and/or constitutive expression of INO1. The Ino^- phenotypes of the SWI/SNF genes are caused by the dependence of INO1 transcription on the SWI/SNF complex (PETERSON et al. 1994 ; PETERSON and TAMKUN 1995 ), which is involved in chromatin remodeling. Furthermore, mutations in histone H4 can bypass the requirement for the SWI/SNF complex, thus permitting INO1 expression (SANTISTEBAN et al. 1997 ) in Swi^- or Snf^- strains. The Ino^- and Opi^- phenotypes described above are all associated with mutations that affect the general transcription apparatus and/or chromatin structure, suggesting that expression of the INO1 gene is highly sensitive to perturbation in the general state of cellular transcription.

In this study, we have demonstrated that mutations at the REG1 locus can also result in constitutive expression of the INO1 gene and that the reg1 mutation confers an Opi^- phenotype. The REG1 gene product has been shown to be necessary for repression of genes such as SUC2 which are under catabolite repression by the glucose response signal transduction pathway. The REG1 gene product is a regulatory subunit of Glc7p, a type 1 protein phosphatase that regulates the SNF1 kinase (TU and CARLSON 1994 , TU and CARLSON 1995 ). Another component of the glucose response pathway is SNF4, a regulatory subunit of the SNF1 kinase (CELENZA and CARLSON 1986 ; JIANG and CARLSON 1996 ). The SNF1 gene encodes a serine-threonine protein kinase required to activate those genes under catabolite repression (CELENZA and CARLSON 1986 ).

In addition to their effects upon glucose-repressible genes, snf1 mutations have Ino^- phenotypes (HIRSCHHORN et al. 1992 ). The INO1 gene and the other coregulated genes, CHO1, OPI3, and CHO2, are all constitutively expressed in the sia1-1 (reg1-600) mutant (Table 3; Figure 2). These data and the observation that snf1 mutants are Ino^- suggest that UAS_INO-containing genes require the action of the SNF1 kinase for their expression and the action of the REG1 gene product for their repression. Thus, INO1 expression and regulation appear to require a fully functioning glucose response pathway. It is curious that insulin regulation of FAS in mammalian cells acts through an E-box element (WANG and SUL 1997 ) homologous to UAS_INO. The yeast FAS (SCHULLER et al. 1995 ) and the acetyl Co-A carboxylase (ACC1) promoters (HASSLACHER et al. 1993 ) also contain UAS_INO sequences. Thus, it is tempting to speculate that the regulatory mechanisms controlling fat metabolism in response to glucose availability in animals and fungi might have had a common ancestry. However, INO1 and other coregulated genes of phospholipid biosynthesis are expressed and regulated by inositol in the presence of glucose, as shown by the experiments reported here, all of which were conducted in glucose-containing medium. Furthermore, in a related report, SHIRRA and ARNDT (1998) report that glucose has little effect on INO1 expression. Thus, it appears that the glucose response signal transduction cascade is required for INO1 expression and regulation. However, the REG1 gene product must function via a mechanism that is distinct from that by which it governs classical catabolite repressible genes such as SUC2, which is not measurably expressed in wild-type cells grown in the presence of glucose.

Furthermore, the expression of INO1 is influenced by at least one other signal transduction cascade. The ire1 and hac1 mutations (NIKAWA and YAMASHITA 1992 ; COX and WALTER 1996 ; NIKAWA et al. 1996 ; COX et al. 1997 ) have also been reported to cause inositol auxotrophy. The IRE1 locus encodes a protein kinase involved in the unfolded protein response pathway and HAC1 encodes a positive regulator of IRE1 (COX and WALTER 1996 ). Regulation of INO1 expression must, therefore, integrate inputs from several transduction pathways.

In an independent study, SHIRRA and ARNDT (1998) report the isolation of suppressors of the inositol auxotrophy of the spt15-328 mutation in the TATA binding protein (TBP). Consistent with our findings that reg1 mutants can suppress the inositol auxotrophy of certain ino4 and ino2 missense alleles, Shirra and Arndt isolated a recessive suppressor of the inositol auxotrophy of spt15-328 that proved to be an allele of REG1. They also identified a dominant suppressor that is an allele of SNF4. Shirra and Arndt also reported that one of their suppressors is an allele of OPI1. However, we found that the opi1 allele does not suppress the inositol auxotrophy of ino4-8 (Table 2D). One explanation for the different effects of opi1 mutants on the Ino^- phenotypes of the ino4-8 and spt15-328 mutations could be that Opi1p represses transcription of INO1 (and other genes whose transcription is dependent on the binding of the Ino2p/Ino4p complex) via an interaction with the TBP. Curiously, however, Shirra and Arndt report that the expression of INO1 transcript is regulated by inositol in the opi1 spt15-328 double mutant. Yet, in the opi1 SPT15 strain, INO1 expression is constitutive (SHIRRA and ARNDT 1998). This result suggests that Opi1p attenuates the level of transcription of INO1 via TBP but does not actually control the regulatory response to inositol.

If this hypothesis is correct, then the regulation in response to inositol by INO1 is mediated by regulatory factors working at a point in the regulatory cascade that precedes the steps mediated by both Opi1p and the TBP. The Ino2p/Ino4p complex is a possible target for such regulation. The ino4-8 mutation is a point mutation, glutamic acid to lysine at residue 79 in the loop region of the bHLH motif (AMBROZIAK 1994 ). The recessive suppressors isolated in this study, sia1-1 (reg600) and sia2-1, proved to have the unusual property that they suppressed not only the inositol auxotrophy of ino4-8 strains and ino4-26 (threonine to isoleucine in the basic region of the bHLH motif; AMBROZIAK 1994 ), but also ino2-419 (arginine to lysine in the loop region of the bHLH motif; NIKOLOFF and HENRY 1994 ). However, neither the sia1-1 nor the sia2-1 mutations were able to suppress either the ino4 or the ino2 mutation (Table 2). Thus, the suppression mechanism appears to depend on some residual function of the mutated Ino2p/Ino4p complex.

Cell extracts prepared from strains carrying ino2-419, ino4-8, or ino4-26 point mutations have very low or undetectable ability to heterodimerize and bind DNA as a complex (AMBROZIAK 1994 ; NIKOLOFF and HENRY 1994 ). However, when wild-type Ino2p is cotranslated in vitro with the ino4-8 or ino4-26 mutant gene products, the heterodimers formed have some residual ability to bind UAS_INO in vitro (AMBROZIAK 1994 ). The mutation in REG1, which deregulates the protein kinase activity of the SNF1 kinase, may lead to strengthening of the mutated residual Ino2p/Ino4p complex permitting it to function, at least partially, in vivo. We propose that Reg1p affects INO1 transcription, via its role in regulating the SNF1/SNF4 complex, by influencing the interaction of Ino2p/Ino4p with each other and, thus, with UAS_INO. This, in turn, affects the interaction of the INO1 promoter with the SPT15 gene product, TBP. opi1 mutations, including opi1, do not suppress ino4-8 and, thus, we believe that the mechanism of Opi1p action does not influence the activity of the Ino2p/Ino4p complex or its binding to UAS_INO. Because opi1 mutations suppress spt15-328, Opi1p could function as a mediator between TBP and its recruitment to UAS_INO by the active Ino2p/Ino4p complex.

	ACKNOWLEDGMENTS

We are indebted to Peggy Shirra and Karen Arndt for ongoing discussion and for providing strains during the progress of this work. This work was supported by a National Institutes of Health grant GM-19629 to S.A.H.

Manuscript received June 10, 1998; Accepted for publication February 10, 1999.

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